The aim of this study was to compare the effect of fermentation pH on protein bioaccessibility of four soymilk curds enriched with tea polyphenols (TP). The curds were generated by fermentation with Weissella hellenica D1501 and the fermentation terminated at different pH values, namely at pH 5.7, 5.4, 5.1, and 4.8 (SMTP-5.7, SMTP-5.4, SMTP-5.1, SMTP-4.8). Particle-size distribution, soluble protein content, gel electrophoresis, and peptides content were monitored at oral, gastric, and intestinal levels. Results showed that SMTP-4.8 was the matrix most resistant to protein digestion in the gastric phase according to the soluble protein content. Similar particle size distribution and protein degradation patterns were observed for these curds in gastric and intestinal phase. However, there was a significant difference (P < 0.05) in the content of small peptides (<10 kDa) at the end of intestinal digestion among the four curds. Overall, terminating fermentation at pH 5.4–5.7 of soymilk curds enriched with TP is recommended.Keywords: electrophoresis; lactic acid bacteria; peptide content; protein bioaccessibility; soymilk;

Ultrasound treatment during proteolysis could facilitate enzymatic hydrolysis. The degree of hydrolysis (DH) and ACE inhibitory activity of hydrolysates are the two most important factors for process control and quality assessment. This study proposed a novel method for in situ and real-time quantification of DH and ACE inhibitory activity during the ultrasonic-assisted enzymolysis process using miniature near infrared (NIR) spectroscopy with a fiber optic probe. The synergy interval partial least squares (Si-PLS) algorithms were studied via variable selection and multivariate calibrations. The results showed that the DH of CGM and ACE inhibitory activity of the CGM hydrolysates (under ultrasonic-assisted hydrolysis) were increased by 5.74% and 8.32%, respectively, compared to those of the control. The calibration model built using Si-PLS between the NIR spectra and chemical data exhibited high coefficient of prediction (Rp) of 0.9006 and root mean square error of prediction (RMSEP) of 2.98% for the DH, and Rp of 0.9516 and RMSEP of 11.20% for the ACE inhibitory activity. This study demonstrates that the miniature NIR spectroscopy technique with fiber optical probe with Si-PLS algorithms has high potential for in situ monitoring during ultrasonic-assisted enzymatic hydrolysis in the protein industry.

•The r-BOX cycle based platform was further optimized.•A growth favorable and efficient acetyl-CoA synthesis method is established.•The information about bottleneck nodes in r-BOX cycle was updated.Medium-chain fatty acids (MCFAs, 6–10 carbons) are valuable precursors to many industrial biofuels and chemicals, recently engineered reversal of the β-oxidation (r-BOX) cycle has been proposed as a potential platform for efficient synthesis of MCFAs. Previous studies have made many exciting achievements on functionally characterizing four core enzymes of this r-BOX cycle. However, the information about bottleneck nodes in this cycle is elusive. Here, a quantitative assessment of the inherent limitations of this cycle was conducted to capitalize on its potential. The selection of the core β-oxidation reversal enzymes in conjunction with acetyl-CoA synthetase endowed the ability to synthesize about 1 g/L MCFAs. Furthermore, a gene dosage experiment was developed to identify two rate-limiting enzymes (acetyl-CoA synthetase and thiolase). The de novo pathway was then separated into two modules at thiolase and MCFA production titer increased to 2.8 g/L after evaluating different construct environments. Additionally, the metabolism of host organism was reprogrammed to the desired biochemical product by the clustered regularly interspaced short palindromic repeats interference system, resulted in a final MCFA production of 3.8 g/L. These findings described here identified the inherent limitations of r-BOX cycle and further unleashed the lipogenic potential of this cycle, thus paving the way for the development of a bacterial platform for microbial production of high-value oleo-chemicals from low-value carbons in a sustainable and environmentally friendly manner.

Resveratrol has been the subject of numerous scientific investigations due to its health-promoting activities against a variety of diseases. However, developing feasible and efficient microbial processes remains challenging owing to the requirement of supplementing expensive phenylpropanoic precursors. Here, various metabolic engineering strategies were developed for efficient de novo biosynthesis of resveratrol. A recombinant malonate assimilation pathway from Rhizobium trifolii was introduced to increase the supply of the key precursor malonyl-CoA and simultaneously, the clustered regularly interspaced short palindromic repeats interference system was explored to down-regulate fatty acid biosynthesis pathway to inactivate the malonyl-CoA consumption pathway. Down-regulation of fabD, fabH, fabB, fabF, fabI increased resveratrol production by 80.2, 195.6, 170.3, 216.5 and 123.7%, respectively. Furthermore, the combined effect of these genetic perturbations was investigated, which increased the resveratrol titer to 188.1 mg/L. Moreover, the efficiency of this synthetic pathway was improved by optimizing the expression level of the rate-limiting enzyme TAL based on reducing mRNA structure of 5′ region. This further increased the final resveratrol titer to 304.5 mg/L. The study described here paves the way to the development of a simple and economical process for microbial production of resveratrol.

