Purification and Characterization of Antioxidative Peptides Derived From Fermented Milk (<Emphasis Type="Italic">Lassi</Emphasis>) by Lactic Cultures

Fermentation of milk with lactic acid bacteria is the most suitable approach to enrich the bioactive peptides in fermented milk products. So in the present study, two sets of fermented milk (lassi) were prepared. The one lassi was prepared using standard Dahi culture NCDC-167(BD₄) and the other one was made with the same Dahi culture combined with Lactobacillus acidophilus NCDC-15 as an adjunct culture. The preparation steps i.e. preheat treatment and incubation period were optimized by using response surface methodology to obtain maximum antioxidant activity. Lassi prepared with adjunct culture using optimized conditions showed an antioxidant activity of 0.66?±?0.01 µM Trolox/mg protein which was significantly higher than that control (0.22?±?0.01 µM Trolox/mg protein). Out of 59 peptide fragments of β casein fermented by L. acidophilus and 24 peptides from control have been identified by LC–MS/MS. Most of the peptides showed the antioxidant activity. The therapeutic potential of fermented milk products could be improved by increased production of bioactive peptides through controlled fermentation using appropriate proteolytic starter strain.     

Trends in the application of Bacillus in fermented foods

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Bacillus species such as Bacillus subtilis and Bacillus amyloliquefaciens are widely used to produce fermented foods from soybeans and locust beans in Asian and West African countries, respectively. Genomic information for B. subtilis strains isolated from Asian Bacillus-fermented foods (BFFs) has been gathered, and the chemical components of fermented products were defined with metabolomic approaches, facilitating the development of new starter strains and the evaluation of health claims. On the other hand, although advanced studies have been performed for some commercially produced BFFs, home-manufactured products still remain to be characterized in rural areas. In West Africa, the microbial flora of BFFs was examined in detail, leading to the isolation of candidates of the starter that produced bacteriocin against Bacillus cereus contaminating the products. These studies may provide a choice of Bacillus strains in food application and increase opportunities for further usage of Bacillus in foods.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Decreased formation of branched-chain short fatty acids in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> by metabolic engineering

Objectives

To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared.

Results

By knockout of the ptb gene, 1.01 g BCFAs kg^?1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg^?1). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg^?1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12.

Conclusions

HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

     

The Production of Xylitol and d-Xylonic Acid by Yeast

Pichia quercibus Phaff et Knapp produced xylitol and d-xylonic acid by aerobic dissimilation of d-xylose at good yield of 40% of sugar consumed. The products were isolated from the fermented broth and identified. It would be interesting that both of xylitol, a reduction product of d-xylose, and d-xylonic acid, an oxidation product, are accumulated in the fermented broth.     

Quantification of the Relative Effects of Temperature,pH, and Water Activity on Inactivation of Escherichia coli in Fermented Meat by Meta-Analysis

