Ozturkoglu-Budak Sebnem, Wiebenga Ad, Bron Peter A, de Vries Ronald P
Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, 3584 CT Utrecht, The Netherlands; Department of Dairy Technology, Faculty of Agriculture, University of Ankara, 06110, Ankara, Turkey.
Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, 3584 CT Utrecht, The Netherlands.
Int J Food Microbiol. 2016 Nov 21;237:17-27. doi: 10.1016/j.ijfoodmicro.2016.08.007. Epub 2016 Aug 13.
We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable availability. Future studies will aim to purify these enzymes to test their suitability for use in similar artisanal cheeses or in large scale commercial cheeses.
我们之前鉴定了奶酪成熟过程中存在的微生物群，并观察到迪夫勒洞穴奶酪中存在较高的蛋白酶和脂肪酶活性。为了确定单个分离菌株对酶活性的贡献，我们研究了一系列代表该微生物群的物种的蛋白水解和脂肪水解能力。总共评估了先前从迪夫勒洞穴奶酪中分离出的17株真菌、5株酵母和18株细菌菌株。分别在脱脂乳琼脂和精神蓝脂肪酶琼脂上进行定性蛋白酶和脂肪酶活性检测，从而筛选出用于定量分析的菌株。对于定量分析，将菌株接种在含有从成熟第一天获得的经辐照的迪夫勒洞穴奶酪的基本培养基上。在16株选定的丝状真菌中，短密青霉、洞穴青霉和奥尔森青霉表现出最高的蛋白酶活性，而总状毛霉是最佳的脂肪酶产生菌。解脂耶氏酵母在蛋白酶和脂肪酶活性方面是表现最佳的酵母。在18株细菌菌株中，分别有14株和11株在琼脂平板上显示出蛋白酶和脂肪酶活性。藤黄微球菌、平流层芽孢杆菌、古老短杆菌、嗜冷嗜冷杆菌和蛋白水解假单胞菌表现出最高的蛋白酶和脂肪酶活性。通过胃蛋白酶抑制剂A的特异性抑制作用，酵母和丝状真菌的蛋白酶主要被鉴定为天冬氨酸蛋白酶，而苯甲基磺酰氟（PMSF）的抑制作用表明大多数细菌酶属于丝氨酸型蛋白酶。我们的结果表明，通常具有高凝乳活性的天冬氨酸蛋白酶主要来源于真菌菌株，因此真菌酶似乎更适合用于奶酪工业。由于本研究中所研究的微生物酶成本低且供应稳定，它们可能是动物源凝乳酶（凝乳酶）的替代品。未来的研究旨在纯化这些酶，以测试它们在类似手工奶酪或大规模商业奶酪中的适用性。
