Effect of addition of inulin and fenugreek on the survival of microencapsulated Enterococcus durans 39C in alginate-psyllium polymeric blends in simulated digestive system and yogurt

Effect of addition of inulin and fenugreek on the survival of microencapsulated Enterococcus durans 39C in alginate-psyllium polymeric blends in simulated digestive system and yogurt

ARTICLEINFO

Article history∶

Received 26 January 2015

Received in revised form

2 April 2015

Accepted 6 April 2015

Available online 15 April 2015

∶

Microencapsulation

Psyllium

Fenugreek

Probiotic

Enterococcus durans 39C

The use of biopolymers for probiotic microencapsulation has been investigated in this paper.The objectives are to enhance its survival rate,colonic release,and stability of these probiotic cultures in digestive condition during storage time.Nine types of biopolymers (alginate-psyllium)blend with different concentration of prebiotic;(inulin or fenugreek) were used as candidate for microencapsulation matrix.One strain of probiotic candidates, namely;Enterococcus durans 39C was used in this study.The microencapsulation of this strain with the respective polymer blend was performed by using a simple extrusion method.All blend of formulations have recorded high encapsulation ef fi ciency at value＞98%.The survival rate of viable probiotic cells under simulated digestive conditions was also high with value above 47%as compared to non-microencapsulated cells.These nine gel formulations also displayed the high survival rate of viable probiotic cells during storage time(28 d).Their release occurred after 2 h in colonic condition and sustained until 12th h of incubation period.An increase of prebiotic effect value added was observed in incorporated inulin and fenugreek formulations.In short,this study revealed that a newherbal-based psyllium and fenugreek polymers have suitable potential as a matrix for probiotic microencapsulation.

?2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1. Introduction

Probiotic is a microorganism that provides bene fi cial health effects.The microorganism must exhibit several characteristics such as resistance against the gastric and intestinal juices and tolerance toward the digestive enzymes degradation[1]. Such challenges can be addressed by microencapsulating the probiotic bacteria within a protective matrix material.The microencapsulation matrix functions not only as a protection against harsh gastrointestinal conditions but also increases the stability and viability of live probiotic culture at various heat/moisture conditions during processing and storage[2,3].

Many polymeric materials can be used as a microencapsulation matrix for probiotic formulation,including alginate, starch,xanthan gum,fat,gelatin,and glycerides derivatives. Among these coating materials,alginate has been recognized as the most commonly used polymer for probiotic microencapsulation in food products,becauseitisnon-toxic, biocompatible,low cost,and easy to apply.Furthermore, alginate has been approved as safe food additive.Nevertheless,alginate is susceptible to adverse chemical conditions such as very low acidic pH and chelating agents,which are commonly excreted during probiotic bacteria fermentation. Additionally,the presence of cations from probiotic fermentation,such as magnesium,sodium,and calcium,causes the destabilization of alginate gelling activity and poor performance as a microencapsulation matrix[4,5].

Polycationic materials such as gelatin,chitosan,&poly-L-lysine can form ion-exchange interaction with the negatively charged alginate structure to improve the stability of the microencapsulating matrix[6,7].The blend of alginate with polycationic biopolymers,particularly for particular polymers with prebiotic functionalities,can be a value added to the probiotic itself.Moreover,prebiotic characteristic is often found in many herbal-based polymers such as psyllium and fenugreek.

Fenugreek gum is a biopolymer extracted from fenugreek seeds.The fenugreek polymer is composed of D-mannan (backbone)andD-galactopyranosyl groups(side chains).The fenugreek gum exhibits several prebiotic effects and has been utilized in animal husbandry and nutritional supplements[8]. In addition,its stability and emulsifying activity[9]make this gel a suitable stabilizer for food industry.Meanwhile,psyllium is extracted from Plantago genus and is a cationic herbal gel in its physiological condition.The gel structure of psyllium is arabinoxylan,which consists of xylose(backbone)and arabinose(side chains)[10,11].Psyllium,as a medicinally active natural polysaccharide,if suitably tailored to prepare the hydrogels that have the potential to act as novel drug delivery devices.Therefore,the present study is an attempt,to synthesize psyllium and poly(AAm)based hydrogels by using N, N-MBAAm as cross linker and ammonium persulfate(APS)as initiator[12].

