To analyze the response mechanisms of Lactobacillus plantarum against manganese starvation stress, different metabolisms from physiology, proteomics and transporters aspects in L. plantarum CCFM 436 were systematically investigated. The kinetics of cell growth (μmax) decreased from 0.310 to 0.256 h−1, while thinner cell morphology was observed by transmission electron microscopy under Mn-starvation conditions. Gas chromatography-mass spectrometry analysis indicated that membrane mobility and compactness increased, with a higher proportion of unsaturated fatty acids and cyclopropane fatty acids. High-performance liquid chromatography analysis showed that intracellular Asp, Glu, and Arg contents, closely related to energy metabolism, were significantly increased. Fourier transform infrared spectroscopy proved that some functional groups (N–H and OC–OH) were significantly affected by Mn starvation. Comparative two-dimensional proteomic analysis identified 73 proteins that differed significantly under Mn starvation conditions. These differentially expressed proteins involved in carbohydrate, amino acid and transcription/translation metabolisms and stress response were categorized as crucial components required to resist manganese starvation stress. Moreover, qRT-PCR analysis proved that MntH 1–5, negatively regulated by MntR, acted as potential Mn importers under Mn starvation conditions. The proposed coordinated mechanism model provides a reference for, and insight into, the intracellular metabolism of LAB strains.

Conjugated linoleic acid (CLA) has been shown to exert various potential physiological properties including anti-carcinogenic, anti-obesity, anti-cardiovascular and anti-diabetic activities, and consequently has been considered as a promising food supplement. Bacterial biosynthesis of CLA is an attractive approach for commercial production due to its high isomer-selectivity and convenient purification process. Many bacterial species have been reported to convert free linoleic acid (LA) to CLA, hitherto only the precise CLA-producing mechanisms in Propionibacterium acnes and Lactobacillus plantarum have been illustrated completely, prompting the development of recombinant technology used in CLA production. The purpose of the article is to review the bacterial CLA producers as well as the recent progress on describing the mechanism of microbial CLA-production. Furthermore, the advances and potential in the heterologous expression of CLA genetic determinants will be presented.

The aim of this study was to investigate the effects of mixed lactic acid bacteria (LAB) against microcystin-LR-exposed hepatotoxicity and oxidative stress in BALB/c mice. 20 LAB strains were randomly divided into three groups and the effects against microcystin-LR (MC-LR) toxicity investigated in vivo. The antioxidative abilities of the three LAB groups were also determined. The results showed that a cocktail of LAB 3 (CLAB3), including Lactobacillus johnsonii ATCC 33200, L. rhamnosus GG, Bifidobacterium adolescentis 1.1290 and B. bifidum CCFM 16, was able to significantly reverse the increased levels of serum ALT and AST, and liver tissue lesions by MC-LR. The alterations of hepatic malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities in the CLAB3 were improved compared to the MC-LR group alone. Moreover, the free radical scavenging and reducing abilities of CLAB3 were higher than other groups. CLAB3 can significantly alleviate MC-LR-induced hepatic damage and improve its oxidative stress. Moreover, the protective effects of the CLAB3 groups are related to its outstanding antioxidative abilities in vitro. LAB can be a promising dietary strategy to prevent cyanobacteria contamination toxicity.

In the present study, a single-stranded DNA (ssDNA) aptamer binding to Bifidobacterium breve with high avidity and selectivity was selected through a whole-bacterium-based systemic evolution of ligands using an exponential enrichment (SELEX) process. Following 12 rounds of selection specific for B. breve, three FAM-labeled aptamers were chosen for flow cytometry analysis and the results revealed that all three aptamers possessed a high binding affinity for B. breve. To obtain the optimal sequence, sequence truncation experiments of these three aptamers were conducted. An aptamer variant BB16-11f with high affinity and selectivity was acquired. In addition, the dissociation constant was significantly reduced to 18.66 ± 1.41 nM. Furthermore, an enzyme linked aptamer assay was developed to prove the potential application of the aptamer BB16-11f in the detection of B. breve. The results showed that the colorimetric assay had a linear relationship between the absorbance at 450 nm and the concentrations of B. breve ranging from 103 cfu mL−1 to 107 cfu mL−1 with a correlation coefficient of 0.98. The limit of detection (LOD) of the assay was 1000 cfu mL−1. Additionally, the developed method was also successfully used to detect B. breve in a milk environment. Taken together, we hold that the developed colorimetric bioassay based on the aptamer BB16-11f is a promising method for the detection of B. breve.

