不同碳氮组合对三角褐指藻兼养生长及脂质积累的影响

为了探讨兼养培养中不同碳氮组合对三角褐指藻(P.tricornutum)生长及脂质积累的影响,以葡萄糖为碳源,氯化铵为氮源,以不同的碳氮组合兼养培养P.tricornutum,用干重法测定P.tricornutum的生物量,三氯甲烷-甲醇法提取脂质,并采用GC分析脂肪酸组成。结果表明,兼养生长中P.tricornutum基于氯化铵消耗的细胞得率系数随着葡萄糖质量浓度的增加而降低,降低了对氮源的需求;在碳氮组合为葡萄糖质量浓度0.5 g/L,氯化铵浓度1.0 mmol/L的条件下,P.tricornutum生物量达最高,为1.25 g/L,脂质含量可达44.6%,不同碳氮组合对P.tricornutum脂质多不饱和脂肪酸(PUFA)和二十碳五烯酸(EPA)含量的影响不显著。     

变形假单胞菌吡咯喹啉醌合成基因簇的克隆与分析

从变形假单胞菌JUIM01中克隆到吡咯喹啉醌(PQQ)合成基因簇,阐明了其基因组成和生物学信息。根据已报道的假单胞菌的基因组进行简并引物设计,采用LA-PCR技术克隆变形假单胞菌的PQQ合成基因簇,对克隆的基因片段进行测序并使用生物信息学方法进行综合分析。结果表明:克隆到的基因片段全长为11 659 bp,其中包括pqqF、pqqA、pqqB、pqqC、pqqD、pqqE、pqqM、pqqH和pqqI共9个基因,编码PQQ生物合成的前体短肽PqqA和合成途径的相关酶;这些基因与荧光假单胞菌Pf0-1的PQQ合成基因簇的基因组成类似,相应基因的序列一致性达41%～94%。本研究中首次从变形假单胞菌中克隆到PQQ合成基因簇,并对其进行生物信息学分析,为变形假单胞菌的PQQ生物合成途径和胞内再生机制的研究奠定了基础,进而为提高2KGA的生产强度提供了理论支撑。     

Effect of growth rate reduction and genetic modifications on acetate accumulation and biomass yields in Escherichia coli

Although acetate biosynthesis in Escherichia coli provides an important intermediary for ATP synthesis, its accumulation inhibits both cell growth and protein production. Since pyruvate provides the largest flux to acetate and is central to the problem of acetate production, acetate accumulation could be reduced or abolished if the pyruvate pool for the TCA cycle was reduced. To examine this possibility, various pyruvate kinase (pyk) and phosphotransferase system (pts) mutants were tested for acetate production in batch cultures with glucose as the only carbon source. The pykA pykF mutant exhibited significant reductions in the specific growth rate and acetate production compared with the wild-type strain. Interestingly, in the case of pts and pts pyk mutants in which increased biomass yields were observed in comparison with the wild-type strain, no acetate production was detected. Therefore, these mutants are potentially useful for higher production of recombinant proteins. The results from the continuous cultivation performed using the wild-type strain at various dilution rates, suggest acetate reduction as a consequence of both genetic changes and growth rate diminutions.     

Evaluation of novel lactose-positive and exopolysaccharide-producing strain of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> for fermented foods

A novel strain of lactic acid bacteria Pediococcus pentosaceus P 773 was isolated from spoiled beer and identified by means of 16S rDNA sequence analysis. The ability to assimilate lactose as a sole carbon source as a specific feature for this strain was detected and confirmed on dairy substrates. In the presence of sucrose containing substrates (sucrose, raffinose) this P. pentosaceus P 773 lactose-positive strain produced a complex of extracellular polysaccharides (Qp = 0.08 g/l/h) with a molecular mass about 2,000 kDa composed by glucose and fructose residues at a ratio 3:1, respectively. These exopolysaccharides were capable to stimulate the growth rate and biomass productivity of common constituent cultures of probiotic dairy starters (Bifidobacterium lactis, Lactobacillus acidophilus, Streptococcus thermophilus) as well as were assimilated as a sole carbon source by these strains. The present study confirmed the presence of lactose-positive and exopolysaccharide-producing strain of P. pentosaceus in natural environment which could be used as a starter culture to impart more functional attributes to fermented food.     

