•Sixteen raw milk samples representative of different Chinese farms were evaluated for psychrotrophic bacteria.•480 isolates were clustered into 85 groups based on RAPD profiles.•Pseudomonas was the most dominant genus followed by Acinetobacter and Flavobacterium.•Pseudomonas fluorescens was the most dominant species followed by Pseudomonas fragi and Pseudomonas psychrophila.•26 species belonging to 14 genera proved to be previously unreported psychrotrophic contaminants in raw milk.Four-hundred and eighty isolates isolated from raw milk samples collected from different farms in China were subjected to random amplified polymorphic DNA analysis and 16S rRNA sequencing for clustering and identification, respectively. All isolates comprised 24 genera and 74 species in total. Pseudomonas was the most prevalent genus, representing 58.8% of all isolates, followed by Acinetobacter (13.3%), Flavobacterium (6.0%) and Sphingobacterium (4.2%). Among the 25 species belonging to Pseudomonas, Pseudomonas fluorescens (15.8%) proved to be the most dominant species, followed by Pseudomonas fragi (7.1%) and Pseudomonas psychrophila (5.4%). Out of these isolates, 12.3% have not been reported in previous studies, highlighting that many psychrotrophic bacteria are rather underexplored in raw milk. In the future, a detailed investigation will be required to characterize these bacterial isolates based on their potential to deteriorate the quality of dairy products.

Chinese traditional fermented foods have a very long history dating back thousands of years and have become an indispensable part of Chinese dietary culture. A plethora of research has been conducted to unravel the composition and dynamics of microbial consortia associated with Chinese traditional fermented foods using culturedependent as well as culture-independent methods, like different high-throughput sequencing (HTS) techniques. These HTS techniques enable us to understand the relationship between a food product and its microbes to a greater extent than ever before. Considering the importance of Chinese traditional fermented products, the objective of this paper is to review the diversity and dynamics of microbiota in Chinese traditional fermented foods revealed by HTS approaches.高通量测序在探究中国传统发酵食品菌群多样性及动态变化中的应用中国传统发酵食品有着上千年的历史,已经成为 中国饮食文化中不可缺少的部分。微生物是发酵 食品的灵魂,为了探究传统发酵食品中的微生物 组成,多种依赖培养和非培养技术都已应用于不 同发酵体系的微生物菌相分析中。高通量测序技 术是近几年兴起的生物学技术,它极大地方便了 环境微生物多样性的研究,促进了我们对复杂微 生物环境的认知,其在发酵食品中的应用提升了 我们对发酵食品品质与微生物关系的认识水平。 本文综述了高通量测序技术在探究我国传统发 酵食品菌相中的应用,总结分析了不同发酵体系 中微生物的组成和动态变化。

•Biofilm forming ability of a wide range of mesophilic and thermophilic sporeformers has been determined•Only Bacillus licheniformis, Anoxybacillus flavithermus and Geobacillus species formed good biofilms on stainless steel in milk•Biofilm formation on polystyrene did not correspond with bacterial adherence on stainless steel surface•Biofilm formation highly depends on strain, substrate, growth medium and type of biofilm (air-liquid or liquid-solid biofilm)•B. licheniformis isolates belonging to the same genetic groups did not show phenotypic similarityBiofilms on the surface of dairy manufacturing plants are potential reservoirs of microbial contamination. These microbial aggregates may harbour pathogenic and spoilage organisms which contaminate dairy products. The biofilm forming capacity of many spore forming isolates of dairy origin has not been given much attention. The present study explored the biofilm forming potential of 148 isolates, comprising mesophilic and thermophilic bacteria, with particular emphasis on Bacillus licheniformis on polystyrene and stainless steel (SS) surfaces. We concluded that only four species are of significance for biofilm development on the surface of SS in the presence of skimmed milk, namely, B. licheniformis, Geobacillus stearothermophilus, Geobacillus thermoleovorans group and Anoxybacillus flavithermus. The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows: 4.8, 5.2, 4.5 and 5.3 log CFU/cm2, respectively. Number of cells recovered from biofilms on 1 cm2 SS coupons increased in the presence of tryptic soy broth (TSB) for all mesophiles including B. licheniformis, while decreased for G. stearothermophilus, G. thermoleovorans group and A. flavithermus. The crystal violet staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk. The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk.Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B. licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups. The work reported in this manuscript will broaden our knowledge on biofilm formation of a large number of dairy isolates and emphasize strain and substrate dependence.