The exopolysaccharide (EPS) production of several Bifidobacterium longum strains isolated from infant and elder feces was determined. The relationship between EPS production and tolerance to artificial gastric and intestinal juices was analyzed. Moreover, priming glycosyltransferase (pGT) gene fragments of these strains were amplified and sequenced. The results indicate that their tolerance correlated well with EPS production, especially the production of cell-surface-bound exopolysaccharide (EPS-b). The EPS-b production by strains isolated from elderly volunteers was found to be significantly higher than that produced by strains isolated from infants. Lastly, the phylogenetic tree of the pGT gene sequence fragments showed that the pGT genes of infant-originated B. longum strains had greater homology than those of elder-originated strains.

•Syntheses of milk-clotting enzyme and exopolysaccharides by BD3526 are aerobic.•Oxygen supply favors the syntheses of milk-clotting enzyme and exopolysaccharides.•Exopolysaccharides accumulations hinder the elevation of milk-clotting activity.•BD3526 milk-clotting enzyme is a potential bacterial coagulant.Paenibacillus spp. BD3526, a novel bacteria species isolated from raw yak (Bos grunniens) milk collected in Tibet, is able to produce metalloproteinase with milk-clotting activity (MCA) in wheat bran broth. To determine the critical factor favoring enzyme productivity, response surface methodology and the Box-Behnken design were adopted to preliminarily optimize BD3526 cultivation conditions for maximum MCA in shaking flasks. The highest degree of MCA (7072 ± 519 SU/mL) was obtained at a liquid volume of 30 mL and 300 r/min, which implies that oxygen supply is crucial for enzyme synthesis. MCA levels were enhanced as agitation speeds and aeration rates were increased in a 7-L scale fermentor. MCA in the supernatant was further boosted by adding hydrogen peroxide (H2O2) to the broth during cultivation. Maximum MCA (14778 ± 1100 SU/mL) and MCA/proteolytic activity (PA) (33000 ± 918) were achieved when cultivation was carried out at an agitation speed of 400 r/min, aeration rate of 5.5 L/min and H2O2 flow of 500 mmol/L at a feeding rate of 2.5 mL/h after 24 h of cultivation.

•Probiotic has significantly increased serum SOD and GSH-PX activities.•The decrease of MDA is significant in probiotic intervention.•Probiotics reduce oxidative stress in aged model mice.This review evaluates the evidence from animal studies for the protective effects of probiotics administration on d-galactose-induced oxidative stress. Of 192 articles identified from Web of Science, Embase and PubMed, 8 articles were included in the meta-analysis. The studies included 280 mice in total. Estimates of plasma superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) were examined using Review Manager Version 5.3. Based on the standardised mean difference (SMD), beneficial effects of probiotics administration were observed in the intervention group compared to the d-galactose model as follows: SOD activity (SMD: 1.59; 95% CI: 0.67, 2.50, P = 0.0007); GSH-PX levels (SMD: 2.93; 95% CI: 1.85, 4.02; P < 10−5); and MDA content (SMD: −2.14; 95% CI: −3.24, −1.05; P = 10−4). This review suggests that probiotics treatment has significant effects on serum SOD activity, GSH-PX activity and MDA content and reduces oxidative stress in aged model mice.

•The response mechanism of L. plantarum in excessive Mn stress was revealed.•EPS was secreted and unsaturated fatty acids of the membrane increased.•Differentially expressed protein via proteomics indicated potential response mechanism.•MntH 4–5, positively regulated by MntR, was identified as key Mn transport elements.As a natural inhabitant of gastrointestinal tract, Lactobacillus plantarum is well known for its health-promoting effects, especially in heavy metal-removal function. The response mechanism integrated physiology and proteomics of Lactobacillus plantarum under excessive manganese stress was systematically performed. More extracellular polymeric substance was secreted, whereas cells preferred to aggregate for resisting manganese stress. The stability of the membrane was maintained by up-regulating the proportion of unsaturated fatty acids, especially cyclopropane fatty acids. The contents of intracellular Asp and Arg, closely related with energy metabolism, decreased under excessive manganese stress. Comparative 2-DE proteomic analysis identified 52 proteins that significantly differed under different levels of manganese stress. The differentially expressed proteins, involved in the carbohydrate, amino acid, stress response and nucleotide metabolisms, were categorized as the crucial components during resisting manganese stress. Moreover, MntH 4 and MntH 5 were identified as the functional elements in regulation of manganese transportation model. The differential expression of six key transporter proteins under different manganese stresses indicated that the MntH 1, 2 and 3 was negatively regulated by MntR, while the regulation of MntH 4 and 5 presented the contrary model. Taken altogether, the study provides new insights into the response mechanism of probiotics to similar environmental stress.Download high-res image (466KB)Download full-size image

