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Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glc p -(1→6)- ( Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glc p -(1→6)-branches ( Leuconostoc citreum , Leuconostoc fallax , Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glc p -(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

Inulin has been widely applied in food, pharmaceuticals, and many other fields because of its versatile physicochemical properties and physiological functions. Previous research showed that inulosucrase from microorganisms could produce higher-molecular-weight inulin than vegetal inulin. Herein, a food-grade recombinant Bacillus subtilis expression system was constructed to produce inulosucrase from Lactobacillus gasseri DSM 20604 without antibiotic resistance genes. The produced inulosucrase was used to biosynthesize inulin with an average molecular weight of 5.8 × 10 6 Da. The physicochemical properties of the produced Lactobacillus inulin were evaluated including microstructure, thermal characteristics, crystallinity, rheological behaviors, and storage stability. By comparing with vegetal inulin and other polymers, the biosynthesized microbial inulin showed some superior properties, such as better gel-forming capability and storage stability in aqueous solution than vegetal inulin. These results opened up possibilities for further investigations aimed at developing microbial inulin in the food industry.

Lactic acid bacteria are known to produce extracellular polysaccharides such as α-glucan, levan, and inulin, which are promising for applications in food systems because of their prebiotic properties. In this work, a novel glucansucrase (GS) Gtf-DSM from Lactobacillus ingluviei DSM 14792 was recombinantly expressed and biochemically characterized as a dextransucrase capable of producing a dextran polysaccharide with four types of linkages, including 69% (α1 → 6), 24% (α1 → 3), 6% (α1 → 4), and 1% (α1 → 2). Intriguingly, the dextransucrase Gtf-DSM had a sequence identity of 99.3% with the reuteransucrase GtfO producing a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. Thus, the dextransucrase Gtf-DSM is a unique target for understanding the linkage specificity of GSs and the investigation of site-directed mutagenesis using Gtf-DSM and GtfO as templates is underway.

Liver diseases alter the gut microbiota, but several lactic acid bacteria can reduce the degree of liver damage. The present study investigated whether Lactobacillus buchneri TCP016 reduces the degree of liver damage by modifying the gut microbiota via its exopolysaccharides (EPSs). First, it was illustrated that the main EPS (EPS016; molecular weight = 8.509 × 10 4 Da) comprised rhamnose, xylose, glucosamine, glucuronic acid, galactose, galacturonic acid, glucose, and mannose in molar ratios of 9.2:3.9:3.8:2.8:2.1:2.0:1.6:1.0. Our data showed that EPS016 alleviated the increase in plasma and hepatic enzyme and cytokine levels, increased superoxide dismutase and glutathione activity, and alleviated bacterial translocation to the liver and mesenteric lymph nodes in vivo. Furthermore, EPS016 ameliorated intestinal mucosal injury and gut flora dysbiosis, thereby decreasing the enrichment of Helicobacteraceae , Lachnospiraceae , and Enterobacteriaceae and increasing the abundance of Lactobacillus , Rikenellaceae , Bacteroidaceae , Bacteroidales _S24-7_group, and Prevotellaceae . These findings indicated that EPS016 inhibits lipopolysaccharides/d-galactosamine-induced liver injury and improves the modification of the gut microbiota.

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD + -dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg -1 , which was close to four times the activity of the probe. Meanwhile, the k cat / K m value of LhDMDH was 28.80 S -1 ·mM -1 , which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.

Hydroxy unsaturated fatty acids (HUFA) can function as antifungal agents. To investigate the antifungal spectrum, that is, the scope of the in vitro fungal-inhibition activities of HUFA and their potential applications, three HUFA were produced by microbial transformation or extracted from plant-seed oils; these compounds included coriolic acid (13-hydroxy-9,11-octadecadienoic acid) from Coriaria seed oil, 10-hydroxy-12-octadecenoic acid from cultures of Lactobacillus hammesii, and 13-hydroxy-9-octadecenoic acid from cultures of Lactobacillus plantarum TMW1.460Δlah. HUFA were purified by high-speed counter-current chromatography (HSCCC), characterized by LC-MS and MS/MS, and their antifungal activities were evaluated with 15 indicator fungal strains. The HUFA had different antifungal spectra when compared with unsaturated fatty acids with comparable structures but without hydroxy groups. The inhibitory effects of HUFA specifically targeted filamentous fungi, including Aspergillus niger and Penicillium roqueforti, whereas yeasts, including Candida spp. and Saccharomyces spp., were resistant to HUFA. The findings here support the development of food applications for antifungal HUFA.