Soybean isoflavones have been one of the potential preventive candidates for antitumor research in recent years. In this paper, we first studied the transformation of soybean isoflavones with the homogenized slurry of Ganoderma lucidum. The resultant transformed products (TSI) contained (703.21±4.35) mg/g of genistein, with transformed rates of 96.63% and 87.82% of daidzein and genistein, respectively, and TSI also could enrich the bioactive metabolites of G. lucidum. The antitumor effects of TSI on human colorectal cancer cell line HTL-9, human breast cancer cell line MCF-7, and human immortalized gastric epithelial cell line GES-1 were also studied. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay showed that TSI could dramatically reduce the viability rates of HTL-9 cells and MCF-7 cells without detectable cytotoxicity on GES-1 normal cells when the TSI concentration was lower than 100 μg/ml. With 100 μg/ml of TSI, HTL-9 cells were arrested in the G1 phase, and late-apoptosis was primarily induced, accompanied with partial early-apoptosis. TSI could induce primarily earlyapoptosis by arresting cells in the G1 phase of MCF-7 cells. For HTL-9 cells, Western-blot and reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that TSI (100 μg/ml) can up-regulate the expression of Bax, Caspase-3, Caspase-8, and cytochrome c (Cyto-c), indicating that TSI could induce cell apoptosis mainly through the mitochondrial pathway. In addition, the expression of p53 was up-regulated, while the expression of Survivin and nuclear factor κB (NF-κB) was down-regulated. All these results showed that TSI could induce apoptosis of HTL-9 cells by the regulation of multiple apoptosis-related genes.通过灵芝菌生物转化大豆异黄酮,得到富含苷元及灵芝活性成分的多因子转化产物,并研究了转化产物对结直肠癌细胞HTL-9 的体外凋亡诱导,初步探讨转化产物的抗癌活性及机理。灵芝是一种珍贵的药用真菌,大豆异黄酮的苷元物质也具有重要的药理活性,本文首次利用灵芝菌液体发酵的匀浆液生物转化大豆异黄酮,所得到的产物中大豆苷元与染料木素转化率高,同时还富集了灵芝菌的活性成分,并对转化产物的抗癌活性及机理进行了初步探讨。首先利用灵芝菌液体发酵的匀浆体系生物转化大豆异黄酮(图1)。其次,对转化产物的抗癌活性进行研究,主要包括对癌细胞存活率(图2)、细胞凋亡(图3)及细胞周期分布(图4)的影响。最后,利用蛋白质印迹(Western-blot)与逆转录聚合酶链反应(RT-PCR)技术对凋亡相关的基因和蛋白进行检测(图5 和表2),初步探讨转化产物的体外抗癌机理。本实验结果显示,转化产物中大豆苷元及染料木素的转化率分别为96.63%和87.82%,其中染料木素的含量可达(703.21±4.35) mg/g,同时转化产物中还富含了灵芝菌的活性成分。其次,对转化产物抗癌活性研究发现,其能有效降低HTL-9 细胞的存活率,可通过将细胞阻滞于G1 期而诱导细胞晚期凋亡。此外,转化产物(100 μg/ml)还可明显上调Bax、Caspase-3、Caspase-8、Cyto-c和p53 的表达量,而Survivin 和NF-κB 表达量发生明显下调。结果表明,转化产物主要通过线粒体途径诱导细胞凋亡,但同时还调控多个与凋亡相关的基因。

