Screening and characterization of ß-galactosidase activity in lactic acid bacteria for the valorization of acid whey.

ß-Galactosidase is an enzyme produced by some strains of lactic acid bacteria (LAB) commonly found in dairy products; however, industrial demand for these enzymes is still low. Acid whey (AW), a lactose-rich byproduct, has large output from cottage cheese and remains unexploited. The purpose of this study was to understand the production mechanism of ß-galactosidase from LAB using AW as a culture medium. First, bioinformatics analysis was conducted on 15 species of LAB. Then, 24 strains were selected and inoculated in de Man, Rogosa, and Sharpe (MRS) broth and in AW medium to compare the bacterial kinetic growth and ß-galactosidase production. Bacterial growth and total protein activity were measured using spectrophotometric techniques. ß-Galactosidase activity was determined by 2 methods: following the hydrolysis of o-nitrophenyl-ß-d-galactopyranoside and of 5-bromo-4-chloro-3-indoyl-ß-d-galactopyranoside (X-gal) in tryptic soy agar plates. The relative expression of the ß-galactosidase gene was performed using real-time quantitative PCR. Despite generally lower growth in AW, 18 strains showed higher ß-galactosidase activity when grown in AW compared with MRS medium. The highest ß-galactosidase activity in AW was in Lactobacillus helveticus strain OSU-PECh-4A, which showed almost 5 times higher activity than average. Analysis of 6 selected strains for expression of the bgal-620 gene found higher overexpression in AW than in MRS, regardless of specific ß-galactosidase activity. Strains of LAB such as OSU-PECh-4A could valorize AW through the production of ß-galactosidase (as an aid to lactose digestion) and production of prebiotic galactooligosaccharides.

Characterization and Evaluation of Proteolysis Products during the Fermentation of Acid Whey and Fish Waste and Potential Applications.

Reduction of waste in the food industry is critical to sustainability. This work represents one strategy of valorizing waste streams from the dairy (acid whey) and fisheries industries (fish waste) using fermentation. The main approach was to characterize the peptides produced by this fermentation under three conditions: (1) fermentation without adding inoculum; (2) with the addition of a single lactic acid bacterial strain; and (3) the addition of a consortium of lactic acid bacteria. Previous results indicated that the rapid acidification of this fermentation was advantageous for its food safety and microbial activity. This work complements our previous results by defining the rate of peptide production due to protein digestion and using two-dimensional (2D) gel electrophoresis and proteomic analysis to give a more detailed identification of the peptides produced from different waste streams. These results provide important information on this process for eventual applications in industrial fermentation and, ultimately, the efficient valorization of these waste streams.

Characterization and Cellular Uptake of Peptides Derived from In Vitro Digestion of Meat Analogues Produced by a Sustainable Extrusion Process.

Whether proteins in meat analogues (MAs) have the ability to provide equivalent nutrition as those in animal meat remains unknown. Herein, a MA was produced by high-moisture extrusion using soy and wheat proteins. The physicochemical properties, in vitro digestion, and cellular uptake of the released peptides were systematically compared between the MA and the chicken breast (CB). The MA showed a higher hardness but a lower degree of texturization than the CB. After simulated digestion, soluble peptides in the MA had a higher molecular weight and higher hydrophobicity. No observable cytotoxicity or inflammatory response to Caco-2 cells was found for both MA and CB digests. The former exhibited less permeability of peptides across Caco-2 cells. Liquid chromatography with tandem mass spectrometry found that the identified peptides in MA and CB digests contained 7-30 and 7-20 amino acid residues, respectively, and they became shorter after cellular transportation. The amino acid composition showed fewer essential and non-essential amino acids in the MA permeate than in the CB permeate.

Identification of Putative ß-Galactosidase Genes in the Genome of Lactobacillus helveticus OSU-PECh-4A.

The Lactobacillus helveticus OSU-PECh-4A strain, from the Ohio State University Parker Chair collection, produces exceptional ß-galactosidase activity using acid whey as a culture medium, compared with a commercial broth. The strain has a genome sequence of 1,834,843 bp, and its GC content is 36.69%. Using InterProScan v5.50-84.0 software, four genes with putative ß-galactosidase function were found.

