Determination of six groups of mycotoxins in Chinese dark tea and the associated risk assessment.

Chinese dark tea is widely enjoyed for its multiple health-promoting effects and pleasant taste. However, its production involves fermentation by microbiota in raw tea, some of which are filamentous fungi and thus potential mycotoxin producers. Accordingly, whether mycotoxins pose health risk on dark tea consumption has become a public concern. In this study, a cleaning method of multi-functional column (MFC) and immunoaffinity column (IAC) in tandem combined to HPLC detection was developed and validated for determining ten mycotoxins of six groups (i.e., aflatoxins of B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, fumonisins of B1, B2, and T-2) in dark teas. The interferences from secondary metabolites were effectively reduced, and the sensitivities and recoveries of the method were qualified for tea matrices. Six groups mycotoxins were determined in 108 samples representing the major Chinese dark teas by using the new method. Subsequently, the dietary exposure and health risks were evaluated for different age and gender groups in Kunming and Pu'er in China and Ulan Bator in Mongolia. The occurrence of zearalenone was 4.63% and that of ochratoxin A was 1.85%, with the other four groups mycotoxins were below the limits of quantification. The hazard index values for the five groups' non-carcinogenic mycotoxins were far below 1.0. The deterministic risk assessment indicated no non-carcinogenic risks for dark tea consumption in the three areas. Probabilistic estimation showed that the maximum value of 95th percentile carcinogenic risk value for the aflatoxins was 2.12 × 10-8, which is far below the acceptable carcinogenic risk level (10-6). Hereby, six groups mycotoxins in Chinese dark tea showed no observed risk concern to consumers.

The Core Role of Bacillus subtilis and Aspergillus fumigatus in Pile-Fermentation Processing of Qingzhuan Brick Tea.

To identify the microorganisms responsible for the formation of the main quality components of Qingzhuan brick tea (QZBT) during solid-state fermentation (SSF), predominant thermoduric strains were isolated from the tea leaves collected during SSF. According to their capability of releasing cellulase, pectase, protease, and polyphenol oxidase, four strains were selected as starter cultures to ferment sun-dried tea leaves during artificially inoculated SSF. According to the major enzymatic activities and quality components content (tea polyphenols, catechins, amino acids, soluble sugar, and theabrownin), it was found that Aspergillus fumigatus M1 had a significant effect on the transformation of polyphenols and Bacillus subtilis X4 could enhance the ability of bioconversion of strain M1. Strain X4 and M1 may be the core microbes responsible for developing these biochemical components of QZBT, as the values of quality components of tea leaves fermented by these two strains for 6 days was very close to that of the sample naturally fermented for 35 days in the tea factory. The results could be significant in developing industrial starters for the manufacture of QZBT and stabilizing the product quality of different batches.

Loading of anthocyanins on chitosan nanoparticles influences anthocyanin degradation in gastrointestinal fluids and stability in a beverage.

The optimal preparation parameters to create anthocyanin-loaded chitosan nanoparticles was predicted using response surface methodology (RSM). A Box-Behnken design was used to determine the preparation parameters that would achieve the preferred particle size and high encapsulation efficiency. The result suggested that the optimized conditions were 2.86mg/mL carboxymethyl chitosan (CMC), 0.98mg/mL chitosan hydrochloride (CHC) and 5.97mg anthocyanins. Using the predicted amounts, the experimentally prepared particles averaged 219.53nm with 63.15% encapsulation efficiency. The result was less than 5% different than the predicted result of 214.83nm particle size and 61.80% encapsulation efficiency. Compared with the free anthocyanin solution, the anthocyanin-loaded chitosan nanoparticles showed a slowed degradation in simulated gastrointestinal fluid. Compared with the free anthocyanin solutions in a model beverage system, the stability of the anthocyanins was increased in the anthocyanin-loaded chitosan nanoparticles.

Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH.

OBJECTIVE: The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148). RESULTS: Cell biomass and biomass yield of L. lactis grown with 4 µg hemin/ml and O2 were higher than those without aeration when the culture pH was controlled at 5-6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5-6.5) was at pH 5.5. CONCLUSION: The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

Exogenous transglutaminase improves multiple-stress tolerance in Lactococcus lactis and other lactic acid bacteria with glutamine and lysine in the cell wall.

OBJECTIVE: To increase the resistance of ingested bacteria to multiple environmental stresses, the role of transglutaminase in Lactococcus lactis and possible mechanisms of action were explored. RESULTS: L. lactis grown with transglutaminase exhibited significantly higher resistance to bile salts, stimulated gastric juice, antibiotics, NaCl, and cold stress compared to the control (cultured without transglutaminase), with no negative influence on cell growth. Transmission electron microscopy revealed that the cell walls of L. lactis cultured with 9 U transglutaminase/ml were approx. 1.9-times thicker than the control. Further analysis demonstrated that the multi-resistant phenotype was strain-specific; that is, it occurred in bacteria with the presence of glutamine and lysine in the peptidoglycan. CONCLUSION: Supplementation of culture media with transglutaminase is an effective, simple, and inexpensive strategy to protect specific ingested bacteria against multiple environmental challenges.

[Improving industrial microbial stress resistance by metabolic engineering: a review].

