Effect of microencapsulation on survival of Biﬁdobacterium BB-12 exposed

8132019 Effect of microencapsulation on survival of Bifidobacterium BB-12 exposed

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Effect of microencapsulation on survival of Bi 1047297dobacterium BB-12 exposedto simulated gastrointestinal conditions and heat treatments

Carlise B Fritzen-Freire Elane S Prudecircncio Stephanie S Pinto Isabella B Muntildeoz Renata DMC Amboni

Universidade Federal de Santa Catarina Departamento de Ciecircncia e Tecnologia de Alimentos Rod Admar Gonzaga 1346 Itacorubi 88034-001 Florianoacutepolis SC Brazil

a r t i c l e i n f o

Article history

Received 4 May 2012

Received in revised form

16 July 2012

Accepted 24 July 2012

Keywords

Microencapsulation

Spray drying

Bi 1047297dobacterium BB-12

Prebiotics

Stress conditions

a b s t r a c t

The aim of this study was to evaluate the survival of Bi

1047297dobacterium

BB-12 microencapsulated by spraydrying with the partial (11) replacement of reconstituted skim milk (RSM) with prebiotics (inulin

oligofructose-enriched inulin and oligofructose) during the encapsulation process (yield) and under

stress conditions such as simulated gastrointestinal conditions and heat treatments The microcapsulesproduced with RSM and inulin and those produced with RSM and oligofructose-enriched inulin showeda higher viability after spray drying and encapsulation yield All the microcapsules produced in this

present study showed the highest survival rate of bi1047297dobacteria after gastric acid 1047298uid and bile salts testswhen compared with the free cells The microcapsules produced with RSM and inulin and those

produced with RSM and oligofructose-enriched inulin also showed higher protection for bi1047297dobacteriaunder simulated gastrointestinal conditions Similarly these microcapsules showed relevant counts of

bi1047297dobacteria under extreme heat conditions 2012 Elsevier Ltd All rights reserved

1 Introduction

To exert health bene1047297ts the concentration of live probiotic

bacteria needs to be of approximately 6 log CFU g1 of the productat the time of consumption (Roy 2005) Some of the major bene1047297tsof probiotics include control of serum cholesterol levels andintestinal infection which bene1047297cially in1047298uence the immune

system improving lactose utilization and anticarcinogenic activity(Nomoto 2005 Shah 2007) Thus probiotic bacteria such as bi1047297-dobacteria have been incorporated into various food products(Fritzen-Freire Muumlller Laurindo amp Prudecircncio 2010 Paseephol amp

Sherkat 2009 Rivera-Espinoza amp Gallardo-Navarro 2010)However a major challenge in relation to the application of pro-biotic cultures in functional foods is the maintenance of viability

during processing (Granato Branco Cruz Faria amp Shah 2010)Moreover since viable and biologically active micro-organisms areusually required at the target site in the host it is essential thatprobiotics withstand the hostrsquos natural barriers such as gastroin-testinal transit (Chen Chen amp Kuo 2007 Kim et al 2008)

Microencapsulation technologies are a promising prospect forintroducing viable probiotic bacteria in foods because the encap-sulation matrix can provide a physical barrier against stress

conditions (Chaacutevarri et al 2010 Nazzaro Fratianni Coppola Sadaamp Orlando 2009) The capsules should also be able to maintaintheirintegrity during passage through the gastrointestinal tract until

they reach their target destination (colon) where they shouldbreak down and release the probiotic bacteria (Ding amp Shah 2009)These technologies are also often used to protect the cells from heattreatment in food processing thus potentially reducing cell injury

and death (Ding amp Shah 2007)Spray drying is a commonly used method for probiotic encap-

sulation (De Vos Faas Spasojevic amp Sikkema 2010) This methodinvolves atomization of an emulsion or a suspension of probiotics

and encapsulating agents in a hot air drying chamber resulting inrapid evaporation of water (Gharsallaoui Roudaut ChambinVoilley amp Saurel 2007) The advantages of spray drying are its

rapidity and relatively low cost The technique is highly reproduc-ible and its most important feature is that it is suitable for industrialapplications (Burgain Gaiani Linder amp Scher 2011) Despite theadvantages claimed for this method and the short residence timein the drying chamber signi1047297cant inactivation of the cells can occur

during the process Thus the choice of an appropriate dryingmedium is crucial because it might enhance the survival rate of bacteria throughout stressful treatments (Silva Freixo Gibbs ampTeixeira 2011) Reconstituted skim milk (RSM) is widely used as

a protective agent in spray drying (Rokka amp Rantamaumlki 2010) butonly few studies have been reported on the application of prebi-otics as a source of coating materials According to Corcoran Ross

Corresponding author Tel thorn55 48 37215366 fax thorn55 48 37219943

E-mail address elaneccaufscbr (ES Prudecircncio)

Contents lists available at SciVerse ScienceDirect

LWT - Food Science and Technology

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e l w t

0023-6438$ e see front matter 2012 Elsevier Ltd All rights reserved

httpdxdoiorg101016jlwt201207037

LWT - Food Science and Technology 50 (2013) 39e44



Fitzgerald and Stanton (2004) prebiotics as inulin and oligo-fructose may potentially be exploited as carrier media for the

purposes of spray drying and may be useful for enhancing probioticsurvival during processing and storage The effect of prebiotics isrelated to selective stimulation for example of Bi 1047297dobacterium in

the gut thereby increasing the hostrsquos natural resistance to invadingpathogens (Coudray Tressol Gueux amp Rayssiguier 2003) BieleckaBiedrzycka and Majkowska (2002) con1047297rmed the appropriatenessof combining prebiotics and probiotics demonstrating greater

effectiveness when compared with probiotics aloneThe aim of this study was to evaluate the survival of Bi 1047297do-

bacterium BB-12 microencapsulated by spray drying with partial

(11) replacement of RSM with prebiotic agents (inulinoligofructose-enriched inulin and oligofructose) during theencapsulation process (yield) and under stress conditions such assimulated gastrointestinal conditions and heat treatments

