FT 5105 FOOD MICROBIOLOGY · ATP NADH NADH NAD+ NAD+ ... cooled to 45C Starter culture is added...

FT 5105 FOOD MICROBIOLOGY POSTGRADUATE INTITUTE OF AGRICULTURE

BY

Terrence Madhujith

Professor in Food Science and Technology

University of Peradeniya

At the of the session, the learner should be able to ◦ Define fermentation

◦ Describe the importance of fermentation as a food preservation method

◦ Describe the factors affecting fermentation

FT 5105 Food Microbiology 2

First explained by Louis Pasteur – fermentation by yeast

Pasteur’s “life without air”

Originates from the Latin word fervere [boiling ]

He explained that fermentation occurs under anaerobic conditions and can bring about CO2, ethanol and other products

Fermentation is considered the oldest food preservation method


Even today fermentation plays a vital role in food preservation

Fermentation occurs naturally

However, uncontrolled fermentation leads to spoilage

Controlled growth may bring about desirable changes.

Fermentation brings about food and beverages

Alcoholic beverages

Industrial products

Pharmaceuticals

Hormones





Fermentation is the metabolic process in which CHO and related compounds are oxidized with the release of energy in the absence of any external electron acceptors

The final electron acceptors are organic compounds therefore, only partial oxidation happens and only little energy is released


• Fermentation is all about CHO and related materials

• However, almost all foods contain fat and protein as well

• These compounds too undergo simultaneous changes

• Thus, a distinction is necessary – Changes of CHO fermentation

– Changes of proteins proteolytic/putrefaction

– Changes in lipids lipolytic


When a food undergoes fermentation all three processes are possible

However, one process usually dominates the others

Which process dominates is decided by ◦ Type of food

◦ Type of MO

◦ Environmental conditions


Alcoholic fermentation of glucose

C6H12O6 → 2 C2H5OH + 2 CO2

glucose ethanol carbon dioxide

Glucose Glycolysis Pyruvic Acid

Acetaldehyde Ethyl Alcohol

CO2

ATP

ATP

NADH

NADH NAD+

NAD+

Pyruvic acid from glycolysis is reduced to lactic acid by NADH, which is oxidized to NAD+.

This commonly occurs in muscle cells. Lactic acid fermentation allows glycolysis to continue by ensuring that NADH is returned to its oxidized state (NAD+).




Result of a positive role of microorganisms

Fermentation has been used for over 5000 years

Fermented foods show more shelf life and nutritional qualities compared to their non-fermented counterparts

Lactic acid bacteria and yeasts play a major role


Dairy Products

Acidophilus milk

Bulgarian buttermilk

cheese

Kefir

Yoghurt

Kumiss


Starter cultures are used

Lactic starter culture MOS convert lactose to lactic acid L. cremoris L. lactis L. diacetilactis Leuconostoc citrovorum

Starter culture can be single or mixed

Usually the cultures are preserved by freeze drying or freezing

pH drops down to 4.3 – 4.5

Titratable acidity increases up to 0.8-1.0%


1. Yoghurt S. thermophilus & L. bulgaricus

are used at 1:1 ratio

Cocci grow faster than rod.

Thus, cocci are mainly responsible for LA production

Rod adds flavor and aroma

Acetaldehyde is mainly responsible for yoghurt flavor

Combination produces more LA and acetaldehyde that they produce when grown alone


Milk is heated to 85-95C

cooled to 45C

Starter culture is added (2%)

Incubate at 42-45C for 3-5h

Place in cups and chill to 4C

Titratable acidity goes up to 0.9%

To achieve this, fermentation is to be stopped at 0.65%

Streptococcus can tolerate pH up to 3.6

Lactobacillus can tolerate only up to 4.2

During fermentation, pathogenic MOs are destroyed. E.g. Coliform


Freshly produced yoghurt contains up to 109 organisms/g

During storage the count drops to 106/g

Acetaldehyde is present at 30-40 ppm level

Diacetyl is present in minute quantities

Both contribute to the flavor


treptococcus salivarius

Lactobacillus delbrueckii

Lactobacillus acidophilus

Lactobacillus casei

Bifidobacterium adolescentis


Hundreds of different varieties produced around the world

Cheese can be defines as consolidated curd of milk solids in which milk fat is entrapped by coagulated casein