The therapeutic effect of psyllium is known in many cultures as treatment and risk reduction in several gut disorders such as constipation,diarrhea,ulcerative colitis,chronic kidney disease,and irritable bowel syndrome[13,14].Likewise, psyllium can be considered superior candidate for the probiotic microencapsulation matrix because of its bene fi ciary effects on both probiotic culture and human gastrointestinal tract health.

In this study,we investigate the potential of alginatepsyllium blend with prebiotic(inulin and fenugreek)for their suitability as microencapsulation matrix for probiotic bacteria.Different concentrations of inulin and fenugreek were blended with alginate-psyllium via a simpleextrusionmethod to microencapsulate Enterococcus durans 39C cultures.Extrusion method in the small scale of production,in comparison with other methods,is low cost,simple,and easy with the high rates of cell viability.Moreover,by using suitable supporting materials,the small size of beads for being added to the food products,can be produced[2,15].Hence,in this study the morphological characteristics,encapsulation ef fi ciencies, survivalrates,andviabilitiesofthe E.durans39Cbacteriaupon microencapsulation with extrusion method at harsh gastrointestinal conditions(stomach acid&alkaline bile)during storage were investigated.

2. Materials and methods

2.1. Materials

E.durans 39C were isolated and identi fi ed from the traditional yogurt(West region of Iran).Fenugreek seeds and psyllium seed husk were purchased from herb market in Tabriz(Tabriz, Iran)and sodium alginate from Sigma-Aldrich(Germany). MRS broth,MRS agar,calcium chloride,hydrochloric acid, sodium hydroxide,Acrylamide(AAm),and sodium hydrogen phosphate were purchased from Merck(Germany).Ammonium persulphate(APS),N,N,-methylene bis acrylamide(NNMBAAm),Inulin and oxgall were obtained from Sigma--Aldrich(Germany).

2.2. Isolation,molecular identi fi cation and characterization of E.durans 39C

E.durans 39C was isolated from 60 samples of traditional yogurt that were randomly collected from the retailers in different parts of Kermanshah province in Iran.This probiotic species was isolated and ampli fi ed through anaerobically growth of MRS broth medium for 24 h at 37°C and spread onMRS agar media similar to mentioned condition[16].The total genomic DNA was extracted by the method described by Leenhouts et al.(1990)with some modi fi cations[17].The 16S rDNA gene ampli fi cation was carried by using the universal bacterial primer pairs namely,F:5′AGAGTTTGATCMTGGCTCAG-3′and R:5′-TACCTTGTTAGGACTTCACC-3′.The PCR program cycles were as follows:denaturation at 95°C for 4 min,32 cycle of:94°C,1 min,58°C,1 min,72°C for 95 s,and the fi nal extension was performed for 5 min in 72°C.The puri fi ed DNAs were sequenced by the Korean sequencing company,Macrogene.The sequencing results were blasted with the deposited sequences in the NCBI and GenBank site (http://blast.ncbi.nlm.nih.gov/Blast.cgi)to identify the isolated bacteria cells.

The agar diffusion well method was utilized to determine the antibacterial activity of the bacterial isolate against some clinically important human pathogens such as Escherichia coli (PTCC 1276),native isolate of E.coli(026),Salmonella typhimurium(ATCC 14028),Staphylococcus aureus(ATCC 25923),Bacillus cereus subsp.kenyae(PTCC 1539),Listeria monocytogenes(PTCC 1163),Klebsiella pneumoniae(PTCC 1053),Shigella fl exneri(PTCC 1234),Pseudomonas aeruginosa(PTCC 1181),Candida albicans (PTCC 5027),Serratia marcesens(PTCC 1187),Enterococcus faecalis(PTCC 1394),Staphylococcus saprophyticus subsp.saprophyticus(PTCC 1440)and Streptococcus mutans(PTCC 1683).To determine the antibiotic susceptibility,the disc diffusion method against some clinically important antibiotics such as chloramphenicol(30 μg),vancomycin(30 μg),tetracycline (30 μg),erythromycin(15 μg),Ampicillin(10 μg),gentamycin (10 μg),clindamycin(2 μg),sulfamethoxazol(25 μg)and penicillin(10,000 units)was exploited[18].

