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Effect of microencapsulation on survival of Lactobacillus plantarum in simulatedgastrointestinal conditions, refrigeration, and yogurt

Graziela Brusch Brinques, Marco Antnio Zchia Ayub

Food Science and Technology Institute, Federal University of Rio Grande do Sul State, Av. Bento Gonalves, 9500, PO Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil

a r t i c l e i n f o

Article history:Received 5 May 2010Received in revised form 20 September2010Accepted 10 October 2010Available online 14 October 2010

Keywords:Lactobacillus plantarumMicroencapsulationViabilityProbiotics

a b s t r a c t

In the present research the survival of free and microencapsulated cells of a new strain of Lactobacillus plantarum BL011 under stress conditions was tested in sodium alginate or pectin, coated with sodiumalginate or chitosan. Results for the simulated gastrointestinal medium (SGT) showed no change in via-bility of cells in relation to the control. However, the simulated gastric medium (GM) drastically reducedthe viability under the tested conditions, with no signicant differences between free and immobilizedcells. Under refrigerated storage viability of immobilized cells were greatly enhanced compared to thefree microorganisms, and the treatments showing the lowest loss of viability were those of 4% (w/v) pec-tin, 3% (w/v) sodium alginate coated with chitosan and a mixture of 2% (w/v) sodium alginate and 2% (w/v) pectin, respectively. Loss of viability of immobilized L. plantarum in 3% alginate coated with chitosan inyogurt was of 0.55 log cycles during 38 days of storage. The results of this study suggest the efciency of immobilization techniques to increase the survival of lactobacilli in yogurt under refrigerated storage.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Lactobacillus plantarum is one of the most widely used lacticacid bacterium, showing a homofermentative metabolism, moder-ate acid tolerance, and is consideredas a GRAS (Generally Regardedas Safe) organism. Many strains of L. plantarum are marketed asprobiotics (De Vries et al., 2006). When a new strain of probioticbacterium is being tested to be used in foods, viability test to gas-trointestinal conditions and to conditions of processing and storageshould be performed ( FAO/WHO, 2001).

The benets promoted by probiotic bacteria are increasingly ex-plored in different uses in various types of foods ( Kailasapathyet al., 2008; Nazzaroet al., 2008; Prado et al., 2008; Souza and Saad,2009; Vasiljevic and Shah, 2008). However, cell viability in theseproducts is often low and the ability to survive and multiply inthe digestive tract strongly inuences the benets that probioticscan produce ( zer et al., 2009). Probiotics have to be metabolicallystable and active in the product, survive the passage of the upperdigestive tract in large numbers, and show the ability to adhereand colonize the intestine system ( Champagne et al., 2005;Mattila-Sandholm et al., 2002; Stanton et al., 2005 ).

Microencapsulation is a promising technique for bacterial cellprotection and several studies have been carried out investigatingthe protective role of this technique against adverse conditions to

which probiotics can be exposed ( Capela et al., 2006; Chen et al.,2006; Heidebach et al., 2010; Mandal et al., 2006; Michida et al.,2006; zer et al., 2008; Sheu and Marshall, 1993; Sultana et al.,2000). One advantage of microencapsulation with hydrocolloidsis that cells are entrapped within the matrix during the formationof the spheres, while in other techniques such as spray drying,freeze drying and uidized bed drying, the microorganisms arecompletely released into the product ( Krasaekoopt et al., 2003).The most widely used matrix for microencapsulation is alginate.However, alginate gels are susceptible to disintegration in the pres-ence of excess monovalent ions, Ca2+ chelating agents, and harshchemical environments, such as those of low pH ( Gserd et al.,1999). Although some works have reported that the presence of fermentable carbohydrates in the gastrointestinal medium signi-cantly enhances survival of probiotic cells to the gastric environ-ment ( Corcoran et al., 2005), so far no studies were carried outusing the technique of microencapsulation by emulsication in or-der to verify the possibility of increasing the viability of L. planta-rum towards gastric medium. Other technique investigated forincreased stability of the microcapsules made with polymers isthe application of coating with polyelectrolytes such as chitosanand polylysine ( Huguet et al., 1996; Krasaekoopt et al., 2004,2006; Mokarram et al., 2009 ). The aims of the present work wereto study the viability of free and microencapsulated L. plantarumunder simulated conditions of gastrointestinal transit, refrigeratedstorage, and in model yogurt systems in order to know whetherthese techniques could improve the availability of probiotics infoods.

