产酯酵母固定化改善酱油风味的研究

以海藻酸钠做载体,氯化钙为增强剂,包埋产酯酵母获得固定化细胞。经过一系列条件试验,找出了固定化产酯酵母的最适作用条件。经小试和中试证实,处理后的酱油酱香浓郁,接近于传统酿造酱油的风味。     

固定化酵母细胞产增香酯的研究

本工作在研究固定化己酸菌的基础上,进一步用海藻酸钙固定化酵母细胞,用活细胞计数法探讨了固定化酵母细胞的存活状态及增殖情况,从促进产酯能力上对固定化酵母和游离酵母进行了比较,并将其应用于白酒发酵之中与已酸菌液共同发酵生成己酸乙酯,可以使普通白酒的主体香气成份提高2.5倍,因此,认为固定化酵母细胞产增香酯在改进白酒质量上将很有前途。     

鲁氏接合酵母对酱油中氨基甲酸乙酯前体物的代谢

【目的】研究鲁氏接合酵母氮源代谢特性,确定鲁氏接合酵母氮源代谢与酱油中氨基甲酸乙酯前体物瓜氨酸和尿素积累的关系。【方法】通过单一氮源培养、偏好型氮源培养和盐胁迫培养,检测不同条件下鲁氏接合酵母对精氨酸、瓜氨酸和尿素的代谢能力。【结果】通过对鲁氏接合酵母氨基酸利用能力的分析,确定了甘氨酸、丙氨酸和天冬酰胺3种氨基酸为鲁氏接合酵母的偏好型氮源。在偏好型氮源存在时,鲁氏接合酵母对尿素和瓜氨酸的利用并不受到抑制,丙氨酸和甘氨酸还能够促进对二者的利用。鲁氏接合酵母在单一氮源培养条件下不会降解精氨酸而积累尿素和瓜氨酸,反而可以大量利用氨基甲酸乙酯的前体物尿素和瓜氨酸。但在盐胁迫下,鲁氏接合酵母利用尿素和瓜氨酸受到阻遏,从而造成酱油中氨基甲酸乙酯前体物不能被充分利用而积累。【结论】盐胁迫阻遏了鲁氏接合酵母对瓜氨酸和尿素的利用,从而造成酱油发酵过程中耐盐细菌所产生的氨基甲酸乙酯前体物的积累。     

高耐性酱油酵母选育及其高密度发酵调控

高耐性酵母广泛应用于食品、酿造、饲料、生物能源等行业,酵母的耐受性对其生产和应用有着决定性影响。高耐性酵母菌种改良是酵母资源利用的关键步骤,高密度发酵是克服耐性酵母产业化的主要瓶颈技术。对传统酿造食品酱油生产中常用的耐高盐酵母菌株的选育、耐性机制及其高密度发酵技术研究进展进行了综述。     

南海局部海洋沉积物中真菌多样性及产酶活性

【目的】从11份南海海洋沉积物中分离耐盐真菌,并对其物种多样性及产酶活性进行研究。【方法】利用平板涂布法分离耐盐真菌,基于形态学和ITS序列的系统进化研究耐盐真菌多样性;利用6种筛选培养基对耐盐真菌进行产酶活性筛选。【结果】分离得到1689株耐盐真菌,共41个形态种。形态学和ITS序列分析表明,这些真菌归于15个属,其中曲霉属(Aspergillus)和青霉属(Penicillium)为优势菌群。对已测序的41株耐盐真菌的产酶活性研究表明,8株产纤维素酶,9株产淀粉酶,5株产复合酶,16株产蛋白酶,3株产脂肪酶,未发现产壳聚糖酶的菌株,其中Acrodontium sp.8m和Aspergillus sp.86b产复合酶的活性相对较高,而Penicillium sp.41m产蛋白酶的活性相对较高。【结论】南海局部海洋沉积物中耐盐真菌丰富,多数菌株具有产酶活性。     

不同氯化钠浓度对Candida etchellsii CICIM Y0600生长和生物特性的影响研究

在酱油发酵过程中增香酵母的代谢产物能显著提高酱油产品的风味和品质.为了提高增香酵母菌在酱醪中的适应性,通过逐步提高氯化钠浓度的方法对酵母菌的耐盐度进行驯化培养.通过分析表明,经过5个批次的驯化,增香酵母菌的耐氯化钠浓度由180 g/L提高到240 g/L.对驯化后的三株耐不同氯化钠浓度的增香酵母菌株进行生物特性研究.结果表明,在200 g/L氯化钠浓度的培养基中,Candida etchellsii CICIM Y0600产游离氨基酸和酸解氨基酸总量分别达到2.01g/L和7.00 g/L.和其他不同氯化钠浓度条件下比较,其产有机酸种类没有变化,挥发性酯类物质含量最高,为0.80 g/L.耐盐度的提高使增香酵母菌更好地适应了在酱油发酵中的高盐度环境.     

