WO2022142381A1 - 矿化微生物粉剂制备方法 - Google Patents

矿化微生物粉剂制备方法 Download PDF

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WO2022142381A1
WO2022142381A1 PCT/CN2021/113383 CN2021113383W WO2022142381A1 WO 2022142381 A1 WO2022142381 A1 WO 2022142381A1 CN 2021113383 W CN2021113383 W CN 2021113383W WO 2022142381 A1 WO2022142381 A1 WO 2022142381A1
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mineralized
preparing
freeze
powder
protective agent
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钱春香
芮雅峰
陆兆文
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东南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

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  • the invention belongs to the interdisciplinary science and technology in the fields of microbiology and civil engineering materials, in particular to a method for preparing a mineralized microbial powder.
  • mineral deposition induced by mineralized microorganisms can block concrete cracks, achieve self-healing effect, and improve concrete durability; it also has a good application in heavy metal-contaminated soil and water bodies, and can deposit heavy metal ions to achieve the purpose of repair;
  • microbial mineralization technology has also been used to treat solid waste, providing a new way for solid waste resource utilization.
  • microbial mineralization technology is green, environmentally friendly, and has no secondary pollution. It has broad application prospects in the field of civil engineering materials. However, in practical applications, there is no microbial powder product that can be widely used.
  • the present application provides a preparation method of mineralized microorganism powder which can be widely used and makes the application process of mineralized microorganisms in the field of engineering materials more convenient and quicker.
  • a method for preparing a mineralized microbial powder according to the present invention comprises the following steps:
  • the mineralized microorganisms can induce the formation of mineralized products, which are harmless to the human body and have good ecological properties.
  • the mineralizing microorganism is one or more of Bacillus colloidii, Bacillus alkalophila, and the like.
  • the components of the medium used for culturing the mineralized microorganism include beef extract and peptone; wherein, each liter of the medium contains 2-5 g of beef extract and 3-8 g of peptone.
  • beef extract provides nitrogen source, carbon source and inorganic salt for the growth of mineralized microorganisms
  • peptone provides nitrogen source, carbon source and growth factor for the growth of microorganisms.
  • the culture conditions were 30 ⁇ 2° C., 170 ⁇ 5 rpm.
  • the centrifugation method is preferably a high-speed centrifugation method, which is characterized in that the operation is simple and quick, such as cleaning and disinfection of instruments, and it is easier to control pollution and has advantages.
  • centrifugal speed of the high-speed centrifugation method is 4000-6000 r/min, and the centrifugal time is 5-15 min.
  • the method for testing the total number of cells includes hemocytometer, flow cytometry or OD value method.
  • step (2) described vacuum freeze-drying technology needs to use freeze-drying protective agent to protect microorganism, and preferred freeze-drying protective agent is glycerol, glucose, L-cysteine salt, skimmed milk powder, Tween-80, One or more combinations of sodium glutamate.
  • the mass fraction of glycerol is preferably 5% to 10%
  • the mass fraction of glucose is preferably 10% to 15%
  • the mass fraction of L-cysteine salt is preferably 10% to 15%
  • the mass fraction of skim milk powder is preferably 10% to 15%
  • the mass fraction of Tween-80 is preferably 2% to 4%
  • the mass fraction of sodium glutamate is preferably 0.5% to 1.5%. with the best protection.
  • the mass fraction of skim milk powder is 10% to 15%
  • the mass fraction of glucose is 10% to 15%
  • the mass fraction of glycerol is 3 to 8%.
  • the mass fraction of sodium glutamate is 0.5% to 1%.
  • the mixing conditions of the freeze-dried protective agent and the bacterial cells include the pH value of the protective agent, the mixing ratio of the protective agent and the bacterial cells, and the mixing equilibrium time of the protective agent and the bacterial cells, and the pH value of the protective agent is preferably 7-9.
  • the mixing ratio of the protective agent and the bacterial liquid is preferably 2:3 to 3:2, and the mixing equilibrium time is preferably 50 to 70 min.
