Effect of microencapsulation with Maillard reaction products of whey proteins and isomaltooligosaccharide on the survival of Lactobacillus rhamnosus

LWT - Food Science and Technology 73 (2016) 37e43

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Effect of microencapsulation with Maillard reaction products of whey proteins and isomaltooligosaccharide on the survival of Lactobacillus rhamnosus Lu Liu a, b, Xiaodong Li a, b, c, *, Yongming Zhu a, b, Awa Fanny Massounga Bora a, b, Yongbo Zhao a, b, Lingling Du a, b, Donghua Li a, b, Weiwei Bi a, b a b c

Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, No. 59 Mucai St., Xiangfang Dist, 150030 Harbin, China College of Food Science, Northeast Agricultural University, No. 59 Mucai St., Xiangfang Dist, 150030, Harbin, China Synergetic Innovation Center of Food Safety and Nutrition, 150030 Harbin, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2015 Received in revised form 7 March 2016 Accepted 16 May 2016 Available online 20 May 2016

Lactobacillus rhamnosus (L. rhamnosus) was microencapsulated with Maillard reaction products of whey protein and isomaltooligosaccharide (WIMRPs) through cold gelation within emulsification system and its survival was evaluated after exposure to simulated gastrointestinal conditions. The Maillard reaction of whey protein and isomaltooligosaccharide (4:1) occurred in wet heating conditions (90
C, 4 h) forming high molecular weight conjugate. The WIMRPs microspheres showed the highest encapsulation yield (88.88%) after cold gelation within emulsification system compared with the microcapsules produced with the mixture of whey protein and isomaltooligosaccharide (WIMIX). The shape of WIMRPs microparticles was more spherical and smooth. The microparticles WIMRPs and WIMIX had mean diameter of 183 mm and 201 mm, respectively. WIMRPs provided better protection of probiotics after exposure of the microparticles to simulated gastrointestinal conditions, with the count of 7.48 and 7.17 log10 cfu/g respectively after 90 min incubation in Simulated Gastric Fluid (SGF) and 3% bile salt solution. The microspheres could be completely released in 60 min. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Microencapsulation Lactobacillus rhamnosus Maillard reaction Whey protein Isomaltooligosaccharide

1. Introduction Probiotics are a group of bacteria described as ‘living microorganisms administered in certain amounts that positively affect the health of the host’. In recent years, there has been a growing demand for use of probiotics in foods. However, probiotics are very sensitive to adverse environments which leads to a low survival rates in products (Cook, Tzortzis, Charalampopoulos, & Khutoryanskiy, 2012). Microencapsulation frequently protects probiotic bacterial cells in functional food applications and might enhance survival during gastrointestinal transit. Microencapsulation materials have gained significant interest over the last decade, such as alginates (Chan, Yim, Phan, Mansa, & Ravindra, 2010), polysaccharides (Argin,

* Corresponding author. Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Food College of Northeast Agricultural University, MuCai Street 59, Xiangfang Zone, Harbin 150030, China. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.lwt.2016.05.030 0023-6438/© 2016 Elsevier Ltd. All rights reserved.

€rst, & Kulozik, 2009). Kofinas, & Lo, 2014), proteins (Heidebach, Fo The utilization of dairy proteins as encapsulants would also be a reasonable approach, since encapsulation of probiotic microorganisms in a dense dairy protein matrix with a pronounced buffering capacity could protect the cells from harsh conditions, such as low pH-values in the human stomach (Heidebach et al., 2009). In previous research, whey protein and microparticulated whey protein concentration were particularly popular as encapsulating materials. These milk protein microspheres showed good protection for Bifidobacterium (Choi, Ryu, Kwak, & Ko, 2010; Livney, 2010; De Castro-Cislaghi et al., 2012). However, the wall material with a single constituent does not effectively allow functional properties that are desired in a specific application (Choi et al., 2010). In addition, the wall material with a single constituent is limited to provide adequate coating for the goal. In order to overcome these problems and offer versatility, a combination of proteins and carbohydrates has been suggested for wall materials during microencapsulation (Gbassi, Vandamme, Ennahar, & Marchioni, 2009; Gebara et al., 2013; Rajam, Karthik, Parthasarathi, Joseph, &

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L. Liu et al. / LWT - Food Science and Technology 73 (2016) 37e43

