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1.
Halbmayr E Mathiesen G Nguyen TH Maischberger T Peterbauer CK Eijsink VG Haltrich D 《Journal of agricultural and food chemistry》2008,56(12):4710-4719
This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( S?rvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry. 相似文献
2.
Nguyen TT Nguyen HA Arreola SL Mlynek G Djinović-Carugo K Mathiesen G Nguyen TH Haltrich D 《Journal of agricultural and food chemistry》2012,60(7):1713-1721
The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ~170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS. 相似文献
3.
Landete JM Rodríguez H de Las Rivas B Muñoz R 《Journal of agricultural and food chemistry》2008,56(12):4497-4503
Aroma is an important sensory parameter of food products. Lactic acid bacteria have enzymatic activities that could be important in the modification of food aroma. The complete genome sequence from Lactobacillus plantarum WCFS1 shows a gene (lp_3054) putatively encoding a protein with benzyl alcohol dehydrogenase activity. To confirm its enzymatic activity lp_3054 from this strain has been overexpressed and purified. Protein alignment indicated that lp_3054 is a member of the family of NAD(P)-dependent long-chain zinc-dependent alcohol dehydrogenases. In lp_3054 all of the residues involved in zinc and cofactor binding are conserved. It is also conserved the residue that determines the specificity of the dehydrogenase toward NAD (+) rather than NADP (+) and, therefore, L. plantarum benzyl alcohol dehydrogenase is less active in the presence of NADP (+) than in the presence of NAD (+). The purified enzyme exhibits optimal activity at pH 5.0 and 30 degrees C. The kinetic parameters K m and V max on benzyl alcohol as a substrate were, respectively, 0.23 mM and 204 mumol h (-1) mg (-1). Besides its activity toward benzyl alcohol, it showed activity against nerol, geraniol, phenethyl alcohol, cinnamyl alcohol, and coniferyl alcohol, all of which are volatile compounds involved in determining food aroma. The biochemical demonstration of a functional benzyl alcohol dehydrogenase activity in this lactic acid bacteria species should be considered when the influence of bacterial metabolism in the aroma of food products is determined. 相似文献
4.
Di Cagno R De Angelis M Limitone A Minervini F Carnevali P Corsetti A Gaenzle M Ciati R Gobbetti M 《Journal of agricultural and food chemistry》2006,54(26):9873-9881
After a large screening on sourdough lactic acid bacteria, exopolysaccharide (EPS)-forming strains of Weissella cibaria, Lactobacillus plantarum, and Pediococcus pentosaceus were selected. After 6 days of incubation at 30 degrees C, the synthesis of EPS in MRS-based broth ranged from 5.54 to 7.88 mg mL-1. EPS had an apparent molecular mass of ca. 104 Da. As shown by carbohydrate consumption, the synthesis of EPS was found from sucrose only. Two types of homopolysaccharides were synthesized: glucans simultaneously with growth and fructans after 1 day of incubation. Two protein bands of ca. 180-200 kDa were in situ detected on SDS-PAGE gels incubated with sucrose. PCR products of ca. 220 bp were found for L. plantarum PL9 (100% of identity to putative priming glycosyltransferase of L. plantarum WCFS1) and W. cibaria WC4 (80% of identity to putative glycosyltransferase, epsD, of Bacillus cereus G9241) by using hybrid primers for the priming gtf genes. Degenerated primers DexreuR and DexreuV showed a unique PCR product, and the predicted amino acid sequences were identical for W. cibaria WC4 and L. plantarum PL9. The sequence had similarity with polysaccharide biosynthesis glycosyltransferases. W. cibaria WC4 or L. plantarum LP9 synthesized ca. 2.5 g kg-1 EPS during sourdough fermentation with sucrose added. Compared to the sourdough started with an EPS-negative strain, the sourdough started with W. cibaria WC4 or L. plantarum LP9 increased the viscosity, and the resulting bread had higher specific volume and lower firmness. The synthesis of EPS by selected sourdough lactic acid bacteria could be considered as a useful tool to replace the additives for improving the textural properties of baked goods. 相似文献
5.
