Further Characterization of a Caffeine-Resistant Mutant of Aspergillus parasiticus

Studies were performed to characterize further Aspergillus parasiticus BCR1, a caffeine-resistant mutant of A. parasiticus NRRL 2999, particularly in regard to its caffeine-dependent production of aflatoxins. The enhanced synthesis of aflatoxins by caffeine was highly specific since neither dimethylxanthines nor purines could substitute for the trimethylxanthine. Caffeine's effects were phase dependent and only increased toxin formation if added early in the microorganism's life cycle. The ability of BCR1 to exclude caffeine appeared dependent on the initial levels of caffeine in the growth medium. Respiration and glucose utilization in the wild type strain were inhibited strongly by caffeine, but BCR1 was resistant to these effects. Comparison of glucose uptake kinetics in the wild type and mutant strains indicated that caffeine inhibition of aflatoxin synthesis in the wild type was not due to a disruption of glucose transport.     

可降解咖啡碱的酵母菌菌种筛选及其培养条件的优化

从贵州省毕节市金沙县茶叶种植基地的土壤中分离得到一株可降解咖啡碱的真菌,经表型特征、内转录间隔区序列测定及系统发育树分析鉴定为酿酒酵母菌(Saccharomyces cerevisiae)同源菌,命名为Y-1。通过单因素试验及响应面试验设计,以咖啡碱降解率为指标,得到响应面回归模型预测方程,根据预测方程,确定菌株Y-1最优的培养条件为:温度31.00℃,发酵时间46.60 h,咖啡碱添加量0.50 mg/mL、菌种添加量6.90%。在此条件下,利用菌株Y-1在培养基中降解咖啡碱,咖啡碱降解率最高可达(59.39±0.28)。     

红曲色素高产菌的诱变选育与发酵优化

红曲红是一种由红曲霉生产的具有较高药用及营养价值的天然色素。为提高红曲红液态发酵产量,利用常压室温等离子体诱变技术(ARTP)对紫红曲霉（Monascus purpureus）LBBE进行诱变并优化了发酵条件。确定ARTP诱变条件为：红曲霉孢子浓度107个/mL、通气量10 SLM、功率80 W、诱变时间42 s。上述诱变条件下,经10轮诱变,红曲霉正向突变率从44.6 %降至3.8%,突变株LBBE-15的红曲红色价达到1 244 U/mL,较出发菌株提高1.26倍。红曲霉培养条件优化确定为：接种体积分数7%、硫酸锰1 g/L、初始pH 3.7。优化后的红曲红色价达到1 376 U/mL。50 L罐分批发酵显示,红曲红色价为642 U/mL。该研究结果为实现工业大规模生产提供了数据参考。     

Caffeine and chlorogenic acids intake from coffee brew: influence of roasting degree and brewing procedure

The influence of coffee cultivar, roasting degree and brewing procedure in the presence and transfer of caffeine and caffeoylquinic acids (CQAs) from ground roasted coffee to the brew was evaluated. Two coffee cultivars were roasted in three roasting degrees and brewed using two different procedures. Compounds were determined simultaneously by HPLC‐DAD. Caffeine levels ranged from 87.3 to 122.5 mg/100 mL for Coffea arabica cv. Catuaí Amarelo and from 123.3 to 192.0 mg/100 mL for C. canephora cv. Apoatã. The sum of CQA isomers ranged from 24.2 to 41.3 mg/100 mL for brews prepared with dark roasted coffee and from 187.7 to 295.6 mg/100 mL for light roasted ones. Brews prepared by boiling showed higher content of the compounds than the corresponding filtered ones. C. arabica cv. Catuaí Amarelo light roasted coffee brew presented the lowest caffeine/CQA ratio, regardless of the brewing procedure used, in comparison with the highest ratio of the dark boiled brews.     

