4. Enkapsulasi Probiotik

7/23/2019 4. Enkapsulasi Probiotik

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Encapsulation of probiotic livingcells :

From laboratory scale to industrialapplications



• Modern consumers expect their foodto be healthy and to prevent illnessas they are increasingly interested intheir personal health

• This explains the reason for a risinginterest in probiotic health-based

products.



Probiotic

• Is a term that means ‘‘for life’’ and definedas ‘‘live microorganisms that beneficiallyaect the host’s health by improving its

microbial balance’’ !"uller# $%&%'• ‘‘(ive microorganisms that# )hen

administered in ade*uate amounts# confer ahealth benefit on the host’’ !"+,/,#0110'

• In fact# probiotic products are importantfunctional foods as they represent about234 of the )orld functional food mar5et



• Probiotic bacteria have been incorporatedinto a )ide range of foods# including dairyproducts !such as yogurt# cheese# ice

cream# dairy desserts' but also in non-dairyproducts !such as chocolate# cereals# 6uices'

• There is poor survival of probiotic bacteriain products containing free probiotic cells

• Providing probiotic living cells )ith aphysical barrier to resist adverseenvironmental conditions



• Microencapsulation !M7' is a po)erful technology)hich has been developed for use in the foodindustry and allo)s the protection of bacterial cells

• 8e*uires some specific processing steps )hich

complicate the manufacture of the food product andincrease its cost.

• Many challenges exist )hen considering M7 ofprobiotic living cells li5e# probiotic strain selection

for its health benefits and *uantity re*uire to havepositives eects# but also stability of the cells duringthe processing steps and storage and finally# eectson sensory properties of the food



Probiotics

• Probiotics are feed and food supplementsthat beneficially aect the host’s health

• The term ‘‘probiotic’’ includes a large range

of microorganisms# mainly bacteria but alsoyeasts.

• 9ecause they can stay alive until theintestine and provide beneficial eects on

the host health# lactic acid bacteria!(+9'#non-lactic acid bacteria and yeasts can beconsidered as probiotics



• (+9 are the most important probiotic 5no)n to havebeneficial eects on the human gastro-intestinal !:I'

tract.• These bacteria are :ram-positive and usually live in a

non-aerobic environment but they also can supportaerobic conditions

• 9ifidobacteria are also :ram-positive and can gro) ata p/ range of ;.3<&.3 but the most importantcharacteristic is the fact that they are strictlyanaerobic

• ,ther (+9 !e.g. (actococcuslactis# 7nterococcus

faecium# etc.' and non-lactic acid bacteria !e.g.7scherichia coli strain nissle' but also some yeasts!e.g. =accharomyces cerevisiae# =accharomycesboulardii# etc.' are also considered as probiotics.

• >ead bacteria# products derived from bacteria or end

products of bacterial gro)th could provide some



• The eects of probiotics are strain-specific

• The probiotic health benefits may be dueto the production of acid andor

bacteriocins# competition )ith pathogensand an enhancementof the immunesystem

• >ose levels of probiotics depend on theconsidered strain#but $12<$1? @"Ag ofproduct per day is generally accepted



• Ingested probiotic strains do notbecome established members of thenormal intestinal Bora but generally

persist only for the period ofconsumption and for a relativelyshort period thereafter



The mechanism of action ofprobiotic bacteria

• Probiotics have been reported to playa therapeutic role by modulatingimmunity# lo)ering cholesterol#

improving lactose tolerance andpreventing some cancers

• 7ects of probioticsC modes of action



1. Related with the modulation of the host’sdefences which is most likely important for the

prevention and treatment of infectious diseaseand also for treatment of intestinal inammation.

.Probiotics may inBuence the immune system by means ofproducts such as metabolites# cell )all components or>D+.

.In fact# these products can be recognised by the host cellssensitive for these because of the presence of a specificreceptor. In this context# the main target cells aregenerally the gut epithelial and the gut associatedimmune cells.