Probiotics have been used to rebuild the antibiotic-induced dysfunction in gut microbiota, but whether the different strains of probiotics result in similar or reverse effects remains unclear. In this study, the different recovery effects of two cocktails (each contains four strains) of Lactobacillus and fructooligosaccharide against cefixime-induced change of gut microbiota were evaluated in C57BL/6J mice. The results show that the use of cefixime caused a reduction in the diversities of the microbial community and led to significantly decreasing to one preponderant Firmicutes phylum, which was difficult to restore naturally in the short term. The gut microbiota compositions of the groups treated with the probiotic cocktails were much more diverse than those of the natural recovery group. The effects of Lactobacillus cocktails against the cefixime-induced gut microbiota change may mainly be due to the beneficial SCFAs production in vivo and also be related to the good cell adhesion properties performed in vitro. Meanwhile, the restoration of the cefixime-induced gut microbiota was significantly different between two Lactobacillus groups since the Lactobacillus strains with high levels of fructooligosaccharide use and better cell adhesion properties performed considerably better than the Lactobacillus strains with high survival rates in the gastrointestinal tract. The contents of short-chain fatty acids in ceca were increased to 26.483 ± 1.925 and 25.609 ± 2.782 μmol/g in the two probiotic cocktail groups respectively compared to 15.791 ± 0.833 μmol/g (P < 0.05) in control group. Moreover, intestinal inflammation was alleviated by administration of the Lactobacillus cocktails. However, fructooligasaccharide administration showed certain effects on gut microbiota restoration (such as an increase of Akkermansia), although its effect on the entire microbiome structure is not so obvious.

The heavy metal cadmium (Cd) is a hazardous pollutant that exerts various toxic effects on aquatic animals. The biomagnifying effects of this non-essential element in the food chain also pose threats to human health. In this study, the protective effect of a dietary probiotic supplementation, Lactobacillus plantarum CCFM8610, on the intestinal microbiota and physiological conditions of Nile tilapia (Oreochromis niloticus) exposed to waterborne Cd was evaluated. Two hundred fish were divided into four groups, i.e., control, probiotic-only, Cd-only and Cd-plus-probiotic. The fish were exposed to waterborne Cd at a level of 1 mg L−1 for 4 weeks and the probiotic was administered twice daily at 108 CFU g−1 in the fish diet. Waterborne Cd exposure caused a profound decline in the gut microbial diversity and marked alterations in the composition of the microbiota. Dietary supplementation with L. plantarum CCFM8610 reversed the changes in the intestinal microbiota composition in the Cd-exposed fish and reduced the abundance of Flavobacterium and Pseudomonas. Compared with the Cd-only group, the probiotic treatment significantly promoted growth performance and prevented the death of the Cd-exposed fish. L. plantarum CCFM8610 supplementation also decreased Cd accumulation and alleviated oxidative stress in the tissues, and reversed the alterations in hemato-biochemical parameters in the blood of fish. The results suggest that L. plantarum CCFM8610 can be considered a safe dietary supplement for the prevention of Cd-exposure-induced problems in aquaculture and food safety.

Aluminum (Al) has various adverse effects on health of humans and animals. The aim of present study was to demonstrate that Lactobacillus plantarum CCFM639 can alleviate the adverse effects on liver and kidney of mice caused by chronic Al exposure. Animals were assigned into control, CCFM639 only, Al only, Al plus CCFM639, and Al plus deferiprone groups. The strain was given by oral gavage for 14 weeks, and Al was introduced via drinking water for the first 8 weeks. Analyses of Al and trace elements levels in feces, blood, and tissues were performed. The biochemical markers (GSH, GPx, SOD, CAT, and MDA) of oxidative stress in livers and kidneys, as well as the levels of ALT, AST, BUN, and CRE in blood, were determined. Our results showed that L. plantarum CCFM639 can significantly reduce Al accumulation in tissues, regulate imbalance of trace elements, and thereby alleviate oxidative stress and pathological changes in hepatic and renal tissues. Therefore, L. plantarum CCFM639 could alleviate Al-induced hepatic and renal injuries, and the possible mechanisms may involve in regulating the imbalance of trace elements.

•L. plantarum CCFM639 can restore the learning and memory deficits of Al-exposed mice.•This probiotic reduced the Al accumulation in brain.•This probiotic restored integrity of tight junction proteins in brain.•L. plantarum CCFM639 demonstrates significant neuroprotective potential in Al-induced neuronal injury.Aluminium (Al) is a neurotoxin that has a diverse range of negative effects on cerebral function. In our previous study, a candidate probiotic, Lactobacillus plantarum CCFM639, was selected for its ability to alleviate Al toxicity in vitro and in vivo. Our aim was to investigate the neuroprotective potential of L. plantarum CCFM639 in Al-induced neuronal injury. L. plantarum CCFM639 or defetoxamine was given to mice by gavage once daily for 14 weeks. During the first 8 weeks, aluminium chloride was administered orally to all mice via drinking water. The results showed that L. plantarum CCFM639 significantly improved memory deficits of Al-exposed mice. This candidate probiotic also reduced the accumulation of Al in the brains, restored integrity of tight junction proteins, alleviated cerebral oxidative stress and decreased the levels of pro-inflammatory cytokines in the mice brain. Thus, L. plantarum CCFM639 demonstrates significant neuroprotective potential in Al-induced neuronal injury.

