Microencapsulation and supply of Bifidobacterium lactis DSM 10140 in fermented traditional African beverages

Abstract

Probiotic foods are intended to supply selected viable microorganisms, for example Lactobacillus acidophilus and Bifidobacterium, to consumers. These organisms, when consumed at the daily intake of 108 , provide benefits beyond basic nutrition. Probiotic (AB) foods generally include fermented dairy products such as yoghurts and cheeses, targeted at the upmarket consumer. However, due to technical problems associated with the foods and the organism, viable Bifidobacterium rarely occur in AB foods. The principle aims of this study were to develop a suitable delivery system for Bifidobacterium to the consumer, and to supply these living organisms in the affordable traditional fermented African beverages, amasi and mahewu. This would provide the benefits of probiotics to the rural African consumer, where malnutrition and gastrointestinal diseases occur. The organism selected for this study was Bifidobacterium lactis DSM 10140, commonly associated with AB starter cultures for yoghurts. The delivery system selected was microencapsulation of B. lactis using a mixture of the generally recognised as safe (GRAS) edible gums, gellan and xanthan. Supply vehicles for the microcapsules to the consumer were amasi and mahewu. Prior to microencapsulation, rheological studies were undertaken to determine whether the gellan-xanthan gum mix would provide a suitable support matrix for microencapsulated B. lactis. This was done using a Paar Physica MGR 300 rotational rheometer with a cone plate 50-2 measuring system. Results indicated that the hydrated gellan-xanthan gum mix behaved as a non-Newtonian material, and the flow curve fitted well to the Herschel-Bulkley model. This demonstrated that the gel was a relatively viscous material with solid properties. The average yield stress of the gel was 1.515 Pa, indicating that the gel was stable, and at lower stresses would behave as a solid. The gel mix would be disrupted by shear stresses associated with mastication and peristalsis. The minimum viscosity of the gel was constant at temperatures between 46°C - 61°C. It was concluded from these data that the gel was suitable for microencapsulation and that microcapsules should only be included in soft foods, which do not require chewing. Temperatures associated with microencapsulation, at minimum gel viscosities, were not lethal to B. lactis. Bifidobacterium lactis cells were incubated under anaerobic conditions (4% H2, 10% CO2, and 86% N2) at 37°C overnight in 250 ml Tryptone-Yeast-Glucose (TYG) broth, and grown to an 00600 0.9 - 1.1. Cells were harvested and washed for microencapsulation using centrifugation. Microencapsulation of the organism was done using a mono-axial extrusion technique together with a superposed airflow, by manually extruding the aqueous gum I cell mix through a 27.5 G bevelled needle, fitted on to a 10 ml syringe. The resultant microdroplets were hardened by free fall into 0.1 M CaCI2 solution. Microcapsules were separated from the CaCI2 solution by filtration through Whatman No.1 filter paper. All procedures were carried out in a laminar flow hood. Results indicated that the method of microencapsulation used in this study was successful. Using a concentrated inoculum of B. lactis, high numbers (lOglO 11-12 etu.g-1 ) of bacteria were incorporated into the microcapsules. Therefore the daily intake would be provided by 0.1 g microcapsules. The diameter and size distribution of microcapsules were determined by laser diffractometry. This showed a maximum microcapsule diameter of 2.22 mm with 50% (w/v) of the microcapsules having a diameter of < 0.637 mm. Although this represents a considerable size variation, this would not adversely affect mouthfeel of the beverages, as only 0.1 g microcapsules would be required to obtain at least 108 B. lactis in any volume of amasi or mahewu. To enumerate immobilised viable B. lactis, two techniques were compared. These involved the use of either a pestle and mortar, or high power ultrasound (HPUS) (20 kHz, 750 W). Results showed that HPUS was superior to the pestle and mortar technique. A short exposure (15 s) to HPUS disrupted the matrix releasing all entrapped etus, whereas when using the pestle and mortar xiii technique, cells remained partially entrapped in the gel. Therefore the pestle and mortar technique yielded lower cfu values than expected. The survival of microencapsulated B. lactis, in 1 M sodium phosphate buffer, was studied as a possible means of supply of microcapsules to industry for incorporation into foods. Microcapsules were stored in the buffer