0260-8774/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.jfoodeng.2010.10.006

Corresponding author. Tel.: +55 51 3308 6685; fax: +55 51 3308 7048.E-mail address: [email protected] (M.A.Z. Ayub).

Journal of Food Engineering 103 (2011) 123128

Contents lists available at ScienceDirect

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2. Materials and methods

2.1. Reagents

All reagents used in this work were of analytical grade and pur-chased from SigmaAldrich (St. Louis, USA).

2.2. Microorganism

A strain of Lactobacillus plantarum , isolated by our group fromSerrano cheese and described elsewhere ( De Souza et al., 2003),was used in this study. This strain was identied as Lactobacillus plantarum BL011 and it is kept as a certied stock at MicrobiologyCulture Collection of BiotecLab (UFRGS, Brazil). Working stocks of cultures were maintained in 20% glycerol suspension frozen at

18 C.

2.3. Microencapsulation of microorganisms

Themethoddescribedby Sheu andMarshall (1993) was adoptedfor encapsulation of L. plantarum cells. Microencapsulation wascarriedout using sodiumalginate (A; A1089.01.AF,Synth, Diadema,Brazil), low-methoxyl citric pectin (P; GENU BTM LM102-AS, PluryQumica, Diadema, Brazil), and blends of A and P (Table 1). It wasalso tested the coating of beads with sodium alginate and chitosan(448869, Chitosan low molecular weight, Aldrich, St. Louis, USA)using the methodology described by Krasaekoopt et al. (2004).The treatments used and their respective abbreviations are shownin Table 1.

2.4. Concentrated cell culture preparation

Erlenmeyer asks (1000 mL) containing 200 mL of the MRSbroth (De Man et al., 1960), were inoculated with 1.5 mL of a glyc-erol stock culture and incubated at 37 C 1 C in a rotatory shaker(MA 830, Marconi, Piracicaba, Brazil) at 180 rpm and grown tooptical density (OD, 600 nm) of 1.0. The cells were harvested bycentrifugation (Hitachi, Himac CR 21E, Tokio, Japan) at 5000 g for10 min at 4 C. Cell pellets were washed twice with 0.9% (w/v) so-dium chloride solution and were nally re-suspended in 10 mL of 0.9% (w/v) sodium chloride solution. These cell suspensions wereused as free cells or were aseptically mixed with 40 mL of polymersolution, according Table 1, and were applied to the immobiliza-tion system. The average cell concentrations of these polymer-pro-biotic mixtures were approximately 5 1012 CFU mL 1.

2.5. Preparation of microencapsulated cell

Cells were microencapsulated by mixing one part cultureconcentrated with four parts polymer solution. The treatmentsused for the immobilization of L. plantarum and their respectiveabbreviations are shown in Table 1. One part of the mixture

(50 mL) was added dropwise to ve parts of vegetable soybeanoil (Soya, Bunge, Gaspar, Brazil) (250 mL in asks of 1000 mL) con-taining Tween 80 (0.2%, w/v), and stirred for 10 min at 200 rpm bymagnetic stirring. After this, 500 mL of calcium chloride (0.05 M forALG or 0.10 M for PEC and ALPE) was quickly and gently added(20 mL s 1, Masterex Peristaltic Pump L/S, Ultrapump2, Model7520-47, Cole-Parmer Instrument Company, Illinois, USA) down

the side of the ask until the water/oil emulsion was broken. Thebeads were formed within 10 min and were collected by centrifu-gation (350 g , 10 min, 4 C). The spheres were washed twice andrecovered under the same centrifugation conditions with 0.9%(w/v) sodium chloride solution and re-suspended in 50 mL of 0.9% (w/v) sodium chloride solution for further assays. The averagecell concentration in the nal solution of the encapsulation processwas 1.5 109 CFU mL 1. The whole procedure was performedusing autoclaved (121 C, 15 min) materials and under sterile con-ditions in a horizontal laminar air-ow cabinet.