•98 L. sanfranciscensis isolates were obtained and genotypically characterized•10 sequence types were revealed by MLST.•The multiplex-RAPD divided the 98 L. sanfranciscensis strains into 6 types.•Geographical origin seemed not related to the genotypic diversity of L. sanfranciscensis.•The combination of MLST and multiplex-RAPD improved the genotypic characterization.Lactobacillus sanfranciscensis is the predominant lactic acid bacteria (LAB) species in Chinese traditional sourdoughs and conduces to the flavor and rheology properties of Chinese steamed bread, a staple food originated in China over 1500 years ago. The aim of this study is to describe the intraspecific diversity of 98 L. sanfranciscensis isolates from 11 Chinese traditional sourdoughs in different regions by multilocus sequence typing (MLST) and multiplex random amplified polymorphic DNA-polymerase chain reaction (multiplex RAPD-PCR). MLST scheme was reduced from six gene fragments (gdh, gyrA, mapA, nox, pgmA and pta) to five (gdh, gyrA, mapA, nox and pta) since the fragment of pgmA displayed only one allele. 10 different sequence types (STs) were revealed by MLST and 6 of them containing 79.8% of the isolates were classified into one clonal complex, demonstrating a close relationship among them. The multiplex-RAPD analysis was performed by employing the combined primers OPL-05 + RD1 and divided the 98 L. sanfranciscensis isolates into 6 types with the similarity level of 70%. According to the result, it seems that the genotypic variations of L. sanfranciscensis strains showed by MLST have no relations to geographical origin. MLST seems to have a higher discriminatory power compared with multiplex-RAPD since it produced more groups, but multiplex-RAPD could help to distinguish some strains in the same ST. Hence, an optimal genotypic characterization of L. sanfranciscensis was obtained under the combination of MLST and multiplex-RAPD analysis, targeting different genetic variations.

Aroma of Chinese steamed bread (CSB) is one of the important parameters that determines the overall quality attributes and consumer acceptance. However, the aroma profile of CSB still remains poorly understood, mainly because of relying on only a single method for aroma extraction in previous studies. Therefore, the objective of this study was to determine the volatile aroma compounds of five different samples of CSB using three different aroma extraction methods, namely solid-phase microextraction (SPME), simultaneous distillation–extraction (SDE), and purge and trap (P&T). All samples showed a unique aroma profile, which could be attributed to their unique microbial consortia. (E)-2-Nonenal and (E,E)-2,4-decadienal were the most prevalent aromatic compounds revealed by SDE, which have not been reported previously, while ethanol and acetic acid proved to be the most dominant compounds by both SPME and P&T. Our approach of combining three different aroma extraction methods provided better insights into the aroma profile of CSB, which had remained largely unknown in previous studies.通过同时蒸馏萃取(SDE)、顶空固相微萃取(SPME)及吹扫捕集(P&T)技术结合气相色谱-嗅闻-质谱(GC-O-MS)技术对传统酸面团制备的馒头特征风味成分进行分析,并初步探讨特征风味形成机理。首次采用不同风味物质分析技术探索传统酸面团制备的馒头关键风味成分。采用同时SDE、SPME 及P&T 方法,对不同传统酸面团制备的馒头特征风味物质进行分析。SDE 结果显示,反式-2-壬烯醛和反式-2,4-癸二烯醛为传统酸面团馒头中关键风味成分。SPME 和P&T 方法表明乙醇和醋酸是关键风味物质。因此,需要结合不同技术方法,全面分析传统酸面团制备的馒头中特征风味成分。

•Two novel ACE-inhibitory peptides (VLSRYP and LRFF) were isolated and identified from the yeast fermented milk.•VLSRYP and LRFF behaved as competitive ACE inhibitors with IC50 values of 36.7 and 116.9 µM, respectively.•The optimum process conditions are determined for the maximum production of ACE-inhibitory peptides.•Response surface methodology was used to achieve the optimal process conditions.•Rotation speed is found to be the most significant factor influencing the ACE inhibition.Angiotensin-converting enzyme (ACE) inhibitory peptides from milk fermented with the yeast Kluyveromyces marxianus Z17 were identified. The optimum process conditions for the production of bioactive peptides were also established. Two novel peptides exhibiting ACE-inhibitory activity were identified from the fermented milk using Sephadex G-15 gel filtration, reversed phase-high performance liquid chromatography and MALDI/TOF-TOF MS/MS. The sequences of the two novel peptides were VLSRYP and LRFF with IC50 values of 36.7 and 116.9 µM, respectively. Lineweaver–Burk plots revealed that both peptides behaved as competitive ACE inhibitors. Response surface methodology was used to determine the optimum process conditions for the production of ACE-inhibitory peptides. The results showed that the most significant factors affecting ACE inhibition were fermentation temperature, inoculum level and rotation speed. The maximum ACE-inhibitory activity (81.23%) was observed at temperature 32 °C, initial pH 6.5, inoculum level 6% and rotation speed of 189 rpm. The peptide content and peptidase activity (carboxypeptidase and aminopeptidase) had a significant positive effect on ACE inhibition, while endoproteinase activity showed an insignificant effect. The results of this study may contribute to the development of a functional beverage with antihypertensive effects.

