Effect of cell-surface components and metabolites of lactic acid bacteria and probiotic organisms on cytokine production and induction of CD25 expression in human peripheral mononuclear cells

In the current study, the relative contribution of cell-surface components (CSC) and cell-free supernatants (CFS) in the immuno-modulatory properties of 17 strains of probiotic and lactic acid bacteria (LAB) was assessed. The production of pro- and antiinflammatory cytokines including IL-2, IL-4, IL-10, IL-12 p70, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β was measured at different time points after stimulation of buffy coat derived-peripheral blood mononuclear cells (PBMC) from healthy donors with CSC and CFS of probiotic and LAB. Results showed that CSC of probiotic and LAB strains induced production of T helper 1 and 2 type cytokines. Transforming growth factor-β was stimulated at highest concentrations, followed by IL-10 and TNF-α. The CFS of all tested bacterial strains induced PBMC for significantly high levels of IL-10 secretion compared with unstimulated cells, but the values were less than lipopolysaccharide-stimulated cells. Cytokines due to CFS stimulation showed declined concentration for IL-2, TNF-α, and IL-4, and complete disappearance of IL-12, IFN-γ, and transforming growth factor-β in the cultured medium at 96 h of incubation. Results of cytokine data demonstrate proinflammatory TNF-α immune responses are mainly directed through cell-surface structures of probiotic and LAB, but antiinflammatory immune responses are mediated both by metabolites and cell-surfaces of these bacteria. The induction of CD4(+)CD25(+) regulatory T cells after stimulation of PBMC with CSC and CFS of probiotic and LAB showed regulatory T cell activity appeared to be influenced both by the CSC and metabolites, but was principally triggered by cell surfaces of probiotic and LAB strains.

Survival and fermentation activity of probiotic bacteria and oxidative stability of omega-3 oil in co-microcapsules during storage

Tuna oil (O) and probiotic bacteria Lactobacillus casei (P) were co-microencapsulated in whey protein isolate (WPI)-gum Arabic (GA) complex coacervate. The co-microcapsules (WPI-P-O-GA), L. casei microcapsules (WPI-P-GA) and tuna oil microcapsules (WPI-O-GA) were converted into powder using spray and freeze drying. The interaction between probiotic bacteria and omega-3 oil in co-microcapsules, particularly in terms of oxidative stability of omega-3 oil and vitality/viability of probiotic bacteria and any synergistic outcome, was studied. The effect of storage temperature (5 and 25 °C) and time (90 days) on the survival and fermentation activity of L. casei and oxidative stability of tuna oil in the microcapsules/co-microcapsules was determined. A synergism between oxidative stability of omega-3 oil and vitality of probiotic bacteria was observed, when they were co-microencapsulated and spray dried. These co-microcapsules will likely have utility in functional food formulations due to simple and cost effective stabilisation and delivery of two important functional ingredients.

Survival, oxidative stability, and surface characteristics of spray dried co-microcapsules containing omega-3 fatty acids and probiotic bacteria

The objective of the study was to determine optimum inlet and outlet air temperatures of spray process for producing co-microcapsules containing omega-3 rich tuna oil and probiotic bacteria L. casei. These co-microcapsules were produced using whey protein isolate and gum Arabic complex coacervates as shell materials. Improved bacterial viability and oxidative stability of omega-3 oil were used as two main criteria of this study. Three sets of inlet (130°C, 150°C, and 170°C) and outlet (55°C, 65°C, and 75°C) air temperatures were used in nine combinations to produce powdered co-microcapsule. The viability of L. casei, oxidative stability of omega-3 oil, surface oil, oil microencapsulation efficiency, moisture content, surface elemental composition and morphology of the powdered samples were measured. There is no statistical difference in oxidative stability at two lower inlet air temperatures (130°C and 150°C). However, there was a significant decrease in oxidative stability when higher inlet temperature (170°C) was used. The viability of L. casei decreased with the increase in the inlet and outlet air temperatures. There was no difference in the surface elemental compositions and surface morphology of powdered co-microcapsules produced under these nine inlet/outlet temperature combinations. Of the range of conditions tested the co-microcapsules produced at inlet-outlet temperature 130–65°C showed the highest bacterial viability and oxidative stability of omega-3 and having the moisture content of 4.93 ± 0.05% (w/w). This research shows that powdered co-microcapsules of probiotic bacteria and omega-3 fatty acids with high survival of the former and high stability against oxidation can be produced through spray drying.

Bioanalytical evaluation of Lactobacillus delbrueckii subsp. lactis 313 cell-envelope proteinase extraction

Lactobacilli cell-envelope proteinases (CEPs) have demonstrated numerous biopharmaceutical applications in the development of new streams of blockbuster nutraceuticals; thus, the development of efficient and commercially viable methods for CEP extraction will promote their full-scale application. In this study, the sub-cellular location of CEPs in Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was identified and the effects of different extraction methods were investigated for their ability to efficiently release CEPs from LDL 313. Significantly high relative proteinase activity of~95% was detected in cell-wall fractions and ~5% activity was observed for osmotic fluids, implying that proteinases in LDL 313 are cell-wall bound. CEPs were released from cell-wall via incubation in calcium-free buffer, indicating the enzyme is liable to self-digestion and ionic misfolding. Of the different extraction methods investigated, the use of 5 M LiCl was the most suitable, under the conditions of experimentation, for releasing high levels of CEPs from LDL 313.