2 Materials and methods

21 Materials

Bi 1047297dobacterium BB-12 (BB-12 Chr Hansen Hoacutensholm

Denmark) was used as the active material for the microcapsulesThe encapsulating agents used were commercial skim milk powder(Molico Nestleacute Satildeo Paulo Brazil) and the prebiotic agents inulin

(Orafti HPX Orafti Tienen Belgium) with degree of polymeriza-tion (DP) 23 oligofructose-enriched inulin (Orafti Synergy1Orafti Tienen Belgium) which is a mixture of oligofructose (DP2e8) and long-chain inulin fraction (DP 10e60) and oligofructose

(Orafti P95 Orafti Tienen Belgium) with DP 2e8 MRS agar (DifcoSparks USA) lithium chloride (Vetec Rio de Janeiro Brazil) sodiumpropionate (Fluka Neu-Ulm Germany) AnaeroGen (OxoidHampshire UK) and bile salts (Oxgall Difco Sparks USA) were

used for the microbiological analysis All the chemicals used wereof analytical grade

22 Sample preparation and microencapsulation

The feed solutions were prepared as described by AnantaVolkert and Knorr (2005) with modi1047297cations Reconstitutedskim milk (RSM) at a concentration of 200 g L 1 was used as the

control medium The prebiotic media used for spray drying con-sisted of an equal ratio of RSM and each of the three prebiotics(ratio of 11 200 g L 1 total concentration) All the media werehomogenized in sterile distilled water and heat treated at 80 C for

30 min Freeze-dried probiotic cells of Bi 1047297dobacterium BB-12 wererehydrated at 25 g L 1 using a 120 g L 1 solution of RSM and frozenas stock solution at 18 C into sterile glass bottles This probioticstock solution was incubated at 37 C for 2 h and after that it was

inoculated (100 mL L 1) into the four feed solutions and then

submitted to spray dryingThe microencapsulation process was performed with a labora-

tory scale spray dryer (Buchi B-290 Flawil Switzerland) at constant

air inlet temperature of 150 2 C and outlet temperature of 55 3 C as described by Fritzen-Freire et al (2012) The micro-capsules containing Bi 1047297dobacterium BB-12 were collected from the

base of the cyclone and placed in sterile plastic bottles Thus fourdifferent microcapsules were obtained through the followingcombination of encapsulating agents M1 (with 200 g L 1 of RSM)M2 (with 100 g L 1 of RSM and 100 g L 1 of inulin) M3 (with

100 g L 1 of RSM and 100 g L 1 of oligofructose-enriched inulin)and M4 (with 100 g L 1 of RSM and 100 g L 1 of oligofructose)

To evaluate the survival rate of bi1047297dobacteria during spraydrying the enumeration was determined before (in the feed solu-

tions) and after (microcapsules M1 M2 M3 and M4) the process

The viable cell counts of the bi1047297dobacteria exposed to simulatedgastrointestinal conditions and different heat treatments were

performed in the free (control) and in the microencapsulatedforms

23 Enumeration of bi 1047297dobacteria

To enumerate the bi1047297dobacteria the samples were seriallydiluted with peptone water (01 g 100 g1) and plated on MRS agarmodi1047297ed with the addition of lithium chloride (02 g 100 g1) and

sodium propionate (03 g 100 g1) as proposed by Vinderola andReinheimer (1999) The plates were incubated in anaerobic jarscontaining AnaeroGen at 37 1 C for 72 h After the incubationperiod the count of viable probiotic cells was carried out and

expressed as log colony-forming units per gram (log CFU g1)

However the entrapped bi1047297dobacteria (1 g) was 1047297rst re-suspendedin 9 mL of phosphate buffer (01 mol L 1 pH 70) followed byhomogenization in a magnetic stirrer for 10 min according to themethod proposed by Sheu Marshall and Heymann (1993) This

treatment ensured the complete release of the entrapped bi1047297do-bacteria from inside the dried particles All the experiments wereperformed in triplicate

24 Encapsulation yield

Encapsulation yield (EY) (g 100 g1) ie survival rate during themicroencapsulation process (Eq (1)) was calculated as proposed by

Picot and Lacroix (2004)

EY frac14 ethN =N 0THORN 100 (1)

where N is the number of viable cells (log CFU g1) of dry matter inthe microcapsules and N 0 is the number of viable cells(log CFU g1) of dry matter in the feed solutions (before drying)

25 Survival of free and microencapsulated bi 1047297dobacteria in

simulated gastric conditions and bile salts

The NGYC medium (120 g 100 g1 non-fat milk 20 g 100 g1

glucose10 g 100 g1 yeastextract and 005 g 100 g1 cysteine) was

used to simulate gastric conditions as described by Iyer andKailasapathy (2005) The free (1 mL) and microencapsulated (1 g)bi1047297dobacteria were added to the NGYC medium that had beenpreviously adjusted to pH 20 pH 30 or 65 (control) with 5 mol L 1

