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1 FORMULATING DAIRY BASED PRODUCT Food Review Seminar: Update on Dairy Industry, Bogor June 2012 USING PRO-PREBIOTIC INGRIDIENTS Ir. Lilis Nuraida, MSc. Ph.D. SEAFAST Center and Departemen Teknologi Pangan dan Gizi Institut Pertanian Bogor Outline Definitions of Pro Pre and Syn biotic Definitions of Pro-Pre and Syn-biotic Application of Probiotic in Dairy Industry Pro- and Pre-biotic in Fermented Dairy Products Pro- and Pre-biotic in non fermented product Lilis Nuraida June 2012 Microencapsulation to improve viability of probiotic
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1

FORMULATING DAIRY BASED PRODUCT

Food Review Seminar: Update on Dairy Industry, Bogor June 2012

USING PRO-PREBIOTIC INGRIDIENTS

Ir. Lilis Nuraida, MSc. Ph.D.SEAFAST Center and

Departemen Teknologi Pangan dan GiziInstitut Pertanian Bogor

Outline

Definitions of Pro Pre and Syn biotic Definitions of Pro-Pre and Syn-biotic Application of Probiotic in Dairy Industry Pro- and Pre-biotic in Fermented Dairy

Products Pro- and Pre-biotic in non fermented

product

Lilis Nuraida ‐ June 2012

Microencapsulation to improve viability of probiotic

2

Probiotic, Prebiotic and SynbioticTerm Definition

Probiotic = for life (G k)

Live microorganisms administered in d t t hi h f l(Greek) adequate amounts which confer a l

health benefits to the host FAO/WHO (2001)

Prebiotic Nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth

d/ ti it f li it d

Lilis Nuraida ‐ June 2012

and/or activity of one or a limited number of bacteria in the colon, and thus improves host health

Synbiotic A mixture of probiotic and prebiotic

Probiotic Market Probiotic foods

The largest share of the market, estimated $13.8 billion in 2008, should reach $17.0 billion in 2013, 4.2 percent growth.

Mostly in dairy products, with yogurts, kefir and cultured drinks representing the major categories of probiotic foods.

Y t d t t d f th l t h f l ti 36 6 t Yogurt products accounted for the largest share of sales, representing 36.6 percent.

Emerging food applications: probiotic cheese, nutrition bars, breakfast cereal, and infant formula.

Probiotic supplements. The second largest share, $1.2 billion in 2007, expected to reach $1.7 in 2013, 5.8

percent growth.

Capsules, tablets and powders, with capsule the largest share of sales, representing 75 percent.

P bi ti i di t

Lilis Nuraida ‐ June 2012

Probiotic ingredients Worth $797.6 million in 2008 and increase to $917 million by the end of 2013, 2.8

percent growth.

Probiotics of the lactobacillus genus accounted for the largest share, representing 61.9 percent of total sales in 2007.

http://www.naturalproductsinsider.com/news/2008/05/global-probiotics-market-worth-196b-by-2013.aspx

3

Prebiotic market Projected to reach nearly $1.2 billion and $225

million, respectively, by the year 2015, in the European and the U S market (Global IndustryEuropean and the U.S. market (Global Industry Analysts).

Rapidly rising in popularity within the functional food market: Applications in dairy products, health drinks, nutrition

bars breakfast cereals beverages bakery products

Lilis Nuraida ‐ June 2012

bars, breakfast cereals, beverages, bakery products, meat products, mineral supplements, weight loss products, green foods, infant food and pet food.

http://www.nutraceuticalsworld.com/contents/view_breaking-news/2010-02-23/report-finds-significant-potential-in-prebiotics-m/

Microflora Species

Lactobacilli Lactobacillus acidophilusL. rhamnosusL reuteri

Probiotic bacteria

L. reuteriL. caseiL. gasseriL. plantarumL. jhonsonii

Bifidobacteria Bifidobacterium bifidumB. longumB. BreveB infantis

Not all species/strains are equalProbiotic

Lilis Nuraida ‐ June 2012

B. infantisB. adolescentis

Enterococci Enterococcus faecalisE. faecium

Lactococci Lactococcus lactis subsp lactis

properties are strain dependentEach strains has to be characterised

4

Viability of probiotic bacteria Viability, physiological and metabolic activity of probiotic

bacteria in a food product at the point of sale are important consideration for their efficacy

They have to survive during shelf life of a food transit through

Probiotic bacteria should be present in a food to minimum concentration of 106 cfu/g or the daily intake should be about 109

cfu/g Fermented Milk and Lactic Acida Bacteria Beverages

Association Japan: minimum 107 cfu/ml to be present in dairy

They have to survive during shelf life of a food, transit through high acidic and alkaline conditions in the gastro-intestinal tract