Improved antimicrobial spectrum of the N-acetylmuramoyl-L-alanine amidase from Latilactobacillus sakei upon LysM domain deletion.

The gene encoding N-acetylmuramoyl-L-alanine amidase in Latilactobacillus sakei isolated from a fermented meat product was cloned in two forms: its complete sequence (AmiC) and a truncated sequence without one of its anchoring LysM domains (AmiLysM4). The objective of this work was to evaluate the effect of LysM domain deletion on antibacterial activity as well the biochemical characterization of each recombinant protein. AmiC and AmiLysM4 were expressed in Escherichia coli BL21. Using a zymography method, two bands with lytic activity were observed, which were confirmed by LC-MS/MS analysis, with molecular masses of 71 kDa (AmiC) and 66 kDa (AmiLysM4). The recombinant proteins were active against Listeria innocua and Staphylococcus aureus strains. The inhibitory spectrum of AmiLysM4 was broader than AmiC as it showed inhibition of Leuconostoc mesenteroides and Weissella viridescens, both microorganisms associated with food decomposition. Optimal temperature and pH values were determined for both proteins using L-alanine-p-nitroanilide hydrochloride as a substrate for N-acetylmuramoyl-L-alanine amidase activity. Both proteins showed similar maximum activity values for pH (8) and temperature (50 °C). Furthermore, structural predictions did not show differences for the catalytic region, but differences were found for the region called 2dom-AmiLysM4, which includes 4 of the 5 LysM domains. Therefore, modification of the LysM domain offers new tools for the development of novel food biopreservatives.

Characterization of antibacterial activity of a N-acetylmuramoyl-L-alanine amidase produced by Latilactobacillus sakei isolated from salami.

Lactic acid bacteria are the predominant group within meat products, whose metabolites such as bacteriocins and peptidoglycan hydrolases inhibit pathogenic or spoilage bacteria. Fermented meat products, as a salami, is a good source to analyze the viable microbiota, due to these products present a low risk to consumer health. The aim of this work was to identify the lactic acid bacteria with broad antibacterial activity present in salami, purify the protein responsible for this activity, achieve antagonistic spectrum and perform the biochemical characterization. Five strains from salami were selected, isolated and identified by 16S rRNA gene sequencing. The antimicrobial activity was evaluated by antagonism assay and zymography, using spoilage microorganisms commonly found in meat products. The strain that showed a broad antibacterial activity was Latilactobacillus sakei and the antibacterial activity was given by a protein with 75-kDa of molecular mass, identified by LC/MALDI-TOF/TOF. The sequence analysis showed 67% of identity with a N-acetylmuramoyl-L-alanine amidase protein with five non-identical LysM domains. The purified protein showed an optimal pH of 8.0 and heat resistance at 80 °C for 10 min. L. sakei strain displayed antibacterial activity against Gram-negative and Gram-positive spoilage microorganisms. The results of this study provide the information to use Latilactobacillus sakei as a starter culture which will provide the necessary metabolites to combat undesirable microorganisms. Additionally, the conditions and properties for the best application and use of the antibacterial protein produced by this strain. This protein may have a potential use in the food industry as a new antibacterial agent.

Improving Human Health with Milk Fat Globule Membrane, Lactic Acid Bacteria, and Bifidobacteria.

The milk fat globule membrane (MFGM), the component that surrounds fat globules in milk, and its constituents have gained significant attention for their gut function, immune-boosting properties, and cognitive-development roles. The MFGM can directly interact with probiotic bacteria, such as bifidobacteria and lactic acid bacteria (LAB), through interactions with bacterial surface proteins. With these interactions in mind, increasing evidence supports a synergistic effect between MFGM and probiotics to benefit human health at all ages. This important synergy affects the survival and adhesion of probiotic bacteria through gastrointestinal transit, mucosal immunity, and neurocognitive behavior in developing infants. In this review, we highlight the current understanding of the co-supplementation of MFGM and probiotics with a specific emphasis on their interactions and colocalization in dairy foods, supporting in vivo and clinical evidence, and current and future potential applications.

Invited review: Acid whey trends and health benefits.