Solid-state fermentation of glutinous rice by Rhizopus oryzae (rice wine or Jiu-niang in Chinese) is a traditional Chinese food as well as a natural enzyme cocktail with the potential to be used in bread making. The objective of this work was for the first time to study the enzyme activities of Jiu-niang and its effect on dough quality and bread staling. The results showed that the principal enzymes in Jiu-niang were amylase, protease and lipase. The Jiu-niang enzyme cocktail significantly improved the volume, texture, and shelf life of wheat breads. The use of an optimal enzyme concentration (addition of Jiu-niang at 10% on flour basis) increased specific volume (19%), reduced crumb hardness (50%), and reduced moisture migration rate and bread staling rate. Using low-field nuclear magnetic resonance (LF-NMR) analysis, proton mobility and starch retrogradation of Jiu-niang bread during storage were determined and discussed. These results indicated that Jiu-niang could be used as a natural quality improver for bread making.

Solid-state fermentation of glutinous rice by Rhizopus oryzae (rice wine or Jiu-niang in Chinese) is a traditional Chinese food as well as a natural enzyme cocktail with the potential to be used in bread making. The objective of this work was for the first time to study the enzyme activities of Jiu-niang and its effect on dough quality and bread staling. The results showed that the principal enzymes in Jiu-niang were amylase, protease and lipase. The Jiu-niang enzyme cocktail significantly improved the volume, texture, and shelf life of wheat breads. The use of an optimal enzyme concentration (addition of Jiu-niang at 10% on flour basis) increased specific volume (19%), reduced crumb hardness (50%), and reduced moisture migration rate and bread staling rate. Using low-field nuclear magnetic resonance (LF-NMR) analysis, proton mobility and starch retrogradation of Jiu-niang bread during storage were determined and discussed. These results indicated that Jiu-niang could be used as a natural quality improver for bread making.

•NMR images visually showed spatial inhomogeneity in water content because of vaporization.•Water content of sausages showed the strongest linear correlation with T2 area.•Linear models overestimated water content and were reliable at high fat content.•Water content in fermented sausages can be possibly measured by LF-NMR in a nondestructive manner.Traditional fermented sausages containing 0%, 25%, 50%, and 75% fat are produced by natural fermentation. Water in sausages was investigated by low-field nuclear magnetic resonance (LF-NMR). T1-weighted phantom magnetic resonance images visually showed spatial inhomogeneity in water content. Center of intensity was brighter than its edges, indicating that water distribution in sausages was inhomogeneous because of vaporization. Low-fat samples showed remarkable spatial inhomogeneity in water content. T2 populations, which were referred to as free water (T23) and immobilized water (T22), shifted toward short relaxation times and gradually merged into one population during fermentation. Relative area percentage of immobilized water (M22) increased, whereas that of free water (M23) decreased. Approximately 95% of water was present as free water and immobilized water. Water content of sausages showed the strongest linear correlation (R2 > 0.92) with T2 area. The linear model showed good accuracy and precision but overestimated water content. Reliability of model decreased as fat content increased because of inhomogeneity in water content of low-fat samples. Therefore, water content can be measured based on sample weight, fat content, and T2 area in a nondestructive manner and regardless of sample size.

•The highest metabolic efficiency of r-BOX was achieved.•NADH was identified as a crucial determinant of process yield.•Fermentative redox balance enabled maximum product synthesis.•Two major factors limiting acetate reduction pathway were identified.Previous studies have made many exciting achievements on pushing the functional reversal of beta-oxidation cycle (r-BOX) to more widespread adoption for synthesis of a wide variety of fuels and chemicals. However, the redox cofactor requirement for the efficient operation of r-BOX remains unclear. In this work, the metabolic efficiency of r-BOX for medium-chain fatty acid (C6-C10, MCFA) production was optimized by redox cofactor engineering. Stoichiometric analysis of the r-BOX pathway and further experimental examination identified NADH as a crucial determinant of r-BOX process yield. Furthermore, the introduction of formate dehydrogenase from Candida boidinii using fermentative inhibitor byproduct formate as a redox NADH sink improved MCFA titer from initial 1.2 g/L to 3.1 g/L. Moreover, coupling of increasing the supply of acetyl-CoA with NADH to achieve fermentative redox balance enabled product synthesis at maximum titers. To this end, the acetate re-assimilation pathway was further optimized to increase acetyl-CoA availability associated with the new supply of NADH. It was found that the acetyl-CoA synthetase activity and intracellular ATP levels constrained the activity of acetate re-assimilation pathway, and 4.7 g/L of MCFA titer was finally achieved after alleviating these two limiting factors. To the best of our knowledge, this represented the highest titer reported to date. These results demonstrated that the key constraint of r-BOX was redox imbalance and redox engineering could further unleash the lipogenic potential of this cycle. The redox engineering strategies could be applied to acetyl-CoA-derived products or other bio-products requiring multiple redox cofactors for biosynthesis.