Lilis Nuraida ‐ June 2012

Association, Japan: minimum 10 cfu/ml to be present in dairy products

National Yoghurt Association, USA specifies 108 cfu/g at the time manufacture

Prebiotic effect of various oligosaccahrides

Carbohydrate Nondigestibility Fermentation Selectivity Prebiotic status

Inulin and oligofructose

Yes Yes Yes Yes

Galactooligosaccharides

Probable ? Yes Yes

Lactulose Probable ? Yes Yes

Isomaltoligosaccharides

Partly Yes Promising No

Lactosucrose NA NA Promising No

Xylooligosacchraides

NA NA Promising No

Lilis Nuraida ‐ June 2012

des

Soybeanoligosaccharides

NA NA NA No

Glucooligosaccharides

NA NA NA No

Roberfroid, 2007

5

Use of Prebiotics in food

Safety of ingredient is a must and good sensory properties desirable

Good prebiotics are stable under heat and Good prebiotics are stable under heat and when dried, can be stored at room T for months

A daily dose of 5-8g/d FOS or GOS has a prebiotic effect in adults

Doses higher than 20 g/day might induce

Lilis Nuraida ‐ June 2012

Doses higher than 20 g/day might induce some side effects, such as increased flatulence or abdominal bloating.

Application of Probiotic in Dairy Industry

Fermented products: Yoghurt and fermented milk drinkYoghurt and fermented milk drink Cheeses

Non-fermented products: Ice cream and milk based dessert Powdered milk for infant Butter, Mayonnaise, Fat spread

Lilis Nuraida ‐ June 2012

6

The choice of probiotic microoganism for production of starter culture

Posses health beneficial effect to human Tolerance to acidity and bile salts The ability of the probiotic

microorganism to grow in the medium to increase the cell number

The robustness of microoganism to

Lilis Nuraida ‐ June 2012

The robustness of microoganism to withstand the freezing or drying of starter culture

Several aspect must be considered during the production of probiotic fermented milk drink

Many probiotic strain grow slowly in non-supplemented milk due to limited proteolytic activity

S l t d ith h d li d t i h Supplemented with hydrolised protein, whey derivatives, or amino acid

The production condition are often unsuitable for their growth Optimum temperature for probiotic isolated from

human is 37oC Conventional yoghurt fermentation done at 42oC

Lilis Nuraida ‐ June 2012

The metabolites of probiotics may be undesirable due to formation off flavor Bifidobacteria produce acetic acid and lactic acid in

the proportion 3:2 which give vinegar like taste

7

Yoghurt and fermented milk drink

Conventional yoghurt is fermented by L. bulgaricus subsp delbruekii and S thermophilussubsp delbruekii and S. thermophilus

not very resistant to the bile released into the small intestinenot maintained alive in the gastrointestinaltract in very high numbers

Bio‐yoghurt is yoghurt contain live probiotic microoganisme, i.e. Lactobacillus acidophillus and 

Lilis Nuraida ‐ June 2012

strains of Bifidobacterium the presence of which may provide beneficial health effect (Laurent‐Hattingh and Viljoen,  2001).

Incorporation of probiotic into fermented milk

Add the probiotic microorganisms together with the starter culture (as Direct to Vat Innoculation/DVIstarter culture (as Direct to Vat Innoculation/DVI culture) Probiotics do not usually grow markedly during mixed

fermentation

The probiotic microorgnisms may be grown in one batch of milk to achieve a high viable count, another batch of milk is fermented with traditional starter

lt

Lilis Nuraida ‐ June 2012

culture. The two batches are then mixed together

Probiotic microorganism(s) may be used as starter culture, the fermentation may be longer

8

Yoghurt fermentation

Homogenized milkTS 12%

Incubation at 43.3, to reach pH 4.8

Prebiotic

Heating and cooling

Addition starterculture

Cooling, agitating,

Packaged in small container

Cooling and Holding

Set yoghurt

Lilis Nuraida ‐ June 2012

Packed in cups

Bulk container

small containerOr cup

y g

ProbioticStirred yoghurt

Factors affecting L. acidophillus and Bifidobacteria in bio-yoghurt

Yoghurt acidity

Most strain of bifidobacteria are sensitive to pH below 4.6

The product should be maintain at pH above 4.6

L. acidophillus is more resistant

Strains

The bifidobacteria should be able to grow in milk. Some lack of proteolytic activity

Co-culture and species interaction

H2O2 produced by L. bulgaricus is detrimental to L. acidophillus

Synergistic growth between L. acidophillus and Bifidobacterium

Lilis Nuraida ‐ June 2012

Dissolved oxygen

Bifidobacteria is strictly anaerob

Storage condition

Low temperature restrict the growth of Lactobacillus and so over-acidification

Bifidobacteria less tolerant to low temperature storage

9

Improvement of survival L. acidophillus and Bifdobacteria in bio-yoghurt...................1

