Isolation and Screening of Lipase-Producing Fungi with Hydrolytic Activity

Lipases are enzymes that can be secreted by several microorganisms, making interesting the biodiversity exploration for searching new microorganisms able to produce these enzymes. Many agro-industrial residues can be used as potential substrates for production of enzymes. The main objective of this work was the isolation and screening of microorganisms with potential to produce lipases. Among 24 fungi, five were selected as good lipase producers using tributyrin on agar plates and solid state fermentation of soybean bran. Two of them were isolated from soil samples, another two from soybean bran, and one from dairy products. These fungi were identified by microcultivation technique as from Penicillium and Aspergillus genera. Through random amplified polymorphic DNA technique, the most promising strains could be genetically discriminated, selecting two fungi as good lipase producers but genetically different. One isolated from soybean bran could hydrolyze efficiently triglycerides with fatty acids with different chain length. Another isolated from dairy products was only effective to hydrolyze triglycerides with long-chain fatty acids. Two distinct groups could be verified by means of this technique, comprising the most productive strains and the lowest or nonproductive ones in terms of hydrolytic activity.     

食用菌膳食纤维功能特性及其应用研究进展

膳食纤维对于人体健康有着重要作用。食用菌作为一种新的膳食纤维资源,将其添加到食品中既能发挥其保健功能又可提升食品的品质,具有广阔的开发前景。本文综述食用菌膳食纤维的组成、功能特性、改性方法及其应用现状,为食用菌膳食纤维的综合开发和利用提供一定的参考。     

Purification and characterisation of a mollusc β-d-(1→3)-glucan hydrolase

Two $\text{β-}\text{d}\text{-(1→3)-}\text{glucan}$ hydrolase enzymes of the crystalline style of the surf clam, Spisula solidissima, have been purified and characterised. Both enzymes are predominantly exo-hydrolases and have similar properties, suggesting that one is formed from the other. Purification and characterisation were effected to establish the optimum conditions for hydrolytic activity. This is a prerequisite for the determination of their potential use in the food industry, such as for lytic activity on microorganism cell walls. For example, one ultimate aim of this work is the formulation of an enzyme preparation capable of releasing protein from yeasts and fungi for human consumption.     

Analytical Methods for Pectin Methylesterase Activity Determination: a Review

Pectin methylesterase is an enzyme with an important in vivo role in plants as well as in food industry. This enzyme catalyzes the hydrolysis of methyl ester bounds in pectin which is one of the main components of cell wall in plants, producing methanol and free carboxylic groups. The effect of pectin methylesterase in food quality has been extensively studied, producing desirable effects in texture improvement as well as undesirable effects in some beverages. Likewise, the low methoxyl pectin produced by this enzyme has characteristics that contribute to formulate best quality food products. Pectin methylesterase is a ubiquitously enzyme that presents multiple isoforms, but is not only present in plants; it is also found in fungi, bacteria, and yeast, which have specific chemical and physical characteristics. The latter makes the task of analyzing the wide variety of these enzymes with its specific characteristics difficult. Based on this enzyme relevance and the aforementioned, multiple methods have been developed in order to evaluate pectin methylesterase activity with different research objectives. In this paper, the importance of the enzyme as well as advantages and drawbacks of the different methods will be discussed besides applications and evolution of these will be mentioned. Additionally, this paper will improve the understanding of the systems used in pectin methylesterase activity analysis.     

Bioactive peptides as natural antioxidants in food products – A review

BackgroundDiseases related to oxidative stress and food quality decay are of major concern worldwide as they can lead to economic losses in both public health and food production. The antioxidant peptides, extracted from food proteins, can be explored as natural new drug and food ingredient.Scope and approachAntioxidant peptides are extracted from non-antioxidant precursor proteins from different origin by the activity of either proteolytic microorganisms or isolated enzymes. In the present review, the main sources of bioactive peptides will be discussed. Moreover, the current strategies to obtain these compounds as well as their health benefits and in vivo biological effects will be evaluated. Considerations for further research and development of strategies to increase the knowledge about this underexplored activity of peptides will be also considered.Key findings and conclusionsBioactive peptides' content and profile differ according to the matrix studied and the method used. The utilization of fermentation processes and enzymes has been established to obtain antioxidant bioactive peptides from proteins, being isolated enzymes the most commonly used method, due to their superior control over releasing and obtaining targeted peptides. Antioxidant peptides have the ability to reduce the formation of oxidative products along with the induction of antioxidant enzymes in vivo. However, at this stage of development more in vivo studies are needed in order to evaluate the specific effects on the health of selected antioxidant peptides. In food technology, successful application in meat products strengthens the role of selected peptides as antioxidant additives, although there is a need to observe the effects of the isolated bioactive peptides in other food matrices along with studies to scale-up its production.     