In this study, Rhizopus oligosporus RT-3, which was first isolated in our group, was used for solid state fermentation of soybeans (R. oligosporus-fermented soybeans, RFS) in a short time (22 h). The effects of fermentation on total phenolic content (TPC), isoflavone composition, antioxidant activity and DNA damage protection of soybeans were investigated. Besides, the effects of different polarity solvents in extracting antioxidant compounds were also tested. The results showed that fermentation significantly enhanced TPC and isoflavone aglycone content, antioxidant activity and DNA damage protection, but decreased the content of isoflavone glucosides. The enhanced antioxidant activity of RFS could be ascribed to the markedly higher levels of TPC and isoflavone aglycones achieved during fermentation. The study also indicated that extraction solvents had significant effects on TPC and isoflavone glucoside and aglycone content, and antioxidant activity. The water extract of RFS showed the highest TPC, ABTS˙+ and hydroxyl radical scavenging activity and chelating ability, whereas the 80% ethanol extract of RFS exhibited the strongest DPPH radical scavenging activity, reducing power and antioxidant capacity determined by a silver nanoparticle-based method. TPC and isoflavone aglycone content were highly correlated with antioxidant activity. In addition, bioaccessibility studies also demonstrated that RFS exhibited higher TPC and isoflavone aglycone bioaccessibility, as well as stronger antioxidant activity compared to non-fermented soybeans. Thus, this study demonstrated that RFS with enhanced TPC and isoflavone aglycone content, antioxidant activity and DNA damage protection which could be considered a good source of natural antioxidants in the prevention of oxidative damage-induced diseases, and it can serve as a nutraceutical and functional food/ingredient in health promotion and disease risk reduction.

•Improved batch pullulan production was obtained under nitrogen-limiting conditions.•The activities of PGM and FKS were increased by nitrogen limitation.•Transcriptional levels of pgm1 and fks were up-regulated by nitrogen limitation.•Nitrogen limitation resulted in more ATP supply for pullulan biosynthesis.Batch culture of Aureobasidium pullulans CCTCC M 2012259 for pullulan production at different concentrations of ammonium sulfate and yeast extract was investigated. Increased pullulan production was obtained under nitrogen-limiting conditions, as compared to that without nitrogen limitation. The mechanism of nitrogen limitation favoring to pullulan overproduction was revealed by determining the activity as well as gene expression of key enzymes, and energy supply for pullulan biosynthesis. Results indicated that nitrogen limitation increased the activities of α-phosphoglucose mutase and glucosyltransferase, up-regulated the transcriptional levels of pgm1 and fks genes, and supplied more ATP intracellularly, which were propitious to further pullulan biosynthesis. The economic analysis of batch pullulan production indicated that nitrogen limitation could reduce more than one third of the cost of raw materials when glucose was supplemented to a total concentration of 70 g/L. This study also helps to understand the mechanism of other polysaccharide overproduction by nitrogen limitation.

•Lactobacillus plantarum B1–6 was able to ferment soy whey.•Antioxidant properties were evaluated in vitro using several methods.•Fermentation enhanced antioxidant activities, total phenolic and isoflavone aglycone contents of soy whey.•Fermented soy whey showed higher protection effects for damaged DNA.•Fermentation process allows to obtain a nutraceutical and functional soy beverage.The antioxidant capacity of fermented soy whey (FSW) was investigated in order to find out their nutraceutical potential, and unfermented soy whey (USW) was used as a control. FSW was prepared using Lactobacillus plantarum B1–6 as a starter. The effects of fermentation on soy whey in terms of total phenolic content (TPC), antioxidant activities and DNA damage protection were investigated. The antioxidant activities of FSW and USW were determined by different standard methods. The results showed FSW extract compared to USW possessed more TPC and isoflavone aglycone contents, and had higher ABTS radical scavenging activity, ferric reducing antioxidant power, reducing power, hydroxyl radical scavenging activity and superoxide radical scavenging activity. Additionally, FSW extract exhibited greater protection against oxidative DNA damage induced by Fenton's reagent. The results suggested that FSW with enhanced antioxidant capacity could provide a functional soy beverage to contribute to the health and nutritional status improvement of consumers.

