Direct spray drying and microencapsulation of probiotic Lactobacillus reuteri from slurry fermentation with whey

Authors


Correspondence

Christopher Beermann, Faculty of Food Technology, Department of Biotechnology, University of Applied Sciences Fulda, Marquardstrasse 35, Fulda, 36039, Germany. E-mail: christopher.beermann@lt.hs-fulda.de

Abstract

Aims

Formulations of dietary probiotics have to be robust against process conditions and have to maintain a sufficient survival rate during gastric transit. To increase efficiency of the encapsulation process and the viability of applied bacteria, this study aimed at developing spray drying and encapsulation of Lactobacillus reuteri with whey directly from slurry fermentation.

Methods and Results

Lactobacillus reuteri was cultivated in watery 20% (w/v) whey solution with or without 0·5% (w/v) yeast extract supplementation in a submerged slurry fermentation. Growth enhancement with supplement was observed. Whey slurry containing c. 109 CFU g−1 bacteria was directly spray-dried. Cell counts in achieved products decreased by 2 log cycles after drying and 1 log cycle during 4 weeks of storage. Encapsulated bacteria were distinctively released in intestinal milieu. Survival rate of encapsulated bacteria was 32% higher compared with nonencapsulated ones exposed to artificial digestive juice.

Conclusions

Probiotic L. reuteri proliferate in slurry fermentation with yeast-supplemented whey and enable a direct spray drying in whey. The resulting microcapsules remain stable during storage and reveal adequate survival in simulated gastric juices and a distinct release in intestinal juices.

Significance and Impact of the Study

Exploiting whey as a bacterial substrate and encapsulation matrix within a coupled fermentation and spray-drying process offers an efficient option for industrial production of vital probiotics.

Introduction

The Food and Agriculture Organization of the United Nations and the World Health Organization underline in their definition of probiotics that living micro-organisms with health-promoting mechanisms have to be administered in adequate amounts to be effective (FAO and WHO 2001). Therefore, dietary formulations of probiotics have to be robust against industrial production processes in a cost-effective way and should maintain a sufficient survival rate after oral uptake and gastrointestinal tract (GIT) transit.

Commonly accepted bacterial counts for dietary products are at least 106 CFU ml−1 (Kailaspathy and Chin 2000). A wide range of health benefits have been described, such as immune response-modulating properties, improving gut barrier function and protecting effects of the colon against pathogens (Anal and Singh 2007). Lactobacillus reuteri strains are probiotic lactic acid bacteria (LAB) regularly applied to milk-related products but also in meat, fruit and vegetable-based products. Furthermore, L. reuteri has been described to be a robust bacterium feasible for large-scale cultivation techniques and has high viability during storage and production (Casas and Dobrogosz 2000).

Probiotic encapsulation is a multi-stage process which includes fermentation, harvesting, resuspension in matrix material and encapsulation. To increase process efficiency and improve the survival of bacteria, this study aimed at developing a microencapsulation process for L. reuteri by spray drying directly from slurry fermentation using whey as a culture substrate and encapsulation matrix. Several encapsulation procedures for bacteria have been established, predominantly emulsion, extrusion and spray drying (Anal and Singh 2007; Rokka and Rantamäki 2010). Both extrusion and emulsion mainly generate wet particles by cross-linking polymer systems which have to be dried to enable handling and storage afterwards (Cook et al. 2012).

Encapsulation by spray drying directly achieves optimal product moisture content, which is between 4 and 7% for storage stability (Ananta et al. 2005). Other main advantages of spray drying are its low cost and fast production of large quantities of viable cells (De Castro-Cislaghi et al. 2012). Hereby, predominant factors for bacterial viability loss in this process are heat, oxygen, as well as mechanical and osmotic forces (Meng et al. 2008). In contrast, encapsulation with spray drying yields dried powder of small particle sizes with a sufficient protection for the core (Anal and Singh 2007; Cook et al. 2012).