Anandharamakrishnan, 2012). Considering the simplicity, low cost, and gentle formulation conditions for high retention of cell viability, extrusion and emulsification techniques are extensively used (Homayouni, Ehsani, Azizi, Yarmand, & Razavi, 2007; Chan et al., 2011). Internal gelation approach overcomes the problem of capsule clumping encountered in external gelation approach, by slowly releasing Ca2þ ions from insoluble calcium sources instead of adding soluble calcium chloride directly. It thus produces smaller and more uniform microcapsules (Poncelet et al., 1995). In emulsification/internal gelation an insoluble or partially soluble salt of calcium is already present inside the droplets of the water in oil emulsion (w/ o) (Gouin, 2004; Chan, Lee, & Heng, 2006). In contrast to heatinduced gelation, cold gelation makes possible to entrap the heatsensitive nutraceuticals at ambient temperatures (Alting, de Jongh, Visschers, & Simons, 2002; Nicolai, Britten, & Schmitt, 2011). Maillard reaction causes changes in flavor and structure due to proteinereducing sugar interactions during food processing which result in the formation of proteinesugar-conjugates. These conjugates show some new properties with multifunctional application possibilities. Maillard-type protein-carbohydrate conjugates are known for their excellent emulsifying properties and have been used to encapsulate volatile oils and flavor compounds (Shah, Davidson, & Zhong, 2012). More importantly, Maillard reaction is a promising approach to generate glycoproteins. Glycosylation offers a new process for improvement of functional properties as well as being a substrate pre-treatment process to control enzymatic digestion in order to generate tailor-made peptides as food additives with important health benefits like probiotics due to glycoprotein resistance to further enzyme hydrolysis (Cheison, Josten, & Kulozik, 2013). It has been suggested that prebiotic as non-digestible food ingredients when added to certain foods may increase the viability of bacteria through gastrointestinal tract and have a beneficial effect on human health (Khalf and Dabour, 2010). Prebiotic and probiotic combination, known as synbiotic have been shown to improve probiotic proliferation in the intestine and help in modifying the gut community (Ozer, 2005). Fructooligosaccharides (FOS) and isomaltooligosaccharides (IMO) have been used as bifidogenic compounds in human food and infant formula (Boehm et al., 2004; Euler, Mitchell, Kline, & Pickering, 2005). Moreover, the conjugation of prebiotic oligosaccharides with a protein could potentially increase their colonic persistence, allowing them to reach the distal colonic region, where most of chronic gut disorders originate (Seo, Karboune, Yaylayan, & L’Hocine, 2012). Compared to microcapsules based on other wall material (Pinto et al., 2015), Maillard reaction product displayed higher colloidal, thermal, oxidative stabilities and protein gel. Both Maillard reaction product and prebiotic (isomaltooligosaccharide) can increase the survival of probiotics under in vitro acidic and bile salt conditions and also in stored food product. Cold gelation within emulsification system produced smaller and more uniform microcapsules and gentle formulation conditions for high retention of cell viability. Therefore, this study aimed to evaluate the effect of microencapsulation with Maillard reaction products of whey proteins and isomaltooligosaccharide on the viability of Lactobacillus rhamnosus during the encapsulation process and under the simulated digestive system. Production of synbiotic microcapsules with the Maillard reaction of whey protein and isomaltooligosaccharide by cold gelation within emulsification system has potential applications in the functional food industry such as in cheese, yogurt and fruit juices.