In recent years, acid whey production has increased due to a growing demand for Greek yogurt and acid-coagulated cheeses. Acid whey is a dairy by-product for which the industry has long struggled to find a sustainable application. Bulk amounts of acid whey associated with the dairy industry have led to increasing research on ways to valorize it. Industry players are finding ways to use acid whey on-site with ultrafiltration techniques and biodigesters, to reduce transportation costs and provide energy for the facility. Academia has sought to further investigate practical uses and benefits of this by-product. Although modern research has shown many other possible applications for acid whey, no comprehensive review yet exists about its composition, utilization, and health benefits. In this review, the industrial trends, the applications and uses, and the potential health benefits associated with the consumption of acid whey are discussed. The proximal composition of acid whey is discussed in depth. In addition, the potential applications of acid whey, such as its use as a starting material in the production of fermented beverages, as growth medium for cultivation of lactic acid bacteria in replacement of commercial media, and as a substrate for the isolation of lactose and minerals, are reviewed. Finally, the potential health benefits of the major protein constituents of acid whey, bioactive phospholipids, and organic acids such as lactic acid are described. Acid whey has promising applications related to potential health benefits, ranging from antibacterial effects to cognitive development for babies to human gut health.

Supercritical CO2 treatment reduces the antigenicity of buttermilk ß-lactoglobulin and its inflammatory response in Caco-2 cells.

ß-Lactoglobulin (ß-LG) is believed to be a common allergen in bovine milk. Buttermilk (BM) powder has abundant contents of milk fat globule membrane and phospholipid, both of which have been demonstrated to have positive effects on brain and cognitive development during early infancy. This study focused on modifying ß-LG in BM via supercritical CO2 (ScCO2) treatment to modify its reactivity to antibodies and thus reduce its antigenicity. Buttermilk powder was treated in a supercritical fluid extraction system with food-grade CO2 at 100, 150, 200, 250, 350, and 400 bar at 2 temperatures, 50 and 75°C. All analyses were completed in a 10% BM suspension (wt/vol). The BM proteins were examined using sodium dodecyl sulfate (SDS)-PAGE, Western blot, ELISA, and periodic acid staining methods. Semi-purified ß-LG was used to evaluate the cytotoxicity, viability, and inflammatory response in the Caco-2 cell line by means of the lactate dehydrogenase assay, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay, and IL-8 production, respectively. The SDS-PAGE showed that the signal intensity of ß-LG bands was reduced by up to 50% after being processed at 250 bar and 75°C for 30 min. Lighter and more diffuse signals were found by Western blot, indicating modification of the protein structure. The ELISA demonstrated that ScCO2 treatment could significantly change ß-LG antigenicity in BM. Sugar moieties in bands corresponding to ß-LG were revealed by periodic acid staining, indicating glycosylation only in samples treated with ScCO2. Caco-2 cells treated with whey proteins had high viability, 24.9% lower inflammation, and no evidence of cytotoxicity compared with untreated cultures. These results showed that reduced antigenicity of ß-LG was caused by lactosylation, which has been reported as a possible pathway to reduce the allergenicity in foods. The denaturation of ß-LG by supercritical fluid processing is a promising way to address milk allergy, which remains a problem requiring more attention and further research.

Characterization of milk and soy phospholipid liposomes and inflammation in 3T3-L1 adipocytes.

Milk phospholipids (PL) are valuable dairy components that appear to impart human health benefits, including improved cognitive function in infants and adults. The commercial food industry uses primarily plant-based sources of PL, such as soy lecithin. However, it remains unclear whether different compositions of PL from different dietary sources, such as milk, convey the same benefits. We hypothesized that PL derived from bovine milk or soy have differing physiological effects in terms of inflammation due to their differences in composition. The objectives of this study were to characterize milk and soy liposomes by their physicochemical properties and composition and to evaluate their effects in vitro by means of inflammatory gene expression analyses. Milk and soy phospholipid large unilamellar vesicles (MPL-LUV and SPL-LUV, respectively) prepared using thin-film hydration coupled with extrusion were similar in terms of structure, size, and stability; however, they differed significantly in composition. The 3T3-L1 adipocytes were selected for this work because adipocytes are the main site of uptake, synthesis, modification, and breakdown of lipids and are important inflammatory mediators in mammalian systems. In this work, these cells exposed to both liposome varieties showed high biocompatibility and low cytotoxicity up to concentrations of 0.5 mg/mL as measured by colorimetric MTT and lactate dehydrogenase assays. Furthermore, SPL-LUV showed trends toward stimulating inflammation compared with MPL-LUV as measured by expression of 2 proinflammatory cytokines, monocyte chemoattractant protein-1 (MCP-1) and IL-6. Expression of MCP-1 significantly increased 1.82-fold relative to the control upon SPL-LUV treatment, with similar trends for IL-6 (increased 1.59-fold). The MPL-LUV showed relatively no change in cytokine expression. The results obtained in this work suggest that the methodology used to prepare LUV and the composition and proportion of milk PL are important in measuring cell physiology changes and inflammatory status in mammalian cells.

