我国部分地区自然发酵豆酱中真菌的多样性和真菌毒素的检测
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摘要
豆酱(Soybean paste)起源于我国,距今已有3000余年的历史。迄今为止,自然发酵的家庭自制豆酱仍然是我国众多地区广为食用的习惯性食品。由于开放式的制作环境,自然发酵豆酱中必然贮藏着丰富的真菌资源;同时大量真菌的滋生导致有害真菌毒素的产生,对食用者的健康还具有潜在的危害。有关豆酱中细菌和酵母菌方面的研究较多,然而,由于真菌分类专业性较强,真菌毒素的检测设备昂贵、操作复杂,一直以来研究甚少。
本文首次对豆酱中的真菌进行了系统的分类学研究,获得25属90种真菌;并对我国不同地区、不同制作方式和不同发酵时期的豆酱中的真菌区系进行了系统的分析,得出了规律性的结论;首次采用国家出入境检验检疫系统应用的先进的免疫亲和柱净化-高效液相色谱法,对可疑样品进行了主要真菌毒素的检测,发现豆酱中存在4种有害真菌毒素,表明家庭自制豆酱具有潜在的安全隐患;同时,对研究获得的曲霉Aspergillus和毛霉Mucor进行了产蛋白酶活性测定,发现62%的Aspergillus和33%的Mucor具有产蛋白酶活性;据此对1株Aspergillus和3株Mucor进行了产酶特性研究,筛选获得的高产蛋白酶菌株有望用于豆酱的工厂化生产或其它经济领域。具体结果如下:
1.豆酱中的真菌种类
采集了东北三省和内蒙古自治区以及河南、河北、四川、安徽、贵州、福建、江西计11个省(自治区)72个地区具有地方特色的家庭自制豆酱样品251份,长春市双阳区昌隆酿造厂根霉纯种发酵生产、大连市黑土地酿造厂米曲霉纯种发酵生产和锦州豆娃酱业有限公司自然发酵生产的豆酱样品26份,以及市场销售的不同品牌的豆酱样品11份。从上述288份豆酱样品中分离和鉴定出真菌25属90种,其中接合菌4属13种,子囊菌3属6种,有丝分裂孢子真菌18属71种。中国新记录种2个,即短链镰孢菌Fusarium brevicatenulatum和嗜粪青霉Penicillium coprophilum;曲霉新变种2个,即黑曲霉金州变种Aspergillus nigervar.jinzhouanum和棒曲霉金色变种A.clavatusvar.aureus。论文对每种真菌作了详细的形态描述及样品来源记录,同时附有显微形态图片,附有曲霉Aspergillus、曲霉有性型属和青霉Penicillium的菌落图版,8种主要的Aspergillus附有分生孢子的扫描电镜照片。
2.豆酱中疑难菌株的鉴定
Aspergillus和Penicillium中的许多种类形态相近,传统形态学的方法难以准确鉴定。黄曲霉A.flavus和米曲霉A.oryzae形态极为相似,但A.flavus可产生黄曲霉毒素(aflatoxins),而A.oryzae通常用于发酵食品生产,故准确的鉴定非常重要。文中对A.flavus和A.oryzae进行了多次反复观察,并与A.oryzae标准菌株进行比较,同时对其ITSrDNA基因序列进行了分析,确认了二者的分类地位。测定了黑曲霉金州变种A.niger var.jinzhouanum和棒曲霉金色变种A.clavatus var.aureus以及嗜粪青霉P.coprophilum的β-tubulin基因序列,并进行序列比对,确定了分类地位。A.niger var.jinzhouanum的分生孢子梗上具有明显的小附枝,此为对Aspergillus的新特征的首次描述,文中对该菌的形态进行了详细描述,并附有扫描电镜照片。
3.自然发酵豆酱中真菌的多样性分析
