Study of the transgalactosylation activity of ß-galactosidase from a new strain Kluyveromyces lactis 3

Beta-galactosidase (EC.3.2.1.23) is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for synthesis of transgalactosylated oligosaccharides that act as prebiotics with several beneficial effects on the consumers. ß-Galactosidase production by Kluyveromyces lactis 3 was studied in shake flask culture. The highest enzymatic activity was obtained at 10-th hour of the fermentation. The optimum temperature for transferase activity was 50°C. When incubated with 30% lactose in 50 mM phosphate buffer (pH 6.0) the enzyme can synthesize up to 41% galacto-oligosaccharides (GalOS). β-Galactosidase from strain Kluyveromyces lactis 3 produces mainly oligosaccharides with degree of polymerization (DP) 6 at 40°C and with DP 3 at 50°C

Physiological studies of Leuconostoc mesenteroides strain NRRL B-1149 during cultivation on glucose and fructose media

Glycosyltransferases are extracellular and cell-associated sucrase enzymes produced mainly by lactic acid bacteria Leuconostoc mesenteroides, oral Streptococcus species and also Lactobacillus species. According to the synthesized polymer (glucan or fructan) in the presence of sucrose, these enzymes are divided into two groups: glucosyltransferases (GTFs) and fructosyltransferases (FTFs). Only Streptococcus, Lactobacillus and Leuconostoc strains are known as producers of both GTFs and FTFs. The enzymes from Lactobacillus and Leuconostoc spp. are implicated in the synthesis of polymers and oligosaccharides (OS) important for human health because of their prebiotic properties and immunomodulating activity. In the present work, we studied the production of extracellular and cell-associated glycosyltransferases by Leuconostoc mesenteroides strain NRRL B-1149 during its growth on media containing glucose or fructose as a main carbon source. The enzyme activities, pH and biomass formation were measured and compared during the cultivation. We have shown that glucose and fructose have not an equal role for enzyme production. The highest extracellular activity was detected at the 4th hour during the cultivation of the strain in medium with fructose – 5.45 U/mg. When the strain was cultivated in medium with glucose, the maximum of extracellular enzyme activity was detected at the 5th hour of the cultivation but the measured activity was about 9 times lower compared to these, obtained after cultivation in fructose medium. The studied strain produced mainly extracellular glycosyltransferases in glucose or fructose medium, which were 92.4% and 97.1% of the total enzyme activity, respectively. In order to characterize the produced enzymes, cell-associated and extracellular enzymes were determined using SDS-PAGE and in situ Periodic Acid Schiff′s staining after incubation with 10% sucrose. When the investigated strain was grown in media with sucrose, glucose or fructose, several types of glycosyltransferases were detected – dextransucrase with molecular weight 180 kDa and two fructosyltransferases, corresponding to 120 kDa and 86 kDa molecular weights

Lukanka, a Semi-Dried Fermented Traditional Bulgarian Sausage: Role of the Bacterial Cultures in its Technological, Safety and Beneficial Characteristics

Background: Production of different fermented meat products is a well-known practice done in different European countries since ancient times. Fermentation of primary materials and/or smoking and salting processes are part of the preservation processes and is important for the formation of final products which is inherent in South European countries. Originally, fermentation of meat products is intended for preservation and safe storage for long periods of time. However, nowadays, gastronomical properties of fermented meat products are essential in obtaining specific flavor, odor, color and structure of the sausages which consumers highly prefer. Emphasis is given on gastronomic characteristics, which results from the various combination of raw meat, specific spices and the natural microbiota or conducted fermentation processes by application of specific starter cultures. Seven bacterial genera (Lactobacillus, Leuconostoc, Staphylococcus, Enterococcus, Lactococcus, Micrococcus and Streptococcus) are commonly used as meat starter cultures. Complex fermentation processes that occur during the ripening of the fermented meat products are the results of the interaction between bacterial starter cultures, remaining enzymes in the muscle and fat tissue and available bacterial enzymes. Objective: The present overview aims to provide information related to the characterization of the specific microbiota associated with lukanka, a naturally-fermented semi-dried Bulgarian sausage. What is the specificity of its fermentation processes; how do different starter and indigenous meat microbiota interfere to form specific final products; what is the role of starter and adjunct cultures in the safety of the products; how is the Bulgarian lukanka classified in the perspective of other Mediterranean dry fermented sausages? These are some of the questions that this review will discuss

Effects of Organic Solvents on Acceptor Reactions for Oligosaccharide Synthesis Catalyzed by Glucansucrase URE 13-300