  • the pH of the protective agent was adjusted using acetic acid and sodium bicarbonate.
  • the thickness of the pre-freeze drying is preferably 3-8 mm, and the specific operation method of pre-freezing is to use an ultra-low temperature refrigerator to pre-freeze for 2 to 3 hours, and the temperature is set to -80 ⁇ 2°C.
  • the drying technology described in this application is preferably a vacuum freeze-drying technology, which is characterized in that: the drying is carried out at a low temperature, and the loss of some volatile components in the material is very small; the growth of microorganisms and the action of enzymes cannot be carried out, so the original properties can be maintained; It can remove more than 95-99% of the water, and the product can be stored for a long time without deterioration.
  • step (3) the storage condition is -68 ⁇ 4°C, and the lower the temperature, the more stable the powder is.
  • the conditions for judging the stability of the powder are enzyme activity and water content.
  • the test method of enzyme activity is conductivity method or colorimetric method; the test method of water content is gravimetric method.
  • the method for verifying the microbial mineralization ability is as follows: adding mineralized microorganisms and metal ions that can be mineralized in deionized water, culturing in a shaking incubator at 25-30°C, taking out after 3-5 days, and carrying out the precipitation product. verify.
  • the metal ions that can be mineralized include one or more of calcium ions, zinc ions, lead ions, etc., wherein the ion concentration is preferably 10-80 mmol/L.
  • the present invention proposes a method for preparing mineralized microbial powder, which is simple to operate, does not affect the activity of mineralized microorganisms, is easy to preserve, can be directly added to engineering materials for use, and can be used for rock Civil engineering, environmental geotechnical engineering, waste residue utilization and cement-based materials.
  • Fig. 1 is powder preparation process flow chart
  • Fig. 2 is a comparison chart of particle size distribution of mineralized products
  • Fig. 3 is the XRD comparison chart of mineralized products
  • Figure 4 is a comparison diagram of the microscopic morphology of the mineralized products.
  • the present invention provides a method for preparing a mineralized microbial powder.
  • the process flow is shown in Figure 1, and the specific operation process is as follows:
  • the microorganisms are inoculated into the culture medium, wherein the medium components include beef extract and peptone, and the culture conditions are 30 ⁇ 2 ° C, 170 ⁇ 5 rpm, and take out after culturing to the logarithmic phase;
  • the centrifuge After the centrifuge is sterilized, high-speed centrifugation is performed on the bacterial liquid, and the centrifugal separation conditions are preferably 4000-6000 rpm, 5-15 min, and a concentrated bacterial liquid is obtained for use.
  • the number of bacteria in the bacterial liquid is (5-7) ⁇ 10 8 CFU/ mL;
  • step 4 Mix the concentrated bacterial liquid in step 2 and the protective agent prepared in step 3 for 50 to 70 minutes according to the ratio of 2:3 to 3:2;
  • the protective agent is prepared with water as a solvent, wherein the mass fraction of skim milk powder is 15%, the mass fraction of glucose is 15%, the mass fraction of glycerol is 5%, and the mass fraction of sodium glutamate is 1%.
  • the pH was adjusted to 8 with acetic acid and sodium bicarbonate adjustment;
  • step (5) powder/microorganism stock solution and calcium chloride in deionized water, so that the total number of cells is 10 8 CFU/mL, and the calcium ion concentration is 25 mmol/L, and placed in a shaking culture at 25 to 30° C.
  • the protective agent is prepared with water as a solvent, wherein the mass fraction of skim milk powder is 10%, the mass fraction of glucose is 15%, the mass fraction of glycerol is 3%, and the mass fraction of sodium glutamate is 1%.
  • the pH was adjusted to 8 with acetic acid and sodium bicarbonate adjustment;
  • step (6) Add the powder of step (5) and calcium chloride in deionized water, so that the total number of cells is 10 8 CFU/mL, and the calcium ion concentration is 25 mmol/L, and placed in a shaking incubator at 25 to 30 °C for 3 days. After taking out, the precipitation product was verified, and the results showed that the powder had mineralization ability.