西索米星生产菌株的诱变选育及工艺优化

利用原生质体常压室温等离子体（ARTP）诱变方法选育西索米星高产菌株,经诱变筛选获得一株高产菌株I4-10,其西索米星的生物效价达到1 389 U/mL,较原始菌株提高了35.4%。通过对高产菌株I4-10进行碳源、氮源优化,确定可溶性淀粉和牛肉粉是突变菌株I4-10的最适碳源和氮源。进一步采用响应面实验设计方法优化发酵培养基,结果表明黄豆饼粉和DL-蛋氨酸的添加量为显著影响因素,经优化其最适添加质量浓度分别为32.78 g/L和1.75 g/L。在此最适工艺下,西索米星的生物效价提高至1 849 U/mL,较原始菌株提高了80%。最后对原始菌及高产突变株中西索米星代谢途径及菌株生长相关的关键酶进行转录水平比较分析,初步解析了突变菌株I4-10高产西索米星的机制。     

常压室温等离子体诱变选育高产脂肪酶解脂耶氏酵母

采用常压室温等离子体(Atmospheric room temperature plasma,ARTP)对产脂肪酶的解脂耶氏酵母菌株YL1进行诱变;通过三丁酸甘油酯平板法、p-NPP法以及酸碱滴定法等筛选得到高产脂肪酶的目标菌株C4,并研究其遗传稳定性。结果表明,解脂耶氏酵母菌株YL1的最佳诱变时间为60s,菌株致死率达97.45%;突变株C4的脂肪酶酶活为13.4 U/mL,较出发菌株提高了82.6%,多代培养后遗传稳定;与出发菌株相比,突变株脂肪酶可使维生素A棕榈酸酯的合成转化率提高36.9%。     

Strain Improvement Through UV and Chemical Mutagenesis for Enhanced Citric Acid Production in Molasses-Based Solid State Fermentation

Aspergillus niger was subjected to UV radiation and chemical mutagenesis to develop its hyper-producing mutants for enhanced citric acid production. Ethyl methane sulfonate (EMS) and Ethidium bromide (EB) were used for chemical mutagenesis of Aspergillus niger. UV, and chemically treated mutants of Aspergillus niger were identified by using 2-deoxy, D-glucose as selective marker. The selected mutants were cultured in solid state fermentation (SSF) of sugarcane molasses medium (10%) using corn cobs, banana stalk, sugarcane bagasse, wheat straw, and wheat bran as carrier substrates. After pH adjustment and sterilization, the triplicate flasks were inoculated with 5 mLof homogenous spore suspensions of selected mutants of A. niger and the flasks were subjected to SSF under still culture conditions. The mutant EB-3 (treated with 1 mg/mL ethidium bromide for 120 min) giving highest citric acid yield (64.2 mg/mL) in 72 h was selected as hyper-producing mutant. Citric acid production process using EB-3 mutant was then optimized to enhance citric acid production by the mutant in SSF. Aspergillus niger EB-3 mutant could produce 67.72 mg/mL citric acid in 72 h using banana stalks as support material under optimum conditions of pH (pH 6), incubation temperature (35°C) and inoculum size (5 mL) in SSF.     

Optimization of beta-carotene production by Rhodotorula glutinis using high hydrostatic pressure and response surface methodology

ABSTRACT:  Rhodotorula glutinis RG6 was treated by high hydrostatic pressure (HHP) of 300 MPa for 15 min for improving its ability of β-carotene production. After the treatments of 5 repeated cycles, the mutant strain RG6p was obtained, β-carotene production of which reached 10.01 mg/L, increased by 57.89% compared with 6.34 mg/L from parent strain RG6. To optimize the medium for β-carotene fermentation by mutant RG6p, a response surface methodology (RSM) approach was used in conjunction with a factorial design and a central composite design, and the maximum yield of β-carotene (13.43 mg/L), an increase of 34.17% compared to the control, was obtained at a pH 6.7 with an optimum medium (40 mL/250 mL) of yeast extract (4.23 g/L), glucose (12.11 g/L), inoculum (30 mL/L), tomato extract (2.5 mL/L), peanut oil (0.5 mL/L), and (NH₄)₂SO₄ (5 g/L).     