采用DPS数据处理系统,对不同地区、不同制作方法、不同发酵时期的家庭自制豆酱的真菌区系和多样性进行了分析,认为豆酱中真菌的多样性特点受环境条件和制作方式影响较大。分析结果表明,青霉Penicillium、曲霉Aspergillus、毛霉Mucor、镰孢菌Fusarium、枝顶孢Acremonium、根霉Rhizopus、帚霉Scopulariopsis、木霉Trichoderma和芽枝孢Cladosporium为豆酱中的优势真菌类群。红曲菌Monacsus在传统发酵食品中具有极其重要的作用,本试验只分离到少量的Monacsus,表明该菌在自然发酵豆酱中不能形成优势。自制豆酱中未分离到A.oryzae而分离到大量的形态上与之相近的A.flavus,表明自制豆酱中不利于有益A.oryzae的生长,这从分类学的角度表明自制豆酱存在安全隐患。在豆酱制作过程中,真菌的种类和数量变化较大。发酵初期Mucor生长占据优势,后期随着酱醅含水量降低,Mucor逐渐被Penicillium、Aspergillus、Fusarium等种类代替,但Mucor伴随着整个豆酱制作过程。Aspergillus在豆酱的盐水发酵和酱醪成熟期均具有优势,说明Aspergillus在豆酱发酵中具有重要作用。制作方式和家庭制作小环境对豆酱的品质影响较大,庄河地区的“部分制曲法”自制豆酱减少了有害杂菌的滋生,值得推广借鉴;制作环境卫生条件差的家庭自制豆酱中可分离到更多有害杂菌,影响豆酱的品质和风味,甚至会威胁食用者的健康。
4.豆酱中主要真菌毒素的检测
对21个家庭自制豆酱样品和2个工厂生产的样品进行了主要真菌毒素的检测,采用免疫亲和柱净化-荧光光度法检测到13个样品中含有黄曲霉毒素(AF),占所检测样品的56.5%;采用免疫亲和柱净化-高效液相色谱法检测到6个样品中含有黄曲霉毒素B_1(AFB_1),占所检测样品的26%,同时分别各有1个样品相应检测到玉米赤霉烯酮(ZEA)、T-2毒素和呕吐毒素(DON),检出率较低。检测结果表明,家庭自制豆酱具有潜在的危害,应改善家庭自制豆酱的制作方法,注意制作环境的安全卫生,减少制作过程中有害杂菌的污染,由此控制和减少豆酱中有害真菌毒素的产生。
5.豆酱中产蛋白酶菌株的筛选对自然发酵豆酱中获得的34株Aspergillus和24株Mucor进行产蛋白酶活性菌株筛选,发现共21株Aspergillus和8株Mucor具有产蛋白酶活性,分别占所测试菌株的62%和33%,表明自然发酵豆酱中的真菌资源具有极大的应用潜力。
6.蛋白酶特性研究对菌株Asp-195v进行了产蛋白酶特性研究,获得了Asp-195v的最佳培养基配比,即麸皮:豆粕为7:3,含水量为40%,pH值为7.0。改变培养温度、培养时间,并考察反应温度、pH值、盐度对蛋白酶活性的影响,发现最适培养温度为28℃,最适培养时间为48h,最适反应温度为50℃,最适反应条件为中性,盐度影响蛋白酶活性,但本菌株对盐有一定的耐受力,适宜于豆酱的发酵环境。
对筛选获得的3株Mucor进行蛋白酶活性测定。结果表明,M-339、M-513和M-808菌株相应在26℃、麸皮:水为1:1.1,28℃、麸皮:水为1:1.2,30℃、麸皮:水为1:1.2时产蛋白酶活性最高。研究所测菌株的产酶活性特点与相应的豆酱发酵环境一致。
Soybean paste is one of the commonest traditional condiments in China.The origin of Chinese soybean paste can be traced back to Zhou Dynasty,about 3,000 years ago.Untill now, the naturally fermented home-made soybean paste was widely consumed by Chinese people. Due to the open fermenting process,there are rich fungi in the home-made soybean paste and it is inevitable to avoid the contamination of mycotoxins and may harm the consumer's health. Many studies were conducted on bacteria and yeast in soybean paste,but its fungal studies were scarce.
