CN1231926A - 含有葡糖淀粉酶和酸性真菌蛋白酶的组合物 - Google Patents

含有葡糖淀粉酶和酸性真菌蛋白酶的组合物 Download PDF

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CN1231926A
CN1231926A CN99103605A CN99103605A CN1231926A CN 1231926 A CN1231926 A CN 1231926A CN 99103605 A CN99103605 A CN 99103605A CN 99103605 A CN99103605 A CN 99103605A CN 1231926 A CN1231926 A CN 1231926A
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O·J·兰特罗
J·J·菲什
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Genencor International Indiana Inc
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Abstract

本发明是关于从含有可发酵的糖或可转化为糖的组分的原料制备乙醇的方法,包括下列步骤:a.在α－淀粉酶存在下液化原料,得到液化的糖化醪;b.在葡糖淀粉酶存在下糖化液化的糖化醪,得到水解的淀粉和糖;c.用酵母发酵水解的淀粉和糖,得到乙醇;d.回收得到的乙醇;将真菌蛋白酶在糖化过程中引入液化的糖化醪中,和/或在发酵过程中引入水解的淀粉和糖中。本发明还涉及含有葡糖淀粉酶和酸性真菌蛋白酶的组合物。

Description

含有葡糖淀粉酶和酸性真菌蛋白酶的组合物
本申请是申请号为92104557.3，申请日为1992年5月16日，发明名称为“制备乙醇的方法”的发明专利申请的分案申请。
本发明涉及用生物学手段制备乙醇的方法。
从全谷物糖化醪发酵制备乙醇的方法是众所周知的。
在Biomass 16(1988)77-87中已经说明向糖化醪中加入碱性蛋白酶可提高足以保持发酵生成乙醇速率的氨基氮：向高梁或买罗高梁中加入从灰绿链霉素产生的蛋白酶和碱性蛋白酶，导至较高的发酵生成乙醇速率。
加拿大专利1143677描述了由淀粉原料，如小麦、大麦或黑麦制备乙醇的方法。该方法包括在复合水解酶存在下淀粉、纤维素和玉米粒中含有的某些其他物质的水解步骤，复合水解酶由真菌木霉素Koningii产生，并含C1的酶、内和外葡聚糖酶、纤维二糖酶、木聚糖酶、β-葡糖苷酶、蛋白酶和一些淀粉酶。
然而，当在发酵桶中有较高含量的干固体糖化醪时，这些方法不能产生乙醇。
本发明的目的是提供提高乙醇生成速度的方法，其中在发酵桶中在较高含量的溶解的干固体糖化醪存在下，酵母可发酵糖化醪，得到较高含量的乙醇。
另一个重要的观察结果是根据本发明的方法从发酵桶中得到的稀釜馏物的粘度比在相同的干固体含量下根据已知方法从发酵桶得到的稀釜馏物的粘度低。实际上，这会有助于减少蒸发器的堵塞和/或允许在较高的固体含量下蒸发稀釜馏物。此外也可预期较低粘度的釜馏物在干燥前易于与酒糟混合。
本发明是关于从含有高含量干固体糖化醪原料制备乙醇的方法，原料中含有可发酵的糖或可转化成糖的组分，上述方法包括下列步骤：
a.在α-淀粉酶存在下液化原料，得到液化的糖化醪：
b.在葡糖淀粉存在下糖化液化的糖化醪，得到水解的淀粉和糖；
c.用酵母发酵水解的淀粉和糖，得到乙醇：
d.回收乙醇；其中将真菌蛋白酶在糖化过程中引入液化的糖化醪中，和/或在发酵过程中引入水解的淀粉和糖中。
用酸性真菌蛋白酶得到好的结果。
用于本发明中的酸性真菌蛋白酶是由真菌产生的酸性蛋白酶，其特征是在酸性条件下有水解蛋白质的能力。一般，酸性真菌蛋白酶由曲霉菌属、毛霉菌属、根霉菌属、念珠菌属、Coriolus、内座壳霉菌属、Entnomophtora、Irpex、青霉菌属、菌核和球拟酵母属衍生。通常，选择的真菌蛋白酶是热稳定的，由下列霉菌衍生：如曲霉菌属，如黑曲霉、A.saitoi或米曲霉；毛霉菌属，如渺毛霉或M.miehei；Endothia，如Parasitica；或根霉菌属，如根霉。优选的酸性真菌蛋白霉由黑曲霉衍生。特别优选的酸性真菌蛋白霉由黑曲霉变种得到，该黑曲霉变种是已知的商标AFP-2000，可从Solvay Enzymes，Inc.买到。
本发明中使用的酸性真菌蛋白酶的量取决于蛋白酶的酶活性。一般，0.001至2.0ml 2％酸性真菌蛋白酶溶液加入450gm调节成含20-33％干固体的悬浮液中。该悬浮液在糖化过程中是液化的糖化醪，和/或在发酵过程中是水解的淀粉或糖。通常，该溶液加入量是0.005至1.5ml。该溶液优选加入量是0.01至1.0ml。
本发明的方法中使用的α-淀粉酶一般是使淀粉中α-(1-4)糖苷键无规定断裂的酶。通常，α-淀粉酶选自微生物酶。这些酶的E.C.号是E.C.3.2.1.1，特别是E.C.3.2.1.1-3。本发明的方法中使用的优选的α-淀粉酶选自热稳定的微生物α-淀粉酶。特别优选的α-淀粉酶由芽孢杆菌衍生。用由商标为TAKA-THERM II的地衣芽孢杆菌衍生的α-淀粉酶得到好的结果，该地衣芽孢杆菌在市场上可从Solvay Enzymes，Inc.买到。
本发明方法中使用的α-淀粉酶量取决于α-淀粉酶的酶活性。一般，0.001至2.0mlα-淀粉酶溶液加入1000gm原料中。通常，该溶液加入量是0.005至1.5ml。该溶液优选加入量为0.01至1.0ml。
本发明方法中使用的葡糖淀粉酶一般是从淀粉的未还原端除去连续的葡糖单元的酶。其可水解淀粉的直链和支链葡糖键。通常，葡糖淀粉酶选自微生物酶。本发明方法中使用的优选的葡糖淀粉酶选自热稳定的真菌葡糖淀粉酶。特别优选的葡糖淀粉酶由曲霉菌属衍生。用商标为DISTILLASE的黑曲霉衍生的葡糖淀粉酶得到好的结果。该黑曲霉在市场上可由Solvay Enzymes，Inc.买到。
本发明方法中使用的葡糖淀粉酶的量取决于葡糖淀粉酶的酶活性。一般，0.001至2.0ml葡糖淀粉酶溶液加入450gm调节至含20-33％干固体的悬浮液中，该悬浮液在糖化过程中是液化的糖化醪，和/或在发酵过程中是水解的淀粉和糖。通常，该溶液加入量是0.005至1.5ml。该溶液优选的加入量是0.01至1.0ml。
本发明方法中使用的酵母一般是面包酵母，也通称子囊酵母或啤酒酵母。用弗雷斯克曼(Fleishmann)面包酵母得到好的结果。
含可发酵的糖或可转化成糖的组分的原料通常是含淀粉的原料，如块茎、根、全磨碎的玉米、玉米棒子、玉米、谷物、小麦、大麦、黑麦买罗高梁和谷子，含糖原料，如废糖蜜，果实原料，甘蔗或甜菜，含纤维素原料，如木材或植物剩余物。该原料优选是含淀粉的原料，如玉米棒子、全磨碎的玉米、玉米、谷粒、买罗高梁或谷物，和其混合物。用玉米棒子、玉米或买罗高梁和其混合物可得到好的结果。
液化、糖化、发酵和回收乙醇的步骤是众所周知的。例如，这些步骤描述在P.Prve，U.Faust，W.Sittig，D.A.Sukatsch编辑的Fundamentals of Biotechnology，第10章，第381-403页中，该书在此以参考文献引用。
糖化和发酵步骤可同时或分开进行。优选糖化和发酵步骤同时进行。当同时进行时，葡糖淀粉酶和酸性真菌蛋白酶可以简单混合的组合物形式引入。含有由黑曲霉(商标DISTILLASE)衍生的葡糖淀粉酶和由黑曲霉(商标AFP-2000)衍生的酸性真菌蛋白酶的该组合物，由Solvay Enzymes，Inc.以商标FERMENZYME销售。
在糖化过程中向液化糖化醪中加入一些营养素，和/或在发酵过程中向水解的淀粉中加入营养素也可是有利的。该营养素的例子是回糟、酵母提取物、玉米浆和其混合物。
在糖化过程中向液化糖化醪中加入一些盐和/或在发酵过程中向水解的淀粉和糖中加入盐也会是有利的。该盐的例子是NaCl和硫酸铵。
在糖化过程中向液化糖化醪中加入一些其他酶和/或在发酵过程中向水解的淀粉和糖中加入其他酶也将是有利的。该酶的例子是纤维素酶、半纤维素酶、磷酸酯酶、内和外葡聚糖酶和木聚糖酶。
通常，在全谷物糖化醪发酵中，乙醇用蒸馏法回收。离心剩余的釜馏物，从稀的釜馏物中除去酒糟固体。然后，稀的釜馏物部分浓缩，得到含有约30-45％固体的糖浆。该糖浆与酒糟部分合并，干燥，得到干的酒糟固体加可溶物(DDGS加可溶物)。DDGS加可溶物以动物饲料销售。在稀的釜馏物浓缩过程中，蒸发器的堵塞是很普遍的问题，必须定期清洗蒸发器。与浓缩稀的釜馏物有关的其他问题是在固体含量30-40％时粘度很高。当冷却时糖浆通常形成凝胶。看来胶凝作用可能是由于蛋白质和淀粉。认为淀粉的存在引起磨碎的玉米悬浮物不完全液化，在蒸馏中可发生一些淀粉的液化。
根据本发明，使用酸性真菌蛋白酶，较高干固体含量的糖化醪可发酵，得到较高含量的乙醇。在较高干固体含量糖化醪存在下，酸性真菌蛋白酶加入谷物糖化醪中，用酵母使其发酵，生成乙醇。
                          实施例1
该实施例说明酸性真菌蛋白酶的存在怎样改进生成乙醇的速度和用酵母发酵全玉米糖化醪达到的乙醇含量。
步骤a：液化
