Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。     

Proteolysis characteristics of Actinomucor elegans and Rhizopus oligosporus extracellular proteases under acidic conditions

Enzymatic hydrolysis of soybean protein isolate by the extracellular proteases from Actinomucor elegans and Rhizopus oligosporus at pH 3.0, 3.5, 5.0, 5.5 and 6.0 was investigated. The activity of the A. elegans protease is lower than that of R. oligosporus, but both proteases exhibit considerable degradation of soybean protein at pH 5.5 and 6.0. The water‐soluble protein content and the degree of hydrolysis of the hydrolysates are increased significantly, and bitterness values are very low. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) reveals that these proteases have different cutting sites on peptide polymers. At pH 5.5, there is a lower content of total free amino acids (39.20 mg per 100 mL; 62.68% hydrophobic amino acids) in the R. oligosporus protease hydrolysate. In conclusion, treatment with R. oligosporus protease at pH 5.5 achieves efficient degradation of soybean protein, suggesting a promising industrial process for making bitterless protein hydrolysates.     

再论如何提高腐乳坯的出率

该文从腐乳酿造工艺流程角度出发,探讨了大豆品质选择、腐乳坯操作、熟浆操作、点浆操作、上榨制坯等工艺环节对腐乳出坯率的影响,为提高腐乳出坯率奠定了一定的基础,为我国腐乳业的发展提供参考。     

Degradation of anti-nutritional factors and reduction of immunoreactivity of tempeh by co-fermentation with Rhizopus oligosporus RT-3 and Actinomucor elegans DCY-1

The effects of solid-state fermentation with four filamentous fungi (Rhizopus oligosporus, Actinomucor elegans, Neurospora crassa and Rhizopus oryzae) and their combination on the degradation of protein allergens and anti-nutritional factors (ANFs) of tempeh were evaluated. Glycinin, β-conglycinin, trypsin inhibitor and flatulence-causing oligosaccharides were significantly hydrolysed by all assayed strains but the level of reduction of ANFs depended on the strain. ELISA was conducted to evaluate IgE immunoreactivity using sera from soy sensitive individuals. Results revealed that co-fermentation of R. oligosporus and A. elegans resulted in the highest reduction in IgE immunoreactivity for all sera used and showed greatest degradations on α’, α, β, acidic subunits (100% hydrolysed) and extensive hydrolysis of flatulence-causing oligosaccharides. Tempeh fermented with R. oligosporus and A. elegans displayed high level of soluble protein and peptide (<10 kD) content. Co-fermentation of R. oligosporus and A. elegans was proved to be effective in producing hypoallergenic tempeh.     

Hypoallergenic buckwheat flour preparation by Rhizopus oligosporus and its application to soba noodle

The effect of Rhizopus oligosporus on the structure and functional properties of buckwheat grain during fermentation was investigated and the production of buckwheat hypoallergenic flour was studied. The spores of R. oligosporus were mixed with steamed buckwheat grains, and they were incubated at 30 °C and 85% relative humidity (RH) for 0, 24, 48 and 72 h. The changes of structure, free amino acids, minerals, phytic acid and allergenic proteins in the grains were determined during the time course of incubation. The 48-h-fermented buckwheat (FeB) was found to be involved in formation of amino acids with higher amounts of total amino acids, and some of them increased 50 times more; including isoleucine, leucine, lysine, valine, glycine, histidine, tyrosine and γ-amino butyric acid (GABA). SDS–PAGE and Western Blot analyses showed that R. oligosporus was very effective to reduce allergenic proteins of buckwheat and could produce the hypoallergenic buckwheat flour. In its application to noodle making, the FeB-substituted wheat flour noodle was softer and had more elasticity than the control without FeB flours (1CW), except for the 10% substitution of FeB. Uncooked and cooked noodles were brownish compared to those of the 1CW. The R. oligosporus could possibly be used to produce hypoallergenic buckwheat with higher functional and good rheological properties, and thus produced FeB flour was considered to become a big prospect for development of new food in the future.     

