155
The influence of bacteriocin-producing probiotic starter cultures on fermentation time and
post-acidification in yoghurt
A thesis submitted for the degree of Master of Science by research
By
Alexandra Stevens B.Sc. (Honours) - Food Technology
2003
School of Molecular Sciences Victoria University
Werribee Campus, Victoria Australia
WER THESIS 637.1476 STE 30001008249122 Stevens, Alexandra The influence of bacteriocin-producing probiotic starter cultures
inoculaated for making yoghurt. The control yoghurt took 3 hours and 20 minutes to
ferment while the Sonicated yoghurt took 4 hours and 25 minutes. The pH of both
yoghurts decreased rapidly over the 4 weeks of storage with the control yoghurt
having the lowest pH. The control had a higher viable count of Z. delbrueckii subsp.
bulgaricus than the sonicated yoghurt. There was a high number of Z. acidophilus
and bifidobacteria in the inoculation and due to the short fermentation time the
increase in their population was only slight. The storage trial showed that the
probiotic bacteria survived the harsh conditions present in yoghurt. The organic acid
concentration was measured. The control batch of yoghurt produced more lactic,
butyric and propionic acid than the experimental batches.
Production of antimicrobial substances by Z. acidophilus was determined against Z.
delbrueckii subsp. bulgaricus. Four strains of Z. acidophilus (La-5, La2404, La2405
and La2406) were used as the producer organism for inhibitory activity against Z.
delbrueckii subsp. bulgaricus using modified spot on lawn and agar well-diffusion
techniques. Two strains of Z. delbrueckii subsp. bulgaricus (Lb2515, Lb2501) were
used as the indicator organism. The four strains of Z. acidophilus produced inhibitory
zones against one strain of Z. delbrueckii subsp. bulgaricus (Lb2515). These
inhibitory zones were confirmed to be bacteriocin as no zones appeared when treated
with proteolytic enzymes.
In order to determine lysis of Z. delbrueckii subsp. bulgaricus with bacteriocin
produced by Z. acidophilus, one strain of Z. acidophilus (La-5) was incubated with
one strain of Z. delbrueckii subsp. bulgaricus (Lb2515) at inoculation levels of 1%,
5% or 10% for 8 hours. Plate counts and P-galactosidase activity were measured.
During incubation, the Z. acidophilus counts increased and the Z. delbrueckii subsp.
bulgaricus counts decreased suggesting that Z. acidophilus was producing bacteriocin
against Z. delbrueckii subsp. bulgaricus. However Z. acidophilus did not inhibit Z.
delbrueckii subsp. bulgaricus enough to stop its growth. Therefore, it was thought,
that if the bacteriocin produced by Z. acidophilus could be concentrated and purified,
this could be added as a supplement to yoghurt, to inhibit growth of Z. delbrueckii
subsp. bulgaricus.
Z. acidophilus was inoculated in MRS broth and incubated for 18 hours. This was
then centrifuged and neutralised to pH 6.0. The broth was filtered using a 30kDa
ultrafiltration unit and was concentrated approximately 50 times, and the bacteriocin
was extracted and purified. This was then added to Z. delbrueckii subsp. bulgaricus at
different rates (1%, 5% and 10%) to examine if any inhibition occurred. The results
showed that concentrated bacteriocin inhibited Z. delbrueckii subsp. bulgaricus. The
10% sample had the lowest viable counts after 10 hours with a 5 log cycle difference.
The next lowest was the 5% sample which also had a 5 log cycle difference. The 1%
sample had a 3 log cycle difference.
The bacteriocin was incorporated in milk during inoculation the youghurt and
probiotic bacteria at \% and 2% levels. The fermentation time of all three yoghurts
was 3 hours. There seemed to be little difference in the growth of Z. delbrueckii
subsp. bulgaricus during fermentation between the three yoghurts. The 1% batch had
the highest viable count followed by the control and then the 2% batch. Z. acidophilus
and B. longum increased in number slightly during fermentation. The control yoghurt
had the highest number of Z. acidophilus followed by the 1% batch and the 2% batch.
The 2%) batch had the highest number of B. longum followed by the control and the
1% batch. During storage the pH dropped considerably in all yoghurts. The numbers
of Z. delbrueckii subsp. bulgaricus decline in all three yoghurts over the 6 weeks of
storage. The probiotic bacteria decreased during storage in all yoghurts.
The analysis of organic acids was performed using the HPLC. The lactic acid
production increased during fermentation and fluctuated during storage in all three
yoghurts. The l%o yoghurt batch had the highest concentration at the end of
fermentation followed by the control and the 2% yoghurt. From these experiments, it
was observed that the bacteriocin did not inhibit Z. delbrueckii subsp. bulgaricus in
the yoghurt. Therefore it was presumed that there was some substance blocking the
activity of bacteriocin in yoghurt.
Z delbrueckii subsp. bulgaricus (1%) was grown with 1%, 5% and 10% levels of
bacteriocin for 8 hours in 12% RSM. The results showed that the bacteriocin had no
effect on the growth of Z. delbrueckii subsp. bulgaricus bacteria.
Ill
2.6. J.2 Bacteriocins produced by bifidobacteria 28
2.6.1.3 Bacteriocins produced by Lactococcus and
Pediococcus. 29
2,6,2 Applications of bacteriocins 30
3.0 MATERIALS AND METHODS 31
3.1 Bacterial Strains 31
3.2 Maintenance of Microorganisms 31
3.3 Media Preparation 32
3.3.1 Peptone water 32
3.3.2 MRS agar and broth 32
3.3.3 Selective medium for Z. aci<iop/zi7w5 32
3.3.4 Selective medium for Lactobacillus delbrueckii subsp.
Bulgaricus 32
3.3.5 Selective medium for 5*.//zerwo/'/jz/Mi' 33
3.3.6 Selective medium for bifidobacteria 33
3.3.7 Preparation of serial dilution for spread and pour plating 33
3.4 Yoghurt Preparation 34
3.4.1 Yoghurt making in general 34
3.4.2 Yoghurt making using commercial bacteria 34
3.4.3 Yoghurt made with commercial bacteria and
Z. acidophilus ond Bifidobacterium infantis 1912 34
3.4.4 Yoghurt made with commercial bacteria and
Z. acidophilus 2Sidi Bifidobacterium longum 1941 36
3.4.5 Yoghurt made with sonicated Lactobacillus
delbrueckii subsp. bulgaricus 36
3.4.6 Yoghurt made with bacteriocin 36
3.5 Time Interval Specification 37
3.6 Analyses 37
3.6.1 pH 37
3.6.2 OD readings 37
3.6.3 Organic acid determination using HPLC 37
3.6.4 Assay of P-galactosidase 38
3.6.5 Microbiology analysis 39
Vlll
3.7 Detection and Assay of Inhibitory Activity 3 9
3.8 Concentrating and Purification of Bacteriocin 40
3.8.1 Concentration bacteriocin using ultra-filtration 40
3.8.2 Purification of bacteriocin 41
3.9 Lysis of Lactobacillus delbrueckii subsp. bulgaricus In
Different Media 41
3.9.1 Lysis of Lactobacillus delbrueckii subsp. bulgaricus
in MRS broth 41
3.9.2 Lysis of Lactobacillus delbrueckii subsp. bulgaricus
in RSM 42
3.9.3 Lysis of Lactobacillus delbrueckii subsp.
bulgaricus in RSM without casein 42
3.10 Growth Curves of Different Lactobacillus acidophilus 42
3.11 Sources of Chemicals, Reagents and Microbiological Media 43
3.11.1 Chemicals and reagents 43
3.11.2 Microbiological media 43
3.12 Equipment and Instmments 43
3.12.1 Anaerobic jars 43
3.12.2 pH 43
3.12.3 Centrifuge 43
4.0 RESULTS AND DISCUSSION 44
4.1 The Effects On Probiotic Bacteria in Yoghurt When
Grown With Commercial Yoghurt Strains 44
4.1.1 Growth characteristics of two different commercial
strains of Lactobacillus delbrueckii subsp. bulgaricus
and iS. thermophilus 44
4.1.2 The effect on probiotic bacteria in yoghurt when
fermented with commercial bacteria 45
4.L2.1 Organic acid production in yoghurt when
fermented with commercial yoghurt strains
and probiotic bacteria (L. acidophilus LA-5
andB. infantis 1912) 46
4.1.2.2 Conclusion 48
ix
4.1.3 Probiotic bacteria in yoghurt using Bifidobacterium
longum 1941 48
4.1.3.1 Organic acid production in yoghurt when
fermented with commercial yoghurt strains
and probiotic bacteria (L. acidophilus and B.
longum 1941) 50
4.1.3.2 Conclusion 51
4.2 The Effect of Sonicating Lactobacillus delbrueckii subsp.
bulgaricus on the Survival of Probiotic Bacteria in Yoghurt 51
4.3 Antimicrobial Substances Produced by Yoghurt and Probiotic
Bacteria 54
4.3.1 Growth characteristics of Lactobacillus acidophilus 54
4.3.2 Screening of Lactobacillus acidophilus against
Lactobacillus delbrueckii subsp. bulgaricus for
bacteriocin production 55
4.3.3 Determinationof inhibitory substance 56
4.3.4 Antagonism between yoghurt and probiotic bacteria 56
4.4 Assessment of ViabiUty of Lactobacillus delbrueckii subsp.
bulgaricus Grown With Various Sizes of Inoculum of
Lactobacillus acidophilus 58
4.5 Purification of Bacteriocin 60
4.6 Concentrated Bacteriocin Grovm With Lactobacillus
delbrueckii subsp. bulgaricus 61
4.7 Bacteriocin Incorporated in Yoghurt Production 63
4.7.1 pH and viable counts 63
4.7.2 Organic acid analysis 66
4.8 Bacteriocin in Milk 69
4.8.1 Bacteriocin in different levels of milk 69
4.8.2 Bacteriocin in different media 70
5.0 CONCLUSION 126
6.0 FUTURE DIRECTION 130
7.0 REFERENCES 131
LIST OF TABLES
Table No. Title Page No. 1 Health benefits of probiotic bacteria 16
2 Yoghurt made with two different commercial strains of 71 Lactobacillus delbrueckii subsp. bulgaricus (Mild and Robust) and S. thermophilus
3 The effect on probiotic bacteria (Z. acidophilus LA-5 and B. 72 infantis 1912) during fermentation when grown with two commercial yoghurt strains.
4 The effect on probiotic bacteria (Z. acidophilus LA-5 and B. 73 infantis 1912) during storage when grown with two commercial yoghurt strains.
5 The concentration of organic acids in yoghurt when fermented with 74 2 different commercial yoghurt strains and Probiotic bacteria (Z. acidophilus LA-5 and B. infantis).
6 The concentration of organic acids in yoghurt during storage when 75 fermented with 2 different commercial yoghurt strains and probiotic bacteria (Z. acidophilus LA-5 and .5. infantis 1912).
7 The effect on probiotic bacteria (Z. acidophilus LA-5 and B. 76 longum 1941) during fermentation when grown with two commercial yoghurt strains.
8 The effect on probiotic bacteria (Z. acidophilus LA-5 and B. 11 longum 1941J during storage when grown with two commercial yoghurt strains.
9 The concentration of organic acids in yoghurt when fermented witl 78 different Commercial yoghurt strains and probiotic bacteria (Z. acidophilus LA-5 and 5. longum 1941).
10 The organic acid concentration in yoghurt during storage when 79 fermented with 2 different Commercial yoghurt strains and probiotic bacteria (Z. acidophilus LA-5 and 5. longum 1941).
11 Concentration of organic acids in commercial yoghurts. 80
12 The effect of sonicating Lactobacillus delbrueckii subsp. 81 bulgaricus 2515 cultures has on fermentation time and the survival of S. thermophilus, L. acidophilus and B. infantis 1912 during fermentation and storage.
xi
13 The effect of sonicating Lactobacillus delbrueckii subsp. 82 bulgaricus 2515 cultures has on production of organic acids in yoghurt during fermentation by probiotic and yoghurt bacteria
14 The effect of sonicating Lactobacillus delbrueckii subsp. 83 bulgaricus 2515 cultures has on organic acid content in yoghurt during storage
15 Average zone of inhibition produced by Z. acidophilus (LA-5, 84 LA-2404, LA-2405, LA-2406) against target organism Z. delbrueckii subsp. bulgaricus (LB-2501 and LB-2515)
16 Sensitivity of bacteriocin produced by Z. acidophilus LA-5 against 85 Z. delbrueckii subsp. bulgaricus 2515 a target organism to various enzymes and pH.
17 Antagonism between yoghurt and probiotic bacteria 86
18 Nature of inhibitory substance produced by yoghurt and probiotic 87 bacteria to various enzymes and pH.
19 Changes in viable counts when Lactobacillus delbrueckii subsp. 88 bulgaricus is grown with different inoculum sizes of Z. acidophilus
20 Changes in viable counts of Lactobacillus delbrueckii subsp. 89 bulgaricus when grown with different levels of concentrated bacteriocin.
21 The effect on viable counts when growing Lactobacillus 90 delbrueckii subsp. bulgaricus with different levels of bacteriocin (1' ' Replicate).
22 The effect on viable counts when growing Lactobacillus 91 delbrueckii subsp. bulgaricus with different levels of bacteriocin (2"" Replicate).
23 The effect on viable counts when growing Lactobacillus 92 delbrueckii subsp. bulgaricus with different levels of bacteriocin (3'"'' Replicate).
24 Effects of incorporation of bacteriocin on survival of ZactoftaczY/MJ' 93 delbrueckii subsp. bulgaricus, S. thermophilus, L. acidophilus and bifidobacteria during fermentation of yoghurt.
25 Effects of incorporation of bacteriocin on survival of Lactobacillus 94 delbrueckii subsp. bulgaricus, S. thermophilus, L. acidophilus and B. longum during weeks 1-3 of storage.
xn
26 Effects of incorporation of bacteriocin on survival of Lactobacillus 95 delbrueckii subsp. bulgaricus, S. thermophilus, L. acidophilus and B. longum during weeks 4-6 of storage.
27 The effects of incorporating bacteriocin in yoghurt on production 96 of organic acid during fermentation.
28 The effects of incorporating bacteriocin in yoghurt on production 97 of organic acids during storage.
29 1% Lactobacillus delbrueckii subsp. bulgaricus grown with 98 different inoculation of bacteriocin in 12% reconstituted skim milk.
30 Effects of bacteriocin produced from Z. acidophilus LA-5 on 99 Lactobacillus delbrueckii subsp. bulgaricus 2515 after growing in different levels of milk.
31 The effects of bacteriocin on the viable counts of Lactobacillus 100 delbrueckii subsp. bulgaricus when grown in 12%) and 3%) skim milk.
32 The effect bacteriocin has on viable counts of Lactobacillus 101 delbrueckii subsp. bulgaricus when grown in different media.
Xlll
LIST OF FIGURES
Figure Title Page No. No.
1 Outline of the stimulation of grov^h of yoghurt bacteria 12
2 Standard procedure for preparation of yoghurt. 35
3 Growth curve of 4 strains of Z. acidophilus over 18 hours as measured 102 by optical density.
4 Changes in cell density of Z. acidophilus and Lactobacillus delbrueckii 103 subsp. bulgaricus when grown together and separately.
5 Changes in p-galactosidase concentration when Lactobacillus 104 delbrueckii subsp. bulgaricus is grown with different inoculation sizes of Z. acidophilus.
6 Changes in viable counts of Z. acidophilus when grown with 105 Lactobacillus delbrueckii subsp. bulgaricus.
7 Changes in viable counts of Lactobacillus delbrueckii subsp. 106 bulgaricus when grown with different inoculum sizes of Z. acidophilus.
8 Zones of inhibition of Z. acidophilus against Z. delbrueckii subsp. 107 bulgaricus.
9 Zones of inhibition of Z. acidophilus against Lactobacillus delbrueckii 108 subsp. bulgaricus, before filtration, cells removed, neutralised to pH 6.0 and treated with catalase.
10 Zones of inhibition of Z. acidophilus ago-insi Lactobacillus delbrueckii 109 subsp. bulgaricus before filtration, treated with proteolytic enzymes.
11 Zones of inhibition of the concentrate obtained by Z. acidophilus 110 against Z. delbrueckii subsp. bulgaricus after passing through a 30kDa MWCO membrane.
12 Zones of inhibition of the permeate obtained by Z. acidophilus against 111 Z. delbrueckii subsp. bulgaricus after passing though a 30 kDa MWCO membrane.
13 Zones of inhibition of the purified bacteriocin suspended in sodium 112 carbonate before dialysis against Z. delbrueckii subsp. bulgaricus.
14 Zones of inhibition of the purified bacteriocin solution after dialysis 113 against Z. delbrueckii subsp. bulgaricus.
15 Zones of inhibition of autoclaved purified bacteriocin solution against 114 Z. delbrueckii subsp. bulgaricus.
xiv
16 Effects of purified bacteriocin (5%) on the growth of Lactobacillus 115 delbrueckii subsp. bulgaricus.
17 Changes in viable counts of Lactobacillus delbrueckii subsp. 116 bulgaricus when grown with different levels of concentrated bacteriocin.
18 Effects on acetic acid production when bacteriocin is incorporated in 117 yoghurt.
19 Effects on butyric acid production when bacteriocin is incorporated in 118 yoghurt.
20 Effects on formic acid production when bacteriocin is incorporated in 119 yoghurt.
21 Effects on lactic acid production when bacteriocin is incorporated in 120 yoghurt.
22 Effects on orotic acid production when bacteriocin is incorporated in 121 yoghurt.
23 Effects on production of propionic acid when bacteriocin is 122 incorporated in yoghurt.
24 Effects on uric acid production when bacteriocin is incorporated in 123 yoghurt
25 1% Lactobacillus delbrueckii subsp. bulgaricus grown with different 124 levels of bacteriocin in 12% RSM
26 The effect bacteriocin has on viable counts of Lactobacillus 125 delbrueckii subsp. bulgaricus when grown with different media
XV
1.0 INTRODUCTION
Consumers are becoming more aware of maintaining their "internal health". The
consumer trend is moving towards taking a preventative approach rather than a
curative approach to modem health problems. Hence, probiotic fiinctional foods are
becoming increasingly popular in the diets of people in Australia, and in other parts of
the Western world. The number of probiotic foods is currently much greater in
Japanese and European markets than in Australia. Therefore, there is potential in
marketing opportunities in AustraUa and production is expected to grow rapidly.
However, studies (HuU et al, 1984; Shioppa et al, 1981; Shah et al, 1995; Shah et
al, 2000) have shown that probiotic bacteria are unstable in yoghurt. Recent surveys
conducted in Australia (Anon 1992; Rybka and Fleet, 1997; Shah et al, 1995; Shah et
al., 2000) and in Europe (Iwana et al., 1993) have shown low viability of probiotic
organisms in commercial preparations which have created a negative image about
these products. The loss of viability is claimed to be due to a variety of factors
including H2O2 and acid produced by yoghurt bacteria, dissolved O2 level in the
product (Dave and Shah, 1997) and lack of nutrients in milk to sustain their growth.
By enhancing the viability and survival of probiotic bacteria, confidence of the
consumers in "probiotic health foods" can be restored.
A number of health benefits associated with the consumption of products containing
live probiotic bacteria such as Lactobacillus acidophilus and Bifidobacterium have
been claimed. Yoghurt starter bacteria {Lactobacillus delbrueckii subsp. bulgaricus
and Streptococcus thermophilus) do not survive or colonise in the gastrointestinal
tract. Hence probiotic organisms are often incorporated into fermented milk products
such as yoghurt. However, propagation of probiotic bacteria, in particular
Bifidobacterium is difficult, as the organisms are fastidious and their numbers decline
during storage due to post-acidificafion by yoghurt bacteria, in particular by Z.
delbrueckii subsp. bulgaricus (Dave and Shah, 1998a). As a result, many yoghurt
manufacturers use starter cultures devoid of Z. delbrueckii subsp. bulgaricus but
containing a mixture of Z. acidophilus. Bifidobacterium and S. thermophilus (ABT
cultures). However, use of ABT starter cultures increases fermentation time
significantly, which is undesirable, given the rigid schedule in modem yoghurt
manufacturing. Z. delbrueckii subsp. bulgaricus produces proteolytic enzymes and is
found to support the growth of probiotic bacteria during fermentation by releasing
growth factors such as amino acids and peptides by hydrolysing milk proteins.