Outbreaks of Escherichia coli infections linked to fermented meats have prompted much research into the kinetics of E. coli inactivation during fermented meat manufacture. A meta-analysis of data from 44 independent studies was undertaken that allowed the relative influences of pH, water activity (a_w), and temperature on E. coli survival during fermented meat processing to be investigated. Data were reevaluated to determine rates of inactivation, providing 484 rate data points with various pH (2.8 to 6.14), a_w (0.75 to 0.986), and temperature (−20 to 66°C) values, product formulations, and E. coli strains and serotypes. When the data were presented as an Arrhenius model, temperature (0 to 47°C) accounted for 61% of the variance in the ln(inactivation rate) data. In contrast, the pH or a_w measured accounted for less than 8% of variability in the data, and the effects of other pH- and a_w-based variables (i.e., total decrease and rates of reduction of those factors) were largely dependent on the temperature of the process. These findings indicate that although temperatures typically used in fermented meat manufacture are not lethal to E. coli per se, when other factors prevent E. coli growth (e.g., low pH and a_w), the rate of inactivation of E. coli is dominated by temperature. In contrast, inactivation rates at temperatures above ∼50°C were characterized by smaller z values than those at 0 to 47°C, suggesting that the mechanisms of inactivation are different in these temperature ranges. The Arrhenius model developed can be used to improve product safety by quantifying the effects of changes in temperature and/or time on E. coli inactivation during fermented meat manufacture.Fermented meats encompass a diverse range of product styles in which raw, ground meat is preserved by the processes of fermentation and drying (or maturation). These products are typically manufactured without a bactericidal heat treatment, and instead, inhibition of growth and inactivation of contaminating pathogens rely upon the collective effects of acid pH, reduced water activity (a_w), and the presence of lactic acid and, potentially, curing salts (nitrate and/or nitrite) and spices. However, pathogenic Escherichia coli can contaminate and survive in fermented meat products at levels sufficient to cause serious illness in consumers, as evidenced by numerous outbreaks of E. coli infections epidemiologically linked to uncooked fermented meat products (8, 9, 54, 64). Knowledge of the kinetics of nonthermal inactivation of E. coli, and the factors affecting it, is important to be able to optimize the safety of fermented meat processes.Several research groups have conducted studies on the survival of pathogenic E. coli during the processing of specific fermented meat products (4, 6, 14, 16, 23, 25). In some cases, the effects of alternative ingredients or processing parameters have also been determined (12, 21, 24, 30). Such investigations have allowed the lethality of a specific fermented meat process to be determined and the microbiological safety of that product, in regards to E. coli, to be described. However, most of these studies have been essentially empirical and product/process specific, i.e., they have not sought to discern key variables or their interactions that influence the extent of inactivation of E. coli or other pathogens during meat fermentation and maturation. Thus, while they are very important and useful, it has been difficult to extrapolate the results of those studies to different fermented meat processes, confounding efforts to assess product safety without the requirement for challenge tests or to give manufacturers the confidence to develop new or modified processes that remain safe.In an attempt to identify the main factors that influence the inactivation of E. coli in fermented meat products, we utilized data that already existed in the scientific literature and reassessed that information via a process similar to meta-analysis. Meta-analysis is a statistical technique that involves amalgamating, summarizing, and reviewing previous quantitative research to identify trends. It is used, albeit rarely in the area of microbiology, as a means to address a wide variety of questions where a reasonable body of primary research studies exists. In a preliminary investigation, based on an analysis of limited published and other data, Ross and Shadbolt (51) observed that inactivation of E. coli in fermented meat processes was dominated by temperature and that pH and a_w levels appeared to be less influential, except insofar as creating conditions inimical to E. coli growth. The objective of the present investigation was to rigorously test that observation by collation and analysis of a large data set describing the inactivation of E. coli during manufacture of fermented meats and to attempt to identify underlying patterns in the responses of E. coli to conditions encountered during fermented meat processes. From the observations of Ross and Shadbolt (51), and because the inactivation of E. coli in fermented meat is not instantaneous, we based our analyses on the rate of E. coli inactivation, calculated from viable count data and processing times reported by a variety of published and unpublished sources. We sought to relate the inactivation rate to reported environmental conditions, including pH, a_w, and temperature, and to summarize the observations in a predictive mathematical model.     

Oxidase-Peroxidase Method of Ethanol Assay in Fermented Musts and Wine Products

A new alcohol oxidase-peroxidase method of ethanol assay in fermented musts and wine products is described and compared to conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined. The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry (correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). The proposed method is less time- and labor-consuming and allows simultaneous analysis of a series of wine samples.     

Effects of <Emphasis Type="Italic">Lactobacillus</Emphasis>-fermented ginger stem on <Emphasis Type="Italic">Salmonella</Emphasis>-infected broiler chicks

Broiler salmonellosis is a major problem for poultry industry. Here, we supplemented broiler feed with 1% of ginger stems (GS) fermented with Lactobacillus paracasei and analyzed the effects on the resistance to Salmonella gallinarum. The chickens were divided into four dietary groups. The control group (C) received the basal diet, and the other chickens received the basal diet supplemented with 0.1% w/w L. paracasei ML-7 (L group), 0.1% ginger stem powder (GS group), or 0.2% fermented ginger stem (FGS group) for 21 days. The dietary groups were further split into two subgroups: one challenged with 1 × 105 CFU/mL S. gallinarum orally administered in 1 mL of saline from days 7 and 14, and one that received 1 mL of saline without bacteria. Both uninfected and S. gallinarum-infected broilers fed with fermented GS (FGS) significantly increased body weight and feed intake, and had lower mortality compared to relative control groups. Furthermore, dietary FGS decreased cecal, Salmonella spp. counts and serum IgA and IgG levels. These results indicate that FGS prevented S. gallinarum colonization and promoted weight gain in broilers, suggesting that FGS supplementation can be effectively used as a replacement of antibiotic growth promoters to prevent Salmonella infection.     