  • the protective agent is prepared with water as a solvent, wherein the mass fraction of skim milk powder is 15%, the mass fraction of glucose is 10%, the mass fraction of glycerol is 3%, and the mass fraction of sodium glutamate is 1%.
  • the pH was adjusted to 8 with acetic acid and sodium bicarbonate adjustment;
  • step (6) Add the powder of step (5) and calcium chloride in deionized water, so that the total number of cells is 10 8 CFU/mL, and the calcium ion concentration is 50 mmol/L. After taking out, the precipitation product was verified, and the results showed that the powder had mineralization ability.
  • the protective agent is prepared with water as a solvent, wherein the mass fraction of skim milk powder is 15%, the mass fraction of glucose is 15%, the mass fraction of glycerol is 5%, and the mass fraction of sodium glutamate is 1%.
  • the pH was adjusted to 8 with acetic acid and sodium bicarbonate adjustment;
  • step (6) Add the powder of step (5) and zinc chloride in deionized water, so that the total number of cells is 10 8 CFU/mL, and the calcium ion concentration is 50 mmol/L, and then placed in a shaking incubator at 25 to 30° C. for 3 days. Take out and verify the precipitated product. The result shows that the powder has mineralization ability.

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Abstract

公开了一种工程材料领域矿化微生物粉剂制备方法，属于微生物学和土木工程材料领域的交叉科学技术。首先将矿化微生物进行培养，当菌液培养至对数期时，使用高速离心机进行菌液浓缩，获得细菌总数为(5-7)×108CFU/mL的浓缩菌液，将浓缩菌液与配制好的保护剂混合后预冻，将预冻菌液通过冷冻干燥技术制备成粉剂，粉剂通过低温方式进行储存，冻干后矿化微生物仍保持矿化能力。方法操作简单，不影响矿化微生物活性，便于保存，可直接掺加到工程材料中使用，可用于岩土工程、环境岩土工程、废渣利用和水泥基材料领域。

Description