Production of volatile compounds by Rhizopus oligosporus during soybean and barley tempeh fermentation

Rhizopus oligosporus Saito can ferment soybeans or cereal grains to tempeh, a sliceable cake with improved nutritional properties. Volatiles produced by different R. oligosporus strains grown on malt extract agar (MEA), barley and soybean were investigated. The effect of co-cultivation with Lactobacillus plantarum on the production of volatiles was also studied. Volatile compounds were collected in situ by headspace diffusion and identified by GC-MS. The ten R. oligosporus strains that had different colony morphologies on MEA produced very similar volatile profiles, except for slight variations among the minor volatile compounds (e.g. sesquiterpenes). Likewise, practically no differences in volatile profiles were observed between three of the strains grown on soybeans. In contrast, the R. oligosporus volatile profile on soybean was different from that on barley from the same strain. Co-cultivation with L. plantarum did not influence volatile production by R. oligosporus. The dominant compounds produced on all three substrates were ethanol, acetone, ethyl acetate, 2-butanone, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Acetaldehyde and 2-methyl-propanal were also produced on MEA and barley, while 2-pentanone, methyl acetate, 2-butanol and 3-methyl-3-buten-1-ol were observed on soybeans. Ethanol, 2-methyl-1-butanol and 3-methyl-1-butanol were the most abundant volatile compounds produced on MEA and barley, while 2-butanone was the dominant volatile metabolite on soybean. The mushroom odour compounds, 3-octanone and 1-octen-3-ol, were only detected from soybean and soybean tempeh.     

序贯设计在毛霉固体发酵工艺优化中的应用

对毛霉AS3.2778固体发酵的工艺条件进行了探讨,利用序贯设计方法对影响产酶的若干因素进行了优化,确立了相对稳定的发酵工艺条件:250mL三角瓶装8g麸皮(其中添加1.23%麦芽糖、1.56%蛋白胨、0.74%KH2PO4、0.06?SO4·7H2O作为辅助营养物),培养基的起始水添加量为0.79mL/g麸皮,发酵温度24.1℃,时间48h.在优化的工艺条件下,可发酵产酶1541U/g干基,比初始发酵水平提高了95.5%.     

Effect of temperature and relative humidity during transportation on green coffee bean moisture content and ochratoxin A production

Changes in temperature, relative humidity, and moisture content of green coffee beans were monitored during transportation of coffee from Brazil to Italy. Six containers (three conventional and three prototype) were stowed in three different places (hold, first floor, and deck) on the ship. Each prototype was located next to a conventional container. The moisture content of the coffee in the container located on the first floor was less affected by environmental variations (0.7%) than that in the hold and on the deck. Coffee located in the hold showed the highest variation in moisture content (3%); in addition, the container showed visible condensation. Coffee transported on the deck showed an intermediary variation in moisture (2%), and there was no visible condensation. The variation in coffee moisture content of the prototype containers was similar to that of the conventional ones, especially in the top layers of coffee bags (2 to 3%), while the increase in water activity was 0.70. This suggests that diffusion of moisture occurs very slowly inside the cargo and that there are thus sufficient time and conditions for fungal growth. The regions of the container near the wall and ceiling are susceptible to condensation since they are close to the headspace with its high relative humidity. Ochratoxin A production occurred in coffee located at the top of the container on the deck and in the wet bags from the hold (those found to be wet on opening the containers at the final destination).     

Impact of relative humidity and temperature on visible fungal growth and OTA production of ochratoxigenic Aspergillus ochraceus isolates on grapes

Aspergillus ochraceus is an ochratoxin A (OTA) producer mould found in grapes and this may contribute to OTA contamination in wines and grape juices. The influence of relative humidity (R.H.; 80, 90 and 100%) and temperature (10, 20 and 30 °C) on visible mould growth on grapes and OTA accumulation after 14 days of incubation by this fungal species has been studied using a full factorial design with three replicates.The two abiotic factors and their interaction (R.H.×temperature) affected significantly the A. ochraceus growth in berries, which was maximum at 90–100% R.H. levels. With regard to the optimum temperature level, it occurred at 30 °C at 80 and 90% R.H., whereas no significant differences were detected at 20 and 30 °C when R.H. was 100%.OTA production by A. ochraceus on grapes was not significantly modified by the assayed levels of temperature and R.H, with a mean value of 3.53 ng g⁻¹.Predictive models of percentage of grapes with visible growth of A. ochraceus isolates under different relative humidity and temperature are presented.     

Characterization of extra- and intracellular phytases from Rhizopus oligosporus used in tempeh production

Rhizopus oligosporus strain CT11K2, commonly used in tempeh (fermented soybean) production produced both extra- and intracellular phytases. The enzymes were isolated from growth media and the cultured mould and partially purified by acetone fractionation, gel filtration on Sephadex G-100 and DEAE-cellulose chromatography. Intracellular phytase activity was higher than that of the extracellular enzyme. Both enzymes showed maximum activity at pH 4.5 and 55 degrees C, suggesting relatively high thermostability. The enzymes were partially inhibited by high concentrations of substrate. The Km and Vmax values of the extracellular phytase were 0.15 mM and 0.076 mumol Pi per min per ml DEAE-cellulose purified enzyme, respectively, and for the intracellular phytase were 0.17 mM and 0.34 mumol Pi per min per ml enzyme, respectively. Extracellular phytases showed inactivation and activation energies for the hydrolysis of phytic acid of approximately 28,300 cal per mol and 6100 cal per mol, respectively, while inactivation and activation energies for the intracellular phytase were approximately 33,200 per mol and 9500 cal per mol, respectively.     