However, their presence is undesirable as the organisms are responsible for post-
acidification which results in loss of viability of probiotic bacteria (Dave and Shah,
1998a).
Bacteriocins are proteinaceous compounds produced by lactic acid bacteria that kill or
inhibit closely related bacteria (Tagg et al., 1976). There is a tremendous interest in
bacteriocins and their role as a preservative in minimally processed foods. They have
been found to inhibit food-borne pathogens such as Clostridium botulinum and
Listeria monocytogenes, the latter being able to grow at refirigeration temperatures
(Montville and Kaiser, 1993). As bacteriocins are protein and "natural", this will
satisfy consumer demand for "fresh" and "preservative free" products.
To date, several Z. acidophilus strains have been studied for the production and
isolation of bacteriocins (Barefoot and Klaenhammer, 1984; Reddy et al., 1983; Toba
et al., 1991) and their antimicrobial effects against various pathogens, and to
understand the role of these organisms. These studies have shown that some strains of
Z. acidophilus produce bacteriocins against Z. delbrueckii subsp. bulgaricus. In a
study by Dave and Shah (1998b), Z. acidophilus produced bacteriocin against seven
strains of Z. delbrueckii subsp. bulgaricus.
This project aimed to develop a process for using the beneficial effects of traditional
yoghurt starters (Z. delbrueckii subsp. bulgaricus and S. thermophilus) for quick
fermentation and enhancing viability of probiotic organisms through the control of
post-acidification using controlled lysis of Z. delbrueckii subsp. bulgaricus by
bacteriocin producing strains of Z. acidophilus. Specifically the aims of the project
were:
1. To investigate ways of lysing Lactobacillus delbrueckii subsp. bulgaricus to
release sufficient growth factors to sustain the growth of selected starter sfrains
of iS". thermophilus in order to reduce fermentation time of the probiotic
organisms (Z. acidophilus ^ind Bifidobacterium sp.).
2. To investigate whether bacteriocin producing strains of Z. acidophilus can be
effectively used to lyse cells of Z. delbrueckii subsp. bulgaricus in situ in order
to control post-acidification,
3. To trial bacteriocin producing strains of Z. acidophilus, together with Z.
delbrueckii subsp. bulgaricus, S. thermophilus and Bifidobacterium sp. in
commercial yoghurt production to evaluate the (a) lysis of Z. delbrueckii
subsp. bulgaricus, (b) viability and survival of Z. acidophilus and
Bifidobacterium sp., (c) reduction in fermentation time and (d) control of post-
acidification.
Chapter 1 and 2 contain a review of literature, Chapter 3 explains the materials and
methods used and Chapter 4 presents and discusses the results. Chapter 5 gives a
summary of results and Chapter 6 and 7 discuss fiiture research direction and a list of
references, respectively.
2.0 LITERATURE REVIEW
2.1 Yoghurt
2.1.1 History
No one knows exactly when or how yoghurt originated, but apparently when the goat
was first domesticated in Mesopotamia about 5000 BC, its milk was stored warm due
to the hot climate and naturally formed a curd. Someone with sufficient courage
tasted this curd and rendered a favourable verdict (Kosikowski, 1997). The practice
of souring milk was eventually refined and incessant curiosity about the agents
causing fermentation, led to the discovery of bacteria (Kosikowski, 1997).
2.1.2 Yoghurt around the world
Yoghurt is very popular in Asia and in many countries such as Iran, fraq and Turkey,
it possesses an importance unequalled elsewhere. Yoghurt is made mainly from
sheep's milk or a combination of sheep and goat's milk and incubated by placing a
blanket over the pan (Kosikowski, 1997). In Chang Tang region of Tibet the Phala
nomads use yak, sheep and goat milk for making yoghurt, which can be churned into
butter, which is added to salty tea and drunk as a nutritious beverage forty times a day
(Kosikowski, 1997).
In certain countries fermented milk foods are favoured over fresh milk for reasons of
safety, better flavour, texture and possible beneficial therapeutic effects. In countries
where inadequate transport, pasteurization and refrigeration facilities exist,
particularly tropical countries, many authorities prefer to sour the milk first. In this
manner the presence of lactic acid bacteria and their metabolic end products
discourage growth of food poisoning and disease producing bacteria.
In Western countries yoghurt appears in different forms. It is eaten as a dessert, a
snack between meals, a complete lunch or as a diet and health food. It is often
flavoured with Suit, honey or vanilla and is available in its natural form (Kosikowski,
1997). The increase in consumption of yoghurt in the Western world owes much to
the development of its health food image (Early, 1998). Marketing strategies have
concentrated on the availability of reduced/lower fat content, calorific content,
extended shelf-life, additive free yoghurt, health promotion of probiotic bacteria and
children's yoghurt, which are milder and sweeter in taste (Early, 1998).
2.1.3 Characteristics
Yoghurt is a fermented milk food that is produced from milk or skim milk plus a
starter culture containing two bacteria {Streptococcus thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus) that produce lactic acid. According to Australian
Standard H8, yoghurt should have a pH 4.5 or below and a titratable acidity greater
than 0.9%) expressed as lactic acid. The delicate flavour in plain yoghurt is achieved
through a bacterial relationship influenced by such factors as acid concentration.
Other factors include volatile flavour components in small amounts such as acetic
acid, diacetyl and acetaldehyde. The latter produced by Z. delbrueckii subsp.
bulgaricus, is a major contributor to the unique flavour of yoghurt (Kosikowski,
1997). Yoghurt has a smooth, light gel texture, however, it can also be produced as a
liquid beverage and a solid frozen dessert.
Yoghurt is often flavoured with fruit, honey and essences and may be dyed with
acceptable food dyes. Stabilisers are often added to give smoothness characteristics,
but no salt is added to yoghurt.
Generally the fat content is about 3%) in Australian yoghurt, when it is made with
whole milk but there are many varieties of reduced fat yoghurt which have 1.7%, and
"no fat" yoghurt, which has a fat content below 0.1%. Yoghurt that is higher in fat
has a much smooth texture and richer flavour than the low fat varieties. The texture
of yoghurt does vary between varieties and with the yoghurt is manufactured.
2.1.4 Manufacture of yoghurt
The manufacturing process of yoghurt can vary from one manufacturer to another.
The composition can vary, as do yoghurt types and types of starter cultures used,
which therefore, affect the principles of manufacture. In this section the two main
types of yoghurt making; set and stirred, will be discussed. However, firstly it is
important to consider the types of cultures that are used in yoghurt making.
2.1.4.1 Yoghurt cultures
The general fiinction of any starter culture should be to produce sufficient lactic acid
in as short a time as possible to ferment milk to pH 4.5 and to give acceptable texture,
viscosity and flavour in the final product. Most commercial manufacture involves the
use of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.
These two bacteria have a relationship that is termed symbiosis (Early, 1998). S.
thermophilus initiates the fermentation process by stimulation of peptides and free
amino acids released from the milk proteins by Z. delbrueckii subsp. bulgaricus. The
lactobacilli in turn are stimulated by formic acid produced by S. thermophilus (Early,
1998).
Streptococci dominate the early stage of yoghurt fermentation. As the redox
potential of the milk medium is reduced and the pH lowered from 6.5 to 5.5, growth
of Z. delbrueckii subsp. bulgaricus is enhanced. Below pH 5.0, lactobacilli dominate
yoghurt fermentation and produce acetaldehyde and lactic acid, yielding the
characteristic yoghurt flavour. Continued acid production lowers yoghurt pH to near
4.6, which induces clotting.
Other bacteria such as Lactobacillus helveticus, and probiotic bacteria such as
Lactobacillus acidophilus, bifidobacteria and Lactobacillus casei are also used in
yoghurt as a replacement for Z. delbrueckii subsp. bulgaricus or as a therapeutic
starter culture.
Lactobacillus delbrueckii subsp. bulgaricus is a robust culture and continues to
ferment during cooling leading to excessive lactic acid production. This results in a
very sour taste. The trend has been to move away from Lactobacillus delbrueckii
subsp. bulgaricus because of the unfavourable flavour and replace it with what is
called an 'ABT' culture which consists of Z. acidophilus, bifidobacteria and S.
thermophilus. Another popular starter culture is 'ABC, which consists of Z.
acidophilus, bifidobacteria and Z. casei. These cultures (ABT and ABC) are used as
adjunct starter cultures, while yoghurt bacteria are used as a primary culture.
Yoghurt cultures {Lactobacillus delbrueckii subsp. bulgaricus and S. thermophilus)
are not natural inhabitants of the intestine and cannot survive under acidic conditions
and bile concentration encountered in the gastrointestinal tract. Therefore, probiotic
bacteria (Z. acidophilus and bifidobacteria) are added to yoghurt to give therapeutic
benefits. Probiotic bacteria will be discussed later in this chapter.
2.1.4.2 Processing
As discussed previously there are many variations in the manufacture of yoghurt. In
this section the general process of yoghurt making will be described paying particular
attention to set and stirred type yoghurts. Set yoghurt is fermented in its retail
container and is undisturbed forming a semi-solid mass. Stirred yoghurt is fermented
in a vessel and the coagulum is broken during the cooling and packaging stage, giving
a very smooth texture (Early, 1998).
For making yoghurt, milk is heated to approximately 85°C for 30 minutes. This step
has several aims: it eliminates or reduces levels of food spoilage microorganisms and
reduces the total microbiological population to a level, which will not compromise the
growth of the starter culture microorganisms. The most important factor in this heat
treatment is that it denatures the whey proteins, P-lactoglobulin and a-lactalbumin, in
order to improve the texture and viscosity of the final product and to assist in the
prevention of whey separation during shelf Hfe (Kosikowski, 1997; Early, 1998).
After heat treatment, the milk is cooled to a suitable temperature of 45°C for
inoculation. At this point, fruit can be added to containers for set type yoghurt. The
warm inoculated milk is then poured on top of the fruit mix and allowed to incubate.
For stirred type yoghurt, the fermentation is carried out in a large vessel, and fhiit is
added after fermentadon (Kosikowski, 1997; Early, 1998). Fermentation is carried
out for both types of yoghurt at 42°C until the pH reaches 4.5, during which the curd
should not be disturbed. The yoghurt curd or 'coagulum' begins to form as more
lactic acid is produced and the iso-electric point of casein (pH 4.6-4.7) is approached
(Early, 1998). Fermentation time does vary between 6-12 hours depending on the
bacteria, batch size and milk season.
For stirred yoghurt, once fermentation time is completed, the curd is broken using
slow speed paddle agitation for no more than 5-10 minutes (Vamam, 1994). This
produces the required body and texture and the yoghurt is then cooled in a two-stage
process. The first stage is to cool the yoghurt to 15-20°C, at which point, fhiit or
other flavours can be added (Vamam, 1994). Second stage cooling is below 5°C,
which is achieved in a cold store (Vamam, 1994). Cooling should be carefully
controlled, since too rapid cooling leads to syneresis or 'wheying off. The cooled,
stirred yoghurt is then filled into containers and must remain chilled until it reaches
the consumer. Yoghurt has a shelf Hfe of approximately 42 days. In the case of set
yoghurt, cooling takes place inside the retail container and is started before the final
pH is reached. Care must be taken when transferring the yoghurt, as excessive
agitation reduces the viscosity and can also cause syneresis (Vamam, 1994).
There are other types of yoghurt, such as thermised yoghurt, which once fermented
the yoghurt is pasteurised to kill all yoghurt bacteria. This increases the shelf life
considerably, often up to 12-15 weeks. However, this practice is not permissible in
Australia as the product is defined as containing live micro-organisms at
commercially viable levels on consumption (Early, 1998).
Drinking yoghurt is manufactured much the same way, but has lower total soUds and
undergoes homogenisation to ftirther reduce viscosity (Vamam, 1994). Frozen
yoghurts may be prepared from conventional set or stirred yoghurts, although there
will be a higher level of sugar and stabilisers, required to maintain the coagulum
during freezing and storage and a small quantity of cream may be added to improve
'mouth-feel'. The yoghurt is frozen in a blast freezer (-20°C) or frozen with aeration
in an ice-cream freezer (Vamam, 1994).
2.2 Yoghurt Bacteria
Yoghurt bacteria include Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus thermophilus. These bacteria are commonly used in the production of
yoghurt and are part of the Lactic acid bacteria group. The characteristics of lactic
acid bacteria and the genus Lactobacillus and Streptococcus are discussed next.
2.2.1 Lactic acid bacteria
Lactic acid bacteria (LAB) are a group of gram-positive bacteria united by a
constellation of morphological, metabolic and physiological characteristics. The
general description of the bacteria included in the group is gram positive, nonsporing,
nonrespiring cocci or rods, which produce lactic acid as the major end product during
the fermentation of carbohydrates (Sahninen and von Wright, 1993). The genera
included in this group are Aerococcus, Carnobacterium, Enterococcus, Lactobacillus,
Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus and
Vagococcus (Salminen and von Wright, 1993).
2.2.2 Genus Lactobacillus
The members of the genus Lactobacillus are gram-positive, non-sporing baciUi, which
can vary from slender long rods to short ones. Lactobacilli have complex nutritional
requirements including energy, which they derive via homo-or heterofermentative
catabolism of carbohydrates, a carbon source, and a variety of nucleotides, amino
acids and vitamins for growth (Hoover and Steenson, 1993; Wood, 1992). This genus
has been subdivided into three subgenera determined by the fermentation end
products. Homofermentative lactobacilli exclusively ferment hexose sugars to lactic
acid by the Embden-Meyerhof pathway. They do not ferment pentose sugars or
gluconate. This group includes Lactobacillus delbrueckii subsp. bulgaricus,
Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, which are starter
cultures. They grow at higher temperatures (>45°C) than lactobacilli in the other
groups and are thermoduric. Z. acidophilus is also a member of this group but is not a
starter culture organism (Marth and Steele, 1998).
Facultatively heterofermentative lactobacilli ferment hexose sugars, either only to
lactic acid or to lactic acid, acetic acid, ethanol and formic acid when glucose is
limited. Pentose sugars are fermented to lactic and acetic acid via the
phosphoketolase pathway. This group includes Lactobacillus casei, which is not
usually used as a starter culture but has beneficial secondary fermentation during
cheese ripening (Marth and Steele, 1998).
Obligately heterofermentative lactobacilli ferment hexose sugars to lactic acid, acetic
acid or ethanol and carbon dioxide using the phosphoketolase pathway. Pentose
sugars are also fermented using this pathway. These lactobacilli can cause
undesirable flavour and gas formation during ripening of cheese. Species include
Lactobacillus kefir (Marth and Steele, 1998).
The organic acids that are produced (lactic and acetic) serve directly as antagonists to
other competing microflora by lowering the pH of the surrounding environment. This
decrease in pH of their environment allows the lactobacilli to effectively compete and
ultimately dominate fermenting ecosystems, since they are more acid tolerant than
other organisms, including many pathogenic and spoilage species, as well as other
lactic acid bacteria (Hoover and Steenson, 1993).
Lactobacilli are widespread in nature and are found in the oral cavity, gastrointestinal
tract, and vagina of humans and animals (Salminen and von Wright, 1993). Many
species of lactobacilli have found applications in the food industry. They are
generally the most acid-tolerant of the LAB and therefore, will terminate many
spontaneous lactic fermentation such as silage and vegetable fermentation.
The species of Lactobacilli, that are important in this project, are Lactobacillus
delbrueckii subsp. bulgaricus and Lactobacillus acidophilus, which belong to the
Thermobacterium group. Lactobacillus delbrueckii subsp. bulgaricus is gram-
positive with very slender and long rods. It has an optimum temperature of 42°C and
grows best under anaerobic conditions; however, it is considered a facultative
anaerobe. It is the bacterium responsible for the production of acetaldehyde, the main
contributor of the characteristic flavour in yoghurt. It is commonly grovm in deMan
Rogosa and Sharpe (MRS) broth and in reconstituted skim milk (RSM) with glucose
and yeast extract and selective media is MRS broth, pH 5.2. Lactobacillus
acidophilus will be discussed later in this chapter.
10
2.2.3 Genus Streptococcus
The members of this genus are gram-positive cocci, forming pairs and chains of cells
when cultured in liquid media (Wood, 1992). The sfreptococci have complex
nutritional requirements that vary between species but do involve amino acids,
peptides, purines, pyrimidines and vitamins as growth factors. Carbohydrates are
fermented with major production of lactic acid and minor amounts of acetic and
formic acids, ethanol and carbon dioxide (Wood, 1992).
Streptococcus thermophilus is the strain used for yoghurt. Its optimum growth
temperature is 37°C and grows aerobically. It can be grown in MRS broth but is
better suited to Ml7 broth in which it can be grown selectively. It also grows well in
RSM with glucose and yeast extract.
2.3 Yoghurt bacteria in yoghurt
S. thermophilus initiates the fermentation process by stimulation of peptides and free
amino acids released from the milk proteins by Z. delbrueckii subsp. bulgaricus the
main amino acid being valine. Milk contains too little of these amino acids and the
cocci which are very weakly proteolytic, form the acids too slowly. S. thermophilus
enhances the growth of the rods by forming formic acid out of pyruvic acids under
anaerobic conditions and by a rapid production of carbon dioxide (Walstra et al,
1999). Figure 1 shows an outline of the stimulation of the growth of yoghurt bacteria
in milk.
11
Lactic acid
Formic acid Peptides + amino acids
Streptococcus thermophilus Lactobacillus delbrueckii subsD. bulearicus
Milk
Figure 1. Outline of the stimulation of growth of yoghurt bacteria
(Source: Walstra e a/., 1999)
As the redox potential of the milk medium is reduced and the pH lowered from 6.5 to
5.5, growth of Z. delbrueckii subsp. bulgaricus is enhanced. Below pH 5.0,
lactobacilli dominate yoghurt fermentation and produce acetaldehyde and lactic acid,
yielding the characteristic yoghurt flavour. Continued acid production lowers yoghurt
pH to near 4.6, which induces clotting. High quality yoghurts have a pH of 4.2 to 4.3
at the time of consumption and possess proper taste and aroma.
2.4 Post-acidiflcation in yoghurt
During refiigerated storage, the lactobacilli in yoghurt continue to produce acid and
pH is lowered to <4.0, which results in an excessively sour product. This process is
known in the industry as "post-acidification". The extra lactic acid produced causes
the pH to decrease further, which makes the environment unsuitable for the survival
of probiotic bacteria and gives a very sour flavour, which is not desirable.
The pH also decreases during storage, due to ongoing metabolic activity of yoghurt
bacteria, in particular by Z. delbrueckii subsp. bulgaricus. It is for this reason that
many yoghurt manufacturers use starter cultures devoid of Z. delbrueckii subsp.
bulgaricus but containing Z. acidophilus. Bifidobacterium sp. and S. thermophilus
12
(ABT). However, the use of ABT starter cultures alone, increase fermentation time
significantly, which is undesirable, given the rigid schedule in modem yoghurt
manufacturing.
Pasteurising the finished yoghurt, to destroy viable starter culture bacteria, can also
prevent post-acidification however, pasteurisation of yoghurt is not permitted in
Australia.
