Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.     

液体乳中嗜冷菌数的测定

研究了液体乳中嗜冷菌的快速菌落计数方法，通过实验得出，样品中嗜冷菌在21℃条件下培养48h，结果表明，该方法快速，准确，简便易行。     

微生物发酵法生产γ-癸内酯

γ-癸内酯是在香料工业中普遍应用的一种风味物质,随着人们对绿色产品的追求,天然γ-癸内酯的生产得到了香精香料行业的重视。本文通过对文献报道的几种产γ-癸内脂的微生物的研究比较发现,YarrowialipolyticaAS2.1405较为适宜发酵转化蓖麻油生产γ-癸内酯,产率约为0.1%。      

Heat-stable proteases from psychrotrophic pseudomonads: secondary structure and heat stability

Circular dichroism (CD) spectral analysis of a purified protease of Pseudomonas fluorescens T16 was carried out at different temperatures to determine the relationship between its secondary structure and its heat-stability. The protease protein was found to be 33% β-structure and 67% random coil. The protein lacked α-helical structure. Unfolding of the enzyme molecule was increased from 25°C, with maximum unfolding at 45°C. The enzyme exhibited maximum inactivation (low temperature inactivation [LTI] phenomenon) at temperatures between 45 and 55°C. At higher temperatures (60–95°C) the protease appears to undergo an additional conformational change to a more ordered stable structure. Metal ions such as Ca²⁺ appeared to be involved in structural stabilization and are required for protection against heat inactivation. The comparisons of amino acid composition, sequence homology, hydrophobicity index and polarities of heat-stable proteases were made to predict their heat-stabilities.     

Production of indole by wine-associated microorganisms under oenological conditions

A high concentration of indole has been linked to ‘plastic-like’ off-flavour in wines, predominantly in wines produced under sluggish fermentation conditions. The purpose of this study was to determine the ability of yeast and bacteria to form indole and whether tryptophan was required for indole accumulation during winemaking. Wine-associated yeast and bacteria species (Saccharomyces cerevisiae, Saccharomyces bayanus, Candida stellata, Hanseniaspora uvarum, Kluyveromyces thermoloterans, Oenococcus oeni, Lactobacillus lindneri, Pediococcus cerevisiae and Pediococcus parvulus) were screened for their potential to generate indole during alcoholic or malolactic fermentation. Tryptophan was required for the accumulation of indole in chemically defined medium, and all yeast and bacteria fermentations were able to accumulate indole. C. stellata showed the greatest potential for indole formation (1033 μg/L) and among the bacteria, the highest concentration was generated by L. lindneri (370 μg/L). Whether primary fermentation is the principle cause of indole formation remains to be determined. We hypothesise that during an efficient fermentation, indole is removed through catabolic metabolism, but, when a sluggish fermentation arises, non-Saccharomyces species might produce excess indole that is still present by end of fermentation.     

Production of aldehyde oxidases by microorganisms and their enzymatic properties

In order to establish an efficient process to decompose environmentally toxic aldehydes, dioxygen-dependent aldehyde oxidase (ALOD) from microorganisms was first sought, and some bacteria and actinomycetes were found to produce the enzyme in their cells. Methylobacillus sp., Pseudomonas sp. and Streptomyces moderates were selected as the representative ALOD-producing strains and their enzymes were partially purified and characterized. The three ALODs could oxidize a wide range of aldehydes including formaldehyde, aliphatic aldehydes, and aromatic aldehydes, though their preferences differ depending on their producing strains. The other enzymatic properties were also determined with regard to their producing strains. Methylobacillus sp. ALOD had the most acidic optimum pH for its activity and stability and Pseudomonas sp. ALOD had the highest stability against heat treatment. Three native ALODs had molecular weights ranging from 140 to 148 kDa and were composed of three subunits of different sizes: large (85 to 88 kDa), medium-sized (37 to 39 kDa) and small (18 to 23 kDa).     

Adherence of psychrotrophic bacteria to dairy equipment surfaces.