HCl or 1 mol L 1 NaOH in 10 mL aliquots The samples were incu-bated at 37 1 C for3 h An aliquotfromeachtreatment was takenhourly for determination of the viable cell counts

The resistance to bile salts was determined by inoculating free

and microencapsulated cells in milkeyeast extract medium(100 g 100 g1 non-fat milk 05 g 100 g1 yeast extract and

005 g 100 g1

cysteine) (Truelstrup Hansen Allan-Wojtas Jin ampPaulson 2002) containing 0 (control) 50 and 100 g L 1 of Oxgall

bile salts The samples were withdrawn after incubation at37 1 C for 0 3 and 6 h to carry out cell counts of thebi1047297dobacteria

26 Survival of free and microencapsulated bi 1047297dobacteria under

heat treatments

The resistance of free and microencapsulated Bi 1047297dobacterium

BB-12 (M1 M2 M3 and M4) to heat treatments (55 65 and75 1 C for 1 and 10 min) was investigated using sterile distilledwater as a suspending medium One gram of microcapsules and1 mL of the free cell suspension were transferred into test tubes

containing 10 mL of distilled water each as suggested by Sabikhi

CB Fritzen-Freire et al LWT - Food Science and Technology 50 (2013) 39e4440



Babu Thompkinson and Kapila (2010) After the heat treatments(water bath) the content was cooled to room temperature

(w25 C)and thenthecell countsof bi1047297dobacteria were performed

27 Statistical analysis

The data analysis was carried out using STATISTICA 70 software

(StatSoft Inc Tulsa USA) Analysis of variance (ANOVA) was used todetermine signi1047297cant differences (P lt 005) amongst the micro-

capsules Differences between means were detected by usingTukeyrsquos test

3 Results and discussion

31 Enumeration of bi 1047297dobacteria and encapsulation yield

The viable cell counts for Bi 1047297dobacterium BB-12 in the feedsolutions (before spray drying) in the microcapsules (after micro-encapsulation ie M1 M2 M3 and M4) produced with differentencapsulating agents and also the encapsulating yields are shown

in Table 1The results showed similar (P gt 005) counts of bi1047297dobacteria in

the different feed solutions On the other hand the counts of bi1047297-

dobacteria and the encapsulation yields were higher (P lt 005) forthe microcapsules produced with reconstituted skim milk (RSM)and inulin (M2) and with RSM and oligofructose-enriched inulin(M3) when compared to the microcapsules produced only with

RSM (M1) and with oligofructose (M4) This result occurred prob-ably because as described by Fritzen-Freire et al (2012) the inulinacts as a thermoprotector for the cells of Bi 1047297dobacterium BB-12

undergoing the spray drying process showing a positive effect ontheir survival during the encapsulation process Moreover Chaacutevezand Ledeboer (2007) reported that inlet and outlet temperaturesare one of the most important causes of bacterial stress and

mortality during the spray drying process and consequently affectthe encapsulation yield Therefore in this present study low

temperatures (inlet temperature of 150 2 C and outlet temper-ature of 55 3 C) were used in order to minimize heat treatmentwhile obtaining a good overall drying result Fu and Chen (2011)also reported that the cell loss during the atomization step could

be attributed to the choice of the appropriate drying medium aswell as to the high water content of the feed solution and the shortresidence time in the drying chamber of the equipment Thereforeas described by those authors the combined effect of these three

factors could protect the bacterial cells from being heated up toa fatal temperature and thus result in good encapsulation ef 1047297-ciency as was veri1047297ed in this present study

In addition the countof viable probiotic cells obtained forall the

microcapsules was above the recommended levels for a probiotic

food ie equal to or greater than 6 log CFU g1 of the productwhich is in accordance with what was reported by Shah (2007)



The effect of acid conditionson the viabilityof the freeand of the

microencapsulated Bi 1047297dobacterium BB-12 is shown in Fig 1 Thisstrain showed a steady loss in viability when exposed to acid

Table 1

Viable cell counts of Bi 1047297dobacterium BB-12 in the feed solutions and in the

microcapsules produced with different encapsulating agents and the encapsulation

yields (EY)

Encapsulating agents

Reconstituted

skim milk

(RSM)

RSM and

inulin

RSM and

oligofructose-

enriched inulin

RSM and

oligofructose

Feed solutions

(log CFU g1)

1473 001a 1448 018a 1437 017a 1403 033a

Microcapsules

(log CFU g1)

1057 005a 1091 007b 1088 002b 990 008a

EY (g 100 g1) 7172 027a 7537 043b 7568 104b 7058 103a

aebMeans standard deviation with different superscript letters in the same line

indicate signi1047297

cant differences (P lt 005) among the studied samples

M1 M2 M3 M4 free cells

initial 1 h 2 h 3 h



120

110

90

80

70

60

100

V i a b l e c e l l s ( l o g C F U g

- 1 )

120

110

100

90

80

70

60

V i a b l e c e l l s ( l o

g C F U g

- 1 )

V i a b l e c e l l

s ( l o g C F U g

- 1 )