Lactobacillus delbrueckii subsp. bulgaricus does have benefits to the yoghurt making
process. If it is excluded the fermentation process becomes very slow and increases
the cost of manufacture. The bacteria also produces proteolytic enzymes that is found
to support the growth of probiotic bacteria (Z. acidophilus and Bifidobacterium)
during fermentation by releasing growth factors such as amino acids and peptides, and
also stimulates production of acetaldehyde, acetic acid, diacetyl and lactic acid.
Supplementation with exogenous nutrients such as yeast extract, tryptone or deMan,
Rogosa and Sharpe (MRS) broth has been found to promote Z. acidophilus and
bifidobacteria growth (Dave and Shah, 1998b). However, the nutrients required for
this (i.e. MRS broth) are either not permitted in yoghurt, contribute an off-flavour or
are too expensive on a commercial basis. If the growth factors from Z. delbrueckii
subsp. bulgaricus could be released into the yoghurt this would be a natural way of
supplementing the probiotic bacteria with nutrients.
2.5 Probiotic bacteria
2.5.3 Definition
According to Parker (1974) probiotic bacteria are 'organisms and substances
produced by these organisms which contribute to intestinal microbial balance'.
Probiotic bacteria was defined by Fuller (1989) as "a live microbial food supplement
which beneficially affects the host in improving its intestinal microbial balance".
Wood (1992) broadened Fuller's definition as 'a probiotic is a mono- or mixed culture
of live microorganisms which, appHed to animal or man, affect beneficially the host
by improving tiie properties of the indigenous microflora'.
13
Probiotic bacteria are lactic acid bacteria that are of human origin, are acid and bile
resistant to survive in the intestine, they are able to adhere and colonise in the
intestinal tract, are antagonistic against carcinogenic and pathogenic bacteria and are
stable during processing and storage (Salminen, 1998).
2.5.4 Lactobacillus acidophilus
Lactobacillus acidophilus is a probiotic bacteria commonly used in yoghurt. It is a
gram-positive rod but is much shorter than Lactobacillus delbrueckii subsp.
bulgaricus. The cells are non-motile and non-spomlating and proteins in the cell wall
may be important in attaching the bacterium to the intestinal wall (Tamime, 1999). Z.
acidophilus requires riboflavin, pantothenic acid, folic acid, and niacin for growth but
not other B vitamins (Tamime, 1999). It has an optimum temperature of 37°C and no
growth occurs below 15°C and the optimum pH is 5.5-6.0. Z. acidophilus is
presented in Group I as obligately homofermentative the same as Lactobacillus
delbrueckii subsp. bulgaricus. It can also be grown in MRS broth and in RSM with
glucose and yeast extract. It can also be grown selectively in MRS with sorbitol or
salicin.
Z. acidophilus has been found to have health-promoting properties, which will be
discussed later in this chapter. It also produces bacteriocins, which are antibiotic-like
substances that may be important in the prevention of pathogenic growth.
Bacteriocins are also discussed later in this chapter.
2.5.5 Genus Bifidobacterium
The members of the genus Bifidobacterium are gram-positive, non-sporing bacilli.
The rods of bifidobacteria often have an irregular shape, with a concave central region
and swollen ends. It is however not unusual to encounter cells that are coccoid or
appear as short bacilli with varying widths. The shape depends very much on the
constituents of the media (Tamime, 1999). Bifidobacteria can utilise ammonium salts
as sole source of nitrogen. These bacteria also produce an enzyme, fhictose-6-
phosphate phosphoketolase (F6PPK), known as "bifidus shunt" and this can be used
14
to identify the genus, however, not all strains produce enough F6PPK for it to be
detectable. The fermentation of two molecules of glucose, leads to two molecules of
lactate, and three molecules of acetate (Tamime, 1999).
There are currently thirty different strains of bifidobacteria which have been isolated
from different sources such as the faeces of humans, animals, birds and sewage, the
human vagina, bees and dental caries (Tamime, 1999). Only six species are of
interest to the dairy industry for the manufacture of fermented dairy products. These
include Bifidobacterium adolescentis, B. breve, B. bifidum, B. infantis, B. lactis and B.
longum that have been isolated from human subjects. This restriction is based on the
assumption that, if an isolate is of human origin, then it should become implanted on
the walls of intestines and metabolise in the colon of another human (Tamime, 1999).
The two species used in this study were B. infantis and B. longum. They grow best at
37°C under anaerobic conditions. The best media is MRS broth with \% L-cysteine
and they can also be grown selectively on MRS-Broth with NNLP and L-cysteine
added to it.
2.5.6 Health benefits of probiotic bacteria
A number of health benefits for products containing live probiotic bacteria (Z.
acidophilus and Bifidobacterium) have been claimed. As a result, these organisms are
increasingly incorporated into dairy products, such as yoghurt. Metchnikoff (1908) in
his book "The Prolongation of Life" proposed the theory that the longevity of the
Bulgarians was in part due to ingesting large quantities of fermented milks containing
lactobacilli. This observation has led to much interest in the role of lactic acid
products in alleviation of human and animal disorders. The benefits offered by Z.
acidophilus and bifidobacteria include improvement in intestinal disorders and lactose
tolerance, antimicrobial properties, reduction in serum cholesterol, antimutagenic and
anti-carcinogenic activities and adherence to intestinal cells (Shah, 2000b; Harding,
1995). These fermented dairy products have also been reported to be effective in the
treatment of diarrhea, constipation, colitis, reducing blood cholesterol, pathogenic
recolonisation of the intestinal fract, flatulence, gastric acidity and gasfroenteritis
15
(Harding, 1995). Table 1 shows a summary of health benefits associated with
probiotic bacteria.
Table 1: Health benefits of probiotic bacteria
Health Benefit Proposed mechanism
Protection against undesirable organisms Production of inhibitory compounds i.e. acids, H2O2 and bacteriocins.
Improved digestion
Control of semm cholesterol
Partial breakdown of protein, fat, carbohydrates and improved bioavailability of nutrients.
Gut microflora can metabolise cholesterol
Protection against cancer
Improved immune system
Improved lactose digestion
Probiotic bacteria can inhibit carcinogens and enzymes involved in converting procarcinogens to carcinogens.
Enhancement of macrophage formation, stimulation of suppressor cells and production of interferon.
Bacteria can produce enzyme to breakdown lactose.
Prevention of constipation
Increased vitamin contents
Improvement in bowel movement and stabilisation of ecological balance in the intestinal tract.
Synthesis of group B vitamins
Control of vaginal infection Inhibition of fimgi and bacteria responsible for the infection.
(Sources: Marth and Steele, 1998; Dave, 1998; Shah, 2000b; Harding, 1995; Salminen
and von Wright, 1993; Fuller, 1992; Goldin and Gorbach, 1987)
16
2.5.4.1 Antimicrobial and antimutagenic properties of probiotic bacteria
Microorganisms that are considered probiotic must have several important
characteristics including their ability to produce antimicrobial substances such as
organic acids (e.g. lactic and acetic acids), hydrogen peroxide and bacteriocins to
suppress the growth of pathogenic and putrefying bacteria.
These probiotic bacteria produce several organic acids such as acetic, lactic and
pymvic acids. Other acids produced in small quantities included citric, hippuric acid,
orotic acid and uric acid (Lankaputhra and Shah, 1998). Lactic and acetic acids are
the major acids produced and these acids account for more than 90% of the acids
produced (Shah, 2000b). It is well documented that organic acids are inhibitory
against coliforms, Salmonella, and Clostridia in vitro, but convincing in vivo
evidence is still lacking (Salminen and von Wright, 1993). Several researchers believe
that lactic acid is the only antimicrobial agent of any importance and that lowering of
pH due to lactic acid or acetic acid produced by these bacteria in the gut has
bacteriocidal or bacteriostatic effect (Shah, 2000b). Lankaputhra and Shah (1998)
studied the levels of acetic, butyric, lactic and pymvic acids produced by the probiotic
bacteria as determined by HPLC technique. All strains produced these acids with
butyric acid being produced by most strains of Z. acidophilus and bifidobacteria.
Some strains of lactic acid bacteria including lactococci, lactobacilli, leuconostoc and
pediococci have the ability to generate hydrogen peroxide during growth and lack of
catalase by these bacteria causes its accumulation in growth media (Shah and Dave,
2002). Accumulation of hydrogen peroxide occurs by the action of superoxide
dismutase in most lactic bacteria or by manganese ions present in high concentrations
in the cytoplasm of bacteria. Hydrogen peroxide has been reported to inhibit the
growth of Staphylococcus aureus, E. coli. Salmonella typhimurium, Clostridium
perfringens, Pseudomonas sp. and other psychrotrophs (Shah and Dave, 2002).
Hydrogen peroxide in the presence of organic acids such as lactic acid is more
inhibitory to bacteria (Lankaputhra and Shah, 1998).
Some strains of Z. acidophilus and bifidobacteria have been reported to show
antimutagenic and anticarcinogenic properties. The mechanism of antimutagenicity of
probiotic bacteria have not been understood or identified so far and the mechanism of
17
antimutagenicity remains speculative. It has been suggested that microbial binding of
mutagens could be the possible mechanism of antimutagenicity (Orrhage et al, 1994).
Lankaputhra and Shah (1998) studied the antimutagenic activity of organic acids
against eight mutagens and promutagens. The study found that butyric acid showed
the highest anitmutagenic activity against all the 8 mutagens or promutagens. Lactic
and pymvic acids showed lower antimutagenic activities and thus it appears that lactic
acid produced by lactic acid bacteria plays a minor role in antimutagenic activity.
Therefore probiotic bacteria which produces butyric acid are more likely to provide
antimutagenic properties.
2.5.4.2 Inhibition of spoilage organisms
Bacteria that produce these inhibitory characteristics described in section 2.5.4.1 show
inhibition against spoilage organisms. Probiotic bacteria show strong antimicrobial
properties against Gram positive bacteria such as Staphylococcus aureus, Clostridium
perfringens than against Gram negative bacteria such as Salmonella typhimurium and
Escherichia coli (Shah, 2000b; Hoover and Steenson, 1993). Much interest has been
focused on evaluating the sensitivity of Listeria monocytogenes to lactic acid bacteria
bacteriocins and it has become a model target bacterium in many studies of food
preservatives (Hoover and Steenson, 1993). Listeria monocytogenes can grow at
refrigeration temperatures and is found in meats and dairy foods which is why there is
such interest in probiotics as preservatives.
Listeria is sensitive to many bacteriocins including nisin. Daba et al (1991) found
that the bacteriocin 'mesenterocin 5' appeared to be specific for Listeria species and
not active against other lactic acid bacteria. Daeschel and Klaenhammer (1985) found
Clostridium botulinum to be inhibited by the bacteriocin of Pediococcus pentosaceus.
Okereke and Montville (1991) investigated it ftirther and observed that some sfrains
of Lactobacillus planarum, L. lactis and P. pentosaceus inhibited Listeria.
Bmno and Shah (2002) investigated the role of bifidobacteria in the inhibition of
pathogenic and pufrefactive microorganisms. In this study four strains of
bifidobacteria {B. infantis, B. pseudolongum and 2 sfrains of B. longum) were grown
18
with different pathogenic bacteria including Escherichia coli, Clostridium
perfringens, C. chauvoei, C. sporogenes, Candida albicans, Enterobacter aerogenes,
Streptococcus agalactiae, S. mitis and S. pyogenes. All bifidobacteria strains
inhibited all strains of pathogenic bacteria when grown together. The inhibition was
found to be due to the lowering of the pH by the bifidobacteria strains from
production of lactic and acetic acids as a result of fermentation of glucose. When the
pH of the supematant was adjusted to neutral the inhibition of growth were absent.
2.5.4.3 Anticarcinogenic activity
Some strains of Z. acidophilus and bifidobacteria have been reported to show
anticarcinogenic properties. The evidence of anticancer effect can be due to decrease
in faecal enzymes involved in conversion of procarcinogens to carcinogens. These
probiotic bacteria also lower levels of harmfiil enzymes such as P-glucosidase and p-
glucuronidase responsible for catalysing the conversion of harmful amines (Lidback
etal, 1992).
Fermented dairy products have also been shown to either inhibit chemically induced
colon tumors or transplantable tumor lines in rodents (Fuller, 1992). Goldin and
Gorbach (1984 and 1987) have found evidence for the anti-tumor effect of Z.
acidophilus. Oral supplementation of a diet containing viable Z. acidophilus of
human origin and bile resistant caused a significant decline in 3 different fecal
bacterial enzymes associated with carcinogenesis. The reduction in fecal enzyme
activity was noted in both humans and rats.
Butyric acid is claimed to prevent carcinogenic effects at molecular (DNA) level
(Smith, 1995; Tanaka et al, 1990 and Yanagi et al, 1993). Yanagi et al (1993)
reported that addition of butyric acid to a diet containing 20% margarine prevented
mammary tumour formation by 7,12-dimethylbenz(a) anthracene in rats. Thus, it
appears that antimutagenic effects of probiotic bacteria may be due to both inhibition
by bacterial cells and production of organic acids, especiaUy butyric acid (Shah,
2000b).
19
2.5.4.4 Lactose intolerance
Lactose intolerance is a condition in which lactose is not completely digested into its
component monosaccharides, glucose and galactose (Shah, 1993). Lactose is cleaved
into its monosaccharides by the enzyme p-D-galactosidase, lactose intolerance results
from a deficiency of this enzyme. Lactose is the principal carbohydrate in milk and
therefore lactose intolerant sufferers do not consume milk or other dairy products.
The traditional cultures used for making yoghurt {Lactobacillus delbrueckii subsp.
bulgaricus and S. thermophilus) have substantial quantities of P-D-galactosidase and
it has been suggested that the consumption of yoghurt may assist in alleviating the
symptoms of lactose intolerance. Kilara and Shahani (1976) have reported that
during fermentation lactobacilli produce lactase, which hydrolyses lactose in milk to
glucose and galactose. Bifidobacteria are resistant to bile, which gives them an
increased chance of colonising the gut, and delivering the enzyme to its site of action
(Hughes and Hoover, 1991). This would benefit consumers who are lactose intolerant
and have to limit their dairy intake.
2.5.4.5. Reduction in serum cholesterol
Cholesterol lowering effects of fermented milk and their culture organisms has been
the subject of a number of studies. Studies have shown that consuming certain
cultured dairy products can help reduce serum cholesterol level. In the 1974 (Mann
and Spoerry, 1974) study revealed that organisms such as Z. acidophilus could have
potential in reducing semm cholesterol in humans. Mann and Spoerry (1974) were
investigating the influence of a surfactant (Tween 20) on semm cholesterol levels.
They found that when they fed a group of men on a high cholesterol diet fermented
milk both groups, that is men who were receiving the surfactant and men who were
not, the semm cholesterol level decreased.
Several animal feeding studies have shown that consumption of milk containing cells
of Lactobacillus acidophilus by animals resulted in lower semm cholesterol levels
than in animals which did not receive milk containing the lactobacilli (Danielson et
al, 1989; GiUilande/a/., 1985; Gmnewald, 1982).
20
Homma (1988) found feeding of fermented milks containing very large numbers of
bifidobacteria (10^ CFU/g) to hypercholesterolemic human subjects resulted in
lowering cholesterol from 3.0 to 1.5g/L of blood semm.
Klaver and Meer (1993) reported that removal of cholesterol from the culture medium
by Z. acidophilus RP32 and other species was not due to bacteria uptake of
cholesterol but resulted from bacterial bile salt deconjugating activity. The
deconjugation of bile acid by the intestinal flora may influence the serum cholesterol
level. The deconjuagted bile acid does not absorb lipid as readily as conjugated
counterpart leading to reduction in cholesterol level.
Research into the potential of Z. acidophilus to exert hypocholesterolemic effects in
humans has indicated tremendous variation among strains of Z. acidophilus isolated
from the human intestinal tract in their ability to assimilate cholesterol (Buck and
Gilliland, 1994). Evaluation of strains of Z. acidophilus currently used commercially
in cultured dairy product in the United States had revealed that none of them are
particularly active with regard to actively assimilating cholesterol in laboratory media
(Gilliland and Walker, 1990). On the other hand, new strains that are very active in
this regard have been isolated from the human intestinal tract and thus they may
provide greater potential for use as a dietary adjunct to assist in controlling semm
cholesterol levels (Buck and GiUiland, 1994).
Z. acidophilus and bifidobacteria actively assimilated cholesterol and other organic
acids. Reports by Gilliland et al. (1985) show that Z. acidophilus itself may take up
cholesterol during the grovyth in the small intestine and make it unavailable for
absorption into the blood stream. The effects of lactic acid bacteria on cholesterol
levels are therefore inconsistent and range from a sigruficant reduction to no
reduction. The exact mechanism is unknown (Shah, 2000b)
2.5.5 Viability of Probiotic bacteria in yoghurt
For therapeutic benefits, the minimum level of probiotics bacteria in yoghurt has been
suggested to be 10^ viable cells per gram of yoghurt to enable them to survive in the
gut and colonise (Tamime and Robinson, 1999). Despite the importance of viability
21
of probiotic bacteria studies have shown that probiotic bacteria are unstable in yoghurt
(Hull et al, 1984; Shioppa et al, 1981). Recent surveys conducted in Ausfralia
(Anon, 1992; Shah et al, 1995) and in Europe (Iwana et al, 1993) have shown poor
viability. Several factors have been claimed to affect the viability of yoghurt and
probiotic bacteria in fermented milk products. The viability of probiotic bacteria in
yoghurt depends on the strains used, interaction between species present, culture
conditions, production of hydrogen peroxide due to bacterial metabolism, final acidity
of the product and the concentrations of lactic and acetic acids. The viability also
depends on the availability of nutrients, grovv^h promoters and inhibitors,
concentration of sugars, dissolved oxygen and oxygen permeation through package,
inoculation level, incubation temperature, fermentation time and storage temperature
(Young and Nelson, 1978; Gilliland et al, 1988; Shah, 1997; Conway et al, 1987;
Shah and Jelen, 1990; Costello, 1993; Bertoni et al. 1994; Shah et al, 1995;
Lankaputhra and Shah, 1995; Lankaputhra et al, 1996b). However the main factors
for loss of viability have been attributed to the decrease in the pH of the medium and
accumulation of organic acids (Shah, 2000a; Hood and Zottola, 1988).
During production of yoghurt, yoghurt bacteria and probiotic bacteria produce organic
acids and the pH is lowered to 4.5 or lower due to legal requirements and in order to
produce good quality yoghurt. The amount of lactic acid could vary at the same pH in
yoghurt due to the buffering effects of ingredients added to yoghurt mixes (Dave,
1998). Also depending on the extent of growth of bifidobacteria, concentration of
acetic acid (which is more toxic compared to lactic acid) would vary in the product
(Dave, 1998).
Z. acidophilus tolerates acidity, however a rapid decrease in their number has been
observed under acidic conditions (Shah and Jelen, 1990, Lankaputhra and Shah,
1995). Bifidobacteria are not as acid tolerant, as the grovyth ceases below pH 5.0,
while the growth of Z. acidophilus ceases at pH 4.0 (Shah, 1997).
In a study by Dave and Shah (1997a) the viability of yoghurt and probiotic bacteria
was assessed during manufacture and 35 days of storage of yoghurt made from four
commercial starter cultures. The viability of Z. acidophilus was affected by the
presence of Lactobacillus delbrueckii subsp. bulgaricus whereas bifidobacteria
22
exhibited better stabiUty in the yoghurt prepared from cultures that contained
Lactobacillus delbrueckii subsp. bulgaricus. The viability of both probiotic
organisms improved when the dissolved oxygen concentration was low in the product
and the storage temperature affected the viabiUty of bifidobacteria but not Z.
acidophilus.