To evaluate the low pH and high bile salt tolerance of isolated strain,each cell culture medium was centrifuged at 4000×g for 5 min.The supernatants were removed,and the cell plates were re-suspended for 2 h in 15 ml of low pH solution(pH 1.8 at 37°C)and high bile salt solution(0.5%w/v oxgall,pH 6.8,and37°C)by a gentle agitation[19].The survival rate was calculated using the following equation:survival rate (%)=(log cfu N1/log cfu N0)×100%,where N1 corresponds to the total clones treated with extra bile salts or acids and N0 corresponds to the total clones before they were incubated under harsh conditions[20].

2.3. Preparation of probiotic cells

The isolated E.durans 39C cultures were ampli fi ed by anaerobic growth of 100 μL respective stock cultures in 15 ml MRS medium for 24 h at 37°C.The cells were harvested by centrifugation(10 min,1200×g)at 4°C,washed and resuspended in phosphate buffer(pH 7.2).The cells were counted to a desirable amount three times in MRS agar using pour plate method,prior to the microencapsulation step.The equal volume of the same viable cell population was divided and used in the microencapsulation step by different biopolymers blend.

2.4. Extraction of fenugreek gel

To extract the fenugreek gel,Goel(2009)method was utilized with some modi fi cations.Brie fl y,20 g of grinded fenugreek seeds were soaked in 50 ml of distilled water(pH 7)at 65°C and stirred for 2 h.A homogenous gel was centrifuged(20 min, 14000×g)to separate the gel phase.The gel was washed twice with distilled water and ready to be applied in microencapsulation matrix[21].

2.5. Extraction of psyllium gel

To extract the psyllium gel,a method previously described by Guo et al.(2008)was adopted with some modi fi cations.Brie fl y, 20 g of psyllium husk was dispersed in 200 ml water at 80°C and mixed for 12 h until pysllium gel was homogenous.Then, it was centrifuged for 20 min at 14000×g to separate the gel phases.The separated gel was dissolved in 50 ml NaOH solution(2 M)and was stirred for 2 h at 37°C followed by centrifugation(15 min,14000×g).The alkaline phase was neutralized by using HCl(2 M)and followed by centrifuging (15 min,14000×g),a yellowish gel was separated and washed twice with distilled water prior to its usage as microencapsulation matrix[11].

2.6. Synthesis of Psy-cl-poly(AAm)

Reaction wascarriedoutwith1gofpsyllium husk, 1.095×102mol/l of APS,known concentration of monomer and cross linker in the aqueous reaction system at 65°C temperature for 2 h.Polymers were stirred for 2 h in distilled water and for 2 h in ethanol to remove the soluble fraction and then were dried in air oven at 40°C.Different polymeric networks[psy-cl-poly(AAm)]were synthesized by varying[AAm] (from 1.41×101mol/l to 7.03×101mol/l)and by varying[N,NMBAAm](from 6.45×103mol/l to 32.40×103mol/l)[22].

2.7. Microencapsulation of probiotic and prebiotics using extrusion technique

The extrusion method was utilized for microencapsulation of E.durans 39C using alginate-psyllium blend with various prebiotic(inulin and fenugreek)concentrations.Alginateencapsulated cells(ALG)andun-microencapsulated bacteria suspended in distilled water were used as the control.The sodium alginate,psyllium,fenugreek gels and inulin were autoclaved(121°C for 15 min)prior to the microencapsulation procedure.The bacterial cultures(10%(w/v))were suspended in 5 ml of 0,0.5,1.0,1.5 and 2%inulin(Sigma-Aldrich,Germany))or fenugreek(Tabriz,Iran)solutions and then mixed with 10 ml of 1.5%(w/v)sodium alginate gel(Sigma-Aldrich, Germany)and 10 ml of 0.5%(w/v)psyllium gel(Tabriz,Iran) solutions.The fi nal prebiotics concentrations were respectively 0,0.1,0.2,0.3,and 0.4%.

10 ml of these solutions were mixed and stirred for 30 min to allow homogeneous mixture.These homogeneous solutions were extruded through a 21-gauge nozzle in 100 ml sterile CaCl2solution(0.5 M)where microencapsulated beads were created.The beads were isolated by fi ltering(using Whatman paper No.1)and washing twice with sterile water before using them in the subsequent experiments and then stored in peptone solution(0.1%(w/v))at 4°C.