第一步是全玉米的液化。磨碎的全玉米可从工业燃料酒精生产厂得到。为了液化，将1740gm磨碎的玉米加入4500ml自来水中。向该悬浮液中加入0.99gm CaCl2·2H2O。然后，悬浮液放入68℃水浴中，pH调至6.2-6.4。在不断搅拌下，0.6mlTaka-ThermRII酶加入悬浮液中，在68℃保温1小时。Taka-ThermRII酶是液体热稳定的微生物(地衣芽孢杆菌变种)α-淀粉酶，在市场上从Solvay EnzymeS，Inc.可买到。在保温过程中观察到不引人注意的胶凝作用。然后，悬浮液放在加热板上，在良好搅拌下加热至沸腾。悬浮液煮沸5分钟后放到90℃水中，保温2小时。煮沸后，向悬浮液中再加入1.2ml Taka-ThermRII酶。悬浮液冷却至25℃，pH用25％H2SO4调至4.6-4.8。干固体含量(DS)用自来水调至20-21％。
步骤b：糖化和发酵
在500ml三角烧瓶中加入450gm步骤a(液化)中得到的液化的玉米糖化醪，同时进行糖化和发酵。表1中所示的合适量的酶与0.8gm弗雷斯克曼面包酵母(7gm，薄片包装)一起加入糖化醪中。在旋转烧瓶混合之前使干酵母水合约10分钟。用Parafilm包覆烧瓶，放在36℃水中，使其发酵适当时间。定时从烧瓶中取出10ml试样用于分析。
分析
定时取样，测定pH。用HPLC测定乙醇和碳水化物含量。在HPLC分析之前离心分离试样，上清液用0.01N H2SO4适当稀释(10倍溶液)，用0.45μ过滤器过滤。用20μl试样在BioRad HPX87H柱中在60℃分离，用0.01N NH2SO4作流动相，流速0.7ml/min。检测器是折射率检测器，用峰面积定量。碳水化物用葡糖标准表示为W/V％DS。甘油和乳酸同样用甘油和乳酸标准表示为W/V％。乙醇用乙醇标准表示为V/V％。
结果
对于该实施例，每个烧瓶中加入0.267ml DistillaseR L-200酶。DistillaseR酶是由黑曲酶变种(可从Solvay Enzymes，Inc.得到)衍生的液体葡糖淀粉酶(AG)的商标名称，每毫升含有200Diazyme单位。如表1中所示，每个烧瓶中加入不同量的酸性真菌蛋白酶(2％AFP-2000酶溶液)。AFP-2000酶是由Solvay Enzymes，Inc.买到的黑曲酶变种衍生的酸性真菌蛋白酶。
表1总结了由于加入酸性真菌蛋白酶(AFP)由全玉米糖化醪同时糖化和发酵得到的结果。该结果表明加入AFP增加了得到乙醇的速度和含量。这些结果表明乙醇收率增加12％。可以看出乙醇收率的增加是较完全发酵的结果。无AFP存在，较多的葡糖不发醇。
有或无AFP存在甘油含量大致相同。既然认为产生了乙醇，在发酵物料中含有蛋白酶时甘油含量实际上较小。在发酵桶中有蛋白酶时pH也稍微下降。乳酸含量未表示出来，但是在所有情况下乳酸含量都小于0.1％。
结果也表明，正如在发酵的初期较低量的不可发酵的糖和增加的葡糖所表明的，增加葡糖淀粉酶的量提高了糖化的速度。增加葡糖含量未提高乙醇生成速度。看来好像发酵速度不受可发酵的碳水化物的限制。
                        实施例2
根据实施例1中给出的方法制备全玉米糖化醪，每个烧瓶加入弗雷斯克曼酵母的量是0.8或1.6gm。对于该实施例，试验3个含量的蛋白酶(酸性真菌蛋白酶)。发酵方法和取样与实施例1中给出的相同。结果列于表2中。
较高含量的酵母(1.6gm)看来导至发酵速度提高，可发酵的糖(葡糖和麦芽糖)含量基本上降低至减少酵母存活力的范围内。在所有情况下，用较低的酵母含量(0.8gm)乙醇收率不高。在酵母含量0.8gm时，加入蛋白酶得到与实施例1相同的结果，即较快的发酵速度，较高的乙醇含量，可发酵糖的较完全的发酵。
                          实施例3
在本实施例中，全玉米糖化醪用酵母提取物强化。也试验AFP的影响。本实施例所用的酵母提取物是已知的，由Universal FoodsCorp.(Milwaukee，WI)得到的，商标Amberex1003。酵母提取物Amberex1003是由酵母蛋白酶的自溶作用产生的水溶性的啤酒酵母提取物。其含有蛋白质、酞、游离氨基酸、维生素、矿物质和微量元素。正如氨基氮与总氮比例30所表明的，在有较高量游离氨基氮时，蛋白质含量(Nx6.25)是56％。
全玉米的液化按实施例1中的方法进行。每个烧瓶中加入450gm20％DS液化糖化醪。其他添加剂在表3中给出，发酵在33℃进行。从发酵桶中取样，按实施例1中描述的方法处理。表3仅总结了发酵过程中的乙醇含量。
酵母提取物的加入表明在发酵过程中提高了乙醇生成的速度(表3)。AFP与酵母提取物一起加入所提高的乙醇收率达到高含量酵母提取物(16ml/烧瓶)的程度。这些结果证明蛋白酶可促进从易于被酵母代谢的谷物蛋白质产生氨基氮。
                     实施例4
一般，在工业燃料乙醇生产中，液化的全玉米糖化醪在发酵前用通常称为回糟的稀釜馏物稀释。加入回糟达到两个目的，将营养素加入糖化醪中，并减少了被浓缩的稀釜馏物体积。在本实施例中全玉米糖化醪从燃料乙醇生产厂得到，以评价酸性真菌蛋白酶对发酵的作用。本实施例中使用的工业糖化醪已经液化，含有常量的葡糖淀粉酶和回糟，稀释为通常的固体含量，用酵母接种。仅有的变量是加入酸性真菌蛋白酶。每个烧瓶含有450gm糖化醪和表4中表示量的AFP。然后，烧瓶放在30℃水浴中发酵。发酵操作用实施例1中描述的HPLC监测，结果总结于表4中。
表4中的结果表明在含有回槽的糖化醪中用AFP的效果与以前只用全玉米糖化醪(无回槽)的效果相同，即快速的乙醇生成速度和较高的乙醇收率。
                        实施例5
前面的实施例表明有加入糖化醪中的酸性真菌蛋白酶的发酵提高了乙醇生成速度和收率。发酵基本上在糖化醪有相同的干固体(DS)浓度下进行。在本实施例中，全玉米糖化醪在各种糖化醪DS含量下，有和无酸性真菌蛋白酶加入时发酵。工业液化的全玉米用自来水稀释成各种DS含量。在每种DS含量下，相应的酶和干酵母按表5中表示的那样加入。糖化醪DS含量通过在强制热空气干燥箱中于100℃将试样干燥过夜来测定。发酵在33℃进行，按实施例1描述的方法取样。结果总结在表5中。
乙醇的结果另总结在表6中。在每个糖化醪含量下，结果表明加入蛋白酶提高了乙醇生成速度和收率。结果还表明仅有蛋白酶存在，可得到15％V/V乙醇。看来好像在约28％DS糖化醪固体下得到最大的乙醇生成，同时还得到完全的发酵。
                       实施例6
实施例5表明在有酸性真菌蛋白酶存在下全谷物发酵提高了乙醇生成速度和得到的乙醇含量(15％V/V)。该实施例表明在各种干固体糖化酶含量下，含有稀釜馏物时，酸性真菌蛋白酶的作用。工业液化的全玉米以一定比例与稀釜馏物混合，以便总的固体含量的8.6％来自稀釜馏物。
发酵条件与实施例5中给出的条件相同，和发酵结果一起总结在表7中。蛋白酶(AFP)的存在提高了乙醇生成速度和相对于对照试验不含蛋白酶的烧瓶得到的乙醇含量。
                      实施例7
该实施例说明糖化过程中AFP的效果。在糖化和发酵分开步骤中的主要区别是在糖化中温度显著高于发酵温度。一般，糖化在60℃进行。全玉米糖化醪按实施例1中的方法液化。然后，液化的糖化醪用两种不同的方法处理。在一种情况(A)下，450gm糖化醪加入烧瓶中，加入合适量的AG和AFP(表8-A中所示)，将烧瓶放入60℃水浴中24小时进行糖化。糖化后，烧瓶冷却至25℃，用干酵母接种，放入33℃水浴中发酵。在第二种情况(B)下，液化的糖化醪加入与A相同的烧瓶中，只加入AG，烧瓶在60℃保温24小时。然后，烧瓶冷却，加入合适量的蛋白酶(AFP)和酵母，放入33℃水浴中发酵。发酵过程通过如实施例1中描述的HPLC分析进行监测。
表8-A总结了在高温糖化和发酵过程中AFP作用的结果。这些结果表明在糖化温度下AFP能水解蛋白质。这足以推定，由于糖化过程中AFP的存在，蛋白酶是活泼的，消化蛋白质。在发酵过程中AFP的存在也有效地水解蛋白质。糖化后加入AFP(B部分)导致如前面的用AFP的实施例中看到的相同的发酵速度增加。在本实施例的两部分的不同发酵时间存在的乙醇含量总结在表9中。结果也表明过量的AFP在增加发酵速度方面看来未得到任何附加的效果。
这些结果表明AFP的活性是足够稳定的，在60℃是有效的。例如这对发酵前进行的糖化是重要的。结果表明AFP和AG可以是混合物，在糖化和发酵中分别加入或同时加入。
                          实施例8
在美国玉米是最广泛使用的发酵制备乙醇的淀粉源。买罗高梁大概是其次使用的淀粉源。在本实施例中，全谷物买罗高梁用于评价AFP对于发酵成乙醇的影响。将全磨碎买罗高梁制成悬浮液，用实施例1中用于全磨碎玉米的相同方法使其液化。也使用实施例1中的发酵条件，即每个烧瓶加入450gm20％DS的糖化醪。同时糖化发酵的条件与实施例1中的相同。酶含量在表10中给出。结果表明当有AFP存在时乙醇生成速度较大，结果与用AFP的玉米糖化醪发酵的结果相似。这些结果表明AFP可将买罗高梁中的蛋白质转化为容易被酵母代谢的氨基氮。
                   实施例9
在本实施例中，由工业燃料乙醇生产厂得到的浓缩的稀釜馏物用葡糖淀粉酶(Distillase)和AFP处理，试图减少糖浆浓度。浓糖浆用水稀释成14.7％DS。三个稀释的糖浆试样在36℃保温过夜：1)对照试样，2)含Distillase试样，3)含AFP试样。保温后，试样在旋转蒸发器中浓缩至30％DS，用Brookfield RVT/100型粘度计在25℃测定粘度。对照试样粘度1455cps，AG和AFP试样粘度分别是835和668/cps。这些结果表明蛋白质和碳水化物有助于粘度。接着将AFP加入糖化醪中，有助于减少稀釜馏物的粘度和可能减少蒸发器的堵塞。
                   实施例10