."inally# the interaction bet)een probiotics and the host’simmune cells by adhesion might be the triggeringsignalling cascade leading to immune modulation



!. "y a direct e#ect on othermicroorganisms which can becommensal and$or pathogenic

• In this case# the therapy and the treatment ofinfections are concerned but restoration of themicrobial balance in the gut is an importantfactor too.

• Probiotics have the ability to be competitive)ith pathogens and therefore allo) forpreventing their adhesion to the intestine

•

7ventually# probiotics have the ability to aectsome microbial products such as toxins andhost products li5e bile salts and foodingredients



E%amples of published healthbene&ts: the gut and the immune

system• In 7urope# gut health has been sho)n to be

the 5ey sector for mar5eting functional foods

• InBammatory bo)el disease is a chronic

recurrent pathology# )hich mainly consistsin ulcerative colitis and @rohn’s disease.

• =ome probiotic strains ! 7.coli Dissle $%$?and (actobacillus rhamnosus ::' can

prevent relapses of inBammatory bo)eldiseases and are able to decrease therecurrence of ulcerative colitis



• + number of probiotic strains areeective in preventing antibiotic-associated diarrhoea and there is

also promising evidence of apreventive eect of probiotics in@lostridium difficile associated

diarrhoea



'ynbiotics: a combination of theprobiotics and prebiotics positives

e#ects• Prebiotics can be defined as ‘non-digestible

food ingredients that# )hen consumed insufficient amounts# selectively stimulate

the gro)th andor activity of one or alimited number of microbes in the colonresulting in documented health benefits’

• Inulin and fructo-oligosaccharides are the

most common prebiotics used# because oftheir resistance against gastric acid andpancreatic enEymes



• =ynbiotic can be defined as ‘a mixture ofprobiotics and prebiotics that beneficially aectsthe host by improving the survival andimplantation of live microbial dietary supplements

in the :I tract# by selectively stimulating thegro)th andor activating the metabolism of one ora limited number of health promoting bacteria#and thus improving host )elfare’.

• =ynbiotics are not only a mixture of probiotics andprebiotics but a synergy bet)een the t)ocomponents



Encapsulation

• Physicochemical or mechanical process toentrap a substance in a material in order toproduce particles )ith diameters of a fe)nano metres to a fe) millimetres

• The encapsulated substance called the corematerial is dispersed in a matrix also namedcoating or shell.

•

This carrier material must be food grade ifused in food industry# and able to form abarrier to protect the encapsulated substance.



+pplications of encapsulation ofbioactive components

• @ontrolling oxidative reaction

• Mas5ing Bavours# colours# andodours

• Providing sustained and controlledrelease

• 7xtending shelf life# etc

• Protect the cells against an adverseenvironment more than controlledrelease



=chematic representation of encapsulationsystemsC !a'reservoir type!b'matrix type!c'coated matrix type



Probiotic encapsulation technologiesCsiEe range provided by each techni*ue



• (o) viability of probiotics in dairy products suchas yogurt and froEen dairy desserts due to theconcentration of lactic acid and acetic acid# lo)

p/# the presence of hydrogen peroxide# and thehigh oxygen content

• 7ncapsulation has been investigated forimproving the viability of microorganisms in both

dairy products and the :I tract• The viability of encapsulated probiotic cells

depend on the physico-chemical properties of thecapsules.