矿化微生物粉剂制备方法 技术领域
本发明属于微生物学和土木工程材料领域的交叉科学技术，具体涉及一种矿化微生物粉剂制备方法。
背景技术
微生物矿化技术在工程材料领域的应用引起了广大学者的关注。由于矿化微生物能诱导生物矿物沉积，且相比于化学方法形成的矿物具有显著胶结能力，可以胶结砂土形成具有一定强度的胶结体，因此可应用于岩土工程，如抑制扬尘、边坡防护等。此外，矿化微生物诱导矿物沉积可封堵混凝土裂缝，达到自愈合的效果，提高混凝土耐久性；在重金属污染的土壤和水体中也有较好的应用，可以沉积重金属离子，达到修复的目的；近年来，微生物矿化技术还被用于处理固体废弃物，为固废资源化利用提供了新途径。
相比传统方法，微生物矿化技术绿色、环保，无二次污染，在土木工程材料领域具有广阔应用前景，但具体实际运用中，还没有可广泛使用的微生物粉剂产品。
发明内容
发明目的：针对上述现有技术，本申请提供了一种可广泛使用，使得矿化微生物在工程材料领域的应用过程更加方便快捷的矿化微生物粉剂的制备方法。
技术方案：本发明所述的一种矿化微生物粉剂制备方法，包括以下步骤：
(1)取矿化微生物培养至对数期后取出，高速离心浓缩菌液，细胞总数为(5～7)×10 8CFU/mL；
(2)将浓缩菌液和冷冻保护剂混合后预冻；
(3)真空冷冻干燥获得矿化微生物粉剂，储存。
步骤(1)中，所述矿化微生物能诱导矿化产物生成，对人体无害且生态性良好。进一步的，所述矿化微生物为胶质芽孢杆菌、嗜碱芽孢杆菌等一种或多种。
步骤(1)中，培养矿化微生物所用培养基成分包括牛肉膏、蛋白胨；其中，每升培养基含有牛肉膏2～5g，蛋白胨3～8g。其中，牛肉膏为矿化微生物生长提供氮源、碳源、无机盐，蛋白胨为微生物生长提供氮源、碳源、生长因子。
进一步的，培养条件为30±2℃、170±5rpm。
所述离心分离方法优选高速离心法，其特点是，操作简便快捷，比如再仪器的清洗和消毒，较易控制污染等方面具有优势。
进一步的，所述高速离心法的离心速度为4000～6000r/min，离心时间为5～15min。
本申请中，所述细胞总数测试方法包括血球计数板计数法、流式细胞仪法或OD值法。
步骤(2)中，所述真空冷冻干燥技术需使用冻干保护剂对微生物进行保护，优选的冻干保护剂为甘油、葡萄糖、L–半胱氨酸盐、脱脂奶粉、吐温-80、谷氨酸钠的一种或几种复配。
当选用单一冻干保护剂时，以水作为溶剂，甘油的质量分数优选为5％～10％，葡萄糖的质量分数优选为10％～15％，L–半胱氨酸盐的质量分数优选为10％～15％、脱脂奶粉的质量分数优选为10％～15％、吐温-80的质量分数优选为2％～4％、谷氨酸钠的质量分数优选为0.5％～1.5％，此时具有最佳保护效果。
当选用冻干保护剂进行复配时，以水作为溶剂，脱脂乳粉的质量分数为10％～15％，葡萄糖的质量分数为10％～15％，甘油的质量分数为3～8％，谷氨酸钠的质量分数为0.5％～1％。
步骤(2)中，所述冻干保护剂与菌体的混合条件包括保护剂pH值、保护剂与菌体混合比例、保护剂与菌体混合平衡时间，保护剂pH值优选为7～9，保护剂与菌液的混合比例优选为2:3～3:2，混合平衡时间优选为50～70min。
进一步的，保护剂的pH值使用乙酸和碳酸氢钠调节。
步骤(2)中，所述预冻冻干厚度优选的为3～8mm，预冻的具体操作方法为，使用超低温冰箱预冻2～3h，温度设置为-80±2℃。
本申请所述干燥技术优选真空冷冻干燥技术，其特点是：干燥在低温下进行，物质中的一些挥发性成分损失很小；微生物的生长和酶的作用无法进行，因此能保持原来的性状；能排除95～99％以上的水分，后产品能长期保存而不致变质等。
步骤(3)中，所述储存条件为-68～4℃，温度越低，粉剂越稳定。
进一步的，对粉剂稳定性的判断条件为酶活性和含水量。酶活性的测试方法为电导率法或比色法；含水量的测试方法为重量测量法。