本实施例说明当AFP加入全谷物糖化醪中时，其减少稀釜馏物粘度的性能。含有葡糖淀粉酶和用酵母接种的工业液化的玉米糖化醪用于本实施例。3个3升试样在36℃发酵60小时。加入糖化醪中的AFP量对照试样为0，另两个试样每升糖化醪为10和20SAPU(每分钟产生的酪氨酸摩尔数)。发酵后用蒸馏回收乙醇。釜馏物用过程过滤器过滤，分离酒糟(滤饼)和稀釜馏物(滤液)。然后，得到的滤液浓缩至50％DS。按实施例9中的方法测定粘度。对照糖浆的粘度是503CP，每升糖化醪10和20单位AFP的试样粘度分别是233和197CP。这些结果表明发酵桶中蛋白质的作用确实减少了稀釜馏物的粘度。
                       表1
       AFP对全玉米糖化醪同时糖化/发酵的影响
                                       HPLC分布
        发酵时间    葡糖  麦芽糖  未发酵碳水化物   乙醇      甘油
        (Hrs)pH    ％w/v   ％w/v      ％w/v       ％w/v      ％w/v1号烧瓶-0.12ml AG，0ml AFP
         3    4.6   2.38   1.36        16.38         1.01     .21
         17   3.9   4.92   2.67         4.68         5.30     .63
         41   3.7   3.30    .18         1.65        10.40     .98
         65   3.8   2.21    .23          .47        12.00    1.05
         95   3.9   2.07    .23          .35        12.15    1.012号烧瓶-0.12ml AG，.1ml AFP
          3   4.6   2.70   1.47       16.25          1.02     .21
         17   3.9   3.21   2.10        4.57          7.27     .68
         41   3.9    .63    .19         .78         12.87     .98
         65   4.0    .39    .23         .34         13.47     .98
         95   4.1    .58    .21         .38         13.52     .97
                              表1(续)
               AFP对全玉米糖化醪同时糖化/发酵的影响
                                         HPLC分布
         发酵时间     葡糖   麦芽糖  未发酵碳水化物   乙醇      甘油
         (Hrs)pH     ％w/v   ％w/v        ％w/v      ％w/v      ％w/v3号烧瓶- 0.12ml AG，.3ml AFP
         3    4.6     2.72    1.43        15.20         .97      .20
        17    3.9     2.41    1.25         4.33        8.51      .68
        41    4.0      .13     .19          .59       13.44      .92
        65    4.1      .15     .20          .39       13.83      .95
        95    4.1      .43     .16          .27       13.74      .924号烧瓶-0.16ml AG，0ml AFP
         3    4.6     3.96    1.68        14.74         .91      .21
        17    3.9     7.39    1.20         3.93        5.53      .67
        41    3.7     3.97     .23          .87       10.50      .95
        65    3.8     2.40     .27          .34       12.17     1.06
        95    3.9     2.40     .26          .34       12.14     1.08
                          表1(续)
           AFP对全玉米糖化醪同时糖化/发酵的影响
                                        HPLC分布
       发酵时间        葡糖    麦芽糖   未发酵碳水化物   乙醇    甘油
       (Hrs)pH        ％w/v    ％w/v        ％w/v       ％w/v   ％w/v5号烧瓶 - 0.16ml AG，.1ml  AFP
          3    4.6    4.38     1.80         14.18         .99     .21
         17    3.9    5.12      .75          3.68        7.28     .71
         41    3.9     .83      .21           .50       12.98    1.02
         65    4.0     .71      .26           .34       13.41    1.00
         95    4.1     .79      .23           .37       13.59    1.026号烧瓶-0.12ml AG，.3ml AFP
          3    4.6    4.72     1.91         13.37        1.07     .21
         17    4.0    4.05      .44          3.41        8.67     .73
         41    4.0     .07      .20           .41       13.68     .96
         65    4.1     .19      .19           .29       13.77     .96
         95    4.2     .54      .21           .42       13.90     .94
                        表1(续)
           AFP对全玉米糖化醪同时糖化/发酵的影响
                                  HPLC分布
      发酵时间    葡糖    麦芽糖  未发酵碳水化物   乙醇      甘油
      (Hrs)pH    ％w/v    ％w/v      ％w/v        ％w/v     ％w/v7号烧瓶-0.20ml AG，0ml AFP
        3     4.7  6.19    2.26       11.41         .96       .21
       17     3.9  9.18     .30        2.68        5.68       .69
       41     3.7  4.14     .19         .64       10.61       .98
       65     3.9  2.56     .27         .31       11.98      1.08
       95     3.9  2.59     .26         .33       12.07      1.058号烧瓶-0.20ml AG，.1ml AFP
        3     4.6  6.79    2.44       10.63         .94       .20
       17     4.0  6.87     .27        2.41        7.53       .75
       41     4.0   .74     .26         .36       13.00      1.01
       65     4.0   .58     .26         .36       13.56      1.05
       95     4.1   .82     .21         .30       13.57      1.04
                      表1(续)
        AFP对全玉米糖化醪同时糖化/发酵的影响
                                   HPLC分布
         发酵时间   葡糖  麦芽糖 未发酵碳水化物   乙醇     甘油