As the main bioactive metabolites of Ganoderma lucidum, triterpenoids have various pharmacological effects. In this paper, the nutritional requirements and culture conditions of a submerged culture of G. lucidum were optimized using the response surface methodology; maximum mycelia biomass and intracellular triterpenoid production reached 1.87 g/100 ml and 93.21 mg/100 ml, respectively, for a culture consisting of wort 4.10% (0.041 g/ml) and yeast extract 1.89% (0.0189 g/ml), pH 5.40. For the first time, we established that wort, which is cheap and abundant, can replace the more commonly used glucose as the sole source of carbohydrate. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), 10 major ganoderic acids were tentatively identified based on the predominant fragmentation pathways with the elimination of H2O and CO2, as well as cleavage of the D-ring.通过优化液体发酵培养基提高灵芝三萜产量,并对其三萜成分进行初步鉴定。麦芽作为啤酒生产原料,含有丰富的糖类及其他营养物质,并且价格低廉,来源广泛。本文首次将麦芽汁作为碳源物质以提高灵芝三萜产量,并对其所含灵芝酸成分进行初步鉴定。以灵芝菌丝生物量和胞内三萜产量为检测指标,对其液体发酵培养基中的碳(C)源和氮(N)源进行筛选,并利用单因素实验确定其相应浓度,再经响应面法优化液体发酵培养基(C 源、N 源、pH)(表1)。另一方面,酸碱转化法纯化灵芝三萜提取物后,利用高效液相色谱-电喷雾-质谱(HPLC-ESI-MS)法初步鉴定灵芝所含灵芝酸成分(表2)。本实验响应面结果显示:当培养基含麦芽汁4.1%,酵母浸出粉1.89%, pH 为5.40 时,灵芝液体发酵菌丝生物量及胞内三萜产量均达到最优,分别为1.87 g/100 ml 和93.21 mg/100 ml。经HPLCESI-MS 鉴定,初步确定其含有10 种灵芝酸成分。

Bacterial diversity of Tibetan kefir grains from different areas in China was investigated by culture-independent metagenomic methods, and comprehensive taxonomic profiling was done. Eleven genera and their proportions were found in all the 4 samples, and they were Lactococcus (40.93–72.02 %), Lactobacillus (10.50–47.89 %), Acetobacter (4.50–14.15 %), Shewanella (1.73–7.58 %), Leuconostoc (0.64–0.84 %), Pseudomonas (0.07–0.63 %), Streptococcus (0.02–0.22 %), Acinetobacter (0.05–0.12 %), Pelomonas (0–0.01 %), Dysgonomonas (0–0.01 %), and Weissella (0–0.01 %). Shewanella, Acinetobacter, Pelomonas, Dysgonomonas, Weissella, and Pseudomonas were the first time found in Tibetan kefir grains. α-Diversity analysis showed that they varied in bacterial richness and diversity while β-diversity showed that the variation of genera composition among sampling sites was small. Principal component analysis showed that Lactobacillus, Lactococcus, and Acetobacter were the main genera which caused the diversity. This study provided theoretical basis for further investigation and application of Tibetan kefir grains.

Biotransformation of phytosterol (PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper. The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology. Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M. neoaurum ZJUVN-08 strain. Results showed that molar ratio of hydroxypropyl-β-cyclodextrin (HP-β-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production. By analyzing the statistical model of three-dimensional surface plot, the optimal process conditions were observed at 0.1 g/L inducer, pH 7.0, molar ratio of HP-β-CD to PS 1.92:1, 8.98 g/L PS, and at 120 h of incubation time. Under these conditions, the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized (5.99 g/L), while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized (84.03%). The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results. It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process.