Rheological properties and volatile composition of fermented milk prepared by exopolysaccharide-producing <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> n.v. Er2 317/402 strain Narine

Fermented milk products were made using a probiotic strain of human origin, Lactobacillus acidophilus n.v. Er2 317/402 strain Narine. The effects of the strain on the texture, rheology, microstructure, and volatile profiling of fermented milk products over a period of 25 days under refrigerated storage conditions were investigated using steady and dynamic rheological tests, confocal laser scanning microscopy (CLSM), and headspace solid-phase microextraction gas chromatography mass spectrometry, respectively. CLSM images confirmed that exopolysaccharides (EPSs) from Narine were large in size and interacted with proteins. EPSs (50 mg) were obtained after 8 h of incubation. Volatome profiling of Narine fermented milk revealed 15 volatile compounds, including 2-heptanone, 2-nonanone, acetoin, isobutyric acid, isovaleric acid, and 2-phenylethanol. EPSs produced from L. acidophilus n.v. Er2 317/402 strain Narine gave the fermented milk a smooth ropy texture. The natural, soft-bodied, drinkable, and pleasant flavor of Narine makes it an attractive food ingredient.     

Milk fermented by Propionibacterium freudenreichii induces apoptosis of HGT-1 human gastric cancer cells

Background

Gastric cancer is one of the most common cancers in the world. The “economically developed countries” life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate.

Methodology/Principal Findings

A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy.

Conclusions/Significance

Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.     

Polymorphism of lactose genes in the dairy yeasts <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>, potential probiotic microorganisms

Molecular karyotyping and Southern blot hybridization were used to investigate chromosomal polymorphism of the LAC genes controlling lactose fermentation in Kluyveromyces marxianus strains isolated from various dairy products and natural sources in Russia and CIS countries. Profound polymorphism of karyotype patterns and accumulation of LAC genes were observed in dairy K. marxianus strains. K. marxianus strains isolated from dairy products intensively fermented lactose at 37°C after one day of cultivation, while non-dairy strains exhibited delayed lactose fermentation or did not ferment it at all. Based on the fermentation tests, twelve K. marxianus strains were selected, which are of interest as potential probiotic microorganisms suitable for further molecular genetic studies and breeding.     

In Vitro and In Silico Analysis of Novel ACE-Inhibitory Bioactive Peptides Derived from Fermented Goat Milk

In the study, two Lactobacillus cultures i.e. L. casei (NK9) and L. fermentum (LF) were studied for their proteolytic activity, di and tripeptidase activity, ACE-inhibitory activity and peptides production under optimized growth condition from fermented goat milk (Capra aegagrus hircus). NK9 and LF were found to be a strong proteolytic culture with 2.0% rate of inoculation after 48 h. LF (10 kDa retentate) produced maximum peptides among all the retentates of the fermented goat milk. Goat milk fermented with NK9 (10 kDa permeates) exhibited peptide sequence i.e. AFPEHK which had ACE inhibitory activity, matched with goat milk protein databases of AHTPDB. However, L. casei (NK9) and L. fermentum (LF) could be explored for the production of ACE inhibitory peptides from fermented goat milk.     

Traditional fermented beverages from Mexico as a potential probiotic source

Fermentation is one of the oldest ways of processing food. Some fermented food is produced industrially, but can also be produced in an artisanal way by certain ethnic groups, called traditional fermented foods. In Mexico, there are a variety of traditional fermented beverages which are produced in an artisanal way. They include those made with maize (atole agrio, pozol, and tesgüino), fruit (tepache and colonche), and obtained by plant fermentation (pulque, tuba, and taberna). These beverages have been used since ancient times for religious and medicinal purposes. The medicinal effect may be due to fermented microorganisms. The presence of beneficial microorganisms known as probiotics provides beneficial effects to consumer health, improving the balance of intestinal host, and reducing the risk of gastrointestinal diseases, mainly. Most probiotics belong to the genus Lactobacillus, but Bifidobacterium, Bacillus, and yeast are also found. Therefore, it is important that the microbiological diversity of the beverages is studied and documented. This review includes information on the microbial diversity and probiotic potential of the most important traditional fermented beverages from Mexico.     