•We study the response of rice starch radicals in microwave electromagnetic field.•We reveal three different components of radical in the treated rice starch samples.•Microwave power level and water content could influence the amount of radicals.•We validate the effect of microwave on chemical bonds of rice starch radicals.Radical generation in rice starch under microwave treatment as well as the related chemical bond changes were investigated by electron paramagnetic resonance (EPR) and Raman spectroscopy. Samples with water activity of 0.4 and 0.7 have been treated and analyzed. It was found that microwave power level and water content could influence the amount of radicals along with the radical components and their contribution. Raman spectra showed corresponding changes in vibrational features of chemical bonds. During storage the signal intensity started to drop after a short period of increase. Rice starch radicals were relatively stable and could exist a long time in room temperature. Through signal simulation, 3 main components were separated from the original spectra and the evolving process was investigated. The main component was the radical located on C1 position in the glucose ring.

Heavy metal cadmium (Cd) is an environmental pollutant that causes adverse health effects in humans. This toxic metal has been detected in a wide range of fruit and vegetables. A strain of lactic acid bacteria, Lactobacillus plantarum CCFM8610, was screened out for its good ability to bind Cd, and this study was designed to investigate the Cd binding properties of this bacterium, and to evaluate its use for removal of Cd from fruit and vegetable juices. Electron microscopy observations and energy dispersive X-ray analysis confirmed that the majority of the Cd was bound to the surface of the bacterial cell. The Cd biosorption of L. plantarum CCFM8610 was strongly pH dependent, and carboxyl and amino groups of the bacterial surface molecules are important in the binding process. The biosorption was fast and efficient, and could be well explained by the Langmuir–Freundlich dual isotherm model (R2 = 0.99) and the pseudo second-order kinetic model (R2 = 0.99). After a 2 h incubation and a simple centrifugation, L. plantarum CCFM8610 treatment removed 67% to 82% of the Cd from nine types of fruit and vegetable juices. Long-period fermentation by L. plantarum CCFM8610 (36 h) also significantly decreased Cd concentrations in the juices (56% to 81%). Our results show that this food-grade bacterial strain could be used as a potential probiotic for Cd removal from fruit and vegetable juices.

Traditional paocai brine (PB) is continuously propagated by back-slopping and contains numerous lactic acid bacteria (LAB) strains. Although PB is important for the quality of paocai (Chinese sauerkraut), the taste features, taste-related compounds of PB-paocai and the effects of LAB communities from PB on the taste compounds remain unclear. An electronic tongue was used to evaluate the taste features of 13 PB-paocai samples. Umami, saltiness, bitterness, sweetness, and aftertaste astringency were the main taste features of PB-paocai. A total of 14 compounds were identified as discriminant taste markers for PB-paocai via gas chromatography–mass spectrometry (GC–MS)-based multimarker profiling. A LAB co-culture (Lactobacillus plantarum, Lactobacillus buchneri, and Pediococcus ethanoliduran) from PB could significantly increase glutamic acid (umami), sucrose (sweetness), glycine (sweetness), lactic acid (sourness), and γ-aminobutyric acid in PB-paocai, which would endow it with important flavor features. Such features could then facilitate starter screening and fermentation optimization to produce paocai-related foods with better nutritional and sensory qualities.

In this study, C57BL/6J mice were fed diets supplemented with different proportions of lactulose (0%, 5%, and 15%) for 2 weeks to study its effects on the luminal and mucosal microbiota. The luminal and mucosal samples of cecum and colon were investigated. After high-lactulose treatment (15%), pH of the luminal contents decreased from 6.90–7.72 to 5.95–6.21 from the cecum to distal colon, and the amount of total short-chain fatty acids in the cecum was significantly increased. The luminal content was mostly dominated by Firmicutes, Actinobacteria, and Bacteroidetes, while the mucus was dominated by Firmicutes, Proteobacteria, and Bacteroidetes. The abundance of Actinobacteria was significantly increased in the content, and Proteobacteria was the most abundant phylum (∼50%) in the mucus after high-lactulose treatment. At the genus level, Bifidobacterium and Akkermansia were both significantly increased in the content, and Helicobacter was the most abundant in the mucus.Keywords: Bifidobacterium; Helicobacter; lactulose; luminal; mucosal;