Several spray-drying processes for dietary applications have been established utilizing different polysaccharides and protein compounds to protect probiotics. The pH value profile of the gastrointestinal passage ranges from 1·9 to 2·5 in the stomach, which is the most harmful for orally applied probiotics, up to pH values between 6·15 and 7·88 in the small intestine and ending up with a slightly acidic pH value in the colon (Cook et al. 2012). Also, digestive enzymes, such as gastric pepsin and pancreatic trypsin and chymotrypsin, pancreatin and bile salts are further antibacterial factors (Gbassi et al. 2011; Doherty et al. 2012).

Whey is a cheap cheese by-product containing 50% of the milk nutrients, predominantly lactose (4·9% of total whey) and whey protein (0·7% of total whey) with the main protein fractions β-lactoglobulin and α-lactalbumin. With its dissimilar nutritional value, whey possesses different interesting bio- and techno-functional properties. Aside from specific lipids, vitamins, minerals and high-energetic lactose, whey has been discussed to be an attractive source of functional proteins and peptides with distinct amino acid profiles (Smithers 2008). To continue, whey is thermostable and possess excellent gelation, water binding, emulsification as well as foam forming properties and foam formation with thermal stability (Foegeding et al. 2002; Smithers 2008). Recent studies suggest that whey protein resists against acidic milieus (Doherty et al. 2012). Gbassi et al. (2009) determined no damaging effect at pH 1·8. The stability against pepsin is more complex as α-lactalbumin breaks down in the presence of pepsin, while the main whey protein β-lactoglobulin stays intact (Gbassi et al. 2009).

Considering the physiological and technological potential, whey might be useful as a bacterial substrate and encapsulation matrix within a coupled fermentation and spray-drying process to offer an efficient option for industrial production of vital probiotics. For this, the growth of L. reuteri in whey was characterized in batch slurry fermentation process coupled with direct encapsulation of bacteria by spray drying. At last, physical product characteristics and bacterial protective properties against GIT conditions were investigated in vitro.

Materials and methods

Bacterial strain

Probiotic Lactobacillus reuteri (DSM 20016) was obtained from the German Collection of Microorganisms and Cell Cultures – Leibnitz Institute DSMZ – and cultured aerobically in 9 ml de Man–Rogosa–Sharpe (MRS) broth (pH 6·2) at 37°C for 24 h.

Growth of Lactobacillus reuteri in whey

To characterize the growth of L. reuteri in whey, approximately 103 CFU ml−1 bacteria were inoculated in a 200-ml-volume fermentation of watery 20% (w/v) whey solution (pH 6·0) with or without 0·5% (w/v) yeast extract as supplement and cultivated in a agitating flask culture at 37°C. Before inoculation, whey solution was sterilized in 80°C water bath for 20 min. Bacterial cell count was measured after 0, 24, 48 and 72 h by direct plate counting as described below.

Batch slurry fermentation of Lactobacillus reuteri in a continuous stirred-tank reactor (CSTR)

The growth of L. reuteri was observed in a laboratory-scale reactor (Biostat A, Sartorius Ltd., Melsungen, Germany). Approximately 104 CFU ml−1 bacteria were inoculated in 1 l batch slurry fermentation of watery 20% (w/v) whey solution with 0·5% (w/v) yeast extract as supplement, tested with adjustment at pH 5·0 and cultivated at 37°C with agitation using one six-wing rotating disc at 200 rpm, respectively. Samples of 5 ml were taken to determine cell counts after 0, 24, 48 and 72 h by direct plate counting as described below.

Microencapsulation of Lactobacillus reuteri in whey

For the spray-drying process, a 48-h-incubated 200-ml slurry culture of L. reuteri with at least 108 CFU ml−1 was applied. The fermented slurry was spray-dried by a laboratory-scale spray dryer (Büchi mini spray dryer B190; Flawil, Switzerland) with two different outlet temperatures maintained at 55 ± 2 and 65 ± 2°C and a flow rate of 500 Nl h−1. Outlet temperatures were reached by adjusting different inlet temperatures (89 ± 1 and 100 ± 1°C) and feed levels from 2 to 4 ml min−1. Spray-dried powder samples were collected from the cyclone and mixed gently.

