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Understanding the components composition characteristics of human milk is conductive for the development of infant formulas. The total fatty acids composition and the stereo-distribution of fatty acids in human milk triglycerides (TGs) from three regions (Zhengzhou, Wuhan and Harbin) of China were analyzed. The total and intrapositional fatty acids composition of human milk TGs showed significant differences among three regions. Zhengzhou, Harbin and Wuhan human milk possessed highest levels of medium-chain fatty acids (MCFA, 6.36 ± 1.46%), short-chain fatty acids (SCFA, 0.10 ± 0.03%) and monounsaturated fatty acids (MUFA, 34.61 ± 0.45%), and polyunsaturated fatty acids (PUFA, 30.34 ± 0.33%), respectively. The preference distribution of fatty acids in human milk TGs was hardly affected by regions, for the relative conservative interpositional levels of each fatty acids in three regions. Not only the specific distribution of sn-2 position, but the esterification of fatty acids in sn-1 and sn-3 positions also had preference. SCFA and MCFA were mainly esterified at sn-3 position, but LCSFA most unlikely distributed at the sn-3 position. The preference esterification of MUFA were sn-1, sn-3 > sn-2, and PUFA were sn-3 > sn-1 > sn-2. Therefore, the optimization of infant formulas not only needs considering total fatty acid composition of TGs but requires thinking over the stereo-distribution of fatty acids.

Hypercholesterolemia drives the development of cardiovascular disease, Supplementation with probiotics that interfere with cholesterol metabolism provide a contribution to disease prevention. 54 strains with good hydrophobicity were measured bile salt hydrolase (BSH) activity and cholesterol removal ability. Evaluated the strains for probiotic potential with the intestinal colonization ability and the resistance to simulated digestion subsequently. 10 strains were finally selected for further characterization. As the potential functional strains, explored their effects on the expression of genes related to cholesterol metabolism and signaling pathways. 6 strains could regulate expression of Niemann–Pick C1-like 1 (NPC1L1) through farnesoid X receptor (FXR)/liver X receptor (LXR) pathway. Among of them, 4 strains could reduce the lipid deposition of ox-LDL-induced macrophages which had the potential abilities to improve atherosclerosis. Based on lipid-lowering abilities and common characteristics of probiotics, Lactobacillus vaginalis FN3 and Bifidobacterium animalis subsp. Lactis F1-7 could be taken as promising probiotic candidates for further in vivo studies and might have commercial value.

Ten lactic acid bacteria (LAB) strains of different species isolated from Chinese traditional sourdough were investigated on their volatile compounds in sourdough fermentation. Sourdoughs fermented with different LAB species were analyzed by principal component analysis (PCA) based on their volatiles. Furthermore, the in situ gene expressions of Lactobacillus sanfranciscensis during sourdough fermentation were investigated by using RNA sequencing for the first time. PCA could discriminate between the sourdoughs fermented by homofermentative and heterofermentative LAB, the former containing more aldehydes and ketones with more than 6 carbon atoms, while the latter featuring ethanol and esters. The Lactobacillus plantarum-fermented sourdough was characterized by high C4–C6 volatiles content, while L. sanfranciscensis showed a unique volatile profile without explicit explanatory compounds. The RNA sequencing suggested that, compared to the sourdough fermented for 6 h, L. sanfranciscensis enhanced carbohydrate metabolism and self-protection activities, but decreased cell proliferation in sourdough at 12 h.

Spray-drying is an efficient drying technique for preparing probiotic powders, but the high temperatures employed during the process results in low survival rates of lactic acid bacteria. This study aimed to enhance spray-drying tolerance of lactic acid bacteria by intracellular delivery of trehalose via electroporation. Diverse electroporation conditions were applied to Lactobacillus delbrueckii ssp. bulgaricus sp1.1 cells exposed to 10% trehalose prior to spray drying with 30% re-constituted skimmed milk. Survival rates of spray-dried bacteria increased with an intracellular trehalose content of ≥3.5 µg/107 colony-forming units (CFU) and reached 100% with intracellular trehalose content of 10.1 µg/107 CFU. The application of two electroporation pulses at 2.5 kV/cm helped increase the survival rate of L. bulgaricus from 38% to 61% after spray drying. Membrane damage and pore formation caused by excessive pulsed electric field treatment resulted in cell death after spray drying. Sufficient intracellular trehalose protected cell walls and membranes from further damage by spray drying. Multi-pulse electroporation of trehalose into lactic acid bacteria prior to spray drying can potentially increase cell viability.