for 21 days at either 4°C or 22°C. Results showed that cell viability was not significantly reduced (p>0.05) at either temperature after 21 days. Hence this form of storage could be used to deliver viable immobilised B. lactis to the food industry. In order to assess the survival of immobilised B. lactis in the GIT, the microcapsules were incubated at 37°C over a period of 240 min in simulated gastric juice (SGJ) (pH 1.5). Viable counts were performed by sampling at regular intervals. A similar study was done in simulated bile and pancreatic juices (BPJ) (pH 6.5). In SGJ, it was demonstrated that there was a significant reduction (3 log cycles) (p<0.05) of free cells after 240 min. However, this trend was not noted for microencapsulated B. lactis. Therefore, the gellanxanthan gel matrix protected B. lactis from the lethal effect of SGJ. In BPJ, no significant difference (p>0.05) was noted for surviving fractions of both immobilised and free B. lactis. Commercial pasteurised amasi (pH 4.4) and mahewu (pH 3.5) were selected as the supply vehicles for the microencapsulated B. lactis. Known numbers of viable microencapsulated and free B. lactis cells were added to both beverages. For most samples, incubation was at either 4°C or 22°C for 21 days in the presence of atmospheric oxygen. In addition, free cells were incubated anaerobically at 22°C. As oxygen is limiting in the microcapsules, these were not incubated under anaerobic conditions. The survival I shelf-life studies of commercial amasi indicated no significant difference (p>0.05) in survival rate between immobilised and free B. lactis cells. The reduction noted for viable counts of immobilised or free B. lactis cells was approximately 1.5 log cycles. Even so, after 21 days viable immobilised B. lactis (1010 0.1 g'l microcapsules) remained in excess of the daily intake 108 , whereas in the free B. lactis cells, the viable count declined to 106 mr1 . Statistical analyses showed that temperature or oxygen presence had little effect on the survival of both immobilised or free B. lactis cells (p>O.05). In mahewu, decline in viability of cells was observed for most samples. However microencapsulation enhanced cell survival at both 4°C and 22°C when compared to free cells. The decrease in viable B. lactis free cells occurred more rapidly (3 log cycles) in mahewu, than in amasi, at both 4°C and 22°C. Throughout the shelf-life studies it was apparent that viable B. lactis cell numbers did not increase. This was advantageous as metabolites associated with B. lactis growth would have adversely altered the taste of both amasi and mahewu. Sensory evaluation of the traditional fermented African beverages, enriched with either viable immobilised or free B. lactis, was done in order to determine consumer response to the product. An analytically trained 12-member taste panel analysed the beverages for colour, texture, and taste. The triangle taste test procedure was used. No differences were detected with regard to texture, and colour of the fermented beverages containing immobilised B. lactis. However, in the fermented beverages containing free cells, a change in viscosity was noted. There was a significant difference (p<O.05) recorded in flavour for both amasi and mahewu containing free B. lactis cells. In the two fermented beverages enriched with immobilised cells, significant (p<O.05) flavour differences were detected in mahewu. However, this was not observed in the amasi samples containing immobilised B. lactis. Therefore, in order to retain the sensory properties of amasi, B. lactis should be supplied in microcapsules. In mahewu, although flavour differences noted were not unpleasant to the panellists, results from this study indicate that the use of commercial flavoured mahewu should be considered as a supply vehicle for microencapsulated B. lactis. Overall, this study demonstrated that immobilisation of B. lactis in gellan-xanthan gum is possible. Microcapsules produced contained high numbers of viable B. lactis, and were suitable for incorporation into soft foods. The gel matrix significantly protected viable cells from harsh conditions associated with SGJ. Although the surviving fraction of immobilised cells, when compared to free cells, was not improved in amasi samples, it is recommended that for technological reasons associated with production of amasi, microencapsulation should be used. In mahewu, microencapsulation enhanced B. lactis survival at both 4°C and 22°C. Therefore immobilisation of B. lactis in mahewu is necessary in order to maintain the daily intake. Immobilised B. lactis should be incorporated into both beverages after fermentation, and pasteurisation.