Assessment of Safety and Probiotic Traits of Enterococcus durans OSY-EGY, Isolated From Egyptian Artisanal Cheese, Using Comparative Genomics and Phenotypic Analyses.

An Enterococcus durans strain, designated OSY-EGY, was previously isolated from artisanal cheese. In this work, comparative genomic and phenotypic analyses were utilized to assess the safety characteristics and probiotic traits of the bacterium. The comparative genomic analysis revealed that the strain is distantly related to potentially pathogenic Enterococcus spp. The genome was devoid of genes encoding acquired antibiotic resistance or marker virulence factors associated with Enterococcus spp. Phenotypically, the bacterium is susceptible to vancomycin, ampicillin, tetracycline, chloramphenicol, and aminoglycosides and does not have any hemolytic or gelatinase activity, or cytotoxic effect on Caco-2 cells. Altogether, these findings confirm the lack of hazardous traits in E. durans OSY-EGY. Mining E. durans OSY-EGY genome, for probiotic-related sequences, revealed genes associated with acid and bile salts tolerance, adhesion, competitiveness, antioxidant activitiy, antimicrobial activity, essential amino acids production, and vitamins biosynthesis. Phenotypically, E. durans OSY-EGY was tolerant to acidic pH (3.0), and presence of 0.3% bile salts. The bacterium showed adhesion capability to Caco-2 cells, cholesterol-lowering effect, DPPH scavenging activity, and antimicrobial activity against several Gram-positive pathogenic bacteria. Based on the current work, we propose that E. durans OSY-EGY is a potentially safe strain with desirable probiotic and antimicrobial traits. Thus, the investigated strain could be a promising candidate for several industrial applications.

Draft Genome Sequence of Lactobacillus rhamnosus OSU-PECh-69, a Cheese Isolate with Antibacterial Activity.

The novel strain Lactobacillus rhamnosus OSU-PECh-69 was isolated from provolone cheese. It produces antimicrobial agents having a molecular mass of 5 to 10 kDa that are active against Gram-positive and Gram-negative bacteria. The strain has a genome sequence of 3,057,669 bp, a GC content of 46.6%, and up to two gene clusters encoding bacteriocins.

Growth of lactic acid bacteria in milk phospholipids enhances their adhesion to Caco-2 cells.

The mechanisms of bacterial adhesion to human cells involve several complex reactions and activation of genes and proteins. It has been reported that the food components in dairy matrices, such as sugar or salt, can decrease bacterial adhesion to Caco-2 cells. However, it has not been evaluated whether the bacteria grown in media supplemented with milk phospholipids (MPL) can increase or decrease the adhesion of these cells. The objective of this work was to evaluate the effects of MPL on the kinetic growth of lactic acid bacteria (LAB) and their functional characteristics as probiotics, expression of surface protein genes, and adherence to Caco-2 cells. Seven LAB strains isolated from various dairy products were characterized. Five of the tested LAB strains were able to grow in a chemically defined medium supplemented with MPL. Lactobacillus reuteri OSU-PECh-48 showed the highest growth rate and the greatest optical density. All of the strains tested showed tolerance to acidic conditions at pH 3.0 and to bile salts at 0.5 and 1% concentrations. Auto-aggregation and cell surface hydrophobicity ability were evaluated, with nonsignificant differences between the strains grown in MPL and without MPL. Gene expression of 6 surface proteins was evaluated in the presence or absence of MPL. Pediococcus acidilactici OSU-PECh-L and OSU-PECh-48 were the strains with highest relative expression of 5 of the 6 genes evaluated. Lactobacillus paracasei OSU-PECh-BA was the strain with the lowest level of expression of surface protein genes. Most of the bacteria tested had increased adhesion to Caco-2 cells after growth in MPL. The bacteria with the highest degrees of adhesion observed were Lactobacillus paracasei OSU-PECh-3B, Pediococcus acidilactici OSU-PECh-L, and Lactobacillus reuteri OSU-PECh-48. The genes Cnb and EF-Tu increased in expression in the presence of MPL in most of the LAB tested. The results obtained in this work demonstrate the high potential of these LAB strains for use as starters or beneficial cultures in fermentation of not only dairy products but also other food fermentation processes, with promising ability to increase residence time in the gut, modify the microbiome, and improve human health.