• Ripened cheese types are produced through further microbial activity

• All hard cheese are ripened by bacteria for 2-16 months – e.g. Cheddar

• Semi hard cheese – by bacteria over 1-8 months – e.g. Gouda

• Semi hard – by mold 2-12 months – e.g. Roquefort and Blue

Four basic steps are involved

1. Curdling of milk

Performed by either adding rennet or through starter culture

Pure of mixed starter cultures can be used

Streptococcus cremoris S. lactis S. thermophilus Lactobacillus lactis L. helveticus L. bulgaricus



Casein gets curdled during this step

LA produced by the starter culture brings pH down

Casein coagulates at pH between 4.64 - 4.78

Casein is present as calcium caseinate

LA forms its salt (Calcium lactate) by reacting with calcium caseinate

Casein gets precipitated when Ca level lowers to a certain extent

Rennin makes Ca paracaseinate

CP formed reacts with free Ca thus casein gets precipitated

pH does not go down

Sometimes a combination of the two are used

2. Draining the curd to remove excess moisture

Pressure may or may not be used depending on the type

3. Salting

4. Ripening



Many varieties except few are ripened. E.g. cottage and cream cheese are not ripened

Roquefort Cheese: The curd is inoculated with Penicillium roqueforti

Inoculated curd is placed in ripening room of which T and RH are carefully controlled

When the fungal growth is adequate the temperature is brought down to control the fungal growth


P. roqeuforti is aerobic, however, can grow even at low oxygen concentrations such as 4.25%

To provide oxygen, holes are made in the curd

Camemberti Cheese

Ripening goes on for 2-4 weeks

Inoculated with Penicillium camemberti

They grow on the rind (outer layer)

The proteolytic enzymes secreted by the fungi soften the cheese


• Thus the texture of cheese changes

• Swiss Cheese:

• Ripening is carried out by – Lactobacillus

– Streptococcus and

– Propionibacterium

• They produce acetic acid, propionic acid and water

• CO2 makes holes in cheese


Swiss cheese – L. bulgaricus + S. thermophilus + Propionibacterium shermanii

Glu Pyruvate lactate propionate acetate CO2

Cheddar Cheese:

Various Streptococci and Lactobacilli are used to ripen cheddar cheese

Limburger Cheese:

Streptococcus, Lactobacillus and Brevibacterium linens are used

The latter is proteolytic and mainly responsible for producing flavor

High salt and low pH conditions are used


Group Variety Primary ripening organism/s

Hard Cheddar Bacteria

Swiss Eye forming Bacteria and other bacteria

Semi hard Brick Bacteria

Roquefort Molds

Gorgonzola Molds

Soft Limburger Bacteria

Cammemberti Mold

Cottage Unripened

cream Unripened


Lactic acid

Imparts a fresh acid flavor to cheese

Assists rennet in coagulating casein

Improve texture

Suppress the growth of pathogens/spoilage Mos

Traces of aroma compounds produced by LABs contribute to the flavor of cheese


Produced by churning pasteurized cream (71C for 30 min)

Butter culture (starter culture) is introduced ◦ Increases yield

◦ Helps develop flavor

S. lactis and S. cremoris – form LA

Leuconostoc dextranicum and L. citrovorum - develop flavor


Acetoin (3-hydroxybutanone) produced by latter is spontaneously oxidized to diacetyl

Acetoin and diacetyl give the characteristic butter flavor

0.25 – 1% starter is used


Milk based drink containing kefir grains

Lactobacillus bulgaricus

L. lactis naturally present in kefir are used

Lactose fermenting yeasts are

also added


Lactobacillus produce LA while yeasts produce alcohol

The final alcohol content is 1-2%

Cow, goat or sheep milk is used

When mare milk is used the product is known as Kumiss


Produced using lactic starter culture

Pasteurized cream

Starter culture

holding until acidity develops

churning

Butter Buttermilk

Commercial cultured buttermilk is prepared by inoculating skim milk with lactic culture