This study was to screen strains which had effects on insulin resistance in vitro and explore the possible mechanism. These strains were tested the ability of colonization, hormone release, lipid accumulation, glucose uptake and immunomodulatory. Three strains of Lactobacillus paracasei 1F-20, Lactobacillus fermentum F40-4 and Bifidobacterium animalis subsp. lactis F1-7 led to results as follows: (1) survived in the harsh conditions. (2) increased the hormone release of glucagon-like peptide 1(GLP-1), peptide YY (PYY) and up-regulated genes expression of proglucagon gene (GCG), PYY; (3) decreased lipid accumulation, down-regulated genes expression of sterol regulatory element binding protein-1C (SREBP-1C), patatin-like phospholipase domain containing 3 (PNPLA3) and up-regulated peroxisome proliferator activated receptor-α (PPARα); (4) increased the uptake of glucose and up-regulated the protein expression of phosphatidylinositol 3- kinase (PI3K) and phosphorylated serine-threonine protein kinase (pAKT). (5) decreased the proinflammatory cytokine IL-6 and TNF-α. It demonstrated that the three strains prevented the development of insulin resistance.

The exploration and evaluation of bacteriocin-producing lactic acid bacteria (LAB) have been one of the powerful means to food preservation. A total of 300 strains were isolated from Chinese traditional fermented milk products. A bacteriocin-producing LAB, named Lactobacillus plantarum J23, was screened and identified. Bacteriocin Lac-B23 from L. plantarum J23 was purified by 80% ammonium sulfate precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography. Molecular weight of bacteriocin Lac-B23 was determined to be approximately 6.73 kDa by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and it was confirmed as a novel bacteriocin by liquid chromatography-mass spectrometry. Moreover, bacteriocin Lac-B23 showed thermal stability when heated at below 100°C for 30 min, pH stability between pH 2.0 and 12.0, and sensitivity to trypsin, proteinase K, and proteinase E. The antimicrobial activity of bacteriocin could be enhanced by addition of Fe2+, Mn2+, and ethyl alcohol, and inhibited by Cu2+, K+, Ca2+, Zn2+, Mg2+, and sodium chloride. The results suggested bacteriocin Lac-B23 to have potential application prospects in the food industry.

Spray drying is a fast and low-cost method of drying lactic acid bacteria, but it features a low survival rate. A pre-treatment was designed to increase the survival by embedding bacteria in basic or acidic proteins with the principle of opposite charges attraction. Transglutaminase (TGase)-induced cross-linking then tightened the cell-protein complexes. The results showed that mixing of basic proteins with acidic proteins and TGase treatment increased the survival rates of spray-dried Lactobacillus bulgaricus sp1.1. 84.78% of cells in the TGase-treated milk basic protein (MBP)-soy protein isolate (SPI) bacterial solution remained after drying. Positively charged MBP adhered to more bacteria (negative charge) and formed tight closed loop-shaped complexes with SPI (negative charge) by electrostatic attraction, and TGase enhanced this attraction by strengthening of the hydrogen, CO and CN bonds and exposing the inner α-helix. Imbedding bacteria in TGase-treated acidic-basic protein can increase efficiency of the current formulation.

Much effort has been devoted to studying the production of Streptomyces transglutaminase (TGase). However, more exploration into the mechanism of TGase biosynthesis is necessary to enhance its production further. The effect of excessive metal stress on Streptomyces mobaraensis's TGase activity, growth rate, and mycelium differentiation were evaluated. To elucidate the regulatory mechanism of TGase production and cell differentiations, a proteomic analysis and qRT-PCR of S. mobaraensis was performed. This study showed that the TGase biosynthesis was enhanced while the cell growth was inhibited under MgCl

In the present study, the effects of exopolysaccharides (EPS) from nine Lactobacillus strains with a high degree of bio-activity on human colon cancer cell line HT-29 were studied. The extracellular polymeric substances from 4 strains, namely K11, M5, SB27 and ×12, displayed desirable anti-proliferative activity against HT-29 cells. Crude and acidic EPS were purified from the 4 strains and the inhibitory effects were further investigated. The crude and acidic EPS from these 4 strains exerted anti-proliferation effects on HT-29 cells in a dose-dependent manner but were nontoxic to Vero cells. Treatment with EPS significantly induced G0/G1 cell cycle arrest and apoptosis of HT-29 cells. Hoechst 33258 staining of acidic EPS-treated HT-29 cells revealed different degrees of morphological changes within the nucleus and the formation of apoptotic bodies. Caspase-3 activity was markedly upregulated in HT-29 cells following treatment with acidic EPS. In addition, acidic EPS from the SB27 strain demonstrated the most robust inhibitory effect on HT-29 cells. The results of the present study suggest that as an inducer of apoptosis EPS has the potential to be applied in the treatment of colorectal cancer.

Summary In this study, we have investigated the microbial diversity and stability in the Tibetan kefir during the primary cultivation and subcultivation processes via a combination of culture-independent and culture-dependent methods. According to the culture-independent methods, the profiles of PCR-DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis) indicated that nine microbial species were predominant at different cultivation stages; seventy four isolates of seven predominant species were obtained and identified via the culture-dependent methods. PCR-DGGE further confirmed that Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus paracasei, Streptococcus thermophilus, Kazachstania unispora and Saccharomyces cerevisiae were the most dominant species in the Tibetan kefir during the process of primary cultivation, among which Lb. kefiri, Lb. paracasei and K. unispora showed relative strong stability during both the processes of primary cultivation and subcultivation. These findings suggested that some isolates of the three species possessed the potentiality of being used in the development of direct vat set (DVS) starters for the production of Tibetan kefir and the related products.

