Tyrosine and phenylalanine catabolism byLactobacillus cheese flavor adjuncts

Bacterial metabolism of Tyr and Phe has been associated with the formation of aromatic compounds that impart barny-utensil and floral off-flavors in cheese. In an effort to identify possible mechanisms for the origin of these compounds in Cheddar cheese, we investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening (pH 5.2, 4% NaCl, 15°C, no sugar) conditions. Enzyme assays of cell-free extracts indicated that L. casei strains catabolize Tyr and Phe by successive, constitutively expressed transamination and dehydrogenation reactions. Similar results were obtained with L. helveticus strains, except that the dehydrogenase enzymes were induced during incubation under cheese-ripening conditions. Micellar electrokinetic capillary chromatography of supernatants from L. casei and L. helveticus strains incubated under simulated cheese-ripening conditions confirmed that Tyr and Phe transamination and dehydrogenation pathways were active in both species and also showed these reactions were reversible. Major products of Tyr catabolism were p-hydroxy phenyl lactic acid and p-hydroxy phenyl acetic acid, while Phe degradation gave rise to phenyl lactic acid, phenyl acetic acid, and benzoic acid. However, some of these products were likely formed by nonenzymatic processes, since spontaneous chemical degradation of the Tyr intermediate p-hydroxy phenyl pyruvic acid produced p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, and p-hydroxy benzaldehyde, while chemical degradation of the Phe intermediate phenyl pyruvic acid gave rise to phenyl acetic acid, benzoic acid, phenethanol, phenyl propionic acid, and benzaldehyde

A complete genome sequence ofLactobacillus helveticus R0052, a commercial probiotic strain

Lactobacillus helveticus R0052 is a commercially available strain that is widely used in probiotic preparations. The genome sequence consisted of 2,129,425 bases. Comparative analysis showed that it was unique among L. helveticus strains in that it contained genes encoding mucus-binding proteins similar to those found in Lactobacillus acidophilus

Tryptophan catabolism by Lactobacillus casei andLactobacillus helveticus cheese flavor adjuncts

Microbial degradation of Trp is thought to promote the formation of aromatic compounds that impart putrid, fecal, or unclean flavors in cheese, but pathways for their production have not been established. This study investigated Trp catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under carbohydrate starvation (pH 6.5, 30 or 37°C, no sugar) and near cheese-ripening (pH 5.2, 4% NaCl, 15°C, no sugar) conditions. Enzyme assays of cell-free extracts indicated that both species of Lactobacillus catabolized Trp to indole-3-lactic acid, and micellar electrokinetic capillary chromatography of culture supernatants showed this reaction occurred via successive transamination and dehydrogenation reactions. Tryptophan decarboxylase activity was also detected in all Lactobacillus cell-free extracts, but tryptamine was not detected in culture supernatants. Micellar electrokinetic capillary chromatography showed that Trp metabolism in Lactobacillus casei LC301 and LC202 was similar under both incubation conditions and that those catabolic reactions were reversible (i.e., conversion of indole-3-lactic acid to Trp). In contrast, Trp catabolism by Lactobacillus helveticus LH212 was only detected under near cheese ripening conditions. Cells of Lactobacillus helveticus CNRZ32 did not catabolize Trp in either condition but did convert indole-3-pyruvic acid to Trp in carbohydrate starvation medium and to Trp and indole-3-lactic acid under near cheese ripening conditions

A Campus Partnership to Foster Compliance with Funder Mandates

Data from federally funded research must now be made publicly accessible and discoverable. Researchers must adhere to guidelines established by federal agencies, and universities must be prepared to demonstrate compliance with the federal mandate. At Utah State University, the Office of Research and Graduate Studies and the Merrill-Cazier Library partnered to facilitate data sharing and create an audit trail demonstrating compliance with the terms of each researcher’s award. This systematic approach uses existing resources such as the grant management system, the institutional repository (IR), and the Library online catalog. This paper describes our process and the first eight months of implementation

Influence of adjunct use andcheese microenvironment on nonstarter lactic acid bacteria populations in Cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7°C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 104 cfu/ml did not completely suppress “wild” NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety

Gas-Forming Nonstarter Lactorbacilli

An obligatory heterofermentative lactic acid bacterium Lactobacillus wasatchii sp. nov. isolated from gassy Cheddar cheese was studied for growth, gas formation, salt tolerance and survival against pasteurization treatments at 63°C and 72°C. Initially, Lb. wasatchii was thought to only use ribose as a sugar source and we were interested in whether it could utilize galactose. Experiments to determine rate and extent of growth and gas production in carbohydrate restricted (CR) de Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions with various combinations of ribose and galactose at 12, 23, and 37°C were conducted with 23°C being the more optimum growth temperature of Lb. wasatchii. When grown on ribose (0.1%, 0.5%, and 1%), maximum specific growth rates (μmax) within each temperature were similar. When galactose was the only sugar, μmax was 2 to 4 times lower than with ribose. At all temperatures, highest final cell densities (OD640) of Lb. wasatchii were achieved in CR-MRS plus 1% ribose, 0.5% ribose and 0.5% galactose, or 1% ribose combined with 1% galactose. Similar μmaxvalues and final cell densities were achieved when 50% of ribose in CR-MRS was substituted with galactose. Such enhanced utilization of galactose in the presence of ribose to support bacterial growth has not previously been reported. It appears that Lb. wasatchii co-metabolizes ribose and galactose, utilizing ribose for energy and galactose for other functions such as cell wall biosynthesis. Co-utilization of both sugars could be an adaptation mechanism of Lb. wasatchii to the cheese environment to efficiently ferment available sugars for maximizing metabolism and growth. As expected, gas formation by the heterofermenter was observed only when galactose was present in the media. Growth experiments with MRS plus 1.5% ribose at pH 5.2 or 6.5, with 0, 1, 2, 3, 4, or 5% NaCl revealed that Lb. wasatchii is able to grow under salt and pH conditions typical of Cheddar cheese (4 to 5% salt-in-moisture, ~pH 5.2). Finally, we found Lb. wasatchii cannot survive LTLT pasteurization but survives HTST lab pasteurization with 4.5 log reduction occurred. The ability of Lb. wasatchii to survive HTST pasteurization and grow under cheese ripening conditions implies that the presence of this nonstarter lactic acid bacteria can be a serious contributor to gas formation and textural defects in Cheddar cheese

Transcriptome Analysis of \u3ci\u3eBifidobacterium Longum\u3c/i\u3e Strains that Show a Differential Response to Hydrogen Peroxide Stress

Consumer and commercial interest in foods containing probiotic bifidobacteria is increasing. However, because bifidobacteria are anaerobic, oxidative stress can diminish cell viability during production and storage of bioactive foods. We previously found Bifidobacterium longum strain NCC2705 had signifi- cantly greater intrinsic and inducible resistance to hydrogen peroxide (H2O2) than strain D2957. Here, we explored the basis for these differences by examining the transcriptional responses of both strains to sub-lethal H2O2 exposure for 5- or 60-min. Strain NCC2705 had 288 genes that were differentially expressed after the 5-min treatment and 114 differentially expressed genes after the 60-min treatment. In contrast, strain D2957 had only 21 and 90 differentially expressed genes after the 5- and 60-min treatments, respectively. Both strains showed up-regulation of genes coding enzymes implicated in oxidative stress resistance, such as thioredoxin, thioredoxin reductase, peroxiredoxin, ferredoxin, glutaredoxin, and anaerobic ribonucleotide reductase, but induction levels were typically highest in NCC2705. Compared to D2957, NCC2705 also had more up-regulated genes involved in transcriptional regulation and more down-regulated genes involved in sugar transport and metabolism. These results provide a greater understanding of the molecular basis for oxidative stress resistance in B. longum and the factors that contribute to strain-to-strain variability in survival in bioactive food products

Genetic and Physiological Responses of \u3ci\u3eBifidobacterium animalis\u3c/i\u3e subsp. \u3ci\u3elactis\u3c/i\u3e to Hydrogen Peroxide Stress