         (Hrs)pH   ％w/v  ％w/v      ％w/v       ％w/v    ％w/v9号烧瓶-0.20ml AG，.3ml AFP
        3    4.7   7.05    2.58      10.27        1.03      .20
       17    4.0   5.26     .21       2.34        8.68      .75
       41    4.0    .07     .23        .38        13.61     .90
       65    4.1    .31     .22        .40        13.86    1.00
       95    4.1   0.52     .21        .42        13.89     .9710号烧瓶-0.25ml AG，0ml AFP
        3    4.6   8.48    3.20       7.59          .96     .20
       17    3.9  10.19     .19       1.86         5.77     .72
       41    3.8   4.07     .24        .41        10.81    1.04
       65    3.9   2.62     .27        .33        11.93    1.11
       95    4.0   2.78     .29        .34        12.20    1.06
                               表1(续)
                  AFP对全玉米糖化醪同时糖化/发酵的影响
                                   HPLC分布
           发酵时间   葡糖   麦芽糖   未发酵碳水化物   乙醇   甘油
           (Hrs)pH   ％w/v     w/v         ％w/v      ％w/v  ％w/v11号烧瓶-0.25ml AG，.1ml AFP
         3    4.6    5.99     2.12         11.49       1.01    .20
        17    4.0    7.76      .17          1.59       7.44    .77
        41    4.0     .88      .27           .34      13.10   1.01
        65    4.1     .90      .26           .36      13.51   1.05
        95    4.2    1.02      .22           .32      13.42   1.0112号烧瓶-0.25ml AG，.3ml AFP
         3    4.6    6.33     2.30         10.43       1.02    .20
        17    4.0    5.97      .17          1.48       8.75    .80
        41    4.0     .07      .22           .36      13.59    .97
        65    4.1     .23      .19           .37      13.82   1.01
        95    4.2     .49      .18           .36      13.91    .97每个烧瓶(500三角烧瓶)含有450gm液化的全玉米糖化醪和所示量的DistillaseL-200(AG)和2％AFP-2000(酸性真菌蛋白酶)。烧瓶放在36℃水浴中。
                            表2
            不同的AFP含量在两种酵母接种含量下对
              全玉米糖化醪同时糖化/发酵的影响
                                    HPLC分布
         发酵时间    葡糖  麦芽糖   未发酵碳水化物    乙醇     甘油
         (Hrs)pH    ％w/v  ％w/v        ％w/v         ％w/v     w/v1号烧瓶-0.12ml AG，0ml AFP，.8gm酶母
            3  4.8   3.37  1.40          15.63          .96     .21
           15  4.1   3.90  3.42           5.81         4.69     .59
           39  3.9   3.10   .29           1.85         9.94     .95
           64  4.0   2.05   .38            .51        12.18    1.05
           89  4.1   2.04   .38            .38        12.15    1.022号烧瓶-0.12ml AG，0.05ml AFP，.8gm酶母
            3  4.8   3.49  1.40          15.37          .92     .20
           15  4.1   3.02  3.27           5.67         5.11     .60
           39  4.0   1.28   .27           1.55        11.16     .93
           64  4.1    .85   .35            .47        13.22    1.01
           89  4.2    .87   .39            .43        13.06    1.01
                                表2(续)
                   不同的AFP含量在两种酵母接种含量下对
                     全玉米糖化醪同时糖化/发酵的影响
                                      HPLC分布
             发酵时间    葡糖    麦芽糖   未发酵碳水化物    乙醇    甘油
            (Hrs)pH     ％w/v    ％w/v         ％w/v       ％w/v    ％w/v3号烧瓶-0.12ml AG，0.10ml AFP，.8gm酶母
            3   4.8      3.79    1.45          14.87         .95      .20
           15   4.1      2.62    3.29           5.66        5.82      .61
           39   4.1       .88     .33           1.25       11.82      .93
           64   4.1       .53     .37            .45       13.56     1.00
           89   4.3       .42     .29            .34       13.35      .984号烧瓶-0.12ml AG，0.25ml AFP，.8gm酶母
            3   4.8      4.05    1.52          15.19        1.01      .20
           15   4.1      1.69    3.01           5.60        6.63      .60
           39   4.1       .52     .30            .94       12.40      .92
           64   4.2       .12     .32            .46       13.68      .94
           89   4.3       .19     .25            .36       13.04      .91
                            表2(续)
              不同的AFP含量在两种酵母接种含量下对
                全玉米糖化醪同时糖化/发酵的影响
                                         HPLC分布
          发酵时间    葡糖  麦芽糖    未发酵碳水化物    乙醇     甘油