2. Materials and methods 2.1. Materials Probiotic strain Lactobacillus rhamnosus 6134 was obtained from the China Center for Industrial Culture Collection. The strain was isolated from human intestinal Lactobacillus strains of healthy subjects and preserved in the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University in China. Isomaltooligosaccharide was provided by Baolingbao Biology Co., Ltd. of Shandong. Whey protein concentrate (WPC-80) used in this study was purchased by Beijing Milky Way Trade Corp., Ltd (Beijing, China). 2.2. Methods 2.2.1. Preparation of Lactobacillus rhamnosus suspension Lactobacillus rhamnosus 6134 was reactivated for 3 times before use. The strain was inoculated into MRS (Huankai Microbial Sci & Tech Co., Ltd, Guangzhou,China) broth and incubated at 37
C for 24 h. The cells were harvested by centrifugation (Allegra™64R, Beckman, German) at 3000 g, 4
C for 10 min and washed twice with saline solution. Afterwards, cells were dispersed into saline solution and used for encapsulation. 2.2.2. Preparation of wall material The isomaltooligosaccharide and whey protein concentrate (WPC-80) were dissolved in deionized water at a weight ratio of 1:4 and the protein concentration was adjusted to 10% (w/v) and then kept refrigerated at 4
C for 12 h. The pH was adjusted to 8.0 with 5 M NaOH. The solution was heated at 90
C for 4 h (WIMRPs), and for 5 min (WIMIX). The solution without isomaltooligosaccharide was heated at 90
C for 5 min to serve as control (CON). All solutions were cooled down and then refrigerated at 4
C before further use. The entire process for the preparation of wall material was performed in triplicate. 2.2.3. SDSepolyacrylamide gel electrophoresis The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSePAGE) was used to confirm covalent modification of biopolymers by Maillard reaction. It was performed according to n, Belloque, Alonso, and Lo pezthe method described by Chico ~ o (2008) with slight modification, using 5% (pH 6.8) stackFandin ing gel and 12% (pH 8.8) separating gel. The samples were native protein, WPCeISO mixture, WPC heat-treated at 90
C for 5 min, mixtures of WPC with isomaltooligosaccharide heat-treated at 90
C for 5 min and for 4 h, respectively. The protein content of all samples was 2 mg/mL. Samples were mixed with sample buffer (4% SDS, 20% glycerol, and 0.125 M TriseHCl, pH 6.8) containing the reductive agent 2-b-Mercaptoethanol in a 4:1 volume ratio. The mixtures were then heated at 100
C for 3 min. Then, the sample was immediately cooled on ice. A molecular weight (Mw) standard, which was composed of a cocktail of proteins (6500e200,000) (takara Biotechnology Co., Ltd. Dalian, China) was also run. Aliquots (10 ml) of the samples were loaded into the wells and initially run at 80 V. When the samples were with in the separation gradient gel, the voltage was increased to 120 V. Subsequently, the gel was stained with 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 6.8% acetic acid mixed solution and destained in a solution containing 5% methanol and 7.5% acetic acid. 2.2.4. Production of microparticles All glassware used in this protocol was sterilized at 121
C for 15 min. 2 mL of L. rhamnosus concentrate was mixed with 28 mL of

L. Liu et al. / LWT - Food Science and Technology 73 (2016) 37e43

the WIMIX or WIMRPs dispersion, to create a wall material solutionecell mixture with about 10 log10 cfu/g of probiotic cultures. The encapsulating process was performed according to the method described by Sadeghi, Madadlou, and Yarmand (2014) with modification. The proteinecell mixture solution was supplemented with 10 mM equivalent calcium chloride and 3.5 g L1 glucono-d-lactone as acidogen. 30 mL of the proteinecell mixture containing L. rhamnosus was added to 150 g of tempered (40
C) sunflower oil in a 200 mL erlenmeyer flask and stirred at a constant speed of 900 rpm with a magnetic stirrer for 120 min. During the process the emulsified droplets of proteinecell mixture were converted into gel particles. Subsequently, the gelatinized microcapsules were separated from the oil by gentle centrifugation (5000 g, 1 min). The supernatant was removed and the sediment was diluted twice with saline solution. 2.2.5. Morphological characterization of the microparticles by optical examination Samples of microcapsules containing L. rhamnosus cells, taken from the proper/appropriate dilution aqueous capsule-slurry was observed with a light biological microscope (Motic BA 300, Motic China Group, China). 2.2.6. Encapsulation yield To count encapsulation yield is according to the method described by Heidebach et al. (2009). The ratio between the protein concentration of the proteinecell mixture (Cpcm) and the resulting capsule-slurry (Cslurry) was taken into account as a dilution factor for the calculation of the encapsulation yield (EY). The encapsulation yield of L. rhamnosus was calculated as follows:

