•Ultrafiltration was used to recovery whey protein after cross-linking at various conditions using TG enzyme.•The ultrafiltration recovery efficiency of the cross-linking protein in whey was increased.•The membrane fouling was decrease indicated membrane operation time can be extended.•Find a new way to improve the whey protein ultrafiltration recovery operation.In whey ultrafiltration (UF) production, two main problems are whey protein recovery and membrane fouling. In this study, membrane coupling protein transglutaminase (TG) catalysis protein cross-linking was investigated under different conditions to find out the best treatment. We found that the optimal conditions for protein recovery involved catalyzing whey protein cross-linking with TG (40 U/g whey proteins) at 40 °C for 60 min at pH 5.0. Under these conditions, the recovery rate was increased 15–20%, lactose rejection rate was decreased by 10%, and relative permeate flux was increase 30–40% compared to the sample without enzyme treatment (control). It was noticeable that the total resistance and cake resistance were decreased after enzyme catalysis. This was mainly due to the increased particle size and decreased zeta potential. Therefore, membrane coupling enzyme catalysis protein cross-linking is a potential means for further use.

•This study can provide a natural source of substitute for antitumour drugs with potential application value.•The substitute was two novel homogeneous exopolysaccharides (EPSs) derived from the L. casei strain isolated from yak milk in the minority area of China.•Their physicochemical characterizations were elucidated systematically by GC–MS, FTIR spectroscopy, methylation analysis and FE-SEM.•They can both induce HT-29 cell apoptosis by the activation of caspase-3 and -8 genes as well as through regulating the pro-apoptotic genes Bad and Bax.Two high molecular weight fractions (LW1 and LW2) of exopolysaccharides (EPSs) produced by Lactobacillus casei SB27 were isolated from yak milk obtained from the Gansu Tibetan area of China. GC–MS, FTIR spectroscopy, methylation analysis and FE-SEM analysis were performed to elucidate the physicochemical characterization of these two fractions, and their in vitro antitumour activities were also evaluated. The molecular weights (Mws) of LW1 and LW2 as determined by HPGPC were 25.10 and 12.34 kDa, respectively. Monosaccharide composition analysis revealed that LW1 and LW2 were mainly composed of galactose (52.4% and 57.4%, mol%) and glucose (29.1% and 22.2%, mol%), respectively. Methylation results showed that the main chain of LW1 likely involves (1 → 4)-linked Galp and (1 → 4)-linked Glcp with its side chains being (1 → 4,6)-linked Galp through the O-6 position connected to the backbone, whereas the main chain of LW2 likely involves (1 → 4)-linked Galp and (1 → 4)-linked Glcp with its side chains being (1 → 3)-Galp through the O-6 position of (1 → 3,6)-Galp linked to the main chain. Evaluation of the microcosmic morphology, as revealed by FE-SEM analysis of the two EPS fractions, showed a sheet-like appearance with a folded surface and a compact structure. The results from in vitro antitumour tests indicated that both LW1 and LW2 could significantly inhibit the proliferation of HT-29 colorectal cancer cells and up-regulated the expressions of Bad, Bax, Caspase-3 and -8 genes. Finally, TEM images revealed the apoptotic morphological changes of HT-29 cells induced by LW1 and LW2. Our results suggested that LW1 and LW2 possess potential not only for use in functional food products but also as a source of natural antitumour drugs.

Most psychrotrophic bacteria have the ability to produce thermoresistant proteases that can destroy the quality of milk and dairy products. To investigate the population dynamics of psychrotrophic bacteria during refrigeration, three raw cows' milk samples (sample A comprising milk from 10 farms in Beijing, sample B comprising milk from 5 farms in Heihe, and sample C comprising milk from 7 farms in Harbin) were refrigerated at 0–5 °C and 5–10 °C. PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) analysis revealed that the bacterial community profiles varied from geographical site to site, and with refrigeration temperature. The dominant psychrotrophic bacteria among the samples after storage were affiliated with the order Pseudomonadales. Following isolation and identification, 8 psychrotrophic isolates were selected as stronger protease producers and their growth and proteolytic activities were assessed. The results indicate that the composition of psychrotrophic bacteria play an important role in the determination of the quality of milk and dairy products.

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 101 cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 102 cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 101 cfu/reaction, which was 102 lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.