2.8. Morphological analysis of the microencapsulated probiotic beadsThe topographicalmorphologicalproperty of microencapsulated beads was observed by placing a sample on a microscope slide and docked on a fl uorescent microscope (Olympus BX61,Japan),which is equipped with U-MWU2 fl uorescence fi lter(excitation fi lter BP 330-385,dichromatic mirror DM 400,emission fi lter LP 420).The average size of the beads was estimated from the mean diameter of 50 beads obtained from each of the gel microcapsules.

2.9. Moisture content and water activity of microencapsulated beads

The moisture content of powdered microencapsulated beads was determined by drying the sample in an oven at 105°C for 12 h.Meanwhile,the water activity of beads was evaluated using a water activity meter(Dewpoint,USA)at maintained temperature(24±0.5°C)[23].

2.10. Encapsulation ef fi ciency(EE)

To determine the encapsulation ef fi ciency,50 mg of each microencapsulated beads was disintegrated in 10 ml phosphate buffer(pH 7.2)at 37°C for 30 min and subsequently the entrapped viable bacteria were counted by the pour plate technique in MRS agar.In pour plate method,the probiotic samples were serially diluted and pour-plated on MRS agar and then incubated anaerobically at 37°C for 24 h.Counts were expressed as number of CFU per gram of product.The encapsulation ef fi ciency was calculated by the following equation:

Where N is the number of entrapped viable bacteria cells and N0 displays the free viable bacteria cells before microencapsulation.The data were expressed as the mean of three counts±standard error[19].

2.11. Survival of microencapsulated cells after incubation in simulated digestive system

To evaluate the conservation ef fi ciencyofmicroencapsulation as well as the viability of microencapsulated bacteria in simulated digestive system,100 mg ofeach microencapsulated beads was separately mixed and incubated for 2 h in 20ml of simulated gastricjuices(pH 1.8 at 37°C)for 0,30, 60,90 and 120 min followed by a placement in simulated intestinal juice containing 0.5%w/v oxgall(pH 6.8 at 37°C for 120 min)with gentle agitation(100 rpm).These treated beads were disintegrated in 10 ml phosphate buffer(pH 7.2)and the viablebacteriawerecounted throughthepourplate technique in MRS agar.The following equation was applied to calculate the survival percentage of treated microencapsulated cells in simulated digestive system:

Survival rate(%)=(log CFU/g beads after treatment/log CFU/g beads before treatment)×100.

Where CFU displays number of colony forming unit on the pour plate agar[19].

2.12. Storage stability of microencapsulated probiotic in yogurt

The storage stability was performed according to a modi fi ed method described by Shi et al.(2013).The stability of unmicroencapsulatedandmicroencapsulatedbacteriawas assessed during 4 weeks storage in yogurt at 4°C.The viability of cells in seven different storage times(1,3,5,7,14,21 and 28 d)wasmeasured.Duringstoragetime,0.5gmicroencapsulated cells at the room temperature by gentle shaking (100 rpm)was dissolved in 5 ml sodium citrate solution (50 mM)with pH 7.5.The released and un-microencapsulated probiotic cells were serially diluted 10 times by using saline solution,and then,50 μl of aliquots was placed on the MRS agar for 24 h anaerobic growth(37°C).The viable(%)rates of probiotic cells were calculated by utilizing the pour plate method in MRS agar[24].Meanwhile,acidity of yogurt containingfreeandmicroencapsulatedprobioticcellswas measured during storage time.

2.13. Release of microencapsulated probiotic

To assess the rate of microencapsulated cell release from beads,the method was previously described by Mandal et al. (2006)was used.Brie fl y,one gram of each bead was incubated in 50 ml of simulated intestine fl uid(SIF)containing 0.1 M monobasic potassium phosphate(this simulates a colonic solution)at condition of pH 7.4,temperature 37°C and gently agitation(100 rpm).The samples at different time intervals were taken and the units of the released bacteria were counted by the pour plate method[25].

2.14. Statistical analyses

All experiments were designed based on completely random design with three replications for each treatment group.The data were analyzed using ANOVA and Duncan test methods, with the signi fi cant differences were considered for means with P＜0.05.Meanwhile,the SPSS statistics19 software was utilized in the data analysis.