Rodríguez H Landete JM Curiel JA de Las Rivas B Mancheño JM Muñoz R 《Journal of agricultural and food chemistry》2008,56(9):3068-3072
It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry. 相似文献
6.
为了筛选出优良亚硝酸盐和生物胺的降解植物乳杆菌,并研究在鱼肉发酵香肠中的应用。该文主要考察了19株植物乳杆菌亚硝酸盐和生物胺的降解活性及相关基因的携带情况,从中筛选出优良菌株,并研究其对鱼肉香肠发酵过程中各种亚硝胺、生物胺、腐败微生物的抑制作用和对其他理化指标的影响。试验结果表明,L.plantarum CP3对亚硝酸盐和生物胺的降解能力最强,分别达到95%和77%,PCR结果显示此菌株中含有亚硝酸盐降解(假定蛋白(hypothetical protein,HP)、谷胱甘肽还原酶(glutathione reductase,GR)、一氧化还原酶(Oxidoreductase,ORD))和生物胺降解的相关基因(multicopper oxidases,suf I),且不含生物胺生成相关基因(hdc,tdc,odc)。GC-MS测定结果显示L.plantarum CP3对发酵香肠中4种亚硝胺-二乙基亚硝胺(N-nitrosodiethylamine,NDEA),二甲基亚硝胺(N-nitrosodimethylamine,NDMA),亚硝基吗啉(N-nitrosomorpholine,NMOR)和甲基乙基亚硝胺(N-nitrosomethylamine,NMEA)的生成都具有显著的抑制作用,联合接种L.saki M4可以协同提高亚硝胺的降解能力。并且,与对照组相比,接种L.plantarum CP3+L.saki M4的鱼肉香肠,发酵48 h后腐胺、尸胺和酪胺的含量分别降低了76.83%,93.33%和88%。另外,接种乳酸菌处理组香肠中的肠杆菌、假单胞菌等腐败菌及pH值、硫代巴比妥酸(thiobarbituric acid,TBA)和挥发性盐基氮(total volatile basic nitrogen,TVB-N)的含量显著低于对照组。该研究结果体现了L.plantarum CP3作为香肠等肉制品发酵剂的良好开发应用前景。 相似文献
7.
植物乳杆菌（Lactobacillus plantarum）能够抑制黄曲霉生长,但起主要抑菌作用的物质尚未明确。该研究采用非靶向代谢组学技术比较分析了8株抑菌活性较好（S组）和8株抑菌活性较差（W组）的L. plantarum发酵上清液。结果显示,两组L. plantarum发酵上清液的代谢组存在显著差异（P<0.05）。通过数据库比对鉴定得到咪唑乙酸、酪氨酸等30个显著差异代谢物（P<0.05）,其中有机酸、脂肪酸等酸性物质较多为22个。通过与已报道的乳酸菌产生的抗真菌物质相比较,找到十八烷酸、吲哚乙酸等结构一致或结构类似物,表明上清液中酸性物质起主要的抑菌作用,且其抑菌活性依赖于低 pH 值的酸性环境。在L. plantarum产生的主要有机酸中,乳酸、乙酸、丙酸的抑菌活性良好,其抑制黄曲霉活性从大到小依次为丙酸、乙酸、乳酸。当乙酸浓度为2.64g/L、丙酸浓度为1.76 g/L时,可完全抑制浓度为106个/mL的黄曲霉孢子生长。综合表明,植物乳杆菌代谢物中有机酸和脂肪酸为主要抑菌物质,且抑菌活性随酸性物质浓度增大而增强。 相似文献
8.