•

In fact# the type and the concentration of thecoating material# particle siEe# initial cell numbersand bacterial strains are some parameters )hichare important to master



• In the case of probiotic encapsulation#the ob6ective is not only to protect the

cells against adverse environment# butalso to allo) their release in a viable andmetabolically active state in the intestine

• The obtained microparticles have to be)ater-insoluble to maintain their integrityin the food matrix and in the upper partof the :I tract and finally# particle

properties should allo) progressiveliberation of the cells during the intestinalphase



Encapsulation technology: (stages

$. Incorporating the bioactive component in a matrix)hich can be li*uid or solid. In case of the core isli*uid# incorporation )ill be a dissolution or adispersion in the matrix )hereas if the core is

solid the incorporation )ill be an agglomeration oran adsorption

0. The li*uid matrix is dispersed )hile a solution ispulverised on the solid matrix

F. =tabilisation by a chemical !polymerisation'# aphysicochemical !gelification' or a physical!evaporation# solidification# coalescence' process



:eneral plan describing steps toproduce microcapsules



)aterials used to encapsulateprobiotic cells

• Daturally derived polysaccharideextracted from various species of algaeand composed of G->-mannuronic andH-(-guluronic acids

• +lginate hydrogels are extensively usedin cell encapsulation and calcium

alginate is preferred for encapsulatingprobiotics because of its simplicity# non-toxicity# biocompatibility and lo) cost

1.

*lginate



>isadvantages

• +lginate beads are sensitive to the acidicenvironment )hich is not compatible forthe resistance of the microparticles in the

stomach conditions• =caling-up of the process is very difficult.

• The microparticles obtained are veryporous )hich is a dra)bac5 )hen the aimis to protect the cells from itsenvironment



The defects can becompensated

• Mixing alginates )ith other polymercompounds

• @oating the capsules by other compound

or applying structural modification of thealginate by using dierent additives

• Mixing alginate )ith starch is commonlyused and it has been sho)n that thismethod results in an improvement ofprobiotic encapsulation eectiveness



!. +ellan gum and %anthan gum

• :ellan gum is a microbial polysaccharidederived from Pseudomonas elodea )hich isconstituted of a repeating unit of four

monomers that are glucose# glucuronicacid# glucose and rhamnose

• + mixture of xanthan<gelan gum has beenused to encapsulate probiotic cells and

contrary to alginate# the mixture presentshigh resistance to)ards acid conditions.



(. ,-arrageenan

• Datural polymer )hich is commonly usedin the food industry

• The technology using the compound

re*uires a temperature comprised bet)een;1 and 31 @ at )hich the cells are addedto the polymer solution.

• 9y cooling the mixture to room

temperature# the gelation occurs and then#the microparticles are stabilised by addingpotassium ions



• The encapsulation of probiotic cells

in 5-carrageenan beads 5eeps thebacteria in a viable state# but theproduced gels are brittle and are notable to )ithstand stresses



/. ellulose acetate phthalate

•

9ecause of having a safe nature# celluloseacetate phthalate is used for controllingdrug release in the intestine

• The advantageC it is not soluble at acidic

p/ !less than 3' but it is soluble at p/higher than 2.

• The encapsulation of probiotic bacteria

using cellulose acetate phthalate providesgood protection for microorganisms insimulated :I conditions



0. hitosan

•

+ linear polysaccharide composed ofglucosamine units )hich canpolymerise by means of a cross-lin5formation in the presence of anionsand polyanions

• This component has not sho)n agood efficiency for increasing cell

viability by encapsulation and it ispreferably use as a coat but not as acapsule



• 7ncapsulation of probiotic bacteria)ith alginate and a chitosan coatingprovides protection in simulated :I

conditions and therefore# it is a good)ay of delivery of viable bacterialcells to the colon

• >isadvantageC seems to haveinhibitory eects on (+9 for example



. 'tarch

•

+ polysaccharide consisting of a largenumber of glucose units 6oinedtogether by glucosidic bonds.

• =tarch consists mainly of amylose# alinear polymer of >-glucopyranose

6oined by H-$-; glucosidic bond andamylopectin# a branched polymer of

glucose 6oined by H-$-; glucosidicbond and H-$-2 glycosidic bond forramification



• 8esistant starch is the starch )hich is notdigested by pancreatic enEymes!amylases' in the small intestine.