α-Galactosidase had applied in food and feed industries for hydrolyzing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to purify the α-galactosidase of Aspergillus foetidus ZU-G1 and compared the biochemical and hydrolytic properties of three major α-galactosidase forms (α-gal I, α-gal II and α-gal III). The molecular mass of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 106.3, 49.7 and 109.9 kDa, respectively. Its optimum reaction temperature was 60 °C and stable below 50 °C. The optimum pH of α-gal I and α-gal III was 5.0 and α-gal II was 4.0. Under 28 °C conditions for 24 h, α-gal I was stable at pH 4.0, α-gal II was stable at pH 6.0, and α-gal III was pH 5.0. α-Galactosidase was completely inhibited by Ag+. CuSO4·5H2O and SDS were powerful inhibitors of α-gal I and α-gal III but had little effect to α-gal II. EDTA did not strongly affect α-gal I and α-gal III, while strongly affect α-gal II. CaCl2·2H2O, MgSO4·7H2O and MnSO4·7H2O were activation for α-gal I, α-gal II and α-gal III. No significant inhibition of enzymes activity was observed in the presence of raffinose, lactose as well as other sugars tested. Synthetic substrate p-nitrophenyl-α-d-galactopyranoside was not preferentially hydrolyzed than natural substrates, such as melibiose, stachyose and raffinose. Under 40 and 50 °C incubation for 1–5 h, the stachyose of soybean milk was degraded by α-gal I, α-gal II and α-gal III and strongly hydrolyzed by α-gal II, and the raffinose of soybean milk was completely hydrolyzed by α-gal II and weakly hydrolyzed by α-gal I and α-gal III. The distinct hydrolytic and biochemical properties of α-gal I, α-gal II and α-gal III further signify the α-galactosidase of A. foetidus ZU-G1 was propitious to soybean milk and related food industry.

Naturally fermented pickles harbour many lactic acid bacteria (LAB). Forty-three LAB strains with conjugated linoleic acid (CLA)-producing ability were isolated from three naturally fermented pickle brines. Of these isolates, lp15 identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis exhibited the highest CLA-producing ability (26.1% conversion) at 48 h in de Man Rogosa Sharpe (MRS) broth in the presence of 100 μg/ml of linoleic acid (LA). Compared to other strains, L. plantarum strain lp15 showed the highest tolerance upon increased levels of LA in the medium, i.e., up to 600 μg/ml. This strain converted about 25% of LA into CLA isomers [predominantly cis-9, trans-11 CLA (9-CLA) and trans-10, cis-12 CLA (10-CLA)], of which 75% was 9-CLA. Interestingly, though the conversion rate of LA into CLA by lp15 remained stable between 100 to 600 μg/ml LA levels in the medium, it dropped sharply at 1000 μg/ml. Taken together, the lp15 strain displayed relatively high LA tolerance with higher conversion rate, which implies that this strain is a valuable candidate for enhancing the CLA content in food-sources like pickles.

Beer barley LTP1 in beer is an important component of beer foam, and it participates in the formation of beer foam. The digestion of beer barley LTP1 by proteinase A from brewing yeast leads to the decline of beer foam stability, especially for the unpasteurized beer. The objective of this study was to construct an industrial brewing yeast strain to secrete recombinant barley LTP1 into fermenting wort during beer fermentation for the foam stability improvement. We constructed barley LTP1 expression cassette and transformed into the host industrial yeast cells to replace partial PEP4 alleles using homologous recombination method. The expression of b-LTP1 was under control of the constitutive yeast ADH1 promoter, and the concentration of recombinant barley LTP1 secreted by recombinants reached 26.23 mg/L after incubation in YEPD medium for 120 h. The PrA activity of the recombinant strain declined compared with the host strain. The head retention of beer brewed with the recombinant industrial strain (326 ± 12 s) was improved when the host strain WZ65 (238 ± 7 s) and the constructed strain S.c-P-1 (273 ± 10 s) with partial PEP4 gene deficiency were used as control. The present study may provide reference for brewing industries and researches on beer foam stability.