Enterococcus faecium Y31 is a class IIa bacteriocin producer found in traditional Chinese fermented foods. The bacteriocin produced by Y31 (bacteriocin Y31) has a broad inhibitory spectrum. Here, we investigated the safety and behaviours of E. faecium Y31 in North-eastern Chinese traditional fermentation paocai (NCTFP) to assess its suitability as an adjunct culture. E. faecium Y31 showed interesting features, such as the absence of virulence determinants (aggregation substance [agg], enterococcal surface protein [esp], endocarditis antigen [efaAfm], gelatinase [gelE], hemolysin [cylA, cylB, cylM], sex pheromone [cpd, cob, ccf], adhesion of collagen [ace], and hyaluronidase [hyl] genes), sensitivity to various antibiotics, lack of haemolytic compounds, and lack of biogenic amine production. E. faecium Y31 could propagate in raw Chinese cabbage and produce bacteriocin Y31. To evaluate its performance as an adjunct culture for NCTFP, changes in the overall quality of control (noninoculated) and experimental NCTFP (inoculated with E. faecium Y31 under previously optimised fermentation conditions) were determined in duplicate fermentations of Chinese cabbage using chemical, microbiological, texture, and sensory evaluations. In the presence of E. faecium Y31, faster fermentation and higher bacteriocin Y31 production were observed than in the control. Additionally, the nitrite concentration of the fermentation solution decreased more rapidly, the total viability of the culture increased, and the lactic acid bacterium population was higher than those in NCTFP produced by spontaneous fermentation. No yeasts and moulds were detected in NCTFP produced by inoculation fermentation or spontaneous fermentation, and the textural properties of these two products were similar. Sensory evaluation results for NCTFP produced by inoculation fermentation were superior to those produced by spontaneous fermentation. Therefore, E. faecium Y31 was sufficient as an adjunct culture to enhance both the hygienic and sensory attributes of NCTFP and its use may help to address the issues inherent in commercial production of NCTFP.

We conducted a comparative in vitro study on the proliferative effects of natural human lactoferrin (nhLF) and bovine lactoferrin (bLF) on osteoblasts. We investigated cell proliferation, cell survival, cell cycle, and mRNA and protein expression of proliferating cell nuclear antigen. Results indicated that treatment with 100 μg/mL of bLF or nhLF promoted the proliferation and sustenance of osteoblasts, and increased the length of the G 2 /M and S phases compared with the untreated osteoblasts. Results of real-time quantitative PCR and Western blot showed that mRNA and protein expression of proliferating cell nuclear antigen by osteoblasts treated with bLF or nhLF were greater than those of the untreated control. At the same concentration, bLF demonstrated a greater effect on osteoblast proliferation than did nhLF. This study provides insights of significance in the utlization of bLF in healthy food formulas.

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 × 10 1 cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 10 2 cfu/reaction. Evaluation of the performance of the method in P. fluorescens -contaminated pasteurized cow milk revealed a detection limit of 7.4 × 10 1 cfu/reaction, which was 10 2 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.

The proteolysis activity, texture, viscosity, particle size and sensory evaluation of 12 isolates identified by 16S rDNA sequence analysis, which were screened from 123 lactic acid bacteria (LAB) isolated from Chinese traditional fermented milk, were evaluated. Proteolytic activity of these selected strains ranged from 2.43 to 11.60 mmol Leu/L of milk. Texture analysis indicated that yogurts of Streptococcus thermophilus were of higher value than those of Lactobacillus delbrueckii at all levels of measurement. Apparent viscosity of yogurt varied considerably among the strains, with values ranging from 4790 to 9870 mPa·s. All the strains showed excellence in firmness, consistency, cohesiveness, viscosity and little difference in the particle size. L. delbrueckii CH9-3 4.0, SB3 and SB25 provided excellent protease activity and could be used as yogurt culture to improve the curd quality. S. thermophilus LBH, Q10 and L. delbrueckii SP1.1 showed high viscosity, and could be mixed with other LAB strains to obtain thick yogurts. Practical Applications The different technological characterizations of lactic acid bacteria could contribute to the different physicochemical and sensorial properties of the fermented dairy products. The studied strains with different properties would provide a well-adapted tool for producing new dairy products or possibly assign to the same traditional products with different physicochemical and sensorial properties through the new specified proteolytic activity, viscosity and flavor.

A gene from Zea mays coding a TGase was expressed in E. coli and identified by Western blotting. Under optimal expression conditions, the production and specific activity of refolded TGZ were 1.41 mg/L and 0.34 U/mg. The activated TGZ was employed to cross-link milk proteins. The yoghurt treated with TGZ showed a lower syneresis, higher apparent viscosity and texture than untreated yoghurt. The properties of TGZ-treated sample were better than those of MTG-treated samples, so TGZ may have potential as an additive in yoghurt manufacture.

