为改善郫县豆瓣成曲酶系活力,对传统酿造酱制品中高产蛋白酶霉菌进行分离鉴定,并以蚕豆瓣为原料,利用单一菌株和复合菌株共培养制曲,比较2种制曲方式主要酶活力。实验中分离到2株高产蛋白酶菌株QM-6和QH-3,经形态学和生物学鉴定分别为米曲霉(Aspergillus oryzae) 和黑曲霉(Aspergillus niger)。米曲霉QM-6和黑曲霉QH-3共培养制曲比单一菌株周期短,且豆瓣成曲曲香浓郁;利用复合菌株制曲,中性蛋白酶、碱性蛋白酶、氨肽酶比单一米曲霉制曲酶活力高,最高分别为1 532、1 164和151 U/g,酸性蛋白酶、纤维素酶和淀粉酶活力最高分别为513、121和574 U/g,比单一米曲霉或黑曲霉制曲提高约1倍。综合制曲过程中的曲料菌丝、孢子、颜色、气味、质地等变化与酶活力比较,米曲霉QM-6和黑曲霉QH-3共培养复合制曲能有效提高蚕豆曲酶系活力,弥补单菌株制曲酶活力不足的缺陷,且成曲品质优良。
In order to improve the enzyme activity of Pixian horsebean paste, fungus with high protease production ability from traditional fermented soy sauce products were isolated and identified. The enzyme activities of two Koji making processes were studied using single strain and multi-strain co-cultivation. Two strains, QM-6 and QH-3, with high-protease production ability were isolated. They were further identified as Aspergillus oryzae and Aspergillus niger, respectively, using morphology and biological analysis. The broad bean koji produced by co-cultivation with A. oryzae QM-6 and A. niger QH-3 had shorter fermentation period and more fragrance, compared with that using single strain cultivation. Moreover, activities of neutral protease, alkaline protease, and aminopeptidase were higher in koji produced by co-cultivation in comparison to those produced by A. oryzae QM-6 alone, with the highest activities of 1 532 U/g, 1 164 U/g, and 151 U/g, respectively. The highest activities of acid protease, cellulase, and amylase were also obtained using co-cultivation (513 U/g, 121 U/g, and 574 U/g, respectively), which were about twice as high as that of single strain fermentation. Based on the changes in mycelium, spores, color, flavor, texture of koji making, and enzyme activities, co-cultivation of A. oryzae QM-6 and A. niger QH-3 in broad bean koji making could effectively improve enzyme activities and qualities of broad bean koji to compensate the shortages of producing koji by single strain fermentation.
[1] 徐炜桢,赵红宇,杨懿,等.郫县豆瓣后发酵过程中细菌群落与呈香物质相关性研究[J]. 食品与发酵工业, 2018, 44(9): 22-28.
[2] 刘云珍,许喜林,刘永琪,等. 豆瓣酱制曲工艺条件优化[J]. 中国酿造, 2016, 35(11):108-111.
[3] HU H L, BRINK J V D, GRUBEN B S, et al. Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi[J]. International Biodeterioration & Biodegradation, 2011, 65(1):248-252.
[4] 林晓华,陈钢,王柳杨. 响应面法优化曲霉型豆豉的双菌种制曲工艺[J]. 食品科学, 2013, 34(3): 233-238.
[5] PENG M Y, LIU J Y, HUANG Y, et al. Effects of a mixed koji culture of Aspergillus oryzae HG-26 and Aspergillus niger HG-35 on the levels of enzymes, antioxidants and phenolic compounds in soy sauce during the fermentation process[J]. Food Science and Technology, 2017, 52(7): 1 585-1 593.
[6] 孟甜,黄韬睿,李玉锋. 响应面法优化郫县豆瓣制曲工艺[J]. 食品科学, 2014, 35(15): 193-197.
[7] 李峰,赵萍,周昌豹,等. 郫县豆瓣高酶活米曲霉选育鉴定及复合菌制曲改善酶系组成研究——郫县豆瓣复合菌制曲改善酶系组成研究[J]. 中国酿造, 2012, 31(12):30-32.
[8] VISHWANATHA K S, RAO A G A, SINGH S A. Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341[J]. Food Chemistry, 2009, 114(2):402-407.