This study is the first systematic research on fungal diversity in naturally fermented soybean paste.The comprehensive analysis of fungal diversity was performed in aspects of paste making regions,methods and fermentation time.It is also the first detection of harmful mycotoxions in soybean paste by high-performance liquid chromatography after immunoaffinity chromatogaphy cleanup.The result shows that the presence of four mycotoxions marks the potential danger of the home-made soybean paste.The study of protease activity of some Aspergillus and Mucor isolates was also conducted,it is found that 62%of Aspergillus and 33%of Mucor possessed protease activity.The protease features of one Aspergillus strain and three Mucor strains were studied.The total results are listed as follows:
The fungal species isolated from soybean paste
Totally 280 samples of soybean pastes were collected and investigated in this study, including 251 samples of home-made products in 72 regions of 11 provinces including Northeastern China,Inner Mongolia,Henan,Hebei,Sichuan,Anhui,Guizhou,Fujian and Jiangxi,26 samples of industry products from Changlong brewing factory in Changchun, Heitudi brewing factory in Dalian and Douwa brewing factory in Jinzhou,and 11 samples from markets.At last 90 fungal species belonging to 25 genera were isolated and identified. Of which 13 species of four genera are Zygomyctes,6 species of three genera Ascomyctes and 71 species of 18 genera Anamorphic fungi.Fusarium brevicatenulatum and Penicillium coprophilum are two newly recorded species for China,Aspergillus niger var.jinzhouanum and A.clavatus var.aureus are named as new taxa for their specific morphology and molecular evidence.Each fungal species was morphologically illustrated and described with microscopic photos.The species of Aspergillus and Penicillium were provided with clony features on standard media.The SEM photos of 8 main Aspergillus species were provided.
The molecular identification of some suspicious fungal strains
Such Aspergillus species as A.flavus and A.oryzae are easily misidentified due to their similar morphological characteristics.It is well-known that A.flavus is the aflatoxins producer and A.oryzae the common fungus used in food industry.In order to correctly identify the two species,the comparative studies were conducted on their cultures on standard media and rDNA ITS sequences.The comparative studies reveal that A.flavus occurred more frequently on soybean paste than A.oryzae.Theβ-tubulin sequence analyses were performed to support the new taxa of A.niger var.jinzhouanum and A.clavatus var.aureus also as P.coprophilum. A.niger var.jinzhouanum is characterized by adnate or verticillate appendages on conidiophore stalks just below the vesicles.The appedages on conidiophore stalks are new descriptive characters for Aspergillus in literature.
The analysis of fungal diversity of naturally fermented soybean pastes
The research data were analyzed with the software of data processing system(2002).The results indicate that the fungal diversity was influenced by the paste making environment and methods.The predominant fungal groups in home-made pastes were Penicillium,Aspergillus, Mucor,Fusarium,Acremonium,Rhizopus,Scopulariopsis,Trichoderma and Cladosporium, while the commercial fungi of Monacsus and A.oryzae were seldom encountered.It impies that home-made soybean pastes were not good media for the two economic fungi.The continous investigation of fungi on home-made pasets shows that the fungal species population and amount had changed during the whole fermenttion course.The fungal population of soybean pastes were dominated by Mucor in early fermentation stage,and then replaced by Penicillium,Aspergillus and Fusarium with the decrease of moisture in pastes. The presence of Aspergillus from brine fermentation until mature stage of soybean pastes indicates its important role palyed in the soybean paste making process.The making methods and microenvironments influenced the quality of the soybean pastes.The method of“partial koji”in Zhuanghe which did not favor the vigorous multiplication of poisonous fungi should be recommended for the home-made soybean pastes.
Detection of main mycotoxins in soybean pastes
The mycotoxin content of 21 home-made samples and two industry samples were examined by high performance liquid chromatography after immunoafinity column clean-up. The results shows that 13 samples of soybean pastes(56.5%)were detected to contain aflaxoxin,six samples(26%)contain aflatoxin B_1.The frequence of ZEA,T-2 and DON contamination was low respectively with only one sample contaminated with each one toxin. The results indicate that the potential danger exists in home-made soybean pastes.
Screening of protease-producing fungal strains in soybean pastes
34 strains of Aspergillus spp.and 24 Mucor spp.isolated from naturally fermented soybean paste were screened and 21 strains(62%)of Aspergillus spp.and 8 strains(33%)of Mucor spp.were detected with high protease activity,it implies that many economic fungi from home-made soybean paste could be commercially utilized in industry.
Studies on the protease feature of protease-producing strains
The protease feature of Asp-195v isolate was studied.The optimum medium is bran: bean cake=7:3 with 40%moisture content and pH 7.0.The optimum conditions for the protease production were 28℃and 48h of incubation and neutral pH condition.The favorable temperature for protease reaction was 50℃.The salt concentration influenced the protease activity.