          (Hrs)pH    ％w/v  ％w/v          ％w/v       ％w/v     ％w/v5号烧瓶-0.12ml AG，0ml AFP，1.6gm酶母
            3  4.7  3.50      1.55         14.54        2.45     .28
           15  4.0  1.91      3.27          5.63        6.31     .71
           30  4.0  1.65       .36          1.42       11.30    1.03
           64  4.0  2.43       .48           .49       11.77    1.03
           89  4.1  2.50       .44           .38       11.97     .996号烧瓶-0.12ml AG，0.05ml AFP，1.6gm酶母
            3  4.7  3.68      1.58         14.67        1.50     .28
           15  4.0  1.34      2.99          5.52        6.66     .69
           39  4.1   .91       .30          1.16       11.79     .95
           64  4.1  1.68       .40           .45       11.53     .97
           89  4.2  1.62       .38           .38       12.50     .95
                              表2(续)
                 不同的AFP含量在两种酵母接种含量下对
                    全玉米糖化醪同时糖化/发酵的影响
                                          HPLC分布
         发酵时间      葡糖   麦芽糖  未发酵碳水化物    乙醇    甘油
         (Hrs)pH      ％w/v   ％w/v         ％w/v      ％w/v    ％w/v7号烧瓶-0.12ml AG，0.10ml AFP，1.6gm酶母
            3   4.7    3.84    1.64         14.01       1.63     .29
           15   4.0     .07    1.25          7.40       7.13     .69
           39   4.2     .70     .35           .85      11.99     .91
           64   4.1    1.12     .29           .35      11.44     .86
           89   4.2    1.22     .31           .35      12.68     .938号烧瓶-0.12ml AG，0.25ml AFP，1.6gm酶母
            3   4.6    3.82    1.65         13.77       1.66     .28
           15   4.0     .07    1.31          6.52       7.89     .69
           39   4.2     .61     .32           .68      12.34     .87
           64   4.2     .95     .28           .40      12.97     .90
           89   4.3    1.08     .02           .36      13.07     .89每个烧瓶含有450gm液化的全玉米糖化醪和所示含量的Distillase，2％AFP和干弗雷斯克曼酵母。烧瓶放在36℃水浴中。
                           表3
         酵母提取物对液化的全玉米糖化醪同时糖化/发酵的影响
                                           乙醇含量％V/V
      AGa  AFPb YEc  干酵母d          发酵时间(Hrs.)烧瓶     (ml)   (ml)  (ml)    (gm)      3      17      41      65      931        .12     0      0      .8     .66    3.28    7.72    9.95   11.522        .12    .1      0      .8     .67    4.78   10.81   12.53   11.873        .12     0      2      .8     .58    4.06    9.47   11.75   12.174        .12    .1      2      .8     .81    5.34   11.41   12.73   12.575        .12     0      4      .8     .75    4.20   10.43   12.67   12.366        .12    .1      4      .8     .74    5.95   11.81   12.74   12.697        .12     0     10      .8     .58    5.87   12.08   12.87   11.918        .12    .1     10      .8     .65    7.11   12.25   12.61   12.369        .12     0     16      .8     .82    7.65   12.23   12.23   12.3610       .12    .1     16      .8     .59    7.77   12.23   12.30   12.11a AG＝Diazyme L-200的体积b AFP＝酸性真菌蛋白酶的2％溶液的体积c YE＝20％Amberex1003的体积d 干酵母＝弗雷斯克曼干啤酒酵母的量每个烧瓶含有450gm液化的全玉米糖化醪。烧瓶放在33℃水浴中。
                           表4
          含有回糟的工业全玉米液化糖化醪的同时糖化/发酵
                            乙醇含量％v/v
        AFPa               发酵时间(Hrs)烧瓶        (m1)       2.5        19.5        43.5         671            0        2.14        5.91       11.13      11.072          .05        2.12        7.32       11.96      13.263          .10        2.18        7.41       12.60      13.89a AFP＝2％AFP-2000溶液的体积每个烧瓶含有450gm糖化醪。发酵在30℃进行。
                             表5
           AFP对各种固体含量的全玉米液化糖化醪同时糖化/发酵的影响
                                    HPLC分布
         发酵时间1 葡糖  麦芽糖  未发酵碳水化物    乙醇    乳酸    甘油
         (Hrs)pH   ％w/v  ％w/v      ％w/v         ％w/v   ％w/v   ％w/v1号烧瓶-23.7％w/w糖化醪DS，0.12ml AGa，0ml AFPb
            3  4.5  2.14   3.12      12.06           .83    .18     .14
           16  3.9  2.72   5.93       4.46          4.41    .18     .52
           40  3.8   .94   1.71       3.43          9.13    .19     .84
           64  3.9   .56    .10       1.06         12.13    .22    1.03
           88  3.8    ND    .09        .38         12.94    .19    1.032号烧瓶-23.7％w/w 糖化醪 DS，0.12ml AG，0.10ml AFP
            3  4.4  2.28   3.17      11.49           .83    .17     .13
