Natural flavor biosynthesis by lipase in fermented milk using in situ produced ethanol.

ABSTRACT: Many flavoring agents on the market are extracted from natural sources or synthesized chemically. Due to the disadvantages of both methods, biotechnology is becoming a promising alternative. In this study, short chain ethyl esters with fruity notes were biosynthesized in UHT whole milk via coupling ethanolic fermentation with lipase (Palatase®) transesterification. Kluyveromyces marxianus, Lactobacillus fermentum and Lb. paracasei were used for fermentation. Milk fat was esterified with in situ produced ethanol by adding lipase at 0, 8 and 24 h of fermentation. Viable cell counts and pH were monitored during 48 h fermentation period. Flavor active ethyl esters, ethanol and free fatty acids were analyzed using headspace SPME-GC. Free fatty acid levels were lower in K. marxianus samples than lactobacilli. K. marxianus produced higher amounts of ethanol and esters than lactic acid bacteria. Viable cell counts decreased after lipase application at 0 and 8 h, possibly due to fatty acid production. Addition of lipase at 24 h resulted in improved cell counts as well as ethanol and ester production in the case of K. marxianus. This study demonstrated that fermenting milk with alcohol producing cultures in conjunction with lipase application can be an alternative to artificial flavorings in fermented milks.

Survival of probiotic strain Lactobacillus paracasei L26 during co-fermentation with S. cerevisiae for the development of a novel beer beverage.

Amidst the rising popularity of craft beers, it would be opportune to develop a novel, unfiltered and unpasteurized sour beer with high probiotic live counts. However, as beer typically contains hop iso-α-acids that prevent the growth and survival of probiotic lactic acid bacteria, the use of suitable fermentation strategies is crucial. The growth, and survival of the probiotic bacterium, Lactobacillus paracasei L26, were assessed during a 10-day co-fermentation period with a brewer's yeast, Saccharomyces cerevisiae S-04, in unhopped wort. Isomerized hop extract was added prior to storage of the beers at 25 °C and 5 °C. During co-fermentation in unhopped wort, L. paracasei L26 maintained high viable cell counts above 8 Log CFU/mL, indicating species compatibility with the yeast. The majority of fermentable sugars were attenuated by S. cerevisiae S-04, with a concomitant production of alcohols and esters. Significant amounts of lactic acid were produced by L. paracasei L26 (P < 0.05). During storage with added isomerized hop extract, maximal probiotic viability enhancing effects were observed in the presence of live S. cerevisiae S-04, in combination with refrigeration. The results suggest that beers could be a vehicle for probiotic delivery under appropriate conditions. This was the first study demonstrating the feasibility of utilizing probiotic lactobacilli as starter cultures in beer brewing.

Saccharomyces cerevisiae EC-1118 enhances the survivability of probiotic Lactobacillus rhamnosus HN001 in an acidic environment.

The present study attempted to partially characterize and elucidate the viability-enhancing effect of a yeast strain Saccharomyces cerevisiae EC-1118 on a probiotic strain Lactobacillus rhamnosus HN001 under acidic conditions using a model system (non-growing cells). The yeast was found to significantly enhance (P < 0.05) the viability of the probiotic strain under acidic conditions (pH 2.5 to 4.0) by 2 to 4 log cycles, and the viability-enhancing effects were observed to be influenced by pH, and probiotic and yeast concentrations. Microscopic observation and co-aggregation assay revealed that the viability-enhancing effect of the yeast could be attributed to direct cell-cell contact co-aggregation mediated by yeast cell surface and/or cell wall components or metabolites. Furthermore, non-viable yeast cells killed by thermal means were observed to enhance the viability of the probiotic strain as well, suggesting that the surface and/or cell wall component(s) of the yeast contributing to co-aggregation was heat-stable. Cell-free yeast supernatant was also found to enhance the viability of the probiotic strain, indicating the presence of protective yeast metabolite(s) in the supernatant. These findings laid the foundation for further understanding of the mechanism(s) involved and for developing novel microbial starter cultures possibly without the use of live yeast for ambient-stable high-moisture probiotic foods.

Biovalorization of Market Surplus Bread for Development of Probiotic-Fermented Potential Functional Beverages.

Bread wastage is a growing concern in many developed countries. This research aimed to explore the biovalorization of market surplus bread for the development of probiotic-fermented beverages in a zero-waste approach. Bread slurries with different initial total solid contents were inoculated with probiotics Lacticaseibacillus rhamnosus GG (LGG) and Saccharomyces cerevisiae CNCM I-3856, alone and in combination. Our results showed that, of all percentages tested, 5% (w/w, dry weight) initial total solid content resulted in better growth of the probiotics and higher cell counts, while the texture of bread slurries with concentrations higher than 5.0% was too thick and viscous for bread beverage developments. In addition, the development of probiotic-fermented bread beverages was feasible on various types of bread. Furthermore, food additives (sweetener and stabilizer) did not affect the growth of LGG and S. cerevisiae CNCM I-3856 in both mono- and co-culture fermentation. During shelf life measurement, co-inoculation of LGG with S. cerevisiae CNCM I-3856 significantly improved the survival of LGG compared to the mono-culture at 5 and 30 °C, demonstrating the protective effects provided by the yeast. Our study suggests the potential of using market surplus bread as raw materials to deliver live probiotics with sufficient cell counts.

Growth, survival, and metabolic activities of probiotics Lactobacillus rhamnosus GG and Saccharomyces cerevisiae var. boulardii CNCM-I745 in fermented coffee brews.