Monitoring Hydroxycinnamic Acid Decarboxylation by Lactic Acid Bacteria Using High-Throughput UV-Vis Spectroscopy.

Hydroxycinnamic acid (HCA) decarboxylation by lactic acid bacteria (LAB) results in the production of 4-vinylplenols with great impact on the sensorial characteristics of foods. The determination of LAB decarboxylating capabilities is key for optimal strain selection for food production. The activity of LAB strains from the Ohio State University-Parker Endowed Chair (OSU-PECh) collection potentially capable of synthesizing phenolic acid decarboxylase was evaluated after incubation with HCAs for 36 h at 32 °C. A high-throughput method for monitoring HCAs decarboxylation was developed based on hypsochromic shifts at pH 1.0. Out of 22 strains evaluated, only Enterococcus mundtii, Lactobacillus plantarum and Pediococcus pentosaceus were capable of decarboxylating all p-coumaric, caffeic and ferulic acids. Other strains only decarboxylated p-coumaric and caffeic acid (6), only p-coumaric acid (2) or only caffeic acid (1), while 10 strains did not decarboxylate any HCA. p-Coumaric acid had the highest conversion efficiency, followed by caffeic acid and lastly ferulic acid. Results were confirmed by HPLC-DAD-ESI-MS analyses, showing the conversion of HCAs into their 4-vinylphenol derivatives. This work can help improve the sensory characteristics of HCA-rich foods where fermentation with LAB was used during processing.

Purification and characterization of a phospholipid-hydrolyzing phosphoesterase produced by Pediococcus acidilactici isolated from Gouda cheese.

Lipolysis occurs during ripening of dairy products as a result of esterase or lipase activity. Lactic acid bacteria (LAB) are considered to be weakly lipolytic bacteria compared with other species. In cheeses with extended ripening periods, lipolytic LAB may have several advantages. Pediococcus acidilactici is a LAB frequently found in fermented dairy products, but no previous reports exist on their production of esterases or lipases. Our interest in the relationship of LAB and enzymatic characterization is due to the multiple reports of the benefits of LAB in the gut microbiome, particularly at the intestinal membrane. Pediococci have been characterized as probiotic and especially active in membrane interactions. The aim of this project was to purify, characterize, and identify the phosphoesterase produced by P. acidilactici originally isolated from Gouda cheese and determine its phospholipid (PL) hydrolysis profile, with a focus on increased absorption of these compounds in the human gut. Native zymograms were performed to identify a protein with lipolytic activity in the intracellular fraction of P. acidilactici. The enzyme was purified via size-exclusion HPLC, concentrated via ultrafiltration, and identified using sequence analysis in liquid chromatography (LC)-MS/MS. The purified fraction was subjected to biochemical characterization as a function of pH, temperature, ion concentration, hydrolysis of different substrates, and PL. A single protein with a molecular weight of 86 kDa and esterase activity was detected by zymography. Analysis of the LC-MS/MS results identified a putative metallophosphoesterase with a calculated molecular weight of 45.5 kDa, suggesting that this protein is active as a homodimer. The pure protein showed an optimal activity between pH 8.0 to 9.0. The optimal temperature for activity was 37°C, and the enzyme lost 15% of activity after incubation at 90°C for 1 h. This enzyme showed activity on short-chain fatty acids and exhibited high hydrolysis of phosphatidylinositol. It also hydrolyzed phosphatidylserine, phosphatidylcholine, and sphingomyelin. Phosphatidylethanolamine was hydrolyzed but with less efficiency. The characteristics and lipolytic actions exerted by this protein obtained from LAB hold promise for a potential strain of esterase or lipase that may exert human health benefits through increased digestibility and absorption of nutrients found in dairy products.