           16  3.9  1.90   5.71       4.16          5.18    .18     .54
           40  3.7   .28    .46       2.87         10.55    .19     .82
           64  4.0    ND    .08        .74         12.84    .21     .93
           88  3.9    ND    .06        .39         12.95    .19     .91
                         表5(续)
          AFP对各种固体含量的全玉米液化糖化醪同时糖化/发酵的影响
                                         HPLC分布
         发酵时间1   葡糖    麦芽糖  未发酵碳水化物     乙醇     乳酸    甘油
          (Hrs)pH     ％w/v    ％w/v        ％w/v       ％w/v     ％w/v   ％w/v3号烧瓶-26.4％ w/w 糖化醪 DS，0.132ml AG，0ml AFP
             3  4.4    2.24    3.49         14.16         .95       .19    .15
            16  4.0    3.43    6.65          5.03        4.74       .20    .56
            40  3.8    1.37    2.16          3.97        9.73       .20    .89
            64  4.0    1.48     .11          1.44       12.74       .21   1.06
            88  3.9     .59     .16           .60       13.04       .20   1.044号烧瓶-26.4％ w/w 糖化醪 DS，0.132ml AG，.11ml AFP
             3  4.4    2.17    3.08         11.51         .81       .17    .13
            16  4.0    2.55    6.14          4.60        5.60       .20    .58
            40  3.9     .58    1.00          3.62       11.39       .21    .89
            64  4.1      ND     .10           .87       14.17       .21   1.02
            88  4.0      ND     .08           .41       13.98       .20    .98
                         表5(续)
       AFP对各种固体含量的全玉米液化糖化醪同时糖化/发酵的影响
                                         HPLC分布
          发酵时间1  葡糖    麦芽糖  未发酵碳水化物    乙醇    乳酸    甘油
          (Hrs)pH    ％w/v    ％w/v     ％w/v          ％w/v   ％w/v   ％w/v5号烧瓶-28.3％ w/w 糖化醪 DS，0.144ml AG，0ml AFP
            3   4.4   2.62    3.48      12.84           .87      .18     .14
           16   3.9   3.88    6.42       4.80          4.67      .20     .53
           40   3.9   2.31    1.93       4.12         10.15      .21     .91
           64   4.2   2.74     .16       1.57         13.20      .22    1.09
           88   3.9   2.19     .20        .79         13.77      .21    1.106号烧瓶-28.3％ w/w 糖化醪 DS，0.144ml AG，0.12ml AFP
           3    4.3   3.24    4.14      14.02         1.02      .20      .16
          16    3.9   2.95    5.67       4.40         5.18      .19      .54
          40    3.9    .74    1.20       3.90        11.84      .21      .92
          64    4.2    .56     .18       1.06        15.00      .22     1.10
          88    4.0    .40     .18        .42        15.30      .22     1.11
                           表5(续)
    AFP对各种固体含量的全玉米液化糖化醪同时糖化/发酵的影响
                                       HPLC分布
       发酵时间1   葡糖    麦芽糖  未发酵碳水化物    乙醇    乳酸  甘油
        (Hrs)pH    ％w/v    ％w/v       ％w/v        ％w/v   ％w/v  ％w/v7号烧瓶-30.6％ w/w 糖化醪 DS，0.156ml AG，0ml AFP
            3    4.4   3.96  4.82       15.92         1.15     .22    .18
           16    4.0   4.65  6.73        5.12         4.74     .21    .54
           40    3.9   3.19  2.40        4.66        10.35     .22    .94
           64    4.2   4.27   .19        2.00        13.21     .23   1.11
           88    4.1   3.59   .22         .92        12.77     .21   1.058号烧瓶-30.6％ w/w 糖化醪 DS，0.156ml AG，0.13ml AFP
            3    4.4   4.36  4.94       14.14         1.12     .13    .17
           16    4.0   3.78  6.10        4.91         5.76     .21    .57
           40    4.0   1.37  1.24        4.16        12.35     .23    .93
           64    4.3   2.16   .23        1.32        15.09     .24   1.08
           88    4.1   2.13   .23         .63        15.59     .21    .98
                      表5(续)
       AFP对各种固体含量的全玉米液化糖化醪同时糖化/发酵的影响
                                       HPLC分布
           发酵时间1  葡糖   麦芽糖  未发酵碳水化物    乙醇    乳酸    甘油
           (Hrs)pH    ％w/v   ％w/v     ％w/v          ％w/v    ％w/v  ％w/v9号烧瓶-32.8％ w/w 糖化醪 DS，0.168ml AG，0ml AFP
             3   4.5    5.32   5.61     15.18           1.18      .24    .19
            16   4.0    5.68   7.07      5.54           4.94      .22    .59
            40   4.0    4.68   2.59      5.16          10.56      .24   1.01
            64   4.3    6.28    .24      2.42          13.37      .25   1.20