The dietary intake of lead (Pb) via contaminated food and drinking water possesses a serious risk to human health. In this study, the Pb2+ binding ability of 25 lactic acid bacteria strains was investigated, and the strain with the highest binding property, Lactobacillus plantarum CCFM8661 (36.66 ± 1.01 %), was selected for further study. The results of electron microscope revealed that most of Pb2+ was bound on the surface of bacterial cells and small amount of Pb2+ was observed in the cells. The comparison of the binding ability of different cellular components also demonstrated that the cell walls (73.79 ± 0.31 %) play a significant role in the Pb2+ binding. Through the study of the binary metals systems, the results showed that L. plantarum CCFM8661 still preferred to bind Pb2+ at the presence of other cations such as Fe2+ and Zn2+. The binding process was well explained by the Langmuir isotherm model (R2 = 0.97), the Langmuir–Freundlich dual isotherm model (R2 = 0.99), and the pseudo-second-order kinetic model (R2 = 0.99).

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l−1, 100 g wet cells l−1, and 25 g LA l−1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l−1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l−1.Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

•Three cell models were used to assay the CAA value of CFS and SS of lactobacilli.•Three cell lines were associated in determining the CAA values of CFSs and SSs.•No correlation between the AA of CFSs and SSs of test lactobacilli in the CAA assay.•Anti-oxidative properties of CFSs and SSs in CAA assay are strain-specific.Among the beneficial effects of Lactobacillus spp., the common mechanism underlying health protection is believed to be related to antioxidant activity. The antioxidant activities of cell-free suspension (CFS) and strain suspension (SS) of 13 Lactobacillus strains derived from 4 species (L. rhamnosus, L. acidophilus, L. casei, and L. fermenti) were determined. The correlation of CFSs and SSs in different cell lines, as well as the correlation among the three cell models (RAW264.7, Caco-2, and EA.hy926 cell lines), were compared. All CFSs and SSs of lactobacilli showed that they significantly inhibited the production of intracellular reactive oxygen species without creating obvious cytotoxic effects in the three mammal cell lines. These results suggested that RAW264.7, Caco-2, and EA.hy926 cell lines could be used as cell models to assay the cellular antioxidant activity of CFS and SS of lactobacilli. Thus, lactobacilli and by-products are a potential source of natural antioxidants and to reduce oxidative stress.

Dietary fatty acids (FA) are increasingly recognized as major biologic regulators and have properties that relate to health outcomes and disease. Conjugated linoleic acid (CLA) is a generic term denoting a group of isomers of linoleic acid (C18:2, n-6) with a conjugated double bond. CLA has attracted increased research interest because of its health-promoting benefits and biological functions. In a variety of studies, CLA has been shown to impact immune function and has protective effects against cancer, obesity, diabetes, and atherosclerosis in animal studies and in different human cell lines. Studies investigating the mechanisms involved in the biological functions of CLA are emerging with results from both in vivo and in vitro studies. Most of the biological effects have been attributed to the c9,t11-CLA and t10,c12-CLA isomers. The purpose of this review is to discuss the effects of CLA on health and disease and the possible mechanisms for CLA activities.

Our previous study confirmed that Lactobacillus plantarum CCFM8610 has protective effects against chronic cadmium (Cd) toxicity in mice, whereas L. bulgaricus CCFM8004 fails to provide similar protection. This study was designed to evaluate the protective effects of soymilk fermented with these lactic acid bacteria, against chronic Cd toxicity in mice, and to give an insight into the mechanism of the conjunct effect of soymilk and these strains. Experimental mice were divided into five groups as control, Cd only, non-fermented soymilk plus Cd, CCFM8610-fermented soymilk plus Cd, and CCFM8004-fermented soymilk plus Cd. The treatment of all groups was carried out for 8 weeks. Levels of Cd were measured in feces and tissues, and alterations in several biomarkers of Cd toxicity were noted. The results showed that non-fermented soymilk gave limited protection against chronic Cd toxicity in mice. However, oral administration of L. plantarum CCFM8610-fermented soymilk was able to increase fecal Cd excretion, reduce tissue Cd burden, alleviate tissue oxidative stress, reverse changes in hepatic and renal damage biomarkers, and ameliorate tissue histopathological changes in mice, indicating that L. plantarum CCFM8610-fermented soymilk could be considered as a dietary therapeutic strategy against chronic Cd toxicity. The treatment of L. bulgaricus CCFM8004-fermented soymilk provided similar protection, although the effects were less significant than for CCFM8610 treatment. The conjunct effects of the strains and the soymilk may be attributed to the increased Cd excretion ability and antioxidative capacity after fermentation.