Technically relevant enzymes and proteins produced by LAB suitable for industrial and biological activity.

Lactic acid bacteria (LAB) are a unique subset of microorganisms that have co-evolved with humans since the beginning of agricultural practices and animal domestication and throughout our never-ending quest for food preservation, digestibility, and flavor enhancement. LAB have historically played a preponderant role in our foods. In this review, we focus on the enzymatic activities and current or potential applications of LAB in our lives. A description of each of the enzymatic systems in LAB is included. Glycosidases, which hydrolyze the most abundant food molecules and as sources of carbon, sustain the lives of organisms on Earth as well as ensure microbial innocuity by the production of lactic acid from the uniquely mammalian carbohydrate, lactose. Lipases and proteases or proteinases are of fundamental importance in food fermentations and in dairy foods for flavor development. Bacteriocins and peptidoglycan hydrolases are part of the enzymatic system of LAB that has evolved to make these bacteria fierce competitors in various microbiomes, which are highly important for the human gut. In this review, we also present an explanation on how the versatility of the genetics of LAB can adapt to the matrix where they are placed with the advantage of not having any toxicity to humans. The systematic study of LAB enzymes has allowed for some unique applications in foods and biopharmaceutical industries. Here, we summarize how different enzyme systems in LAB are classified, and thus, facilitate much-needed further studies to understand the fundamentals and translate them into applications to improve our lives.

Lactic acid bacteria isolated from dairy products as potential producers of lipolytic, proteolytic and antibacterial proteins.

Regular consumption of fermented dairy products helps maintain a healthy microbiota and prevent gut dysbiosis-linked diseases. The lactic acid bacteria (LAB) present in food enhance the digestibility of proteins, moderate the release of fatty acids, and support human health through inhabiting the gastrointestinal tract. These desirable properties of LAB are attributed, in part, to their metabolic processes involving enzymes such as lipases, proteases, and antibacterial proteins. The LAB strains presenting higher enzymatic activities may offer improved functionality for applications in foods. The first aim of this work was to isolate and identify LAB from diverse dairy products and select those with enhanced enzymatic activities. Secondly, this work aimed to investigate the subcellular organization and identity of these enzymes after semi-purification. Out of the total 137 LAB strains isolated and screened, 50.3% and 61.3% of the strains exhibited lipolytic and proteolytic activities, respectively. Seven strains displaying high enzymatic activities were selected and further characterized for the cellular organization of their lipases, proteases, and antibacterial proteins. The lipolytic and proteolytic activities were exhibited predominantly in the extracellular fraction; whereas, the antibacterial activities were found in various cellular fractions and were capable of inhibiting common undesirable microorganisms in foods. In total, two lipases, seven proteases, and three antibacterial proteins were identified by LC-MS/MS. Characterization of LAB strains with high enzymatic activity has potential biotechnological significance in fermentative processes and in human health as they may improve the physicochemical characteristics of foods and displace strains with weaker enzymatic activities in the human gut microbiota.

Biochemistry and regulatory functions of bacterial glucose kinases.

Glucokinases (Glks) are enzymes widely distributed in all three domains of life. They are located at the beginning of the glycolytic pathway and are responsible for the glucose phosphorylation from various phosphate group donors such as ATP, ADP and polyphosphate. So far, there are eight crystallized Glks, and at least one belongs to each of the three reported Glk families. Structural studies have elucidated the mechanism for Glk action and multimerization. Cloning, overexpression and biochemical characterization have demonstrated the wide diversity of these enzymes. As reported for various microorganisms, in addition to their catalytic activity, some Glks, possessing ROK (Repressor Orf Kinases) motifs, also display a regulatory role. This function has been associated to the mechanisms of carbon catabolite regulation, morphological differentiation and antibiotic production. The present review covers the classification, detailed tertiary structure, mechanism of action, biochemical characterization and some regulatory aspects of bacterial Glks.