Joseph et al (1998) studied antagonism between yoghurt and probiotic bacteria
isolated from commercial starter cultures and commercial yoghurts using modified
spot on lawn and agar well diffuison assays. Zones of inhibition for two
Bifidobacterium isolates were observed with all Z. acidophilus sfrains. The isolates of
Z. acidophilus were resistant and did not show inhibition by any of the four groups of
microorganisms. Dave and Shah (1997a) and Shah and Ly (1999) also observed
antagonistic relationships between yoghurt and probiotic bacteria
There are certain growth factors that probiotic bacteria require to grow. Milk is
considered to be a less than optimal medium for the growth of bifidobacteria. The
essential factor which is lacking in cow's milk but present in human milk is A^-acetyl-
D-glucosamine-containing saccharides which are known as the bifidus factors
(O'Brien et al, 1960; GHck et al, 1960; Kurmann, 1988; Rasic and Kurmann, 1983;
Poch and Bezkorovainy, 1988). Lactulose (4-0-P-D-galactopyransyl-D-fi-uctose) also
has a growth promoting effect on bifidobacteria (Mizota et al, 1987; Park et al,
1988).
Kosikowski (1982) suggested the use of sterile milk supplemented with 0.5%) Bacto-
liver, 0.05% MgS04 and 0.001% cysteine for growth of bifidobacteria in milk.
Marshall et al (1982) fortified milk with whey protein and threonine to provide the
bifidobacteria with nutritious medium and lower redox potential. Anand et al (1985)
reported good growth of B. bifidum in sterile skim milk supplemented with !%>
dextrose and 0.1%) yeast extract.
Oxygen content is also a critical factor for bifidobacteria as it is anaerobic organism.
During yoghurt production oxygen can easily invade and dissolve in the milk (Shah,
2000a). To exclude oxygen during the production of bifidus milk products, special
23
equipment is required to provide an anaerobic environment. Oxygen can also enter
the product through packaging materials during storage (Shah, 2000a).
2.5.6 Improving viability of probiotic bacteria
It is important that the cells remain viable throughout the projected shelf life of a
product so that when consumed the product contains sufficient viable cells. One of
the important characteristics of probiotic bacteria is their ability to survive the acid in
the human stomach and bile in the intestine. Several investigators have studied the
survival of Z. acidophilus and bifidobacteria in the presence of acid and bile salts.
Clark et al. (1993) studied the survival of .5. infantis, B. adolescentis, B. longum and
B. bifidum in acidic conditions and reported that B. longum survived the best. Clark
and Martin (1994) reported that B. longum tolerated bile concentrations of as high as
4.0% whereas Ibrahim and Bezkorovainy (1993) found B. longum to be the least
resistant to bile.
Many strains of Z. acidophilus and Bifidobacterium sp. lack the ability to survive
harsh conditions and may not be suitable for use as dietary adjuncts in fermented
foods. Lankaputhra and Shah (1995) have shown that of six strains of lactobacilli,
three Z. acidophilus strains survived best under acidic conditions. Two strains
of Z. acidophilus showed the best tolerance to bile. Among the nine strains of
Bifidobacterium sp., B. longum and B. pseudolongum survived best under acidic
conditions. B. longum, B. pseudolongum and B. infantis showed best tolerance to bile.
However B. infantis survived poorly in acidic conditions and therefore may not be
suitable for inclusion as dietary adjuncts. Therefore the selection of appropriate
strains on the basis of acid and bile tolerance would help improve viability of these
probiotic bacteria strains.
Probiotic bacteria may require the incorporation of micronutrients in yoghurt. Dave
and Shah (1998b) investigated the effects of cysteine, whey powder, whey protein
concentrate, acid casein hydrolysates and tryptone on the viability of Streptococcus
thermophilus, Lactobacillus acidophilus and bifidobacteria. It was observed that the
addition of cysteine, whey protein concentrate, acid casein and tryptone improved the
viability of bifidobacteria but whey powder failed to improve viability. Sodium
24
dodecyl sulfate-PAGE and amino acid analysis suggested that a nitrogen source in the
form of peptides and amino acids improved the viability of bifidobacteria.
Shah and Lankaputhra (1997) sonicated Z. delbrueckii subsp. bulgaricus to release
growth factors to support probiotic bacteria. They found that the probiotic bacteria
numbers were 2 log cycles higher in yoghurt made with mptured yoghurt bacteria and
was still above the recommended level during 6 weeks of storage.
Z. acidophilus produces a bacteriocin against Z. delbrueckii subsp. bulgaricus, which
could be used to lyse the bacteria and release the intracellular contents. This would
also be a natural way of preserving the product.
2,6 Bacteriocin
Lactic acid bacteria produce a wide variety of antimicrobial proteins including peptide
antibiotic, antibiotic-like substances, bacteriocins and bacteriocin-like substances for
the inhibition of food-home pathogens and spoilage organisms (Shah and Dave,
2002). Among the antibiotic like substances, nisin is well characterised. Tagg et al.
(1976) defined bacteriocins as 'proteinaceous compounds that show antimicrobial
activity against closely related species'. This definition holds tme for the majority of
bacteriocins, however it is now evident that bacteriocins may act beyond closely
related species or those confined to the same ecological niche (Shah and Dave, 2002).
Bacteriocins share a number of characteristics and many studies on antimicrobial
proteins produced by lactic acid bacteria frequently cite these criteria. There are six
criteria suggested by Tagg et al. (1976) however these should not be used as inflexible
criteria as it has become increasingly clear that few antimicrobial proteins fit all six
criteria (Hoover and Steenson, 1993). The six criteria include
• Bacteriocins must be proteins
• Bacteriocins must be bactericidal
• Bacteriocins must have specific binding sites
• Bacteriocins must be plasmid mediated
• Bacteriocins must be produced by lethal biosynthesis
25
• Bacteriocins must be active against a narrow spectrum of closely related
bacteria
Many of these criteria are not essential in the definition of bacteriocins. There are
many bacteriocins that are not produced by lethal biosynthesis as they are produced as
proteins in the grovyth phase without the lysis of the producing organism.
Klaenhammer (1993) concluded that there are only two tme requisites for
bacteriocins: their proteinaceous nature and their lack of lethaUty to cells, which
produce them.
Biochemical and genetic studies of bacteriocins produced by lactic acid bacteria have
now defined four major classes (Klaenhammer, 1993). Class I bacteriocins are
membrane-active and heat stable peptides. They contain lantibiotics that are small
ribosomally synthesised polypeptides containing modified amino acids such as
lanthionine and 3-methyl-lanthionine (Shah and Dave, 2002; Marth and Steele, 1998).
Nisin produced by Lactococcus lactis subsp. lactis is the most prominent lantibiotic
(Hoover and Steenson, 1993). Class I bacteriocin peptides undergo post-translational
modifications.
Class II bacteriocins are small hydrophobic peptides which are moderately heat stable
(Hoover and Steenson, 1993). This class does not contain unusual amino acids such
as lanthionine (Marth and Steele, 1998). To date, many bacteriocins belonging to
class II have been identified and characterised such as Lactacin F and B, Lactocin 27,
Camobacteriocins, Brevicin 37 Pediocin PA-1, Sakacin P, Curvacin A and Enterocin
A (Shah and Dave, 2002; Hoover and Steenson, 1993; Marth and Steele, 1998). Class
II bacteriocin peptides do not undergo post-translational modifications. Class II
bacteriocins have two sub-groupings: class Ila bacteriocins are effective against
Listeria and thus have potential as antimicrobial agent in food and feed. The majority
of bacteriocins produced by Z. acidophilus are heat stable, low molecular mass, non-
lanthiobiotic peptides, which belong to class II.
Class III bacteriocin contain large heat labile peptides. There appear to be numerous
members representing this class among the lactobacilli, including helveticin J,
acidophilucin A, lactacin A and B and caseicin 80 (Hoover and Steenson, 1993).
26
Class IV bacteriocins are complex bacteriocins formed by the association of
bactericidal proteins with one or more other essential chemical moieties (Shah and
Dave, 2002).
Most bacteriocins produced by LAB have narrow antibacterial spectmm confined to
species related to producer organisms, whereas some bacteriocins are active against
Listeria sp. and other food-home pathogenic and spoilage organisms. Most
bacteriocins are hydrophobic and hence can be bound by lipids and phospholipids
(Shah, 2002).
The antimicrobial activity of bacteriocins is due to increases in the permeability of the
cytoplasmic membrane of target cells causing the dissipation of the proton motive
force or disturbing membrane transport and thus inhibiting energy production and
biosynthesis of proteins. The mechanism of action of nisin involves binding to the
peptidoglycan layer, causing destabilisaton of the membrane by the formation of
pores which allow leakage of ions such as a potassium and magnesium and dissipation
of the proton motive force (Shah and Dave, 2002).
2.6.1 Bacteriocins produced by Lactic acid bacteria
2.6.1.1 Bacteriocins produced by L. acidophilus
Among lactobacilli Z. acidophilus has been regarded as a good candidate for use as a
dietary adjunct. The chemical nature and stmcture of antibacterial substances
produced by Z. acidophilus have been studied.
Dave and Shah (r997b) and Joseph et al. (1998) have shown that some strains of Z.
acidophilus produce bacteriocins against Z. delbrueckii subsp. bulgaricus. In a study
by Dave and Shah (1997b), Z. acidophilus (LA-1) produced bacteriocin against seven
strains of Z. delbrueckii subsp. bulgaricus (2501, 2505, 1515, 2517, 2519, LB-3 and
LB-4), one strain each of Z. casei, (2603), Z. helveticus (2700) and L. Jugurti (2819).
To date several Z. acidophilus strains have been studied for the production and
isolation of bacteriocins (Barefoot and Klaenhammer, 1984; Reddy et al, 1983; Toba
et al, 1991) and to study their antimicrobial effects against various pathogens.
27
Lactocidin has been identified and purified by chromatography on a silicic acid
column from an active fraction in the acid soluble fraction of cultures of Z.
acidophilus. Lactocidin has a broad antibiotic spectmm against Gram negative and
Gram positive bacteria (Shah and Dave, 2002).
Barefoot and Klaehammer (1983) and Barefoot et al. (1994) observed Lactacin B
which is produced by Lactobacillus acidophilus. It is heat stable and only detected in
cultures maintained at pH 5.0 to 6.0. The bacteriocin was found to inhibit Z.
delbrueckii subsp. bulgaricus, L. helveticus and L. lactis. It was also observed that
lactacin B only demonstrated antagonism against lactobacilli.
Shah and Dave (1999) investigated the bacteriocin acidophilicin, which is produced
by Z. acidophilus LA-1. It was found to be heat stable and had a molecular mass of
54kDa. This bacteriocin was isolated and purified using a two-stage fractionation
with ammonium sulfate. It was also found that bacteriocin production was affected
by pH but showed activity over a wide range of temperatures.
2.6.1.2 Bacteriocins produced by bifidobacteria
Bifidobacteria have a higher antibacterial activity compared to lactobacilli. However,
most bifidobacteria do not produce any antibacterial substances other than lactic acid
and acetic acid. Only few reports are available on the nature of the antimicrobial
activity of bifidobacteria and studies on the chemical nature and stmcture of
antibacterial substances produced by bifidobacteria are still in infancy stage (Shah and
Dave, 2002).
Yildirim and Johnson (1998) and Yildirim et al (1999) reported on the Bifidocin B a
bacteriocin produced by Bifidobacterium bifidum NCFB 1454. This bacteriocin was
found to be resistant to organic solvents and heat, and showed activity after storage at
-20°C and -70°C for 3 months. With a molecular mass of about 3.3 kDa Bifidocin B
was active against some food-home pathogens and food spoilage bacteria such as
Listeria, Enterococcus, Bacillus, Lactobacillus Leuconostoc and Pediococcus species.
The bacteriocin was active against Gram positive bacteria but not Gram negative
bacteria.
28
Anand et al. (1984) and Anand et al (1985) reported a bacteriocin named bifidin,
which is produced by B. bifidum 1452. This bacteriocin was found to be heat stable at
100°C for 30 min and inhibited the growth ofE. coli. Bacillus cereus, Staphylococcus
aureus. Micrococcus flavus and Pseudomonas fluorescens.
2.6.1.3 Bacteriocins produced by Lactococcus and Pediococcus
There are many bacteriocins that are produced by Lactococcus and Pediococcus. The
best characterised and have found ways in food application are nisin and pediocin.
Nisin is produced by Lactococcus lactis subsp. lactis and is one of the most studied
bacteriocin. Nisin was discovered in 1928 and has received commercial application in
the food industry (Shah and Dave, 2002). It is used in processed cheese, hard cheese,
milk, yoghurt, cottage cheese, bacon and smoked fish (Marth and Steele, 1998; Shah
and Dave, 2002). Nisin is heat stable and can be added before heat processing or
canning of foods and as it is a polypeptide any residues remaining in foods are
digested. It can inhibit Bacillus subtilis, Salmonella typhimurium, E. coli, C.
sporogenes, C. tyropbutyricum Listeria Staphylococcus, Lactobacillus, Micrococcus,
Pediococcus and Leuconostoc (Hoover and Steenson, 1993). Nisin causes cellular
death by affecting cytoplasmic membrane and proton motive force (Marth and Steele,
1998; Shah and Dave, 2002; Hoover and Steenson, 1993). It is part of class I as it
contains the amino acids lanthionine and is therefore called a lantibiotic (Marth and
Steele, 1998).
Pediocin is produced by Pediococcus pentosaceus (previously known as Pediococcus
cerevisiae) and Pediococcus acidilactici has also been reported. These bacteria
inhibit growth of some strains of Gram positive bacteria including Pediococcus,
Lactobacillus, Leuconostoc and Bacillus (Marth and Steele, 1998; Shah and Dave,
2002; Hoover and Steenson, 1993). The inhibitory substance produced by P.
pentosaceus is known as pediocin A and that produced by P. acidilactici is called
pediocin AcH (Hoover and Steenson, 1993). Both pediocins have been used in food
systems such as the production of soft cheeses to control the growth of Listeria
monocytogenes (Marth and Steele, 1998; Shah and Dave, 2002).
29
2.6.2 AppHcations of bacteriocins
There is a tremendous interest in bacteriocins and their role as a preservative in
minimally processed foods. The discovery of psychrotrophic pathogens such as
Listeria monocytogenes that grow at refrigeration temperatures has cast doubt over the
safety of minimally processed refrigerated foods (Hoover and Steenson, 1993).
Bacteriocins have been found to inhibit food-home pathogens such as Listeria and
Clostridium botulinum and as bacteriocins are protein and "natural", this will satisfy
consumer demand for "fresh" and "preservative free" products (Montville and Kaiser,
1993).
Today only nisin has found practical application and is currently being used
worldwide. Pediocin has also received industrial attention for control of Listeria. The
bacteriocins of Lactobacillus acidophilus have received much attention and could
possibly be another dietary adjunct. The bacteria is currently added to yoghurt for
their health promoting benefits and to date several Z. acidophilus strains have been
studied for the production and isolation of bacteriocin and their antimicrobial effects
against various pathogens. Such Z. acidophilus strains (e.g. LA-1) could be used to
lyse Z. delbrueckii subsp. bulgaricus to release intracellular enzymes in order to
catalyse the production of essential peptides and amino acids. This would then act as
growth factors for S. thermophilus, L. acidophilus and Bifidobacterium i.e., in situ
production of growth factors without post-acidification. Morgan et al. (1997) studied
the effect of increasing starter cell lysis in cheddar cheese using a bacteriocin-
producing adjunct. Cheeses manufactured with the bacteriocin-producing adjunct
exhibited increased cell lysis, elevated concentrations of free amino acids and higher
sensory evaluation scores than cheese manufactured without the adjunct.
30
3.0 MATERIALS AND METHOD
3.1 Bacterial Strains
Pure cultures of Streptococcus thermophilus 2014, Bifidobacterium infantis 1912
(VUP 13518) and Bifidobacterium longum 1941 (VUP 13514) were obtained from
Victoria University Culture Collection. Lactobacillus acidophilus (LA-5, 2504, 2505,
2506) and Lactobacillus delbrueckii subsp. bulgaricus 2515 and 2510 were obtained
from Chr. Hansen Pty. Ltd. (Bayswater, AustraUa). Two freeze dried commercial
cultures "Robust" and "Mild" were obtained from National Foods Ltd. The strain
numbers have been concealed for confidential reasons. "Robusf contains a
Streptococcus thermophilus and very robust Lactobacillus delbrueckii subsp.
bulgaricus culture. "Mild" contains a Streptococcus thermophilus and a mild
Lactobacillus delbrueckii subsp. bulgaricus.
3.2 Maintenance of Microorganisms
Working cultures were maintained in 12%o reconstituted skim milk (RSM)
supplemented with \% glucose and 0.5%) yeast extract (RGY) or in DeMann, Rogosa
and Sharpe broth (MRS broth). Bifidobacterium required \% L-cysteine as well.
All lactic acid bacteria were stored as Hquid stock cultures in RSM supplemented with
glucose (l%o) and yeast extract (0.5%)). Aliquots were taken and mixed with 20%
glycerol and stored in cryovials at -80°C and -20°C until required. When required, a
vial was thawed and grown for 24-48 hours. Three transfers were carried out before
bacteria were used and bacteria were grown for 18 hours after each transfer. After 20
transfers a fresh culture was taken from original frozen stock culture to avoid changes
in morphology.
31
3.3 Media preparation
3.3.1 Peptone water
Peptone water (0.15%)) was prepared by dissolving 1.5g of peptone medium (Oxoid,
Australia) in 1 litre of distilled water and dispensing 9 mL aliquots into McCartney
bottles followed by autoclaving at 121°C for 15 minutes at 15 psi. Sterile peptone
water was stored at room temperature.
3.3.2 MRS agar and broth
MRS broth was used for growing Z. acidophilus, Lactobacillus delbrueckii subsp.
bulgaricus and S. thermophilus. MRS broth and Wo L-cysteine hydrochloride was
used to grow bifidobacteria. To prepare MRS agar (Oxoid, Australia) lOg of
bacteriological agar (Oxoid, Australia) was added to the broth, which was sterilised at
121°C for 15 minutes at 15 psi.
3.3.3 Selective medium for Z. acidophilus
For selective enumeration of Z. acidophilus, MRS-sorbitol agar was used. This was
based on the method developed by Lankaputhra and Shah (1996a). To prepare MRS-
sorbitol agar, 26g of MRS broth without carbohydrate (Amyl media), 40 mL of MRS
supplement (Amyl media) and 1 Og of agar were mixed and 900mL of water was
added. This was then autoclaved at 121 °C for 15 minutes at 15 psi. A 10 %> sorbitol
(Oxoid) solution was made and 100 mL was filter sterilised into 900mL of MRS agar
(no carbohydrates) in a laminar flow. Once pouring was complete these plates were
incubated anaerobicaUy at 37°C for 72 hours.
3.3.4 Selective medium for Lactobacillus delbrueckii subsp. bulgaricus
For selective enumeration of Lactobacillus delbrueckii subsp. bulgaricus, MRS agar
at pH 5.2 was used. To prepare this medium, 52g of MRS broth (Oxoid) and lOg of
bacteriological agar were mixed with 1 L of water. When dissolved, the pH was
32
adjusted to 5.2 using 5M NaOH. This was autoclaved at 121 °C for 15 minutes at 15
psi. After pouring these plates were incubated anaerobicaUy at 42°C for 72 hours.
3.3.5 Selective medium for S. thermophilus
For selective enumeration of S. thermophilus. Ml7 agar was used. To prepare Ml7
agar, 23g of M17 broth was mixed with lOg agar, 50mL of M17 supplement and 950
mL of distilled water. It was autoclaved at 121 °C for 15 minutes and 15 psi and once
cooled and poured these plates were incubated aerobically at 37°C for 24 hours.
3.3.6 Selective medium for bifidobacteria
For selective enumeration of bifidobacteria MRS- NNLP (nalidixic acid, neomycin
sulphate, lithium chloride and paromomycin sulphate) (Sigma Chemicals Company)
was used. To prepare MRS-NNLP agar, 52g of MRS broth was mixed with lOg of
agar and IL of water. After autoclaving (121°C for 15 minutes and 15 psi) and
cooling to 45°C, filter sterilized 1% NNLP and 1% L-cysteine hydrochloride were
added. After pouring and setting, plates were incubated anaerobicaUy at 37°C for 72
hours.