Lactobacilli are associated with multiple health-protective effects. One beneficial effect is their antioxidant activity, which needs to be measured using efficient biologically relevant assays. In this study, a cellular antioxidant assay (CAA) was used to determine the cellular anti-oxidative properties of 10 Lactobacillus strains from 5 species (L. rhamnosus, L. plantarum, L. acidophilus, L. casei, L. fermenti) in hepatocellular carcinoma (HepG2) cells and the results were compared with those obtained with the traditional 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging assay. The results from these two methods showed no obvious correlation at three concentrations (107–109 colony-forming units per mL). A further study was performed to evaluate the protective effects of the strains against H2O2-induced oxidative stress in HepG2 cells and the results showed greater consistency in the data obtained with CAA assay than in those from the DPPH radical scavenging assay. The findings indicate that CAA may be a better choice for the detection of the antioxidant activity of Lactobacillus strains.

Aspergillus oryzae 100-8 and the parental strain A. oryzae 3.042 are used in soy sauce fermentation in China. The growth rate of A. oryzae 100-8 is faster than A. oryzae 3.042, and the soy sauce flavors obtained with A. oryzae 100-8 fermentation are better than those obtained with A. oryzae 3.042. In this study, comparisons were made through biomass, reactive oxygen species (ROS) and gas chromatography-mass spectrometry (GC-MS) measurements, and the reasons for these differences were investigated through transcriptome and qRT-PCR analysis. The analysis indicated that several unique genes are closely associated with hyphal growth and flavor formation, as demonstrated by changes in the expression levels of these genes. These unique genes regulate hyphal growth and flavor formation in soy sauce koji fermentation.

Fructooligosaccharides (FOS) are one of the most studied prebiotics, selectively stimulating the growth of health-promoting bacteria in the host. However, there is increasing evidence that commensal gut bacteria, such as Bacteroides fragilis, Clostridium butyricum, Enterobacter cloacae, and even the pathogenic Escherichia coli BEN2908, are also able to metabolize FOS in vitro, and in some cases, FOS displayed adverse effects. Therefore, it is necessary to identify FOS-metabolizing species that are present in the gut. Unlike previous studies focusing on individual strains, this study used the traditional culture method combined with an alignment search on the gut bacteria database established from the Human Microbiome Project (HMP). The alignment results showed that homologous proteins for FOS transporters and glycosidases were distributed in 237 of the 453 strains of gut bacteria. La506 msmK encoding the ATP-binding protein and Aec45 fosGH1 encoding glycoside hydrolase were most widely distributed, in 155 and 55 strains, respectively. Seven of eight strains with both transporters and glycosidases were proven to be capable of metabolizing FOS, while five strains without either transporters or glycosidases were not. Fifteen species isolated from human feces and 11 species from the alignment search were identified to be FOS-metabolizing, of which Cronobacter sakazakii, Marvinbryantia formatexigens, Ruminococcus gnavus, and Weissella paramesenteroides are reported here for the first time. Thus, alignment search combined with the culture method is an effective method for obtaining a global view of the FOS-metabolizing bacteria in the gut and will be helpful in further investigating the relationship between FOS and human gut bacteria.

Fructo-oligosaccharides (FOS) are usually regarded as a type of prebiotic, favorably stimulating the growth of bifidobacteria and lactobacilli. However, they are not the specific substrates for these target species, and other bacteria, such as Streptococcus, Escherichia, and Clostridium, have been shown to be able to utilize FOS. Previous studies have mainly investigated only a few bacteria groups, and few reports analyzed the global effects of FOS on intestinal microbial communities. In this study the effects of FOS on gut bacteria in mice were investigated through a 16S rRNA metagenomic analysis. In the FOS-low group, the abundance of Actinobacteria significantly increased and that of Bacteroidetes decreased after FOS diet (5%) for 3 weeks. In the FOS-high group, Enterococcus was promoted and levels of Bifidobacterium and Olsenella both notably increased after FOS diet (25%) and the microbiota tended to revert to initial structure 2 weeks after FOS treatment ceased. The most striking observation was that Olsenella became a dominant genus comparable with Bifidobacterium after FOS treatment, and one strain of Olsenella, isolated from mice feces, was confirmed, for the first time, to be capable of using FOS. The results indicated that metagenomic analysis was helpful to reveal the FOS effects on the global composition of gut communities and new target for future studies.