Prevention over acidification:

Applying heat shock before addition of probiotic culture

Lowering storage temperatur to 3 4oC Lowering storage temperatur to 3-4oC

Improving buffering capacity by the addition of whey protein concentrate

Modification of incubation temperature

Incubation temperature of 37oC favours the growth of bifidobacteria

Rate of innoculation

Lilis Nuraida ‐ June 2012

Some probiotic bacteria grow poorly in milk use a large innoculum size or concentrated inoculum

Selection of starter culture

Improvement of survival L. acidophillus and Bifidobacteria in bio-yoghurt...................2

Two-stage fermentation

Acid and hydrogen peroxyde produce by yoghurt starter culture may b d t i t l t bi ti lt ddi bi ti b t i ftbe detrimental to probiotic culture adding probiotic bacteria after fermentation or adding yoghurt starter culture at later stage

Addition of growth promoting substances

Supplementation of casitone, casein hydrolysate, fructose, whey protein concentrate improved viability of L. acidophillus

Cystein, acid hydrolysate and tryptone improved viability of bifidobacteria

Lilis Nuraida ‐ June 2012

Microencapsulation

Type of packaging container

Bifidobacteria is anaerobic, while lactobacilli is microaerophilic

10

Effect prebiotic on bio-yoghurt

Addition of oligosaccharides potentially enhance viability bifidobacteria: Use of lactulose in fermented milk improved quality of

f t d ki ilk b L id hil L hfermented skim milk by L. acidophilus, L. rhamnosus, Lactobacillus bulgaricus and Bifidobacterium lactis in co-culture with Streptococcus thermophilus.

Increased the counts of all probiotics, with particular concern to B. lactis (bifidogenic effect)

Inulin addition to co-cultures and cocktail:

Lilis Nuraida ‐ June 2012

Enhanced products firmness, Increase in microbial growth induced by metabolic

interactions among lactic acid bacteria and partial inulinmetabolization.

(Oliviera et al., 2011)

Probiotic Cheeses Natural cheese has proven to be a good carrier for

probiotic cultures.

Studies have suggested that consuming probiotics in a cheese matrix is favorable for the viability of probioticsy pthrough the digestive tract.

Desirable properties of probiotic bacteria to be incorporated to cheese:

Must survive the entire shelf-life of cheese.

Must not produce metabolites that are detrimental to the quality of cheese

Lilis Nuraida ‐ June 2012

Should not interfere with the normal activity of other essential microorganisms in the cheese

Should be compatible and not produce antimicrobial compounds

Should be able to grow on starter culture media

11

Introduction of probiotic microorganism into cheeses

Introduce as adjunct cultures together with lactic starter cultures

Risk of losing large numbe rof cells to whey or domination of lactic starter cultures

Addition of microencapsulated probiotic

Protected the probiotic from degradation duirng teh aging period

Addition of dried probiotic cultures during salting of curd on semi-hard and hard cheese

Addition of freeze dried culture to mtrix of Cheddar cheese following cheddaring and salting

Addition of fermented cream dressing in cotage cheese

Lilis Nuraida ‐ June 2012

Cream dressing is added for flavour and texture development

Two stage fermentation:

Fermentation with probiotic bacteria for 2 h followed by fermentation with starter culture

The lactic starter culture grow faster than probiotic bacteria

Standardized milk

Starter culture

Whey draining

CheddaringProbiotic

Rennet

Curd Cutting

Milling

SaltingProbiotic

Lilis Nuraida ‐ Juni 2012

Cooking Hooping and Pressing

The manufacturing stage of Cheddar Cheese (Tamime et al., 2007)

12

Ice cream and frozen milk based dessert

The ice cream matrix might be a good vehicle for probiotic culture due to its composition (milk protein, fat, lactose and other compounds)other compounds)

During manufacturing ice cream, freezing involves vigorously agitating to incorporate air Detrimental to probiotic bacteria: Lactobacilli are microaerophilic, bifidobacteria are

anaerobic Freeze stress must be considered with respect to viability

Lilis Nuraida ‐ June 2012

Freeze stress must be considered with respect to viability during manufacture and extended storageImpact of probiotic bacteria on flavor should be considered as ice cream is not fermented product

Incorporating probiotic into ice cream

Direct, i.e. blend the ice cream mix and probiotic cells prior to freezing

Involve fermentation of the milk for proliferation of probiotic bacteria prior to blending with ice cream mix frozen yoghurt ice creamprior to blending with ice cream mix frozen yoghurt ice cream