120

110

100

90

80

70

60

a

b

c

Fig 1 Survival of Bi 1047297dobacterium BB-12 free and microencapsulated with different

encapsulating agents under in vitro acid conditions at 37 1 C (a) pH 65 (b) pH 30

and (c) pH 20 (M1) microcapsule with reconstituted skim milk (RSM) (M2)

microcapsule with RSM and inulin (M3) microcapsule with RSM and oligofructose-

enriched inulin (M4) microcapsule with RSM and oligofructose Error bars repre-

sent the mean of the results of the experiment

CB Fritzen-Freire et al LWT - Food Science and Technology 50 (2013) 39e44 41



conditions However the microcapsules containing bi1047297dobacteriasurvived very well (P lt 005) after exposure to in vitro acid

conditions when compared with the free cells Also a decrease of approximately 4 log was noted in the number of free cells after 3 hof incubation at pH 2 when compared to decreases of about2 login

the all microencapsulated bi1047297dobacteria under similar conditionsThe microcapsules produced with RSM and inulin (M2) and thoseproduced with RSMand oligofructose-enriched inulin (M3) showedhigher (P lt 005) count when incubated in pH 30 and 20 after 3 h

of exposure when compared to the microcapsules produced onlywith RSM (M1) and with oligofructose (M4) These results suggestthat inulin had a better effect on the protection of the bi1047297dobacteria

during the gastric simulation Barclay Ginic-Markovica Cooper andPetrovsky (2010) reported that inulin might be ideal to transportsubstances to the colon since it is stable to the range of pH and ionicstrength in the human gastrointestinal tract Moreover

Mantzouridou Spanou and Kiosseoglou (2012) also state that theinulin molecules are not hydrolyzed by the human gastrointestinaltract enzymes and hence provide a bene1047297cial effect on healthacting as dietary 1047297ber Apart from such properties Ann et al (2007)

claim that the use of prebiotic substrates such as inulin asencapsulating agents may promote more bene1047297cial effects

including the reduction of diarrhea and the inactivation of patho-gens in the gastrointestinal tracts of both humans and animals

The survival of free and microencapsulated Bi 1047297dobacterium BB-12 was also monitored for up to 6 h after exposure to milkeyeastmedium containing 50 and 100 g L 1 bile salts (Fig 2) Theresults indicate a similar trend that was found under high acidconditions From the initial counts of free and microencapsulated

bi1047297dobacteria the numbers declined steadily as the bile concen-tration and time of incubation increased Moreover the decreaserate was much greater in the free cells The microcapsules lesssusceptible to Oxgall were those produced with RSM and inulin

(M2) and those produced with RSM and oligofructose-enrichedinulin (M3) with decreases of 239 and 232 log CFU g1 inviability after 6 h of incubation and at 100 g L 1 bile salts

respectively On the other hand the free cells showed the largest(P lt 005) decreases in their viability under the same conditions(371 log CFU g1) These results are in accordance with those ob-tained by Chandramouli Kailasapathy Peiris and Jones (2004) andIyer and Kailasapathy (2005) who used the same concentrations of

bile salts as those employed in this present study and noted thatencapsulated probiotic bacteria can survive in higher numbers thanthe free probiotic cells

Finally the results obtained in this present study showed that

microencapsulation with RSM and the prebiotics inulin andoligofructose-enriched inulin could provide good protection forbi1047297dobacteria undergoing the gastric acid 1047298uid and bile salts tests


heat treatments

The survival of free and microencapsulated bi1047297dobacteria

exposed to temperatures of 55 65 and 75 1 C are shown inTable 2 The free cells of Bi 1047297dobacterium BB-12 were very sensitiveto heat treatment their numbers decreased from 1103 log cycles to

1000 log at 55 1 C and less than 600 log at 65 1 C and75 1 C after 10 min Differences between the decrease in thecounts of the free and of the encapsulated cells ( P lt 005) werenoted This result occurred probably because as described by

Corcoran Stanton Fitzgerald and Ross (2008) the excessive heatunfolds the higher order structure of macromolecules such asprotein and nucleic acid of bacterial cells breaks the linkagebetween monomeric units and eventuallycauses the destruction of

the monomers leading to bacterial death

The microcapsules produced only with RSM (M1) survived well

at 55 1 C for 10 min with anaverageloss of only015 log CFU g1

compared to microcapsules containing prebiotics (M2 M3 and M4)with an average loss of 044 051 and 034 log CFU g1 respectively

after 10 min However at 65 1 C all microcapsules showeda similar reduction (P lt 005) in viable cell count Meanwhilenone of the microcapsules showed any probiotic count(gt600 log CFU g1) after exposure for 10 min at 75 1 C These

results indicate that the encapsulating agents used in this presentstudy offered little protection for the probiotic bacteria after thistimetemperature combination Barclay et al (2010) suggest that inthe presence of water an increase in temperature leads to an

increase in hydrolysis of inulin that follows 1047297

rst- or pseudo-1047297

rst-



- 1 )

initial 2 h 4 h 6 h



- 1 )



- 1 )

120

110

100

90

80

70

60

120

110

100

90

80

70

60

120

110

100

90

80

70

60

a

b

c


encapsulating agents in the presence of (a) 00 g L 1 (b) 50 g L 1 and (c) 100 g L 1 bile

salts after 6 h exposure at 37 1 C (M1) microcapsule with reconstituted skim milk

(RSM) (M2) microcapsule with RSM and inulin (M3) microcapsule with RSM and

oligofructose-enriched inulin (M4) microcapsule with RSM and oligofructose Error

bars represent the mean of the results of the experiment




order kinetics at both neutral and acid pHs Those authors alsoreported that the inulin hydrolysis rate can be considered insig-

ni1047297cant for processing time frames up to w60 C but can be morerelevant at higher temperatures as those used in this present study