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.     

辣木籽油食用安全性毒理学评价

以SD大鼠和昆明系小鼠为试验动物进行急性毒性、遗传毒性和30 d喂养试验,研究辣木籽油的食用安全性。结果表明:急性毒性试验未发现动物有明显中毒症状与死亡;遗传毒性试验结果均为阴性;30 d喂养试验发现当喂食含有6%、12%辣木籽油饲料的雌、雄大鼠的血糖水平显著降低,喂食含有12%辣木籽油饲料的雄性大鼠的总蛋白水平显著降低,其他各项血液生化指标均无明显变化。因此,辣木籽油是安全无毒的可食用油脂。     

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.     

Reduction of soybean oligosaccharides and properties of alpha-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected] JK55

This study was undertaken to investigate the potential for reducing non-digestive oligosaccharides (NDO) in soy foods, as well as the influence of exogenous conditions on intracellular α-galactosidase (α-Gal) producing lactic acid bacteria. Two strains, Lactobacillus curvatus R08 and Leuconostoc mesenteriodes JK55, showed the highest levels of raffinose degrading activity at over 40 U mL⁻¹, and presented maximum activities during the stationary phase in a medium where raffinose was the only carbon source. Raffinose was the most effective inducer, followed by melibiose, and galactose; the enzymes were partially inhibited by fructose and sucrose. On the other hand, limited activity was observed in glucose. The strains displayed optimum activity levels at neutral pH and a 35–37 °C temperature range. The α-Gal activities of L. curvatus R08 and Leu. mesenteriodes JK55 were maintained at pH 6.5–10.0. The activity of the α-Gal enzyme was stable in a relatively broad range of temperatures from 0 to 40 °C for 3 h. In soymilk, Leu. mesenteriodes JK55 and L. curvatus R08 completely hydrolyzed the NDO after 18–24 h of fermentation. The abilities of L. curvatus R08 and Leu. mesenteriodes JK55 to degrade raffinose sugars and, particularly, to produce organic acids from sugar, could contribute to reductions in the anti-nutritional properties of soy, and to the accumulation of compounds with beneficial properties during food processing. Furthermore, this study provides the optimum conditions to induce α-Gal from these strains.     

利用CRISPR-Cas9和Cre/loxP系统删除34个非必需基因构建L-苏氨酸生产菌

L-苏氨酸是一种被广泛应用于食品、饲料及医药等领域的必需氨基酸。大肠杆菌以葡萄糖为碳源合成L-苏氨酸的过程中,一些非必需基因的转录和翻译会消耗碳源。利用CRISPR-Cas9和位点特异性重组系统Cre/loxP,敲除了大肠杆菌MG1655基因组中puuE和ynaI区间的34个非必需基因（共30.372 kb）,获得了突变菌MG003,然后通过高表达L-苏氨酸合成途径中关键基因thrA*、thrB和thrC以及L-苏氨酸转运酶编码基因rhtA和rhtC提高L-苏氨酸产量。与对照菌MG1655/pFW01-thrA*BC相比,MG003/pFW01-thrA*BC生长加快,L-苏氨酸产量提高25.5%;MG003/pFW01-thrA*BC-rhtA的L-苏氨酸产量提高了43.3%;MG003/pFW01-thrA*BC-rhtC的L-苏氨酸产量提高了74.5%。研究结果表明,大肠杆菌基因组中34个非必需基因的删除有利于其提高L-苏氨酸合成能力。     

Effects of different food commodities on larval development and α-amylase activity of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

The present work was undertaken to study the influence of four commodities (wheat flour, dates, sorghum and barley) on Plodia interpunctella post-embryonic development. Larval weight, larval mortality, pupation and adult emergence were recorded. The study also aimed to find out the effect of these commodities on protein and glycogen production as well as on α-amylase activity. Results indicated that the weight of fourth instar larvae placed on dates increased gradually. Percentage mortality was low. Pupation and adult emergence were delayed. In contrast, the weight of larvae placed on wheat flour, sorghum or barley remained low. Pupation and adult emergence occurred sooner than among those placed on dates and the percentage mortality was highest for larvae placed on barley. Results also showed that protein content and α-amylase activity were lower for larvae placed on dates than for those placed on other commodities. The biochemical composition of different commodities showed that dates are a rich source of glucose, while their protein and starch contents were very low as compared to the other commodities. In contrast, wheat flour, sorghum and barley contained large amounts of starch and protein and low amounts of glucose. Thus, the reduction in α-amylase activity was probably due to the high levels of glucose in dates.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.     