            88   4.2    5.86    .27      1.26          13.14      .23   1.1410号烧瓶-32.8％ w/w 糖化醪 DS，0.168ml AG，.14ml AFP
             3   4.4    5.00   5.37     14.09           1.14      .22    .17
            16   4.0    4.59   6.38      5.24           5.72      .21    .60
            40   4.0    2.63   1.57      4.75          12.73      .25   1.00
            64   4.3    4.66    .27      1.84          14.86      .26   1.15
            88   4.3    4.75    .28       .98          14.16      .24   1.061含有450gm糖化醪的所有烧瓶用0.8gm干弗雷斯克曼酵母接种并在33℃保温。a AG＝每个烧瓶中加入的Distillase L-200的体积。b AFP＝每个烧瓶中的2％AFP溶液的体积。
                              表6
            在表5中的各种糖化醪固体含量下乙醇含量的总结
 糖化醪                   乙醇％v/v烧瓶  ％DS    AFP    3hr    16hr    40hr   64hr    88hr1    23.7     -     .83    4.41    9.13   12.13   12.942    23.7     +     .83    5.18   10.55   12.84   12.953    26.4     -     .95    4.74    9.73   12.74   13.044    26.4     +     .81    5.60   11.39   14.17   13.985    28.3     -     .87    4.67   10.15   13.20   13.776    28.3     +    1.02    5.18   11.84   15.00   15.307    30.6     -    1.15    4.74   10.35   13.21   12.778    30.6     +    1.12    5.76   12.35   15.09   15.599    32.8     -    1.18    4.94   10.56   13.37   13.1410   32.8     +    1.14    5.72   12.73   14.86   14.16
                         表7
      AFP对含有回糟的液化的玉米糖化醪同时糖化/发酵的影响
     糖化醪   AG    AFP                 乙醇  ％v/v烧瓶1   ％DS    (ml)   (m1)    3hr    16hr    40hr    64hr    88hr1       24.2    .120    0      .88    4.68    9.82    11.83   11.652       24.2    .120    0.10   .87    5.34   11.15    12.18   11.813       26.33   .132    0      .94    4.99   10.06    12.83   11.824       26.33   .132    0.11   .93    4.87   11.10    13.21   12.935       28.6    .144    0      .92    5.12   10.77    13.92   13.516       28.6    .144    0.12  1.02    5.85   12.01    14.34   13.82
                          表7(续)
     AFP对含有回糟的液化的玉米糖化醪同时糖化/发酵的影响
   糖化醪   AG    AFP            乙醇％v/v烧瓶1  ％DS   (ml)   (ml)   3hr    16hr   40hr    64hr    88hr7      31.0  .156    0      1.27   5.75   11.05   13.90   13.428      31.0  .156    0.13   1.19   5.65   11.77   15.16   14.439      32.6  .168    0      1.28   5.86   11.20   13.90   13.7210     32.6  .168    0.14   1.20   6.50   12.90   15.32   14.901 所有烧瓶含有450gm糖化醪，用0.8gm干弗雷斯克曼酵母接种并在33℃保温。
                            表8-A
    用AFP糖化和全玉米糖化醪的发酵，发酵前在60℃有AFP存在的糖化
                                        HPLC分布
           发酵时间1   葡糖   麦芽糖  未发酵碳水化物    乙醇    甘油
           (Hrs)pH     ％w/v   ％w/v       ％w/v         ％w/v   ％w/v1号烧瓶-0.12ml AGa，0ml AFPb，.8gm酶母
            3   4.5    16.57    .46        1.54           .69      .15
           20   3.8     8.95    .32        1.21          5.60      .78
           44   3.9     2.96    .30         .90         16.22     1.09
           70   4.0      .05    .26         .51         12.21     1.182号烧瓶-0.12ml AG，.10ml AFP，.8gm酶母
            3   4.4    15.64    .53        1.63           .75      .16
           20   3.7     6.17    .37        1.32          7.45      .86
           44   4.0      .04    .39         .81         12.88     1.14
           70   4.0      .01    .29         .42         12.25     1.073号烧瓶-0.12ml AG，，12ml AFP，.8gm酶母
            3   4.4    15.78    .62        1.74            .83     .16
           20   3.8     5.65    .37        1.24           6.79     .79
           44   4.0      .04    .38         .81          12.62    1.12
           70   4.0      .02    .30         .42          12.62    1.08
                          表8-A(续)
    用AFP糖化和全玉米糖化醪的发酵，发酵前在60℃有AFP存在的糖化
                                    HPLC分布
        发酵时间1  葡糖   麦芽糖  未发酵碳水化物   乙醇    甘油
        (Hrs)pH    ％w/v   ％w/v       ％w/v       ％w/v   ％w/v4号烧瓶-0.12ml AG，.15ml AFP，.8gm酶母
            3  4.4  16.51   .60         1.74         .69     .15
           20  3.8   6.04   .36         1.36        6.90     .82
           44  4.2    .04   .37          .83       12.81    1.12
           70  4.1    .01   .30          .41       12.42    1.055号烧瓶-0.12ml AG，20ml AFP，.8gm酶母