所述微生物矿化能力验证方法为：在去离子水中掺加矿化微生物和可被矿化的金属离子，置于25～30℃振荡培养箱中培养，3～5d后取出，对沉淀产物进行验 证。
进一步的，所述可被矿化的金属离子包括钙离子、锌离子、铅离子等的一种或几种，其中离子浓度优选为10～80mmol/L。
有益效果：与现有技术相比，本发明提出一种矿化微生物粉剂制备方法，该方法操作简单，不影响矿化微生物活性，便于保存，可直接掺加到工程材料中使用，可用于岩土工程、环境岩土工程、废渣利用和水泥基材料领域。
附图说明
图1为粉剂制备工艺流程图；
图2为矿化产物粒径分布对比图；
图3为矿化产物XRD对比图；
图4为矿化产物微观形貌对比图。
具体实施方式
为更好的理解本发明，将结合具体实施例进一步阐述本发明所述内容。
本发明提供一种矿化微生物粉剂制备方法，工艺流程如图1所示，具体操作过程如下：
1.将微生物接种到培养基进行培养，其中培养基成分包括牛肉膏和蛋白胨，培养条件为30±2℃、170±5rpm，培养至对数期后取出；
2.离心机消毒后对菌液进行高速离心分离，离心分离条件优选为4000～6000rpm、5～15min，获得浓缩菌液待用，菌液中细菌数为(5～7)×10 8CFU/mL；
3.按照保护剂配方配制保护剂，使用乙酸和碳酸氢钠调节将pH调节至7～9范围内待用；
4.将步骤2浓缩的菌液与步骤3配制的保护剂按照2:3～3:2的比例混合50～70min；
5.将混合后菌液放入超低温冰箱预冻2～3h，冻干厚度为3～8mm；
6.将预冻好的菌液放入真空冷冻干燥机中，经过24h的冻干获得冻干粉剂，密封后放入超低温冰箱储存。
7.在去离子水中掺加矿化微生物和可被矿化的金属离子，置于25-30℃振荡培养箱中培养，3-5d后取出，对沉淀产物进行验证。
实施例1
一种矿化微生物粉剂制备方法：
(1)称取牛肉膏3g/L，蛋白胨5g/L，配置成液体培养基，在121℃条件下灭菌30min后接种胶质芽孢杆菌，在中国典型培养物保藏中心的保藏编号为CCTCC No:M 2012406，在30±2℃、170±5rpm条件下培养；
(2)将培养至对数期的菌液倒入离心管置于高速离心机中。设置离心速度4000r/min，离心时间5min，获得浓缩菌液，测得细菌总数为5.233×10 8CFU/mL；
(3)以水作为溶剂配制保护剂，其中脱脂乳粉的质量分数为15％，葡萄糖的质量分数为15％，甘油的质量分数为5％，谷氨酸钠的质量分数为1％，使用乙酸和碳酸氢钠调节将pH调节至8；
(4)将配制的保护剂与离心所得的浓缩菌液按1:1的体积平衡混合60min，然后放入超低温冰箱中预冻2h，冻干厚度为5mm；
(5)将预冻好的菌液放入真空冷冻干燥机中，经过24h的冻干获得冻干粉剂，密封后放入超低温冰箱储存，性质稳定；
(6)在去离子水中掺加步骤(5)粉剂/微生物原菌液和氯化钙，使得细胞总数为10 8CFU/mL，钙离子浓度为25mmol/L，置于25～30℃振荡培养箱中培养，3d后取出，根据图2可见，以冻干粉剂培养的菌液与冻干前菌液形成的矿化产物沉淀粒径相差不大；根据图3中XRD结果可见，两图中的曲线在2θ为12.6°、32.9°和59.4°处都有3个特征衍射峰，由此可以判断菌液与粉剂所形成的矿化产物的成分是一致的；根据图4中SEM结果可见，菌液与粉剂形成的矿化产物沉淀在形貌上基本没有差别，由此可见，本发明涉及的工程材料矿化微生物粉剂制备方法具有较好的应用效果。
实施例2
一种矿化微生物粉剂制备方法：
(1)称取牛肉膏3g/L，蛋白胨5g/L，配置成液体培养基，在121℃条件下灭菌30min后接种胶质芽孢杆菌，在中国典型培养物保藏中心的保藏编号为CCTCC No:M 2012406，在30±2℃、170±5rpm条件下培养；