Mixed cultures of food-grade probiotic bacteria and enteric bacteria demonstrate both synergism and inhibition on menaquinone production

The interaction between probiotic (Enterococcus spp., Lactobacillus spp., and Lactococcus spp.) and enteric (Bacteroides spp., Escherichia coli, and Salmonella spp.) bacteria with respect to menaquinone production was examined. Menaquinones were measured in cell pellets by high-pressure liquid chromatography and the main homologues produced were MIK_7–11. The growth of both Bacteroides and E. coli cultured with the 3 probiotics was significantly inhibited with concomitant reduction in menaquinone production. The vitamin K status of humans could be affected by consumption of probiotic dairy foods via the contribution made by gut microflora.

Consumer acceptability and antioxidant potential of probiotic-yogurt with spices

The study focuses on developing novel probiotic yogurts containing spices with acceptable sensory properties, therapeutic levels of probiotics and with beneficial antioxidant capacity. Eight types of yogurts with added spice oleoresins (cardamom, cinnamon and nutmeg) and probiotics [Lactobacillus acidophilus strain 5(LA5), or Bifidobacterium animalisssp.Lactis (Bb12)] were produced. Two successive consumer sensory taste panels (n = 54) using a nine point hedonic scale were conducted to evaluate the acceptability of the yogurts. Viable counts of probiotics and antioxidant capacity of yogurt samples at 1, 7, 14 and 28 days of storage at 4 °C were monitored. The probiotic-yogurt products containing spices showed good sensory properties, with the best results obtained with cardamom oleoresin (with LA5 or Bb12). The presence of spice oleoresins (cardamom, nutmeg and cinnamon) did not affect the probiotic population (LA5or Bb12) in yogurt during 4 weeks of refrigerated storage. The antioxidant capacity (with LA5or Bb12) over the storage period was also maintained.

Quick and low cost immobilization of proteinases on polyesters: comparison of lactobacilli cell-envelope proteinase and trypsin for protein degradation

Cell-envelope proteinases (CEPs) are a class of proteolytic enzymes produced by lactic acid bacteria and have several industrially relevant applications. However, soluble CEPs are economically unfavorable for such applications due to their poor stability and lack of reusability. In a quest to prepare stable biocatalysts with improved performance, CEP from Lactobacillus delbrueckii subsp. lactis 313 and trypsin (as a model enzyme) were immobilized onto nonwoven polyester fabrics in a three-step protocol including ethylenediamine activation and glutaraldehyde crosslinking. Immobilization gave protein loading yields of 21.9% (CEP) and 67.7% (trypsin) while residual activity yields were 85.6% (CEP) and 4.1% (trypsin). The activity of the immobilized enzymes was dependent on pH, but was retained at elevated temperatures (40-70°C). An increase in Km values was observed for both enzymes after immobilization. After 70 days of storage, the immobilized CEP retained ca. 62% and 96% of initial activity when the samples were stored in a lyophilized form at -20°C or in a buffer at 4°C, respectively. Both immobilized CEP and trypsin were able to hydrolyze proteins such as casein, skimmed milk proteins and bovine serum albumin. This immobilization protocol can be used to prepare immobilized biocatalyst for various protein degradation processes.

Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases, for protein degradation

Enzymatic hydrolysis is a widely used approach to improve the functional, nutritionaland physiological properties of food proteins. In this study, cross-linked enzyme aggre-gates (CLEAs) have been prepared from cell-envelope proteinases (CEPs) of Lactobacillusdelbrueckii subsp. lactis 313 and their proteolytic properties have been evaluated using severalfood proteins. We have optimized cross-linking conditions including ammonium sulphateconcentration, incubation temperatures, agitation speed, glutaraldehyde cross-linker con-centration, reaction time and the addition of proteic feeders. Particularly, the presence ofBSA improves retained activity of cross-linked CEP aggregates (CLCEPAs) from 21.5% to 40.9%.Blocking unreacted cross-linking groups on aggregates is important to enhance recyclabil-ity of CLCEPAs. CLCEPAs had attractive thermal stability at 50◦C and it showed enhancedcatalytic activity over long-term storage after lyophilization. We have demonstrated thatCLCEPAs has proteolytic properties on different food proteins including complex (chickenegg albumin, skimmed-milk protein), fractionated (bovine casein, whey protein isolate), andpurified (bovine serum albumin) proteins. Being the first report of CLEAs from lactobacilliCEPs, this study demonstrates the feasibility of using LDL 313 CLCEPAs for degradation ofvarious food proteins.