紫外诱变枯草芽孢杆菌选育葡萄糖-1-磷酸高产菌株

对出发菌株进行紫外诱变以获得葡萄糖-1-磷酸产量高且遗传稳定性好的枯草芽孢杆菌菌株。以葡萄糖-1-磷酸的发酵产量为考察指标,采用HPLC-MS检测方法,对出发菌株Bacillus sublitis XH-13进行三轮紫外诱变,并对获得的高产菌株连续传代6次。结果表明,枯草芽孢杆菌突变菌株UV3-8的葡萄糖-1-磷酸产量最高,达到6.85 g/L,是出发菌株产量的1.54倍,且遗传稳定性好。菌株Bacillussubtilis XH-13_UV3-8的营养简单、葡萄糖-1-磷酸产量高且遗传稳定性好,具有产业化应用前景。     

CAFFEINE CONTENT IN COLAS FROM NEW YORK STATE AND ONTARIO

Thirty-nine canned soft drinks obtained from New York (NY) State and from Ontario in 1986 were analyzed for caffeine. The major brands of cola drinks from either location differed little in caffeine concentration, with mean values of 92.5 μg/mL for NY State and 86.5 μg/mL for Ontario. However, different can volumes contributed toward the difference in caffeine content per can of cola beverages obtained in NY State and Ontario. The mean caffeine values for the "regular colas" were 34.3 and 21.5 mg/can (concentration range: 2.3–133.4 μg/mL and 0.1–104.9 μg/mL) for the NY State and Ontario products, respectively. The range of caffeine content for all products was from 0.8 to 50.8 mg/can for the NY State colas and from 0.03 to 29.4 mg/can for the Ontario products. Comparison with earlier reports (for USA from 1979:47.3 mg/can; for Ontario from 1976: 40 mg/can) indicated a general decrease in the amount of caffeine in all types of cola beverages in the last 7 years.     

PGK1, the gene encoding the glycolitic enzyme phosphoglycerate kinase,acts as a multicopy suppressor of apoptotic phenotypes in S. cerevisiae

In a previous paper we reported the construction of a S. cerevisiae strain lacking the essential gene LSM4, which could survive by the introduction of a truncated form of the orthologous gene from Kluyveromyces lactis. This strain showed apoptotic hallmarks and other phenotypes, including an increased sensitivity to caffeine and acetic acid. The suppression of the latter phenotype by overexpressing yeast genes allowed the isolation of PGK1, the gene encoding the glycolytic enzyme phosphoglycerate kinase. This gene restored normal ageing, oxygen peroxide resistance and nuclear integrity in the mutant. Other phenotypes, such as caffeine sensitivity and glycerol utilization, were also suppressed. Copyright © 2009 John Wiley & Sons, Ltd.     

A dark brown roast coffee blend is less effective at stimulating gastric acid secretion in healthy volunteers compared to a medium roast market blend

Coffee consumption sometimes is associated with symptoms of stomach discomfort. This work aimed to elucidate whether two coffee beverages, containing similar amounts of caffeine, but differing in their concentrations of ^βN‐alkanoyl‐5‐hydroxytryptamides (C5HTs), chlorogenic acids (CGAs), trigonelline, and N‐methylpyridinium (N‐MP) have different effects on gastric acid secretion in healthy volunteers. The intragastric pH after administration of bicarbonate with/without 200 mL of a coffee beverage prepared from a market blend or dark roast blend was analyzed in nine healthy volunteers. Coffee beverages were analyzed for their contents of C5HT, N‐MP, trigonelline, CGAs, and caffeine using HPLC‐DAD and HPLC‐MS/MS. Chemical analysis revealed higher concentrations of N‐MP for the dark brown blend (87 mg/L) compared to the market blend coffee (29 mg/L), whereas concentrations of C5HT (0.012 versus 0.343 mg/L), CGAs (323 versus 1126 mg/L), and trigonelline (119 versus 343 mg/L) were lower, and caffeine concentrations were similar (607 versus 674 mg/mL). Gastric acid secretion was less effectively stimulated after administration of the dark roast blend coffee compared to the market blend. Future studies are warranted to verify whether a high ratio of N‐MP to C5HT and CGAs is beneficial for reducing coffee‐associated gastric acid secretion.     