2.6. Coating with chitosan and sodium alginate

The chitosan and sodium alginate solutions were preparedaccording to Krasaekoopt et al. (2004). Briey, the beads of immo-bilized L. plantarum BL011, prepared as described in item 2.5, wereimmersed in 100 mL of chitosan solution and shaken at 100 rpm,37 C for 40 min on an orbital shaker for coating. The chitosan-coated beads were collected by centrifugation (350 g , 10 min,4 C).The pellet was washed twice with 0.9% (w/v) sodiumchloridesolution and ressuspended in 50 mL of 0.9% (w/v) sodium chloridesolution. For the sodium alginate coating, the washed immobilizedL. plantarum BL011 beads were mixed with 100 mL of a 0.17% (w/v)sodium alginate solution and placed in a rotatory shaker at100 rpm, 37 C for 20 min. The same procedures described for thecoating with chitosan were used here for beads recover.

2.7. Resistance to gastrointestinal media

2.7.1. Preparation of simulated gastric and intestinal juicesThe simulated juices were prepared according to Charteris et al.

(1998) and Michida et al. (2006). Simulated gastric juices (SGJ)were prepared by suspending pepsin (P7000, 1:10,000) in sterilesodium chloride solution (0.5%, w/v) to a nal concentration of 3 g L 1 (1038 U mL 1) and adjusting the pH to 2.0 with concen-trated HCl or sterile 0.1 mol L 1 NaOH. Simulated intestinal juices(SIJ) were prepared by suspending pancreatin USP (P-1500) in ster-ile sodium chloride solution (0.5%, w/v) to a nal concentration of 1 g L 1, with 4.5% bile salts (Oxoid, Basingstoke, UK) and adjustingthe pH to 8.0 with sterile 0.1 mol L 1 NaOH. Both solutions wereltered for sterilization through a 0.22 l m membrane.

2.7.2. Cell tolerance to gastrointestinal

The tolerance of free and immobilized cells of L. plantarum onsimulated gastric and intestinal juices was determined using the

Table 1

Treatments used in the immobilization of L. plantarum .

Trial Abbreviation

Free cells SIMOBeads prepared with sodium alginate 3% (w/v) ALGBeads prepared with citric pectin 4% (w/v) PECBeads prepared with mixture of sodium alginate 2% (w/v) and citric pectin 2% (w/v) ALPEBeads prepared with sodium alginate 3% (w/v) coating with sodium alginate solution 0.17% (w/v) ALGABeads prepared with citric pectin 4% (w/v) coating with sodium alginate solution 0.17% (w/v) PECABeads prepared with mixture of sodium alginate 2% (w/v) and citric pectin 2% (w/v) coating with sodium alginate solution 0.17% (w/v) ALPEABeads prepared with sodium alginate 3% (w/v) coating with chitosan solution 0.4% (w/v) ALGQ Beads prepared with citric pectin 4% (w/v) coating with chitosan solution 0.4% (w/v) PECQ Beads prepared with mixture of sodium alginate 2% (w/v) and citric pectin 2% (w/v) coating with solution 0.4% (w/v) ALPEQ

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adapted method described by Charteris et al. (1998). The testswere performed using a series of sterile Falcon tubes of 15 mL,one for each sampling point (see times of sampling bellow). Twodifferent conditions were tested: in the rst and second tests,0.4 mL of the suspension of either immobilized or free cells weremixed with 1.8 mL of SGJ or SIJ, gently mixed and incubated for120 min at 37 C 1 C. The control for these tests (CONT) was

done by incubating 0.4 mL of either free or immobilized cells in1.8 mL sterile sodium chloride solution (0.5 %, w/v) for 120 minat 37 C 1 C. After the addition of free cells or immobilized toSGJ and SIJ, the pH of these were corrected to 2.0 and 8.0, respec-tively, with sterile 0.1 mol L 1 NaOH or concentrated HCl. Aliquotsof 1 mL were removed at 0, 30, 60, and 120 min (for all trials) forthe determination of total viable counts.

2.8. Resistance to refrigerated storage

The viability of L. plantarum under refrigeration was evaluatedby incubating 0.4 mL (approximately 1.5 109 cells mL 1) of freeor immobilized cells suspension added of 1.8 mL of sterile sodiumchloride solution (0.5%, w/v) and kept in the refrigerator at 4 1 C.Aliquots of 1 mL were taken every other day for 38 days to deter-mine the total number of viable cells. The immobilized beads weredissolved in the appropriate buffer solution and they were used todetermine the total number of viable cells.