• 8esistant starch can reach the colon )hereit )ill be fermented

• This specificity provides good entericdelivery characteristic that is a betterrelease of the bacterial cells in the largeintestine

• 9y its prebiotic functionality# resistantstarch can be use by probiotic bacteria inthe large intestine



• 8esistant starch is an ideal surface

for the adherence of the probioticcells to the starch granules

• This can enhance probiotic delivery

in a viable and a metabolically activestate to the intestine



2. +elatin• + protein gum# )hich ma5es a thermo reversible gel

and )as used for probiotic encapsulation# alone orin combination )ith other compounds.

• >ue to its amphoteric nature# it is an excellentcandidate for cooperation )ith anionic

polysaccharides such as gellan gum.• These hydrocolloids are miscible at a p/ higher than

2# because they both carry net negatives chargesand repel each other.

•

The net charge of gelatin becomes positive )henthe p/ is ad6usted belo) the isoelectric point andthis causes the formation of a strong interaction)ith the negatively charged gellan gum



3. )ilk proteins

•

Datural vehicles for probiotics cellsand o)ing to their structural andphysico-chemical properties# theycan be used as a delivery system

• Proteins have excellent gelationproperties and this specificity hasbeen recently exploited to

encapsulate probiotic cells



4ispersion methods

• 'pray drying C + solution containingthe probiotic living cells and the

dissolved polymer matrix is prepared.• The polymer matrices are generally

gum arabic and starches because they

tend to form spherical microparticlesduring the drying process

1.*tomi5ation

' d i



• *dvantages: the rapidity and the

relatively lo) cost of the procedure.• highly reproducible and suitable for

industrial applications

• 4isadvantageC has a small field ofapplication but the main problem isthe use of high temperature )hich is

not compatible )ith the survival ofbacteria

'pray drying



• In order to improve probiotic survival#

protectants can be added to the media priorto drying.

• "or example# granular starch improvesculture viability during drying and storage#

soluble fibre increase probiotic viabilityduring storage and trehalose is athermoprotectant.

• =pray-dried capsules can be coated by an

additional layer in order to give a protectionagainst acidic environment of the stomach orto reduce the deleterious eect of bile salts



'pray Free5e drying

• Probiotic cells are in a solution )hich is

atomiEed into a cold vapour phase of acryogenic li*uid such as li*uid nitrogen.

• This step generates a dispersion of froEendroplets. "roEen droplets are then dried in afreeEe dryer

• +dvantagesC providing controlled siEe# largerspecific surface area than spray-dried capsules

•

>isadvantagesC use of high energy# the longprocessing time and the cost )hich is F1<31times more expensive than spray-drying

=chematic presentation of the spray drying procedure The



=chematic presentation of the spray-drying procedure. Thesolution is pressured and then atomiEed to form a ‘‘mist’’ intothe drying chamber. The hot gas !air or nitrogen' is blo)n inthe drying chamber too. This hot gas allo)s the evaporation of

the solvent. The capsules are then transported to a cycloneseparator for recovery



!. Emulsi&cation

• 7mulsification is a chemical techni*ue toencapsulate probiotic living cells and usehydrocolloids !alginate# carrageenan and

pectin' as encapsulating materials• The principleC relationship bet)een the

discontinuous and the continuous phases

• "or encapsulation in an emulsion# an

emulsifier and a surfactant are needed. +solidifying agent !calcium chloride' is thenadded to the emulsion

Emulsi&cation and ionicgeli&cation

=chematic presentation of the emulsification procedure. +



p psmall volume of the cell-polymer suspension !i.e.# thediscontinuous phase' is added to a large volume of vegetableoil !i.e.# the continuous phase'. The mixture is thenhomogeniEed to form a )ater-in-oil emulsion. ,nce the )ater-

in-oil emulsion is formed# the )ater-soluble polymer must bein solubilised to form tiny gel particles )ithin the oil phase



• +dvantagesC the emulsion techni*ueis easy to scale-up and gives a highsurvival rate of the bacteria#capsules have a small diameter