3. Results and discussion

3.1. Molecular identi fi cation and characterization

The PCR-ampli fi ed 1500 bp fragment of 16S rDNA gene of this isolate was sequenced and blasted with the deposited sequences in GenBank.Isolate with 99-100%homology was identi fi ed as E.durans 39C by considering the threshold values of taxonomical studies(97%)[26].This strain was isolated from traditional yogurt.According to our results and FAO/ WHO guidelines,identi fi cation of Enterococcus strains by sequencing of 16S rDNA can be considered as an accessible and suitable technique[27].The threshold value for taxonomical studies is around 97%,hence,16S rDNA sequencingwith 99-100%homology was performed for phylogenetic clustering as a valid and accurate technique[26].

Probiotics must have some characteristics to be effective and to improve the host health such as resistance to gastrointestinal acid and bile,susceptibility against the antibiotics, and showing the high anti-microbial activity.Based on scienti fi c agreements,the characterization and assessment of probiotics properties must be performed by standard in vitro experiments[28].E.durans 39C displayed a favorable antipathogen activity and acceptable antibiotic susceptibility.E. durans 39C inhibited the growth of 7 indicator pathogens including E.coli(PTCC 1276),S.aureus(ATCC 25923),K.pneumoniae(PTCC 1053),S. fl exneri(PTCC 1234),P.aeruginosa(PTCC 1181),S.marcesens(PTCC 1187),and S.mutans(PTCC 1683). Moreover,E.durans 39C was sensitive or semi-sensitive to all examined antibiotics.But its tolerance to low pH(32%)and high bile salt concentrations(47%)were weak,hence,microencapsulation by different biopolymer matrices was performed to compensate for this weakness.

3.2. Morphological analysis of microencapsulated probiotic beads

Nine types of gel formulations(Table 1)were designed to improve encapsulation ef fi ciency,gastrointestinal survival, colonic release rates,and storage stability of E.durans 39C. Meanwhile,alginate-encapsulated cells(2%(w/v))were used as control.The beads harvested from the microencapsulation experiment,by extrusion step,were observed under a light microscope.The morphologicalanalysis,revealed that alginate-psyllium blends with various inulin and fenugreek concentrations show a spherical or elliptical shape(Fig.1) with polymer act as protective layers for the bacteria(Fig.1C). Moreover,in microencapsulation blends,prebiotic particles were integrated in polymer network(Fig.1D).Meanwhile,the microscopy images by optical microscope demonstrated that in all these nine gel formulations,prepared beads entrapped the bacterial cells(Fig.1E).

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The average diameters(based on 50 beads)for alginate, alginate-psyllium,alginate-psyllium blend with inulin,and alginate-psyllium blend with fenugreek were 830-1100 μm, 240-330 μm,210-380 μm and 380-780 μm,respectively(Table 1).The mean diameters of beads containing alginate-psyllium or alginate-psyllium blend with inulin(F1-F5)were signi ficantly(P＜0.05)smaller than beads containing alginatepsyllium blend with fenugreek(F6-F9).In alginate-psyllium blend with fenugreek,the mean diameters of beads containing lower concentration of fenugreek(F6 and F7)were significantly smaller than high concentration ones such as F8 and F9(P＜0.05)(Table 1).

In this study,the different concentrations(1-2%)of prebiotics(inulin and fenugreek)and alginate-psyllium blend were investigated[29].According to the previous researchers (Lot fi pour et al.(2012)and Chandramouli et al.(2004)),low concentrations(less than 1%(w/v))of alginate used as the supporting gel or their mixture with other gels resulted in low viscosity and crosslinking sites did not create a spherical and an uniform encapsulated beads shape.Whereas,the extrusion of alginate or their mixture with other gels through a syringe at the high concentrations(more than 2%(w/v))was dif fi cult due to high viscosity[19,30].Hence,the present study selected 1.5%(w/v)sodium alginate+0.5%(w/v)psyllium gels as the optimum concentrations of biopolymer matrices according to the formulations previously designed by Krasaekoopt and Watcharapoka,(2014).

From the morphological analysis,alginate-psyllium blends illustrated spherical or elliptical shape beads.The similar beads with two protective layers were observed by Lot fi pour et al.(2012).The spherical or elliptical shape makes consumption,industrial production,and packaging of beads easier[31,32].The beads from alginate-psyllium+fenugreek blend exhibited elliptical shape.The similar results were reported in alginate[33]and collagen-alginate[34].