3.3.7 Preparation of serial dilution for spread and pour plating
One gram of sample was added to 9mL of 0.15%o peptone water and vortexed. Then
ImL of this dilution was transferred to a second bottle of 9 mL of 0.15% peptone
water and series of ten-fold dilutions were prepared (10^ to 10''). Enumeration was
carried out using the pour plate technique or spread plate technique. DupUcate plates
were then incubated at appropriate temperatures. Plates containing 25 to 250 colonies
were counted and recorded as log of colony forming units per gram of sample.
33
3.4 Yoghurt Preparation
3.4.1 General yoghurt making
All yoghurt was made as follows with various changes, which will be explained, later.
The process for yoghurt making is shown in Figure 2. Milk for yoghurt making was
made by dissolving 12%» reconstituted skim milk powder in distilled water. This was
heated to 85°C for 30 minutes. The milk was allowed to cool to 45°C and then
inoculated with starter culture. The inoculated milk was then poured intolOOmL cups
and incubated at 42°C. The pH was measured every 2 hours until a pH of 4.5 was
reached. The yoghurt was then removed and stored at 4°C for 4-6 weeks for storage
trials.
3.4.2 Yoghurt making using commercial cultures
Two freeze dried commercial cultures "Robust" and "Mild" were obtained from
National Foods Ltd. The "Robust" culture was added at the rate of 14g per 500L
while the "Mild" culture was added at the rate of 17.2g per 500L. The yoghurts were
made the same as described in Section 3.4.1 and were stored at 4°C for 4 weeks. The
viability of yoghurt bacteria and the pH changes were assessed during fermentation
and storage.
3.4.3 Yoghurt made with commercial culture and Z. acidophilus LA-5 and
Bifidobacterium infantis 1912
The culture was diluted first in 100 ml of 12% RSM for better dispersion and were
added at the same rate as in 3.4.2. Probiotic bacteria, Z. acidophilus LA-5 and
Bifidobacterium infantis 1912 were grown for 18 hours in RSM supplemented with
glucose and yeast extract and added at a rate of l%o. The yoghurts were made as
described in section 3.4.1 and were stored at 4°C for 4 weeks. The viabUity of
yoghurt bacteria and probiotic bacteria was assessed during fermentation and storage
and changes in pH were monitored.
34
12% SMP dissolved in water.
^ r
Heat mixttire to 85°C Hold for 30 minutes
> r
Cool milk to 45 °C
1 '
Inoculate with starter culture
^ r Fill cups with inoculated milk
^ r
Incubate at 42°C
^ r
When yoghurt reaches pH 4.5 store at 4°C
Figure 2. Standard procedure for preparation of yoghurt.
35
3.4.4 Yoghurt made with commercial bacteria and Z. acidophilus and
Bifidobacterium longum 1941
This yoghurt was prepared as described in section 3.4.3 with Bifidobacterium longum
1941 replacing Bifidobacterium infantis 1912. Yoghurts were stored at 4°C for 6
weeks. The viability of yoghurt and probiotic bacteria and pH change were assessed
during manufacture and storage.
3.4.5 Yoghurt made with sonicated Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus delbrueckii subsp. bulgaricus was grown in MRS broth at 42°C for 18
hours, centrifuged and cells were collected and suspended in sterile Milli Q water.
The cells were washed and centrifuged three times. The final volume was made up to
lOmL. A Branson Sonifier 450 (Branson Ultrasonics Corporation, Eagle Road,
Danbury, CT, USA) was used to sonicate the cells. The sonicator was set for 50%
output and samples were sonicated for 4 minutes, with a period of 30 seconds for
cooling after each minute of sonication. The samples were kept in an ice bath and the
temperature did not rise above 15°C.
A control yoghurt was made using unsonicated cultures of S. thermophilus 2014, Z.
acidophilus LA-5, B. infantis 1912 and Lactobacillus delbrueckii subsp. bulgaricus
2515 at a rate of 1%) each. The experimental yoghurt contained S. thermophilus 2014,
Z. acidophilus LA-5, B. infantis 1912 and sonicated Lactobacillus delbrueckii subsp.
bulgaricus 2515 at a rate of 1% each. The yoghurts were made as described in section
3.4.1 and were stored at 4°C for 4 weeks. The viability of yoghurt and probiotic
bacteria and pH change were assessed during manufacture and storage.
3.4.6 Yoghurt made with bacteriocin
Yoghurt was prepared as described in section 3.4.1 with the following modifications.
The control batch was prepared as above, while two experimental batches were made
that included the same bacteria plus \% and 2% concentrated bacteriocin respectively.
36
The viability of yoghurt and probiotic bacteria and pH change were assessed during
manufacture and storage.
3.5 Time interval speciHcation
The "0 h" time represents the observations taken immediately after the addition of
starter culture in the milk. The "end of fermentation" represents the observations
taken after yoghurt reached pH 4.5 before transfer into cold storage. The "Week 1",
"Week 2", etc. represent the analysis of yoghurt after that many weeks of storage.
3.6 Analyses
3.6.1 pH
The pH values of the yoghurt and media were measured at 20°C using a HI 8417 pH
meter (Hanna Instruments, New South Wales, Australia). Calibration was done daily
using fresh pH 4.0 and 7.0 standard buffers.
3.6.2 OD readings
Optical density was measured using a Pharmacia spectrophotometer (LKB Biochrom,
England) at 620nm.
3.6.3 Organic acid determination using HPLC
The organic acids analysed included lactic acid, acetic acid, orotic acid, propionic
acid, butyric acid, formic acid and uric acid. The acids were quantified using High
Performance Liquid Chromatography (HPLC, Varian Australia Pty. Ltd., Mulgrave,
Australia) according to the modified method by Scalabrini et al. (1998) and Shin et al
(2000) with some modifications.
For extraction of acids, 5g of yoghurt samples were diluted with 4.93mL of 0.009N
H2SO4 and 70^L of 15.8M HNO3 was added. This was mixed and digested for 30
37
minutes. A 1ml aliquot was centrifuged at 14,000 rpm (5415C centrifuge. Crown
Scientific, Germany) for 10 minutes. The supematant was pipetted off and placed
into a HPLC vial and capped. Samples were stored at 4°C until required.
Standards of each acid were made using distilled water except for orotic and uric
acids, which were dissolved in O.IM NaOH and measured using the HPLC. A
standard curve was produced from the area obtained. Single standard and a mixed
standard were also injected to help determine retention time. The retention time for
orotic acid 7.9-8.1 min, lactic acid was 12.8-13.1 min, formic acid 13.2-13.6 min, uric
acid 14.2-14.7 min, acetic acid 14.8 min, propionic acid 17.4-18.0 min and butyric
acid 21.0-21.7 min. The standard curve regression coefficients were 1 for lactic,
acetic, propionic, butyric and formic acids and 0.9998 for orotic acid and 0.9996 for
uric acids.
The levels of organic acids were measured using a Aminex HPX-87H ion exchange
column with a SSI 505LC-column oven at 65°C. A Varian 9100 autosampler, a
Varian 9012 solvent delivery system and a Varian variable wavelength UV-Vis 9050
detector was used. Varian Star software (Varian, Australia) was also used. Detection
was carried out at 210nm with a sample injection rate of 50uL and a mn time of 30
minutes. The mobile phase was 0.009N H2SO4 with a flow rate of 0.6mL per minute.
3.6.4 Assay of P-galactosidase
To determine the enzyme activity, a modified version of the method by Shah and
Jelen, (1990) was used. Solutions of 0.005M o-nitrophenyl-P-D-galactopyranoside
(ONPG) were prepared with 0.1% phosphate buffer at pH 7, and 250)iL of sample
culture was incubated with 1.25mL of ONPG solution for 15 min at 37°C. The
reaction was stopped by adding ImL of IM cold sodium carbonate. The amount of o-
nitrophenyl released was measured with a specfrophotometer at 420nm against a
blank consisting of 1.25mL ONPG, 250^L MRS broth no culture and ImL of IM
sodium carbonate. The unit of lactase activity was estimated as the enzyme which
liberated one jumole o-nitrophenol from ONPG per min per gram samples at 37°C.
38
3.6.5 Microbiological analysis
The pour plate method as mentioned in section 3.3.7 was used to determine viabiUty
of yoghurt and probiotic bacteria. Selective enumeration was used as described in
sections 3.3.2 to 3.3.6.
3.7 Detection and assay of inhibitory activity
The detection of inhibitory activity was done by the spot on lawn technique (Dave,
1997). Twenty-five milHHtres of 1% agar was poured into a sterile petri dish. When
set, wells were cut using the end of a cut 6mm pipette. The bottom of the wells was
sealed with sterile 0.9%) agar. Fifty microUtres of an active bacteriocin producing
organism were put in the wells. The plates were left for 2 hours for diffusion. The
wells were then filled with P/o agar and then overlaid with approximately lOmL of
0.9% agar seeded with P/o indicator organism. Plates were incubated at 37°C for 24
hours. If any inhibitory substances were present, a clearing zone formed around the
weUs.
The nature of the inhibitory substance produced was determined by the well-diffusion
technique (Dave, 1997). Agar (0.9%) was cooled to 45°C after autoclaving. This
agar was inoculated with P/o of active indicator bacteria and 25mL of it was poured
into a petri dish and wells were cut in the solidified agar. The producer organism was
centrifuged (4500 rpm, 12 minutes, 4°C) (5415C Centrifijge, Crown Scientific,
Germany) and the supematant was filter sterilised using a 0.45)im membrane. The
supematant was divided into three portions for different treatments. The first portion
was untreated, the second portion was neutralised to pH 6.0 using 5M NaOH and the
third portion was neutralised (pH 6.0) and treated with catalase (final concentration
0.05-0. Ifig/mL) and incubated for 2 hours in a 37°C water bath. Fifty microUtres of
each sample was pippetted into wells and left for 2 hours for diffusion. Plates were
incubated at 37°C for 24 hours.
The purpose of neutralising and treating with catalase was to remove the inhibitory
effects caused by acid and hydrogen peroxide. If a zone still appeared after these
39
treatments then the next step was taken to determine if the inhibitory substance was a
bacteriocin.
Plates were prepared with 0.9%) agar that had been seeded with 1% indicator bacteria.
When solidified, 6mm wells were cut as before. The producer organism was
centrifuged and filtered as above to obtain a cell free extract. The supematant was
neutralised (pH 6.0) and treated with catalase and one of the following enzymes,
trypsin (final concentration Img/mL), papain (0.5mg/mL), proteinase K (0.2mg/mL)
and cmde protease (Img/mL). The treated samples were incubated for 2 hours at
37°C. Fifty microlitres of sample was added to the wells and left to diffuse for 2
hours and then incubated at 37°C for 24 hours. If no zone appeared, it was
determined that the inhibitory substance was sensitive to proteolytic enzymes. This
would confirm that a bacteriocin was present, as it is proteinaceous in nature.
3.8 Concentrating and Purification of Bacteriocin
3.8.1 Concentration bacteriocin using ultra-filtration
Z. acidophilus was grown for 18 hours in MRS broth at 37°C. This was centrifiiged at
5000rpm for 15min at 4°C (Beckman J2-HS centrifuge, Beckman Instmments Inc.,
Palo Alto, CA, USA) to remove cells. The supematant was neutralised to pH 6.0 and
was concentrated using a ultra-filtration (UF) unit with molecular weight cut off
(MWCO)of30kDa.
A Vivaflow 50 unit (Vivascience, Germany) was used to filter MRS broth for samples
less than 5L. Two Vivaflow 200 units were used for samples between 5L and lOL.
These units were used with a Millipore pump (Millipore Corporation, MA, USA) and
an Easy load head (Masterflex, USA). The pump was kept constant at lOOkPa.
The concentration ratio for samples less than 5L was approximately 20 and for
samples more than 5L was approximately 50.
40
3.8.2 Purification of bacteriocin
The bacteriocin was fractionated with ammonium sulphate according to the method of
Dave and Shah (1997). A 40%o saturation was used with 243g of ammonium sulphate
added to IL of liquid. Ammonium sulphate was added slowly and each addUion of
salt was made only after the previously added amount had completely dissolved. This
was then left to stir for 2 hours at 4°C. The mixture was then centrifuged (Beckman
J2-HS centrifiige, (Beckman Instmments fric, Palo Alto, CA, USA) at 8000 rpm for
10 minutes. The precipitate was removed from the supematant and weighed for yield
determination. The precipitate was then dissolved in O.IM sodium citrate buffer (pH
6.0) at a rate of 0.1 g of protein per millilitre of sodium citrate buffer. This solution
was dialysed using 14000-16000 MWCO dialysis tubing against O.OOIM sodium
citrate buffer (pH 6.0) for 2 days at 4°C. The bacteriocin solution was then
autoclaved at 121 °C for 15 minutes and was stored at 4°C for immediate use or -20°C
for storage.
3.9 Lysis of Lactobacillus delbrueckii subsp. bulgaricus in different media
3.9.1 Lysis of Lactobacillus delbrueckii subsp. bulgaricus in MRS broth
Lactobacillus delbrueckii subsp. bulgaricus was grown with different levels of
bacteriocin in MRS broth. The levels of bacteriocin used were 1%, 5% and 10% and
these were grown with 1%) of Lactobacillus delbrueckii subsp. bulgaricus. A control
of 1% Lactobacillus delbrueckii subsp. bulgaricus without bacteriocin was also used.
Once the MRS broth was inoculated, they were incubated for 10 hours at 42°C.
Samples were taken at hours 0, 6 and 10 to measure the viable counts of Lactobacillus
delbrueckii subsp. bulgaricus.
The similar experiment was conducted except, this time lower levels of bacteriocin
were used. The levels of bacteriocin used were: 0.5%, l%o and 2% and these were
grown with P/o Lactobacillus delbrueckii subsp. bulgaricus. A control of 1%
Lactobacillus delbrueckii subsp. bulgaricus without bacteriocin was again used.
Once the MRS broth was inoculated, it was incubated for 8 hours at 42°C and samples
41
were taken every 2 hours to measure the viable counts of Lactobacillus delbrueckii
subsp. bulgaricus.
3.9.2 Lysis of Lactobacillus delbrueckii subsp. bulgaricus in RSM
This experiment is similar to the method described in section 3.9.1. The levels of
bacteriocin used were: P/o, 5%o and 10%) and these were grovm with 1% of
Lactobacillus delbrueckii subsp. bulgaricus. A control of P/o Lactobacillus
delbrueckii subsp. bulgaricus was also made. Once the 12%o RSM was inoculated and
incubated at 42°C for 8 hours, samples were taken every 2 hours to measure viable
counts of Lactobacillus delbrueckii subsp. bulgaricus.
3.9.3 Lysis of Lactobacillus delbrueckii subsp. bulgaricus in RSM without
casein
Three percent RSM was prepared and the casein was removed according to the
method by Uemura et al. (1998). Hydrochloric acid (4M) was added to the 3%) RSM
until the pH reached 4.5 where casein precipitated. The RSM was then centrifuged at
13000 rpm for 10 min and the supematant was separated and neutralised to pH 6.5.
The casein free medium was then autoclaved at 12PC for 15 minutes.
3.10 Growth curves of different strains of Lactobacillus acidophilus
Four different strains of Z. acidophilus were grown in MRS broth over 18 hours at
37°C. The bacteria were inoculated using the formula
0.1 X no. ofmL
OD reading of active culture
After inoculation, the samples were placed in a 37°C incubator and OD readings and
plate counts were taken every 2 hours beginning at 0 hour and ending at 18 hours.
OD readings were measured using a spectrophotometer at 620nm.
42
3.11 Sources of chemicals, reagents and microbiological media
3.11.1 Chemicals and reagents
All chemicals and reagents were obtained from Sigma-Aldrich Chemicals (Australia).
3.11.2 Microbiological media
All microbiological media were obtained from Oxoid Ltd. (Hampshire, England) or
from Amyl-Media Pty. Ltd. Dandenong, Australia.
3.12 Equipment and Instruments
3.12.1 Anaerobic jars
Anaerobic jars with forty two plate capacity and catalysts were obtained from Oxoid.
H2 and CO2 generating sachets (Oxoid) were used to create an anaerobic environment.
3.12.2 pH
The pH of yoghurt and media was measured using a HI 8417 pH meter (Hanna
Instmments, New South Wales, Australia).
3.12.3 Centrifuge
Beckman J2-HS centrifuge (Beckman Instmments Inc., Palo Alto, CA, USA) was
used for centrifuging large samples of about 10-lOOOmL. For volumes smaller than
2mL, a 5415C centrifuge (Crown Scientific, Germany) was used. For samples
between 2-50mL and requiring an rpm of less than 4000 a Sorvell RT7 bench top
centrifuge (Dupoint) was used.
43
4.0 RESULTS AND DISCUSSION
4.1 The effects on probiotic bacteria in yoghurt when grown with
Commercial yoghurt strains
4.1.1 Growth characteristics of two different commercial strains of
Lactobacillus delbrueckii subsp. bulgaricus and S. thermophilus.
Two commercial strains of yoghurt were obtained from National Foods Ltd. and
yoghurt was made according to the method described in section 3.4.2. The results are
shown in Table 2. The yoghurt inoculated with the "Mild" culture took 9 hours to
ferment and the yoghurt inoculated with the "Robust" culture took 6.5 hours. The
Mild yoghurt had a much lower count of Lactobacillus delbrueckii subsp. bulgaricus
than the Robust yoghurt in the initial stage of fermentation but did increase by 2 log
during fermentation. The Robust yoghurt increased by 3 log. The counts of S.
thermophilus were similar in both yoghurts but the Mild yoghurt had a higher count at
the end of fermentation due to the longer fermentation time.
During, storage the viable counts of Lactobacillus delbrueckii subsp. bulgaricus in the
Mild yoghurt decreased slightly in the week 1 but were stable up to week 4. The S.
thermophilus counts were also stable throughout the storage period. The pH of the
Mild yoghurt decreased by 0.1 in the first week of storage and then remained stable
for the next three weeks.
The viable counts of Lactobacillus delbrueckii subsp. bulgaricus in the Robust culture
increased in the first week of storage, which is normal, as this bacteria can grow at
lower temperatures. The counts then decreased slightly during week 2 and week 4 of
storage but remained in the same log scale. The counts of S. thermophilus increased
during storage but also stayed in the same log scale. The pH of the Roust yoghurt
decreased considerably in the first week of storage and dropped again during the
second week. It, however, did increase in week 4.
The Robust culture had a higher viable count of Lactobacillus delbrueckii subsp.
bulgaricus in the initial inoculation which is why the fermentation time was less than
that for Mild bacteria. The pH of the Robust yoghurt was much lower during storage
44
than that for the Mild yoghurt which is possibly due to the higher population of
Lactobacillus delbrueckii subsp. bulgaricus in the Robust yoghurt. Lactobacillus
delbrueckii subsp. bulgaricus and S. thermophilus in both yoghurts remained stable
during storage, although the Lactobacillus delbrueckii subsp. bulgaricus in the Robust
yoghurt decreased slightly and this could be due to the lower pH of the yoghurt. Dave
and Shah (1997a) also observed a decrease in Lactobacillus delbrueckii subsp.
bulgaricus during storage, which may have been due to a drop in pH.
4.1.2 The effect on probiotic bacteria in yoghurt when fermented with
commercial bacteria
Lactobacillus acidophilus LA-5 and Bifidobacterium infantis 1912 were inoculated
with commercial bacteria in yoghurt as described in section 3.4.3. The results of
viable counts and pH are presented in Tables 3 and 4. The Mild yoghurt took 7 hours
to ferment and the Robust yoghurt took 5 hours. The Mild yoghurt had a higher
population of S. thermophilus than the Robust yoghurt at the beginning of
fermentation but at the end of fermentation the Robust yoghurt had higher S.
thermophilus counts. There was a slightly higher viable count of Lactobacillus
delbrueckii subsp. bulgaricus in the Robust yoghurt and this increased by 2 log at the
end of fermentation. The variable count in the Mild yoghurt increased 1 log scale
during incubation.