  Abstract   Background and Objective: Glucansucrases from GH70 family are effective transglucosylases, able to use non-carbohydrate acceptors. Glycosylation of flavonoids or terpenoids increases their water-solubility and bioavailability. Enzymatic glycosylation by glucansucrases can be improved by addition of organic solvents to the reaction media. Thus, the aim of the study was to assess effects of menthol, carvacrol and thymol solubilized in organic solvents on the activity of glucansucrase URE 13-300 and transferase reaction. Material and Methods: Several organic solvents were assessed for their effects on glucansucrase activity using DNS method. Kinetic parameters in presence of the most appropriate solvents were evaluated as well. Thymol, carvacrol and menthol were solubilized in DMSO and their effects on the enzyme activity was assessed. Dynamic of oligosaccharides synthesis in aqueous-organic media was investigated using high-performance liquid chromatography. Results and Conclusion: Maltose-derived oligosaccharides synthesized by glucansucrase URE 13-300 showed degrees of polymerization from 3 to 6 in presence of organic solvents, as well as in presence of buffer alone. Their concentrations did not differ significantly in each of the reactions in aqueous-organic media. Furthermore, kinetic parameters showed adjacent Km values with 5% solvents compared to the control reaction in buffer. These findings revealed that the overall synthesis of glucooligosaccharides was not altered by the organic solvents, nevertheless they changed the product distribution throughout the transferase reactions. These moderate effects of the selected organic solvents were important requirement for the glycosylation of biologically active compounds for use in the food industry. Conflict of interest: The authors declare no conflict of interest

Metabolic Profiling of Xylooligosaccharides by Lactobacilli

Three lactic acid bacteria (LAB) strains identified as Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus sakei isolated from meat products were tested for their ability to utilize and grow on xylooligosaccharides (XOSs). The extent of carbohydrate utilization by the studied strains was analyzed by HPLC. All three strains showed preferences for the degree of polymerization (DP). The added oligosaccharides induced the LAB to form end-products of typical mixed-acid fermentation. The utilization of XOSs by the microorganisms requires the action of three important enzymes: β-xylosidase (EC 3.2.1.37) exo-oligoxylanase (EC 3.2.1.156) and α-L-arabinofuranosidase (EC 3.2.1.55). The presence of intracellular β-D-xylosidase in Lb. brevis, Lb. plantarum, and Lb. sakei suggest that XOSs might be the first imported into the cell by oligosaccharide transporters, followed by their degradation to xylose. The studies on the influence of XOS intake on the lipids of rat liver plasma membranes showed that oligosaccharides display various beneficial effects for the host organism, which are probably specific for each type of prebiotic used. The utilization of different types of oligosaccharides may help to explain the ability of Lactobacillus strains to compete with other bacteria in the ecosystem of the human gastrointestinal tract

In vitro Digestion: Exploring the probiotic abilities and metabolization of human milk oligosaccharides by two strains of Limosilactobacillus fermentum isolated from breast milk

Human breast milk (HBM) serves as the most optimal nourishment for infants, not only providing essential nutrition but also boasting a rich array of immune components. These include secretory antibodies, immune cells, antimicrobial proteins (like lactoferrin and lysozyme), cytokines, and human milk oligosaccharides. Today, the presence of a specific microbiome in human milk is known. Our primary research involves the examination of certain probiotic properties displayed by two strains of Limosilactobacillus fermentum, which were isolated from breast milk. Furthermore, our goal was to evaluate their ability to metabolize breast milk-derived oligosaccharides through an in vitro digestion simulation system. The in vitro model simulating gastrointestinal digestion was performed according to INFOGEST method with some modification. We used various molecular techniques to isolate and identify strains from breast milk. Additionally, we employed different biochemical analyses to determine specific enzyme activities, and we also assessed the fundamental probiotic characteristics of two strains Limosilactobacillus fermentum. We found good probiotic characteristics in the examined strains, as well as favorable growth properties in the presence of specific human milk oligosaccharides. In this regard, we believe that breast milk represents a rich source for isolating potential probiotic strains

Optimization of the expression of levansucrase L17 in recombinant E. coli

Levansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose

Metabolic profiling of probiotic strain Lactobacillus delbrueckii subsp. bulgaricus L14 cultivated in presence of prebiotic oligosaccharides and polysaccharides in simulating in vitro gastrointestinal tract system

AbstractThis study examined the effect of lactulose, galactooligosaccharide, fructooligosaccharide, inulin, and β-glucan on the probiotic strain Lactobacillus delbrueckii subsp. bulgaricus L14, cultivated in an in vitro gastrointestinal system model. We analyzed the degree of hydrolysis of the studied prebiotic oligosaccharides in condition of simulated gastric fluid. The results showed that lactulose had the highest resistance, galactooligosaccharide underwent hydrolysis, and fructooligosaccharide was the most sensitive. Among the polysaccharides, fructose was released from inulin and glucose from β-glucan. Short-chain oligosaccharides and metabolites derived from studied prebiotic oligosaccharides and polysaccharides, supported the growth of probiotic strain L14, which showed the highest growth with fructooligosaccharides and β-glucan as carbohydrate sources. The profile of the activated enzymes secreted by the probiotic strain L14, indicated their inducible character. Beta-galactosidase was activated in the presence of lactulose and GalOS, inulinase was activated in the presence of inulin and fructooligosaccharides, and β-glucosidase was activated in the presence of β-glucan fragments. Analysis of the produced organic and short-chain fatty acids showed that the typical representative of the homofermentative lactobacilli Lb. delbrueckii subsp. bulgaricus L14 changes its metabolism from a homofermentative to a heterofermentative type, best expressed in the presence of lactulose, galactooligosaccharide, and β-glucan

Multi-Biomarker Assessment in Common Carp (Cyprinus carpio, Linnaeus 1758) Liver after Acute Chlorpyrifos Exposure

L