L-精氨酸高产菌株的亚硝基胍诱变选育和种子培养基的优化研究

为了筛选L-精氨酸的高产菌株,采用亚硝基胍对出发菌株A TCC 14067 （Brevibacterium flavum）进行诱变,结合抗精氨酸结构类似物D-精氨酸,S-甲基半胱氨酸平板抗性筛选高产菌株,并采用正交试验优化了种子培养基.结果显示,经过1 mg/mL的亚硝基胍诱变处理4min后,采用14 mg/mL的D-精氨酸抗性平板和8mg/mL的S-甲基半胱氨酸抗性平板筛选获得精氨酸高产菌株,由于解除精氨酸自身的反馈调节,L-精氨酸积累增大,产酸量达37.2 g/L,比野生菌株提高了128.2％,得到的高产菌株遗传性状稳定.通过对种子培养基进行正交试验优化,确定最终培养基配方为葡萄糖3.0％,玉米浆2.0％,硫酸铵2.0％,KH2PO4 0.10％,MgSO4·7H2O 0.05％,尿素0.15％,在此发酵条件下,L-精氨酸产量达37.8 g/L.     

L-瓜氨酸生产菌株的筛选及鉴定

通过选择性培养基初筛(利用精氨酸双水解酶筛选培养基)和复筛(高效液相色谱法测定L-瓜氨酸质量浓度),从杭州市勾庄地区西瓜大棚的土壤中筛选获得45株产L-瓜氨酸菌株.其中一株细菌(菌株编号11)转化L-精氨酸生产L-瓜氨酸的能力最强,产生的L-瓜氨酸浓度最高,0.344 g湿菌,在60 mL转化体系中转化一天,可将20 g/L的底物L-精氨酸转化为9.31g/L的L-瓜氨酸.对11号菌株分别进行形态学观察,生理生化试验及分子生物学鉴定,初步鉴定该菌株为粪肠球菌(Enterococcus faecalis).     

L-精氨酸高产菌的诱变育种及其摇瓶产酸条件

以亚硝基胍(NTG)逐级处理钝齿棒杆菌(Corynebacterium crenatum)JDN28,分别选用D-精氨酸、甲基半胱氨酸为结构类似物进行抗性选育,获得一株L-精氨酸的高产菌株YDM403(His-,SGr,D-Argr,methyl-Cysr).在含葡萄糖120 g/L的优化培养基中,摇瓶发酵96 h,L-精氨酸达28～32 g/L,较出发菌株提高了113％.     

利用木糖生产L-乳酸高产菌太空诱变育种

利用太空诱变育种技术对利用木糖产L-乳酸的野生菌株进行了诱变,通过富集、平板筛选、液体发酵筛选、遗传稳定性实验,最终得到一株正向突变菌株Lt-s.该菌株在5L发酵罐水平上,以8%的木糖为底物,于52℃发酵48h,L-乳酸产量达到70.0g/L,糖酸转化率为87.0%,L-乳酸产量较出发菌株提高约12.0%.该菌株的获得为利用木质纤维素生产L-乳酸奠定了一定基础.     

基因组改组快速提高谷氨酸棒杆菌L-鸟氨酸产量

以谷氨酸棒杆菌ATCC13032为出发菌,应用基因组改组技术快速提高L-鸟氨酸产量。经过紫外线、亚硝基胍和甲基磺酸乙酯分别诱变处理,获得6株产量有所提高的突变株,以此构建用于基因组改组的候选菌库。考察培养基成分对原生质体再生率的影响。经过两轮的灭活原生质体递推式融合,以磺胺胍和氟化钠为双抗性筛选标记,共筛选出2株遗传性能稳定的改组菌株。其中改组菌株F2-6摇瓶发酵72h,积累L-鸟氨酸产量为2.99g/L,是出发菌株的13.6倍。结果表明基因组改组技术能够在短期内使谷氨酸棒杆菌的L-鸟氨酸产量得以提高。     

钝齿棒状杆菌产赖氨酸突变株的选育

用N-甲基-N′-硝基—N—亚硝基胍(NTG)对钝齿棒状杆菌(Corynebacterium crenatum)W L1.0245进行了诱变处理,获得了高丝氨酸、蛋氨酸、亮氨酸及丙氨酸营养缺陷型和S-(2-氨基乙基)-L-半胱氨酸(AEC)抗性突变株,并经摇瓶筛选出几株能产生L-赖氨酸的钝齿棒状杆菌AEC抗性株、高丝氨酸缺陷型株和高丝氨酸缺陷型兼AEC抗性突变株。TR-275菌株(Thr~-AECr)在筛选培养基中发酵累积L-赖氨酸盐酸盐25.6mg/ml(对葡萄糖的转化率25.6%)。     