Protection of the probiotic cells againts freeze damage is important (use cryoprotectant)

Freeze dried/ Spray Dried encapsulated

Ice cream mix Natural yoghurt containing probiotic

Prebiotic

Lilis Nuraida ‐ June 2012

probioticg p

Blend, Freeze and Stored

Fermented frozen dessertPro or synbiotic ice cream

13

Problems during processing ice-cream containing probiotic culturesStep Problem Solutions

Formulation with fruit

l /j i

•High acidity of the final product may lead to decreased probiotic survival

•S i di t h i hibit

•Preferably, use of fruit pulp/juices with a lower natural acidity

•Ch k f i hibit ti it f th pulp/juice •Some ingredients may have inhibitory activity against probiotic strains

•Check for inhibitory activity of the food ingredients to be used against the probiotic strains to be employed

Fermentation Probiotic bacteria show some loss in viability at low pH values

•Control of the pH during the fermentative process

•Increased inoculum concentration•Selection of strains tolerant to low pH values

Lilis Nuraida ‐ June 2012

Beating Oxygen represents a factor of toxicity for probiotic bacteria

Selection of oxygen-tolerant strains

Storage Stress induced by freezing reduces the viability of probiotic bacteria by at least 1 log cycle

•Increased inoculum concentration•Avoid temperature oscillations during storage of the product

Cruz et al., 2009

Stability of the probiotic cultures during ice-cream storage

Probiotic cultures wee capable maintaining their stability in frozen food productsstability in frozen food products

Addition of prebiotic (inulin and oligofructose) : Higher overrun for the ice-cream mix with inulin Less changes in melting properties Firmer during storage Increase probiotic survival during storage of the ice-

t i i li f t

Lilis Nuraida ‐ June 2012

cream containing oligofructose

Improvement stability: the use of microencapsulation of cultures and the supplementation with prebiotics

Cruz et al., (2009)

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Application of probiotic in powdered/dried dairy products

Liquid materials + liquid ingredient

Spray drying

Dry base

Lilis Nuraida ‐ June 2012

Powdered dairy product

Mixing Dry ingredient

Dried encapsulated

probiotic

Microencapsulation: a process whereby the cells are retained within

Microencapsulation Technique to improved viability

Protection againts adverse conditions in the product

whereby the cells are retained within the encapsulating membrane

Improving viability, survival during freezing, stability during storage

Entrapment in gelatin, calcium alginat, xanthan-gellan or vegetable gums

E l t d ll b d i d t

Lilis Nuraida ‐ June 2012

Encapsulated cells can be dried to produce cell powder/granule (freeze drying, spray drying, fluidized bed drying)

Protection againts deleterious conditions in the upper GI tract

15

Technique for microencapsulation

Atomization:Spray drying Spray drying

Spray Chilling

Extrusion Technique

Emulsion Technique

Freeze Drying

Lilis Nuraida ‐ June 2012

Technique for microencapsulation Atomization

Spray drying is a commonly used method of encapsulation in the food industry:

controlled by means of the product feed gas flow and temperature controlled by means of the product feed,gas flow and temperature

Carrier: polysaccharides, lactose, proteins, skim milk, gelatin, soluble starch and gum arabic

Skim milk has proved to be a better wall material than gelatin, soluble starch and gum arabic

Spray Chilling:

the atomization step is similar to spray drying, but the solidifcationf l ti l i b d th i j ti f ld i i t th l

Lilis Nuraida ‐ June 2012

of gel particles is based on the injection of cold air into the vessel

Freeze drying

Cryoprotectan: fructose, lactose, mannose, monosodium glutamate, sorbitol, trehalose

16

Technique for microencapsulation Extrusion Technique adding microrganisms to a hydrocolloid solution, and

extruding the cell suspension through a syringe needle to free-fall into a hardening solut ion or setting bath

Supporting materials: alginate

Emulsion Technique Adding small volume of the cell-polymer suspension

(discontinuous phase to a large volume of a vegetable oil (continuous phase), homogenized to form a water-in-oil

Lilis Nuraida ‐ June 2012

( p ), gemulsion, cross-linked to form insoluble tiny gel particles within the oil phase

Supporting materials: K-karagenan, locust bean gum, cellulose actetate pthalate, alginate, chitosan and gelatin

Extrusion Technique Emulsion Technique

Lilis Nuraida ‐ Juni 2012

Krasaekoopt et al., 2003

17

Example of microencapsulated probiotics application in Dairy Products

Lilis Nuraida ‐ June 2012

Burgain et al. 2012

Example of microencapsulated probiotics application in Dairy Products

Lilis Nuraida ‐ June 2012Burgain et al. 2012


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