为了研究在培养基中添加微量生长因子对菌株冷冻干燥存活率的影响,该研究以植物乳植杆菌LIP-1（Lactiplantibacillus plantarum LIP-1）为研究对象,探究培养基中添加尿嘧啶对菌株冷冻干燥存活率的影响及其作用机制。结果表明与未添加尿嘧啶的空白对照组相比,在培养基中添加0.05 g/L尿嘧啶能够显著提高植物乳植杆菌LIP-1的活菌数与冻干存活率（P＜0.05）。对其作用机制进行探究,发现尿嘧啶的加入能够促使菌株合成更多的尿苷二磷酸（P＜0.05）,从而使肽聚糖含量显著增加（P＜0.05）,进而提高细胞壁的稳定性;同时尿嘧啶的加入抑制了乳清酸向尿苷二磷酸的转化,使胞内的乳清酸含量显著升高（P＜0.05）,菌株利用乳清酸的抗氧化能力减轻了活性氧对细胞膜不饱和脂肪酸的氧化程度,进而减少了细胞膜的损伤程度。结果表明植物乳植杆菌LIP-1通过代谢尿嘧啶减轻了细胞壁与细胞膜受到的冻干损伤,提高了菌株的冻干存活率。研究结果为提高菌株冷冻干燥存活率提供了新的方法和思路。 相似文献
9.
以PMI基因替换植物表达载体pCAMBIA1301中的hpt基因以及pBI121中的GUS基因，构建了以PMI基因为选择标记基因的植物表达载体pCAMBIA1301PMI和pBIPMI，并导入根癌农杆菌EHA105中。研究了两种表达载体对雪柑上胚轴的转化，在培养基附加25 g•L-1甘露糖和5 g•L-1蔗糖为碳源的选择压下，pCAMBIA1301PMI的转化率为27.7％，pBIPMI的转化率为12.7％，对再生植株进行了氯酚红检测和PCR检测，建立了以PMI/甘露糖为选择系统的雪柑转基因体系。 相似文献
10.
乳酸菌胞外多糖能显著改善发酵乳制品及食品的流变学和质构特性。为进一步了解乳酸菌胞外多糖的生物合成途径及调控机制,本研究对参与植物乳杆菌C88胞外多糖生物合成基因簇的部分序列进行了克隆和鉴定。根据GenBank中己报道植物乳杆菌基因序列的保守区域设计特异性引物,扩增出植物乳杆菌C88生物合成蛋白基因（cps4A）序列,并通过染色体步移方法克隆了植物乳杆菌C88参与胞外多糖合成基因簇的部分序列（4．9kb）。利用生物信息学方法预测基因簇中6个阅读框的结构和功能,结果表明该序列与己报道的乳酸杆菌胞外多糖生物合成基因具有高度的同源性（〉96％）;对各阅读框功能预测分析发现,这6个基因主要编码参与胞外多糖合成中的多糖合成蛋白、糖链长度检测蛋白、UDP-葡萄糖-4-异构酶和糖基转移酶。本研究将为利用基因工程方法调控多糖的合成和产量提供理论依据。 相似文献
11.
水稻OsHsfA7基因RNA干扰载体的构建及遗传转化研究 总被引：1，自引：1，他引：0
为研究水稻热激转录因子基因OsHsfA7的功能及其在水稻耐热育种方面的应用价值,本文通过构建水稻OsHsfA7基因RNA干扰载体获得功能抑制变异材料,从反向遗传学进行基因的功能分析。扩增OsHsfA7c DNA3'编码区470bp片段,分别以反向和正向插入到中间载体pBSK连接的GUS片段两侧,然后将得到的RNA干扰片段克隆到改造的植物表达载体p1301M,构建以CaMV35S启动子驱动表达的水稻OsHsfA7基因RNA干扰表达载体。将该载体转入根癌农杆菌EHA105后,采用农杆菌介导法进行了水稻日本晴品种的遗传转化,获得了23株具有潮霉素抗性的转基因植株,其中12株经GUS染色呈蓝色并且DNA检测10株已插入目的片段,RNA检测其中6株OsHsfA7基因的表达水平下降甚至未检出。结果说明本研究构建的OsHsfA7基因RNA干扰载体对该基因表达沉默是有效的。 相似文献
12.