Psychrotrophic bacteria isolated from raw milk were tested for their ability to adhere to steel, two types of rubber, and glass, materials employed in the construction of milking equipment. The adherence assays were carried out by exposure of the materials to radioactively labelled bacteria in both a buffering solution (Ringer's) and milk. The degree of adherence of Gram-positive bacteria was lower (P less than 0.001) than that of Gram-negative bacteria. Glass was the material least prone to bacterial adherence (P less than 0.001); there were no significant differences between the other three materials. Milk was found to inhibit adhesion significantly (P less than 0.05), this inhibition being more evident with the most adherent bacteria. There was no statistically significant correlation between bacterial surface hydrophobicity and adherence. Our results suggest that intrinsic bacterial adherence cannot be considered a relevant factor in the contamination of milking equipment.     

Cold-active lipolytic activity of psychrotrophic Acinetobacter sp. strain no. 6

A lipolytic bacterium, strain no. 6, was isolated from Siberian tundra soil. It was a gram-negative coccoid rod capable of growing at 4 degrees C but not at 37 degrees C and was identified as a psychrotrophic strain of the genus Acinetobacter. Strain no. 6 extracellularly produced a lipolytic enzyme that efficiently hydrolyzed triglycerides such as soybean oil during bacterial growth even at 4 degrees C; it degraded 60% of added soybean oil (initial concentration, 1% w/v) after cultivation in LB medium at 4 degrees C for 7 d. Thus, the bacterium is potentially applicable to in-situ bioremediation or bioaugumentation of fat-contaminated cold environments. We partially purified the lipolytic enzyme from the culture filtrate by acetone fractionation and characterized it. The enzyme preparation contained a single species of cold-active lipase with significant activity at 4 degrees C, which was 57% of the activity at the optimum temperature (20 degrees C). The enzyme showed a broad specificity toward the acyl group (C8-C16) of substrate ethyl esters.     

Intracellular proteases of Thermoactinomyces vulgaris. 2. Biochemical characterization of proteases in cell extracts

The intracellular proteases (IP) of the cellular extract of Thermoactinomyces vulgaris are characterized as to the biochemical properties compared with the corresponding extracellular proteases (EP). According to that, the storage stability and the temperature and pH behaviour (optimum, stability) of both of the proteases are identical. Nevertheless, differences were detected between IP and EP after the action of several effectors and different substrates. As could be seen after a column chromatographic separation of the IP of the cellular extract, it is composed of at least 3 proteases, two of them are serine proteases which can be inhibited moreover unspecifically by p-chloromercuri benzoate. The purified proteases (IP) are very instable and therefore not yet characterized in detail.     

Volatile organic compounds associated with milk spoilage by psychrotrophic bacteria

The volatile organic compounds of milk contaminated with psychrotrophic bacteria were studied by HS‐SPME and GC/MS. Pseudomonas fluorescens PS14, Pseudomonas fragi PS55, Pseudomonas mosselii PS39, Pseudomonas rhodesiae PS62 and Serratia marcescens S92 were inoculated in sterilised milk (2.5% fat) stored at either 5 °C or 10 °C. A total of 47 volatile organic compounds (VOCs) belonging to seven chemical groups were identified in the spoiled milk. Volatile organic compound patterns peculiar to the inoculate bacterial strains were highlighted. 3‐Methylbutan‐1‐ol, 2 methylpropan‐1‐ol, 3‐hydroxybutan‐2‐one, butan‐2,3‐dione, butanoic and hexanoic acids were revealed as potential chemical spoilage indexes of milk spoilage due to the activity of the five psychrotrophic strains studied.     

蛋白酶对豆乳的凝固作用研究

以凝固时间和凝固强度为指标,考察了13种蛋白酶的豆乳凝固能力,发现胃蛋白酶、凝乳酶和风味蛋白酶没有豆乳凝固活性,Alcalase、木瓜蛋白酶和菠萝蛋白酶具有较强的豆乳凝固能力。各种蛋白酶的最适豆乳凝固温度范围比最适水解温度范围高20℃左右,豆乳凝固能力随pH的降低而增强。40℃和70℃下用蛋白酶作凝固剂得到的豆乳凝块破断强度最大为105.2Pa和148.2Pa,小于用氯化钙作凝固剂的豆乳凝块的破断强度(40℃,142.4Pa和70℃,198.4Pa),而且用酶凝固的豆乳凝块黏度较大,乳清不易排出。Alcalase具有较强的豆乳凝固能力,而且形成的豆乳凝块质地细腻,无苦味,具有一定的实际应用价值。     