鼠李糖乳杆菌利用不同益生元的增殖及耐受性研究

作者旨在通过对比鼠李糖乳杆菌（Lactiplantibacillus rhamnosus）与不同益生元组合后,在生长情况、pH和胆盐耐受性及乳酸产量方面的变化筛选出鼠李糖乳杆菌与益生元的最佳组合。结果表明,鼠李糖乳杆菌GG（L. rhamnosus GG, LGG）、鼠李糖乳杆菌HN001在以低聚半乳糖为唯一碳源生长时,最大生物量分别为1.253、1.552,最大比生长速率分别为0.316、0.290 h^-1,乳酸产量分别为3.654、10.914 g/L,与其他益生元组合相比,表现出显著优势。为验证这两种组合在不同胁迫条件下的生长情况,进一步测定了其在不同pH和胆盐质量浓度下的耐受性。这两种组合在1 g/L胆盐条件下的最大生物量分别为0.712和0.694,在pH 4.0条件下的最大生物量分别为0.639和0.728,与其他组合相比存在极显著差异。该研究结果有助于推动鼠李糖乳杆菌与低聚半乳糖组合在食品、医疗等领域的广泛应用,为后续益生菌-益生元组合产品的开发提供了理论依据。     

Comparison of antimicrobial resistance in Salmonella and Campylobacter isolated from turkeys in the Midwest USA

The current study was carried out to determine the antimicrobial resistance profiles and evaluate some molecular characteristics of a set of Salmonella and Campylobacter isolates recovered from production line turkeys in the Midwest region of the United States. A total of 94 birds identified as being positive for both Salmonella and Campylobacter spp. were selected for study. All Salmonella isolates were examined for antimicrobial resistance using the methods employed in the National Antimicrobial Resistance Monitoring System (NARMS). Campylobacter isolates were subjected to similar analysis using the Etest^®. In addition, polymerase chain reaction (PCR) was carried out to determine the presence of the antimicrobial resistance associated genes, integrase (int1), class 1 integrons (Salmonella and Campylobacter) and a multidrug efflux pump (Campylobacter spp.). Results from the study showed that the Salmonella and Campylobacter isolates examined displayed resistance to a number of antimicrobials, with Salmonella and Campylobacter isolates being resistant to at least three antimicrobials while some isolates showed resistances to 6 or 8 different antimicrobials. In addition, 68.1% of the Salmonella isolates tested were found to be positive for the class I integrase gene (int1), 28.7% possessed a 1000 bp gene cassette and 17% possessed an 800 bp gene cassette. All Campylobacter isolates were negative for int1, but 36.2% tested positive for the Campylobacter multidrug efflux pump (CmeB). A considerable number of Salmonella and Campylobacter isolates tested displayed varying degrees of antimicrobial resistance as well as the presence of some factors associated with the carriage and persistence of antimicrobial resistance. Similarities in the types of antimicrobial resistance observed in Campylobacter and Salmonella strains was evident. The results of this study suggest that prescribing practice at the farm level may be a factor in promoting antimicrobial resistance in more than one species of organism. Such practices may, therefore, contribute to the potential health risk for consumers should micro-organisms carrying multiple antimicrobial resistances enter the food chain. This study may be one of the first to report on the incidence of the multidrug efflux pump (CmeB) in Campylobacters recovered from processed turkeys. The antimicrobial resistance and molecular characteristics of Salmonella and Campylobacter is discussed.     

Effects of addition of anka rice on the qualities of low-nitrite Chinese sausages

Anka rice (AR), previously inoculated with Monascus purpureus, was added during manufacturing of low-nitrite Chinese sausages. Chemical compositions and water activities of sausages were not affected. “L”, “a”, and “b” values of sausages with less nitrite (25 ppm) and 0.5% AR added were not significantly different from those with more nitrite (100 ppm) added. Colours of the sausages without AR were light red whereas those with AR added were darker red. Addition of AR did not inhibit lipid oxidation. Higher VBN (volatile basic nitrogen) values of the samples with AR added were observed. With addition of AR, the nitrite degrading rate was retarded. Microbial counts of the sausages with AR added were significantly higher than those of the controls (100 ppm nitrite). The low-nitrite Chinese sausage with addition up to 1.5% AR was acceptable when stored at 4 °C for 28 days.     