Chicoric acid (ChA) and caftaric acid (CafA) were identified as bioactive components of chicory and have been ascribed a number of health benefits. This study investigated the hydrolysis of ChA and CafA with enzymes and a probiotic bacterium Lactobacillus johnsonii (La1). Esterase from Aspergillus japonicus (24 U/mg) hydrolyzed 100% of ChA (5 mM) and CafA (5 mM) after 3 h, at pH 7.0 and 37 °C. Under the same reaction conditions, 100% hydrolysis of ChA and CafA was achieved with a spray-dried preparation of La1. The addition of La1 (100 mg/mL, 3.3 E9 cfu/g) to CafA solution in a gastrointestinal model (GI model) resulted in 65% hydrolysis of CafA. This model simulates the physicochemical conditions of the human gastrointestinal tract. No hydrolysis of CafA was observed after passage through the GI model in the absence of La1. The results of this study support the hypothesis that ChA and CafA are degraded by gut microflora before absorption and metabolization.

β-Galactosidases (β-Gal) of lactic acid bacteria produce oligosaccharides from lactose when suitable acceptor carbohydrates are present. This study aimed to elucidate the structure of oligosaccharides formed by galactosylation of N-acetylglucosamine (GlcNAc) and fucose. Crude cellular extract of Lactobacillus bulgaricus and LacLM of Lactobacillus plantarum were used as sources of β-Gal activity. Disaccharides obtained by galactosylation of GlcNAc were identified as Gal-β-(1→4)-GlcNAc or Gal-β-(1→6)-GlcNAc by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and comparison with external standards. Trisaccharides were identified as Gal-β-(1→6)-Gal-β-(1→[4 or 6])-GlcNAc by LC-MS, analysis of the MS/MS spectra of selected in-source fragment ions, and their relative retention times. LC-MS analysis revealed the presence of five galactosylated fucosides, but their linkage type could not be identified, partly due to the lack of reference compounds. β-Gal of lactic acid bacteria may serve as suitable tools for the chemoenzymatic synthesis of therapeutic oligosaccharides.

Many studies have investigated the effects of pH, temperature, and salinity on the surface-active properties of various surfactants, although in most cases the variables have been studied separately, without considering the effects of any interactions between them. In the present study, a Box-Behnken factorial design was applied to study the effects of pH, temperature, and salinity on the surface-active properties of a biosurfactant produced by Lactobacillus pentosus. The data obtained enabled development of a second-order model describing the interrelationships between operational and experimental variables, by equations including linear, interaction, and quadratic terms. The variable that had the greatest effect on the surface-active properties of the biosurfactant was pH. Moreover, at pH 3-5.5, decreases in salinity and temperature acted synergistically, reducing the surface tension of the biosurfactant; at pH 8, the same effect was observed with increasing salinity and temperature.

Tapping mode atomic force microscopy (TM-AFM) has been used to analyze the supramolecular structure and conformation of the (1-->3)(1-->2)-beta-D-glucan produced by Lactobacillus suebicus CUPV221 isolated from cider. Solutions for TM-AFM observation were prepared by dispersing the solid glucan in distilled water and in alkaline aqueous solutions. It was found that from the distilled water at 10 mg/L or higher concentrations, the (1-->3)(1-->2)-beta-D-glucan forms networks. The heat resistance of the networks depends on the concentration. From the alkaline aqueous solutions, different supramolecular structures were observed depending on the pH. From the weakest alkaline solution, a fairly rough morphology with a high density of spikelike growth features was revealed. As the ionic force of the medium increased, the sizes of the spikelike growth features diminished, and even many disaggregated fibers could be found. At 0.4 M NaOH (pH 13.16), the aggregates had disappeared almost totally. NaOH aqueous solutions (0.1 and 0.4 M) were used to carry out the study of conformation. At 0.1 M NaOH, the aggregates were partially detached, and many free microfibers were found to which a helical conformation could be assigned due to their stiffness and rodlike character. At 0.4 M NaOH, the beginning of the dissociation of the helical structures was seen.