Amidst rising demand for non-dairy probiotic foods, and growing interest in coffees with added functionalities, it would be opportune to ferment coffee brews with probiotics. However, challenges exist in maintaining probiotic viability in high-moisture food products. Here, we aimed to enhance the viability of the probiotic bacteria, Lactobacillus rhamnosus GG, in coffee brews by co-culturing with the probiotic yeast, Saccharomyces cerevisiae var. boulardii CNCM-I745. The yeast significantly enhanced the viability of L. rhamnosus GG, as bacterial populations beyond 7 Log CFU/mL were maintained throughout 14 weeks of storage at 4 and 25 °C. In contrast, the single culture of L. rhamnosus GG suffered viability losses below 6 Log CFU/mL within 10 weeks at 4 °C, and 3 weeks at 25 °C. Growth and survival of S. boulardii CNCM-I745 remained unaffected by the presence of L. rhamnosus GG. Volatile profiles of coffee brews were altered by probiotic metabolic activities, but co-culturing led to suppressed generation of diacetyl and ethanol compared to single cultures. Probiotic fermentation did not alter principal coffee bioactive compounds and antioxidant capacities; however, declines in peroxyl radical scavenging capacities were observed after ambient storage. Overall, we illustrate that yeasts are effective in enhancing probiotic bacterial viability in coffee brews, which may be useful in developing shelf stable probiotic food products.

Targeted and Nontargeted Metabolomics of Amino Acids and Bioactive Metabolites in Probiotic-Fermented Unhopped Beers Using Liquid Chromatography High-Resolution Mass Spectrometry.

Beer is one of the most popular beverages in the world. The increased popularity of craft beers has led to the development of unique beers that are alcohol-free, gluten-free, low calorie, or with functional properties through fermentation with probiotic microorganisms. In this study, functional unhopped beers were evaluated by utilizing probiotics (Lacticaseibacillus paracasei Lpc-37 and ibSium Saccharomyces cerevisiae CNCM I-3856) as starter cultures. The metabolites produced by probiotics were investigated using a nontargeted metabolomics approach and identified against metabolomics databases (Kyoto Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Database (HMDB), Yeast Metabolome Database (YMDB), METLIN tandem mass spectrometry (MS/MS)). Derivatives of branched-chain (leucine) and aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were enriched (one-way analysis of variance (ANOVA) p < 0.05) in probiotic-fermented unhopped beers, especially tryptophan metabolites. In addition, the synergistic effects of yeast-lactic acid bacteria (LAB) interactions led to further enrichment of higher acids such as (S)-(-)-2-hydroxyisocaproic acid, phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid. The potential pathways for the formation of novel bioactive tryptophan metabolites (indole and indoleacrylic acid) by LAB were elucidated. Altogether, probiotic LAB-fermented unhopped beer showed the highest antioxidant capacity and total phenolic content. This work provides the basis for the discovery of bioactive metabolites in probiotic-fermented foods.

Growth, survival, and metabolic activities of probiotic Lactobacillus spp. in fermented coffee brews supplemented with glucose and inactivated yeast derivatives.

Amidst rising interest in non-dairy probiotic foods, and growing global coffee consumption patterns, it would be opportune to ferment coffee brews with probiotics, yet it remains unexplored. In this study, we aimed to develop a fermented coffee beverage rich in live probiotics, by supplementing nutrient-deficient coffee brews with glucose and inactivated yeast derivatives. This was followed by fermentation with single probiotic bacteria cultures (Lactobacillus rhamnosus GG, L. paracasei Lpc-37, L. plantarum 299v, and L. acidophilus NCFM), and subsequent storage at 4 and 25 °C. We demonstrated that nutrient supplementation was essential in supporting probiotic growth and survival in coffee brews, as viabilities above 7 Log CFU/mL could not be sustained longer than 2 weeks in non-supplemented coffees. In contrast, viabilities above 7 Log CFU/mL were maintained for 10 weeks by L. rhamnosus GG and L. paracasei Lpc-37 in supplemented coffees stored under refrigeration. Probiotic metabolic activities led to consumption of glucose, glutamic acid, and alanine, with simultaneous formation of lactic acid, 3-methylbutanoic acid, and diacetyl. Nevertheless, endogenous coffee volatiles, bioactive components, and antioxidant capacities were retained. Overall, we illustrate the potential functionalities of probiotic fermented coffee brews, arising from high probiotic live counts and retention of major coffee bioactive components.

Influence of commercial inactivated yeast derivatives on the survival of probiotic bacterium Lactobacillus rhamnosus HN001 in an acidic environment.

This study evaluated the influence of three inactivated yeast derivatives (IYDs) used in wine production, namely OptiRed®, OptiWhite® and Noblesse®, on the viability of the probiotic strain Lactobacillus rhamnosus HN001 in an acidic environment. Addition of the IYDs at 3 g/L significantly enhanced the survival of the probiotic bacteria by 2.75-4.05 log cycles after 10-h exposure in a pH 3.0 buffer. Acid stress assay with IYD components obtained after centrifugation and filtration revealed that water-soluble compounds were responsible for improving the acid tolerance of L. rhamnosus HN001 for all three preparations. Differences in protective effect amongst the IYDs on L. rhamnosus HN001 were observed when permeates and retentates of the water-soluble extracts, obtained through ultrafiltration with a 2 kDa membrane, were assayed against the lactic acid bacterium. Chemical analysis of the water-soluble components suggests that low molecular weight polysaccharides, specific free amino acids and/or antioxidants in the 2 kDa permeates could have contributed to the enhanced survival of L. rhamnosus HN001 during acid stress. The contrast amongst the 2 kDa retentates' viability enhancing property may have been attributed to the differences in size and structure of the higher molecular weight carbohydrates and proteins, as the survival of the probiotic did not relate to the concentration of these compounds. These results suggests that oenological IYDs could potentially be applied to probiotic foods for enhancing the acid tolerance of the beneficial microorganisms, and consequently prolonging the shelf life of these products.