Cholesterol lowering activity of mango ginger (<Emphasis Type="Italic">Curcuma amada</Emphasis> Roxb.) in induced hypercholesterolemic rats

Mango ginger (Curcuma amada Roxb.) is a spice commonly consumed in Asian countries. Health beneficial hypotriglyceridemic property of mango ginger has been reported earlier. In this investigation, the anti-hypercholesterolemic influence of dietary mango ginger was evaluated in experimental rats. Dietary mango ginger powder (10%) or its equivalent of 10 mg% curcumin-containing portion was fed along with 1% cholesterol supplemented diet in Wistar rats for 5 weeks. The treatment countered the liver and serum total and LDL + VLDL associated cholesterols and increased the HDL associated cholesterol while it had no influence on cholesterol levels in animals, maintained on normal diet. Dietary C. amada and its curcumin-free portion were effective in lowering liver cholesterol in animals, maintained on basal diet, while the curcumin-containing component of C. amada was ineffective. The biliary secretion of total lipids and bile acids was increased by dietary C. amada and both of its components. While biliary cholesterol was increased in animals fed with whole mango ginger, no such increases were noticed in groups fed with either components of mango ginger. Cholesterol absorption in ligated rat intestinal loops was not affected by mango ginger or either of its components. Thus, the present study has evidenced that the spice mango ginger possesses beneficial anti-hypercholesterolemic activity in hypercholesterolemic situation. This information is complementary to the earlier report on the health beneficial hypotriglyceridemic influence of this spice.     

A papaya-specific gene, papain, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic papayas

Genetically modified (GM) papaya lines have been approved for commercialization and are widely cultivated in many countries. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM papayas, one papaya (Fructus Caricae Carica papaya L.) species specific gene, papain, was selected and validated as suitable for using as an endogenous reference gene in transgenic papaya PCR detection. In this article, both qualitative and quantitative PCR assays for the papain gene were assayed with 11 different papaya varieties and identical amplification products were obtained with all of them. No amplified fragments could be detected when DNA samples from 18 kinds of other species were used as templates, which demonstrated that this system was specific for papaya. In real-time quantitative PCR analysis, the detection limit was as low as 10 pg of DNA. Southern blot analysis confirmed that the papain gene was two copies in the tested papaya varieties and no allelic variation was testified among these tested papaya varieties. In addition, the common used exogenous genes of the coat protein (CP) and the replicase (RP) were also assayed in qualitative and real-time quantitative PCR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Wentao Xu and Weibin Bai contributed equally to this work.     

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.     

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl- -glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H₂/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.     

Molecular biological researches of Kuro-Koji molds, their classification and safety

To assess the position of Kuro-Koji molds in black Aspergillus, we performed sequence analysis of approximately 2500 nucleotides of partial gene fragments, such as histone 3, on a total of 57 Aspergillus strains, including Aspergillus kawachii NBRC 4308, 12 Kuro-Koji molds isolated from awamori breweries in Japan, Aspergillus niger ATCC 1015, and A. tubingensis ATCC10550. Sequence results showed that all black Aspergillus strains could be classified into 3 types, type N which includes A. niger ATCC 1015, type T which includes A. tubingensis ATCC 10550, and type L which includes A. kawachii NBRC 4308. Phylogenetic analysis showed these three types belong to different clusters. All 12 Kuro-Koji molds isolated from awamori breweries were classified as type L, thus we concluded type L represents the industrial Kuro-Koji molds. We found all type L strains lack the An15g07920 gene which is required for ochratoxin A biosynthesis in black Aspergillus. This sequence is present in the genome of A. niger CBS 513.88 and has homology to the polyketide synthase fragment of A. ochraceus which is involved in ochratoxin A biosynthesis. Based on the industrial importance and the safety of Kuro-Koji molds, we propose to classify the type L strains as Aspergillus luchuensis, as initially reported by Dr. Inui.