This work demonstrates that the biosurfactant produced by Lactobacillus pentosus from grape marc hydrolysates can be successfully employed in reducing the water repellence of hydrophobic substrates, rather than chemical surfactants, as it can be produced from low-cost residual materials and it is less toxic than chemical surfactants. The method employed to measure the water repellence of the 11 plant substrates, consisting of pine bark, peat, and composts from various origins (biodegradable fraction of municipal solid waste, green waste, sewage sludge, manure, pine bark, and grape marc), was the molarity of ethanol droplet method (MED). Peat, pine bark, and the composts obtained from grape marc and pine bark were severely hydrophobic, having contact angles over 104 degrees , whereas the composts from municipal solid waste were less hydrophobic, with contact angles under 101 degrees . When hydrophobic substrates were treated with the biosurfactant from L. pentosus, the water repellence of the plant substrates was reduced in all but two cases (the least hydrophobic composts), achieving in most of the cases results better than those obtained using chemical surfactants.

Se-enriched Lactobacillus bulgaricus (L. bulgaricus) was generated by administration of sodium selenite (0, 1, 4, 8, 16, 32, and 64 mg/L, respectively) in MRS medium and enriched selenium manifestation in L. bulgaricus was investigated using transmission electron microscopy and energy-dispersive X-ray spectrometry and alterations of essential elements and amino acids in the organism were evaluated. We demonstrate that administration of sodium selenite in the dosage of 1-16 mg/L is suitable for selenium enrichment in L. bulgaricus and can enhance nutritive value in the organism by elevating the contents of essential elements including P, Mg, Mn, Zn, Ca, and total amino acids as well as reducing selenite to insoluble elemental selenium, an electron-dense and amorphous Se (0) granule, thereby depositing it both in the cytoplasm and in the extracellular space of L. bulgaricus. Thus, Se-enriched Lactobacillus can provide a potential dietary source of nontoxic selenium and functional regulator used for food and medical industry.

Viable, heat-and acid-killed Lactobacillus rhamnosus strain GG (LGG) has shown high binding properties with zearalenone (ZEN). To identify the type of chemical moieties and interactions involved in binding with the ZEN, LGG was subjected to different chemical and enzymatical treatments, prior to the binding experiments. Pretreating the viable, heat- and acid-killed bacteria with m-periodate significantly decreased ZEN binding, suggesting that ZEN binds predominantly to carbohydrate components. Pretreatment with Pronase E had no effect on the ability of viable cells to bind ZEN, however, a reduction in the binding of ZEN by heat- and acid-killed cells, suggesting that the new binding sites exposed by heat or acid are proteins in nature. Pretreatment with urea also decreased binding, suggesting that hydrophobic interactions play a role in ZEN binding. The binding of ZEN in concentrations ranging from 0.79 to 62.82 microM and its subsequent dissociation by repetitive aqueous washes was also studied. The binding sites of the bacteria were not saturated by the maximum ZEN concentration studied.

Two innovative microwave hydrolysis procedures for rapidly detecting meso-diaminopimelic acid in lactic acid bacteria have been developed. Whole-cell hydrolysis was performed both in liquid phase and vapor phase with 6 N HCl in sealed vessels using a microwave oven equipped with pressure and temperature probes. The presence or absence of meso-diaminopimelic acid determined by TLC, after the application of liquid- and vapor-phase microwave hydrolysis procedures, gave the same qualitative results as those obtained by traditional hydrolysis. These standardized microwave hydrolysis procedures permit a drastic reduction in hydrolysis time, from 16-20 h to less than 10 min and, consequently, in the total time of meso-diaminopimelic acid analysis (less than 90 min). Microwave hydrolysis in vapor phase is particularly convenient because, besides reducing hydrolysis time, it also eliminates the successive troublesome step of HCl removal.