The present study was conducted to explore a novel strategy to enhance angiotensin I-converting enzyme (ACE) inhibitory activities of navy bean by preparation of navy bean milk (NBM) which was then subjected to fermentation of four lactic acid bacteria (LAB) strains, namely, Lactobacillus bulgaricus, Lactobacillus helveticus MB2-1, Lactobacillus plantarum B1-6, and Lactobacillus plantarum 70810. With the exception of L. helveticus MB2-1, the other three selected strains had good growth performances in NBM with viable counts increased to log 8.30–8.39 cfu ml−1 during 6 h of fermentation, and thus were selected for the following investigations. Protein contents of NBM significantly reduced when treated with L. bulgaricus and L. plantarum B1-6, and the electrophoresis patterns showed the preferable proteins for LAB strains to hydrolyze were α- and β-type phaseolins, whereas γ-type phaseolin was resistant to hydrolysis. RP-HPLC analysis demonstrated all fermented NBM had higher intensities of peaks with retention times between 2.5 and 3.5 min indicative of formation of small peptides. All fermented NBM showed higher ACE inhibitory activity compared to the unfermented ones, for which 2 h, 3 h, and 5 h were found to be the optimum fermentation periods for respectively L. plantarum 70810, L. plantarum B1-6 and L. bulgaricus, with IC50 values of 109 ± 5.1, 108 ± 1.1, and 101 ± 2.2 μg protein ml−1. The subsequent in vitro gastrointestinal simulation afforded all fermented extracts reduced IC50 values and the extracts fermented by L. plantarum B1-6 exerted the lowest IC50 value of 21 ± 2.1 μg protein ml−1. The research has broadened our knowledge bases on the effect of LAB fermentation on the degradation of navy bean proteins and the capacity to release ACE inhibitory peptides. The approach was promising to obtain probiotic products with potential to serve as functional ingredients targeting hypertension.

Solid-state fermentation (SSF) of starchy grain is a traditional technique for food and alcoholic beverage production in East Asia. In the present study, low-field nuclear magnetic resonance (LF-NMR) was introduced for the elucidation of water dynamics and microstructure alternations during the soaking, steaming, and SSF of glutinous rice as a rapid real-time monitoring method. Three different proton fractions with different mobilities were identified based on the degree of interaction between biopolymers and water. Soaking and steaming significantly changed the proton distribution of the sample. The different phases of SSF were reflected by the T2 parameters. In addition, the variations in the T2 parameters were explained by the microstructure changes of rice induced by SSF. The fermentation time and T2 parameters were sigmoidally correlated. Thus, LF-NMR may be an effective real-time monitoring method for SSF in starch systems.

A novel cell-bound exopolysaccharide (c-EPS) was isolated from Lactobacillus helveticus MB2-1 by ultrasonic extraction, anion exchange, and gel filtration chromatography before being structurally characterized. The c-EPS is a heteropolysaccharide with an average molecular weight of 1.83 × 105 Da and is composed of glucose, mannose, galactose, rhamnose, and arabinose at a molar ratio of 3.12:1.01:1.00:0.18:0.16. Methylation analysis and nuclear magnetic resonance analysis revealed that the c-EPS is a linear glucomannogalactan containing repeating units of →6)-β-d-Manp-(1 → 3)-β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 4)-α-d-Galp-(1→ and trace amounts of Rhap-(1→ and (1 → 4)-Arap residues. Complex formation with Congo red demonstrated a triple-strand helical conformation for the c-EPS. Scanning electron microscopy of the c-EPS revealed many regular feather-like structural units. Topographical examination of c-EPS by atomic force microscopy revealed that the c-EPS formed rounded-to-spherical lumps with different sizes and chain formations. Furthermore, preliminary in vitro tests revealed that c-EPS significantly inhibited the proliferation of HepG-2, BGC-823, and especially HT-29 cancer cells.

•LHEPS-1 could significantly inhibit the growth of human colon cancer Caco-2 cells.•Structural feature of LHEPS-1 was elucidated by methylation, GC–MS and 1D/2D NMR.•LHEPS-1 was a kind of moderate branching glucomannogalactan.•A possible structure of LHEPS-1 was proposed.•Anti-colon cancer activity was correlated with structure of LHEPS-1.The present study aimed at investigating the potential anti-colon cancer activity of three purified exopolysaccharides fractions (LHEPS-1, LHEPS-2 and LHEPS-3) from the Lactobacillus helveticus MB2-1. The experimental evidence showed that LHEPS-1 significantly inhibited cell proliferation of human colon cancer Caco-2 cells in both time- and concentration-dependent manners. In contrast, no significant improvements of the inhibitory effects of LHEPS-2 and LHEPS-3 on Caco-2 cells were observed with increasing sample concentrations or prolonged incubation time. Furthermore, the structure of LHEPS-1 was elucidated using methylated analysis, gas chromatography–mass spectroscopy (GC–MS) and nuclear magnetic resonance spectroscopy (NMR), including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR). Results indicated that the LHEPS-1 consisted of a decasaccharide repeating unit with the following structure (n≈122)：Our results suggested that the LHEPS-1 produced by L. helveticus MB2-1 might be suitable for using as natural anti-colon cancer drugs and functional foods ingredients.