The probiotic bacteria viable count was higher in the Mild yoghurt than in the Robust
yoghurt at the end of fermentation. This would be due to the longer incubation time
for the Mild yoghurt. The inoculation size of the bifidobacteria in the Robust yoghurt
was lower than that for the Mild yoghurt which could be why this bacteria did not
increase in number as much as the Mild yoghurt did.
The storage trial results over 4 weeks are presented in Table 4. The pH of the Mild
yoghurt was stable during the storage trial with a slight drop at week 3. The pH of the
Robust yoghurt decreased during week 1 to week 3 but went up again at week 4.
The count of S. thermophilus decreased slightly in both yoghurts during storage and
had very similar counts at the end of the storage periods. The Lactobacillus
45
delbrueckii subsp. bulgaricus viable counts in the Mild yoghurt decreased until week
3 when there was a 1 log increase and in week 4 the counts decreased again. It is not
known what caused this increase in week 3. The Lactobacillus delbrueckii subsp.
bulgaricus in the Robust yoghurt decreased throughout the storage trial with a 1 log
drop in week 4.
In the Mild yoghurt, the Z. acidophilus was stable until week 3 when the population
decreased slightly and then decreased over 1 log scale in week 4. The bifidobacteria
increased nearly 2 log during the 4 weeks of storage. This was unexpected as
probiotic bacteria have problems with viability in yoghurt but the Mild bacteria may
have improved the environment for the bifidobacteria. In the Robust yoghurt, the Z.
acidophilus decreased 3 log over the 4 weeks with a sharp drop at week 4. The
bifidobacteria fluctuated slightly during the storage trial.
In general, the Mild yoghurt had a higher population of probiotic bacteria than the
Robust culture. This is possibly due to the milder Lactobacillus delbrueckii subsp.
bulgaricus bacteria and the higher pH in the Mild yoghurt. The pH drop in the Robust
yoghurt is higher and the "Robust" culture is possibly creating a difficult environment
for the probiotic bacteria to survive. Dave and Shah (1997a) observed in some
commercial strains that Z. acidophilus remained well within the recommended limit
throughout a 35 day storage period when Lactobacillus delbrueckii subsp. bulgaricus
was absent from fermentation. When Z. acidophilus was fermented with
Lactobacillus delbrueckii subsp. bulgaricus viability of Z. acidophilus was lost after
20 days. They also observed that bifidobacteria was not as affected by Lactobacillus
delbrueckii subsp. bulgaricus as Z. acidophilus was and the bifidobacteria remained
above the recommended limit for 35 days of storage.
4.1.2.1 Organic acid production in yoghurt when fermented with commercial
yoghurt strains and probiotic bacteria (L. acidophilus LA-5 and B.
infantis 1912).
The organic acid content of the yoghurts made with commercial yoghurt strains and
probiotic bacteria (Z. acidophilus LA-5 and B. infantis 1912) is presented in Tables 5
and 6. There was no acetic acid produced in either yoghurt as the bifidobacteria was
46
only fermented for no more than 7 hours and it requires over 12 hours to produce
acetic acid.
The lactic acid production in both yoghurts increased during fermentation and during
storage. The Robust yoghurt produced significantiy more lactic acid than the Mild
yoghurt, which would be due to the higher Lactobacillus delbrueckii subsp.
bulgaricus count in the Robust yoghurt. Dave and Shah (1997a) also observed an
increase in lactic acid concentration during storage in yoghurts grown with
Lactobacillus delbrueckii subsp. bulgaricus, S. thermophilus and probiotic bacteria
and without Lactobacillus delbrueckii subsp. bulgaricus.
In the Robust yoghurt butyric acid was found at the end of fermentation and increased
in the first week of storage and then remained stable. There was no butyric acid
produced in the Mild yoghurt.
Formic acid was found at the beginning of fermentation in both yoghurts. In the Mild
yoghurt, the formic acid concentration increased during fermentation and then
decreased during storage. There was no formic acid detected in the Robust yoghurt
after the initial sample. Formic acid is used as a growth factor for the starter bacteria
and since the "Robust" culture contains quick growing Lactobacillus delbrueckii
subsp. bulgaricus the formic acid would have been used up during fermentation
(Walstra et al, 1999). The "Mild" culture, however, is not as fast growing and may
not require the same amount of formic acid.
The concentration of orotic acid was not significantly different and decreased slightiy
during fermentation and then remained constant throughout storage. Orotic acid is
used as a growth factor by the yoghurt starter cultures, most probably by
Lactobacillus delbrueckii subsp. bulgaricus (Tamime and Robinson, 1999).
There was no propionic acid produced in the Mild yoghurt but in the Robust yoghurt
propionic acid was detected after fermentation that increased in the first week of
storage and remained constant for the rest of the storage trial. Propionic acid is
produced by the yoghurt starter bacteria as a volatile flavour compound (Tamime and
47
Robinson, 1999). ft is possible that the "Mild" culture was not able to produce any
propionic acid, as it was a fairly weak culture.
Uric acid is present in milk as a result of normal bovine biomedical processes and
does not change during fermentation or storage (Navder et al, 1990). This result is
reflected in both yoghurts shown in Tables 5 and 6. The concentration remained
between 32 and 35|j.g/g of yoghurt during fermentation and storage. The resuUs were
however significantly different in week 1 and 4.
4.1.2.2 Conclusion
This experiment has shown that the viability of probiotic bacteria is effected by the 2
different commercial cultures. The Mild culture appears to give a good environment
for the bifidobacteria but not the Z. acidophilus. The Robust culture does not provide
a good environment for either bacteria as the viable counts were below the population
required to give any health benefit (approx. 1x10^).
This experiment was repeated using a different strain of bifidobacteria, which is
supposed to be more stable.
4.1.3 Probiotic bacteria in yoghurt using Bifidobacterium longum 1941
Lactobacillus acidophilus LA-5 and Bifidobacterium longum 1941 were inoculated
with commercial bacteria in yoghurt as described in section 3.4.4. The results of
viable counts and pH are presented in Tables 7 and 8. The Mild yoghurt took 7 hours
to ferment and the Robust yoghurt took 5 hours. The Mild yoghurt had a higher
population of S. thermophilus than that of the Robust yoghurt at the beginning of
fermentation but at the end of fermentation the Robust yoghurt had higher S.
thermophilus counts. There was a higher viable count of Lactobacillus delbrueckii
subsp. bulgaricus in the Mild yoghurt, however, there was a slight increase in
population at the end of fermentation. This is possibly due to the weak nature of the
bacteria. The viable count in the Robust yoghurt increased 2 log scale during
incubation, as a result there was a shorter fermentation time.
48
The probiotic bacteria viable count was higher in the Mild yoghurt than in the Robust
yoghurt at the end of the fermentation. This was also seen in the previous experiment.
This would be due to the longer incubation time for the Mild yoghurt and the "Mild"
starter culture. In the Mild yoghurt the Z. acidophilus viable counts increased by 1
log and the B. longum viable counts increased by 2 log. In the Robust yoghurt, the Z.
acidophilus counts increased by 1 log and the B. longum only sHghtly increased in
number.
The storage trial results are presented in Table 8. The pH of the Mild yoghurt
decreased during storage but not as much as that of the Robust yoghurt. There was a
slight increase in pH at week 6 but the reason for this is unclear.
The S. thermophilus remained constant in both yoghurts during storage and had very
similar counts. The Lactobacillus delbrueckii subsp. bulgaricus counts in the Mild
and Robust yoghurts decreased until week 3 when there was a 4 log decrease at week
4. There could have been a problem with the selective agar or a problem with the
refrigerator as in week 5 the counts increased 2- 31og.
In the Mild yoghurt, the Z. acidophilus viable count fluctuated for the first 4 weeks of
storage and then dropped to 1.21x10 . The Z. acidophilus did not perform well in the
Robust yoghurt and the populations decreased rapidly during storage. B. longum also
fluctuated during storage in both yoghurts, particularly in the Robust yoghurt
dropping to 3.3x10^* at week 3 but then increased to 1.01x10^. The Mild yoghurt
finished the storage trial with a viable count for bifidobacteria of 1.65x10^.
The Mild yoghurt had a higher population of probiotic bacteria than the Robust
culture which was also seen in the previous experiment and again this is possibly due
to the milder Lactobacillus delbrueckii subsp. bulgaricus bacteria and the higher pH
in the Mild yoghurt. The "Robust" culture is possibly creating a difficult environment
for the probiotic bacteria to survive.
49
4.1.3.1 Organic acid concentration in yoghurt when fermented with commercial
yoghurt strains and probiotic bacteria (L. acidophilus LA-5 and B.
longum 1941).
The organic acid concentration of the yoghurts made with commercial yoghurt strains
and probiotic bacteria (Z. acidophilus LA-5 and B. longum 1941) are presented in
Tables 9 and 10. There was no acetic acid produced in either yoghurt as the
bifidobacteria was only fermented for no more than 7 hours and ft requires over 12
hours to produce acetic acid. There was no butyric acid produced in the Mild yoghurt
but in the Robust yoghurt butyric acid was found at the end of fermentation and
increased throughout storage.
Formic acid was found in both yoghurts at the beginning of fermentation. In the Mild
yoghurt the formic acid increased during fermentation and then decreased during
storage. There was no formic acid detected in the Robust yoghurt after fermentation.
The lactic acid production in both yoghurts increased during fermentation and during
storage. The Robust yoghurt had a significantly higher concentration of lactic acid
than the Mild yoghurt up to week 3. During weeks 4-6 the difference in concentration
was not significantly different.
The concentration of orotic acid was significantly different at the end of fermentation
and in weeks 1-3. The concentration decreased during fermentation in both yoghurts
and then remained constant throughout storage.
There was no propionic acid produced in the Mild batch but in the Robust yoghurt
propionic acid was detected after fermentation and then increased in the first week of
storage and then remained constant. Uric acid was detected in both yoghurts and
remained constant during fermentation and storage. The concentration was
significantly different in at the end of fermentation and in weeks 3, 4 and 6.
The organic acids produced by the experimental yoghurts were compared to organic
acids produced in commercial yoghurts. Table 11 presents the organic acid content in
three different commercial yoghurts. The commercial yoghurts had more lactic acid
than the experimental yoghurts, possibly due to a longer fermentation time at the
50
factory. There were similar amounts of uric acid in the commercial yoghurts and the
experimental batches. This was as predicted as uric acid is normally present in milk.
There were higher amounts of orotic acid and butyric acid in the commercial
yoghurts. The butyric acid was considerably higher as there was none detected in the
Mild yoghurts and the most in the Robust yoghurt was 90.12^glg of yoghurt. There
was no formic or acetic acid detected in any of the commercial yoghurts and only the
Ski yoghurt detected any propionic acid.
4.1.3.2 Conclusion
The results of the organic acid concentration in these yoghurts are consistent with the
production of organic acid in the yoghurts described in section 4.1.2. The Robust
culture produced more lactic acid than the Mild culture in both experiments and
formic acid was used up during fermentation of the Robust culture but was only
slowly used in the Mild yoghurt. There was no butyric acid produced in the Mild
yoghurt but this was produced by the Robust culture. This shows that the Mild
yoghurt is a slow grower where as the Robust culture is very fast.
These experiments also show that the viability of probiotic bacteria is affected by the
2 different commercial cultures. The probiotic bacteria in both experiments did not
survive the full storage trial. The Mild culture appears to give a better environment for
the bifidobacteria, but not the Z. acidophilus. However, the fermentation time with
Mild cultures is much longer. The Robust culture does not provide a good
environment for either bacteria as the viable counts were below the population
required to give any health benefit (approx. 1x10^). However, the fermentation time is
shorter.
4.2 The effect of sonicating Lactobacillus delbrueckii subsp. bulgaricus on
the survival of probiotic bacteria in yoghurt
ft is known that Lactobacillus delbrueckii subsp. bulgaricus releases grovv^h factors
such as amino acids and peptides. These have been found to support the growth of
probiotic bacteria (Z. acidophilus and Bifidobacterium) during fermentation. If the
51
growth factors from Z. delbrueckii subsp. bulgaricus could be released into the
yoghurt this would be a natural way of supplementing the probiotic bacteria with
nutrients.
In order to allow Lactobacillus delbrueckii subsp. bulgaricus to release its growth
factors, the bacteria was sonicated according to the method described in section 3.4.5
prior to yoghurt making. The results of pH and variable counts are presented in Table
12 and the organic acid concentration results are in Table 13 and 14.
The control yoghurt took 3 hours and 20 minutes to ferment while the sonicated
yoghurt took 4 hours and 25 minutes. The pH of both yoghurts decreased rapidly
over the 4 weeks of storage with the control yoghurt having the lowest pH. If the
Mild and Robust yoghurts are compared to these ones the fermentation time is quite
different. The Robust yoghurt had a fermentation time of 5 hours and in this
experiment the fermentation times are much shorter. This is probably due to the use
of fresh cultures. Freeze dried cultures need time to activate since they are coming
from such a cold environment to a warm one where as the fresh bacteria has been
grown for 18 hours at optimum temperature.
S. thermophilus increased by 1 log in both the control and the experimental yoghurt
during fermentation. During storage the bacteria numbers remained relatively
constant. The control had a higher viable count of Lactobacillus delbrueckii subsp.
bulgaricus than the sonicated yoghurt which is to be expected as the Lactobacillus
delbrueckii subsp. bulgaricus was sonicated, however, it was thought that the
sonication would have decreased the amount of bacteria even further. The bacteria
may be fairly strong and have only been damaged and able to repair itself during
fermentation. The bacteria numbers during storage decreased every week but were
more noticeable in Week 4 in the sonicated yoghurt when it decreased by nearly 1 log.
The probiotic bacteria performed very well in both yoghurts. There was a high
number of Z. acidophilus and bifidobacteria in the inoculation and due to the short
fermentation time there was not a large increase in population. The storage trial
showed that the probiotic bacteria survived the harsh conditions that were present.
The pH of the control and experimental yoghurt were both very low and yet the
52
probiotic bacteria were stable throughout the 4 weeks of storage. It is difficult to
explain as it was thought that the sonicating the Lactobacillus delbrueckii subsp.
bulgaricus had provided the probiotic bacteria with the nutrients to grow but then the
control yoghurt would have a much lower population of probiotic bacteria. It could
be concluded that the Z. acidophilus and bifidobacteria had been very active in the
inoculating culture since the number of colonies is very high compared to the other
inoculations that were done in previous experiments. The inoculations in both
yoghurts were in the 10^ log scale where the Mild and Robust yoghurts discussed in
section 4.1.2 and 4.1.3 were in the 10^ log scale.
Shah and Lankaputhra (1997) sonicated Z. delbrueckii bulgaricus to release growth
factors to support probiotic bacteria. They found that the probiotic bacteria were 2 log
cycles higher after fermentation in yoghurt made with mptured yoghurt bacteria and
was still above the recommended level during 6 weeks of storage.
The organic acid concentration is presented in Tables 13 and 14. There was no acetic
acid produced by either yoghurt, as there was not enough time for the bifidobacteria to
produce it. Butyric acid was present at the initial stage of fermentation and then
increased during fermentation and during storage. The control produced significantly
more butyric acid than the sonicated yoghurt.
Formic acid was present at the beginning of fermentation but disappeared at the end
of fermentation and none was detected during storage. The control had significantly
higher content of lactic acid during fermentation and storage. The concentration
increased during fermentation and the first week of storage, and remained stable
during storage. The same pattem occurred in the experimental yoghurt.
Orotic acid decreased during fermentation in both yoghurts. The sonicated yoghurt
had a higher concentration of orotic acid during storage but was only significantly
different in weeks 2 to 4.
Propionic acid was detected at the initial stage of fermentation in the control batch
and then its level increased. During the first week of storage there was an increase in
concentration of propionic acid but then the level fluctuated throughout the rest of the
53
storage, hi the experimental yoghurt there was no propionic acid detected at the
beginning of fermentation but some was produced during the fermentation. There
was an increase in propionic acid during storage but this was significantly lower than
the control. Uric acid was present in both yoghurts and the experimental batch was
significantly different at the end of fermentation and in weeks 2, 3 and 4.
The organic acid content shows that there were differences in the fermentation of
these yoghurts. The control produced more lactic, butyric and propionic acid than the
experimental. This could be due to the bacteria growing faster and higher population
of Lactobacillus delbrueckii subsp. bulgaricus, which would cause the shorter
fermentation time. The experimental yoghurt had more orotic acid, which would
mean the bacteria did not use as much of it during fermentation. There was only a
slightly lower population of Lactobacillus delbrueckii subsp. bulgaricus in sonicated
product but perhaps the bacteria were repairing themselves during fermentation hence
the longer fermentation time.
Sonication of the Lactobacillus delbrueckii subsp. bulgaricus could possibly improve
the viability of probiotic bacteria in yoghurt however it would be expensive and
impractical. The use of fresh bacteria is also impractical in a manufacturing plant. Z.
acidophilus is known to produce bacteriocin against Lactobacillus delbrueckii subsp.
bulgaricus and if this could be utilised to lyse Lactobacillus delbrueckii subsp.
bulgaricus and release growth factors for probiotic bacteria then this could be a more
practical way of improving yoghurt.
4.3 Antimicrobial substances produced by yoghurt and probiotic
bacteria
4.3.1 Growth characteristics of Lactobacillus acidophilus
To help select a Z. acidophilus strain for further experiments, four different strains of
Z. acidophilus were grown over 18 hours to determine growth pattems. Optical
density was measured at 620nm. The strains used were: Z. acidophilus LA-5, Z.
acidophilus 2404, Z. acidophilus 2405 and Z. acidophilus 2406. The growth pattem is
shown in Figure 3.
54
As the result show, Z. acidophilus LA-5 and LA2404 had the highest growth at 37°C
in MRS broth. Z. acidophilus LA2406 also showed good groyv^h, however, the
growth was slower as compared to strains LA-5 and LA2404. LA2405 did not grow
well and had a final OD reading of only half as that of LA-5. This strain may be
fairiy weak as the initial inoculation required 3.2mL to reach an OD reading of 0.1 as
compared to the other strains of 1.8mL. LA-5 and LA2404 seem to be very robust
and find the conditions optimal for growth. In general, the lag phase was between 0
and 2 hours and then all strains continued into the log phase between 2 and 6 hours.
After 6 -8 hours, all strains entered the stationary phase.
4.3.2 Screening of Lactobacillus acidophilus against Lactobacillus delbrueckii
subsp. bulgaricus for bacteriocin production
Four strains of Z. acidophilus were screened for antimicrobial activity against two
strains of Lactobacillus delbrueckii subsp. bulgaricus. The fourZ. acidophilus strains
(LA-5, 2404, 2405 and 2406) were used against Lactobacillus delbrueckii subsp.
bulgaricus 2515 and Lactobacillus delbrueckii subsp. bulgaricus 2501. The spot-on-
lawn test as described in section 3.7 was used to determine if any antimicrobial
substances were present. The inhibitions of Lactobacillus delbrueckii subsp.
bulgaricus by Z. acidophilus strains are presented in Table 15. The largest average
zone was produced by LA-5 against Lactobacillus delbrueckii subsp. bulgaricus
2515. Z. acidophilus-2AOA also showed a large zone against Lactobacillus delbrueckii
subsp. bulgaricus 2515. LA2405 showed the smallest zones, which also showed
weak growth (Figure 3). This would indicate that this strain is very weak and would
be of no benefit in this study. Z. acidophilus produced zones against Lactobacillus
delbrueckii subsp. bulgaricus 2501 with the largest zone being produced by Z.
acidophilus-LA5. From this study, it was determined that Z. acidophilus-LA-5
produced an antimicrobial substance against Lactobacillus delbrueckii subsp.
bulgaricus 2515 and the next step was taken to determine what kind of antimicrobial
substance was present.