Enumeration of surviving cells

To validate the survival rate of bacteria during drying process, cell counts were observed before and after spray drying. To detect the viable number of bacteria, direct plate counting was used. Samples were serially diluted in Ringer's solution (pH 7·0) and plated on MRS agar (pH 6·2). After 48-h incubation at 37°C under anaerobic conditions, the cell counts were expressed in CFU g−1. Spray-dried samples were previously rehydrated in Ringer's solution at a solid content of 20% (w/v), and the solution was used to determine cell survival. Cell counts were expressed as mean values of duplicate measurements.

Moisture content and particle size of spray-dried powders

The moisture content of spray-dried powders was determined using the moisture analyser (MA 40, Sartorius AG, Gottingen, Germany) at 80°C drying temperature. Data are expressed as mean value of double-tested 1-g product.

Particle size was analysed using the Mastersizer 2000 (Malvern Instruments Ltd., Malvern, Worcestershire, UK). Data are expressed as mean value of triple determinations.

Survival of encapsulated Lactobacillus reuteri during storage

The spray-dried product was stored in plastic tubes at 4°C. Survival of encapsulated L. reuteri during storage was determined directly following the drying process and after 1 week and 4 weeks of storage, by performing direct plate counting as described above.

Survival and release of encapsulated and nonencapsulated Lactobacillus reuteri in simulated gastrointestinal conditions

To estimate the tolerance of encapsulated cells to simulated digestive conditions, an assay was adapted by using a modified version of the method according to Picot and Lacroix (2004). The cultures were exposed to simulated gastric juice (pH 1·9) and afterwards to simulated small intestinal juice (pH 7·5) at 37°C. Simulated gastric juice preparation contained pepsin (0·304 g l−1) (porcine gastric mucosa, P7012, Sigma-Aldrich, Taufkirchen, Germany) dissolved in sterile 0·1 mol l−1 HCl/1 mol l−1 NaOH to adjust pH to 1·9. Simulated pancreatic juice preparation contained pancreatin (19·5 g l−1) (porcine pancreas, P1750, Sigma-Aldrich) dissolved in sterile sodium phosphate buffer (0·02 mol l−1, pH 7·5) adjusted with 0·1 mol l−1 HCl/1 mol l−1 NaOH to pH 7·5. A concentrated bile salt solution contained bile extract powder (150 g l−1) (bile bovine, B3883, Sigma-Aldrich) in sterile distilled water.

To examine the survival of encapsulated L. reuteri, 5·0 g of the dried product containing 107 CFU g−1 bacteria was gently mixed with 30 ml of pepsin preparation (0·26 g l−1, 37°C) in a 50-ml sterile plastic tube. The resulting dispersion was incubated at 37°C in a 250 rpm shaking thermo element (ThermoMixer and BlockThermostate, HLC BioTech, Bovenden, Germany). After 30 min, the reaction was stopped by raising the pH to 7·5. A sample of 1·5 ml was taken and kept on ice before viable cell counts were determined. A volume of 2·5 ml of concentrated sodium phosphate buffer (0·5 mol l−1, pH 7·5) and 1·0 ml of the concentrated bile salt solution were added (3·33 g l−1). After adjusting the pH to 7·5 and filling the volume to 40·5 ml with sterile distilled water, 4·5 ml of the simulated pancreatic juice (1·95 g l−1) was added to make the final volume of the tube to 45 ml. At different time intervals (1, 2, 3, 5 h), 1·5-ml aliquots were taken and placed on ice before bacterial enumeration. Except gentle shaking, no dispersion step was conducted to estimate the release properties of the microcapsules at different time intervals.

