拟南芥籽油超声波辅助提取工艺的响应面法优化

利用响应面法对拟南芥籽油超声波辅助提取工艺进行优化。在单因素试验基础上采用中心组合（Box-Behnken）试验设计方法,研究提取时间、超声功率、提取温度及其交互作用对拟南芥籽油提取率的影响。试验确定的超声波辅助提取拟南芥籽油的最佳条件为：料液比1∶6,提取时间55 min,超声功率400 W,提取温度55℃。在最佳条件下,拟南芥籽油提取率为38.01%。     

一起米酵菌酸食物中毒致死事件的调查

目的 调查米酵菌酸食物中毒发生的原因,为预防和控制由此引发的食物中毒提供科学依据。方法 通过实验室快速检测确定致病因子,根据致病因子发病潜伏期推断可疑餐次及食物,结合流行病学调查、食品/环境卫生学调查、采样及检测结果,判定中毒餐次及食物。结果 流行病学调查提示食物与发病之间存在关联,病例发病潜伏期及临床表现符合米酵菌酸食物中毒发病特点,留样食物和病例血液检出米酵菌酸。结论 中毒餐次为2020年7月19日晚餐,中毒食物为炒河粉,原因是某食品厂在生产、运输等环节存在违规操作,使河粉受到细菌污染,细菌增殖并产毒,导致此次食物中毒。     

半理性设计提高甲酸脱氢酶（CbFDH）活力及热稳定性

甲酸脱氢酶(formate dehydrogenase,FDH）是NADH循环再生的最佳酶之一,广泛应用于食品、医药和化工等行业。但是野生型甲酸脱氢酶普遍存在酶活低、催化效率差等缺点,导致产品转化率较低,影响产品的工业化生产。为了获得具有更佳催化性能的甲酸脱氢酶,作者以博伊丁假丝酵母(Candida boidinii）来源的甲酸脱氢酶为模板,利用HOTSPOT WIZARD v3.1进行三维结构模拟预测,构建了P68G、Q197K两个突变体,比酶活较野生型分别提高了11%和33%。这是由于P68G氨基酸残基侧链的苯环被氢取代,减少了甲酸盐底物进入口袋的空间位阻;而Q197K侧链酰胺基突变为胺丁基增强了酶的柔性。然而这两个突变点对甲酸脱氢酶的热稳定性产生了负面影响,因此在I239位引入半胱氨酸突变与C262构成二硫键以提高其热稳定性,最终获得一株热稳定性显著提高,比酶活较野生型提高31%、较I239C提高45%的突变株CbFDH Q197K/I239C。通过半理性预测蛋白质结构提高了甲酸脱氢酶的活力和热稳定性,为高效构建性能稳定、还原力强的甲酸脱氢酶提供了的理论基础。     

Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana

While the majority of human listeriosis cases appear to be linked to consumption of processed ready-to-eat foods (e.g., deli meats), a few listeriosis outbreaks have been linked to consumption of contaminated vegetables. In this study, we assessed four isolates representing the major Listeria monocytogenes lineages for their abilities to attach to and grow on Arabidopsis thaliana, a well-characterized plant model. When plants were dipped for 5min into 3ml of water containing 8.8logCFU of L. monocytogenes and rinsed repeatedly, L. monocytogenes was recovered from the leaves at densities from 1.52 to 2.17logCFU/cm(2). Ten days after exposure, bacterial numbers had increased over initial numbers by 2.60-2.95logCFU/cm(2). Using L. monocytogenes expressing GFP, bacteria were visualized in the intercellular spaces of A. thaliana leaves, suggesting internalization through stomata. These data indicate that L. monocytogenes can rapidly attach to and multiply on plant surfaces and colonize intercellular spaces in A. thaliana leaves where it may be protected from sanitation treatments. When A. thaliana seeds were exposed to L. monocytogenes, between 4.23 and 4.57logCFU/cm(2) were recovered from leaves 7 days post-germination, suggesting that contaminated seeds can produce contaminated plants. Overall, our study demonstrates that prevention of L. monocytogenes contamination of plants throughout growing stages is critical, consistent with recommendations for other produce-transmitted foodborne pathogens.     