Softening of gluten by wheat proteases

Experimental evidence has been obtained to support the hypothesis that gluten softening is a result of peptide bond scission catalysed by proteolytic enzymes. Extensive softening of gluten is observed even though very few peptide bonds are broken. These effects will probably be more noticeable if the wheat from which the flour is milled has begun to germinate. It is suggested that endogenous bacterial and mould proteases will not usually play a significant part in modem breadmaking processes.     

Production of poly-beta-hydroxybutyric acid by microorganisms accumulated from river water using a two-stage perfusion culture system

The perfusion culture system using a shaken ceramic membrane flask (SCMF) was employed to accumulate microorganisms separated from river water and to produce poly-beta-hydroxybutyric acid (PHB). Using a two-step culture method with a single SCMF, river microorganisms were cultured by separately feeding four representative carbon sources, n-propanol, lactic acid, methanol, and formic acid. After 140 h culture, the cell concentration and PHB content respectively reached 43 g/l and 35% when a propanol medium was fed. Using a two-stage perfusion culture with twin SCMFs, the seed cell mass was increased in the first SCMF and then supplied to the second flask for PHB production. As a consequence, the cellular PHB content rose to 51% in the second SCMF, while the cell concentration gradually increased to 25 g/l after 175 h perfusion culture. These results demonstrated the utility of the two-stage perfusion culture system for developing a cheap means of producing PHB coincident with wastewater treatment.     

Modification of plant proteins by immobilized proteases

A potential application of plant proteins could be a replacement of animal proteins now in use in the food industry on the basis of certain specific functional properties plant proteins have. Modification of the chemical structure of selected plant proteins is needed to replace more expensive animal proteins as food ingredients that have specific functional characteristics. Structure modification may be achieved by physical, chemical, or microbiological methods, or by a combination of these. Immobilized enzyme techniques offer significant advantages for protein modification. Knowledge of the molecular properties of plant proteins is essential to understand the basis of protein functionality, to modify proteins so that they acquire desirable functional properties, and to predict potential applications of modified plant proteins. This paper reviews all the above mentioned aspects of plant protein chemistry and potential utilization.     

蛋白酶酶解酱油沉淀的工艺研究

对蛋白酶酶解低盐固态和原池浇淋2种酿造工艺的酱油沉淀进行了研究,分析了中性蛋白酶的加酶量、pH值、酶解时间、料液比对蛋白质水解度的影响,并通过正交试验优化了中性蛋白酶酶解酱油沉淀的工艺条件,确定了工艺参数.其最佳工艺条件为原池浇淋酱油沉淀的最佳酶解条件为料液比1∶3,酶解6h,加酶量为4000U/g原料,pH值为6.5,水解度可达到43.8％;低盐固态酱油沉淀的最佳酶解条件为料液比1∶3,酶解8h,加酶量为6000U/g原料,pH值为6.5,水解度可达到53.8％.方差分析表明,4因素均对水解度有显著影响.利用中性蛋白酶可较有效地酶解酱油沉淀.     

双酶水解制备大豆多肽的研究

研究了双酶分步水解和双酶混合同步水解制备大豆多肽的效果,对双酶分步作用的加酶顺序以及在加第二种酶之前是否需要使第一种酶失活进行了讨论.     

原料乳中嗜冷菌的检测

针对原料乳中嗜冷菌的一种快速检测方法进行了初步研究。由于嗜冷菌能在乳中产生大量的耐热性脂肪酶,所以我们在脂肪酶活与嗜冷菌数之间建立了一种线性关系,进而通过4-硝基苯酚游离释放法测定脂肪酶的活力来得到嗜冷菌数。     

微生物凝乳酶的研究进展

介绍了凝乳酶的分子特性和凝乳机理,综述了微生物凝乳酶的研究进展,最后介绍了利用基因工程生产凝乳酶和蛋白质工程改造凝乳酶的研究状况.并对凝乳酶的未来生产与开发作了展望.