An enterocin-producing Enterococcus faecium T1 was isolated from Chinese Tibet cheese. The enterocin was purified by SP-Sepharose and reversed phase HPLC. It was identified as unique from other reported bacteriocins based on molecular weight (4629 Da) and amino acid compositions; therefore it was subsequently named enterocin T1. Enterocin T1 was stable at 80–100 °C and over a wide pH range, pH 3.0–10.0. Protease sensitivity was observed to trypsin, pepsin, papain, proteinase K, and pronase E. Importantly, enterocin T1 was observed to inhibit the growth of numerous Gram-negative and Gram-positive bacteria including Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes. Take together, these results suggest that enterocin T1 is a novel bacteriocin with the potential to be used as a bio-preservative to control Pseudomonas spp. in food.

Lactobacillus paracasei subsp. paracasei M5L (L. paracasei M5L) was isolated and co-cultured with HT-29 colon cancer cells to study its anti-colorectal cancer effects and mechanism. Using the MTT assay we found that L. paracasei M5L significantly inhibits HT-29 cell proliferation. Morphological and biochemical characteristics of apoptosis were observed and confirmed by hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM). Lactobacilli could change the cell cycle distribution and induce calreticulin (CRT) translocation from the endoplasmic reticulum to the surface of the cytomembrane. We also determine that vast reactive oxygen species (ROS) were generated, while the activities of superoxide dismutase (SOD) and catalase (CAT) were noticeably diminished following L. paracasei M5L treatment. This study reveals that L. paracasei M5L induces apoptosis in HT-29 cells through ROS generation followed by CRT accompanied endoplasmic reticulum (ER) stress and S phase arrest. These results provide new insights into the possible molecular mechanism of L. paracasei M5L as a novel probiotic with the potential for further application.

Three strains of Lactobacillus delbrueckii subsp. bulgaricus were selected for their proteinase properties in order to improve milk gel firmness. The respective proteinases were purified by ultra-filtration, anion exchange, and hydrophobic interaction chromatographies. The 3 purified proteinases were determined to have molecular masses of about 39, 40, and 52 kDa. The optimal activities of the purified enzymes occurred at pH 6.0 and 40 °C. They are metallopeptidases, activated by Fe2+, inhibited by Ba2+, Zn2+, Mn2+, Xi2+, Fe3+, Cu2+ and EDTA, and serine proteinases which are inhibited by PMSF.

Transglutaminases (TGases) are a family of enzymes that catalyze the cross-linking of proteins and are widely used in the food industry to improve the texture of dairy, meat, and bread products. Zea mays transglutaminase (TGZ) is a new type of TGase with a wide potential. TGZ was expressed in the yeast Pichia pastoris under an alcohol oxidase promoter. Maximal expression of recombinant TGZ was achieved by inducing recombinant GS115 (pPIC9K-tgz) in BMMY medium using 1.5% methanol for 96 h. Secreted TGZ was initially separated using Superdex 200 resin and further purified on cation exchange resin. The activity of TGZ following purification was 0.32 U/mg of protein. The polymerization effect of TGZ on casein catalyzed by recombinant TGZ was slightly lower than the effect of microbial transglutaminase (MTG). TGZ is a new potential additive for the food industry.

•Qula casein was hydrolyzed by different enzymes for the production of ACEI peptides.•Hydrolysates with the highest ACEI activities were selected for LC-MS/MS analysis.•Four QSAR models were established to predict potential ACEI peptides.•Molecular docking studies explored potential ACEI activities of selected peptides.•PFPGPIPN, KYIPIQ and LPLPLL were chemically prepared to determine ACEI activities.Qula casein derived from yak milk was hydrolysed using various enzymes. Hydrolysates were withdrawn at different hydrolysis phases and were determined to their degree of hydrolysis (DH) and ACE (angiotensin I-converting enzyme) inhibitory (ACEI) activities. Using a 3 kDa ultra-filtration membrane, hydrolysates were fractioned into two ranges of molecular weight and permeated fractions were further investigated. A Lineweaver-Burk plot was used to explore the ACEI kinetics of the hydrolysates. Additionally, the peptides in the hydrolysates were identified using LC-MS/MS. We established four quantitative structure-activity relationship (QSAR) models for predicting potential ACEI peptides. Using in silico analysis, four novel ACEI peptides were identified and a molecular docking study further explored the potential ACEI activities. Based on the docking results, three new peptides (PFPGPIPN, KYIPIQ and LPLPLL) were chemically synthesized and their IC50 values were determined. In conclusion, our study suggests that Qula casein may be a valuable source of ACEI peptides.