Nevertheless it was evident that the microcapsules producedwith RSM and inulin (M2) and those produced with RSM and

oligofructose-enriched inulin (M3) showed the highest counts(P lt 005) of bi1047297dobacteria in extreme heat conditions ie at75 1 C for 1 min In addition the microcapsules produced withRSM and oligofructose (M4) showed the lowest resistance to heat

treatments Lian Hsiao and Chou (2002) reported that besidesdifferences in their chemical characteristics encapsulating agentshave different physical properties Furthermore according toTaacuterrega Rocafull and Costell (2010) long-chain inulin with a high

degree of polymerization (used in the microcapsules M2 and M3)is more thermally stable and less soluble thus rendering higherprotection against Bi 1047297dobacterium BB-12 The results of this presentstudy suggest that microencapsulation using inulin and

oligofructose-enriched inulin may enhance thermal resistance of the Bi 1047297dobacterium BB-12

4 Conclusions

The use of the prebiotics inulin and oligofructose-enrichedinulin improved both the survival of Bi 1047297dobacterium BB-12 afterspray drying and the encapsulation yield of the microcapsules

These prebiotics also conferred better protection for the bi1047297do-

bacteria submitted to simulated gastrointestinal conditions andextreme heat treatments

This study shows that microencapsulation by spray drying can

be used as an effective method for maintaining the survival of

Bi 1047297dobacterium BB-12 exposed to stress conditions In addition theresults showed that inulin and oligofructose-enriched inulin arethe most appropriate prebiotics to be used as partial replacement of

reconstituted skim milk (RSM) providing greater resistance tomicroencapsulated bi1047297dobacteria

Acknowledgments

The authors gratefully thank the Fundaccedilatildeo de Amparo agrave Pes-quisa e Inovaccedilatildeo do Estado de Santa Catarina (FAPESC) for their

1047297nancial support and also Clariant for providing the prebiotics

References

Ananta E Volkert M amp Knorr D (2005) Cellular injuries and storage stability of spray dried Lactobacillus rhamnosus GG International Dairy Journal15 399e409

Ann E Y Kim Y Oh S J Imm J Y Park D J Han K S et al (2007) Microen-capsulation of Lactobacillus acidophilus ATCC 43121 with prebiotic substratesusing a hybridisation system International Journal of Food Science and Tech-nology 42 411e419

Barclay T Ginic-Markovica M Cooper P amp Petrovsky N (2010) Inulin e

a versatile polysaccharide with multiple pharmaceutical and food chemicaluses Journal of Excipients and Food Chemicals 1 27e50

Bielecka M Biedrzycka E amp Majkowska A (2002) Selection of probiotics andprebiotics for synbiotics and con1047297rmation of their in vivo effectiveness FoodResearch International 35 125e131

Burgain J Gaiani C Linder M amp Scher J (2011) Encapsulation of probiotic livingcells from laboratory scale to industrial applications Journal of Food Engi-

neering 104 467e

483Chandramouli V Kailasapathy K Peiris P amp Jones M (2004) An improvedmethod of microencapsulation and its evaluation to protect Lactobacillus spp insimulated gastric conditions Journal of Microbiological Methods 56 27e35

Chaacutevarri M Marantildeoacuten I Ares R Ibaacutentildeez F C Marzo F amp Villaraacuten M D C (2010)Microencapsulation of a probiotic and prebiotic in alginateechitosan capsulesimproves survival in simulated gastro-intestinal conditions International Jour-nal of Food Microbiology 142 185e189

Chaacutevez B E amp Ledeboer A M (2007) Drying of probiotics optimization of formu-lation and process to enhance storage survival Drying Technology 25 193e201

Chen M J Chen K N amp Kuo Y-T (2007) Optimal thermotolerance of Bi 1047297do-bacterium bi 1047297dum in gellanealginate microparticles Biotechnology and Bioen-

gineering 98 411e419Corcoran B M Ross R P Fitzgerald G F amp Stanton C (2004) Comparative

survival of probiotic lactobacilli spray-dried in the presence of prebioticsubstances Journal of Applied Microbiology 96 1024e1039

Corcoran B M Stanton C Fitzgerald G amp Ross R P (2008) Life under stress theprobiotic stress response and how it may be manipulated Current Pharma-ceutical Design 14 1382e1399

Coudray C Tressol J C Gueux E amp Rayssiguier Y (2003) Effects of inulin-typefructans of different chain length and type of branching or intestinal absorp-tion and balance of calcium and magnesium in rats European Journal of Nutrition 42 91e98

De Vos P Faas M M Spasojevic M amp Sikkema J (2010) Encapsulation forpreservation of functionality and targeted delivery of bioactive food compo-nents International Dairy Journal 20 292e302

Ding W K amp Shah N P (2007) Acid bile and heat tolerance of free and micro-encapsulated probiotic bacteria Journal of Food Science 72 M446eM450

Ding W K amp Shah N P (2009) An improved method of microencapsulation of probiotic bacteria for their stability in acidic and bile conditions during storage

Journal of Food Science 74 M53eM61Fritzen-Freire C B Muumlller C M O Laurindo J B amp Prudecircncio E S (2010) The

in1047298uence of Bi 1047297dobacterium Bb-12 and lactic acid incorporation on the prop-erties of Minas Frescal cheese Journal of Food Engineering 96 621e627

Fritzen-Freire C B Prudecircncio E S Amboni R D M C Pinto S S Negratildeo-Murakami A N amp Murakami F S (2012) Microencapsulation of bi1047297dobacteriaby spray drying in the presence of prebiotics Food Research International 45306e312

Table 2

The effect of exposure to 55 65 and 75 1 C for 0 1 and 10 min on the survival of bi1047297dobacteria free and microencapsulated with different encapsulating agents