Lambert JM Weinbreck F Kleerebezem M 《Journal of agricultural and food chemistry》2008,56(18):8360-8364
The interest in efficient intestinal delivery of health-promoting substances is increasing. However, the delivery of vulnerable substances such as enzymes requires specific attention. The transit through the stomach, where the pH is very low, can be detrimental to the enzymatic activity of the protein to be delivered. Here, we describe the microencapsulation of the model enzyme bile salt hydrolase (Bsh) using whey protein-gum arabic microencapsulates for food-grade and targeted enzyme delivery in the proximal region of the small intestine. Furthermore, the efficacy of enteric coating microencapsulates for site-specific enzyme delivery was compared in vitro with living Lactobacillus plantarum WCFS1 bacteria that endogenously produce the Bsh enzyme. Microencapsulates allowed highly effective protection of the enzyme under gastric conditions. Moreover, Bsh release under intestinal conditions appeared to be very efficient, although in the presence of pancreatin, the Bsh activity decreased in time due to proteolytic degradation. In comparison, L. plantarum appeared to be capable to withstand gastric conditions as well as pancreatin challenge. Delivery using encapsulates and live bacteria each have different (dis)advantages that are discussed. In conclusion, live bacteria and food-grade microencapsulates provide alternatives for dedicated enteric delivery of specific enzymes, and the choice of enzyme to be delivered may determine which mode of delivery is most suitable. 相似文献
13.
14.
以甘薯品种栗子香的胚性悬浮细胞为受体材料, 用根癌农杆菌介导法,获得了表达bar基因的抗除草剂转基因甘薯植株。农杆菌菌株LBA4404携带含有bar基因的双元载体pCAMBIA3300。共计450个胚性细胞团用于遗传转化。在添加2 mg/L 2,4－D、100 mg/L Carb和0.5 mg/L PPT 的固体MS培养基上选择培养8周后,得到了19个PPT抗性愈伤组织。将这19个抗性愈伤组织转移到添加1 mg/L ABA、100 mg/L Carb和0.5 mg/L PPT 的固体MS 培养基上,其中的10个抗性愈伤组织共再生出103株拟转基因植株。PCR分析表明,其中的69株为转基因植株。Southern blot分析表明,bar基因稳定整合到转基因植株的基因组中。除草剂喷洒试验结果表明,转基因植株具有高度除草剂抗性。 相似文献
15.
不同发酵处理对玉米粉加工特性及淀粉粒结构的影响 总被引：1，自引：1，他引：0
以拓宽玉米粉在食品领域的应用为目标,该研究以玉米粉为原料,研究了发酵处理对其色泽、水合特性、热特性等的影响,并从淀粉结构方面进行了相应的机理分析。结果表明：植物乳杆菌、甜酒曲及两者的混合发酵剂和不同发酵条件的处理,对玉米粉的加工特性影响明显。与对照处理相比,植物乳杆菌发酵处理24 h显著降低（P<0.05）玉米粉的水结合能力、吸水性指数、水溶性指数及膨胀势,分别为9.87%、10.7%、35.2%和13.0%。先接入甜酒曲发酵12 h再接入植物乳杆菌,总发酵时间36 h可使玉米粉的亮度提高7.41%,混菌发酵玉米粉的吸水性、膨胀性比植物乳杆菌单菌种发酵有所增加。与对照处理相比,植物乳杆菌发酵玉米粉24 h使焓值降低25.8%,先接入甜酒曲发酵12 h再接入植物乳杆菌进行混菌发酵比植物乳杆菌单菌种发酵可增加原料的焓值。发酵处理后玉米粉的粒径显著减小,淀粉的官能团位置并未发生改变,但会影响分子排列的有序度和双螺旋度,电镜分析显示发酵破坏了淀粉粒结构的完整性,颗粒表面出现孔洞和裂纹。发酵处理会影响玉米粉的加工特性,其变化同淀粉的变化密切相关。应用发酵技术处理玉米粉以改变其水合特性将为更加合理地在食品领域应用玉米粉提供新的思路。从水合特性的角度看,发酵处理后的玉米粉将可更好地应用于焙烤食品、面条、搅团、玉米糊等食品中,进一步拓宽玉米粉在食品领域的用途。 相似文献
16.