单一及混合乳酸菌发酵对番木瓜汁品质的影响

目的：探讨植物乳杆菌Lactobacillus plantarum （Lp）、鼠李糖杆菌Lactobacillus rhamnosus （Lr）、嗜酸乳杆菌Lactobacillus acidophilus （La）及两两混合菌（Lp+Lr、Lp+La、Lr+La）对番木瓜汁发酵后植物化学、微生物学、风味特性和功能特性的影响,并开发出一种具有改善功能和促进健康特性的番木瓜益生菌发酵饮料。方法：通过测定发酵过程中pH值、可溶性固形物、可滴定酸、活菌数、葡萄糖、果糖、蔗糖、有机酸和挥发性化合物的变化,考察各菌发酵过程中总酚、总黄酮含量和抗α-葡萄糖苷酶、抗酪氨酸酶的能力。结果：在72 h的发酵过程中,单一菌株培养和混合菌株培养的变化相似,pH值显著降低,酸度显著提高;乳酸和琥珀酸含量均显著提高,柠檬酸和苹果酸含量显著降低。发酵后,木瓜汁中总酚类物质和总黄酮含量均有所增加。Pearson相关分析表明,酚类物质的代谢可能有助于增强α-葡萄糖苷酶和酪氨酸酶的抑制作用。所有发酵组中,由植物乳杆菌单菌发酵的番木瓜汁乳酸、总酚、总黄酮含量最高,对α-葡萄糖苷酶和酪氨酸酶的抑制作用最强。未经发酵的番木瓜原汁中主要检出6种挥发性化合物,丁酸含量最大为89.742%,其次为芳樟醇。发酵后,从6组发酵果汁中共鉴定出25种主要挥发性成分,其中丁酸含量显著降低,减少了番木瓜不愉快气味的产生。所有发酵中,Lp单菌产生的挥发性成分最多（18种）,混合菌Lp+Lr的较少（5种）。虽然混合菌Lp+Lr存在的活菌数最多[7.61 lg（CFU/mL）],但总体看来,单一菌种Lp可能更可取。结论：单菌种植物乳杆菌发酵可以改善番木瓜汁的感官特性,并增强其潜在的功能特性。     

An enzyme possessing both glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase from Antrodia camphorata

Glutathione-dependent formaldehyde dehydrogenase (GFD or GSH-FDH) plays important roles in formaldehyde detoxification and antioxidation. A gene encoding GFD from Antrodia camphorata was identified based on sequence homology. The deduced amino acid sequence of 378 amino acid residues is conserved among the reported GFDs. To characterise the Ac-GFD, the coding region was subcloned into a vector pET-20b(+) and transformed into Escherichia coli. The recombinant GFD was expressed and purified by Ni²⁺-nitrilotriacetic acid Sepharose. This purified enzyme showed a single band on a 10% SDS–PAGE. The enzyme retained 50% GFD activity after heating at 50 °C for 5 min. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) presumably to safeguard against nitrosative stress. The K_m values for S-hydroxymethylglutathione and S-nitrosoglutathione were 1.20 and 0.28 mM, respectively.     

Organization and localization of the dnaA and dnaK gene regions on the multichromosomal genome of Burkholderia multivorans ATCC 17616

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for fundamental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH-dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating sequence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE-orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the sigma32-dependent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome.     

Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica. The P. methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80-88%). In P. methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose. Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium. These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth.     

Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain

Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae consists of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. We determined the effect of SDH deficiency on the productivity of organic acids in a sake yeast strain Kyokai no. 9. The SDH activity of single disruptants was retained at 30-90% of that of the wild-type strain, but the activity disappeared in double disruptants of the SDH1 and SDH2 or SDH1b (the SDH1 homologue) genes. Two double disruptants showed no growth on a medium containing glycerol as the sole carbon source, while the single disruptants could utilize glycerol. These results indicate that double disruption of the SDH1 and SDH2 or SDH1b genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The sdh1 sdh1b disruptant showed a marked increase in succinate productivity of up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions. Under both static and sake brewing conditions, the productivity of these organic acids in the disruptants was virtually unchanged from that in the wild-type strain. Furthermore, SDH activity was undetectable in the wild-type and the disrupted strains under static conditions. These results suggest that SDH activity contributes to succinate production under shaking conditions, but not under static and sake brewing conditions.     

Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108

The relationship between quinoprotein alcohol dehydrogenase (ADH) and NAD-dependent ADH was studied by constructing quinoprotein ADH-deficient mutants. Quinoprotein ADH-deficient mutants were successfully constructed from Acetobacter pasteurianus SKU1108 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and also by adhA gene disruption with a kanamycin cassette. The NTG mutant exhibited a complete loss of its acetate-producing ability and acetic acid resistance, while the disruptant also exhibited a loss of its acetic acid resistance but retained a weak ADH activity. The immunoblot analysis of quinoprotein ADH indicated that there are no appreciable ADH subunits in the membranes of both mutant strains. The NTG mutant grew better than the wild-type strain in ethanol-containing medium, despite the absence of quinoprotein ADH. In the mutant, the activities of two NAD-dependent ADHs, present in a small amount in the wild-type strain, markedly increased in the cytoplasm when cultured in a medium containing ethanol, concomitant to the increase in the activities of the key enzymes in TCA and glyoxylate cycles. The disruptant showed a poorer growth than the wild-type strain, producing a lower amount of acetic acid in ethanol culture, and it induced one of the two NAD-dependent ADHs and some of the acetate-assimilating enzymes induced in the NTG mutant. This study clearly showed that quinoprotein ADH is extensively involved in acetic acid production, while NAD-dependent ADH only in ethanol assimilation through the TCA and glyoxylate cycles in acetic acid bacteria. The differences between the NTG mutant and the disruptant are also discussed.     