Temp (C) Time (min) Viability (log CFU g1)

M1 M2 M3 M4 Free cells

55 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1046 008aA 1080 002bcA 1076 004cA 973 004dAB 1058 004acB

10 1042 007aA 1047 002aB 1037 004aB 956 004bB 1000 005cC

65 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1030 002aB 1061 004bB 1051 004bB 923 004cB 909 004dB

10 987 003aC 1012 004bC 1007 003bC 901 004cC NP

75 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011b

1 1007 003aB 1042 003bB 1048 005bB 918 001cB NP

10 NP NP NP NP NP

NP frac14 not probiotic enumeration (minor than 6 log CFU g1)aedMeans standard deviation with different superscript letters in the same line indicate signi1047297cant differences (P lt 005) among the studied microcapsules in the same timeand temperatureAeCMeans standard deviation with different superscript letters in the same column indicate signi1047297cant differences (P lt 005) among times studied for same microcapsule in

the same temperature evaluated

M1 microcapsule with reconstituted skim milk (RSM)

M2 microcapsule with RSM and inulin

M3 microcapsule with RSM and oligofructose-enriched inulin

M4 microcapsule with RSM and oligofructose




Fu N amp Chen X D (2011) Towards a maximal cell survival in convective thermaldrying processes Food Research International 44 1127e1149

Gharsallaoui A Roudaut G Chambin O Voilley A amp Saurel R (2007) Appli-cations of spray-drying in microencapsulation of food ingredients an overviewFood Research International 40 1107e1121

Granato D Branco G F Cruz A G Faria J A F amp Shah N P (2010) Probioticdairy products as functional foods Comprehensive Reviews in Food Science andFood Safety 9 455e470

Iyer C amp Kailasapathy K (2005) Effect of co-encapsulation of probiotics withprebiotics on increasing the viability of encapsulated bacteria in simulated

gastrointestinal conditions and in yoghurt Journal of Food Science 70M18eM23

Kim S J Cho S Y Kim S H Song O J Shin I S Cha D S et al (2008) Effect of microencapsulation on viability and other characteristics in Lactobacillus acid-ophilus ATCC 43121 LWT e Food Science and Technology 41 493e500

Lian W C Hsiao H C amp Chou C C (2002) Survival of bi1047297dobacteria after spraydrying International Journal of Food Microbiology 74 79e86

Mantzouridou F Spanou A amp Kiosseoglou V (2012) An inulin-based dressingemulsion as a potential probiotic food carrier Food Research International 46 260e269

Nazzaro F Fratianni F Coppola R Sada A amp Orlando P (2009) Fermentativeability of alginate-prebiotic encapsulated Lactobacillus acidophilus and survivalunder simulated gastrointestinal conditions Journal of Functional Foods 1319e323

Nomoto K (2005) Review prevention of infections by probiotics Journal of Bioscience and Bioengineering 100 583e592

Paseephol T amp Sherkat F (2009) Probiotic stability of yoghurts containing Jerusalem artichoke inulins during refrigerated storage Journal of FunctionalFoods 1 311e318

Picot A amp Lacroix C (2004) Encapsulation of bi1047297dobacteria in whey protein-basedmicrocapsules and survival in simulated gastrointestinal conditions and inyoghurt International Dairy Journal 14 505e515

Rivera-Espinoza Y amp Gallardo-Navarro Y (2010) Non-diary probiotic productsFood Microbiology 27 1e11

Rokka S amp Rantamaumlki P (2010) Protecting probiotic bacteria by microencapsu-lation challenges for industrial applications European Food Research Tech-nology 231 1e12

Roy D (2005) Technological aspects related to the use of bi1047297dobacteria in dairyproducts Lait 85 39e56

Sabikhi L Babu R Thompkinson D K amp Kapila S (2010) Resistance of micro-encapsulated Lactobacillus acidophilus LA1 processing treatments and simulatedgut conditions Food Bioprocess Technology 3 586e593

Shah N P (2007) Functional cultures and health bene1047297ts International Dairy Journal 17 1262e1277

Sheu T Y Marshall R T amp Heymann H (1993) Improving survival of culturebacteria in frozen desserts by microentrapment Journal of Dairy Science 76 1902e1907

Silva J Freixo R Gibbs P amp Teixeira P (2011) Spray-drying for the production of dried cultures International Journal of Dairy Technology 64 321e335

Taacuterrega A Rocafull A amp Costell E (2010) Effect of blends of short and long-chaininulin on the rheological and sensory properties of prebiotic low-fat custardsLWT e Food Science and Technology 43 556e562

Truelstrup Hansen L Allan-Wojtas P M Jin Y L amp Paulson A T (2002) Survivalof free and calcium-alginate microencapsulated Bi 1047297dobacterium spp in simu-lated gastro-intestinal conditions Food Microbiology 19 35e45

Vinderola C G amp Reinheimer J A (1999) Culture media for the enumeration of Bi 1047297dobacterium bi 1047297dum and Lactobacillus acidophilus in the presence of yoghurtbacteria International Dairy Journal 9 497e505