The small sizes of beads in alginate-psyllium and alginatepsyllium+inulinformulations,do not change the texture and structure of food products and can easily be prescribed[35]. On the other hand,according to one research by Sohail et al. (2011),the smaller sizes of alginate beads(10-40 μm)were reported[2].These high variations in sizes of beads could be due to different polymer concentration and composition[36]. Thehigh mean diametersizesofbeadsin alginatepsyllium+fenugreek formulations can change the products' structure in texture sensitive dairy products such as cream [37]but there is no limitation for pharmaceutical applications in oral delivery.Adding the higher concentration of fenugreek into alginate-psyllium gel yielded the adherent mixtures with the high viscosity.Since fenugreek polymer consists of D-mannan chains withD-galactopyranoyl side-chains structure,can anticipate that it exhibits higher viscosity value as compared to psyllium(with a polymer made-up of xylosechains with arabinose structure).Thus,a possible explanation for successful larger sizes of bead with uniform elliptical shapes in alginate-psyllium+fenugreek than those of alginate-psyllium+inulin formulations.These results is consistent with other researches that in the extrusion method, decrease in the viscosity of supporting gels led to the preparation of smaller beads[19,38].

3.3. Moisture content and water activity of microencapsulated beads

The physicochemical characteristics(moisture content and water activity)of microencapsulated beads prepared by various formulations are discussed in this section.The moisture content of all prepared formulations was lower than 3.28%(w/w).There were no signi fi cant differences for moisture content of these nine gel formulations and control(ALG). The water activity values of alginate-psyllium+fenugreek blends(F6,F7,F8 and F9)were signi fi cantly(P＜0.05)lower thanother formulations.Ontheother hand,alginate-psyllium blend(F1)and control(ALG)showed the highest water activity value(P＜0.05)than other blends(Table 1).

Eratte et al.(2015)and Gardiner et al.(2000)observed the low content of moisture and water activity was same as our results during microencapsulation of probiotics.It was reported that low residual water contents and water activity can improve the storability and stability of powdered-beads containing probiotic bacteria[23,39].

3.4. Encapsulation ef fi ciency(EE)of microencapsulated probiotic beads

The bacterial counts for E.durans 39C before microencapsulation and after disintegrating of microcapsules in the phosphate buffer(pH 6.8)was used to calculate the encapsulation ef fi ciency.Table 1 represents encapsulation ef fi ciency of all beads from alginate(control),alginate-psyllium,alginatepsyllium+inulin,and alginate-psyllium+fenugreek blends. The results indicated successful entrapment of viable bacteria cells(＞98%)in all the prepared microcapsules formulations. According to results,there were no signi fi cant differences for encapsulation ef fi ciency of these nine gel formulations and control.

Because of high encapsulation ef fi ciency(＞98%)observed in our results,the ef fi cient viable probiotic cells(1.5×108CFU/ g)can bereleased.Thesameratesofencapsulation ef fi ciencies(close to 100%)via the extrusion method were reported by other researchers[37,40].According to our results, the encapsulation ef fi ciency was formulation-independent, while some references reported that polymer composition and concentration could affect the encapsulation ef fi ciency [41,42].

3.5. Survival of microencapsulated cells in simulated digestive system

As shown in Table 2 and Fig.2,the un-microencapsulated E. durans 39C was very sensitive to the simulated digestive condition and their viability was lost dramatically.The initial cell count for untreated E.durans 39C before low pH and bile salt treatment was 9.71±0.03 log CFU/g that declined to 3.12±0.03 log CFU/g after incubation and the survivalrate was around 32%.

As predicted,all the microencapsulated formulations showed signi fi cantly high survival rate after exposure to acid and bile condition(P＜0.05),but the best results were observed for microencapsulated cells with F9,F8 and F5(Table 2).All these nine gel formulations had signi fi cantly higher survival rates than alginate-encapsulated beads(control)after exposure to simulated digestive condition(P＜0.05).The survival rates for E.durans 39C microencapsulated in alginate-psyllium blend with 2%fenugreek,1.5%fenugreek,and 1.5%inulin were 79%,74%and 73%respectively.The survival rate for alginate-psyllium blend with 2%fenugreek(F9)was signi ficantly(P＜0.05)higher than other formulations.After 2 h incubation in simulated digestive condition,F9 gel formulation showed 1.96 log decrease in the bacterial CFU counts while with other formulations,there was a steady decrease around 2.48-6.59 log in the cell CFU counts were absorbed(Fig.2).