A rapid and selective HPLC method was developed for the simultaneous analysis of acetoin (ACT) and tetramethylpyrazine (TMP). The chromatography was performed on a Zorbax SB-C18 column at 45 °C, with an aqueous mobile phase (1% acetic acid and 0.05% trifluoroacetic acid in water, pH 2.5)–(methanol) (45:55, v/v). The flow rate was 0.8 mL/min and UV detection wavelength was 297 nm. This method permits the simultaneous determination of ACT and TMP in fermentative foods with detection limits of 5.625 and 0.033 μg/mL, respectively. The recovery was 96.03% for ACT and 92.06% for TMP. Correlation coefficients were greater than 0.9999 for the two compounds. The linearity ranges for ACT and TMP were in the range of 0.02–20 mg/L and 0.12–80 μg/mL, respectively. The proposed method could be used for routine quality control of foods, beverages, natural products, or pharmaceuticals. Current data suggest that the content of TMP in vinegars is positively correlated with that of ACT.

The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

Potato starch was esterified with octenyl succinic anhydride (OSA) in aqueous slurry systems and the major factors affecting the esterification were systematically investigated. The physico-chemical properties of the products were determined by means of Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction and a viscosity analyser (VA). The results indicated that suitable parameters for the preparation of OSA starch from potato in aqueous slurry systems were as follows: concentration of starch slurry, 35% (in proportion to water, w/w), reaction period, 3 h, pH of reaction system, 8.0, reaction temperature, 35 °C, amount of OSA, 3% (in proportion to starch, w/w), OSA dilution-fold, 5. The degree of substitution (DS) was 0.017 and the reaction efficiency (RE) was 72 ± 1.8%. FT-IR spectroscopy showed characteristic absorption of the ester carbonyl groups in the OSA starch at 1724 cm−1. SEM and X-ray diffraction revealed that OSA groups acted by first attacking the surface and some pores formed, but OSA modification caused little change in the crystalline pattern of potato starch (DS 0.045). Apparent pastiness measurement indicated that the starch derivatives gelatinized within a shorter time to achieve higher viscosities over the range of designed concentration of OSA potato starch.

Hypocrellin production using submerged cultivation of the medicinal fungus Shiraia bambusicola revealed that both glucose and (NH4)2SO4 were optimal carbon and nitrogen sources. Hypocrellin production increased with increasing initial glucose concentration within the range of 10–50 g l−1 and (NH4)2SO4 concentration in the range of 1–2 g l−1. The effects of carbon and nitrogen concentration were optimized using central composite experimental design and response surface analysis; maximum hypocrellin production (196.94 ± 6.93 mg l−1) was achieved using 45.7 g l−1 glucose and 1.93 g l−1 (NH4)2SO4.

We investigated the higher structure of konjac glucomannan (KGM) in the amorphous state and solution using a laser particle size analyzer and a water activity meter. The results show that the thermodynamic structures of native KGM were primarily composed of the lamella structure units, which involve both granular crystalline and amorphous regions, and that the connection zones of such units contained both loose and tight aggregation regions. The value of surface tension (σ) of native KGM, resting with the density of its hydroxyl groups’ self-association, was an important parameter to analyze the higher structures of native KGM in the thermodynamic swelling model of native KGM.

Proteinase A (PrA), encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant (36 h) and PrA-negative mutant (48 h) was significantly extended, compared with the wild type strain (24 h) (P<0.05), but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking cultivation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption of S. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 °C the reduced cell viability rate (RCVR) was 10% for the wild type S. cerevisiae and 90% for both mutant strains (P<0.01), suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 °C to 70 °C, the wild type S. cerevisiae had significantly lower relative glucose consumption rate (RGCR) (61.0% and 80.0%) than the partial mutant (78.0% and 98.5%) and the complete mutant (80.0% and 98.0%) (P<0.05), suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1s), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Optimization of a process for extracting astaxanthin from Phaffia rhodozyma by acidic method was investigated, regarding several extraction factors such as acids, organic solvents, temperature and time. Fractional factorial design, central composite design and response surface methodology were used to derive a statistically optimal model, which corresponded to the following optimal condition: concentration of lactic acid at 5.55 mol/L, ratio of ethanol to yeast dry weight at 20.25 ml/g, temperature for cell-disruption at 30 °C, and extraction time for 3 min. Under this condition, astaxanthin and the total carotenoids could be extracted in amounts of 1294.7 μg/g and 1516.0 μg/g, respectively. This acidic method has advantages such as high extraction efficiency, low chemical toxicity and no special requirement of instruments. Therefore, it might be a more feasible and practical method for industrial practice.