Purification and characterisation of a mollusc hydrolase

Two hydrolase enzymes of the crystalline style of the surf clam, Spisula solidissima, have been purified and characterised. Both enzymes are predominantly exo-hydrolases and have similar properties, suggesting that one is formed from the other. Purification and characterisation were effected to establish the optimum conditions for hydrolytic activity. This is a prerequisite for the determination of their potential use in the food industry, such as for lytic activity on microorganism cell walls. For example, one ultimate aim of this work is the formulation of an enzyme preparation capable of releasing protein from yeasts and fungi for human consumption.     

食药用菌液体发酵产品在食品领域应用进展

食药用菌中富含多种营养和活性成分，应用液体发酵技术可获得品质稳定、产量可控的功能性原料。文章总结了食药用菌液体发酵产品在食品领域中的应用现状，以及食药用菌液体发酵产品在食品领域的相关规定，并对食药用菌液体发酵原料在食品领域推广应用、相关规定的完善和未来加工产品开发的应用前景进行了展望。     

Toxigenic penicillia spoiling frozen chicken nuggets

Frozen chicken nuggets are classified as pre-prepared frozen meals. These products are convenient to consumers as they are easy to prepare and allow for long storage by freezing. Over the years, spoilage of frozen food products caused by fungi has been a continual problem for the food industry since mold can develop when frozen foods are allowed to attain temperatures of − 10 °C, or above. The growth of fungi on the food surface results in economic losses and represents a hazard to public health due to the possibility of mycotoxin production. The aim of this study was to identify the species of filamentous fungi involved in the spoilage of frozen chicken nuggets and determine their ability to produce mycotoxins under laboratorial conditions. A total of 7 samples of frozen chicken nuggets were analyzed by dilution plating in potato dextrose agar (PDA). These products had been returned by customers due to visible mold growth on their surface. The predominant species found were Penicillium glabrum, Penicillium polonicum, Penicillium manginii, Penicillium crustosum, Penicillium commune, and Penicillium solitum. Analysis of the profile of secondary metabolites was carried out in HPLC after growing the isolates in Czapek yeast autolysate agar (CYA) and yeast extract agar and sucrose (YESA) and extracting the extrolites with a solution of ethyl acetate, dichloromethane, methanol, and formic acid. Some isolates of these species showed an ability to synthesize mycotoxins such as cyclopiazonic acid citreoviridin, roquefortine C, penitrem A, and verrucosidin under standard conditions. Considering the occurrence of fungal spoilage in frozen food and the potential hazard involved, more studies on psychrophilic fungi growth in foods stored at low temperatures are necessary.     

大型食药用真菌深层发酵研究进展

食药用真菌的研究已成为现代农业产业关注的新焦点。发酵工程是生物工程中的一个重要方面。食药用真菌应用发酵工程的原理生产逐渐成为现代工业发展的趋势。本文综述了大型食药用真菌深层发酵的历史、发酵的工艺与特点。大型食药用真菌深层发酵的应用包括:制备液体菌种用于食药用真菌栽培、将发酵产物提取后用于制备保健食品和药品、代谢产物生产食品或饲料蛋白、制备保健美容化妆品、培育食药用真菌子实体等。食药用真菌利用深层发酵法生产食用菌菌丝体或者其代谢产物都可以在短期内得到。其生产效率远远大于农业栽培法,此外,深层培养能够更方便有效地从发酵液中提取其代谢产物。因此,深层培养将成为现代食药用真菌生产的趋势。     

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 (11) Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. PRACTICAL APPLICATION: Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry.     

食用菌富集重金属因素及其控制技术研究进展

食用菌是一类大型真菌,具有生长快、生物转化率高等特性,可将无机物高效、快速转化为有机可利用的功能性食物资源,富硒、富锗、高氨基酸等功能食用菌产品愈来愈风靡世界,但研究发现食用菌在快速转化生物质的同时,也大量富集了对人体有害的重金属、毒素等物质。论文综述了食用菌吸收和富集重金属基本原理、影响重金属在食用菌中富集的主要因素及控制和减低食用菌富集重金属的主要措施,这对进一步认识食用菌转化生物质过程、富集有害重金属的机理有理论指导意义,也对生产功能性食用菌,为人类提供健康食品具有实践价值。     

Isolation and Characterization of Two Types of Xyloglucanases from a Phytopathogenic Fungus,Verticillium dahliae

Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 3.2.1.151).     