Consumer interest in probiotic bifidobacteria is increasing, but industry efforts to secure high cell viability in foods is determined by these anaerobes’ sensitivity to oxidative stress. To address this limitation, we investigated genetic and physiological responses of two fully sequenced Bifidobacterium animalis subsp. lactis strains, BL-04 and DSM 10140, to hydrogen peroxide (H2O2) stress. Although the genome sequences for these strains are highly clonal, prior work showed they differ in both intrinsic and inducible H2O2 resistance. Transcriptome analysis of early stationary phase cells exposed to a sub-lethal H2O2 concentration detected significant (P2O2 stress resistance might be due to a mutation in a BL-04 gene encoding long chain fatty acid-coA ligase. To explore this possibility, membrane fatty acids were isolated and analyzed by GC-MS. Results confirmed the strains had significantly different lipid profiles; the BL-04 membrane contained higher percentages of C14:0 and C16:0, and lower percentages of C16:1n7 and C18:1n9. Alteration of the DSM 10140 membrane lipid composition using modified growth medium to more closely mimic that of BL-04 yielded cells that showed increased intrinsic resistance to lethal H2O2 challenge, but did not display an inducible H2O2 stress response. Results show deliberate stress induction or membrane lipid modification can be employed to significantly improve H2O2 resistance in B. animalis subsp. lactis strains

Survival of microencapsulated probiotic Lactobacillus paracasei LBC-1e during manufacture of Mozzarellacheese and simulated gastric digestion

An erythromycin-resistant strain of probiotic Lactobacillus paracasei ssp. paracasei LBC-1 (LBC-1e) was added to part-skim Mozzarella cheese in alginate-microencapsulated or free form at a level of 108 and 107 cfu/g, respectively. Survival of LBC-1e and total lactic acid bacteria (LAB) was investigated through the pasta filata process of cheese making (in which the cheese curd was heated to 55°C and stretched in 70°C-hot brine), followed by storage at 4°C for 6 wk and simulated gastric and intestinal digestion. This included incubation in 0.1 M and 0.01 M HCl, 0.9 M H3PO4, and a simulated intestinal juice consisting of pancreatin and bile salts in a pH 7.4 phosphate buffer. Some reductions were observed in both free and encapsulated LBC-1e during heating and stretching, with encapsulated LBC-1e surviving slightly better. Changes in total LAB losses during heating and stretching did not reach statistical significance. During storage, a decrease was observed in total LAB, but no statistically significant decrease was observed in LBC-1e. Survival during gastric digestion in HCl was dependent on the extent of neutralization of HCl by the cheese, with more survival in the weaker acid, in which pH increased to 4.4 after cheese addition. The alginate microcapsules did not provide any protection against the HCl. It is interesting that survival of the encapsulated LBC-1e was greater during incubation in H3PO4 than in the HCl gastric juices. Proper selection of simulated gastric digestion media is important for predicting the delivery of probiotic bacteria into the human intestinal tract. Neither free nor encapsulated LBC-1e was affected by incubation in the pancreatin-bile solution. Based on the level of probiotic bacteria in cheese needed to provide a health benefit and its survival during simulated gastric digestion, as determined in this study, it should theoretically be possible to lower the amount that needs to be ingested in cheese by up to a factor of 103 compared with other fermented dairy foods or when consumed as supplements

Advances in startercultures and cultured foods

With 2005 retail sales close to $4.8 million, cultured dairy products are driving the growth of dairy foods consumption. Starter cultures are of great industrial significance in that they play a vital role in the manufacturing, flavor, and texture development of fermented dairy foods. Furthermore, additional interest in starter bacteria has been generated because of the data accumulating on the potential health benefits of these organisms. Today, starter cultures for fermented foods are developed mainly by design rather than by the traditional screening methods and trial and error. Advances in genetics and molecular biology have provided opportunities for genomic studies of these economically significant organisms and engineering of cultures that focuses on rational improvement of the industrially useful strain. Furthermore, much research has been published on the health benefits associated with ingesting cultured dairy foods and probiotics, particularly their role in modulating immune function. The aim of this review is to describe some of the major scientific advances made in starter and non-starter lactic acid bacteria during the past 10 yr, including genomic studies on dairy starter cultures, engineering of culture attributes, advances in phage control, developments in methods to enumerate lactic acid bacteria and probiotics in dairy foods, and the potential role of cultured dairy foods in modulation of immune function