Torreya grandis is a rare tree species of economic value. Its seeds are rich in lipids, although their use is limited by their high cost. Recent studies demonstrated that certain endophytic fungi can produce metabolites similar to those produced by the host plant. In the present study, endophytic fungal strains capable of producing lipids were isolated from wild Chinese Torreya grandis. Among these, the XF-38 strain had the highest lipid content at 17.68%. Comparative analysis of the fatty acid composition of lipids produced by endophytic fungi and Torreya seeds by GC-MS showed that the two sources shared some of the same components, including linoleic acid, oleic acid, and sciadonic acid (5c, 11c, 14c-eicosatrienoic acid). The morphology and internal transcribed spacer rDNA of the XF-38 strain were determined, and a BLAST search showed that this strain belongs to Bionectria ochroleuca. Finally, generation of a growth curve and determination of the lipid content of strain XF-38 confirmed that lipid production was related to the amount of mycelium, and the lipid content resched 3.1 g/L in the microcrystalline cellulose meduim.

In the present study, a released exopolysaccharide (r-EPS1) from L. plantarum 70810 was modified by acetylation, phosphorylation and carboxymethylation. Scanning electron micrograph (SEM) and thermogravimetric analysis (TGA) showed the r-EPS1 derivatives had different surface morphology and thermal behavior. Compared with r-EPS1, the derivatives exhibited stronger antioxidant and antitumor activities. The study provided experimental evidences that chemical modification could be an effective way to improve the bioactivity of exopolysaccharide from L. plantarum 70810. It is noted that these derivatives could be explored as novel potential antioxidant and antitumor agents.

•The crude polysaccharides (EPS) from L. helveticus MB2-1 have high viscosity.•Isolation and characterization of three EPS fractions from L. helveticus MB2-1.•Three purified EPS fractions had similar average molecular weight (about 2 × 105 Da).•The EPS exhibited high antioxidant and metal ion chelating activities in vitro.•The acidic EPS from L. helveticus MB2-1 had potentials as natural antioxidant.In the present study, the crude polysaccharides (EPS) from Lactobacillus helveticus MB2-1 were fermented with reconstituted whey medium and further fractionated on DEAE-52 cellulose and Sephadex G-100 chromatography to afford three purified fractions of EPS-1, EPS-2 and EPS-3. Then, their preliminary structures and antioxidant activities in vitro were investigated. Three purified EPS fractions (EPS-1, EPS-2 and EPS-3) had similar molecular weight distributions (about 2 × 105 Da), and it was composed of galactose, glucose and mannose with a molar ratio of 1.33:2.75:1.00, 1.00:1.43:9.34 and 1.17:1.00:2.96, respectively. In addition, the antioxidant activity of EPS-1, EPS-2 and EPS-3 was evaluated with the in vitro scavenging abilities. The results indicated that both crude and purified EPS showed strong scavenging activities on three kinds of radical and chelating activities on ferrous ion, and their antioxidant activities decreased in the order of crude EPS > EPS-3 > EPS-2 > EPS-1.

•LHEPS-2 exhibited strong inhibitory activity against gastric cancer BGC-823 cells.•Structure of LHEPS-2 from L. helveticus MB2-1 was investigated.•Methylation and 1D/2D NMR were used to elucidate the structural feature of LHEPS-2.•LHEPS-2 was found to contain a galactomannan backbone with a glucomannan side chain.•A possible structure of LHEPS-2 was proposed.In this study, we reported that three purified exopolysaccharides fractions (LHEPS-1, LHEPS-2 and LHEPS-3) and crude LHEPS from the Lactobacillus helveticus MB2-1 inhibited the proliferation potential of human gastric cancer BGC-823 cells. We found that all the LHEPS fractions and crude LHEPS exerted concentration and time dependent inhibitory activities in vitro on BGC-823 cells. LHEPS-2 exhibited higher antiproliferative activity against BGC-823 cells in vitro than LHEPS-1, LHEPS-3 and crude LHEPS. With the consideration of the outstanding antiproliferative effect of LHEPS-2 on BGC-823 cells, more fine structural characterization of LHEPS-2 was further identified. The structure of LHEPS-2 was elucidated by methylated analysis, GC–MS, 1H NMR and 13C NMR spectroscopy, together with two-dimensional (2D) COSY, TOCSY, HSQC, HMBC and NOESY spectroscopy. Results showed that the LHEPS-2 was consisted of the following repeating units:Figure options