复合诱变定向选育腺苷生产菌

以枯草芽孢杆菌HJ-9（Xan）为出发菌株，根据代谢工程原理，采用原生质体紫外诱变和微波诱变相结合的方法，定向筛选出了具有黄嘌呤缺陷、腺嘌呤脱氨酶阴性、磺胺胍抗性的腺苷生产菌HS-5，其摇瓶发酵产量由出发菌株的1.06mg／m：L，提高到3．15mg／mL。     

红曲霉发酵高温豆粕高产可溶性多肽

通过利用红曲霉发酵高温豆粕产生可溶性多肽,确定菌株对变性蛋白质有一定的分解作用;并采用紫外诱变红曲霉,经初筛和复筛,最终筛选出能高产可溶性多肽的菌株。结果表明：红曲霉发酵可分解高温豆粕中的蛋白质,并且产生分子质量介于7.8～20.1 kD的可溶性多肽。发酵120 h可产生（13.41±0.20） mg/mL的可溶性多肽,是原豆粕的3.60 倍。15 W紫外线灯35 cm处,搅拌条件下照射50 s,红曲霉的致死率为（82.70±2.20）%。在此诱变条件下得到一株高产可溶性多肽的突变红曲霉0501100菌株。该菌株在发酵豆粕96 h时产生的可溶性多肽含量达到（17.20±0.18） mg/mL,是原菌株在同等发酵时间条件下产生可溶性多肽的1.47 倍,是原豆粕的4.61 倍,缩短了发酵时间并且有较好的遗传稳定性。     

Determination of the caffeine contents of various food items within the Austrian market and validation of a caffeine assessment tool (CAT)

The caffeine content of 124 products, including coffee, coffee-based beverages, energy drinks, tea, colas, yoghurt and chocolate, were determined using RP-HPLC with UV detection after solid-phase extraction. Highest concentrations of caffeine were found for coffee prepared from pads (755?mg?l^?1) and regular filtered coffee (659?mg?l^?1). The total caffeine content of coffee and chocolate-based beverages was between 15?mg?l^?1 in chocolate milk and 448?mg?l^?1 in canned ice coffee. For energy drinks the caffeine content varied in a range from 266 to 340?mg?l^?1. Caffeine concentrations in tea and ice teas were between 13 and 183?mg?l^?1. Coffee-flavoured yoghurts ranged from 33 to 48?mg?kg^?1. The caffeine concentration in chocolate and chocolate bars was between 17?mg?kg^?1 in whole milk chocolate and 551?mg?kg^?1 in a chocolate with coffee filling. A caffeine assessment tool was developed and validated by a 3-day dietary record (r ^2?=?0.817, p?<?0.01) using these analytical data and caffeine saliva concentrations (r ^2?=?0.427, p?<?0.01).     

Reduction of Methanol in Brewed Wine by the Use of Atmospheric and Room‐Temperature Plasma Method and the Combination Optimization of Malt with Different Adjuncts

