Impact of microencapsulated peptidase (Aspergillus oryzae) on cheddar cheese proteolysis and its biologically active peptide profile.

We investigated the delivery of calcium-alginate encapsulated peptidase (Flavourzyme(®), Aspergillus oryzae) on proteolysis of Cheddar cheese. Physical and chemical characteristics such as moisture, pH and fat content were measured, and no differences were found between control and experimental cheese at day 0. SDS-PAGE analysis clearly showed that proteolysis of α and k casein was significantly accelerated after three months of maturity in the experimental cheese. A large number of low molecular weight peptides were found in the water soluble fraction of the experimental cheeses and some of these peptides were new. N-terminal amino acid sequence analysis identified these as P(1), Leu-Thu-Glu; P(3), Asp-Val-Pro-Ser-Glu) and relatively abundant stable peptides P(2), P(4), Arg-Pro-Lys-His-Pro-Ile; P(5), Arg-Pro-Lys-His-Pro-Ile-Lys and P(6). These peptides were mainly originated from αs1-CN and ß-CN. Three of the identified peptides (P(1), P(2), P(3) and P(4)) are known to biologically active and P(1) and P(3) were only present in experimental cheese suggesting that experimental cheese has improved health benefits.

Diversity analysis of commensal porcine Escherichia coli - associations between genotypes and habitat in the porcine gastrointestinal tract.

Diversity studies of enteric Escherichia coli have relied almost entirely on faecal isolations on the assumption that they are representative of flora found throughout the gastrointestinal tract. The authors have addressed this belief by analysing isolates obtained from the duodenum, ileum, colon and faeces of pigs. E. coli isolates were obtained from eight pigs and characterized using multi-locus enzyme electrophoresis and PCR-based screening for a range of factors thought to be associated with intestinal and extra-intestinal disease. There are four main genetic groups of commensal E. coli (A, B1, B2, D). Group A strains represented 76 % of the isolates from the duodenum, ileum and colon compared to 58 % of the strains isolated from faeces. A nested molecular analysis of variance based on the allozyme and virulence factor screening results showed that differences among individual pigs accounted for 6 % of the observed genetic diversity, whilst 27 % of the genetic variation could be explained by clonal composition differences among gut regions. Finally, the absence of virulence genes in these commensals indicates that they may be suitable as a probiotic consortium, particularly if they also display increased adherence to enterocytes and antagonistic activity against pathogenic strains of E. coli.

The role of oxygen in the viability of probiotic bacteria with reference to L. acidophilus and Bifidobacterium spp.

The various therapeutic benefits of Lactobacillus acidophilus and Bifidobacterium spp. have resulted in their increased incorporation into dairy foods such as yoghurts. Currently however, the efficacy of these probiotic bacteria is limited by their poor survival during the shelf life of yoghurt. Oxygen toxicity is widely considered to be responsible for the cell deaths of these bacteria. The intestinal origins and the microaerophilic and anaerobic characteristics of L. acidophilus and Bifidobacterium spp. respectively, can render them susceptible to oxygen contained in the food products. This review discusses the influence of the dissolved oxygen in yogurt on the viability of these bacteria. Suggested techniques to protect these probiotic bacteria from oxygen toxicity are evaluated. Although the problem of oxygen toxicity in probiotic bacteria is regarded as significant, little is known however about the cellular interaction of these bacteria with oxygen. This review summarizes what is known about the biochemistry of oxygen toxicity in these bacteria. The various metabolic and biochemical responses of L. acidophilus and Bifidobacterium to oxygen are examined. Additionally, the importance of NADH oxidase and NADH peroxidase in the oxygen tolerance of these bacteria is evaluated and assays used to measure their cellular concentrations are discussed.

An improved method for the determination of NADH oxidase in the presence of NADH peroxidase in lactic acid bacteria.

The complexity of the coupled NADH oxidase-NADH peroxidase enzyme system in lactic acid bacteria makes it difficult to simultaneously determine the individual levels of both these enzymes spectrophotometrically. This study describes an improved assay to accurately determine low concentrations of NADH oxidase from enzyme suspensions containing NADH oxidase and NADH peroxidase. For the standardisation of the assay, pure NADH oxidase and NADH peroxidase were mixed in various proportions and the percentage recovery was estimated by both the currently available assay as well as by the improved assay reported in this study. The recovery of NADH oxidase using the currently available assay ranged from as low as -200% to as high as +102% as against 90-102% in the improved assay. The recovery percentage of NADH peroxidase ranged from 91% to 112% in both assays. The slopes of NADH oxidation by cell-free extracts of six lactic acid bacteria were also measured by both assays for the estimation of NADH oxidase and NADH peroxidase levels. The improved assay can further distinguish between NADH-H(2)O oxidase and NADH-H(2)O(2) oxidase and was successfully applied to identify the type of NADH oxidase in the lactic acid bacteria tested.

Microencapsulation of probiotic bacteria: technology and potential applications.

In the recent past, there has been an explosion of probiotic health-based products. Many reports indicated that there is poor survival of probiotic bacteria in these products. Further, the survival of these bacteria in the human gastro-intestinal system is questionable. Providing probiotic living cells with a physical barrier against adverse environmental conditions is therefore an approach currently receiving considerable interest. The technology of micro-encapsulation of probiotic bacterial cells evolved from the immobilised cell culture technology used in the biotechnological industry. Several methods of micro-encapsulation of probiotic bacteria have been reported and include spray drying, extrusion, emulsion and phase separation. None of these reported methods however, has resulted in the large numbers of shelf-stable, viable probiotic bacterial cells necessary for use in industry for development of new probiotic products. The most commonly reported micro-encapsulation procedure is based on the calcium-alginate gel capsule formation. Kappa-carrageenan, gellan gum, gelatin and starch are also used as excipients for the micro-encapsulation of probiotic bacteria. The currently available equipment for micro-encapsulation is not able to generate large quantities of uniform sized micro or nano capsules. There is a need to design and develop equipment that will be able to generate precise and uniform micro or nano capsules in large quantities for industrial applications. The reported food vehicles for delivery of encapsulated probiotic bacteria are yoghurt, cheese, ice cream and mayonnaise. Studies need to be done on the application of micro-encapsulation of probiotic bacteria in other food systems. The number of probiotic supplements will increase in the future. More studies, however, need to be conducted on the efficacy of micro-encapsulation to deliver probiotic bacteria and their controlled or targeted release in the gastrointestinal tract.