Frias J Song YS Martínez-Villaluenga C González de Mejia E Vidal-Valverde C 《Journal of agricultural and food chemistry》2008,56(1):99-105
Food allergy has become a public health problem that continues to challenge both the public and the food industry. The objective of this research was the detection and quantification of the major human allergenic soy proteins and to study the reduction in immunoreactivity and improvement of amino acid content after fermentation of soybean flour. Fermentation was carried out in the solid state of cracked seeds inoculated with Aspergillus oryzae, Rhizopus oryzae, and Bacillus subtilis and in the liquid state of milled soybean flours fermented naturally by microorganisms present only in the seeds or by inoculation with Lactobacillus plantarum. ELISA and Western blot were used to quantify IgE antibody response, and HPLC was used to identify and quantify total amino acids. L. plantarum fermented soy flour showed the highest reduction in IgE immunoreactivity (96-99%) depending upon the sensitivity of the plasma used. Among the solid fermented products, the lowest reduction in immunoreactivity was obtained when mold strains, R. oryzae and A. oryzae, were used (66 and 68%, respectively, for human plasma 97.5 kUA/L). Among the solid fermented products, those inoculated with B. subtilis yielded a 81 and 86% reduction in immunoreactivity against both human plasma 97.5 IgE kUA/L and human pooled plasma samples, respectively. When soybean was subjected to liquid fermentation, most of the total amino acids increased significantly ( p < or = 0.05). In solid fermentation with R. oryzae, only Ala and Thr content improved. Fermentation can decrease soy immunoreactivity, and there is potential of developing nutritious hypoallergenic soy products. 相似文献
17.
Sánchez-Patán F Tabasco R Monagas M Requena T Peláez C Moreno-Arribas MV Bartolomé B 《Journal of agricultural and food chemistry》2012,60(29):7142-7151
Lactobacillus plantarum IFPL935 was incubated with individual monomeric flavan-3-ols and dimeric A- and B-type procyanidins to identify new metabolites and to determine the effect of compound structural features on bacterial growth and catabolism. Complex extracts rich in A-type proanthocyanidins and phenolic acids from cranberry were also tested. The results showed that L. plantarum IFPL935 exhibited higher resistance to nongalloylated monomeric flavan-3-ols, A-type dimeric procyanidins, and cranberry extract than to (-)-epicatechin-3-O-gallate and B-type dimeric procyanidins. Despite these findings, the strain was capable of rapidly degrading (-)-epicatechin-3-O-gallate, but not A- or B-type dimeric procyanidins. However, it was able to produce large changes in the phenolic profile of the cranberry extract mainly due to the catabolism of hydroxycinnamic and hydroxybenzoic acids. Of most relevance was the fact that L. plantarum IFPL935 cleaved the heterocyclic ring of monomeric flavan-3-ols, giving rise to 1-(3',4'-dihydroxyphenyl)-3-(2″,4″,6″-trihydroxyphenyl)propan-2-ol, activity exhibited by only a few human intestinal bacteria. 相似文献
18.
为了构建具有干扰效应的靶向Survivin和CDK1基因及两者串联的短发夹样RNA（short hairpin RNA,shRNA）表达载体,并研究其靶向干扰对鼻咽癌细胞CNE-2生物学行为的改变。本研究通过构建pU6-SurvivinshRNA、pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重组载体,用脂质体Lipofectamine 2000法将其转染入CNE-2细胞株中,应用RT-PCR方法初步筛选具有干扰效应的重组载体,逆转录实时荧光定量PCR（RT-qPCR）检测Survivin、CDK1 mRNA的基因表达水平,Western blot检测Survivin和CDK1蛋白表达以及噻唑蓝溴化四唑（methyl thiazolyl tetrazolium,MTT）检测癌细胞增殖活性的变化。结果表明：成功构建具有干扰效应的shRNAs表达载体;联合或单基因Survivin、CDK1的shRNAs表达载体干扰CNE-2细胞后,其对应的mRNA和目的蛋白表达均降低,CNE-2癌细胞增殖活性降低,联合基因shRNAs表达载体具有一定程度的干扰增强作用。 相似文献
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Schwekendiek A Spring O Heyerick A Pickel B Pitsch NT Peschke F de Keukeleire D Weber G 《Journal of agricultural and food chemistry》2007,55(17):7002-7009
Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering. 相似文献
20.