55
4.3.3 Determination of inhibitory substance
To determine the inhibitory substance present the well difftision test (Section 3.7) was
used. The cell free extract was treated with sodium hydroxide, catalase, papain,
proteinase K and cmde protease in order to understand the nature of the antimicrobial
substance. Zone of inhibition could be produced by lactic acid, and tteatment with
sodium hydroxide, will neutralize the acid effect and if the zone still appears the effect
may be due to H2O2 or bacteriocin. Table 16 shows the zones of inhibition by Z.
acidophilus against Lactobacillus delbrueckii subsp. bulgaricus. There were zones
present in the neutralized sample, which determines that acid, was not the cause of
inhibition. The sample treated with catalase also had a zone, which suggested that
hydrogen peroxide was not the cause of inhibition either. The zones disappeared
when samples were treated with protein enzymes, which confirmed an active protein
compound was involved in the inhibition of Lactobacillus delbrueckii subsp.
bulgaricus. According to Tagg et al. (1976) bacteriocins are bactericidal or
bacteriostatic compounds containing a biologically active protein moiety. Thus in this
study Z. acidophilus-LAS produced one or more bacteriocins against Lactobacillus
delbrueckii subsp. bulgaricus.
Dave and Shah (1997b) and Joseph et al. (1998) have shown that some strains of Z.
acidophilus produce bacteriocins against Z. delbrueckii subsp. bulgaricus. In a study
by Dave and Shah (1997b), Z. acidophilus (LA-1) produced bacteriocin against seven
strains of Z. delbrueckii subsp. bulgaricus (2501, 2505, 1515, 2517, 2519, LB-3 and
LB-4).
4.3.4 Antagonism between yoghurt and probiotic bacteria
This step aimed to determine if there was any antagonism between yoghurt and
probiotic bacteria. The spot-on-lawn test (section 3.7) was used to determine any
inhibition and the well diffusion test (section 3.7) was used to determine the type of
inhibition. The results of the spot-on-lawn test are presented in Table 17 and the
results of the well diffusion test are in Table 18.
56
As shown in Table 17, Z. acidophilus-LA-5 produced an inhibitory substance against
Z. delbrueckii subsp. bulgaricus 2515, Bifidobacterium longum 1941, and
Bifidobacterium infantis 1912. Lactobacillus delbrueckii subsp. bulgaricus 2515
inhibited Z. acidophilus LA-5, both bifidobacteria strains, but not S. thermophilus. S.
thermophilus produced inhibitory substances against both strains of bifidobacteria but
not against Lactobacillus delbrueckii subsp. bulgaricus. There appeared to be a zone
against Z. acidophilus LA-5, but it was unclear. Bifidobacterium infantis 1912 and
Bifidobacterium longum 19Al showed slight inhibition to Z. acidophilus,
Lactobacillus delbrueckii subsp. bulgaricus and S. thermophilus.
Table 18 shows the nature of inhibitory substances produced by yoghurt and probiotic
bacteria to various enzymes and pH. Lactobacillus delbrueckii subsp. bulgaricus, S.
thermophilus, B. infantis and B. longum showed slight inhibition to Z. acidophilus.
Even after acid and catalase effect were removed, there were still zones present, but it
is interesting that the zones still appeared even after treatment with enzymes. This
means that no proteinaceous compounds were present and therefore no bacteriocin
was detected. The substance would have been a bacteriocin like substance (BLIS).
The cause of these zones is unknown. Z. acidophilus produced a zone against
Lactobacillus delbrueckii subsp. bulgaricus and this was proven to again be
bacteriocin as there were no zones present when treated with proteolytic enzymes.
There were no zones of inhibition observed by other probiotic or yoghurt bacteria
Hsted in Table 17.
The zones in Table 17 are bigger than these presented in Table 18. For example in
Table 17 Z. acidophilus produced a 16.67mm zone against Lactobacillus delbrueckii
subsp. bulgaricus but in well diffusion test (Table 18) there was only a 9.67mm zone
before it was treated. These results are similar to those reported by Joseph et al.
(1998) that a transfer of organisms, in the eariy stationary phase (18 hours) into a
fresh medium with optimum nutrients and favourable pH, could have enhanced the
production of the BLIS on the solid agar medium. Eckner (1992) reported that
antimicrobial substances sometimes are only produced on soHd media.
57
4.7 Assessment of viability of Lactobacillus delbrueckii subsp. bulgaricus
grown with various inocula sizes of Lactobacillus acidophilus
The effect of bacteriocin producing Z. acidophilus on Lactobacillus delbrueckii subsp.
bulgaricus was observed. Various inocula sizes of Z. acidophilus were grown with
Lactobacillus delbrueckii subsp. bulgaricus, and cell density (Figure 4), P-
galactosidase (Figure 5) and viable counts (Table 19) were measured. The
experiment, as described in Section 3.5, used the inoculum sizes of 1, 5 and 10%).
Figure 4 shows the changes in the cell density of Z. acidophilus and Lactobacillus
delbrueckii subsp. bulgaricus. It shows that the sample inoculated with 10% Z.
acidophilus had a higher cell density reading followed by the 5% sample, 1% sample
and then the Z. acidophilus control and the Lactobacillus delbrueckii subsp.
bulgaricus control, respectively. This is consistent as the 10%) sample contains more
bacteria than the other samples. The 5%o sample had less growth than the 10%) and the
1% sample had less than the 5% but more than the controls. Z. acidophilus had a
higher cell density than that of Lactobacillus delbrueckii subsp. bulgaricus, which is
probably due to the difference in growth temperature. The experiment was conducted
at 40°C as it is in between the optimum temperatures of Z. acidophilus (37°C) and
Lactobacillus delbrueckii subsp. bulgaricus (42°C). This temperature was chosen to
give Lactobacillus delbrueckii subsp. bulgaricus the opportunity to grow, as it does
not perform as well when grown below optimum temperature.
The changes in P-galactosidase are shown in Figure 5. This experiment was used to
measure the lysis of Lactobacillus delbrueckii subsp. bulgaricus. It was thought, that
as Lactobacillus delbrueckii subsp. bulgaricus produces more P-galactosidase than Z.
acidophilus and P-galactosidase is intracellular, a higher reading would show, that
Lactobacillus delbrueckii subsp. bulgaricus was being lysed by Z. acidophilus.
However, Figure 5 shows that the Lactobacillus delbrueckii subsp. bulgaricus control
had the lowest amount of p-galactosidase except at the eighth hour when it had the
same as the 1% sample and only marginally lower than the Z. acidophilus control.
This is possibly due to the differences in growth temperature, as this experiment was
not conducted at the optimum temperattire of Lactobacillus delbrueckii subsp.
58
bulgaricus. The pattem in the first two hours of this graph shows that the 10%) sample
produced the highest amount of P-galactosidase, followed by the 5% sample, 1%
sample, LA control and the LB control. During the next two hours (4* hour), the 10%)
sample slowed down sUghfly and the 5%o sample became the highest. In the sixth
hour, the P/o batch shows the highest amount of p-galactosidase, closely followed by
the 5%) sample and the LA control. In the eighth hour, the LA control had the highest
amount followed by the P/o sample and the LB control and then the 10%) and 5%
samples have the lowest amount of p-galactosidase. This change in pattem where the
lowest inoculations had the highest amount of P-galactosidase could be due to no
competition. The LA and LB control are not fighting another bacteria for nutrients
and can grow without competition, even the P/o sample that are competing with each
other, do not have as much bacteria to fight against, as the inoculation was quite low.
The 5%o and 10% samples had a high inoculation and they are competing with each
other. Even though Lactobacillus delbrueckii subsp. bulgaricus is not growing at
optimum temperature, it still is a robust bacterium and will fight for the nutrients.
Another possibility is due to the high inoculation of the 5%, and particularly the 10%
sample, as it will mn out of nutrients before the other samples. This means that not as
much P-galactosidase can be produced, as the bacteria maybe saving energy to
survive.
The changes in viable counts of Z. acidophilus when grown with Lactobacillus
delbrueckii subsp. bulgaricus are shown in Table 19 and Figures 6 and 7. The growth
of Z. acidophilus started off with different inoculations but after eight hours, they had
very similar counts, which is possibly due to only a certain amount of nutrients being
available, and the 10%) sample would have used the nutrients up more quickly, than
the P/o sample. The Lactobacillus delbrueckii subsp. bulgaricus counts followed the
same pattem. The initial counts are slightiy different, but after eight hours the 1% and
5% samples are very similar and the 10% sample had a lower viable count and had
slightly decreased in the eighth hours. The Lactobacillus delbrueckii subsp.
bulgaricus counts were lower than the Z. acidophilus viable counts, but are still very
high and if in yoghurt would still continue to grow and decrease the pH ftirther. It
does appear that Lactobacillus delbrueckii subsp. bulgaricus does decrease slightiy in
the presence of a high inoculation and this may have been more obvious if the growth
59
continued for several more hours. This, however, would not be feasible in yoghurt
making.
This experiment showed that Z. acidophilus did not decrease the viable count or lyse
Lactobacillus delbrueckii subsp. bulgaricus enough to slow the production of lactic
acid to stop post-acidification.
4.5 Purification of bacteriocin
As the experiment in section 4.4 shows Z. acidophilus does not inhibit Lactobacillus
delbrueckii subsp. bulgaricus enough to stop its growth. Therefore, it was thought,
that if the bacteriocin produced by Z. acidophilus could be concentrated, purified, and
added as a supplement to yoghurt, this would inhibit Lactobacillus delbrueckii subsp.
bulgaricus. The process of ultra-filtration of bacteriocin is described in section 3.8.1.
Dave and Shah (1997) have reported that the bacteriocin produced by Z. acidophilus
LA-5 had a molecular weight of approximately 50 kDa. Therefore an ultra-filtration
unit, with a molecular weight cut off of 30 kDa was used. This retained the
bacteriocin in the retentate and was concentrated approximately 50 times, when the
initial solution was either 10 or 15L.
Once the cmde bacteriocin was concentrated, the bacteriocin was purified by the
method described in Section 3.8. The yield of protein extracted from the concentrate
was approximately 0.17%) of the total volume fUtered.
In this experiment, the well difftision method (section 3.7) was used to observe
inhibition in the media before fiftering, in the concentrate and permeate, after
purifying, before and after dialysis and after autoclaving. The zones of inhibition can
be seen in Figures 8-15. Zones appeared in all samples, except the permeate. This
confirms that the molecular weight of the bacteriocin was greater than 30kDa. ft also
shows that the bacteriocin can sustain autoclaving as a zone still appeared after the
sample had been autoclaved (Figure 15).
60
To make the usage of bacteriocin more practical, attempts were made to dry the
bacteriocin into a powdered form. Freeze-drying was attempted several times but
without success. The bacteriocin tumed into a sticky matter and the last bit of
moisture could not be removed even after freeze drying for three days. Drying in a
vacuum oven was also tried but without success. A similar sticky matter, found in the
freeze drying, was also observed in oven dried samples. It appeared that the protein in
some way was trapping moisture.
Morgan et al. (2001) developed a method to spray dry lacticin 3147; a bacteriocin
produced by Lactococcus lactis. This bacteriocin was produced by fermenting Z.
lactis for 24 hours keeping, the pH at 6.5. This fermentate was then pasteurised,
evaporated to 40% total solids, and then spray dried. This powder was tested in
yoghurt, cottage cheese and soup. The lacticin powder inhibited Listeria
monocytogenes and Bacillus cereus in all three products, when added at a rate of 10%).
This method of concentration would be very beneficial to the industry but this
equipment was not available for this project. The lacticin 3147 however, is not heat
stable and lost considerable amount of activity when autoclaved. The bacteriocin
produced by Z. acidophilus LA-5 did withstand autoclaving, and inhibited
Lactobacillus delbrueckii subsp. bulgaricus, which will be discussed later in this
chapter.
4.7 Concentrated bacteriocin grown with Lactobacillus delbrueckii subsp.
bulgaricus
Concentrated bacteriocin was grown with Lactobacillus delbrueckii subsp. bulgaricus
to observe any inhibition. The method used is described in Section 3.9.1. Preliminary
trials were conducted first to observe any changes. Lactobacillus delbrueckii subsp.
bulgaricus (P/o) was grown with 5% bacteriocin in MRS broth for 8 hours. Viable
counts were measured every 2 hours and the resuUs showed (Figure 16) that
bacteriocin appeared to inhibit Lactobacillus delbrueckii subsp. bulgaricus.
The next experiment used different bacteriocin levels, to see which one would inhibit
Lactobacillus delbrueckii subsp. bulgaricus more. The levels used were: 1%, 5% and
10%, and viable counts were measured at 0, 6 and 10 hours. Table 20 and Figure 17
61
show the results of the three replicates. The resufts show that certainly die
concentrated bacteriocin inhibited Lactobacillus delbrueckii subsp. bulgaricus more
than Z. acidophilus did, when grown with Lactobacillus delbrueckii subsp.
bulgaricus. Figure 17 shows that there was a 3 to 5 log cycle difference between the
control and the experimental batches. The 10%) sample did have the lowest viable
count after 10 hours with a 5 log cycle difference. The next lowest was the 5%
sample which also had a 5 log cycle difference. The P/o sample had a 3 log cycle
difference. This experiment showed that Lactobacillus delbrueckii subsp. bulgaricus
is certainly inhibited by concentrated bacteriocin, produced by Z. acidophilus, and the
more bacteriocin added, the more inhibition was observed, which was to be expected.
It was observed that, \% sample did inhibit Lactobacillus delbrueckii subsp.
bulgaricus, and therefore it was decided to lower the inoculation of bacteriocin, as
incorporating 5%o and 10%) would not be economical in industry. •
In the next set of experiments, the inoculation sizes used were: 0.5%, 1% and 2%.
The three replicates are shown in Tables 21 to 23. It can be seen from the three tables
that the 2% batch did inhibit Lactobacillus delbrueckii subsp. bulgaricus the most,
followed by the 1% and the 0.5% samples. The three replicates are presented rather
than one to show that the bacteriocin used lost activity over time. The three
experiments were conducted within a week and the bacteriocin was prepared fresh
and then stored at 4°C until required. During these 7 days the activity of bacteriocin
dropped. The first replicate (Table 21) showed that there was a 4 log cycle difference
between the control and the 2% batch, a 2 log cycle difference between the control
and the 1% batch and a 1 log cycle difference between the control and the 0.5% batch.
In the second replicate (Table 22) the difference between the control was 2 log for the
2% batch, 1 log for the P/o batch and the 0.5%) batch was in the same log cycle as the
control. The third replicate (Table 23) shows that there was a 1 log cycle difference
between the control and all the experimental batches. This shows a marked decrease
in activity over the three experiments in only 7 days when the bacteriocin was stored
at 4°C.
62
4.7 Bacteriocin Incorporated in Yoghurt Production
4.7.1 pH and viable counts
Yoghurt was made as described in section 3.4.6. The levels of bacteriocin chosen
were 1% and 2%) and this was added to the yoghurt at the same time as the bacteria.
Samples were taken at the beginning and at the end of fermentation for viable count
analysis and the pH was monitored at the start of fermentation, 2 hours after and then
every half hour, until a pH of 4.5 was reached. The yoghurt was then stored at 4°C
and pH and viable counts were monitored every week for 6 weeks. All samples taken
were frozen to measure organic acid concentration.
The results of the viable counts and pH during fermentation and storage are shown in
Tables 24, 25 and 26, respectively. The results of organic acid production during
fermentation are shown in Tables 27 and 28 and Figures 18 to 24.
The fermentation time of all three batches of yoghurts was 3 hours, which is
considered as fast for yoghurt making. There was no difference in time between the
batches at all. The pH at the end of fermentation was 4.44 for the control 4.39 for the
yoghurt containing P/o bacteriocin and 4.32 for that containing 2%) bacteriocin. There
is very little difference between these pH values. A possible reason for the slightly
lower value in the 2%o batch is the time it took to measure all of the replicates and
during this short duration the 2%) batch continued to ferment and the pH decreased
slightly.
The viable counts show that S. thermophilus grew about 1 log in all yoghurts,
Lactobacillus delbrueckii subsp. bulgaricus grew 1 log and Z. acidophilus and B.
longum stayed in the same log cycle. There appeared to be little difference in the
growth of Lactobacillus delbrueckii subsp. bulgaricus between the three batches of
yoghurts. The 1% batch had the highest viable counts followed by the control and
then the 2% batch. This means that the added bacteriocin did not inhibit
Lactobacillus delbrueckii subsp. bulgaricus.
63
S. thermophilus grew very well with the 1% having the highest viable count followed
by the control and then the 2%o batch, which is the same as for the Lactobacillus
delbrueckii subsp. bulgaricus counts. Z. acidophilus and B. longum slightly increased
in number. The control yoghurt had the highest number of Z. acidophilus, then the
1% batch then the 2% batch. The 2% batch had the highest number of B. longum
followed by the control and the 1% batch. In all batches the probiotic bacteria only
grew slightly staying within the same log cycle except for B. longum in the 2% batch,
which just grew to 1.00x10^. The probiotic bacteria did not increase in number
further because of the short fermentation time. Probiotic bacteria are known to grow
slowly in milk and there was not enough time for the bacteria to increase in number.
It has been said that for probiotic bacteria to have any therapeutic effect in the gut the
number of these bacteria must be above 1.00x10^ CFU/mL of yoghurt. There was
only just enough Z. acidophilus and not quite enough B. longum in the yoghurt to
cause any therapeutic effect.
During storage the pH dropped considerably in all yoghurts in the first week and in
the 2^^ week, but went up in week 3 and stayed relatively stable up to week 6. It is
unclear why the pH of yoghurt dropped so much in week 2 but it could be due to the
high number of Lactobacillus delbrueckii subsp. bulgaricus present.
S. thermophilus in all the three yoghurts remained constant during storage. The viable
counts remained in the 10 -log cycle and only fluctuated slightly. Lactobacillus
delbrueckii subsp. bulgaricus for all three yoghurts declined in number over the 6
weeks of storage period. In week 1, the counts dropped slightly and in week 2 the
counts were still within the 10^-log cycle but did decrease. In week 3, the counts
decreased in the control and in the 2% batch decreased by 1 log. The P/o batch did go
up, but only very little. In week 4, the control sample went down by 1 log and the P/o
and 2% batches dropped 2 log. This is a fairly substantial decrease. It is possible that
the bacteriocin did help to kill the Lactobacillus delbrueckii subsp. bulgaricus in this
week. This may be why there is such a large decrease in counts in this week and also
why the control did not decrease as much. In week 5 the confrol decreased by 3 log as
did the 1% batch but the 2% batch decreased by 2 log. hi week 6, all yoghurts
decreased 1 log and all had counts in the lO' log cycle. Therefore, Lactobacillus
delbrueckii subsp. bulgaricus in all three batches decreased from 10^ to lO' in 6
64
weeks. The decrease in population of Lactobacillus delbrueckii subsp. bulgaricus as
compared to that ofS. thermophilus was also observed by Kim et al (1993).