EYð%Þ ¼

Ccpm cfu g 1 capsule slurry  Cslurry cfu g1 protein  cell mixture

2.2.7. Survival of free and microencapsulated L. rhamnosus under low pH conditions A low pH (pH ¼ 2.0) stability study of Free, WIMIX and WIMRPS microparticles was carried out according to the method describe by Ma et al. (2008). Those microspheres containing L. rhamnosus (0.50 g) were added into the tubes containing 4.5 mL of SGF (pH 2.0) and incubated at 37
C for 10, 30, 60, 90 and 120 min. At the specified time interval, samples were taken out for analysis. Free L. rhamnosus was 10 times serially diluted with saline solution and 100 ml aliquots were plated on MRS agar plate. Microspheres containing L. rhamnosus were recovered from Simulated Gastric Fluid (SGF), samples were analyzed to evaluate the count of free and microencapsulated L. rhamnosus viable cells. To enumerate the entrapped probiotic cells, encapsulated L. rhamnosus (0.50 g) was dissolved in 4.5 mL, 50 mM sodium citrate solution at pH 7.5, a mechanical method for the capsule disintegration was therefore applied. Cell release was carried out by homogenizing (Ultra-Turrax; IKA, T18 basic, Germany) diluted capsule-slurry for 45 s at 10,000 rpm. Released L. rhamnosus was 10 times serially diluted with saline solution and 100 ml aliquots were plated on MRS agar plate. Colony forming units (CFU) were determined after incubation (48 h, 37
C). 2.2.8. Survival of free and microencapsulated L. rhamnosus under bile salt conditions The stability of encapsulated and free L. rhamnosus was tested in bile salt solution. Suspensions of free L. rhamnosus (0.5 mL) or WIMIX and WIMRPS microspheres containing L. rhamnosus (0.50 g) were placed in a tube containing 4.5 mL bile salt solution (3%, w/v)

39

and incubated at 37
C for 0, 30,60 and 90 min. WIMIX and WIMRPS microspheres were collected at each time interval. Viable cells of L. rhamnosus were determined according to procedures described in Section 2.2.7. 2.2.9. Release study of encapsulated L. rhamnosus Release profile of encapsulated L. rhamnosus in WIMIX and WIMRPS microspheres was studied in Simulated Intestine Fluid (SIF: pH 6.8, 50 mM KH2PO4). The microspheres containing L. rhamnosus (0.50 g) were added to conical plastic tubes containing pre-warmed SIF and incubated at 37
C with shaking at 100 rpm. At predetermined time point, 100 ml aliquots were taken out and immediately assayed for the amounts of released L. rhamnosus. The same volume of the fresh medium was added to replace the volume of the withdrawn sample. The cumulative amount of released rate was plotted against time. 2.3. Statistics analysis For all experiments, three independent assays were carried out. One-way analysis of variance was used to study the differences between means, with a significant level at a  0.05. Data analysis was carried out with SPSS (Windows version 12.0, SPSS Inc., Chicago, IL, USA). All data are presented as mean ± standard error of means. 3. Results and discussion 3.1. SDS-PAGE electrophoresis Fig. 1 shows the electrophoresis SDS-PAGE pattern of WPC and ISO before and after mixing and the part conjugation processes. No difference between the bands in Lanes 0, 1, and 2 was observable, possibly due to the glycosylation with little change in molecular weights. In lanes 3 and 4, the three prominent native bands including bovine serum albumin (BSA), b-lactoglobulin (b-Lg) and a-lactalbumin (a-La) lightly disappeared. The gradual disappear in

Fig. 1. SDS-PAGE pattern of WPC and ISO before and after mixing and the part conjugation processes. Lane 0 is a native WPC, Lane 1 is the mixture of WPC and ISO. Lane 2 is WPC heat-treated 90
C for 5 min (CON), Lanes 3 is the mixture of WPC and ISO heat-treated at 90
C for 5 min (WIMIX), Lane 4 is the mixture of WPC and ISO heat-treated at 90
C for 4 h (WIMRPs). Numbers against protein markers indicate rounded molecular weights (Da).

L. Liu et al. / LWT - Food Science and Technology 73 (2016) 37e43

the protein bands depends on the reaction time. In lane 4, the molar mass distribution of the WPC displayed shifts toward higher Mw and increasingly visible, this phenomenon could confirm the formation of the high molecular weight conjugate caused by Maillard reaction during the conjugation process.