鲁氏接合酵母盐胁迫适应机制及组学研究方法进展

鲁氏接合酵母(Zygosaccharomyces rouxii)是一种耐渗透酵母,被广泛应用于食品酿造生产领域。然而,其耐高渗和耐高盐特性通常会导致食品腐败,给企业造成了极大的经济损失。该文以鲁氏接合酵母盐胁迫适应响应机制为出发点,系统地阐述了高盐胁迫条件对鲁氏接合酵母表型特性的影响,概括了鲁氏接合酵母不同层次上的高盐胁迫响应机制。同时,探究了基因组、转录组和蛋白质组等技术在酵母耐盐机制上的应用,以期为后续研究提供新思路,有助于企业解决食品腐败问题,减少经济损失。     

固定化酵母发酵酱油的新工艺

用把酵母吸附固定在陶瓷载体上的生物反应器来生产酱油,所要求的全部时间被缩短至8天但不影响产品质量,使用酵母为接合酵母(Zygosaccharomyes.rouxii)和假丝酵母(Candida versatilis).前者经历乙醇发酵并且产生2—苯乙醇,后者产生4—乙基—愈创木酚.最近,除传统工艺外,固定化酶和固定化微生物的生物反应器在食品行业的使用已经普遍.已经研究了不同于传统而使用固定化细胞生物反应器发酵酱油的新工艺.     

双酶水解紫贻贝制备酵母调味料研究*

利用复合蛋白酶、复合风味蛋白酶、中性蛋白酶对紫贻贝进行双酶水解,水解效果比较后选择使用复合蛋白酶和复合风味蛋白酶作为复合水解酶,同时采用产酯酵母发酵技术制备调味料,通过电位滴定法测定单菌株和多菌株发酵对双酶水解贻贝肉产总酯的影响。结果表明:产酯酵母1274在双酶水解后,接种量为5%,发酵温度28℃,发酵时间72h时总酯含量为0.65%,相同条件下,产酯酵母1274和1202多菌株发酵时总酯含量为0.78%,经产酯酵母发酵后的调味料,酯香味浓郁,给予产品以发酵特有的风味。     

高盐稀醪酱油发酵原油中微生物区系研究

对传统的高盐稀醪酱油发酵过程中的正常原油和异常原油中的微生物区系进行了分析,并对主要的细菌和酵母菌进行了菌种鉴定。同时,初步探讨了温度对原油中微生物区系的影响。实验结果表明,正常原油和异常原油中细菌总数、芽孢菌数、肠道杆菌、乳酸菌数和厌氧菌数没有明显差异,而酵母菌数和耐盐菌数有明显的差异。正常原油中优势酵母为球拟酵母属(Torulopsis)和酵母属(Saccharomyces),分别占酵母总数的55.9%和35.3%。异常原油中优势酵母为毕赤酵母属(Pichia)、假丝酵母属(candida)和酵母属(Saccharomyces),分别占酵母总数的62.8%、17.9%和9.0%。     

Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g soluble starch and 2.0 g NaNO(3), 100ml seawater, initial pH 6.0, after fermentation at 24.5 degrees C for 30 h. The protease had the highest activity at pH 9.0 and 45 degrees C.     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco

Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.     

Pilot testing ofKloeckera apiculata for the biological control of postharvest diseases of citrus

The efficacy of the yeastKloeckera apiculata, strain 34-9, in controlling postharvest decay of citrus fruit was evaluated in small-scale and pilot tests in commercial packinghouse. Kloeckera apiculata grew efficiently on different media and maintained its antagonistic activity against spore germination ofPenicillium italicum. In small-scale experiments with citrus fruits dipped in the yeast cell suspension, the development of decay in citrus was effectively inhibited. The yeast was compatible with a mixture of low concentration of a commonly chemical fungicide. In packinghouse tests, combining the yeast with 40 mg/kg Carbendazim (MBC) resulted a reduction in the incidence of decay to a level equal to that of the commercial treatment of 200 mg/kg MBC. The efficacy of the strain 34-9 could also be maintained under packinghouse conditions at a cell concentration of the yeast antagonist as low as 10⁶ cells/ml. No significant difference in the efficacy ofK. apiculata was found in either the drench or the spray application systems tested in citrus packinghouse. Scanning electron microscopy revealed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of strain 34-9 against citrus blue mould may be related to its capability to compete withPenicillium italicum, for space and nutrients and /or involvement of directly antagonist of the yeast on the fungus.     

