FORMULATING DAIRY BASED PRODUCT USING PRO-PREBIOTIC INGRIDIENTS

SEAFAST Center and Departemen Teknologi Pangan dan Gizi Institut Pertanian Bogor

Ir. Lilis Nuraida, MSc. Ph.D.

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

Products Pro- and Pre-biotic in non fermented product Microencapsulation to improve viability of probiotic

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Probiotic, Prebiotic and Synbiotic

Term Probiotic = for life (G k) (Greek) Definition Live microorganisms administered in adequate d t amounts t which hi h confer f a beneficial health effect to the host FAO/WHO (2001) Nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or d/ activity ti it of f one or a limited li it d number of bacteria in the colon, and thus improves host health A mixture of probiotic and prebiotic
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Prebiotic

Synbiotic

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.

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

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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 Industry Analysts). Rapidly rising in popularity within the functional food market:
Applications in dairy products, health drinks, nutrition bars,

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

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

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Probiotic bacteria
Microflora Lactobacilli Species Lactobacillus acidophilus L. rhamnosus L reuteri L. L. casei L. gasseri L. plantarum L. jhonsonii Bifidobacterium bifidum B. longum B. Breve B infantis B. B. adolescentis Enterococcus faecalis E. faecium Lactococcus lactis subsp lactis

Bifidobacteria

Enterococci Lactococci

Not all species/strains are equal Probiotic properties are strain dependent Each strains has to be characterised LilisNuraida June2012

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, food transit through high acidic and alkaline conditions in the gastro-intestinal tract

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

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Prebiotic effect of various oligosaccahrides

Carbohydrate Inulinand oligofructose Galactooligosacc harides Lactulose Isomaltoligosacc harides Lactosucrose Xylooligosacchrai des Soybean oligosaccharides Nondigestibility Yes Probable Probable Partly NA NA NA Fermentation Yes ? ? Yes NA NA NA Selectivity Yes Yes Yes Promising Promising Promising NA Prebioticstatus Yes Yes Yes No No No No

Glucooligosaccha NA rides

NA

NA

No

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Roberfroid,2007

Use of Prebiotics in food

Safety of ingredient is a must and good

sensory properties desirable 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 some side effects, such as increased flatulence or abdominal bloating.
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Application of Probiotic in Dairy Industry

Fermented products:
Yoghurt and fermented milk drink Cheeses

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

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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 withstand the freezing or drying of starter culture
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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 Supplemented S l t d with ith h hydrolised d li d protein, t i whey h 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 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
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Yoghurt and fermented milk drink

ConventionalyoghurtisfermentedbyL.bulgaricus

subspdelbruekiiandS. S thermophilus
notveryresistanttothebilereleasedinto thesmall

intestine notmaintainedaliveinthegastrointestinal tractinveryhighnumbers

Bioyoghurtisyoghurtcontainliveprobiotic

microoganisme,i.e.Lactobacillusacidophillus and strainsofBifidobacterium thepresenceofwhichmay providebeneficialhealtheffect(LaurentHattinghand Viljoen,2001).

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Incorporation of probiotic into fermented milk

Add the probiotic microorganisms together with the

starter 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

culture. lt
The two batches are then mixed together

Probiotic microorganism(s) may be used as starter

culture, the fermentation may be longer

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Yoghurt fermentation
Prebiotic
Homogenized milk TS 12% Incubation at 43.3, to reach pH 4.8

Heating and cooling

Cooling and Holding

Cooling, agitating,

Addition starter culture

Set y yoghurt g

Packaged in small container Or cup

Packed in cups

Bulk container

Probiotic

Stirred yoghurt LilisNuraida June2012

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 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

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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 3-4 4o C 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 Some probiotic bacteria grow poorly in milk use a large innoculum size or concentrated inoculum Selection of starter culture

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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 be detrimental ti t lt to probiotic bi ti culture lt adding ddi probiotic bi ti b bacteria t i after ft 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 Microencapsulation Type of packaging container Bifidobacteria is anaerobic, while lactobacilli is microaerophilic

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Effect prebiotic on bio-yoghurt

Addition of oligosaccharides potentially enhance viability

bifidobacteria: Use of lactulose in fermented milk improved quality of f fermented t d skim ki milk ilk b by L. L acidophilus, id hil L L. rhamnosus, h Lactobacillus bulgaricus and Bifidobacterium lactis in coculture with Streptococcus thermophilus. Increased the counts of all probiotics, with particular concern to B. lactis (bifidogenic effect) Inulin addition to co-cultures and cocktail: Enhanced products firmness, Increase in microbial growth induced by metabolic interactions among lactic acid bacteria and partial inulin metabolization.
(Oliviera et al., 2011)

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ProbioticCheeses
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 y of p probiotics through 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 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 LilisNuraida June2012

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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 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

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Standardized milk

Whey draining

Probiotic

Starter culture

Cheddaring

Rennet

Milling

Curd Cutting

Probiotic

Salting

Cooking

Hooping and Pressing

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

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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) During manufacturing ice cream, freezing involves vigorously agitating to incorporate air Detrimental to probiotic bacteria: Lactobacilli microaerophilic, bifidobacteria anaerobic 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
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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 i t to blending bl di with ith ice i cream mix i frozen f yoghurt h t ice i cream Protection of the probiotic cells againts freeze damage is important (use cryoprotectant)

Prebiotic

Freeze dried encapsulated probiotic

Ice cream mix

Natural yoghurt containing gp probiotic

Blend, Freeze and Stored Pro or synbiotic ice cream Fermented frozen dessert LilisNuraida June2012

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Problems during processing ice-cream containing probiotic cultures

Cruz et al., 2009

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Stability of the probiotic cultures during ice-cream storage

Probiotic cultures wee capable maintaining their

stability 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 icecream containing t i i oligofructose li f t 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

Dried encapsulated probiotic

Mixing

Dry ingredient

Powdered dairy product LilisNuraida June2012

Microencapsulation Technique to improved viability

Microencapsulation: a process

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 Encapsulated E l t d cells ll can b be d dried i dt to produce cell powder/granule (freeze drying, spray drying, fluidized bed drying)

Protection againts deleterious conditions in the upper GI tract

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Technique for microencapsulation

Atomization: Spray drying Spray Chilling Extrusion Technique Emulsion Technique Freeze Drying

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Technique for microencapsulation

Atomization Spray drying is a commonly used method of encapsulation in the

food industry: controlled by means of the product feed feed,gas 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 solidifcation of f gel l particles ti l i is b based d on th the i injection j ti of f cold ld air i i into t th the vessel l Freeze drying Cryoprotectan: fructose, lactose, mannose, monosodium glutamate, sorbitol, trehalose LilisNuraida June2012

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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 p ( phase), ), homogenized g to form a water-in-oil emulsion, 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
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Extrusion Technique

Emulsion Technique

Krasaekoopt et al., 2003

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Terima kasih
lilis@seafast.org ; lilis@nuraida.com

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