Fitzgerald and Stanton (2004) prebiotics as inulin and oligo-fructose may potentially be exploited as carrier media for the

purposes of spray drying and may be useful for enhancing probioticsurvival during processing and storage The effect of prebiotics isrelated to selective stimulation for example of Bi 1047297dobacterium in

the gut thereby increasing the hostrsquos natural resistance to invadingpathogens (Coudray Tressol Gueux amp Rayssiguier 2003) BieleckaBiedrzycka and Majkowska (2002) con1047297rmed the appropriatenessof combining prebiotics and probiotics demonstrating greater

effectiveness when compared with probiotics aloneThe aim of this study was to evaluate the survival of Bi 1047297do-

bacterium BB-12 microencapsulated by spray drying with partial

(11) replacement of RSM with prebiotic agents (inulinoligofructose-enriched inulin and oligofructose) during theencapsulation process (yield) and under stress conditions such assimulated gastrointestinal conditions and heat treatments

2 Materials and methods

21 Materials

Bi 1047297dobacterium BB-12 (BB-12 Chr Hansen Hoacutensholm

Denmark) was used as the active material for the microcapsulesThe encapsulating agents used were commercial skim milk powder(Molico Nestleacute Satildeo Paulo Brazil) and the prebiotic agents inulin

(Orafti HPX Orafti Tienen Belgium) with degree of polymeriza-tion (DP) 23 oligofructose-enriched inulin (Orafti Synergy1Orafti Tienen Belgium) which is a mixture of oligofructose (DP2e8) and long-chain inulin fraction (DP 10e60) and oligofructose

(Orafti P95 Orafti Tienen Belgium) with DP 2e8 MRS agar (DifcoSparks USA) lithium chloride (Vetec Rio de Janeiro Brazil) sodiumpropionate (Fluka Neu-Ulm Germany) AnaeroGen (OxoidHampshire UK) and bile salts (Oxgall Difco Sparks USA) were

used for the microbiological analysis All the chemicals used wereof analytical grade

22 Sample preparation and microencapsulation

The feed solutions were prepared as described by AnantaVolkert and Knorr (2005) with modi1047297cations Reconstitutedskim milk (RSM) at a concentration of 200 g L 1 was used as the

control medium The prebiotic media used for spray drying con-sisted of an equal ratio of RSM and each of the three prebiotics(ratio of 11 200 g L 1 total concentration) All the media werehomogenized in sterile distilled water and heat treated at 80 C for

30 min Freeze-dried probiotic cells of Bi 1047297dobacterium BB-12 wererehydrated at 25 g L 1 using a 120 g L 1 solution of RSM and frozenas stock solution at 18 C into sterile glass bottles This probioticstock solution was incubated at 37 C for 2 h and after that it was

inoculated (100 mL L 1) into the four feed solutions and then

submitted to spray dryingThe microencapsulation process was performed with a labora-

tory scale spray dryer (Buchi B-290 Flawil Switzerland) at constant

air inlet temperature of 150 2 C and outlet temperature of 55 3 C as described by Fritzen-Freire et al (2012) The micro-capsules containing Bi 1047297dobacterium BB-12 were collected from the

base of the cyclone and placed in sterile plastic bottles Thus fourdifferent microcapsules were obtained through the followingcombination of encapsulating agents M1 (with 200 g L 1 of RSM)M2 (with 100 g L 1 of RSM and 100 g L 1 of inulin) M3 (with

100 g L 1 of RSM and 100 g L 1 of oligofructose-enriched inulin)and M4 (with 100 g L 1 of RSM and 100 g L 1 of oligofructose)

To evaluate the survival rate of bi1047297dobacteria during spraydrying the enumeration was determined before (in the feed solu-

tions) and after (microcapsules M1 M2 M3 and M4) the process

The viable cell counts of the bi1047297dobacteria exposed to simulatedgastrointestinal conditions and different heat treatments were

performed in the free (control) and in the microencapsulatedforms

23 Enumeration of bi 1047297dobacteria

To enumerate the bi1047297dobacteria the samples were seriallydiluted with peptone water (01 g 100 g1) and plated on MRS agarmodi1047297ed with the addition of lithium chloride (02 g 100 g1) and

sodium propionate (03 g 100 g1) as proposed by Vinderola andReinheimer (1999) The plates were incubated in anaerobic jarscontaining AnaeroGen at 37 1 C for 72 h After the incubationperiod the count of viable probiotic cells was carried out and

expressed as log colony-forming units per gram (log CFU g1)

However the entrapped bi1047297dobacteria (1 g) was 1047297rst re-suspendedin 9 mL of phosphate buffer (01 mol L 1 pH 70) followed byhomogenization in a magnetic stirrer for 10 min according to themethod proposed by Sheu Marshall and Heymann (1993) This

treatment ensured the complete release of the entrapped bi1047297do-bacteria from inside the dried particles All the experiments wereperformed in triplicate

24 Encapsulation yield

Encapsulation yield (EY) (g 100 g1) ie survival rate during themicroencapsulation process (Eq (1)) was calculated as proposed by

Picot and Lacroix (2004)

EY frac14 ethN =N 0THORN 100 (1)

where N is the number of viable cells (log CFU g1) of dry matter inthe microcapsules and N 0 is the number of viable cells(log CFU g1) of dry matter in the feed solutions (before drying)



The NGYC medium (120 g 100 g1 non-fat milk 20 g 100 g1

glucose10 g 100 g1 yeastextract and 005 g 100 g1 cysteine) was

used to simulate gastric conditions as described by Iyer andKailasapathy (2005) The free (1 mL) and microencapsulated (1 g)bi1047297dobacteria were added to the NGYC medium that had beenpreviously adjusted to pH 20 pH 30 or 65 (control) with 5 mol L 1

HCl or 1 mol L 1 NaOH in 10 mL aliquots The samples were incu-bated at 37 1 C for3 h An aliquotfromeachtreatment was takenhourly for determination of the viable cell counts