Low-field nuclear magnetic resonance (LF-NMR) was introduced for the elucidation of tofu in the present study. After multiexponential analysis of relaxation decays, three water fractions centered at about 1.5–2.6, 24–114, and 132–305 ms were detected and identified as T2b, T21, and T22, respectively. Principal component analysis (PCA) of the data revealed that sample aggregation was dependent on solubility of coagulants and contained anions. Stepwise centrifugation and microwave drying were employed as dehydration methods. Significant correlations were observed between T21 and T22 relaxation times and water-holding capacity (WHC) in both dehydration processes, which implied LF-NMR measurements could be an efficient method for determination and prediction of tofu’s water-holding capacity. Ten linear equations that could be applied in prediction of WHC for tofu were reported. LF-NMR was suggested to be a powerful tool for the study of tofu.

A novel food-grade strain Lactobacillus plantarum 70810 producing β-galactosidase with high transgalactosylation activity was isolated from Chinese paocai. The galactooligosaccharides (GOS) were synthesized by using this enzyme with a maximum yield of 44.3 % (w/w) from 400 g/L lactose at 45 °C for 10 h. The β-galactosidase from this strain was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. It was a heterodimer arrangement of approximately 105 kDa composed of two subunits of 35 and 72 kDa. The optimal pH of the purified β-galactosidase was 8.0 for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis, and optimal temperature was 60 °C and 55 °C, respectively. Its Km and Vmax values for oNPG and lactose were 0.89 ± 0.05 mM, 194 ± 3.0 μmoL/min/mg protein, and 9.88 ± 0.16 mM, 15.88 ± 0.21 μmoL/min/mg protein, respectively. This enzyme was slightly inhibited by the hydrolysis products, that is, glucose and galactose. Since the β-galactosidase from L. plantarum 70810 exhibited higher transgalactosylation activity, strong affinity for lactose and low end-product inhibition, it was suggested to be a potential candidate for the synthesis of prebiotic GOS.

Forty-four lactic acid bacteria (LAB) isolated from traditional Sayram ropy fermented milk (SRFM) in southern Xinjiang of China. Further two strains were selected based on their high viscosity-producing activity. Based on the API 50 CHL strip and 16S rDNA sequence analysis, strain MB 2-1 was a Gram-positive, rod-shaped Lactobacillus helveticus and strain MB 5-1 was identified as Streptococcus thermophilus. Both the two LAB strains were grown in the milk fermentations for pure and mixed cultures and were evaluated for their growth, acidification properties, EPS production, and ability to increase the apparent viscosity of fermented milk, respectively. L. helveticus MB 2-1 displayed a high increasing in viable cell counts and the acidifying capacity in pure cultures, whereas the relatively high EPS production and viscosity-producing capacity detected in pure culture with S. thermophilus MB 5-1 as starter culture. In addition, the mixed culture of the two strains showed a higher cell growth, EPS production, and high viscosity-producing capacity at 37 °C. Values of apparent viscosity were 4.03- and 2.41-fold higher in mixed culture than for pure cultures of L. helveticus MB 2-1 and S. thermophilus MB 5-1, respectively. There was a positive correlation between the viscosity and high molecular weight EPS production with pure and mixed cultures. Our data showed two strains combination, with high viscosity-producing and acidifying capacity, can be used in mixed cultures for the manufacture of fermented milk with improved functional properties.

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS–PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg2+ and Cu2+. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for αs-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.Graphical abstractIn this study, purification and characterization of a milk coagulating enzyme from ginger rhizomes are described. This enzyme potentially has application in the cheese industry as a rennet substitute.Research highlights► We purify and characterize plant coagulant from ginger rhizomes. ► The enzyme belongs to cysteine proteases. ► The purified enzyme shows higher milk clotting activity than calf rennet. ► The enzyme may have potential application as a rennet substitute.