3.2. Formation and characterization of capsules The shape of freshly prepared microcapsules are shown in Fig. 2. In contrast to this, the whey protein beads were of less spherical in shape (Fig. 2a), WIMRPs solutions undergo a homogeneous internal gelation process, leading to droplet-like spherical capsules and smooth-surface (Fig. 2b). Maillard reaction made greater contribution to the emulsifying property (Hiller & Lorenzen, 2010; Li et al., 2014). The emulsion method is a well known technology for the microencapsulation of probiotic cells. A small volume of the hydrocolloidecell mixture (discontinuous phase) is emulsified into a larger volume of vegetable oil (continuous phase). Once a waterin-oil-emulsion is formed, the hydrocolloidecell mixture must be insolubilized to form small beads within the oil phase. The process is usually carried out by magnetic stirrer. Gelation is induced at ambient temperature through reduction of the electrostatic repulsion by lowering the pH toward the isoelectric point of proteins. Thus, the gelation kinetic is heterogeneous, which often leads to capsules of irregular shape (Muthukumarasamy, Allan-Wojtas, & Holley, 2006). The freshly made beads by two wall materials were treated and observed with optical microscope at the same way. The emulsifying properties of WIMRPs solutions are mainly due to its ability to lower the interfacial tension between oil and water phase. A spherical shape is generally favored because of the reduced exposure of surface compared with irregular shaped particles. The microparticles WIMRPs, WIMIX and Control had mean diameter of 183 mm, 201 mm and 118 mm, respectively. Microcapsule size with a very wide range have been reported by several researchers and the optimum microcapsule size is a compromise between the effectiveness of encapsulation and the sensory properties (Nag, Han, & Singh, 2011). Microspheres with large size provide more protection than those with small size, but large size microspheres have a negative sensory impact on the product. The particle size of microspheres is an important characteristic for the stability and efficiency of encapsulation. Some references indicated that microspheres with sizes ranged from 250 mm to 1 mm could cause negative sensory in food products (Hansen, Allan-Wojtas, Jin, & Paulson, 2002). In our study, the similar size can be seen for both wall material capsules about range from 100 to 200 mm, so our

beads was just within this limit.

3.3. Encapsulation yield Fig. 3 showed results for encapsulation yields of three difference microspheres containing L. rhamnosus by emulsification technique coupled with internal cold gelation process. Free cells suspension used for encapsulation was around 10 log10 cfu/g. Numbers of cell encapsulated in microspheres were ranged from 8.84 log10 cfu/g to 9.11 log10 cfu/g for different microsphere formulations. Therefore, a high encapsulation yield in WIMRPs microspheres was obtained in this study. The WIMRPs microspheres showed the higher encapsulation yield (88.88%) after cold gelation within emulsification system compared with the microcapsules produced with the mixture of whey protein and isomaltooligosaccharide (87.63%) and Control (85.13%). There was significant difference between WIMRPs and whey protein microspheres (p < 0.05). The high encapsulation yield observed in our study may be due to WIMRPS containing hydrophilic-hydrophobic balanced glycoprotein, it is able to lower

95

Encapsulation yield (%)

40

90

B

AB

A

WIMIX

WIMRPs

85 80 75 70 65 60 CON

Different microspheres Fig. 3. Encapsulation yields of three difference microspheres containing L. rhamnosus by emulsification technique coupled with internal cold gelation process. The error bars represent standard deviations. Different letters labelling bar graphs of the sample categories indicate that mean values are significantly different (P < 0.05). (CON stands for whey protein was heat-treated at 90
C for 5 min. WIMIX stands for the mixture of whey protein and isomaltooligosaccharide was heat-treated at 90
C for 5 min. WIMRPs stands for the mixture of whey protein and isomaltooligosaccharide was heat-treated at 90
C for 4 h).

Fig. 2. Optical WIMix-microsphere(a), WIMrps-microsphere(b) microsphere obtained by a gelation/emulsification method.