2.9. Production of yogurt supplemented with L. plantarumimmobilized in calcium alginate beads coated with chitosan

The model yogurt used in this work was produced as shown inFig. 1, using as starter cultures Yo-Flex YF-L812 (Christian Han-sen, Hoersholm, Denmark) composed of Streptococcus thermophilusand L. delbrueckii subsp. bulgaricus . A volume of 1.8 mL of yogurtwas placed in sterile Falcon tubes of 15 mL and added of 0.4 mL of L. plantarum immobilized in ALGQ. The tubes were stored underrefrigeration at 4 1 C for 38 days. The immobilized beads weredissolved and used to determine the total number of viable cellson selective differential medium for L. plantarum (LPSM) describedby Bujalance et al. (2006), and composed of (in g L 1): bacterialpeptone (10), beef extract (10), yeast extract (5), D-sorbitol (20),ciprooxacin (0.0004), sodium acetate (5), ammonium citrate (2),potassium phosphate (2), magnesium sulfate (0.1), manganese sul-fate (0.05), bromocresol purple (0.02) and agar (15). Ciprooxacin

was sterilized by ltration through a membrane lter of 0.22 l mbefore being added to the cooled medium. The pH of the mediumwas 6.0 0.1. When solidied, LPSM was a purple color and whenL. plantarum grows in this medium yellow color develops aroundthe colonies due to acidication.

2.10. Solubilization of polymer beads

The encapsulated cells on ALG, PEC, ALPE, ALGA, PECA, and APEA were released dissolving 1 mL of bead suspensions in 9 mL 0.1 M phosphate buffer, pH 7.5, followed by shaking for 10 minon a rotatory shaker at 37 C and 180 rpm. The chitosan coatingbeads (ALGQ, PECQ, ALPEQ) were dissolved using 1 mL of the incu-bated material added of 9 mL of sodium citrate buffer (0.1 M), pH6.0, with 10 glass beads, diameter of 0.45 mm, used as an aid inthe physical dissolution of the microcapsules, for 10 min on a rota-tory shaker at 37 C, 180 rpm. The formed solution was then usedto determine the number of viable cells.

2.11. Determination of total viable counts

Total viable counts of L. plantarum were determined by a pourplate method using MRS agar (except to yogurt trials) after serial10-fold dilutions in peptone water. Plates were incubated at37 C for 48 h.

2.12. Data analysis

ANOVA and Tukey0s mean comparison tests ( p 6 0.05) wereused to evaluate the data obtained from the test using the Statisti-ca 7.0 (Statsoft, Tulsa, USA). All experiments and analyses were runin triplicates.

3. Results and discussion

3.1. Resistance to gastrointestinal media

The results showing the in vitro viability of probiotics when ex-posed to SGJ and SIJ are presented in Figs. 2 and 3. These experi-ments aimed at evaluating their tolerance towards models of stomach pH, bile salts, and enzymes present in the upper gastroin-testinal tract ( Champagne et al., 2005; Stanton et al., 2005 ). Fig. 2shows the survival curves of free and immobilized L. plantarum

Fig. 1. Flow diagram for the model yogurt production used in this work.

Fig. 2. Variation in the number of viable cells of L. plantarum BL011 subjected to the

SIJ and the respective controls. (j ) SIMO; (d ) SIJ without immobilization; (N )control ALG; ( ) SIJ ALG; (s ) control ALPE; (e ) SIJ ALPE.

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BL011, in ALG and ALPE when they were incubated with the SIJ. SIJdid not shown any negative inuence over cell viability comparedto the controls except for the SIMO trial (95% condence level),where there was a decrease of 0.2 and 0.4 log for control and SIJ,respectively. Michida et al. (2006) found that free cells of L. planta-rum resisted to exposure to SIJ. Mandal et al. (2006) showed de-creased viability after 12 h of free cells of Lactobacillus caseiNCDC-298 from 9.45 to 7.29 log CFU mL 1 and from 9.34 to5.60 log CFU mL 1 when they were exposed to 1% and 2% bile salts,respectively. After immobilization in alginate, these authors re-ported improved viability, which was proportional to the concen-tration of alginate used. Martoni et al. (2007) veried an increasein viability of 0.91.0 log CFU mL 1 when L. plantarum 80BSH + strain was exposed to simulated intestinal conditions for a10 h incubation time. Since our results showed that SIJ did not im-paired L. plantarum BL011 viability, this test was not used for theother immobilization techniques.