Surface-layer associated proteins (SLAP) that envelop Lactobacillus paracasei ssp. paracasei M5-L and Lactobacillus casei Q8-L cell surfaces are involved in the adherence of these strain to the human intestinal cell line HT-29. To further elucidate some of the properties of these proteins, we assessed the yields and expressions of SLAP under different incubation conditions. An efficient and selective extraction of SLAP was obtained when cells of Lactobacillus were treated with 5 M LiCl at 37°C in aerobic conditions. The SLAP of Lactobacillus M5-L and Q8-L in cell extracts were visualized by SDS-PAGE and identified by Western blotting with sulfo-N-hydroxysuccinimide-biotin–labeled HT-29 cells as adhesion proteins. Atomic force microscopy contact imaging revealed that Lactobacillus strains M5-L and Q8-L normally display a smooth, homogeneous surface, whereas the surfaces of M5-L and Q8-L treated with 5 M LiCl were rough and more heterogeneous. Analysis of adhesion forces revealed that the initial adhesion forces of 1.41 and 1.28 nN obtained for normal Lactobacillus M5-L and Q8-L strains, respectively, decreased to 0.70 and 0.48 nN, respectively, following 5 M LiCl treatment. Finally, the dominant 45-kDa protein bands of Lactobacillus Q8-L and Lactobacillus M5-L were identified as elongation factor Tu and surface antigen, respectively, by liquid chromatography-tandem mass spectrometry.

A total of 32 indigenous lactic acid bacteria (LAB) isolates were previously obtained from 7 samples of traditional dairy products in specific ecological niches throughout the northwestern area of China. Among the 32 isolates, 9 strains of LAB were selected for their potential in fermented dairy products and identified by 16S rDNA sequence analysis. In this study, these 9 isolates were combined each other and 8 combinations were selected according to their fermentation characteristics in yoghurt production. The effects of these 8 combinations on acidification, post acidification, texture and sensory quality of yoghurt were analysed. Results indicated that Streptococcus thermophilus SP1.1 combined with Lactobacillus delbrueckii ssp. bulgaricus 3 4.5 was the best combination and better than the commercial starter culture in terms of yoghurt fermentation time and sensory quality, suggesting that the combination has potential as a commercial starter to improve the quality of yoghurt.

A total of 138 lactobacillus strains were obtained from the traditional fermented foods of minority nationalities or infant faeces, respectively. The lactobacillus strains were screened for anticancer effects and probiotic potential. The results showed that 10 strains exerted anti-proliferative activity and higher adhering capability on HT-29 cells. They were then screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that the 4 strains (cell walls and cytoplasm extracts) displayed the high anti-proliferative activity and the large extent of DNA strand breakage in individual cells. Through the selected procedure, cell walls that were extracted from X12, M5 and K14 strains induced apoptosis in HT-29 cells. Further investigation confirmed that apoptosis-inducing ability of cell wall extracts was attributed to the breakdown of mitochondrial membrane potential, which is a known initiation of apoptotic mitochondrial pathway. Cell walls from X12, M5 and K14 strains, were determined to be less harmful to noncancerous Vero cells than to human colon cancer HT-29 cells. These findings suggested that X12, M5 and K14 strains opposing the ability to induce HT-29 cells apoptosis, and cell wall extracts were involved in this apoptosis induction.

•The whole-genome sequence of E. faecalis LD33 was performed using Illumina Hiseq and the PacBio RSII platform.•E. faecalis LD33 has more clusters of microcin than other sequenced E. faecalis.•When exogenous heme was added, E. faecalis LD33 could undergo respiration to achieve a high biomass.Enterococcus faecalis LD33 strain was originally isolated from traditional naturally fermented cream in Inner Mongolia of China. Its complete genome sequence was carried out using the Illumina Hiseq and the PacBio RSII platform. The genome only has a circular chromosome and a GC content of 37.58%. Other core information shown in the genome sequencing results further insight on this bacterium’s genetic elements for bacteriocin production and the genes related to respiratory chain.