After incorporation of inulin or fenugreek with alginatepsyllium blend around 19-32%of increase in the survival rates was observed.On the other hand,by increase in concentration of prebiotic from 0.5 to 2%,a dramatically increase in the cell survival rates was observed which probably was duetoprotection abilityofprebiotics.Meanwhile,the improving effects of fenugreek in simulated digestive condition(21-32%)were higher than inulin(19-26%).The higher protection ability for fenugreek can be explained due to denser membrane of beads formed by the strong structure of fenugreek.

Gelation and crosslinking of alginate molecules mainly resulted from binding of consecutive blocks of guluronic acid on individual or different alginate molecules induced by the divalent cations except magnesium.The chemical composition andthe MWof alginate affectedthe acid permeabilityand the stability of the alginate/biopolymer membrane.The complex with the fl exible alginate polymers complemented a fragility of the synthesized blend.The acid permeability of the alginate blended with biopolymers strongly depends on the molar ratio of precursors,the sequence of addition and the contact time of each precursor[43].

3.6. Storage stability of microencapsulated probiotic in yogurt

Microencapsulated beads(10%)were added into yogurt on the day of their preparation.The probiotics in yogurt were enumerated periodically after 1 d in the cold room until 4 weeks.Log CFU/g for free and microencapsulated E.durans 39C during the storage time in yogurt is shown in Fig.3.The free probiotic cells displayed a dramatic decrease in their cell viability.The cell viability for free E.durans 39C dropped from 9.52 to 2.83 log CFU/g.The highest rates of decrease were found in the fi rst week while in other three weeks a decrease with the low slope was observed.This probably was due to temperature shock in the fi rst week and the subsequent adaptation process.

Results indicated that microencapsulated E.durans 39C in these nine gel formulations and alginate(control)displayed signi fi cantly high viability at the storage time(P＜0.05).The alginate(ALG)and alginate-psyllium gel formulations(F1)had a moderate protection with around 0.06 log decrease in CFU/g but the excellent viability of microencapsulated cells(＞105%) was observed with other formulations.Meanwhile,the gel formulations with the high concentration of prebiotics(F4,F5, F8 and F9)had a signi fi can(P＜0.05)higher protection ability compared to the lower concentration of prebiotics(F2,F3,F6 and F7).Moreover,addition of inulin and fenugreek slightly increased the acidity of yogurt approximately 12-88%(Fig.4).

The probiotic carrier products such as yoghurt usually are stored at the fridge temperature for one month;hence,the stability experimentswere carriedoutinmentioned conditions [44,45].Un-microencapsulated E.durans 39C cells displayed a dramatic decrease in their cell viability during 28 d storage at 4°C.The same result was found by Shi et al.(2013)where the cell viability of free Lactobacillus bulgaricus after one month storage dropped from10 to 2.3 log CFU/g.Results indicated that microencapsulated cells in these nine gel formulations displayed signi fi cantly high viability at the storage time(P＜0.05). The excellent protection ability for high concentration of inulin and fenugreek can be explained due to growth stimulation activity and denser membrane of beads formed by the strong structure of prebiotics.In this study,the higher concentration of prebiotics(＞2%)could form denser membranes and resulted in better protection,but extrusion of microencapsulationblend with high concentration through the gage nozzles was dif fi cult and decreased the encapsulation ef fi ciency.

3.7. Release of microencapsulated probiotic

The time dependent release of microencapsulated E.durans 39C in the simulated colonic solution and their comparison with the free cells and alginate-encapsulated cells(control) are showed in Fig.5.The initial count of probiotic cells used for release test was 1×107CFU/g.All microencapsulated E. durans 39C with alginate-psyllium blend with fenugreek (F6-F9)could be released partially(25-38%)from the beads after 1 h.But,the complete release was observed after 2 h.Informulations with the low concentration of inulin(F1 and F2), more than 97%of probiotic cells were released after 1 h,while the complete release was observed after 2 h.On the other hand,the high concentration of inulin(≥1.0%)(F3-F5) signi fi cantly reduced the release rate of probiotic cells(0.6-1 log CFU/g)in the fi rst h of release,but the complete release was observed after 2 h.