Curd is broken through agitation – this is called cultured buttermilk

Sour cream is prepared by homogenized and pasteurized light cream with lactic culture

Aroma is due to diacetyl and sourness is due to LA

Culture Action Diary Product

L. delbrueckii bulgaricus

Acid and flavor Bulgarian butter milk

L. Acidophilus Acid Acidophilus milk

Leuconostoc cremoris

Flavor Cultured buttermilk, sour cream, cottage cheese

S. lactis diacetylactis

Acid and flavor Sour cream, buttermilk

L. lactis and bulgaricus and yeast

Acid and alcohol Kefir & Kumiss

S. faecalis Acid and flavor Swiss and Cheddar cheese

• Two functions – Leavening

– Flavor development – yeasts do not produce flavor compounds in significant amounts but bacteria do.

• Yeasts are used for leavening

• Bakers add extra amount of yeast to encourage fermentation and thereby to reduce time spent

• Sugar naturally present in flour is enough, but to boost the activity of yeast sugar is added


Yeasts ferment sugar to CO2

Released CO2 helps in softening the

dough

Their activity starts immediately after mixing them with dough and continues until heat in the oven kills enzymes

During fermentation dough conditioning also happens

San Francisco sourdough bread and similar bread types – both bacteria and yeast used


• Historically, barm [mother sponge] was used as the source of starter culture

• L. sanfrancisco – souring

• S. exiguus – leavening

Idli

• Produced by fermenting pre-soaked rice and black gram overnight and steaming

• L. mesenteroides predominates in Idli

• Strains of S. cerevisiae are used

• Good strains – Produce a large no. of cells

– Stable and viable

– Produce CO2 rapidly

• Original mother culture is increased in number in few steps to final seed culture

• Finally in culture tanks, the seed culture cells are concentrated into a ‘cream’ through centrifugation


This heavy suspension is added into the mash

E.g. sugar cane or beet molasses

Mineral salts and other additives are added to enhance the growth of yeasts

E.g. ammonium slats, urea, malt sprouts, phosphates, vitamins, vegetable extracts etc.

pH is adjusted to 4.3- 4.5

Mixtures are maintained at 30C

Mixture is aerated rapidly


• Molasses are added gradually to maintain sugar content

• After 4-5 budding cycles the mixture is centrifuged to produce a thick ‘cream’

• ‘cream’ is filter pressed to remove excess liquid

• The mass of yeast is made into pellets, cakes after adding little amount of vegetable oil

• Active dry yeast is made by drying to reduce MC to 8%


Fermented Sausages Dry Semi dry Intermediate

Dry sausages – ground meat is mixed with curing agents and seasonings

Mixture is stuffed in casings Incubate for varying periods Starters or natural flora can be used Fermentation time is reduced when starters

are used


Curing mixture contains Glucose

Nitrites/nitrates

During incubation sausages are stored at low RH conditions – 60%

Time varies from 2wk to 6 months

pH goes down to 4.8

L plantarum

L brevis

L buchneri


Semi-dry sausages follow the steps but subjected to less drying

These are usually heated or smoked and cooked before eating

E.g. Bologna, Summer sausages


Uneviscerated fish is mixed with salt at 1:3 ratio

Placed in fermentation tanks and allowed to liquefy for 6 m

Liquid is collected, filtered and ripened for further 3m

Halophilic microbes grow


Fish sauce is a dark colored, clear liquid with a distinct aroma

Streptococcus, Micrococcus and Bacillus predominate

Fish proteases too contribute to liquefication.