Functional Characterization of Potential Probiotic Lactic Acid Bacteria Isolated from <Emphasis Type="Italic">Kalarei</Emphasis> and Development of Probiotic Fermented Oat Flour

Considerable variations among probiotics with respect to their health benefitting attributes fuel the research on bioprospecting of proficient probiotic strains from various ecological niches especially the poorly unexplored ones. In the current study, kalarei, an indigenous cheese-like fermented milk product, and other dairy-based sources like curd and raw milk were used for isolation of lactic acid bacteria (LAB). Among 34 LAB isolates, 7 that could withstand simulated gastrointestinal (GI) conditions were characterized for functional probiotic attributes, viz. adhesion ability, aggregation and coaggregation, extracellular enzyme producing capability, antibacterial activity against pathogens and antibiotic resistance. The isolate M-13 (from kalarei) which exhibited most of the desirable probiotic functional properties was identified as Lactobacillus plantarum based on 16S ribosomal DNA sequence analysis and designated as L. plantarum M-13. The sequence was submitted to GenBank (accession number KT592509). The study presents the first ever report of isolation of potential probiotic LAB, i.e. L. plantarum M-13 from indigenous food kalarei, and its application for development of potential probiotic fermented oat flour (PFOF). PFOF was analysed for parameters like viability of L. plantarum M-13, acidity and pH. Results show that PFOF serves as a good matrix for potential probiotic L. plantarum M-13 as it supported adequate growth of the organism (14.4 log cfu/ml after 72 h of fermentation). In addition, appreciable acid production by L. plantarum M-13 and consequential pH reduction indicates the vigorous and active metabolic status of the potential probiotic organism in the food matrix. Thus, study shows that fermented oat flour may possibly be developed as a potential probiotic carrier especially in view of the problems associated with dairy products as probiotic vehicles.     

Fermentation of L-tartrate by a newly isolated gram-negative glycolytic bacterium

Enrichments on L-tartrate from a freshwater lake sediment yielded a pure culture of anaerobic bacterium designated strain 16Lt1. The rod-shaped organism was motile, did not form spores, and had a gram-negative wall structure. No cytochromes were detected. The mol % G+C of the DNA was 58. The new strain was microaerotolerant, and grew optimally at 30°C and neutral pH in freshwater medium. A wide range of carbohydrates was fermented, with formate, acetate, ethanol, lactate and succinate being the end-products detected. L-tartrate and citrate were fermented to formate, acetate and CO₂. L-tartrate was fermented by the dehydratase pathway, and glucose by the Embden-Meyerhof-Parnas pathway. Fumarate was reduced, but nitrate, sulfate, sulfur and thiosulfate were not used as terminal electron acceptors. Glucose metabolism was constitutive, whereas L-tartrate-degrading activity was inducible. When glucose and L-tartrate were both present as substrates, growth was diauxic with glucose being metabolized first. The growth rate and growth yield were higher on glucose than on L-tartrate. Strain 16Lt1 has been deposited with the Deutsche Sammlung von Mikroorganismen as Bacteroides sp. DSM6268.     

Enhancement of interferon-β production with sphingomyelin from fermented milk

A fermented milk, Kefir, contains an active substance which enhances IFN- secretion of a human osteosarcoma line MG-63 treated with a chemical inducer, poly I: poly C. The active substance in the fermented milk was identified to be sphingomyelin (SpM) by a combined use of a fast atom bombardment mass spectrometry (FAB-MS) and a fast atom bombardment tandem mass spectrometry (FAB-MS/MS). SpM from fermented milk (F-SpM) was a mixture of four molecular species of SpMs having C₂₁-, C₂₂-, C₂₃- and C₂₄-fatty acids. F-SpM enhanced the IFN secretion 14 times, SpMs from other sources also enhanced moderately (2–3 times). Sphingosine and lysosphingomyelin also enhanced the activity but ceramide and cerebroside did not.Abbreviations IFN- interferon- - SpM sphingomyelin - Lyso-SpM lysosphingomyelin - SpS sphingosine - FAB-MS fast atom bombardment mass spectrometry - FAB-MS/MS fast atom bombardment tandem mass spectrometry