Selection and evaluation of seafood-borne psychrotrophic lactic acid bacteria as inhibitors of pathogenic and spoilage bacteria

In this study, inhibitory psychrotrophic lactic acid bacteria were isolated and investigated for future use in biopreservation of seafood products. Screening of 5575 colonies isolated from various seafood products resulted in the selection of 132 colonies presenting inhibitory properties. Among them, 52 isolates had characteristics of LAB and showed growth at 15 °C but not at 30 °C. The inhibition spectrum of these 52 isolates against 14 target strains (Gram-positive and -negative) showed inhibition of typical seafood spoiling and pathogenic bacteria and enabled the formation of seven interesting clusters. Sequencing of the 16S rRNA gene of a representative isolate from each cluster identified three Leuconostoc gelidum, two Lactococcus piscium, one Lactobacillus fuchuensis and one Carnobacterium alterfunditum. Theses strains did not produce histamine nor tyramine, and showed no particular antibiotic resistance profile. Growth rate as a function of temperature was tested for one L. piscium and one L. gelidum isolate and confirmed their psychrotrophic behavior. One out of seven isolates showed bacteriocin-like activity. The inhibition mechanisms of the other isolates are still unknown but may be due to competition for substrate. Absence of a bacteriocin-like component could be a positive point to gain rapid authorization for food application in France. This collection of LAB is now ready for testing on products.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

Characterization of Lactobacillus isolates from fermented olives and their bacteriocin gene profiles

Near one hundred isolates of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum from table olives were studied. Strains were genotyped by rep-PCR. Although the technique failed to differentiate some isolates at the species level, it proved a robust and easy procedure that could be useful for distinguishing between related strains of L. paraplantarum, L. pentosus and L. plantarum from a large pool of unrelated strains of these species. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnB, plnC, plnD, plnE/F, plnF, plnI, plnJ, plnK, plnG and plnN in most isolates of the three species. Sequences of bacteriocin genes present in L. paraplantarum and L. pentosus were homologous to L. plantarum genes. Through a discriminating analysis of the bacteriocin gene profiles, it was possible to establish a relationship between the origin of isolation and the LAB isolates, regardless of species.     

Ascospore inactivation and germination by high pressure processing is affected by ascospore age

The effects of age on high pressure resistance of the ascospores of heat resistant moulds Byssochlamys fulva, B. nivea, Neosartorya fischeri and N. spinosa were determined. Ascospores were harvested from cultures grown for 3–15 weeks at 30 °C on malt extract agar. Following filtration and determination of concentration, the ascospores were subjected to high pressure processing (HPP) at 600 MPa for 10 min in 0.1 M citrate phosphate buffer (pH 4 and 6) and mango puree (pH 5). The results supported our hypothesis that age (maturity) affects high pressure resistance of ascospores of heat resistant moulds. A reduction of log₁₀ 2.5 cfu mL^− 1 was achieved for three week old ascospores ofB. nivea whereas for nine week old ascospores only a half log reduction was achieved. Similar results were observed for B. nivea and N. fischeri. The HPP treatment caused activation of ascospores of N. spinosa, with older ascospores showing increased activation.

Industrial relevance

The observation of activation of some ascospores by HPP, indicates that HPP alone is insufficient for elimination of these problematic spoilage microorganisms. HPP would need to be combined with other hurdles in order to produce high quality pressure-treated shelf-stable fruit products.     

抑制副溶血性弧菌的乳酸菌筛选鉴定及其生物学特性研究

目的:丰富功能性乳酸菌资源库,寻找具有副溶血性弧菌拮抗能力的优良乳酸菌出发菌株。方法:采用牛津杯抑菌试验筛选具有广谱抑菌潜力的乳酸菌菌株,通过生长代谢性能、胃肠液耐受性能、耐盐性、抗生素敏感性、抑菌谱等指标探讨其生物学特性。结果:以副溶血性弧菌为指示菌,筛选得到6株乳酸菌,经形态学、生理生化、分子生物学鉴定,分别归类于类干酪乳酪杆菌、发酵黏液乳杆菌和植物乳植物杆菌。其中,类干酪乳酪杆菌A1抑菌活性最佳,24 h内菌落总数超过1×10⁹ CFU/mL,发酵液pH值稳定在4.1左右,经人工模拟胃液处理2 h后,存活率为54.61%,再经人工模拟肠液处理8 h后,存活率仍可达45.46%,经10%NaCl胁迫处理24 h后,活菌总数>1×10⁵ CFU/mL。同时,类干酪乳酪杆菌A1细菌素粗提物对13种致病菌呈良好抑菌活性,具有广谱抑菌潜力,且对8种常见抗生素未见耐药性。结论:筛选得到了能够抑制副溶血性弧菌且生物学特性优良的类干酪乳酪杆菌A1。