• A canned or bottled made of shredded cabbage leaves

• Preserved mainly by lactic acid generated during fermentation

• Normal cabbage flora is used for fermentation

• 2.25% salt is added and mixed well with cabbage leaves

• Anaerobic conditions are maintained inside the container


• Contents are well packed to release as much air as possible

• This helps in achieving anaerobic conditions inside

• The container is filled up to the top and weight is applied at the top

• This helps in bringing juice out and submerging the leaves in juice

• Otherwise molds and yeasts grow on the surface leading to spoilage


Fermentation commences in a day or two and continues for 3-6 weeks

When the fermentation process is complete ◦ All fermentable CHOs are used up

◦ Typical aroma is developed

◦ Acidity rises to 1.8-2.2%

◦ pH is between 3.2 -3.5


• The final product is stable when stored at low temperature and free of oxygen

• If temperature rises the product darkens and flavor is lost

• Three steps are involved in fermentation

• 1. fermentation by Leuconostoc mesenteroides – Consumes sugars producing lactic acid, acetic acid, ETOH, mannitol and CO2

• Pleasant odor is mainly developed by this


CO2 generated helps bringing in anaerobic conditions quickly

When acidity reaches 1% they start to die off

2. Then Lactobacillus plantarum takes over

They mainly produce lactic acid

When acidity goes up they too die off

3. Then Lactobacillus brevis takes over


• L. brevis can tolerate any acidity that can develop naturally

• A homofermentator thus no CO2 is produced

• During fermentation lactic acid and acetic acid is formed at 4:1 ratio

• L mesenteroides needs low temperature thus it is important to provide low temperature at the onset to develop good flavor


Cucumbers are fermented in large wooden or plastic vats

Cucumber is placed in 5% brine

Strength is increased over 6-9 wks up to 15% every week

Salt extracts water from cucumber thus salinity goes down gradually


Acidity will reach 0.5-1%

pH is 3.8

Aerobacter aerogenes predominate

Produces CO2 and H2 vigorously

They live for about 1 week and die off

Then LABS predominate The main bacterium is L. plantarum

LABs are involved Leuconostoc mesenteroides P. cerevisiae Lactobacillus brevis and Strptococcus faecalis


Yeasts are found at all stages of fermentation

Their peak population is found in the third week

When brine fermentation is complete and salt concentration reaches 15% the mixture is microbiologically stable


Green and black olives

Fully mature fruits are harvested and placed in shallow vats

Olives are first treated with 2% lye solution to remove bitter taste

Alkali is washed away with water

Fruits are placed in barrels and brine (12%) is added

The fruits are graded, sorted and packed in bottles filled with 6-9% brine and 0.5% LA


Olives are mostly pitted to remove the pits and stuffed with pimento

Spoilage of olives: A. Scum formation – due to growth of yeasts.

The whitish - cream colored scum develops on the brine surface

They utilize lactic acid as the energy source B. zapatera spoilage due to Clostridium C. Butyric acid fermentation: Sugars are

converted to butyric acid through fermentation


Yeast spots: White spots develop just beneath the surface of fruits

Softening: Believed to be done by proteolytic molds

Discoloration: prevented by adding lactic acid. Promoted by iron thus iron vessels should never be used for olive processing


Fermentation starts spontaneously

Natural flora is used

Brine from previous well fermented olives is used

First to appear is Aerobacter aerogenes

They produce hydrogen and CO2

Then LABS predominate ◦ L. mesenteroides

◦ P. cerevisiae

◦ L. plantarum


• They produce lactic acid

• More LA can be obtained by adding small quantity of sugar

• This should not be done at the onset as sugar addition promotes excessive gassing

• Fermentation continues until all soluble CHOs are used up

• At this stage the product is fully cured

• It is stable as long as kept out of air

• Final pH reaches 3.8-4

• LA content should be above 0.5%


Soybean and wheat flour are inoculated with A. oryzae or A. soyae

Leave for 3days

This produces a large amount of fermentable matter

Fungal covered material is mixed with 18% NaCl and water and incubate at RT for 6-12m