Isolation of l-methionine-enriched mutant of a methylotrophic yeast,Candida biodinii No. 2201

Six strains of methylotrophic yeast were examined for production of l-methionine-enriched cells. Candida biodinii (Kloeckera sp.) No. 2201, which accumulated 0.54 mg/g-dry cell weight (DCW) of free l-methionine (pool methionine), was selected as the parental strain for breeding l-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradation. A mutant strain, E500-78, which was resistant to 500 μg/ml of dl-ethionine, accumated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were o-ptimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.     

从老抽酱醪中分离耐盐性酵母的研究

以老抽酱醪为实验材料进行耐盐性酵母菌种分离,并做菌种鉴定。分析了在不同盐度条件下耐盐性酵母菌的生长情况和生长过程中培养基总糖的消耗,可以发现实验得到的酵母在22％（质量与体积}E）盐度下依然能够良好生长。结果表明,实验分离出的No．2菌在同级盐度的条件下的生长量要明显高于No．1菌,但在乙醇产率方面,两株菌在相同的含盐量为16％（质量与体积比）的麦芽汁培养基中发酵8d,No．1菌的乙醇产率为3．1％（体积比）,No．2菌的乙醇产率2．9％（体积比）。     

Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase

We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product. High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis. The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E. coli. Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter. Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E. coli strain TG2. Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography. Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested. The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E. coli enzyme. E. coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml. At the same concentration of pseudomonic acid A, E. coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1%. The ability of yeast IRS to rescue E. coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E. coli tRNA(ile).     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

Streptomyces griseolus # 182--a novel organism producing oligomycin antibiotics. Taxonomy, fermentation, and isolation]

Target screening of natural immunosuppressors resulted in isolation of a strain of Streptomyces griseolus (No. 182) producing a complex of antifungal antibiotics. The strain proved to be an aerobe with the growth temperature of 26 to 28 degrees C. Morphological features and physiological properties of the strain were studied. Scanning electron microscopy revealed smooth, oval spores 1.10-1.25 mu in size. The findings showed that the strain belonged to Streptomyces griseolus. Unlike the previously described organisms producing the oligomycin complex the new strain formed straight or twisted sporophores and did not produce melanoid pigment or soluble pigment when grown on the Gauze mineral agar medium No. 1. The procedures for biosynthesis and chemical recovery of the antibiotic complex from the mycelium are described. The complex was shown to include 3 components at a ratio of 80:15:5 identified as oligomycins A, B and C respectively. The oligomycin complex was highly active against Aspergillus niger 137, Tolypocladium inflatum, Fusarium ocsisporum, Curvularia lunata 645 and Trichoderma alba F-32 (MIC 0.1-1.0 mcg/ml). The activity against yeast and bacterial cultures was observed only when the doses were higher than 100 mcg/ml.     

Theanine production by coupled fermentation with energy transfer using gamma-glutamylmethylamide synthetase of Methylovorus mays No. 9

Gamma-glutamylmetylamide synthetase (GMAS) of Methylovorus mays No. 9, produced by Eschericia coli AD494 (DE3) harboring pET21aGM, formed theanine from glutamic acid and ethylamine with coupling of the reaction with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn(2+) to the reaction mixture, whereas Mg(2+) was less effective. Increases to a certain level in the concentrations of GMAS and the substrates in the mixture were effective in increasing theanine formation, but high concentrations of ethylamine (900 mM or more) inhibited yeast sugar fermentation, and eventually decreased theanine formation. The inhibitory effect of ethylamine was restored by increasing the concentration of potassium phosphate buffer in the mixture. Approximately 600 mM (110 mg/ml) theanine was formed in 48 h in an improved reaction mixture containing 600 mM sodium glutamate, 600 mM ethylamine.HCl, 300 mM glucose, 200 mM potassium phosphate buffer (pH 7.0), 30 mM MgCl(2), 5 mM MnCl(2), 5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of yeast cells. The yield of theanine was 100% on the substrates (glutamic acid and ethylamine) and also on the energy source (glucose consumed).