The changes in the counts of Z. acidophilus in yoghurts during manufacture and
storage are presented in Tables 24, 25 and 26. In the first week of storage, Z.
acidophilus in the control batch decreased considerably dropping 2 log. It then
continued to drop considerably for the rest of the storage period where it only had
47.5 CFU/mL at week 6. The 1% batch had a similar decrease in its Z. acidophilus
population in the first week of storage with a 1 log drop and also continued to drop
during the next 5 weeks. The Z. acidophilus decreased at a slower rate the confrol
batch as it did not drop to 70 CFU/mL until week 4 where as the control fell to 38.3
CFU/mL in week 3. The 2%o batch decreased sHghtly in its first week of storage and
then followed a similar pattem to the 1% batch, as it did not drop to 28 CFU/mL until
week 3. The incorporation of bacteriocin may have given the Z. acidophilus a better
chance of survival in the yoghurt. The pH of the yoghurts decreased considerably in
week 2 and this is possibly why the Z. acidophilus dropped several log cycles in week
2. However during the rest of the storage period there was no sudden drop in pH and
all the yoghurts had very similar pH values so pH may not have been the reason why
there was a different rate of Z. acidophilus decrease in the yoghurts. The other factor
that might have affected the viability of Z. acidophilus could be either antagonism by
yoghurt organism. Rybka and Kailasapathy (1995) also observed less viability of Z.
acidophilus in yoghurt with Lactobacillus delbrueckii subsp. bulgaricus.
In the first week of storage, B. longum decreased slightly but remained fairly stable
until week 3 when it dropped 1 log cycle in all yoghurts. In week 4 the counts all
went up 1 log cycle but dropped again in week 5 and then remained in the same log
cycle in week 6. The bifidobacteria seemed to be stable in the yoghurt even though
the population was not high enough to cause any therapeutic effect in the gut. Several
bifidobacteria strains have shown tolerance to low pH (Lankaputhra et al, 1996b).
Martin and Chou (1992) observed that viability of Bifidobacterium sp. was species
and strain dependent and the viability greatiy varied amongst them. The presence of
Lactobacillus delbrueckii subsp. bulgaricus can also increase the viabiUty of
bifidobacteria. Lactobacillus delbrueckii subsp. bulgaricus is known for its
proteolytic nature (Shankar and Davies, 1976) and the free amino acids produced by
65
this organism in yoghurt could be used by other organisms and would have promoted
the growth of probiotic bacteria (Dutta et al, 1973; Singh et al, 1980). Most
bifidobacteria have been found to be weakly proteolytic and free amino acids are
essential for most bifidobacteria (Klaver e? al, 1993). Therefore, tt is expected that
the presence of Lactobacillus delbrueckii subsp. bulgaricus nught be beneficial for
the growth of bifidobacteria during manufacture of yoghurt.
4.7.2 Organic acid analysis
The analysis of organic acids was performed using the HPLC according to the method
described in Section 3.6.3. The changes in organic acid production are presented in
Tables 27 and 28 and Figures 18 to 24. Figure 21 shows the concentration of lactic
acid produced in yoghurt. During fermentation the concentration of lactic acid
increased 10 fold. The 1% yoghurt had the highest concentration at the end of
fermentation followed by the control and the 2%o yoghurt but the two experimental
yoghurts were not significantly different as compared to the control. During storage
the lactic acid concentration in the control increased up to week 2 and then decreased
slightly. It then went up again in week 4 and continued to increase for the next two
weeks.
The lactic acid production in the 1 % batch continued to increase during storage until
week 3. In week 4 and 5, there was a decrease and then a slight increase in week 6.
In the 2% batch, the lactic acid concentration increased in week 1 but then decreased
and fluctuated for the rest of the storage period. Although Figure 21 shows that the
lactic acid production did fluctuate during storage the experimental yoghurts were
significantiy higher in week 1 and for the rest of the storage trial there was no
significant difference.
Acetic acid (Figure 18) was only detected in the control yoghurt and only during
storage. The first week showed a concentration of 15.97|ag/g and week 6 had a
concentration of 16.76jj.g/g and there were only small fluctuations in between. The
other samples did not show any acetic acid. Acetic acid was produced by
bifidobacteria but only after 12 hours. This is why there was no acetic acid detected
66
during fermentation. Factors that could have influenced the production could be the
bacteriocin was affecting the bifidobacteria in some way or the conditions in the
control were more favourable to the bacteria than the other two batches.
Butyric acid (Figure 19) was detected at the end of fermentation in all yoghurts. The
acid then increased during the storage period. The P/o yoghurt had a sigiuficantly
higher concentration of butyric acid during storage than the control and the 2%
yoghurt was not significantly different. As the results show the starter bacteria
produced butyric acid.
Lankaputhra and Shah (1998) studied the levels of acetic, butyric, lactic and pymvic
acids produced by the probiotic bacteria as determined by HPLC. All strains
produced these acids with butyric acid being produced by most strains of Z.
acidophilus and bifidobacteria. Lankaputhra and Shah (1998) also studied the
antimutagenic activity of organic acids against eight mutagens and promutagens. The
study found that butyric acid showed the highest antimutagenic activity against all the
8 mutagens or promutagens. Therefore probiotic bacteria, which produce butyric acid,
are more likely to provide antimutagenic properties.
Formic acid (Figure 20) was present in milk at the beginning of fermentation but by
the end had decreased considerably. The 2% yoghurt was significantly lower than the
control at the end of fermentation. During storage no formic acid was detected. This
shows that formic acid must be used as a growth factor for the starter bacteria.
S. thermophilus produced formic acid, which was also reported by Veringa et al
(1968) and Bottazzi et al. (1971). It was found that the production of formic acid
stimulated the growth of Lactobacillus delbrueckii subsp. bulgaricus. It also can
induce the proteolytic activity of Lactobacillus delbrueckii subsp. bulgaricus in milk
so that it became able to hydrolyse p -lactoglobulin, and asl and p-casein as
compared to only P-casein without the formic acid (Moreira et al, 1997). The
stimulatory effect of formic acid remains unnoticed in intensely heated milk because
in this milk formic acid had been formed by decomposition of lactose. The
production of formic acid by the cocci is, however, essential in industrial practice,
67
where more moderate heat treatments of yoghurt milk are applied (Walsfra et al,
1999).
The changes in orotic acid production in yoghurt are presented in Figure 22. There
was a decrease in the concentration of orotic acid during fermentation and then during
storage the concentration fluctuated slightly but stayed relatively constant. The P/o
yoghurt was significantly lower than the control in weeks 2, 4 and 5 and the 2%o
yoghurt was significantly lower at the beginning of fermentation and in weeks 2 and
5. Orotic acid is used during fermentation as a grov^h factor, which can be seen in
Figure 22 where there is a decrease in concentration during the three hours of
fermentation. Orotic acid is metabolised by the yoghurt starter cultures, most
probably by Lactobacillus delbrueckii subsp. bulgaricus (Tamime and Robinson,
1999). It has been found that orotic acid can be reduced by up to 50%o in milk during
the manufacture of yoghurt (Tamime and Robinson, 1999; Navder et al, 1990). The
reduction was not the same in the yoghurts in this experiment, which is probably due
to a rapid fermentation. Orotic acid possessed some significant therapeutic properties,
since it plays an important role in the biosynthesis of nucleic acids and the lowering of
semm cholesterol (Femandez-Garcia and McGregor, 1994).
Figure 23 shows the production of propionic acid in yoghurt. There was no propionic
acid detected in the milk at the beginning of fermentation, however, the production
began during fermentation and continued during storage. The 1% yoghurt was not
significantly different to the control but the 2% yoghurt was significantly lower. The
yoghurt starter bacteria produce propionic acid as a volatile flavour compound
(Tamime and Robinson, 1999).
The production of uric acid is presented in Figure 24. Uric acid is already present in
milk and remained fairly constant throughout fermentation and storage for all three
yoghurts. This result was also observed by Navder et al. (1990) where uric acid
content was not significantly altered after fermentation. Uric acid is present in milk as
a resuU of normal bovine biomedical processes (Navder et al, 1990).
From these experiments, it was observed that the bacteriocin has not inhibited
Lactobacillus delbrueckii subsp. bulgaricus in the yoghurt. The viable counts of
68
Lactobacillus delbrueckii subsp. bulgaricus are very similar between the three
yoghurts at the end of fermentation and this suggests no inhibition has occurred. The
pH and fermentation times are also very similar and this means the bacteriocin has not
lysed Lactobacillus delbrueckii subsp. bulgaricus to reduce the amount of acid
produced which would in tum have allowed the pH to remain stable and the
fermentation time may have been slower. The lactic acid production is very similar
for all three yoghurts suggesting that the Lactobacillus delbrueckii subsp. bulgaricus
is very active. Therefore it is hypothesised that there is some substance that is
blocking the bacteriocin in yoghurt and the next experiments pursue this hypothesis.
4.8 Bacteriocin in milk
Lactobacillus delbrueckii subsp. bulgaricus {Wo) was grown with P/o, 5% and 10%)
levels of bacteriocin for 8 hours as described in section 3.9.2. Samples were taken
every 8 hours to measure viable counts and these results are presented in Figure 25
and Table 29. The results show that the bacteriocin had no effect on the Lactobacillus th
delbrueckii subsp. bulgaricus bacteria. All the counts at the 8 hour are very similar
and have grown the same as the control with out any inhibition observed.
When these results are compared to the results in Table 20 and Figure 17 when the
bacteriocin was grown with Lactobacillus delbrueckii subsp. bulgaricus in MRS broth
there is a difference observed. The Lactobacillus delbrueckii subsp. bulgaricus when
grown in MRS broth had a 3 log cycle drop. This suggests that there is something in
the RSM that is blocking the bacteriocin activity.
4.8.1 Bacteriocin in different levels of milk
To determine if it is the milk that caused the loss of activity in bacteriocin, different
milk media were made containing 3%), 6% and 12%o RSM. Z. acidophilus (1%) was
grown with Lactobacillus delbrueckii subsp. bulgaricus (P/o) for 6 hours. The spot-
on lawn technique was used to observe any inhibition against Lactobacillus
delbrueckii subsp. bulgaricus. The results are presented in Table 30. It was observed
that the 12% milk batch did not produce any zones and when incubation continued
after 18 hours there still was no zone observed. Dave and Shah (1999) found that
69
bacteriocin was not observed in 12% RSM milk until after 22 hours of incubation.
The 6%) batch showed a zone after 6 hours of incubation and the 3% batch showed a
zone after 2 hours of incubation. These results indicate that there is something present
in milk that is interfering with the bacteriocin activity.
In the next experiment 12%) RSM and 3% RSM were inoculated with P/o
Lactobacillus delbrueckii subsp. bulgaricus and different levels of bacteriocin and
grown for 18 hours. The purpose was to see if concentrated bacteriocin would have
some effect on Lactobacillus delbrueckii subsp. bulgaricus in a lower concentration
of milk. The results are shown in Table 31. It was observed that the bacteriocin had
no effect on the viable counts of Lactobacillus delbrueckii subsp. bulgaricus. The
viable counts were in the same log cycle; however, the batch with 10%) and 5%
bacteriocin grown in the 12%o milk had a higher count than the LB control. The only
difference that could be seen was the viable counts between the 12%) milk and the 3%
milk controls. The viable counts of Lactobacillus delbrueckii subsp. bulgaricus were
1 log lower in the 3% sample. This was probably due to the lack of nutrients in the
milk.
4.8.2 Bacteriocin in different media
It has been confirmed that there is something present in milk that is blocking the
activity of bacteriocin. In this experiment casein was removed to see if it is the cause
of this decrease in activity. Three percent milk was made and casein was removed
using hydrochloric acid as described in Section 3.9.3. The results are presented in
Table 32 and Figure 26. It was observed that in the MRS broth the bacteriocin did
inhibit the growth of Lactobacillus delbrueckii subsp. bulgaricus. The batch with no
casein but with bacteriocin was higher than its control but was lower than the sample
that did contain casein. There does not seem to be any differences between the batch
with casein or without, therefore this experiment indicates that casein may not be the
substance blocking the bacteriocin.
70
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5.0 CONCLUSION
This project investigated ways of lysing Lactobacillus delbrueckii subsp. bulgaricus
to release growth factors. This was to promote the growth of probiotic bacteria, to
reduce fermentation time for yoghurt making and to control post-acidification.
Sonication of the Lactobacillus delbrueckii subsp. bulgaricus could possibly improve
the viability of probiotic bacteria in yoghurt and also control post-acidification. The
levels of both L. acidophilus and bifidobacteria remained higher than the
recommended viable count required for health benefits during 4 weeks of storage.
However, it would be expensive and impractical to sonicate Lactobacillus delbrueckii
subsp. bulgaricus in a manufacturing plant.
L. acidophilus is known to produce bacteriocin against Lactobacillus delbrueckii
subsp. bulgaricus and if this could be utilised to lyse Lactobacillus delbrueckii subsp.
bulgaricus and release gro"wth factors for probiotic bacteria then this could be a more
practical way of improving the viability of probiotic bacteria and controlling post-
acidification.
Different strains of L. acidophilus and Lactobacillus delbrueckii subsp. bulgaricus
were grown against each other on agar plates to determine which strain would be most
suitable. It was determined that L. acidophilus-hA-S produced an antimicrobial
substance against Lactobacillus delbrueckii subsp. bulgaricus 2515 and these strains
were used in the proceeding experiments. The antimicrobial substance was confirmed
to be a bacteriocin based on the treatment with NaOH, catalase and proteolytic
enzymes.
To test the activity of the bacteriocin producing strain of L. acidophilus against
Lactobacillus delbrueckii subsp. bulgaricus 2515, three different inoculation levels of
L. acidophilus were grown with Lactobacillus delbrueckii subsp. bulgaricus. The
levels used were 1%, 5% and 10%. The Lactobacillus delbrueckii subsp. bulgaricus
in the 10% sample had the lowest viable count and had started to decrease at the 8*
hour of fermentation but the count was still very high. This experiment showed that
126
Lactobacillus delbrueckii subsp. bulgaricus does decrease slightly in the presence of a
high inoculation of L. acidophilus and this may have been more obvious if the
fermentation continued for several more hours. This, however, would not be feasible
in yoghurt making therefore L. acidophilus does not decrease the viable count or lyse
Lactobacillus delbrueckii subsp. bulgaricus enough to slow the production of lactic
acid to stop post-acidification.
In order to test the effectiveness of bacteriocin produced by L. acidophilus, the
organism was grown for 18 hours in MRS broth and then centrifuged, neutralised and
concentrated. Protein was then extracted from the concentrate and dissolved in
sodium carbonate. This was then added to Lactobacillus delbrueckii subsp.
bulgaricus at different inoculation levels and grown for 10 hours. The inoculation
sizes used were 1%, 5%o and 10%, and viable counts were measured at 0, 6 and 10
hours. The results show that the concentrated bacteriocin inhibited Lactobacillus
delbrueckii subsp. bulgaricus more than L. acidophilus did. The 10% batch had the
lowest viable count Lactobacillus delbrueckii subsp. bulgaricus after 10 hours with a
5 log cycle difference as compared to the control. This experiment showed that
Lactobacillus delbrueckii subsp. bulgaricus was inhibited by concentrated bacteriocin
produced by L. acidophilus, and the more bacteriocin added, the more inhibition was
observed, which was to be expected.
It was observed that, 1% batch inhibited Lactobacillus delbrueckii subsp. bulgaricus,
and therefore it was decided to lower the inoculation of bacteriocin, as incorporating
5% and 10%) would not be economical for industry. The inoculation sizes used in the
next experiment were 0.5%), 1% and 2%o. The 2% batch inhibited Lactobacillus
delbrueckii subsp. bulgaricus the most. However, the bacteriocin lost activity over
time. This could pose a problem for manufacturing, as the bacteriocin may not be
reliable and would have to be used fresh for each batch of yoghurt.
The bacteriocin was then used at different rates in yoghurt with probiotic bacteria.
Bacteriocin was added at a rate of 1% and 2%. From these experiments, it was
observed that the bacteriocin did not inhibit Lactobacillus delbrueckii subsp.
bulgaricus in the yoghurt. The viable counts of Lactobacillus delbrueckii subsp.
bulgaricus were very similar between the control and experimental yoghurts at the
127
end of fermentation and this suggested that no inhibition occurred. The pH and
fermentation times are also very similar and this indicated the bacteriocin had not
lysed Lactobacillus delbrueckii subsp. bulgaricus, as the fermentation time may have
been slower. Therefore, it was thought that there was some substance that was
blocking the activity of bacteriocin in yoghurt.
Lactobacillus delbrueckii subsp. bulgaricus (1%) was grown with l%o, 5% and 10%)
levels of bacteriocin for 8 hours in 12%) RSM. The results show that the bacteriocin
had no effect on the Lactobacillus delbrueckii subsp. bulgaricus bacteria. All the til
counts at the 8 hour were very similar and grew the same as the control with out any
inhibition observed.
To determine if it was the milk that caused the loss of activity in bacteriocin, different
milk media were made containing 3%, 6% and 12% RSM. It was observed that the
12% milk sample did not produce any zones whereas the 3% and 6%) batches did.
This suggested that there was something in milk that was blocking the activity of
bacteriocin, possibly the protein (bacteriocin) interacted with casein.
Casein was removed to see if it was the cause of the blocking of the bacteriocin.
Three percent milk was made and casein was removed. There did not appear to be any
difference between the sample with casein or without, but it was observed that in the
MRS broth the bacteriocin inhibited L. delbrueckii subsp. bulgaricus. Therefore this
experiment indicated that casein was not the substance blocking the bacteriocin.
This study has shown that the use of Lactobacillus delbrueckii subsp. bulgaricus does
increase the fermentation time of yoghurt and would be very beneficial to
manufacturers. It does, however, increase post-acidification. Sonication could be one
way of controlling this but would be expensive and impractical. This study has shown
that bacteriocin produced by L. acidophilus inhibited the growth of Lactobacillus
delbrueckii subsp. bulgaricus and could be a useful way of improving yoghurt. When
the bacteriocin is concentrated and purified it is very active against Lactobacillus
delbrueckii subsp. bulgaricus when it is grown in MRS broth. However, in milk there
appears to be a protective substance blocking the bacteriocin. This substance was not
casein as shown by experiments.
128
The use of bacteriocin in yoghurt could be beneficial but there are issues that need to
be addressed. The bacteriocin can lyse Lactobacillus delbrueckii subsp. bulgaricus
and would certainly help probiotic bacteria, as there would be a better environment
for them to survive. Post-acidification would also be controlled, as Lactobacillus
delbrueckii subsp. bulgaricus would not be able to produce as much lactic acid. The
fermentation time would be shorter due to the presence of Lactobacillus delbrueckii
subsp. bulgaricus and this would be very important to yoghurt manufacturers. The
investigation into bacteriocin should be continued, as it will be an important
supplement to manufacturers.
129
6.0 FUTURE DIRECTION
This project aimed to determine if bacteriocin produced by L. acidophilus could lyse
Lactobacillus delbrueckii subsp. bulgaricus and use this technique in yoghurt making.
Lactobacillus delbrueckii subsp. bulgaricus can produce necessary growth factors for
probiotic bacteria to utilise and improve viability of probiotic bacteria.
This project determined that bacteriocin inhibited Lactobacillus delbrueckii subsp.
bulgaricus when grown in MRS broth, but not in 12%) RSM. There is something in
milk that blocked the activity of bacteriocin. It is possible that a protein is blocking
its inhibitory activity. Future projects should aim at looking at determining as to what
is blocking the activity of bacteriocin. The structure of bacteriocin should be looked
at. There could be a bonding site on the bacteriocin and it may be possible to block
this site before adding to milk.
The addition of bacteriocin should also be investigated. It would be impractical to
add the bacteriocin as a liquid as it has been found that it loses activity over time.
Stability trials of bacteriocin should be conducted and other methods of application
should be explored. Freeze drying was attempted without success as the product
became sticky and would not dry completely.
Morgan et al. (2001) developed a method to spray dry lacticin 3147; a bacteriocin
produced by Lactococcus lactis. This powder was tested in yoghurt, cottage cheese
and soup; however, lacticin 3147 is not heat stable and lost considerable amount of
activity when autoclaved. This method of concentration would be very beneficial to
the industry and would be suitable for bacteriocin produced by L. acidophilus LA-5 as
it does withstand autoclaving temperatures.
130
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