Fig. 3 shows the variation in the number of viable cells of L. plantarum BL011 subjected to the SGJ. Exposure to SGJ resultedin a drastic decrease in the total number of survivors and therewas no signicant difference between treatments at 95% con-dence. The encapsulation on the tested materials was not effectivein protecting the microorganisms. Sultana et al. (2000) found a sig-nicant drop in viability when probiotics were exposed to pH 2,and immobilization failed to protect the microorganisms fromthese adverse conditions. Gbassi et al. (2009) reported that, after

90 min of incubation, three different strains of L. plantarum encap-sulated in calcium alginate showed a total loss of viability. How-ever, when the same authors used alginate matrix coating withwhey protein, there was an increase in survival, demonstratingthat the technique was effective for the protection of the probioticstrains. Mandal et al. (2006) observed large decrease in the viabil-ity of free cells when subjected to pH 1.5, with only a small in-crease in viability after immobilization. Krasaekoopt et al. (2004)found that encapsulation with alginate coated with chitosan wasthe best treatment to protect studied Lactobacillus for all conditionstested, but no treatment promoted the protection of B. bidum toacidic conditions. Mokarram et al. (2009) showed that L. acidophi-lus and L. rhamnosus exposed to SGJ had higher viability whenencapsulated in calcium alginate with double coating sodium algi-

nate, suggesting that there were a reduction of pore size and distri-bution of gastric juice in the spheres, thus hindering the interaction

between cells with the gastric juice. Martoni et al. (2007) observedsmall losses of cell viability at pH 2.5 and 3.0, with 1.09 and0.6 log CFU mL 1 reductions, respectively, when L. plantarum 80BSH + was exposed to simulated stomach conditions for 4 h. Thesame authors reported a linear decrease in viability of cells whenexposed at pH 2.0, with 8.98 log CFU mL 1 reduction after 4 h,while at pH 1.5, after 30 min of exposure, cells completely lost

their viability.It is important to note that the juices used in simulated testsreported in the literature differ widely among them. For the simu-lated gastric environment, most authors only use sodium chloridesolutions with adjusted pH to the desired value ( Lee and Heo,2000), with few reports with the addition of enzymes ( Charteriset al., 1998; Michida et al., 2006). The same happens for simulatedintestinal juice, with many studies showing the use of sodiumchloride solution with different concentrations of bile salts ( Man-dal et al., 2006), but seldom with the addition of pancreatin tothese solutions ( Michida et al., 2006).

3.2. Resistance to refrigerated storage

Experiments were performed in order to evaluate the efciencyof immobilization to reduce the losses in viability of probiotics un-der refrigeration and the results showing the survival of L. planta-rum BL011 under 38 days of incubation at 4 oC are presented inTable 2. Results show that at the end of this time, the immobilizedcells in PEC and ALGQ had the lowest loss of viability. Even withthe release of some cells during storage due to the collapse of beads, treatment with PEC showed better viability under refriger-ation. Thus, improving the stability of the beads during storagecould reduce the loss of cells to the medium and positively affectviability. The ALGQ system showed good stability and there waslittle loss of viability of L. plantarum BL011 during the storage per-iod. Krasaekoopt et al. (2003) commented that special treatments,such as coating of beads, are used to improve the properties of it. In

these cases, the cross linking with cationic polymers, coating withother polymers, mixed with starch and incorporation of additivescould improve the stability of the spheres.

One of the properties for a given microorganism to be consid-ered probiotic is its capacity to survive storage as a formulatedproduct ( FAO/WHO, 2001; Kosin and Rakshit, 2006). In general,fermented products containing added probiotics should be storedunder refrigeration at 4 C. Our results, therefore, suggest thatimmobilization is promising technology to improve viability of probiotics in formulated products.

Fig. 3. Change in the number of viable cells of L. plantarum BL011 subjected to SGJbetween 0 and 120 min. (white bar) initial number of cells; (black bars) nalnumber of cells.

Table 2

Reduction of the number of viablecells of L. plantarum during 38 daysof refrigerated storage at 4 C.