            3  4.5  15.80   .48         1.63         .73     .15
           20  3.8   5.81   .35         1.37        7.70     .87
           44  4.0    .03   .36          .80       12.38    1.08
           70  4.0    .02   .30          .45       12.76    1.07a AG＝每个烧瓶中加入的Distillase L-200的体积。b AFP＝2％AFP-2000溶液的体积。
                              表8-B
                  用AFP糖化的全玉米糖化醪的发酵
           在发酵前在60℃糖化，在发酵过程中有AFP存在
                                      HPLC分布
         发酵时间1  葡糖   麦芽糖   碳水化物    乙醇    甘油
          (Hrs)pH   ％w/v   ％w/v     ％w/v     ％w/v   ％w/v6号烧瓶-0.12ml AGa，.10ml AFPb，.8gm酶母
            3   4.4   15.70  .38       1.30      .62    .14
           20   3.8    7.60  .31       1.10     6.76    .84
           44   4.0     .09  .35        .71    12.39    1.10
           70   4.1     .02  .30        .41    12.97    1.107号烧瓶-0.12ml AG，12ml AFP，.8gm酶母
            3   4.4   16.08  .40       1.44      .65     .14
           20   3.7    6.69  .29       1.19     6.32     .78
           44   4.1     .04  .35        .75    12.43    1.08
           70   4.1     .02  .28        .42    12.76    1.08
                            表8-B(续)
                   用AFP糖化的全玉米糖化醪的发酵
             在发酵前在60℃糖化，在发酵过程中有AFP存在
                                    HPLC分布
       发酵时间1    葡糖   麦芽糖   碳水化物    乙醇    甘油
       (Hrs)pH      ％w/v   ％w/v      ％w/v    ％w/v   ％w/v8号烧瓶-0.12ml AG，.15ml AFP，.8gm酶母
           3    4.4   15.49    .49      1.62      .60     .14
          20    3.8    6.41    .35      1.39     7.13     .85
          44    4.1     .61    .34       .80    12.51    1.08
          70    4.1     .02    .27       .44    12.78    1.059号烧瓶-0.12ml AG，.20ml AFP，.8gm酶母
           3    4.4   15.84    .50      1.64      .62     .14
          20    3.8    6.05    .35      1.59     7.11     .82
          44    4.12    .04    .33       .81    12.42    1.07
          70    4.11    .03    .26       .41    12.15    1.01a AG＝每个烧瓶中Distillase L-200的体积。b AFP＝每个烧瓶中2％AFP溶液的体积。
                           表9
                 表8-A和B中乙醇含量的总结
                                 乙醇％v/vAFP    糖化前   糖化后    3hrs    20hrs    44hrs    70hrs0       -        -       .69     5.60     10.22    12.21.10ml    +        -       .75     7.45     12.88    12.25.10ml    -        +       .62     6.75     12.39    12.97.12ml    +        -       .83     6.79     12.62    12.62.12ml    -        +       .65     6.32     12.43    12.76.15ml    +        -       .69     6.90     12.81    12.42.15ml    -        +       .60     7.13     12.51    12.78.20ml    +        -       .73     7.70     12.38    12.76.20ml    -        +       .62     7.11     12.42    12.15
                      表10
     AFP对全买罗高梁糖化醪同时糖化/发酵的影响
                                       HPLC分布
          发酵时间    葡糖   麦芽糖   未发酵碳水化物   乙醇     甘油
          (Hrs)pH    ％w/v   ％ w/v        ％w/v      ％w/v    ％w/v1号烧瓶-0.12ml AG，0ml AFP，.8gm酶母
           3   4.3    2.44     1.40        15.54        .84      .18
          17   3.9    1.09     3.63         8.16       5.07      .61
          41   3.7    1.06      .77         3.84       9.76      .94
          70   3.8     .10      .31          .91      11.94     1.032号烧瓶-0.12ml AG，0.1ml AFP，.8gm酶母
           3   4.3    2.85     1.47        15.37        .85      .18
          17   3.9     .75     3.38         7.31       5.78      .58
          41   3.8     .41      .32         2.40      11.19      .86
          70   3.8     .06      .22          .70      12.58      .913号烧瓶-0.12ml AG，0.2ml AFP，.8gm酶母
           3   4.3    2.69     1.40        14.45        .75      .17
          17   3.9     .80     2.90         7.70       6.41      .60
          41   3.8     .19      .27         2.16      11.34      .84
          70   3.8     .06      .20          .74      12.65      .87每个烧瓶含有450gm液化的买罗高梁糖化醪，所示量的Distillase L-200(AG)和2％AFP-2000。烧瓶放在33℃水浴中。

Claims (2)

1.含有葡糖淀粉酶和酸性真菌蛋白酶的组合物。
2.根据权利要求1的组合物，其中葡糖淀粉酶由黑曲霉衍生，酸性真菌蛋白酶由黑曲酶衍生。
CN99103605A 1991-05-17 1999-03-01 含有葡糖淀粉酶和酸性真菌蛋白酶的组合物 Pending CN1231926A (zh)

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DE69226772D1 (de) 1998-10-01
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