Rhizopus oligosporus and yeast co-cultivation during barley tempeh fermentation--nutritional impact and real-time PCR quantification of fungal growth dynamics

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold storage of tempeh (P<0.01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents and compositions, and did not reduce phytate contents. Slight increases of vitamins B(6) and niacinamide, and slight decreases of B(1) and biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh. The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus, real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous substrate such as barley tempeh.     

Growth of lactic acid bacteria and Rhizopus oligosporus during barley tempeh fermentation

The zygomycete Rhizopus oligosporus is traditionally used to ferment soybean tempeh, but it is also possible to ferment other legumes and cereals to tempeh. The traditional soybean tempeh harbours a multitude of microorganisms with potentially beneficial or detrimental effects on quality. Lactic acid bacteria (LAB) have positive effects on the safety of soybean tempeh, but the effects of LAB on R. oligosporus growth have not been investigated. We have developed a cereal grain tempeh by fermenting pearled barley with R. oligosporus ATCC 64063. Four LAB species, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri and Lactococcus lactis were assessed for their growth abilities and their effects on R. oligosporus growth during barley tempeh fermentation. Growth of LAB was assayed as colony forming units (cfu), while growth of R. oligosporus was measured as ergosterol content and hyphal length. The two fungal measurements highly correlated (r=0.83, P<0.001, n=90). The ergosterol content of fungal mycelia ranged from 11.7 to 30.1 mg/g fungal dry matter. L. plantarum multiplied from 4.8 to 7.4 log cfu/g dry tempeh and L. fermentum increased from 4.4 to 6.8 log cfu/g during 24 h incubation at 35 degrees C. L. reuteri and L. lactis had significantly slower growth, with increases from 4.8 to 5.6 log cfu/g and 5.0 to 5.4 log cfu/g, respectively. The growth of R. oligosporus and the final pH (4.9) in barley tempeh were not significantly influenced by any of the LAB investigated.     

豆渣毛霉发酵条件的研究

以氨基酸态氮含量和感官评分为指标,考察了水分含量、接种量、发酵温度和时间对豆渣毛霉发酵的影响,优化了其发酵条件,并比较了发酵前后豆渣主要成分的变化。结果显示,豆渣毛霉固态发酵的最优条件为:在水分含量为70%的豆渣中接种其质量的2%毛霉菌悬液,搅匀后于28℃下发酵4天。发酵后的豆渣色泽棕黄,质地松软,入口细腻,滋味鲜美,发酵风味浓郁;其水分和粗蛋白含量明显降低,氨基酸态氮含量和总酸含量升高。     

雅致放射毛霉和运动发酵单胞菌混合发酵霉豆渣研究

研究雅致放射毛霉和运动发酵单胞菌混合发酵霉豆渣的条件,并在最优发酵条件下测定产品的pH值、氨基态氮含量、总粗蛋白含量及质构在发酵过程中的变化。通过正交试验确定霉豆渣发酵的最优条件为：豆渣蒸煮15min后,在100g豆渣中接入105CFU/mL雅致放射毛霉孢子悬液1.0mL及107CFU/mL运动发酵单胞菌悬液0.2mL,28℃培养3d。在发酵过程中,霉豆渣的pH值从5.12上升至8.40后缓慢下降至8.09;氨基态氮含量则先迅速上升后缓慢下降;总粗蛋白含量逐渐下降。发酵过程使霉豆渣的硬度和咀嚼性明显提高。     

少孢根霉发酵菠萝果渣促进粗多酚溶出的研究

以干燥的菠萝果渣为实验原料,通过单因素实验考察氮源种类、氮源添加量、水分含量、麦麸添加量、发酵温度、发酵时间对少孢根霉发酵菠萝果渣促进粗多酚溶出的影响,确定出适宜的发酵工艺条件,该条件下粗多酚的溶出率比用水直接浸提增加126.28%.同时HPLC分析表明,发酵前后果渣中提取的多酚类物质其种类几乎不变,只是含量显著增加.     