[9] SOUZA P M, WERNECK G, ALIAKBARIAN B, et al. Production, purification and characterization of an aspartic protease from Aspergillus foetidus[J]. Food & Chemical Toxicology, 2017, 109:1 103-1 110.
[10] RAHARDJO Y S P, SIE S, WEBER F J, et al. Effect of low oxygen concentrations on growth and α-amylase production of Aspergillus oryzae in model solid-state fermentation systems[J]. Biomolecular Engineering, 2005, 21(6):163-172.
[11] 余培斌,陈亮亮,张波,等. 双菌种制曲改善黄酒麦曲品质的研究[J]. 食品与发酵工业, 2012, 38(9):1-6.
[12] 魏景超. 真菌鉴定手册[M]. 上海:科学技术出版社, 1979: 405-642.
[13] 陈静,郝伟伟,王春梅,等. 产β-葡萄糖苷酶真菌的筛选鉴定、纯化及酶学性质分析[J]. 食品科学, 2013, 34(5):191-196.
[14] 赵婷婷,卢倩文,宋菲菲,等. 1株产香真菌的筛选及其协同米根霉对米酒发酵的影响[J]. 食品科学, 2017, 38(14):42-48.
[15] 曾林,谭霄,张庆,等.生物转化γ-氨基丁酸酿酒酵母的筛选及其在桑葚酒酿造中的应用[J]. 食品与发酵工业, 2017, 44(6):122-128.
[16] DEY T B, BANERJEE R. Purification, biochemical characterization and application of α-amylase produced by Aspergillus oryzae, IFO-30103[J]. Biocatalysis & Agricultural Biotechnology, 2015, 4(1):83-90.
[17] 丁祖志. 原料预处理工艺对蚕豆酱品质的影响[D]. 无锡:江南大学, 2011.
[18] ZHAO J X, JIANG Q X, XU Y S, et al. Effect of mixed kojis on physiochemical and sensory properties of rapid-fermented fish sauce made with freshwater fish by-products[J]. Food Science and Technology, 2017, 52(9): 2 088-2 096.
[19] 张青,许学书,谢静莉,等. 米曲霉蛋白酶动力学特性的研究[J]. 食品科学, 2007, 28(6):213-217.
[20] BELMESSIKH A, BOUKHALFA H, MECHAKRA-MAZA A, et al. Statistical optimization of culture medium for neutral protease production by Aspergillus oryzae. Comparative study between solid and submerged fermentations on tomato pomace[J]. Journal of the Taiwan Institute of Chemical Engineers, 2013, 44(3): 377-385.
[21] MUSHTAQ Q, IRFAN M, TABSSUM F, et al. Potato peels: A potential food waste for amylase production[J]. Journal of Food Process Engineering, 2017, 40(4): 369-376.
[22] SHI C, WU Y, FANG D, et al. Effect of nanocomposite packaging on postharvest senescence of Flammulina velutipes[J]. Food Chemistry, 2017, 246:414-421.
[23] CHIEN H C, LIN L L, CHAO S H, et al. Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae[J]. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2002, 1 576(1-2): 119-126.
[24] SAHNOUN M, KRIAA M, ELGHARBI F, et al. Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: optimization of culture conditions[J]. International Journal of Biological Macromolecules, 2015, 75:73-80.
[25] 赵强强. 酱油制曲及螺旋藻酱油生产工艺的研究[D]. 青岛:中国海洋大学, 2012.
[26] XU D, PAN L, ZHAO H, et al. Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger[J]. Journal of Industrial Miorobiology & Biotechnology, 2011, 38(9):1 255-1 265.
[27] 李幼筠. “郫县豆瓣”剖析[J]. 中国酿造, 2008, 27(6):19-23.
[28] 何胜华,罗聪,李海梅,等. 米曲霉KFRI 888产中性蛋白酶、α-淀粉酶酶学性质的研究[J]. 食品工业科技, 2006, 27(9):49-50.
[29] 张艳芳. 多菌株制曲促进酶系优化与提高酱油质量的研究[D]. 无锡:江南大学, 2009.
[30] KIM K M, LIM J, LEE J J, et al. Characterization of Aspergillus sojae isolated from meju, Korean traditional fermented soybean bricks[J]. Journal of Microbiology & Biotechnology, 2016, 27(2):251-261.