常温常压等离子体诱变选育高产L-异亮氨酸谷氨酸棒杆菌

以谷氨酸棒杆菌（Corynebacterium glutamicum）23798为原始菌株,对其进行常温常压等离子体（ARTP）诱变,以磺胺胍抗性和氨基酸与茚三酮特异显色为筛选标记,以期得到高产L-异亮氨酸的诱变谷氨酸棒杆菌,并对其遗传稳定性进行研究。结果表明,原始菌株23798经过ARTP诱变处理180 s后,经0.4 mg/mL磺胺胍抗性筛选、多孔板高通量筛选、发酵培养复筛,选育出一株高产L-异亮氨酸诱变谷氨酸棒杆菌（Corynebacterium glutamicum）B1。该菌株在摇瓶中发酵培养48 h,L-异亮氨酸产量达18.5 g/L,比原始菌株提高62.03%,且遗传性状稳定。     

谷氨酸棒杆菌ATCC 14067中L-精氨酸合成途径关键酶的过表达对L-精氨酸合成的影响

谷氨酸棒杆菌中与精氨酸合成相关的酶主要是由2个操纵子基因簇argCJBDFR和argGH编码合成。此外,在L-精氨酸合成途径的前体物谷氨酸和氨甲酰磷酸的合成中,由gdh、icd、gltA、carAB 4个基因分别编码的谷氨酸脱氢酶、异柠檬酸脱氢酶、柠檬酸合酶、氨甲酰磷酸合成酶是谷氨酸和氨甲酰磷酸合成的关键酶。研究以解除了L-精氨酸的别构抑制作用的谷氨酸棒杆菌为出发菌株,利用3种不同抗性的表达质粒分别对这些关键酶的编码基因进行串联共表达。在最终得到的13个菌株中,L-精氨酸产量最高的菌株是出发菌株L-精氨酸产量的6.23倍,这表明串联过表达菌株L-精氨酸合成途径中,不同节点关键酶的编码基因可以更有效地提高菌株中L-精氨酸的产量,为进一步改造L-精氨酸的合成途径、优化L-精氨酸的产量奠定了基础。     

亚硝基胍法诱变选育高产苯乳酸菌株

以自主分离的植物乳杆菌LY-78为出发菌株,通过亚硝基胍法诱变,以抑菌效价为检测指标,采用双层平板拮抗法和牛津杯琼脂扩散法筛选突变菌株。对筛选的突变菌株进行发酵试验,采用反相高效液相色谱法定量检测苯乳酸含量,然后根据产苯乳酸含量的多少,确定最佳菌株。试验结果表明,亚硝基胍诱变质量浓度为1.0 mg/mL、诱变时间为40 min时所获得的突变菌株NY-34具有较高的苯乳酸产量,即648 mg/L,比诱变前(246 mg/L)提高2.63倍。     

高产L-精氨酸谷氨酸棒状杆菌的构建

以产L-精氨酸诱变菌株谷氨酸棒状杆菌（Corynebacterium glutamicum）AJC为出发菌株,采用基因组编辑技术对其进行改造。 首先,敲除阻遏蛋白ArgR和FarR,解除反馈阻遏作用;然后,敲除乳酸脱氢酶编码基因ldh和整合鸟氨酸乙酰转移酶编码基因argJ,阻 断乳酸合成途径和增加前体物;最后,敲除谷氨酸分泌蛋白编码基因NCgl1221和整合乙酰谷氨酸激酶基因argB,减弱L-谷氨酸的胞 外分泌,筛选一株L-精氨酸高产菌株。 结果表明,获得一株高产L-精氨酸菌株AJC-4（C. glutamicum AJCΔargRΔfarRΔldh::PtufargJ ΔNCgl1221::PsodargB）,该菌株在5 L发酵罐中发酵64 h后,L-精氨酸产量和糖酸转化率分别为78.0 g/L和0.38 g/g,较出发菌株AJC分 别提高21.9%、18.8%;副产物乳酸和L-谷氨酸积累量分别为0.11g/L、0.16 g/L,较出发菌株AJC分别降低96.8%、96.1%。     

Effect of the shape of rice starch granules on flour characteristics and gluten‐free bread quality