Metabolic engineering is a technologic platform for industrial strain improvement and aims not only at modifying microbial metabolic fluxes, but also improving the physiological performance of industrial microbes. Microbes will meet multiple stresses in industrial processes. Consequently, elicited gene responses might result in a decrease in overall cell fitness and the efficiency of biotransformation. Thus, it is crucial to develop robust and productive microbial strains that can be integrated into industrial-scale bioprocesses. In this review, we focus on the progress of these novel methods and strategies for engineering stress-tolerance phenotypes referring to rational metabolic engineering and inverse metabolic engineering in recent years. In addition, we also address problems existing in this area and future research needs of microbial physiological functionality engineering.

Optimization of a GC-MS metabolic fingerprint method and its application in characterizing engineered bacterial metabolic shift.

Metabolomics influences many aspects of life sciences including microbiology. Here, we describe the systematic optimization of metabolic quenching and a sample derivatization method for GC-MS metabolic fingerprint analysis. Methanol, ethanol, acetone, and acetonitrile were selected to evaluate their metabolic quenching ability, and acetonitrile was regarded as the most efficient agent. The optimized derivatization conditions were determined by full factorial design considering temperature, solvent, and time as parameters. The best conditions were attained with N,O-bis(trimethylsiyl) trifluoroacetamide as derivatization agent and pyridine as solvent at 75 degrees C for 45 min. Method validation ascertained the optimized method to be robust. The above method was applied to metabolomic analysis of six different strains and it is proved that the metabolic trait of an engineered strain can be easily deduced by clustering analysis of metabolic fingerprints.

[The function of glutathione/glutathione peroxidase system in the oxidative stress resistance systems of microbial cells].

The physiological roles of the glutathione(GSH)/glutathione peroxidase(GPx) system in protecting microbial cells against oxidative stress were reviewed. In eukaryotic model microbe Saccharomyces cerevisiae,this system is obligatory in maintaining the redox balance and defending the oxidative stress. However, the GSH/GPx system only conditionally exists in prokaryotes. Namely,for those prokaryote bacteria containing glutathione reductase and GPx, e.g. Haemophilus influenzae and Lactococcus lactis, by taking up GSH, they might develop a conditional GSH-dependent GPx reduction system, which conferred cells a stronger resistance against oxidative challenge.

Glutathione protects Lactococcus lactis against acid stress.

Previously we showed that glutathione (GSH) can protect Lactococcus lactis against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739-5745, 2003). In the present study, we show that the GSH imported by L. lactis subsp. cremoris SK11 or produced by engineered L. lactis subsp. cremoris NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting L. lactis strains capable of producing or importing GSH.

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host.

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H(2)O(2)-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H(2)O(2) (150 mM) and a superoxide generator, menadione (30 microM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H(2)O(2)-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.

[Glutathione plays an anti-oxidant role in Lactococcus lactis].

To assess the physiological function of GSH in resistance to oxidative stress in Lactococcus lactis ssp. cremoris NZ9000, the recombinant strain NZ9000 (pNZ3203) capable of producing GSH was used as the experiment materials. The anti-oxidant role of glutathione was observed under higher H2O2 dosage, i.e., 150 mmol/L H2O2 treatment for 15 min. The resistance of NZ9000 (pNZ3203) cells grown for 3 h, 5 h, and 7 h (nisin-induced for 1 h, 3 h and 5 h) were 1.8-fold, 2.6-fold, and 2.9-fold that of NZ9000 (pNZ8148) cells, respectively. In addition, the survival of NZ9000 (pNZ3203) cells grown for 5 h (nisin-induced for 3 h), upon treatment of 20 mmol/L menadione for 60 min, was 6.2-fold that of NZ9000 (pN8148) cells. Therefore, introduction a new biosynthetic pathway of glutathione could confer higher resistance to oxidative stress on L. lactis NZ9000.

Heterologous leaky production of transglutaminase in Lactococcus lactis significantly enhances the growth performance of the host.

This study describes a novel strategy to improve the growth performance of Lactococcus lactis by heterologous production of food-grade transglutaminase. The mtg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into L. lactis subsp. cremoris NZ9000. The leaky expression of the mtg gene from the nisA promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of L. lactis, which is an important step for the purposes of producing proteins of commercial interest using L. lactis as a host.

[Influence of expression of transglutaminase on the growth of Lactococcus lactis].

To improve the aerobic growth performance of Lactococcus lactis subsp. cremoris NZ9000, the gene mtg encoding the mature microbial transglutaminase was amplified from the chromosomal DNA of Streptoverticillium mobaraense and then cloned into the nisin-inducible expression vector pNZ8148. The resulting plasmid pFL001 was transformed into strain NZ9000 by electroporation. Compared with strain NZ9000 harboring pNZ8148 (the control strain), strain NZ9000 harboring pFL001 (the recombinant strain) had a remarkably improved aerobic growth performance. When grown aerobically under non-pH-controlled conditions, the maximal biomass of the recombinant strain reached 4.13 g/L, which was 11-fold higher than the growth of the control strain (0.34 g/L). When grown aerobically with the pH controlled at 6.5 +/- 0.1, the maximal biomass of the recombinant strain reached 4.73 g/L, which was an 80% increase compared with the growth of the control strain. In addition, the efficiency of biomass synthesis relative to glucose consumption (Y(x/s)) of the recombinant strain, 71.7 g of biomass per mol of glucose, was 1.6-fold higher than that of the control strain. The significantly improved growth performance of the recombinant strain might be attributed to the expression of mtg in the recombinant strain, which might increase intracellular pH and save part of the energy(ATP) that was originally used for pumping the cytoplasmic H+, and as a consequence, the energy used for growth increased accordingly.