In order to purify proteinase from Lactobacillus delbrueckii subsp. bulgaricus, chromatography methods including ion-exchange and hydrophobic interaction were combined. Chromatographic conditions were structured and optimized. The final sample volume, flow rate, and length of gradient for anion exchange chromatography were 20 mL, 5 mL/min and 10 column volume length of gradient, in that order. Besides, the flow rate and length of gradient for hydrophobic interaction chromatography were 1 mL/min and 10 column volume. Proteinase was purified up to 43-fold with a specific activity of 54.4 U/mg protein, and the recovery of 46.6%. The molecular weight of the purified proteinase was about 40 kDa.

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.

UNLABELLED Traditional fermented vegetables have inconsistent quality and high nitrite content, whereas a commercial starter culture could overcome these problems. A total of 34 lactic acid bacteria strains were screened from 2 homemade naturally fermented Chinese cabbages. Fermented characters of single starter or mixed cultures were examined, including acidification, growth rate, and nitrite depletion ability in the fermented culture. As a result, the combined starter culture (Lactobacillus delbrueckii IWQ and Lactobacillus paracasei J21) was selected. The fermented Chinese cabbage resulted from this mixed starter culture had higher sugar content residue (30.5 mg/mL for mixed culture and 22 mg/mL for control). And the titratable acid of the cabbage fermented by the mixed starter was 110°T, which was twice higher than that of control sample. The selected starter culture also had a better texture and sensory qualities than that the starter cultures from a local fermented Chinese cabbage plant, especially for its significantly lower nitrite content (3.00 mg/kg for mixed culture and 4.49 mg/kg for control). The findings of this study form a database for further studies on the development of starter cultures for fermented cabbage production and could replaced the local plant starter culture. PRACTICAL APPLICATION LABs were screened from traditional fermented food. The fermented characteristics of selected LABs were better than that of starter cultures from a local Chinese cabbage plant. Selected LAB can decrease nitrite content in fermented cabbage. The Chinese cabbage fermented by the selected LAB had the good flavor and texture.

In the present study, a YGNGV-motif-based assay was developed and applied. Given that there is an increasing demand for natural preservatives, we set out to obtain lactic acid bacteria (LAB) that produce bacteriocins against Gram-positive and Gram-negative bacteria. We here isolated 123 LAB strains from 5 types of traditional Chinese fermented food and screened them for the production of bacteriocins using the agar well diffusion assay (AWDA). Then, to acquire LAB producing class IIa bacteriocins, we used a YGNGV-motif-based assay that was based on 14 degenerate primers matching all class IIa bacteriocin-encoding genes currently deposited in NCBI. Eight of the LAB strains identified by AWDA could inhibit Gram-positive and Gram-negative bacteria; 5 of these were YGNGV-amplicon positive. Among these 5 isolates, amplicons from 2 strains (Y31 and Y33) matched class IIa bacteriocin genes. Strain Y31 demonstrated the highest inhibitory activity and the best match to a class IIa bacteriocin gene in NCBI, and was identified as Enterococcus faecium. The bacteriocin from Enterococcus avium Y33 was 100% identical to enterocin P. Both of these strains produced bacteriocins with strong antimicrobial activity against Listeria monocytogenes, Escherichia coli, and Bacillus subtilis, hence these bacteriocins hold promise as potential bio-preservatives in the food industry. These findings also indicated that the YGNGV-motif-based assay used in this study could identify novel class IIa bacteriocinogenic LAB, rapidly and specifically, saving time and labour by by-passing multiple separation and purification steps.

Lactic acid bacteria (LAB) starter cultures can be processed by many drying techniques, among which spray drying has great potential. However, injuries of LAB during spray drying lead to a low survival rate. The microbial injuries of LAB during spray drying are specifically induced by dehydration inactivation, thermal inactivation, and balance between the two. To reduce the drying temperature, new drying techniques based on spray drying were tried; that is, low-temperature vacuum–spray drying and spray–freeze drying.

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.

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.

BACKGROUND Transglutaminases catalyze post-translational modification of proteins by e-(γ-glutamyl) links and covalent amide bonds. Research on properties and applications of plant transglutaminases is less developed than in animals and micro-organisms. In a previous study, optimized Zea mays transglutaminase was purified from recombinant Pichia pastoris strain. The main objective of the present study was to characterize this enzyme and assess its effect on the properties of yoghurt. RESULTS The purified recombinant transglutaminase presented a Km of 3.98 µmol L−1 and a Vmax of 2711 min−1 by the fluorometric method. The enzyme was stable after incubation for 30 min below 50 °C and over a broad pH range of 5–8 at −20 °C for 12 h. The results showed that the crosslinking reaction catalyzed by this enzyme could effectively improve the properties of full and non-fat yoghurts. Also, the properties of non-fat yoghurt could be improved similar to the full-fat product by recombinant transglutaminase. CONCLUSION The application of recombinant transglutaminase in yoghurt indicated that this enzyme could be used as a substitute for microbial transglutaminase in the production of yoghurt, thus providing experimental evidence for the future application of plant transglutaminases in the food industry. © 2013 Society of Chemical Industry