• >isadvantagesC it provides large siEe

range and shape

• The emulsion procedure enables theproduction of the targeted

microcapsules siEe by variation ofagitation speed and the )ateroilratio



• The gel beads can be introduced into

a second polymer solution to createa coating layer that provides addedprotection to the cell or may be giveimproved organoleptic properties



Emulsi&cation and en5ymatic geli&cation

• ,ne problem )ith classical encapsulation

technologies is the use of coatings suchalginate# 5-carrageenan# gellan-gum orxanthan )hich are not allo)ed in dairyproducts in some countries

• The solution can be the use of mil5 proteins in)hich probiotics )ill be encapsulated bymeans of an enEymatic induced gelation.

• Mil5 proteins have excellent gelation properties

and they are natural vehicles for probiotics• This method gives )ater insoluble and

spherical particles.

'chematic presentation of the



pmicroencapsulation of probiotic cells bymeans of rennet-gelation of milk proteins



Emulsi&cation and interfacial polymerisation

• The techni*ue re*uires the formation of an

emulsionC the discontinuous phase contains ana*ueous suspension )ith the probiotic cells andthe continuous phase is an organic solvent.

• To initiate the polymerisation reaction# a

biocompatible agent )hich is soluble in thecontinuous phase# is added.

• The droplets obtained containing probiotic cellsare enveloped in a thin and strong membrane

• Interfacial polymerisation is used to encapsulatemicroorganisms in order to improve theirproductivity in fermentation



(. E%trusion method

• Is a physical techni*ue toencapsulate probiotic living cells anduses hydrocolloids !alginate andcarrageenan' as encapsulating

materials• The M7 of probiotic cells by extrusion

consists in pro6ecting the solution

containing the cells through a noEEleat high pressure.

7xtrusion technologiesC simple needle droplet-generator that usually is



g p p g yair driven !a' and pinning dis5 device !b'. The probiotic cells are addedto the hydrocolloid solution and dripped through a syringe needle or anoEEle spray machine in the form of droplets )hich are allo)ed to free-fall into a hardening solution such as calcium chloride



• *dvantages: extrusion is a simple andcheap method that uses a gentle

operation )hich causes no damage toprobiotic cells and gives a high probioticviability.

•

The technology does not involvedeleterious solvents and can be doneunder aerobic and anaerobic conditions.

• 4isadvantage: it is difficult to use inlarge scale productions due to the slo)formation of the microbeads.

Encapsulation by coating and



Encapsulation by coating andagglomeration• In spray-coating# the core material

needs to be in a solid form and is 5eptin motion in a specially designed vessel

• +dvantageC easy to scale up# )hich is

)hy it is mostly use to encapsulateprobiotics for nutraceuticals for example

• =pray coating is particularly adapted togive multilayer coatings

• =pray coating is a technology )hich isdifficult to master

.coating material is sprayed over the core material and solidifies tof l h f Th



form a layer at the surface. Theli*uid coating material can be in6ected from many angles over thecore materialC Buid-bed top spray coating !a'#Buid-bed bottom spraycoating )ith the urster device !b'#

and Buid bed tangential spray coating !c'



Probiocap

• >eveloped by Institut 8osell and(al’food !@anada'

• @oating freeEe-dried (+9 )ith fatty

acids.

• The technology allo)s strains toresist harsh eects of temperature#

gastric acidity# and compression



>uaolac

• >eveloped by @ell 9iotech !Jorea'

• + dual coating technology for (+9C The first layerof coating is made of soypeptides and the secondlayer is made of cellulose and gum

• The technology allo)s an increase in probioticviability during processing shelf life and duringtheir passage through the :I tract

• 9ased on a p/-dependant release mechanism

)hich protects the cells against acidic environmentin the stomach and release the coating in the p/-neutral environment of the intestines



Encapsulated probiotics in food products

• To avoid negative sensorial impactsof microcapsules in foodK it isdesirable to obtain a siEe belo) $11