The worldwide increase in cheese consumption combined with a scarcity of rennet as well as ethical concerns have resulted in a global interest for natural milk coagulants from plant sources. In this study, the influence of ginger protease in comparison to calf rennet on the physicochemical, microbiological, and sensory characteristics of Peshawari cheese manufactured from cow’s milk was examined. For most of the physicochemical parameters (fat, protein, lactose, acidity, pH), and the main groups of microorganisms (total viable, enterobacteria, Lactobacilli, and molds and yeasts) investigated, no significant (P > 0.05) differences were observed between the two cheeses made by using different coagulants. However, significantly lower (P < 0.05) levels of moisture and higher levels of soluble nitrogen were observed in the cheese produced by ginger protease compared to that made using calf rennet. The main sensory attributes (appearance, body texture, and flavor) were significantly enhanced (P < 0.05) in Peshawari cheese prepared with ginger protease. Importantly, no bitterness was noted by the sensory panel in the Peshawari cheese made with ginger protease. The results reveal that the ginger protease may have potential application for the manufacture of Peshawari cheese.世界范围内奶酪消费量不断增加, 而传统凝乳剂小牛皱胃酶供应相对不足以及出于宗教方面的考虑, 使得一些植物来源的凝乳酶研究引起了广泛兴趣。本文以牛奶为原料, 比较了生姜蛋白酶和小牛皱胃酶作为凝乳剂制备的 Peshawari 奶酪在物化特性、微生物特性以及感官特性方面的差异。结果表明, 用两种凝乳酶制备的奶酪, 其大部分的物化特性指标 (脂肪、蛋白质、乳糖、酸度、pH) 以及主要的微生物指标 (活菌总数、肠道菌、乳酸菌、真菌和酵母) 均无明显差异 (P > 0.05)。但以生姜蛋白酶为凝乳剂制备的奶酪其含水量明显低于用小牛皱胃酶生产的奶酪 (P < 0.05), 相反可溶性氮 (SN) 的含量前者却明显高于后者 (P < 0.05) 。此外, 结果还显示用生姜蛋白酶为凝乳剂制备 Peshawari 奶酪可以明显改善主要的感官特性(外观、质构、风味) (P < 0.05), 更重要的是, 以这种方式生产的 Peshawari 奶酪没有苦味。这一研究表明生姜蛋白酶作为植物来源的凝乳剂在 Peshawari 奶酪生产中具有潜在应用价值。

This study aimed to characterize the dominant microflora in shubat, a special fermented product prepared from unheated two-humped camel milk. In the seven investigated samples of shubat, lactic acid bacteria and yeasts were the dominant microorganisms with the number ranging from log 6.8 to 7.6 cfu/ml and from log 4.3 to 4.7 cfu/ml, respectively. Using phenotypic and molecular methods, a total of 48 LAB isolates were identified as Lactobacillus sakei, Enterococcus faecium, Lactobacillus helveticus, Leuconstoc lactis, Enterococcus feacalis, Lactobacillus brevis and Weissella hellenica, whereas 15 yeast isolates were identified as Kluyveromyces marxianus, Kazahstan uiosporus, and Candida ethanolica. Results showed also that Lactobacillus and Enterococcus as well as Kluyveromyces were the predominant genera, with the most frequently isolated species being Lactobacillus sakei and Enterococcus faecium as well as Kluyveromyces maxius, respectively. This is the first report on the characterization of dominant microflora of shubat in China.

A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography. The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg2+ and Fe2+ ions the activity of the enzyme increased, whereas EDTA and Cu2+ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents.

An endophytic Xylaria sp., having broad antimicrobial activity, was isolated and characterized from Ginkgo biloba L. From the culture extracts of this fungus, a bioactive compound P3 was isolated by bioactivity-guided fractionation and identified as 7-amino-4-methylcoumarin by nuclear magnetic resonance, infrared, and mass spectrometry spectral data. The compound showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus [minimal inhibitory concentrations (MIC) 16 μg·ml−1], Escherichia coli (MIC, 10 μg·ml−1), Salmonella typhia (MIC, 20 μg·ml−1), Salmonella typhimurium (MIC, 15 μg·ml−1), Salmonella enteritidis (MIC, 8.5 μg·ml−1), Aeromonas hydrophila (MIC, 4 μg·ml−1), Yersinia sp. (MIC, 12.5 μg·ml−1), Vibrio anguillarum (MIC, 25 μg·ml−1), Shigella sp. (MIC, 6.3 μg·ml−1), Vibrio parahaemolyticus (MIC, 12.5 μg·ml−1), Candida albicans (MIC, 15 μg·ml−1), Penicillium expansum (MIC, 40 μg·ml−1), and Aspergillus niger (MIC, 25 μg·ml−1). This is the first report of 7-amino-4-methylcoumarin in fungus and of the antimicrobial activity of this metabolite. The obtained results provide promising baseline information for the potential use of this unusual endophytic fungus and its components in the control of food spoilage and food-borne diseases.