The purpose of this study was to optimise the incubation conditions for cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) production by Lactobacillus acidophilus F0221 and evaluate the effect of possible intestinal nutrients on c9, t11 CLA production. Growth in Mann Rogosa Sharp broth supplemented with cysteine and containing 0.5 g L−1 linoleic acid at pH 6.5 with anaerobic incubation at 37 °C for 40 h were found to be the optimal conditions for CLA production. Galactosaccharide, arabinogalactan, galactose and glucose had a higher promoting effect on CLA production (96.19–123.89 μg mL−1) than other carbohydrates. Acetic acid had a higher promoting effect on CLA production (130.98 μg mL−1) than other short chain fatty acids. These results provide detailed parameters for the production of c9, t11 CLA by L. acidophilus F0221 and are valuable for further understanding of c9, t11 CLA formation in the human intestine by this strain.

The interest of probiotics as remedy for a broad number of infectious diseases has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In this study, five selected Lactobacillus isolated from human intestinal and ferment milk were preliminarily identified by 16S rDNA gene sequencing and assessed the ability to inhibit the adhesion of enteropathogens using HT-29 cells model through a process which may be related to specific components of the bacterial surface. Surface layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. Removal of S-layer proteins from the Lactobacillus (treated with 5 M LiCl) reduced inhibition activity as revealed in exclusion, competition and displacement assays, which suggested that S-layer proteins had involved in the adhesion of probiotics. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant band which was approximately 60 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 43.2 % of the total amino acid for the Lactobacillus paracasei M7. So, these probiotics could be used in the health-promoting food products, which could prevent the diarrhea caused by pathogens.

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.

Stress-mediated bioprocess is a strategy designed to enhance biological target productivity. In this study, an attempt was made to enhance transglutaminase (TGase) production by Streptomyces mobaraensis by using different stress conditions including heat shock, alcohols and salt stress. Results showed that the effects of stress on TGase production depended on the type applied. For heat shock, TGase production (1.32 U/ml) was recorded maximum in the culture treated at 48 h post inoculation in water bath at 60 °C for 1 min. For alcohols treatment, the maximum activity of TGase (1.77 and 1.75 U/ml) was obtained when 3% methanol was added into the medium at 0 or 24 h of fermentation. However, a 3.5-fold increased production of TGase (3.8 U/ml) was observed in the medium supplemented with 0.2 mol/l MgCl2 compared with the basic medium at the beginning of fermentation. In conclusion, TGase production from S. mobaraensis was improved by heat shock, methanol and salt stress treatments, MgCl2 stress was the most effective.

In this study, we first tested the capacity for eight different salts as stress-mediated bioprocesses in the production of transglutaminase (TGase). A significant effect on the cell growth and TGase production was obtained with the highest yield of TGase being observed at 96 h of incubation (4.3 U/ml) when the basic medium was supplemented 0.10 M MgCl(2), as opposed to that observed with the basic medium control (2.1 U/ml at 120 h). Data from Western blot assays showed that transformation of pro-TGase to its mature enzyme occurred more rapidly in MgCl(2) medium. Furthermore, total protease, metalloprotease, and serine protease were also synthesized at a faster rate in the medium containing MgCl(2). The results demonstrate that MgCl(2) enhanced the production of key proteases involved in the activation of TGase biosynthesis. To explore the mechanism, viability assay was performed. The results show that MgCl(2) induced the mycelia differentiation, decreased cell growth rate, and stimulated cell death. We argue that TGase production was promoted by the stimulation of mycelium differentiation induced by MgCl(2) stress.

Cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) producing bacteria have attracted much attention as novel probiotics which have shown beneficial effects on host health. However, bile salts are able to inhibit bacterial growth and c9, t11 CLA production. For recovering growth and c9, t11 CLA production of Lactobacillus acidophilus F0221 in the presence of bile salts, Tween series (Tween 20, Tween 40, Tween 60 and Tween 80) were added in growth culture containing 0.3% oxgall. Results showed that the viable counts were significantly (P < 0.05) recovered to 8.58–8.75 log CFU/mL in the presence of all Tween treatments. However, recovery of c9, t11 CLA production was only demonstrated in the presence of Tween 80 (72.89 μg/mL). Stepwise increasing oxgall in a concentrations range from 0.1% to 0.9% according to human intestinal physiological environments, Tween 80 still showed significant (P < 0.05) recovery ability on growth (8.91–8.04 log CFU/mL) and c9, t11 CLA (69.22–34.27 μg/mL) production. The effect of Tween 80 on growth and production was also investigated in the presence of different types of bile salts (sodium salts of cholic acid (CA), deoxycholic acid (DCA), chendeoxycholic acid (CDCA), glycocholic acid (GCA) and taurocholic acid (TCA)). Results showed that Tween 80 could significantly (P < 0.05) recover c9, t11 CLA production in the presence of all types of bile salts, but the Tween 80 could only significantly (P < 0.05) recover viable counts of the strain in the presence of CA, DCA and CDCA. This recovery ability could be attributed to the protection of leakage of intracellular material. Additionally, although bile salts inhibited growth and c9, t11 CLA production by the growing cell, it promoted the c9, t11 CLA production by the resting cell.