The protease activities of 3 Mucor strains were investigated.It was found that the optimum incubation conditions for protease production of the M-339,M-513 and M-808 were bran:water=1:1.1 at 26℃;bran:water=1:1.2 at 28℃and bran:water=1:1.3 at 30℃respectively.The feature of protease production for each Mucor isolate was in accordance with their fermentation environment.
本文首次对豆酱中的真菌进行了系统的分类学研究,获得25属90种真菌;并对我国不同地区、不同制作方式和不同发酵时期的豆酱中的真菌区系进行了系统的分析,得出了规律性的结论;首次采用国家出入境检验检疫系统应用的先进的免疫亲和柱净化-高效液相色谱法,对可疑样品进行了主要真菌毒素的检测,发现豆酱中存在4种有害真菌毒素,表明家庭自制豆酱具有潜在的安全隐患;同时,对研究获得的曲霉Aspergillus和毛霉Mucor进行了产蛋白酶活性测定,发现62%的Aspergillus和33%的Mucor具有产蛋白酶活性;据此对1株Aspergillus和3株Mucor进行了产酶特性研究,筛选获得的高产蛋白酶菌株有望用于豆酱的工厂化生产或其它经济领域。具体结果如下:
1.豆酱中的真菌种类
采集了东北三省和内蒙古自治区以及河南、河北、四川、安徽、贵州、福建、江西计11个省(自治区)72个地区具有地方特色的家庭自制豆酱样品251份,长春市双阳区昌隆酿造厂根霉纯种发酵生产、大连市黑土地酿造厂米曲霉纯种发酵生产和锦州豆娃酱业有限公司自然发酵生产的豆酱样品26份,以及市场销售的不同品牌的豆酱样品11份。从上述288份豆酱样品中分离和鉴定出真菌25属90种,其中接合菌4属13种,子囊菌3属6种,有丝分裂孢子真菌18属71种。中国新记录种2个,即短链镰孢菌Fusarium brevicatenulatum和嗜粪青霉Penicillium coprophilum;曲霉新变种2个,即黑曲霉金州变种Aspergillus nigervar.jinzhouanum和棒曲霉金色变种A.clavatusvar.aureus。论文对每种真菌作了详细的形态描述及样品来源记录,同时附有显微形态图片,附有曲霉Aspergillus、曲霉有性型属和青霉Penicillium的菌落图版,8种主要的Aspergillus附有分生孢子的扫描电镜照片。
2.豆酱中疑难菌株的鉴定
Aspergillus和Penicillium中的许多种类形态相近,传统形态学的方法难以准确鉴定。黄曲霉A.flavus和米曲霉A.oryzae形态极为相似,但A.flavus可产生黄曲霉毒素(aflatoxins),而A.oryzae通常用于发酵食品生产,故准确的鉴定非常重要。文中对A.flavus和A.oryzae进行了多次反复观察,并与A.oryzae标准菌株进行比较,同时对其ITSrDNA基因序列进行了分析,确认了二者的分类地位。测定了黑曲霉金州变种A.niger var.jinzhouanum和棒曲霉金色变种A.clavatus var.aureus以及嗜粪青霉P.coprophilum的β-tubulin基因序列,并进行序列比对,确定了分类地位。A.niger var.jinzhouanum的分生孢子梗上具有明显的小附枝,此为对Aspergillus的新特征的首次描述,文中对该菌的形态进行了详细描述,并附有扫描电镜照片。
3.自然发酵豆酱中真菌的多样性分析
采用DPS数据处理系统,对不同地区、不同制作方法、不同发酵时期的家庭自制豆酱的真菌区系和多样性进行了分析,认为豆酱中真菌的多样性特点受环境条件和制作方式影响较大。分析结果表明,青霉Penicillium、曲霉Aspergillus、毛霉Mucor、镰孢菌Fusarium、枝顶孢Acremonium、根霉Rhizopus、帚霉Scopulariopsis、木霉Trichoderma和芽枝孢Cladosporium为豆酱中的优势真菌类群。红曲菌Monacsus在传统发酵食品中具有极其重要的作用,本试验只分离到少量的Monacsus,表明该菌在自然发酵豆酱中不能形成优势。自制豆酱中未分离到A.oryzae而分离到大量的形态上与之相近的A.flavus,表明自制豆酱中不利于有益A.oryzae的生长,这从分类学的角度表明自制豆酱存在安全隐患。在豆酱制作过程中,真菌的种类和数量变化较大。发酵初期Mucor生长占据优势,后期随着酱醅含水量降低,Mucor逐渐被Penicillium、Aspergillus、Fusarium等种类代替,但Mucor伴随着整个豆酱制作过程。Aspergillus在豆酱的盐水发酵和酱醪成熟期均具有优势,说明Aspergillus在豆酱发酵中具有重要作用。制作方式和家庭制作小环境对豆酱的品质影响较大,庄河地区的“部分制曲法”自制豆酱减少了有害杂菌的滋生,值得推广借鉴;制作环境卫生条件差的家庭自制豆酱中可分离到更多有害杂菌,影响豆酱的品质和风味,甚至会威胁食用者的健康。
4.豆酱中主要真菌毒素的检测
对21个家庭自制豆酱样品和2个工厂生产的样品进行了主要真菌毒素的检测,采用免疫亲和柱净化-荧光光度法检测到13个样品中含有黄曲霉毒素(AF),占所检测样品的56.5%;采用免疫亲和柱净化-高效液相色谱法检测到6个样品中含有黄曲霉毒素B_1(AFB_1),占所检测样品的26%,同时分别各有1个样品相应检测到玉米赤霉烯酮(ZEA)、T-2毒素和呕吐毒素(DON),检出率较低。检测结果表明,家庭自制豆酱具有潜在的危害,应改善家庭自制豆酱的制作方法,注意制作环境的安全卫生,减少制作过程中有害杂菌的污染,由此控制和减少豆酱中有害真菌毒素的产生。
5.豆酱中产蛋白酶菌株的筛选对自然发酵豆酱中获得的34株Aspergillus和24株Mucor进行产蛋白酶活性菌株筛选,发现共21株Aspergillus和8株Mucor具有产蛋白酶活性,分别占所测试菌株的62%和33%,表明自然发酵豆酱中的真菌资源具有极大的应用潜力。