Moromi is pressed to release soy sauce

L. delbrueckii Zygosaccharomyces rouxii carry out anaerobic

fermentation Some other fermented Products Tempeh Miso Ogi Gari


• Fermented soybean

• Soybean is soaked and then cooked

• Inoculated with tempeh from a previous batch

• Wrap in banana leaves and leave for 1-2 days at ambient temperature

• Mold grow on tempeh and as a result it becomes a ‘cake’

• Rhizopus oligosporus R. arrhizus, R. oryzae

• Wheat too can be used


Fermented soybean product common in Japan

Steamed soy is mixed with koji and salt

White miso – results in one week

Brown miso – fermented for a long time – up to 2 years

Aspergillus oryzae is used

Miso is a thick paste used for soups, sauces, spreads etc.


• Popular in Nigeria

• Produced from corn

• Corn is soaked for 2- 3days and wet milled

• Corynebacterium predominated during steeping and it is responsible for diastatic activity

• Lactobacilllus plantarum and S. cerevisiae are also responsible for fermentation

• Used as the first weaning food


Fermented cassava

Used in Wet Africa

Cyanogenic glucosides are released as hydrocyanic acid

Grated cassava (peeled) is left for 3-4 days for fermentation

Fermentation occurs in two stages


in stage 1 – Corynebacterium manihot ferments starch and produces acids thus the pH goes down. CG is released during this stage

In stage 2 – Geotrichum grows and develop flavor


Ale – top fermented product by S. cerevisiae Lager – bottom fermented by S. uvarum

Malted beverages produced by brewing

Yeasts convert fermentable sugars to ethanol and

CO2

Yeasts do not produce enough amylases

Thus, it is essential to convert starch into

fermentable sugars


First step is malting

Barley is soaked and germinated for 5 days and kiln dried

Malt is a good source of amylases Alpha amylase – increase sugar content Beta amylase – liquefy

Kiln-dried product is ground to form a powder [malt milling]


In the next step mashing

Objective is to make more fermentable substances

Hydrolysis occurs

Cereal adjuncts are added

Temperature is raised to 60C – at this temp saccharification occurs



Insoluble matter settles down and the liquid part is separated ‘ Wort’

Hops are added to wort and boiled for 2.5h

Hops add bitter taste due to bitter acids

Also adds resins such as humulone, cohumulone and adhumulone



• Resins are effective against g(+)ves

• Next step is fermentation

• S. cerevisiae [top fermenting]or

uvarum [bottom fermenting] is added into wort

• Fermentation is carried out at 3-14C for 8-14 days

• FT 5105 Food Microbiology 84

Yeasts produce EtOH and CO2

In addition, little amounts of glycerol, acetic acid, aromatic esters too are produced

Aging/ maturation

Adds flavor

Improves clarity


Defects Turbidity Off flavors Poor physical characters

Beer infections

Sarcina sickness:

Caused by

P cerevisiae Lactobacillus pastorianus L. diastaticus and Zymomonas anaerobium


Obesumbacterium proteus produce parsnips odor

Acetobactor and Gluconobactor also produce sourness


• Traditionally made out of grapes

• Can also be made from fruit juices,

berries etc.

• Grapes are harvested when they have the correct sugar content

• Fruits are crushed and SO2 treated - must


• Wine yeast is added – S. cerevisiae ellipsoideus

• Mixture is fermented at 24-27C for about a week.

• High temp deactivate yeast while low temp permit LABs to grow

• Must is to be cooled as fermentation generates heat


• When the first fermentation is over clear liquid is siphoned off to another tank

• Secondary fermentation continues in this tank for about 10 days at about 25C

• At the end again clear portion is siphoned off

• Wine can be flash-pasteurized to precipitate proteins


• Cooled for few days and filtered

• Placed in oak or redwood tanks and stored for months

• Periodically wine is racked

• In the initial fermentation stage, aeration is preferred to allow yeast growth

• Later on anaerobic conditions are favored


Starch from steamed rice is hydrolyzed by A. oryzae

Fermentation is carried out by Saccharomyces sake over 30 – 40 days

Product contains 12-15% alcohol and 0.3% LA

LA is produced by LAB


Seeds are fermented in tanks for 10-12 days

During fermentation liquids generated and temperature rises to 50C

Fermentation occurs in two stages

In the first stage sugars from acidic pulp is converted to alcohol

Then alcohol is oxidized to acetic acid

Yeasts play an important role


• Acidity – Low pH is better

• Sugar content – dry wines are spoilt by bacteria

• Alcohol concentration

• Tannins

• Amount of SO2 – 100-750 ppm

• storage temperature

• Availability of air – oxygen favors unnecessary yeast films.