L. Liu et al. / LWT - Food Science and Technology 73 (2016) 37e43

the interfacial tension. Other particle size of microspheres is an important characteristic for encapsulation yields. The encapsulation yield of microspheres prepared by emulsification method can be affected by several factors such as stirred speed, polymer concentration and ratio of polymer and probiotic. Generally, encapsulation yield was increased significantly with the increase of the polymer’s degree of crosslinking network structure. Many references reported that encapsulation of probiotic with prebiotic could affect the encapsulation yield. Sultana et al. (2000) reported Incorporation of a prebiotic (Hi-Maize starch) in alginate beads could improve the encapsulation efficiency of Lb. casei. Choi et al. (2010) reported the yields of microspheres for Conjugated linoleic acid (CLA) increased 3% depending on Maillard reaction products (WIMRPs) produced by whey proteins and maltodextrin. 3.4. Survival of free and encapsulated L. rhamnosus in SGF The pH stabilities of free and encapsulated L. rhamnosus in SGF are shown in Fig. 4. Viability of L. rhamnosus could not be found within 10 min in SGF pH 2.0 (results not shown). As many articles have been reported, most of free probiotics are easy to be damaged by stomach acid. Sohail et al. reported more than 6 log10 cfu/g of Lactobacillus acidophilus was lost in pH 2.0 SGF for 20 min (Sohail, Turner, Coombes, Bostrom, & Bhandari, 2011). WIMIX microcapsules and WIMRPs microcapsules were resistant to simulated gastric conditions. In Fig. 4, the viability of L. rhamnosus were 7.32 and 7.52 log10 cfu/g at pH 2.0 (p < 0.05) as shown in 60 min, more than 7 log10 cfu/g L. rhamnosus survived after 90 min, being within  rdoba, 2001). A the requirements for probiotics benefits (Hotel & Co similar explanation for higher survival of encapsulated cells was found by Heidebach et al. (2009), who encapsulated probiotics using transglutaminase-induced caseinate gelation. The survival of the viable cells in the simulated gastric environment was higher in WIMRPs microcapsules as compared to WIMIX microcapsules. The WIMRPs microencapsulate tended to provide better protection (p < 0.05). Whey protein microsphere had poor viability of encapsulated L. rhamnosus compared to the other two formulations. Similar results have been reported by others, the buffering effect of

Log10/g microspheres

10 CON WIMIX WIMRPs

9

8

7

41

whey proteins contributed to higher survival rates for bifidobacteria encapsulated in a mixed gel in comparison to free cells when rin, Vuillemard, & Subirade, 2003). Excellent exposed to SGJ (Gue pH tolerance of encapsulated L. rhamnosus was probably due to the fact that a denser hydrogel network formed by MPRS could reduce the diffusion rate of acid into the microspheres and the addition of IMO increased the resistance of these probiotics to low pH. 3.5. Survival of free and encapsulated L. rhamnosus in bile salt solution The effect of bile salt solution on the viability of free and encapsulated L. rhamnosus was presented in Table 1. In this work, free L. rhamnosus totally lost their viability in bile salt solution after 30 min exposure, probably due to the loss of cell wall integrity as a result of the action of the bile salts. Probiotics were sensitive to bile salt solution. Therefore, the diffusion of bile salt into the microcapsules may be limited. This will protect encapsulated probiotics from interacting with the bile salt. The results shown in Table 1 clearly indicated that WIMIX, WIMRPs microspheres could provide a good protection against the damage of the bile salt solution compared to free L. rhamnosus (p < 0.05). The viable amounts of encapsulated L. rhamnosus were reduced to 8.85, 7.53 and 7.17 log10 cfu/g microspheres after exposure to 3% bile salt solution for 30, 60, 90 min, respectively. Around 3.1 and 2.4 log10 cfu/g microspheres were reduced after 90 min exposure, respectively. The reason could be that the structured trapping MRPs matrix was more resistant to the effects of bile salt solution. In this study, high bile salt solution tolerance was observed for encapsulated cells when compared with free cells. It was difficult to make any comparison with our findings because different researchers used different concentrations and sources of bile salts. Several studies reported that encapsulated probiotic bacteria could survive better than free probiotic cells in 1% and 2% bile salts solution (Shi et al., 2013) and in 0.5% and 1% bile solution (X. Y. Li, Chen, Sun, Park, & Cha, 2011). 3.6. Release characteristic of encapsulated L. rhamnosus in SIF Encapsulated L. rhamnosus should be released in SIF before it can confer healthy function to human body. In controlled-release systems, the polymer network is controlled by several physicochemical phenomena such as polymer water uptake, gel layer formation, and polymeric chain relaxation (Kim, La Flamme, & Peppas, 2003; Llabot, Manzo, & Allemandi, 2004). The WIMIX and WIMRPs with different formulations were exposed to the simulated intestinal fluid to study the cell release characteristic of the capsules. Table 1 Survival of free and encapsulated L. rhamnosus after treatment in simulated bile concentrations of 3% for 30, 60 and 90 min (log10 cfu/g).