The isolation and screening of lactic acid bacteria (LAB) from natural sources have been one of the powerful means to obtain strains for the food industry. A total of 275 indigenous isolates were obtained from 43 samples of traditional fermented foods in specific ecological niches throughout the northwestern China, and among which 13 strains of LAB were selected for their potential in food preservation and production. Among the 13 isolates, Lactobacillus (10) was dominant over Lactococcus (3). The distribution of the isolates was as follows: Lactobacillus paracasei ssp. paracasei (J23, M10, M20, M22), Lactobacillus rhamnosus (J20, M18), Lactococcus lactis ssp. lactis (X20, Q7), Lactobacillus casei (Q1, Q12), Lactobacillus plantarum (J11), Lactococcus lactis ssp. cremoris (X8), Lactobacillus delbrueckii ssp. bulgaricus (Q5). All 13 isolates produced bacteriocin with a broad inhibitory spectrum against selected Gram-positive as well as Gram-negative pathogenic and spoilage species. Biochemical analysis revealed that they possessed high acidification and coagulation activity. Several strains possessed the high activity of 2 or 3 technological characteristics, related to novel starters and food preservatives.

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7-3.6logCFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry.

Thirty-six Lactobacilli, isolated from Chinese artisanal fermented milks, were evaluated for potential use as adjunct cultures. All isolates presented low acidification rate. Proteolytic activities of these Lactobacilli ranged from 17.7 to 48.5 mg Gly/L milk, and strain SB5 showed the highest activity. Aminopeptidase activities ranged from 15.1 to 71.0 U/mg. Strains M18L2, SB33 and SB29 were 71.0, 68.6 and 68.5 U/mg, respectively. Autolytic activities of isolates in simulated cheese-like buffer were between 10% and 45%. Strains SB9 and SB11 showed the two highest values. In summary, the aforementioned six strains could be good candidates as adjunct cultures in cheese.

The cDNA encoding β-1,3(4)-glucanase, named PsBg16A, from Paecilomyces sp. FLH30 was cloned, sequenced, and over expressed in Pichia pastoris, with a yield of about 61,754 U mL⁻¹ in a 5-L fermentor. PsBg16A has an open reading frame of 951 bp encoding 316 amino acids, and the deduced amino acid sequence of PsBg16A revealed that it belongs to glycoside hydrolase family 16. The purified recombinant PsBg16A had a pH optimum at 7.0 and a temperature optimum at 70 °C, and randomly hydrolyzed barley β-glucan, lichenin, and laminarin, suggesting that it is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-glucans.

Milk has positive effects on bone growth. However, the effect of skim milk powder (SMP) on bone properties has not been reported. This study investigated the effect of SMP on bone loss in ovariectomized (OVX) rats. Forty female Sprague-Dawley rats were ovariectomized and another 10 rats received a sham operation. The OVX rats were randomly separated into 4 groups: OVX control, OVX SMP1 (SMP at 0.04 g/d), OVX SMP2 (SMP at 0.20 g/d), and OVX SMP3 (SMP at 0.40 g/d). Skim milk powder was supplied in the rat diet for 12 wk, and the rats were gavaged once per day. The effects of SMP on calcium content and bone mineral density of femur were determined by atomic absorption spectrophotometry and dual-energy x-ray absorptiometry, respectively. Compared with the control, SMP at all dose levels tested had no particular effect on weight:length, calcium content, or bone mineral density of femurs. It was demonstrated that SMP (0.04 to 0.40 g/d) had no positive effect on bone loss in OVX rats, probably because the heat treatment used during SMP processing caused a loss of biological activity in the protein.

To screen lactic acid bacteria for starter cultures in cheese production, 21 Lactococcus strains previously isolated from natural fermented milk and koumiss made by herdsman families in the Xinjiang, Gansu, and Qinghai provinces of China were evaluated for optimal growth temperature, acidification activity, proteolytic activity, aminopeptidase activity, and autolytic activity. All isolates presented low acidification rates, and the pH value did not reach 5.3 after 6h of inoculation in sterile reconstituted skim milk at 30°C. Strains X9C2 and T7C showed the highest proteolytic activity of 24.67 and 23.58mg of glycine/L of milk, respectively. For aminopeptidase activity, strains X9C2 and T1C2 displayed the highest activities of 30.56 and 27.70 U/mg of protein using l-leucine- p -nitroanilide as substrate, respectively. Autolytic activity in simulated cheese-like buffer ranged from 7.45 to 34.76%, and strains Q14C2 and Q16C showed the highest values of 34.76 and 34.20%, respectively. Collectively, one main finding is that some technological characteristics of Lactococcus isolates from Chinese traditional fermented products varied greatly. Some isolates with potentially important properties could be valuable for application as starter cultures of cheese or could constitute a mixed culture.