The effect of three different rice varieties with different starch shapes (Seolgaeng (SG), round starch structure; Samkwang (SK), polygonal starch structure and Boramchan (BRC), polygonal starch structure) on rice flour characteristics and gluten‐free bread baking quality was investigated. Rice flours were produced by dry milling and passed through a 200 mesh sieve. Electron microscopy revealed that the structure of SG grains, with round starch granules, possessed larger void spaces than SK and BRC, composed of polygonal starch granules. For this reason, SG grain had low grain hardness and consequently, it was milled to a fine flour with low damaged starch content. The thermo‐mechanical properties were determined by Mixolab, which revealed that SG was gelatinised rapidly and maintained high viscosity after gelatinisation. These characteristics gave SG flour the ability to build up bread structure without gluten. Specific volume and crumb hardness of gluten‐free rice breads made of SG, SK and BRC flours were 3.37, 3.11 and 2.12 mL g^?1 and 2.61, 2.76 and 6.46 N, respectively. The SG flour with round starch structure is appropriate for making gluten‐free rice breads.     

山楂枸杞保健酸奶的功能性研究

目的:探索“山楂枸杞保健酸奶”(简称“SG酸奶”)对大、小鼠降血脂和免疫调节的功能。方法:将大、小鼠分别建立动物病理模型组、SG酸奶组和普通酸奶组,测定其各组动物的血脂(TC、TG、LDL-C、HDL-C)、巨噬细胞吞噬功能、胸腺、脾指数及溶血素(HC50)等指标,观察其相应的功效。结果:SG酸奶组与动物病理模型组和普通酸奶组比较,具有明显的降血脂和免疫调节的功能(p≤0.05～0.001)。结论:SG酸奶有着较好降血脂和免疫调节的功能,它具有保健型乳制品开发的良好的前景。     

Isoflavone content and apoptotic effect in HT-29 cancer cells of a soy germ extract

Colon cancer is one of the leading causes of death and a major public health problem in western countries. We examined the isoflavone content of 70% ethanol extract of soy germ (SG) and its apoptotic effect in HT-29 colon cancer cells. Our results showed that the major isoflavones of the SG extract were daidzein and genistein, and it effectively induced apoptosis in HT-29 cancer cells. In addition, nuclear factor-kappa B (NF-κB) expression was reduced in cells treated with the SG extract, which reduced cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) expression. These combined effects ultimately resulted in apoptosis via caspase-3 activation. In addition, daidzein and genistein, the major isoflavones of SG extract, also exerted the apoptotic effect against HT-29 cancer cells. These results suggest that the inhibition of NF-κB expression is the most important factor in the induction of apoptosis in HT-29 cells treated with the SG extract.     

Effects of silica gel on reduction in gluten during several beer brewing stages

Beer is an alcoholic beverage made with barley as a basic raw material, and therefore, it is not suitable for people with coeliac disease. During the brewing process, the elimination of the haze‐active precursor is performed at the stabilisation stage. Some breweries use silica gel (SG) as stabilisation precursor. This work presents the studies conducted to identify the stage where the addition of SG is more convenient in order to reduce gluten, and the effect on the yeast nutritional value due its incorporation. The incorporation of SG during fermentation allows the reduction in more gluten than the others brewery stages studied, without changing significantly the yeast nutritional value of beer. Furthermore, the use of SG promotes the reduction in polysaccharides which obstruct filtration.     

雪莲果超声波辅助渗透脱水工艺参数的优化

以渗透脱水温度、时间、蔗糖质量分数、超声波功率和处理时间为因素,以失水率(water loss,WL)和固形物增加率(sugar gain,SG)为指标,通过单因素试验,研究雪莲果的渗透脱水工艺参数。以渗透脱水温度、时间、蔗糖质量分数、超声波处理时间为因素,以WL、SG和二者比值(WL/SG)为指标,通过二次回归正交旋转组合试验设计建立雪莲果超声波辅助渗透脱水过程中各响应值(WL、SG和WL/SG)与各因素之间的回归方程,并得到超声波辅助渗透脱水的最优工艺参数。结果表明,超声波辅助处理可显著提高雪莲果渗透脱水效果;影响WL的因素主次顺序是温度＞时间＞蔗糖质量分数＞超声波处理时间;影响SG的因素主次顺序依次是渗透脱水时间＞超声波处理时间＞温度＞蔗糖质量分数;影响SG/WL的因素主次顺序是渗透脱水时间＞蔗糖质量分数＞超声波处理时间＞温度。雪莲果超声波辅助渗透脱水的最佳工艺参数为渗透脱水温度41℃、时间1.7h、蔗糖质量分数60.18%、超声波处理时间35min。在此组合参数条件下,SG/WL平均值为0.059。     

Efficacy of Salmonella enteritidis-immune lymphokines on horizontal transmission of S. arizonae in turkeys and S. gallinarum in chickens.