6.蛋白酶特性研究对菌株Asp-195v进行了产蛋白酶特性研究,获得了Asp-195v的最佳培养基配比,即麸皮:豆粕为7:3,含水量为40%,pH值为7.0。改变培养温度、培养时间,并考察反应温度、pH值、盐度对蛋白酶活性的影响,发现最适培养温度为28℃,最适培养时间为48h,最适反应温度为50℃,最适反应条件为中性,盐度影响蛋白酶活性,但本菌株对盐有一定的耐受力,适宜于豆酱的发酵环境。
对筛选获得的3株Mucor进行蛋白酶活性测定。结果表明,M-339、M-513和M-808菌株相应在26℃、麸皮:水为1:1.1,28℃、麸皮:水为1:1.2,30℃、麸皮:水为1:1.2时产蛋白酶活性最高。研究所测菌株的产酶活性特点与相应的豆酱发酵环境一致。
Soybean paste is one of the commonest traditional condiments in China.The origin of Chinese soybean paste can be traced back to Zhou Dynasty,about 3,000 years ago.Untill now, the naturally fermented home-made soybean paste was widely consumed by Chinese people. Due to the open fermenting process,there are rich fungi in the home-made soybean paste and it is inevitable to avoid the contamination of mycotoxins and may harm the consumer's health. Many studies were conducted on bacteria and yeast in soybean paste,but its fungal studies were scarce.
This study is the first systematic research on fungal diversity in naturally fermented soybean paste.The comprehensive analysis of fungal diversity was performed in aspects of paste making regions,methods and fermentation time.It is also the first detection of harmful mycotoxions in soybean paste by high-performance liquid chromatography after immunoaffinity chromatogaphy cleanup.The result shows that the presence of four mycotoxions marks the potential danger of the home-made soybean paste.The study of protease activity of some Aspergillus and Mucor isolates was also conducted,it is found that 62%of Aspergillus and 33%of Mucor possessed protease activity.The protease features of one Aspergillus strain and three Mucor strains were studied.The total results are listed as follows:
The fungal species isolated from soybean paste
Totally 280 samples of soybean pastes were collected and investigated in this study, including 251 samples of home-made products in 72 regions of 11 provinces including Northeastern China,Inner Mongolia,Henan,Hebei,Sichuan,Anhui,Guizhou,Fujian and Jiangxi,26 samples of industry products from Changlong brewing factory in Changchun, Heitudi brewing factory in Dalian and Douwa brewing factory in Jinzhou,and 11 samples from markets.At last 90 fungal species belonging to 25 genera were isolated and identified. Of which 13 species of four genera are Zygomyctes,6 species of three genera Ascomyctes and 71 species of 18 genera Anamorphic fungi.Fusarium brevicatenulatum and Penicillium coprophilum are two newly recorded species for China,Aspergillus niger var.jinzhouanum and A.clavatus var.aureus are named as new taxa for their specific morphology and molecular evidence.Each fungal species was morphologically illustrated and described with microscopic photos.The species of Aspergillus and Penicillium were provided with clony features on standard media.The SEM photos of 8 main Aspergillus species were provided.