• If there is no inherent acidity, then acidity has to be developed quickly through fermentation

• If exposed to oxygen/air, microbial growth on the surface can oxidizes acids. Thus, acidity goes down. So does the preservative action.

• This can potentially happen in ripening cheddar cheese


Certain yeasts produce alkaline by products such as NH3 as a result of their regular metabolism

This is purposely encouraged in the production of Limburger cheese



A

B

C

D E

F

A-B - Germicidal action

B-C - S. lactis

C-D - Lactobacillus

D-E - molds (oxidize

acids) + yeasts

(produce NH3)

E-F - Proteolytic spore

former bac.

Acid

ity %

Time

Sequence of changes in raw milk in relation to acid concentration

Proteolytic action brings pH of milk higher than that of the original milk

During E-F region, the curdle made during B-D is digested and the curdle becomes gassy


• Alcohol too imparts a preservative action

• Alcohol content depends on initial sugar content, type of yeast, temp of fermentation and oxygen conc.

• Most yeasts cannot tolerate >12-15% (v/v)

• Natural wines contain 9-13%

• Fortified wines contain up to 20%


• Microbes in a mixed fermentation dominate depending on the fermentation temperature

• Thus, temperature may have a direct effect on the microbial products which in turn control the quality of the final product

• E.g. In sauerkraut production – Leuconostoc mesenteroides – acetic acid, LA, alcohol

and CO2 – Lactobacillus cucumeris – LA when L. mesenteroides

leaves

FT 5105 Food Microbiology

10

0

– Lactobacillus pentoaceticus & plantarum – produce more LA when both leave

• L. mesenterodes require cool temp <21C

• If temp is >21C – Lactobacilli outgrow

• Acidity creates by Lactobacilli further prevents mesenteroides growth

• Thus, in sauerkraut making, low temp is given first.


10

1


10

2

Acid

ity %

Time (days)

Sequence of acid fermentation in sauerkraut production

L. mesenteroides

L.curcumeris

L. Pentoaceticus

& L. plantarum

MOs may have different oxygen requirements for their growth and fermentation

E.g. S. cerevisiae (Bakers’ yeast)

and S. ellipsoideus (Wine yeast) grow best under aerated conditions while they ferment sugars rapidly under anaerobic conditions


10

3

• Thus, in commercial production of yeast, air is bubbled through molasses

• In baking – large dough masses provide a relatively anaerobic condition

• Vinegar production is done in two steps

• 1. Sugars alcohol

Anaerobic conditions


10

4

Alcohol Acid

Aerobic conditions

Aerobic conditions are provided by bubbling air through the ferment

This occurs in a vinegar generator until acetic acid conc. Reaches 4%


10

5

• Mos can be separated on the basis of salt tolerance

• LA producers in pickles, olives, sauerkraut etc. tolerate up to 10-18% salt

• Many proteolytic and spoilage Mos can not tolerate above 2.5%

• Adding salts to above ferment gives advantage for LA producers


10

6

• Generating acids make the envt. More unfavorable for others

• Slat also draws sugar and water from vegetables

• Water drawn from vegetables dilutes the brine solution thus, salt level is to be added gradually


10

7

Sauerkraut - 2-2.5%

Olives – 7-10%

Gherkin – 15-18%

Cheese curd is salted to prevent the growth of proteolytic bac. During long ripening periods


10

8

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FT 5105 FOOD MICROBIOLOGY · ATP NADH NADH NAD+ NAD+ ... cooled to 45C Starter culture is added...

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