Chemical feature based pharmacophore models were generated for an angiotensin converting enzyme (ACE) inhibitory peptide using the Discovery Studio 2.0 pharmacophore modeling approach. The pharmacophore hypothesis selected has five features (one negative ionizable region, one hydrogen bond donor, one hydrogen bond acceptor and two hydrophobic functional groups). Additionally, ACE inhibitory hexapeptide previously obtained from silkworm pupae protein was optimized to target the ACE based on the selected pharmacophore. The results suggest that tri-peptide (thr-val-phe) may be structural determinant of ACE activity. Docking studies further provided confidence for the validity of the selected pharmacophore model to perform structure optimization of the ACE inhibitory peptide.

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cultures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.

Fermentation of Phaffia rhodozyma is a major method for producing astaxanthin, an important pigment with industrial and pharmaceutical application. To improve astaxanthin productivity, single factor and mixture design experiments were used to investigate the effects of nitrogen source on Phaffia rhodozyma cultivation and astaxanthin production. Results of single factor experiments showed nitrogen source could significantly affect P. rhodozyma cultivation with respect to carbon source utilization, yeast growth and astaxanthin accumulation. Further studies of mixture design experiments using (NH4)2SO4, KNO3 and beef extract as nitrogen sources indicated that the proportion of three nitrogen sources was very important to astaxanthin production. Validation experiments showed that the optimal nitrogen source was composed of 0.28 g/L (NH4)2SO4, 0.49 g/L KNO3 and 1.19 g/L beef extract. The kinetic characteristics of batch cultivation were investigated in a 5-L pH-stat fermentor. The maximum amount of biomass and highest astaxanthin yield in terms of volume and in terms of biomass were 7.71 mg/L and 1.00 mg/g, respectively.

The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 °C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0∼6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.

In this study, the anti-proliferative effect of cell-free fraction of Tibetan kefir on human gastric cancer cell line SGC7901 in vitro was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a dose-dependent antiproliferative effect, and when the concentration of Tibetan kefir cell-free fraction was more than 8 mg mL−1, a distinct anti-proliferation of SGC7901 cells was observed. Flow cytometric analysis was carried out to detect cell cycle arrest and apoptosis. SGC7901 cells treated with a cell-free fraction of Tibetan kefir were inhibited in the G1/S phase, and both early and late apoptosis could be detected. To further investigate the mechanism of action of cell-free fraction of Tibetan kefir on apoptosis of SGC7901 cells, expression of human bcl-2 and bax genes were measured by qPCR. Apoptosis induced by Tibetan kefir was effected through up-regulation of bax and down-regulation of bcl-2.

Tibetan kefir grains are naturally-existing yogurt starter and have some unique benefits. In this study, response surface analysis was used to optimize their biomass production. Results showed that optimal culture conditions were skim milk concentration, 41.6%; temperature 30.05°C; inoculation amount, 1.86%; time, 20 h and shaker rotating speed, 0r/min, at which the growth rate was 14.33%, 39.4% more than initial. In addition skim milk concentration, temperature and inoculation amount were proved to be significant factors on biomass production.

Aspergillus niger as a cell factory could produce multi-hydratases, and pH level has a significant effect on enzymatic activity. In this study, the activities and stabilities of the multi-hydratases produced by Aspergillus niger zju-Y1 were evaluated under the various pH levels of digestive process in poultry, respectively. The results show that the high activities and stabilities of the multi-hydratases under the simulated pH levels of poultry digestive process endue it the satisfactory characteristics as feedstuff additives of high quality, and the strain Aspergillus niger zju-Y1 could be used as a good producer of enzymes applied in feedstuff.