For comparison, nonencapsulated bacteria cultured in MRS broth (37°C, 24 h) were washed and resuspended in sterile 0·85% saline. Five millilitres of cell suspension containing approximately 106 CFU ml−1 bacteria was added to 30·0 ml of pepsin preparation (0·26 g l−1, 37°C) in a 50-ml sterile plastic tube and maintained at 37°C in a shaking thermo element, as described above. The rest of the procedure was same as that described for the encapsulated cells.

Enumeration of bacteria from samples taken during simulated digestion with encapsulated and nonencapsulated cells was carried out by direct plate counting as described above. The percentage surviving bacteria was calculated as percentage survival = N/N0 × 100, where N0 represents the number of bacteria in the inoculum and N is the viable number of bacteria in the digestion juice.

Statistical analysis

Data were evaluated by GraphPad 5 Prism software (GraphPad Software, Inc., La Jolla, CA, USA). P-values were determined by one-sided nonparametric Wilcoxon–Mann–Whitney U-test for tolerance to simulated gastric juice and by two-sided nonparametric Wilcoxon–Mann–Whitney U-test for storage, moisture content and particle size. P-values ≤ 0·05 were defined as significant.

Results

Growth of Lactobacillus reuteri in whey

The initial experiment was accomplished to define the growth capacity of L. reuteri (DSM 20016) in whey. For this, a watery 20% (w/v) whey solution with or without 0·5% (w/v) yeast extract as supplement was tested. Under both culture conditions, the stationary phase of bacterial growth kinetics of batch culture was reached after 48 h of fermentation in agitating flask at 37°C. After 72-h culturing, the end cell counts in pure whey solution increased 4 log cycles, whereas the bacterial counts increased 5 log cycles by supplementing 0·5% yeast extract to whey.

Batch slurry fermentation of Lactobacillus reuteri in a CSTR

Yeast extract–supplemented whey solution was tested using batch slurry fermentation in a CSTR. As sufficient agitation of whey slurry was reached at 200 rpm, stationary growth phase was reached after 24 h of cultivation with 1·7 × 109 CFU g−1 bacteria.

Survival of Lactobacillus reuteri in whey encapsulation after spray drying

Lactobacillus reuteri was spray-dried at 55 and 65°C temperatures using the whole slurry fermentation with whey directly as feed. Loss of viable bacteria after spray drying is illustrated in Fig. 1. For all experiments, whey solutions with 1·6 (± 1·5) x 109 CFU g−1 bacteria were applied to the encapsulation process. Dried product contained 2·5 (± 1·7) x 107 CFU g−1 bacteria directly after processing independently from used outlet temperatures.

Figure 1.

Cell counts of Lactobacillus reuteri before and after spray-drying process with (image_n/jam12293-gra-0001.png) 55°C and (image_n/jam12293-gra-0002.png) 65°C outlet temperatures and during storage for 7 and 28 days at 4°C storage temperature. The data shown represent the mean of at least three measurements.

Characterization of encapsulated particles

To compare physical characteristics, moisture and particle size of the spray-dried products were determined (Table 1). The moisture content of dehydrated products is of importance for product and bacterial stability while storage. Optimal moisture content is between 4 and 7% for storage (Ananta et al. 2005). Powder manufactured in this study had a mean moisture content of about 6·5 (±0·7)%. There is no significant difference in moisture content of the powder produced at 55 or 65°C outlet temperature of the process. The obtained particles had a mean diameter of 5·5 (±0·3) μm at 55°C and 4·9 (±0·3) μm at 65°C again with insignificant differences with regard to the outlet temperatures. Achieved size is big enough to encapsulate rod-shaped L. reuteri cells with about 2 μm length (Muthukumarasamy et al. 2006).

Table 1. Moisture content and particle size of spray-dried powder directly from slurry fermentation in whey 20% (w/v) with 0·5% (w/v) yeast extract supplementation
Process outlet temperature (°C)Moisture (%)Particle size (μm)
  1. Results for 55 and 65°C are the mean of triplicate trials.