Allergic asthma caused by house dust mite (HDM) is becoming a public health problem. Specific immunotherapy is considered to be the only curative treatment, but it is always associated with IgE-mediated side effects in the therapy process. A few studies showed that the disruption of allergen IgE epitopes could reduce IgE reactivity and thus reduce allergenic activity. In this study, a hypoallergenic derivative of the major HDM allergen Der p2 was constructed by genetic engineering. This derivative was confirmed to have a considerably reduced IgE reactivity compared with Der p2. For its application in vivo, recombinant Lactococcus lactis (LL-DM) was engineered to deliver the Der p2 derivative to the intestinal mucosal surface. Oral administration of LL-DM significantly alleviated Der p2-induced airway inflammation, as shown by reduced inflammatory infiltration and a reduction in Th2 cytokines in bronchoalveolar lavage. This protective effect was associated with an up-regulation of specific IgG2a and a decrease in IL-4 level in the spleen which may affect specific IgE response. Moreover, the levels of regulatory T cells in the mesenteric lymph nodes and spleen were markedly increased in mice fed with LL-DM, suggesting that LL-DM can inhibit allergic responses via the induction of regulatory T cell. Our results indicate that the Der p2 derivative is a promising therapeutic molecule for specific immunotherapy and recombinant lactic acid bacteria could be developed as a promising treatment or prevention strategy for allergic diseases.

Lactobacillus rhamnosus CCFM1107 was screened for high antioxidative activity from 55 lactobacilli. The present study attempted to explore the protective properties of L. rhamnosus CCFM1107 in alcoholic liver injury. A mouse model was induced by orally feeding alcohol when simultaneously treated with L. rhamnosus CCFM1107, the drug Hu-Gan- Pian (HGP), L. rhamnosus GG (LGG), and L. plantarum CCFM1112 for 3 months. Biochemical analysis was performed for both serum and liver homogenate. Detailed intestinal flora and histological analyses were also carried out. Our results indicated that the administration of L. rhamnosus CCFM1107 significantly inhibited the increase in the levels of serum aminotransferase and endotoxin, as well as the levels of triglyceride (TG) and cholesterol (CHO) in the serum and in the liver. Glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were elevated while the levels of malondialdehyde (MDA) were decreased. The enteric dysbiosis caused by alcohol was restored by increasing the numbers of both lactobacilli and bifidobacteria and decreasing the numbers of both enterococci and enterobacter. Histological analysis confirmed the protective effect of L. rhamnosus CCFM1107. Compared with the other lactobacilli and to the drug Hu-Gan-Pian, there is a high chance that L. rhamnosus CCFM1107 provides protective effects on alcoholic liver injury by reducing oxidative stress and restoring the intestinal flora.

Lactulose has been known as a prebiotic that can selectively stimulate the growth of beneficial bifidobacteria and lactobacilli. Recent studies have indicated that Streptococcus mutans, Clostridium perfringens, and Faecalibacterium prausnitzii are also able to utilize lactulose. However, the previous studies mainly focused on the utilization of lactulose by individual strains, and few studies were designed to identify the species that could utilize lactulose among gut microbiota. This study aimed to identify lactulose-metabolizing bacteria in the human gut, using in silico and traditional culture methods. The prediction results suggested that genes for the transporters and glycosidases of lactulose are well distributed in the genomes of 222 of 453 strains of gastrointestinal-tract bacteria. The screening assays identified 35 species with the ability to utilize lactulose, of which Cronobacter sakazakii, Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas putida were reported for the first time to be capable of utilizing lactulose. In addition, significant correlations between lactulose and galactooligosaccharide metabolism were found. Thus, more attention should be paid to bacteria besides bifidobacteria and lactobacilli to further investigate the relationship between functional oligosaccharides and gut bacteria.

Peanut allergy (PNA) has becoming a non-negligible health concern worldwide. Thus far, allergen-specific immunotherapy aimed at inducing mucosal tolerance has widely been regarded as a major management strategy for PNA. The safety profiles and the intrinsic probiotic properties of lactic acid bacteria (LAB) render them attractive delivery vehicles for mucosal vaccines. In the present study, we exploited genetically modified Lactococcus lactis to produce peanut allergen Ara h 2 via different protein-targeting systems and their immunomodulatory potency for allergic immune responses in mice were investigated. By comparison with the strain expressing the cytoplasmic form of Ara h 2 (LL1), the strains expressing the secreted and anchored forms of Ara h 2 (LL2 and LL3) were more potent in redirecting a Th2-polarized to a non-allergic Th1 immune responses. Induction of SIgA and regulatory T cells were also observed at the local levels by orally administration of recombinant L. lactis. Our results indicate that allergen-producing L. lactis strains modulated allergic immune responses and may be developed as promising mucosal vaccines for managing allergic diseases.

A. oryzae 3.042 (China) and A. oryzae RIB40 (Japan) used for soy sauce fermentation show some regional differences. We sequenced the genome of A. oryzae 3.042 and compared it to A. oryzae RIB40 in an attempt to understand why different features are shown by these two A. oryzae strains. We predict 11 399 protein-coding genes in A. oryzae 3.042. The genomes of these two A. oryzae strains are collinear revealed by MUMmer analysis, indicating that the differences are not obvious between them. Several strain-specific genes of two strains are identified by genome sequences’ comparison, and they are classified into some groups, which have the relationship with cell growth, cellular response and regulation, resistance, energy metabolism, salt tolerance, and flavor formation. A. oryzae 3.042 showed stronger potential for mycelial growth and environmental stress resistance, such as the genes of chitinase and quinone reductase. Some genes unique to A. oryzae RIB40 were related to energy metabolism and salt tolerance, especially genes for Na+ and K+ transport, while others were associated with signal transduction and flavor formation. The genome sequence of A. oryzae 3.042 will facilitate the identification of the genetic basis of traits in A. oryzae 3.042, and accelerate our understanding of the different genetic traits of the two A. oryzae strains.

Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.

In this study, a naturally unsecretory intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was attempted to be secreted with different types of secretion signals in Bacillus subtilis. The results showed that Nsp can be secreted efficiently by all selected Sec-type signal peptides. Nsp was successfully exported when fused to Tat-type signal peptides but less efficient than Sec-type. The fusion protein with the non-classical extracellular proteins can be detected in the cell and extracellular milieu. This study further demonstrated that the mature protein plays an important role in protein secretion. Moreover, these results indicated that Nsp could be a useful tool to understand the individual roles of mature proteins and signal peptide in protein secretion, to evaluate the effect of conformation of mature proteins on their export pathway when coupled with Tat-type signal peptide, and to seek the signal of non-classical secretory proteins.

BackgroundCurrently, microbial fermentation method has become the research hotspot for acetoin production. In our previous work, an acetoin-producing strain, Bacillus subtilis SF4-3, was isolated from Japanese traditional fermented food natto. However, its conversion of glucose to acetoin was relatively low. In order to achieve a high-efficient accumulation of acetoin in B. subtilis SF4-3, main medium components and fermentation conditions were evaluated in this work.ResultsThe by-products analysis showed that there existed reversible transformation between acetoin and 2,3-butanediol that was strictly responsible for acetoin production in B. subtilis SF4-3. The carbon sources, nitrogen sources and agitation speed were determined to play crucial role in the acetoin production. The optimal media (glucose·H2O 150 g/L, yeast extract 10 g/L, corn steep dry 5 g/L, urea 2 g/L, K2HPO4 0.5 g/L, MgSO4 0.5 g/L) were obtained. Furthermore, the low agitation speed of 300 r/min was found to be beneficial to the reversible transformation of 2,3-butanediol for acetoin production in B. subtilis SF4-3. Eventually, 48.9 g/L of acetoin and 5.5 g/L of 2,3-butanediol were obtained in a 5-L fermenter, and the specific production of acetoin was 39.12% (g/g), which accounted for 79.90% of the theoretical conversion.ConclusionsThe results indicated acetoin production of B. subtilis SF4-3 was closely related to the medium components and dissolved oxygen concentrations. It also provided a method for acetoin production via the reversible transformation of acetoin and 2,3-butanediol.

To improve the microbiological security of food products, 285 lactic acid bacteria were isolated from koumiss and pickles, and their antimicrobial properties were characterised. Among the strains, Pediococcus acidilactici P9, a strain isolated from a local pickled vegetable, produced an antagonistic substance that inhibited Listeria monocytogenes and Shigella. The substance was heat (121 °C for 20 min) and pH (2.0–10.0) stable, but sensitive to proteolytic enzymes. The production of bacteriocins started at the exponential phase and reached its maximum at the stationary phase, indicating that it was growth related. P. acidilactici P9 also showed an ability to protect HT-29 from invasion by L. monocytogenes and the inhibition rate reached 35.89%. P. acidilactici P9 showed a potential for application in food products as a biopreservative.

In this study, a milk-clotting enzyme (MCE) isolated from Paenibacillus spp. BD3526 was purified and characterized. The MCE was purified 8.9-fold with a 10.11% recovery using ammonium sulfate precipitation and anion-exchange chromatography and the specific milk-clotting activity (MCA) reached 6791.73 SU/mg. The enzyme was characterized as a 35 kDa metalloproteinase, and the zymogen of which was encoded by a 1671 bp gene named zinc metalloproteinase precursor (zmp) with a predicted molecular weight of 59.6 kDa. The optimal temperature for MCA and proteolytic activity (PA) was 65 °C and 60 °C, respectively. The enzyme was stable over a pH range of 5.0–9.0 and at temperatures below 50 °C. The MCA was completely inactivated when the enzyme was heated at 60 °C for 30 min, and the PA was totally inactivated for 20 and 10 min when the enzyme was heated at 55 °C and 60 °C, respectively. The BD3526 enzyme was preferentially active towards κ-casein (κ-CN) and β-casein (β-CN), as determined by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), whereas the hydrolysis of αs-casein (αs-CN) was slow and comparable to that caused by chymosin and asparatic acid proteinase from Rhizomucor miehei. The cleavage site of the metalloproteinase in κ-CN was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.