Methanol, often generated in brewed wine, is highly toxic for human health. To decrease the methanol content of the brewed wine, atmospheric and room‐temperature plasma (ARTP) was used as a new mutagenesis tool to generate a mutant of Saccharomy cescerevisiae with lower methanol content. Headspace gas chromatography was used to determine the identity and concentration of methanol with butyl acetate as internal standard in brewed wine. With 47.4% higher and 26.3% positive mutation rates were obtained, the ARTP jet exhibited a strong effect on mutation breeding of S. cerevisiae. The mutant S. cerevisiae S12 exhibited the lowest methanol content, which was decreased by 72.54% compared with that of the wild‐type strain. Subsequently, the mutant S. cerevisiae S12 was used to ferment different combinations of malt and adjuncts for lower methanol content and higher alcoholic content. It was shown that the culture 6#, which was 60% malt, 20% wheat, and 20% corn, was the best combinations of malt and adjuncts, with the lowest methanol content (104.8 mg/L), and a relatively higher alcoholic content (15.3%, v/v). The optimal malt–adjunct culture 6#, treated with the glucoamylase dose of 0.04 U/mg of grain released the highest reducing sugars (201.6 mg/mL). It was indicated that the variation in reducing sugars among the combinations of malt and different adjuncts could be due to the dose of exogenous enzymes.     

胆固醇氧化酶高产菌株的复合诱变选育

为进一步提高产酶水平,本实验采用超声波联合亚硝基胍的复合诱变方法处理胆固醇氧化酶产生菌(甾短杆菌),通过1mg/mL亚硝基胍和超声(200W,50kHz,35min)同时处理细胞悬液,获得一株红色突变株,其产酶活力从0.5U/mL提高到1.21U/mL,酶活提高了140%。证明该联合诱变手段对甾短杆菌产胆固醇氧化酶具有良好的诱变效果。     

产共轭亚油酸植物乳杆菌的筛选、鉴定与诱变

从传统泡菜中筛选得到1 株乳酸菌lp15 能够合成共轭亚油酸(CLA),经16S rRNA 全序列分析法和API 系统鉴定法鉴定为植物乳杆菌(Lactobacillus plantarum)。利用人工诱变方法,以lp15 为出发菌株,采用紫外线、硫酸二乙酯(DES)依次诱变处理,经进一步液体发酵复筛获得多株突变菌株,CLA合成能力较出发菌株提高了29.3%～52.2%。其中lp15-2-1 突变菌株为CLA 生成能力最高菌株,MRS 培养液中添加0.2mg/mL LA 培养48h,CLA 产量达30.13μg/mL。经气相色谱(GC)检测分析,产物中cis9, trans11- 共轭亚油酸(c9, t11-CLA)占76.5%,trans10, cis12-共轭亚油酸(t10,c12-CLA)占23.5%。     

Factors Contributing to Antilisterial Effects of Raw Egg Albumen

Sensitivity of Listeria monocytogenes strain Scott A and Brie-1 to several factors in raw egg albumen was investigated. A concentration of ca 15% of albumen in trypticase soy broth was listeristatic after 24 hr at 35°C, and listericidal effects were observed at higher concentrations. Supplementation of albumen with iron or biotin did not reverse the inhibition. Preheating of albumen (50–80°C) caused progressive loss of antilisterial effects. Supplementation of broth with lysozyme (>1 mg/mL) produced antilisterial effects that were enhanced at pH 9; conalbumin (>6 mg/mL) suppressed cell growth, while ovomucoid (>2 mg/mL) was inhibitory only at pH 9. Results inferred that antilisterial effects of albumen were caused primarily by lysozyme and were enhanced by ovomucoid, conalbumin, and alkaline pH.     

甜酒曲中产糖化酶根霉菌株紫外诱变选育

从甜酒曲中分离出产糖化酶根霉菌株,通过平板菌落初筛、测定HE值和摇瓶复筛测糖化力,筛选出1株HE值大且糖化力高的优良出发菌株,对其进行紫外线诱变处理,选育得到优良变异株,该菌株的HE值为1.76,糖化力为1281mg/(h ·g),分别较出发菌株高出5.4%、59.5%。对此优良变异株的培养条件进行优化,获得最佳的培养条件为：培养温度为30℃,培养时间为3d,添加玉米粉质量浓度为8g/100mL,在此条件下糖化力为1317mg/(h ·g)。