雅致放射毛霉产孢培养基及产孢条件研究初报

雅致放射毛霉作为一种重要的发酵真菌,广泛应用在霉豆渣发酵和腐乳酿造中,研究其产孢条件对制作直投式菌种具有重要意义。初步筛选了雅致放射毛霉产孢的6种合成培养基和8种天然培养基,同时对碳源、氮源以及碳氮比对雅致放射毛霉产孢的影响进行了研究。实验表明,MEA培养基、小麦麸皮培养基上其孢子产量较高,分别为第9天的1.664×107cfu/mL和第6天的2.04×107cfu/cm2(成活率为51.67%)。以麦芽糖、淀粉、葡萄糖和硝酸钾、硫酸铵分别为碳氮源有利于其产孢。在0.05水平上碳氮比对雅致放射毛霉产孢影响显著,碳源对其产孢影响不显著,碳源为1.92g/L,碳氮比为10:1条件下雅致放射毛霉孢子产量最高。     

对硝基苯酚法对雅致放射毛霉脂肪酶特性的研究

以不同碳链长度的对硝基苯酚酯为底物对雅致放射毛霉（Actinomucor elegans AS3.27）脂肪酶的酶学特性和其发酵的腐乳在发酵过程中脂肪酶活力的变化进行研究,结果表明：分别以对硝基苯酚正辛酸酯（pNPC）和对硝基苯酚棕榈酸酯（pNPP）为反应底物所测得的脂肪酶酶学特性有所不同。以pNPC和pNPP为反应底物时,雅致放射毛霉脂肪酶的最适作用温度分别为30、35℃,最适作用pH为7.5、7.0,最适盐浓度都为0.20mol/L。以两种底物分别测定雅致放射毛霉接种的腐乳在发酵过程中的脂肪酶活力,均显示在后期发酵过程中脂肪酶活力呈现出不断下降的趋势,然而在第20～25d脂肪酶活力呈现出小幅度的反复。使用碱滴定法以聚乙二醇-橄榄油为底物进行验证,得到相同的趋势。     

方便包装腐乳产气原因分析及对策

研究了方便包装腐乳的工艺流程设计以及腐乳后发酵产气因素对发酵过程控制的影响,分析了如食盐浓度、防腐剂、包装材料、后发酵等因素与腐乳产气的相关性,试验结果表明:腐乳后发酵产气是一个必然的过程,添加防腐剂和不同的包装材料对腐乳产气现象的影响不太明显,高浓度的食盐有使后发酵产气滞后的效果,入坛发酵后再包装的腐乳,产气现象明显减少,为方便型腐乳生产提供了依据.     

Effect of temperature and relative humidity on yellowing rate of paddy

The effects of temperature and relative humidity (or water activity) in storage chambers on yellowing rate of paddy were investigated and then an empirical equation for predicting the yellowing rate was developed. Paddy was conditioned using saturated salt solutions at relative humidities ranging from 0.80 to 0.95 and temperatures of 35, 45, 55, 60 and 65°C. The yellowing rate was found to follow the zero order kinetics. The yellowing constant value (k) increased exponentially with temperature and increased linearly with water activity. The magnitude of apparent activation energy varied from 130–145 kJ/mol. A predictive equation for determining yellowing rate was ln k=−δa_w−/T+(γa_w)/T where a_w was water activity (valid from 0.80 to 0.95), T was absolute temperature (valid from 308 to 338 K) and , δ, and γ were constants. The results of variance analysis showed that temperature, water activity and their interaction significantly influenced the yellowing rate of paddy.     

Effects of zeolite formulations on the mortality and progeny production of Sitophilus oryzae and Oryzaephilus surinamensis at different temperature and relative humidity levels

The insecticidal potential of two natural zeolite formulations, Zeocem and Mycostop, was tested against the rice weevil Sitophilus oryzae (L.) and the sawtoothed grain beetle Oryzaephilus surinamensis (L.) on wheat, at the dose rates of 0.25, 0.50, 0.75, and 1 g/kg. The bioassays were carried out under laboratory conditions at three temperature levels (20, 25, and 30^οC) and two relative humidity (r.h.) levels (55, 75%). Mortality was recorded after 7, 14, and 21 d of exposure and progeny production was recorded 65 d later. Complete (100%) mortality was achieved only for O. surinamensis adults with Zeocem for most temperature and r.h. combinations tested. Progeny production for this species was extremely low. In contrast, mortality for S. oryzae was much lower and did not exceed 87% after 21 days at any of the combinations tested, while progeny production was high. For O. surinamensis exposed to Mycostop, mortality after 21 days did not exceed 97% for all temperature and r.h combinations, while overall progeny production was low. Moreover, S. oryzae mortality was generally low and did not exceed 56%, while progeny production for this species was high for most temperature and r.h. combinations. Despite the fact that some general trends were evident, the effect of temperarature and r.h. in zeolite efficacy was often found inconclusive. The results of the present study illustrated the insecticidal potential of zeolites for the contol of certain stored product insect species, which requires additional investigation.