Trial Log10 (N 0/N )

PEC 1.95 0.08ALGQ 2.17 0.49ALPE 2.96 0.20 ,bALPEQ 3.62 0.22b,cPECQ 3.64 0.10b,cALPEA 3.85 0.10b,cALG 4.35 0.33c,dALGA 5.28 0.92dPECA 5.40 0.71dSIMO 6.73 0.13e

N 0 and N mean CFU mL 1 on rstand 38th day, respectively.a,b,c,d,eEqual letters indicates no

statistical difference at 95% of condence.


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3.3. Resistance of L. plantarum BL011 immobilized in calcium alginatebeads coated with chitosan in yogurt

ALGQ immobilization was chosen to verify the viability of L. plantarum BL011 in yogurt under refrigeration and results canbe seen in Fig. 4. Each curve in Fig. 4 represents the change inthe population of L. plantarum BL011 with the storage time relatedto the initial population. Results show that ALGQ in yogurt pre-sented good cell viabilities, with losses of only 0.55 log cycles,compared to 6.73 log cycles for the free cells and 2.17 log cyclesfor ALGQ, both in sodium chloride solution. Cell count of the prod-uct remained above 10 9 CFU mL 1, indicating that this productwould conform to the recommended minimum requirements of

several countries that dairy products should contain at least 107109 CFU of probiotic cultures per product portion for packing-

claiming of probiotic qualities ( ANVISA, 2008; Pagano, 1998; Stan-ton et al., 2005). Although the amount of spheres used in this workwere well above the acceptable quantities for real yogurt formula-tions, these results suggest the efciency of the technique in orderto increase survival of lactobacilli in refrigerated yogurt. Moreover,the Lactobacillus plantarum BL011 strain compared very well withother probiotic bacteria reported in the literature concerning theirviability under refrigeration in yogurt ( Kailasapathy, 2006; Picotand Lacroix, 2004). Adhikari et al. (2000) found that non-encapsu-lated Bidobacterium longum in yogurt showed reductions of viabil-ity of 7870% for the two strains tested when stored for 30 daysunder refrigeration, while for the encapsulated cells with c -carra-

geenan showed no difference in bacterial population during refrig-erated storage. Finally, Krasaekoopt et al. (2006) found an increaseof 1 log in the viability of encapsulated alginate chitosan-coatedcells of L. acidophilus 547 and L. casei 01 compared against free cellsin yogurt under refrigeration at 4 C for 28 days.

4. Conclusions

The viability of both free and immobilized L. plantarum BL011was not affected by the SIJ. However, the SGJ drastically reducesthe viability of L. plantarum under all tested conditions, and whileresistance to simulated gastric juice was low, a viable populationremained in the samples, showing a good resistance to the acidconditions tested. The microencapsulation in ALGQ was effective

in maintaining the stability of the probiotic under storage at refrig-eration temperature and when this method of encapsulation was

used in yogurt, the viability was approximately four times higherwhen compared to cells kept in saline suspension. This work dem-onstrated the potential for using the isolated strain of L. plantarumBL011 since it showed to be stable and keep viability in presence of the simulated intestinal juice and storage under refrigeration.However, it is important to note that we tested high concentrationsof immobilized cells in the model yogurt and lower concentrations

should be tested, specially considering the sensorial aspects of theproduct. These are two interesting characteristics that must be metfor the commercial implementation of probiotics in dairy and otherfood products.

Acknowledgements

The authors wish to thank CNPq (Brazilian Bureau for Science &Technology Research) for the nancial support of this work.

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Fig. 4. Viability of L. plantarum BL011 under refrigerated storage (4 1 C). N0 is theinitial number of cells and N is the number of for each time t (CFU mL 1). -d - freecells; -j - ALGQ in sodium chloride solution (0.5%, w/v); -N - ALGQ in yogurt.

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http://www.anvisa.gov.br/alimentos/comissoes/tecno_lista_alega.htmhttp://www.anvisa.gov.br/alimentos/comissoes/tecno_lista_alega.htmhttp://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdfhttp://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdfhttp://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdfhttp://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdfhttp://www.anvisa.gov.br/alimentos/comissoes/tecno_lista_alega.htmhttp://www.anvisa.gov.br/alimentos/comissoes/tecno_lista_alega.htm

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