556·1 ± 0·85·5 ± 0·3
656·8 ± 0·34·9 ± 0·3

To describe product stability during storage at 4°C, survival of encapsulated bacteria was determined after 1 week and 4 weeks. The bacterial counts of the produced capsules decreased by 1 log cycle after a storage period for 4 weeks (Fig. 1).

Survival and release of encapsulated and nonencapsulated Lactobacillus reuteri in simulated gastrointestinal conditions

To ensure the physio-functional benefits of L. reuteri, a sufficient bacterial survival rate along the stomach and intestine passage is essential to enable probiotic colonization of the colon. Figure 2 examines the survival of free and encapsulated cells after exposure to artificial digestive juices, first 30 min in simulated gastric juice and afterwards in simulated intestinal juice for a maximum of 5 h in vitro.

Figure 2.

Percentage release and survival of (■) encapsulated and (●) nonencapsulated Lactobacillus reuteri in simulated gastric juice for 30 min and afterwards in simulated intestinal juice for a maximum of 5 h. The data shown represent the mean of at least three measurements.

Inoculation in artificial gastric juice resulted in a decline in viable cell counts of L. reuteri. Observed cell counts after 30 min were 12% of the applied bacteria for free cells and about 26% for encapsulated ones. After changing to simulated intestinal juice, the number of viable cell counts for nonencapsulated bacteria remained constant. In contrast, cell counts for encapsulated ones increased during exposure to intestinal juice due to a distinct release of encapsulated bacteria reaching a survival rate of 54% after 3 h. Thus, after 5 h in digestive juices, nonencapsulated L. reuteri revealed 86% loss of living cells, whereas processed bacteria revealed a loss of 54% under simulated gastrointestinal conditions. Therefore, survival rate of L. reuteri increased approximately 32% with encapsulation in whey matrix compared with nonprocessed ones.

The statistical analysis testified the difference between the reduction in cell counts of free and encapsulated cells to be significant. Results show an improved survival rate of L. reuteri in encapsulated forms.

Discussion

Aside lactose, vitamins and several minerals, whey contains a complex protein composition predominated by β-lactoglobulin and α-lactalbumin. These proteins possess a distinct amino acid profile and are a significant nitrogen source for bacteria, whereas whey lactose is a relevant carbohydrate source. In this study, the nutritive value of whey and the distinct characteristics of solubility, denaturation, dissociation and aggregation dependent on pH value and temperature of these components were used to cultivate probiotic L. reuteri (DSM 20016) for a coupled encapsulation process within this matrix by spray drying instead of harvesting the bacteria out of the fermentation medium and resuspending in encapsulation material. In this study, L. reuteri reached cell counts of 2·2 × 108 CFU g−1 in pure whey. On the one hand, bacterial β-galactosidase activity is necessary to metabolize lactose, which is generally shown by L. reuteri (Hidalgo-Morales et al. 2011). Most LABs are able to digest whey due to the high proteolytic activity (Pescuma et al. 2012). However, L. reuteri possesses low proteolytic activity (Hidalgo-Morales et al. 2011), and therefore, supplementation with extra nitrogen sources might improve growth. Parente and Zottola (1991) suggested supporting LAB cultivation in native whey with nutritive complex additives such as yeast extract. Furthermore, an increased whey protein concentration to raise nitrogen source for the bacteria has been described to be helpful (Bury et al. 1998). In this study, the bacterial growth could be improved to maximum cell counts of up to 2·7 × 109 CFU g−1 with yeast extract supplementation.