Lm

$ @h !T bl $'



$. @heese !Table $'

• >ue to a relative high p/ !p/3.3'# being a good

carrier of probiotic microorganisms• Its good buering capacity and its relatively high

fat content may oer a protection to probioticbacteria against enEymatic degradation and

acidic environment of the :I tract• Immobilised Bifidobacterium bifidum )ith an

emulsification techni*ue and the obtainedgelbeads )ere froEen and lyophilised. The

addition of the immobilised cells in the cheese)as not uniform# but the survival ofbifidobacteria in the cheese )as not aected

E%amples of encapsulated



p pprobiotics and their applicationsin cheese.



• The cells remained viable until 0; )ee5s anddid not aect the Bavour and the Bavour

intensity# texture and appearance of thecheese

• @an be explained by a lac5 of bifidobacteriametabolism. To produce acetic and lactic acid#

bifidobacteria need substrates such as lactosebut in this case it )as not available

• The lo) temperature ripening !2-? @' avoidsbifidobacterial gro)th# )hose optimal gro)th

temperature is F? @# but they remain viable.• The bacteria can stay alive for at least 2

months



• =pray-drying is another technologyused to encapsulate probiotics before

their incorporation into @heddar cheese• The spray-dried culture )as stable for ?

)ee5s )hile it )as 5ept at room

temperature and during refrigeration as)ell

• +dvantagesC the cost-eectiveness and

the applicability to large scaleproduction compared to traditionalmethods li5e freeEing or freeEe-drying



0. ogurt• The incorporation of probiotic living cells

in yogurt enhances its therapeutic value

• /o)ever# there is poor level of probioticviability in yogurt because of the lo) p/

!;.0-;.2'.• =tudies have sho)n that the use of

encapsulated probiotic bacteria )as betterfor their survival

• Many authors used encapsulated probioticcells to incorporate into yogurts !Table 0'.

E%amples of encapsulated probiotics



and their applications in yogurt.



• The encapsulation of probiotic cells in beadscomprised of gellan<xanthan gum mixtures isa)ay to increase their tolerance to acidicenvironments

• /o)ever# bacteria have lo) metabolic activityand there is poor acetic acid produced

•

This compound gives a sour sensory propertyto yogurt and the lac5 of it is considered as adefect

• 7ven though the M7 gives a protection to

bifidobacteria in yogurt# the sensory *ualityaected )hich is a ma6or problem forconsumer acceptance



• Probiotic cells can be encapsulated )ithprebiotic ingredients !e.g. 8esistant

starch' or cryoprotectants !e.g. glycerol'to improve their viability

• It has been sho)n that this techni*ueenhances probiotic survival in theproduct but not under simulated :Iconditions

• The co-encapsulation is another )ay to

enhance probiotic viability and consistsin encapsulating t)o dierent probioticbacterial strains together



• 7ncapsulation and co-encapsulation)ere found to increase survival and in

particular# freeEe-dried cells afterencapsulation survived better in theyogurt.

• Thus# yogurt can be a good probioticcarrier if the cells are encapsulated.

• The protected bacteria )hich are in aviable state at the moment of

consumption# )ill survive through the :Itract and arrive in the intestine in aviable state



• @oating the capsule )ith chitosan givesbetter protection to probiotic cells than

alginate )hen considering survival inyogurt and in simulated :I conditions

• + beneficial eect of M7 on probiotic cellsurvival occurs )hen there is oxygen inthe medium# and the micro environmentin this case has a lo)er oxygen level.

• M7 of probiotics for addition into yogurts

appears to prevent losses of oxygen-sensitive strains more than protecting thecells against acidic environment

7xamples of encapsulated probiotics



p p pand their applications in froEen dairy

dessert.

7xamples of encapsulated probiotics



p p pand their applications in various food

systems

7xamples of the use of encapsulated



7xamples of the use of encapsulatedprobiotics for industrial applications



Documents

4. Enkapsulasi Probiotik