Salmonella arizonae (SA) and S. gallinarum (SG) are of economic importance to international poultry production because of their pathogenesis in young poultry during the first week after hatching. Previous studies from our laboratory have shown immune lymphokines (ILK) produced by S. enteritidis (SE)-immunized chickens provide protection against SE organ invasion in day-old chickens and turkey poults. Previous studies have also demonstrated that SG organ invasion was significantly decreased by administration of ILK to broiler chicks. The objective of the present study was to determine the effect of ILK on the incidence of horizontal transmission of SA in turkey poults and of SG in broiler chicks. The effect of ILK administration on horizontal transmission of SA in poults and SG in chicks was assessed in a seeder/contact model. Seeders were challenged with the appropriate bacterium (SA turkeys, SG chicks), contacts were either untreated or administered ILK. Seeders and contacts cohabited within an experimental group throughout the experiment. Mortality and organ invasion as a result of horizontal transmission were determined. There were no significant differences in mortality between non-treated and ILK-treated contact poults. In contrast, SG was extremely pathogenic to young broiler chicks. Non-treated contact chicks had a mortality rate of approximately 68% whereas significant (P < 0.05) reduction in mortality was demonstrated in the contact chicks treated with ILK (15%). Horizontal transmission, as determined by organ invasion, of SA to contact turkey poults and SG to contact broiler chicks was also significantly (P <0.05) decreased by immunoprophylactic administration of ILK. Bacterial recovery of SA from the liver/spleen and the cecal tonsil in contact poults and SG from contact chicks treated with ILK was dramatically reduced when compared to non-treated contact poults and chicks. Our results strongly suggest the immunoprophylactic administration of SE-immune lymphokines to young turkey poults and broiler chicks significantly reduces the horizontal transmission of Salmonella in poultry. These results suggest the possibilities of using a non-vaccine immunologically-based preventative strategy against Salmonella in poultry.     

Purification of stevia extract by chitosan precipitation and reversed-phase chromatography

To improve the shortness of the current purification technology of steviol glycosides (SG) extract, a two-step purification procedure consisting of chitosan precipitation and reversed-phase chromatography (RPC) was investigated. The impurity-to-SG ratio in the SG extract obtained from non-destructive soaking was 5:1 ~ 6:1. It was found that 80.90% of the impurities could be removed by RPC using 20% (v/v) ethanol solution as the eluent with a high SG recovery of 96.98%. When 25% ethanol was applied, the SG recovery was reduced to 71.23%. An adsorption test using D280 anionic resin suggested that the impurities were negatively charged; thus, chitosan was used to co-precipitate the impurities. An optimal decolourisation result of ~ 70% was obtained at 0.06 g·g⁻¹ (w/w, to solids in extract) chitosan addition, shaking at 28 °C for 1 h. Afterwards, the extract was further purified by RPC. The total removal rate of impurities was increased to 91.20%, and the SG recovery was 85.15%.     

The effect of steaming on the free amino acid contents and antioxidant activity of Panax ginseng

This study was carried out to investigate changes in the free amino acid contents and antioxidant activity of Panax ginseng induced by steaming at different temperatures. For this purpose, white ginseng (WG), red ginseng (RG, ginseng steamed at 100 °C) and ginseng steamed at 120 °C (SG) were prepared using an autoclave. Most free amino acids were decreased significantly by steam treatment, with the greatest reduction observed in SG. Total content of free amino acids, 17.9 mg/g in WG was reduced to 12.2 mg/g in RG and 2.79 mg/g in SG. As for Arg which is the most predominant amino acid in ginseng, the content, 10.4 mg/g in WG, decreased significantly to 1.38 mg/g in SG. In particular, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), a well-known neurotoxin, was reduced by 92.9% in SG. In contrast, the level of Maillard reaction products (MRPs) increased with steam treatment, which indicates that the reduction of most amino acids is attributed to the extent of the Maillard reaction. Based on MRPs being useful antioxidants, we assayed the scavenging activity against free radicals produced by 2,2′-azobis-(2-amidinopropane) dihydrochloride (AAPH). The radical scavenging activity of a ginseng extract increased with steam treatment, with the most potent activity in SG. Further, MRPs-rich fraction in SG showed powerful antioxidant activity, which indicates MRPs are major contributors to antioxidant activity enhanced by steam treatment.