6

Time (min)

5 0

10

30

60

90

Time (min) Fig. 4. The survival of free and three difference microspheres encapsulated L. rhamnosus in Simulated Gastric Fluid (SGF) pH 2.0. (CON stands for whey protein was heat-treated at 90
C for 5min. WIMIX stands for the mixture of whey protein and isomaltooligosaccharide was heat-treated at 90
C for 5min. WIMRPs stands for the mixture of whey protein and isomaltooligosaccharide was heat-treated at 90
C for 4 h. Viability of L. rhamnosus could not be found within 10 min in SGF pH 2.0 so results not shown).

0 30 60 90

Number of viable cells (log10 cfu/g) Free cells

WIMIX

10.94 ± 0.15 x x x

9.55 8.42 6.97 6.49

± ± ± ±

0.13Aa 0.36Ba 0.21Cb 0.38Cb

WIMRPs 9.56 8.85 7.53 7.17

± ± ± ±

0.23Aa 0.16Ba 0.19Ca 0.19Ca

Values shown are means ± standard deviations. n ¼ 3 sets of data analyzed in duplicate. X indicates cell concentration <1 log10 cfu/g. Means in rows with different small letters are significant differences (p < 0.05); Means in columns with big letters are significant differences (p < 0.05). WIMIX stands for the mixture of whey protein and isomaltooligosaccharide was heat-treated at 90
C for 5 min. WIMRPs stands for the mixture of whey protein and isomaltooligosaccharide was heat-treated at 90
C for 4 h.

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L. Liu et al. / LWT - Food Science and Technology 73 (2016) 37e43

The release characteristic of encapsulated L. rhamnosus in SIF is showed in Fig. 5. Encapsulated L. rhamnosus in microspheres could be released quickly. Both encapsulated L. rhamnosus could be released from microspheres in 60 min (8.90 and 9.13 log10 cfu/g). The release mechanism was probably due to the swellingeerosion of whey protein-isomaltooligosaccharide network in SIF. Similar results were reported, L. rhamnosus GG in whey protein microbeads release 6.2 log10 cfu/g after 5 min, while the complete was achieved following 30 min incubation in simulated ex vivo intestinal fluid (Doherty et al., 2012). Generally, polymer dense not only affected the protection of L. rhamnosus against acid, but also affected the release profile of encapsulated L. rhamnosus. Wall material dense network structure enhanced the protection of L. rhamnosus against acidic medium while release rate was reduced. However, the release rate of encapsulated L. rhamnosus from WIMRPs microspheres was not significant for comparison with WIMIX microspheres (p > 0.05). It probably was due to that lowly cross-linked protein/saccharide complexes not effect on sterically shielding peptide bonds against proteolysisemay be not the cause of impaired in vitro digestibility of Maillard modification (Hiller & Lorenzen, 2010). Anticipation, the WIMRPs microspheres release started much sooner and faster in the vivo intestine microflora condition, and the wall material also can provide probiotic nutrition.

4. Conclusions The microencapsulation of L. rhamnosu using Maillard reaction products of whey protein and isomaltooligosaccharide (4:1) was found to be the most effective in maintaining the viability of bacteria during exposure to gastrointestinal environmental conditions. The WIMRPs microspheres showed the higher encapsulation yield (88.88%) compared with the microcapsules produced with the WIMIX and the shape of WIMRPs microparticles was more spherical and smooth. The WIMRPs microspheres show the potential as a new delivery carrier for oral administration of bioactive compounds. Further researches also have to be carried out to evaluate the cell viability and investigate technological properties of probiotic strains after microencapsulation during the storage of food environment.

10

Log10/g microspheres

9 8 7 WIMIX WIMRPs

6 5 4 3 2 1 0 0

20

40

60

80

100

Time (min) Fig. 5. Release of encapsulated L. rhamnosus from WIMIX and WIMRPs microspheres in Simulated Intestine Fluid (SIF).

Acknowledgements This research was supported by Heilongjiang Province Education Department General Project (12541027); The Research and Development Program of the Early Part of Technological Achievement Industrialization of Heilongjiang Province Universities(1253CGZH28); Harbin Municipal Science and Technology Innovation special talent returning from overseas (2012RFLXN033).

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