A sensitive protocol based on thin-layer chromatography (TLC) was developed to screen qualitatively bile salt hydrolase (BSH)-active lactobacilli. The sodium salts of glycocholic acid and taurocholic acid were used as substrates, and bacterial BSH activity was confirmed by detecting cholic acid as a product of the bile conjugates using a TLC assay with direct visual observation. Forty-five lactobacilli isolated from human fecal samples were tested for BSH activity by the TLC assay, a conventional plate assay, and a quantitative colorimetric assay. With the TLC and quantitative colorimetric assays, the same 24 BSH-positive strains were detected. No false-positive or false-negative results were detected by the TLC assay. However, only 20 BSH-positive strains were detected with the conventional plate assay. Compared with the conventional plate assay, the TLC assay is more sensitive for the detection of BSH activity of lactobacilli and, thus, more suitable for screening of BSH-active lactobacilli of human origin.

The objective of this study was to evaluate the in vitro immunomodulating capacity of Lactobacillus coryniformis subsp torquens T3L (L. coryniformis T3L) isolated from traditional fermented yak’s milk in Tibet, China, and Lactobacillus paracasei supsp. paracasei M5L (L. paracasei M5L)isolated from kumiss in Sinkiang, China was used as control. The effects of live bacteria, cell wall and genomic DNA of the two Lactobacillus strains on human peripheral blood mononuclear cells (PBMCs) proliferation, production of interleukin-12 (IL-12 p70), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and natural killer (NK) cell activity were assessed. The live bacteria, cell wall and genomic DNA of the two lactobacilli exerted proliferative effects on PBMCs. Live bacteria at 1 × 106 c.f.u. ml−1, cell wall at 20 μg protein ml−1 and DNA at 50 μg DNA ml−1 of the strainS induced the secretion of IL-12 (p70), IFN-γ and TNF-α by PBMCs. NK cell activities increased after cultivation of PBMCs with live bacteria, cell wall and DNA of the strains. Overall, these results demonstrate that the live bacteria, cell wall and genomic DNA of the two Lactobacillus strains exhibit immunomodulating activity.

A method based on colony PCR was developed for rapid screening of class IIa bacteriocin-producing lactic acid bacteria. The principle of the method is to detect the presence of bacteriocin gene by using specific degenerate primers to amplify a 3 kb fragment from bacteriocin structural gene to histidine kinase gene regions. Degenerate primers were designed according to the conserved amino acid sequences (YGNGVXCXXXXCXV and LDNAIE) contained in class IIa bacteriocins. This assay showed agreement with the conventional well-diffusion method. These findings indicate that the method proposed in this study is considerably more rapid and sufficient for screening of class IIa bacteriocin-producing bacterial isolates in environmental samples.

Inulin has become an attractive ingredient in the food industry because of its nutritional and functional characteristics. Extraction of inulin from Jerusalem artichoke and the effects of its enzymatic hydrolyzate on the textural and sensorial characteristics of yogurt were investigated. The extraction yield (98%) of inulin was obtained with dry Jerusalem artichoke powder. The thin-layer chromatography and the ion chromatogram analysis of the enzymatic hydrolyzate revealed that inulinase hydrolyzed inulin into fructooligosaccharides with degrees of polymerization from 2 to 10. Utilization of inulin and hydrolyzate in yogurts improved their textural properties. Sensorial evaluation of yogurts showed that yogurts with inulin or its hydrolyzate had a better body and smoother texture than yogurts with sucrose only. The findings of this study suggest that inulin or its hydrolyzate can partially replace sucrose in formulations in the food industry.

As an initial investigation to improve the insoluble yeast β-1, 3-glucan solubility, a novel β-glucanase from Trichoderma viride TP09 was purified in the culture supernatant and partially characterized. By 70% saturation ammonium sulfate and chromatography on DEAE-Sepharose CL-6B column, β-glucanase was purified 28.7-fold, with recovery of 45.2% of the initial activity. The molecular weight of this enzyme was estimated to be 54.6 KD by SDS-PAGE. The optimum pH and the optimum temperature for the enzyme were 5.0 and 50 °C, respectively. The enzyme showed high stability within the range of pH 3.0–5.0 and thermostability between 30 and 70 °C. The enzyme activity was inhibited by Fe3+, Mg2+, Mn2+, Cu2+, and stimulated by Zn2+, Ca2+, Fe2+. Substrate specificity studies revealed the enzyme to be a β-1, 3–1, 4-glucanase. The β-glucanase showed preference for β-1, 3 linkage and β-1, 4 linkage, but had no activity on α-1, 4 and α-1, 6 linkage. The above results indicated that the enzyme extracted from T. viride TP09 of the beer-making sewage could be used as a potential predominant tool to enhance solubility of the insoluble yeast β-1, 3-glucan. These findings may lead to an enhanced solubility and expedite the progress of application in immunotherapy.