The molecular identification of some suspicious fungal strains
Such Aspergillus species as A.flavus and A.oryzae are easily misidentified due to their similar morphological characteristics.It is well-known that A.flavus is the aflatoxins producer and A.oryzae the common fungus used in food industry.In order to correctly identify the two species,the comparative studies were conducted on their cultures on standard media and rDNA ITS sequences.The comparative studies reveal that A.flavus occurred more frequently on soybean paste than A.oryzae.Theβ-tubulin sequence analyses were performed to support the new taxa of A.niger var.jinzhouanum and A.clavatus var.aureus also as P.coprophilum. A.niger var.jinzhouanum is characterized by adnate or verticillate appendages on conidiophore stalks just below the vesicles.The appedages on conidiophore stalks are new descriptive characters for Aspergillus in literature.
The analysis of fungal diversity of naturally fermented soybean pastes
The research data were analyzed with the software of data processing system(2002).The results indicate that the fungal diversity was influenced by the paste making environment and methods.The predominant fungal groups in home-made pastes were Penicillium,Aspergillus, Mucor,Fusarium,Acremonium,Rhizopus,Scopulariopsis,Trichoderma and Cladosporium, while the commercial fungi of Monacsus and A.oryzae were seldom encountered.It impies that home-made soybean pastes were not good media for the two economic fungi.The continous investigation of fungi on home-made pasets shows that the fungal species population and amount had changed during the whole fermenttion course.The fungal population of soybean pastes were dominated by Mucor in early fermentation stage,and then replaced by Penicillium,Aspergillus and Fusarium with the decrease of moisture in pastes. The presence of Aspergillus from brine fermentation until mature stage of soybean pastes indicates its important role palyed in the soybean paste making process.The making methods and microenvironments influenced the quality of the soybean pastes.The method of“partial koji”in Zhuanghe which did not favor the vigorous multiplication of poisonous fungi should be recommended for the home-made soybean pastes.
Detection of main mycotoxins in soybean pastes
The mycotoxin content of 21 home-made samples and two industry samples were examined by high performance liquid chromatography after immunoafinity column clean-up. The results shows that 13 samples of soybean pastes(56.5%)were detected to contain aflaxoxin,six samples(26%)contain aflatoxin B_1.The frequence of ZEA,T-2 and DON contamination was low respectively with only one sample contaminated with each one toxin. The results indicate that the potential danger exists in home-made soybean pastes.
Screening of protease-producing fungal strains in soybean pastes
34 strains of Aspergillus spp.and 24 Mucor spp.isolated from naturally fermented soybean paste were screened and 21 strains(62%)of Aspergillus spp.and 8 strains(33%)of Mucor spp.were detected with high protease activity,it implies that many economic fungi from home-made soybean paste could be commercially utilized in industry.
Studies on the protease feature of protease-producing strains
The protease feature of Asp-195v isolate was studied.The optimum medium is bran: bean cake=7:3 with 40%moisture content and pH 7.0.The optimum conditions for the protease production were 28℃and 48h of incubation and neutral pH condition.The favorable temperature for protease reaction was 50℃.The salt concentration influenced the protease activity.
The protease activities of 3 Mucor strains were investigated.It was found that the optimum incubation conditions for protease production of the M-339,M-513 and M-808 were bran:water=1:1.1 at 26℃;bran:water=1:1.2 at 28℃and bran:water=1:1.3 at 30℃respectively.The feature of protease production for each Mucor isolate was in accordance with their fermentation environment.
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