PCR ITS-RFLP: A useful method for identifying filamentous fungi isolates on grapes

Restriction digestion analysis of the ITS products was tested as an easy method to identify isolates of filamentous fungi on grapes. Endonucleases SduI, HinfI, MseI, HaeIII were used. Endonucleases BfmI, Cfr9I, Hpy188I, MaeII or PspGI were used as necessary to complete discrimination. The 43 species studied generated 42 different composite profiles. Only the species P. thomii and P. glabrum gave the same composite profile. 96.3% strains tested could be differentiated to the species level with only four enzymes. Hundred ninety nine strains of filamentous fungi were isolated from various vineyards in Burgundy and identified by this method. Penicillium (58.5%) was the genus the most frequently isolated and no strains of the genus Aspergillus was isolated. P. spinolusum was the most isolated species of Penicillium (22.70%). The species C. cladiosporioides, B. cinerea, E. nigrum, A. alternata, T. koningiopsis, P. diplodiella, C. herbarum, A. alternatum, T. cucumeris and F. oxysporum were also isolated. This technique is a rapid and reliable method appropriate for routine identification of filamentous fungi. This can be used to screen large numbers of isolates from various environments in a short time. This is the first exhaustive study of fungal diversity at species level in vineyard.     

食用菌抗氧化活性成分及其抗氧化作用机制研究进展

食用菌不仅味道鲜美,而且具有多种生物活性,其中最重要的活性之一就是抗氧化活性。食用菌中的抗氧化活性成分主要包括多糖类、多酚类、萜类和硒类等。文章综述了国内外食用菌抗氧化成分及其抗氧化作用机制的研究现状,旨在进一步发掘和利用食用菌的食药用价值,扩大其在食品和医药领域的应用范围。     

食用菌产胞外纤维素半纤维素降解酶类的研究进展

我国是食用菌生产大国。食用菌可以分泌降解纤维素、半纤维素和木质素相关的酶,能够利用秸秆、木屑、棉籽壳等多种农业废弃物,具有广泛的应用前景。本文以食用菌所产的主要胞外水解酶类纤维素酶和木聚糖酶为研究对象,从其种类、发酵条件、理化性质和基因克隆等方面综述了食用菌产胞外水解酶的研究现状。     

Pyrrolizidinalkaloide in Honig und Pollen

Pyrrolizidine alkaloids (PA) are secondary plant constituents which comprise about 370 different structures, occurring in two major forms, a tertiary form and the corresponding N-oxide. PA containing a 1,2-double bond are pre-toxins and metabolically activated by the action of hepatic P-450 enzymes to acute toxic and genotoxic pyrroles. Beside the acute toxic effects, the genotoxic and tumorigenicity potential of PA was demonstrated in some eukaryotic model systems. Recently, the potentially PA contamination of food and feeding stuff attracted recurrent great deals of attention. Humans are exposed to these toxins by consumption of herbal medicine, herbal teas, dietary supplements or food containing PA-plant-material. In numerous studies the potential threat to human health by PA is stated. In pharmaceuticals, the use of these plants is regulated. Considering the values observed especially in authentic honey from PA producing plants and pollen products, the results provoke the discussion of an international regulation of PA in food.     

Degradation of Ochratoxin A by Proteases and by a Crude Enzyme of Aspergillus niger

Ochratoxin A is a mycotoxin present in food commodities as cereals, wine, coffee, figs, dried vine fruits or beer and in feeds for animals. The enhancement of its conversion into ochratoxin α is considered to be a way to reduce its presence in the body and, therefore, its toxicity. In this paper we report the ability of several commercial proteases to hydrolyze ochratoxin A into ochratoxin α in different amounts. After an incubation period of 25 h., a significant hydrolytic activity at pH 7.5 for Protease A (87.3%), and for Pancreatin (43.4%) was detected. At pH 3.0, a weak hydrolytic activity was detected for Prolyve PAC (3%). None of the other commercial enzymes tested were able to hydrolyze ochratoxin A in the tested conditions. Also, the isolation of an enzyme extract from an Aspergillus niger strain with very strong ochratoxin A hydrolytic activity at pH 7.5 (99.8%) is reported. This activity is similar to the activity detected in Protease A. Data about the inhibition effect of ethylenediaminetetraacetic acid and phenylmethanesulfonyl fluoride on the involved hydrolytic enzymes showed that enzymes involved in ochratoxin A hydrolysis are metalloproteins.     

Antimicrobial enzymes: Applications and future potential in the food industry

Antimicrobial enzymes are ubiquitous in nature, playing a significant role in the defense mechanisms of living organisms against infection by bacteria and fungi. Hydrolytic antimicrobial enzymes function by degrading key structural components of the cell walls of bacteria and/or fungi, whereas antimicrobial oxidoreductases exert their effects by the generation in situ of reactive molecules. The potential of these enzymes in food preservation is still far from realized at present.