The heat-resistant lipases from psychrotrophic bacteria play a major role in the deterioration of dairy products during storage and shelf life. The aim of this study was to analyze the predominant psychrotrophic bacteria and their lipases with the relevance to preserve the quality of dairy products. Three bovine raw milk samples collected from North China were screened for the presence of lipolytic psychrotrophic bacteria. Overall, 21 psychrotrophic isolates were selected as potential lipase producers and further identified as Pseudomonas. Although each geographical area was characterized by its own unique species, the predominant lipolytic psychrotrophic specie found in all three geographic areas was Pseudomonas fragi. Crude lipases from selected strains were resistant to conventional heat treatments. The results suggested that the monitoring of the dominant psychrotrophic species that were able to produce heat-stable lipase could offer a sensitive and efficient tool for the quality preservation of milk quality and related products. Practical applications The heat-resistant lipases from psychrotrophic bacteria play a major role in the deterioration of dairy products during storage and shelf life. The article presents the identification of lipolytic psychrotrophic bacteria isolated from raw milk in North China and the heat-resistant property of their lipase in order to select the typical strains. Considering this, assessment of the psychrotrophic bacteria had the ability of producing the heat-resistant lipase, and compared their ability of producing lipase and the heat-resistant property of the lipase. Therefore, these analyses can lead to develop better strategy for control of the quality of the dairy products and guarantee dairy products safety.

Traditional fermented vegetables have inconsistent quality and high nitrite content, whereas a commercial starter culture could overcome these problems. A total of 34 lactic acid bacteria strains were screened from 2 homemade naturally fermented Chinese cabbages. Fermented characters of single starter or mixed cultures were examined, including acidification, growth rate, and nitrite depletion ability in the fermented culture. As a result, the combined starter culture (Lactobacillus delbrueckii IWQ and Lactobacillus paracasei J21) was selected. The fermented Chinese cabbage resulted from this mixed starter culture had higher sugar content residue (30.5 mg/mL for mixed culture and 22 mg/mL for control). And the titratable acid of the cabbage fermented by the mixed starter was 110°T, which was twice higher than that of control sample. The selected starter culture also had a better texture and sensory qualities than that the starter cultures from a local fermented Chinese cabbage plant, especially for its significantly lower nitrite content (3.00 mg/kg for mixed culture and 4.49 mg/kg for control). The findings of this study form a database for further studies on the development of starter cultures for fermented cabbage production and could replaced the local plant starter culture.LABs were screened from traditional fermented food. The fermented characteristics of selected LABs were better than that of starter cultures from a local Chinese cabbage plant. Selected LAB can decrease nitrite content in fermented cabbage. The Chinese cabbage fermented by the selected LAB had the good flavor and texture.

A resting cell system was developed for bacteriocin Lac-B23 production from Lactobacillus paracasei J23. The resting cell medium contained (g/L): Glucose 20, Sodium acetate 5.0, MnSO4 0.25 MgSO4 0.5, Ammoniumhydrogencitrate 1.0, KH2PO4 1.0. The resting cell incubation time and temperature were 20 h and 37 °C and the effects of exogenous factors, including amino acids, glycerol, pyruvic acid, and α-ketoglutaric acid were investigated. Cys and Gly could stimulate the production of bacteriocin, while no stimulus effect was observed for Glu, Tyr and Ala. Glycerol and pyruvic acid increased bacteriocin production and the optimum concentrations were 1% and 30 g/L, respectively. Bacteriocin could act as an inducer of its own biosynthesis. These findings are of importance for the further study of bacteriocin biosynthesis regulation and for the improvement of bacteriocin production yields.

Transglutaminases catalyze post-translational modification of proteins by ε-(γ-glutamyl) links and covalent amide bonds. Research on properties and applications of plant transglutaminases is less developed than in animals and micro-organisms. In a previous study, optimized Zea mays transglutaminase was purified from recombinant Pichia pastoris strain. The main objective of the present study was to characterize this enzyme and assess its effect on the properties of yoghurt.The purified recombinant transglutaminase presented a Km of 3.98 µmol L(-1) and a Vmax of 2711 min(-1) by the fluorometric method. The enzyme was stable after incubation for 30 min below 50 °C and over a broad pH range of 5-8 at -20 °C for 12 h. The results showed that the crosslinking reaction catalyzed by this enzyme could effectively improve the properties of full and non-fat yoghurts. Also, the properties of non-fat yoghurt could be improved similar to the full-fat product by recombinant transglutaminase.The application of recombinant transglutaminase in yoghurt indicated that this enzyme could be used as a substitute for microbial transglutaminase in the production of yoghurt, thus providing experimental evidence for the future application of plant transglutaminases in the food industry.

The four wild Lactobacillus rhamnosus strains were examined in vitro for resistance to simulated gastro and intestinal juices, adhesion to HT-29 cells, antagonistic activity against enteric pathogens and immunomodulating activity. The strains L. rhamnosus SB5L, J5L and IN1L were able to survive in simulated gastro juice while the strain L. rhamnosus SB31L lost viability exposed to simulated gastro juice for 3 h. The four strains had high viability in simulated small intestinal juice with little loss (