After complete releasing,log CFU/g for released E.durans 39C in alginate(ALG)and alginate-psyllium formulation(F1), were almost stable ranged from 7 to 7.3 and at P＞0.05 level,no signi fi cant changes were observed.On the other hand,after complete release,remarkable and increasing rates of release for E.durans 39C in alginate-psyllium blend with prebiotics were observed.The log CFU/g values for alginate-psylliumblend with fenugreek(F6-F9)were increased from 7 to 9.6 which was signi fi cantly(P＞0.05)higher than the releasing rate in alginate-psyllium blend with inulin(6.9-8.6).

An effective microencapsulated system must release the probiotic cells at the appropriate time and in an adequate amount to gain their health promoting effects.Therefore, releasing of probiotic cells from the microencapsulated beads in the colonic pH solution is a crucial step for characterization of these beads[25].The polymer concentration and composition affect the release of microencapsulated probiotic cells [19,24].All microencapsulated E.durans 39C with alginatepsyllium blend or low concentration of inulin could be released completely from the beads after one h.The early release for these formulations probably can be explained by the easily erosion of loose networks in the alginate and psyllium.On the other hand,the adding of fenugreek to alginatepsyllium blend signi fi cantly reduced the release rate of probiotic cells.The same results were observed by other researchers[37,40,46].The decreased release probably was due to denser membrane of beads covered by fenugreek.But, Mandal et al.(2006)found that by increasing the gel concentration,no signi fi cant change in the release of probiotics was observed[25].After complete releasing,no signi fi cant changes in bacterial growth were observed for alginate and alginatepsyllium formulations.The same results were reported by Lot fi pour et al.(2012)and Mandal et al.(2006)for constantly release of microencapsulated probiotics in alginate[19,25]. But,the signi fi cant increasing rates of release(P＞0.05)were observed for prebiotic incorporated formulations.It probably was due to strong stimulating effects of inulin and fenugreek on the growth of E.durans 39C,hence,as a potential prebiotic they can improve the release and delivery of probiotic cells to the active sites and consequently enhance the probiotic population in the colon.

4. Conclusion

Microencapsulation of E.durans 39C probiotic bacteria using nine gel mixtures of alginate-psyllium blend with various inulin and fenugreek concentrations was successful.The highest encapsulation ef fi ciency,viability of cell in digestive conditions during storage time,also the increased release rates of probiotic cells in colonic condition throughout the 12 h period was observed for alginate-psyllium blend with fenugreek(F9)formulation.This study showed that herbalbased polymer such as psyllium and fenugreek can be used as a blend with alginate polymer as a matrix for probiotic formulation.They offer added advantages of being prebiotic towards the enhancement of probiotic bacterial growth in the gastrointestinal environment.

Acknowledgement

The fi nancial supports of the University Putra Malaysia are gratefully acknowledged.

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Babak Haghshenasa,*,Yousef Namia,Minoo Haghshenasb, Abolfazl Barzegaric,Simin Shari fic,Dayang Radiahd,Rozita Roslia, Norha fi zah Abdullahd,**

aInstitute of Biosciences,University Putra Malaysia,43400 UPM Serdang,Selangor,MalaysiabSchool of Medicine,Shahid Beheshti University of Medical Sciences,Tehran,Islamic Republic of IrancTabriz University of Medical Sciences,Molecular Medicine and Therapy Lab,Research Center for Pharmaceutical Nanotechnology,Tabriz,Islamic Republic of IrandChemical and Environmental Engineering Department,Faculty of Engineering,University Putra Malaysia, 43400 UPM Serdang,Selangor,Malaysia

*Corresponding author.Institute of Biosciences,University Putra Malaysia,43400 UPM Serdang,Selangor,Malaysia.Tel.:+60 389472215; fax:+60 389472101.

**Corresponding author.Chemical and Environmental Engineering Department,Faculty of Engineering,University Putra Malaysia,43400 UPM Serdang,Selangor,Malaysia.Tel.:+60 389466295;fax:+60 386567120.

E-mail addresses:bhaghshenas2010@gmail.com(B.Haghshenas),nha fi zah@upm.edu.my(N.Abdullah).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2015.04.001

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