For encapsulation, bacteria in stationary phase with reduced proliferation rate, less physiological activity and increased resistance to stress seem to be most appropriate for spray drying. Corcoran et al. (2004) revealed approximately 50% higher survival rates of L. reuteri within spray-drying processes if bacteria were taken from stationary-phase cultures. Further, for spray-drying processes, matrices with 20% solid content are recommended (Desmond et al. 2001; Ananta et al. 2005). In consequence, to couple cultivation and spray-drying process, bacterial growth up to stationary phase should be possible in 20% whey slurry fermentation. In this study, L. reuteri reached the stationary phase at least after 24 h in a slurry fermentation in CSTR.

Generally, spray drying is an effective way to produce probiotic encapsulation with high survival rates and improved resistance during gastric transit (Malmo et al. 2011; Paéz et al. 2012). One critical factor for bacterial survival within the drying process is the exposure to hot air which leads to heat and osmotic stress for the cells. Otherwise, high temperatures are required to facilitate sufficient water evaporation along the process as dried product contains at best moisture of 4 and 7% for good stability of dried implementations throughout storage (Ananta et al. 2005). Hereby, heat sensitivity of the bacterial strain and hygroscopic properties of the encapsulation material are important factors (Ananta et al. 2005; Golowczyc et al. 2011). Survival of heat-sensitive L. paracasei during spray drying ranged from 97% (outlet temperature 70–75°C) to 0% (outlet temperature 120°C) (Gardiner et al. 2000). Other Lactobacilli strains spray-dried in skim milk at 85°C outlet temperature reached higher survival rates (loss of 0·16–0·95) (Paéz et al. 2012). Malmo et al. (2011) revealed a loss of 1·6–3·1 log of L. reuteri (DSM 17938) after spray drying with alginate matrix at 72°C outlet temperature. Higher survival rates of spray-dried L. reuteri (KUB-AC5) were measured at 70°C outlet temperature with a variation of 83–93% survival depending on the growth media (Hamsupo et al. 2005). Taking the distinct heat sensitivity of bacteria into consideration, the viability can be enhanced by lowering the outlet temperature (Lian et al. 2002; Meng et al. 2008). In this study, both outlet temperatures 55 and 65°C lead to sufficient dry powder with a negligible viable cell decrease of 2 log cycles (2·13% mean survival rate) after spray drying due to dehydration and thermal damages of bacterial cell structures (Fig. 1).

Although small-sized particles are less affecting textural and sensorial food qualities, the particle size must at least enable total bacterial encapsulation (Chen and Subirade 2006). In this study, the process produced small particles sized 5 μm in mean (Table 1), which are large enough to encapsulate L. reuteri cells (Anal and Singh 2007). Generally, decreasing moisture content correlates with an increasing temperature in the drying process (Lian et al. 2002; Anandharamakrishnan et al. 2008). In this study, however, neither particle size nor moisture content of capsule parameters were significantly affected by chosen outlet temperatures of the drying process. Desmond et al. (2001) described the possibility that milk proteins are useful to protect probiotic cultures during heating. Furthermore, lactose from whey protein might synergistically improve the micro-organism-protecting properties of these proteins. Young et al. (1993) discovered synergistic effect of whey proteins and carbohydrates for microencapsulation by spray drying. They described higher microencapsulation efficiency using the combination of whey protein and carbohydrates. Additionally, an increased thermotolerance of LAB resulting in high survival rates during storage was observed if growth media contained lactose (Carvalho et al. 2004). The protection of dehydrated biomaterials by sugars is mainly due to hydrogen bonds with the proteins when water is removed (Rokka and Rantamäki 2010). And lactose has been discussed to stabilize the bacterial cell wall by inducing a crusty glass phase (Rosenberg and Sheu 1996). Also, Rokka and Rantamäki (2010) underlined that skim milk and lactose, often in combination with milk proteins, are widely used as protective agents within spray-drying processes.