The objective of this study was to evaluate the antioxidant activity of cultivated fruiting bodies of an endophytic Xylaria sp. (strain number YX-28), from Ginkgo biloba. The results indicated that the methanol extract exhibited strong antioxidant capacity in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) analysis and β-carotene–linoleic acid model system. Total phenolic and flavonoid contents extracted by different solvents were determined using Folin–Ciocalteu procedure and the flavonoid–aluminium method. The results showed that total phenolic and flavonoid contents were the highest in methanol extract (54.51 ± 1.05 mg gallic acid equivalent/g dry weight and 86.76 ± 0.58 mg rutin equivalent/g dry weight), while the hexane extract was the lowest (9.71 ± 0.57 mg GAE/g dw and 10.14 ± 0.76 mg RE/g dw, respectively). The correlation coefficients from regression analysis showed a positive relationship between total phenolic content in the extracts and DPPH activity (R2 = 0.7336), as well as between total flavonoid content and DPPH activity (R2 = 0.9392). Furthermore, GC/MS method was used to confirm the presence of phenolics with antioxidant activity in the methanol extract and resulted in the identification of 41 compounds, esters, phenolics, alkanes, carboxylates and alcohols being the main components. In conclusion, cultivated fruiting bodies of Xylaria sp.YX-28 may have potential as natural antioxidant.

•A novel solid state fermentation with Cordyceps militaris was used to produce fermented mung beans.•Phenolic content and antioxidant activities of mung beans were enhanced by solid state fermentation.•Fermented mung beans showed higher protection effects for damaged DNA.•Phenolics were correlated with the antioxidant activities.•A solid state fermentation process could obtain a novel nutraceutical and functional food.In this study, solid state fermentation of mung beans by Cordyceps militaris SN-18 (C. militaris-fermented mung beans, CFMB) was performed. The effects of fermentation on phenolic content, antioxidant activity, and DNA damage protection of mung beans through various polarities solvent extracts were determined. Results showed that phenolic content, antioxidant activities, and DNA damage protection of mung beans were significantly enhanced by fermentation. The water extract of CFMB exhibited the highest phenolic content, DPPH radical scavenging activity, ferric reducing antioxidant power, reducing power, and DNA damage protection. During fermentation, shikimic acid, chlorogenic acid, vanillic acid, rutin, sinapic acid, and luteolin of mung beans were significantly increased. A clear correlation was found between phenolic content and antioxidant activity. Thus, CFMB with enhanced phenolic compounds, antioxidant activity, and DNA damage protection may be effective in the prevention of oxidative damage-induced disease, and it can serve as a novel nutraceutical and functional food in health promotion.Download full-size image

The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78–84% similarity, and yeasts 80–92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide.

•The moisture reduction of fermented sausages was affected by fermentation time, but not by rose polyphenols.•Addition of rose polyphenols inhibited lipid oxidation and biogenic amine formation.•Rose polyphenols can selectively modify the growth rate and richness of lactic acid bacteria.Rose polyphenols (RPs) have antioxidant and antibacterial activities. In order to evaluated the application of RPs in meat products, the effect of RPs on the safety of fermented sausages was investigated. In the present study, RPs inhibited the increase of pH. Phenylethylamine, putrescine, cadaverine, tyramine and spermidine can be detected in sausages, while spermine and tryptamine were not observed. Sausages containing RPs had lower thiobarbituric acid reactive substance values and biogenic amines than the control. Moreover, RPs decreased the total bacterial count but increased growth rate of lactic acid bacteria. The effect of RPs on oxidation, biogenic amines and bacterial growth is dependent on the concentration. High-throughput DNA sequence analysis revealed that operational taxonomical units (OTUs) of samples without PRs were higher than those containing 3 mg/g RPs, and 323 OTUs shared between two batches. Especially, a higher level of Lactobacillales OTUs was observed in the 3 mg/g batches. These results indicated that RPs could be used to improve the safety of fermented sausage by inhibiting lipid oxidation, biogenic amines formation and spoilage bacterial growth, and increasing growth rate and richness of lactic acid bacteria.

•We sequenced the complete genome of Lactobacillus helveticus MB2-1.•The complete genome of L. helveticus MB2-1 contains a single circular chromosome.•L. helveticus MB2-1 genome has a unique gene cluster for EPS biosynthesis.•The EPS gene cluster of L. helveticus MB2-1contains 21 EPS-related genes.Lactobacillus helveticus MB2-1 is a probiotic bacterium producing exopolysaccharides (EPS), which was isolated from traditional Sayram ropy fermented milk in southern Xinjiang, China. The genome consists of a circular 2,084,058 bp chromosome with no plasmid. The genome sequence indicated that this strain includes a 15.20 kb gene cluster involved in EPS biosynthesis. Genome sequencing information has provided the basis for understanding the potential molecular mechanism behind the EPS production.