The resistance to bile salts was determined by inoculating free

and microencapsulated cells in milkeyeast extract medium(100 g 100 g1 non-fat milk 05 g 100 g1 yeast extract and

005 g 100 g1

cysteine) (Truelstrup Hansen Allan-Wojtas Jin ampPaulson 2002) containing 0 (control) 50 and 100 g L 1 of Oxgall

bile salts The samples were withdrawn after incubation at37 1 C for 0 3 and 6 h to carry out cell counts of thebi1047297dobacteria


heat treatments

The resistance of free and microencapsulated Bi 1047297dobacterium

BB-12 (M1 M2 M3 and M4) to heat treatments (55 65 and75 1 C for 1 and 10 min) was investigated using sterile distilledwater as a suspending medium One gram of microcapsules and1 mL of the free cell suspension were transferred into test tubes

containing 10 mL of distilled water each as suggested by Sabikhi





























Table 1



yields (EY)


Reconstituted

skim milk

(RSM)

RSM and

inulin

RSM and

oligofructose-

enriched inulin

RSM and

oligofructose

Feed solutions

(log CFU g1)

1473 001a 1448 018a 1437 017a 1403 033a

Microcapsules

(log CFU g1)

1057 005a 1091 007b 1088 002b 990 008a

EY (g 100 g1) 7172 027a 7537 043b 7568 104b 7058 103a





initial 1 h 2 h 3 h



120

110

90

80

70

60

100


- 1 )

120

110

100

90

80

70

60


g C F U g

- 1 )

V i a b l e c e l l

s ( l o g C F U g

- 1 )

120

110

100

90

80

70

60

a

b

c


























heat treatments













rst-



- 1 )

initial 2 h 4 h 6 h



- 1 )



- 1 )

120

110

100

90

80

70

60

120

110

100

90

80

70

60

120

110

100

90

80

70

60

a

b

c

















4 Conclusions








Acknowledgments



References







neering 104 467e















Table 2




55 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1046 008aA 1080 002bcA 1076 004cA 973 004dAB 1058 004acB

10 1042 007aA 1047 002aB 1037 004aB 956 004bB 1000 005cC

65 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1030 002aB 1061 004bB 1051 004bB 923 004cB 909 004dB

10 987 003aC 1012 004bC 1007 003bC 901 004cC NP

75 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011b

1 1007 003aB 1042 003bB 1048 005bB 918 001cB NP

10 NP NP NP NP NP




























































Table 1



yields (EY)


Reconstituted

skim milk

(RSM)

RSM and

inulin

RSM and

oligofructose-

enriched inulin

RSM and

oligofructose

Feed solutions

(log CFU g1)

1473 001a 1448 018a 1437 017a 1403 033a

Microcapsules

(log CFU g1)

1057 005a 1091 007b 1088 002b 990 008a

EY (g 100 g1) 7172 027a 7537 043b 7568 104b 7058 103a





initial 1 h 2 h 3 h



120

110

90

80

70

60

100


- 1 )

120

110

100

90

80

70

60


g C F U g

- 1 )

V i a b l e c e l l

s ( l o g C F U g

- 1 )

120

110

100

90

80

70

60

a

b

c


























heat treatments













rst-



- 1 )

initial 2 h 4 h 6 h



- 1 )



- 1 )

120

110

100

90

80

70

60

120

110

100

90

80

70

60

120

110

100

90

80

70

60

a

b

c

















4 Conclusions








Acknowledgments



References







neering 104 467e















Table 2




55 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1046 008aA 1080 002bcA 1076 004cA 973 004dAB 1058 004acB

10 1042 007aA 1047 002aB 1037 004aB 956 004bB 1000 005cC

65 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1030 002aB 1061 004bB 1051 004bB 923 004cB 909 004dB

10 987 003aC 1012 004bC 1007 003bC 901 004cC NP

75 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011b

1 1007 003aB 1042 003bB 1048 005bB 918 001cB NP

10 NP NP NP NP NP



















































heat treatments













rst-



- 1 )

initial 2 h 4 h 6 h



- 1 )



- 1 )

120

110

100

90

80

70

60

120

110

100

90

80

70

60

120

110

100

90

80

70

60

a

b

c

















4 Conclusions








Acknowledgments



References







neering 104 467e















Table 2




55 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1046 008aA 1080 002bcA 1076 004cA 973 004dAB 1058 004acB

10 1042 007aA 1047 002aB 1037 004aB 956 004bB 1000 005cC

65 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1030 002aB 1061 004bB 1051 004bB 923 004cB 909 004dB

10 987 003aC 1012 004bC 1007 003bC 901 004cC NP

75 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011b

1 1007 003aB 1042 003bB 1048 005bB 918 001cB NP

10 NP NP NP NP NP










































4 Conclusions








Acknowledgments



References







neering 104 467e















Table 2




55 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1046 008aA 1080 002bcA 1076 004cA 973 004dAB 1058 004acB

10 1042 007aA 1047 002aB 1037 004aB 956 004bB 1000 005cC

65 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011bA

1 1030 002aB 1061 004bB 1051 004bB 923 004cB 909 004dB

10 987 003aC 1012 004bC 1007 003bC 901 004cC NP

75 0 1057 005aA 1091 007bA 1088 002bA 990 008cA 1103 011b

1 1007 003aB 1042 003bB 1048 005bB 918 001cB NP

10 NP NP NP NP NP


























































Documents

Effect of microencapsulation on survival of Biﬁdobacterium BB-12 exposed