As dietary products contain at least 106 CFU ml−1 bacteria, the survival of probiotics during storage is important to assess product applicability (Kailaspathy and Chin 2000). Different studies have illustrated that the bacterial survival rate was most sufficient at 4°C storage temperature (Corcoran et al. 2004; Silva et al. 2011). In this study, the examined loss in viable cells was at maximum 1 log cycle after a storage period of 4 weeks at 4°C (Fig. 1). Depending on the outlet temperature, the viable counts during storage decreased at 65°C by about 1 log cycle and at 55°C by <1 log cycle, which might be due to physiological heat stress response and disturbed metabolic mechanisms. Also, Hamsupo et al. (2005) obtained an approximately decreased survival rate of 1 log cycle at 4°C storage for 2 months with skim milk spray-dried L. reuteri (KUB-05). Even for sensitive, strictly anaerobic bifidobacteria, whey possesses excellent cell-protective properties at 4°C storage (De Castro-Cislaghi et al. 2012).

To fulfil all health beneficial functions, probiotic bacteria have to reach the terminal ileum and colon alive. The highly acidic stomach and bile salts secreted into the duodenum harm the ingested bacteria (De Castro-Cislaghi et al. 2012). Whey proteins remain stable at acidic pH due to aggregation, whereas after isoelectric point (pH 4·6), solubility improves with increasing pH (Pelegrine and Gasparetto 2005; Gbassi et al. 2009; Dissanayake et al. 2013). Recent studies already have shown that whey protein–based microencapsulation protects bacteria against acid stress, allowing the cells to survive in the stomach and improve cell survival of probiotic LAB in the human GIT (Rokka and Rantamäki 2010; De Castro-Cislaghi et al. 2012). In this study, the survival rate of whey-microencapsulated L. reuteri in simulated gastric juices is 32% higher compared with nonencapsulated ones (Fig. 2). In contrast, no significant increase in survival of calcium alginate–encapsulated L. reuteri under simulated gastric conditions was found by Sultana et al. (2000) and Malmo et al. (2011).

Interestingly, the lactose in whey protein again seems to support this protective effect synergistically. López-Rubio et al. (2012) underlined that bacteria encapsulated with carbohydrate only were less protected compared with whey protein–encapsulated ones. Different combinations of alginate with starch for extrusion and emulsion revealed increased survival of microencapsulated L. reuteri strains in simulated gastric juice (2 h, pH 1·5) compared with free cells (Muthukumarasamy et al. 2006). In contrast, Lactobacilli microencapsulated in alginate and pectin were not protected against acidic milieu (Brinques and Ayub 2011).

Aside from stomach and bile acids, digestive enzymes of the duodenum are relevant. Doherty et al. (2012) have demonstrated that whey protein capsules are pepsin resistant. Whereas β-lactoglobulin is not digestible by gastric pepsin, distinct peptide chains of whey α-lactalbumin and bovine serum albumin are degraded (Gbassi et al. 2009). In this study, neither did free nor did encapsulated bacteria reveal significant loss of living cells after 5-h incubation in artificial intestinal juice. These results are in accordance with a study by Muthukumarasamy et al. (2006), which demonstrated that several strains of L. reuteri survived in bile juice for several hours. Dommels et al. (2009) also reported the resistance of L. reuteri to bovine bile.

However, increased cell counts of encapsulated L. reuteri during incubation in artificial intestinal juice revealed a release of encapsulated bacteria due to a pH value shift from 1·9 of gastric juice to 7·5 of intestinal juice and probably due to whey protein degradation by pancreatin (Fig. 2). Accordingly, Chen and Subirade (2006) described a release of whey protein capsule core material into simulated intestinal juice with pancreatin.

In conclusion, whey exploited as growth substrate and encapsulation matrix within a coupled fermentation and spray-drying process might offer an efficient option for industrial production of vital probiotics. The resulting microcapsules remain stable during storage and reveal adequate survival in simulated gastric juices and a distinct release in intestinal juices. While lactose is supportive but also problematic in this coupled process, like unwanted caramelization or stickiness to the dryer wall, the influence of the whey-protein-to-lactose ratio on the process and product characteristics remains to be investigated.

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