(2)将培养至对数期的菌液倒入离心管置于高速离心机中。设置离心速度5000r/min，离心时间5min，获得浓缩菌液，测得细菌总数为5.453×10 8CFU/mL；
(3)以水作为溶剂配制保护剂，其中脱脂乳粉的质量分数为10％，葡萄糖的质量分数为15％，甘油的质量分数为3％，谷氨酸钠的质量分数为1％，使用 乙酸和碳酸氢钠调节将pH调节至8；
(4)将配制的保护剂与离心所得的浓缩菌液按1:1的体积平衡混合70min，然后放入超低温冰箱中预冻2h，冻干厚度为6mm；
(5)将预冻好的菌液放入真空冷冻干燥机中，经过24h的冻干获得冻干粉剂，密封后放入超低温冰箱储存，性质稳定；
(6)在去离子水中掺加步骤(5)粉剂和氯化钙，使得细胞总数为10 8CFU/mL，钙离子浓度为25mmol/L，置于25～30℃振荡培养箱中培养，3d后取出，对沉淀产物进行验证，结果表明，粉剂具有矿化能力。
实施例3
一种矿化微生物粉剂制备方法：
(1)称取牛肉膏5g/L，蛋白胨5g/L，配置成液体培养基，在121℃条件下灭菌30min后接种胶质芽孢杆菌，在中国典型培养物保藏中心的保藏编号为CCTCC No:M 2012406，在30±2℃、170±5rpm条件下培养；
(2)将培养至对数期的菌液倒入离心管置于高速离心机中。设置离心速度5000r/min，离心时间10min，获得浓缩菌液，测得细菌总数为5.475×10 8CFU/mL；
(3)以水作为溶剂配制保护剂，其中脱脂乳粉的质量分数为15％，葡萄糖的质量分数为10％，甘油的质量分数为3％，谷氨酸钠的质量分数为1％，使用乙酸和碳酸氢钠调节将pH调节至8；
(4)将配制的保护剂与离心所得的浓缩菌液按3:2的体积平衡混合50min，然后放入超低温冰箱中预冻2h，冻干厚度为5mm；
(5)将预冻好的菌液放入真空冷冻干燥机中，经过24h的冻干获得冻干粉剂，密封后放入超低温冰箱储存，性质稳定；
(6)在去离子水中掺加步骤(5)粉剂和氯化钙，使得细胞总数为10 8CFU/mL，钙离子浓度为50mmol/L，置于25-30℃振荡培养箱中培养，3d后取出，对沉淀产物进行验证，结果表明，粉剂具有矿化能力。
实施例4
一种矿化微生物粉剂制备方法：
(1)称取牛肉膏5g/L，蛋白胨5g/L，配置成液体培养基，在121℃条件下灭菌30min后接种嗜碱芽孢杆菌，中国工业微生物菌种保藏管理中心菌株保藏编 号为CICC 23037，在30±2℃、170±5rpm条件下培养；
(2)将培养至对数期的菌液倒入离心管置于高速离心机中。设置离心速度6000r/min，离心时间10min，获得浓缩菌液，测得细菌总数为5.616×10 8CFU/mL；
(3)以水作为溶剂配制保护剂，其中脱脂乳粉的质量分数为15％，葡萄糖的质量分数为15％，甘油的质量分数为5％，谷氨酸钠的质量分数为1％，使用乙酸和碳酸氢钠调节将pH调节至8；
(4)将配制的保护剂与离心所得的浓缩菌液按1:1的体积平衡混合60min，然后放入超低温冰箱中预冻2h，冻干厚度为5mm；
(5)将预冻好的菌液放入真空冷冻干燥机中，经过24h的冻干获得冻干粉剂，密封后放入超低温冰箱储存，性质稳定；
(6)在去离子水中掺加步骤(5)粉剂和氯化锌，使得细胞总数为10 8CFU/mL，钙离子浓度为50mmol/L置于25～30℃振荡培养箱中培养，3d后取出，对沉淀产物进行验证，结果表明，粉剂具有矿化能力。

Claims (8)

  1. 一种矿化微生物粉剂制备方法，其特征在于，包括以下步骤：
    (1)取矿化微生物培养至对数期后取出，高速离心浓缩菌液，细胞总数为(5～7)×10 8CFU/mL；
    (2)将浓缩菌液和冷冻保护剂混合后预冻；
    (3)真空冷冻干燥获得矿化微生物粉剂，储存。
  2. 根据权利要求1所述的矿化微生物粉剂制备方法，其特征在于，步骤(1)中，每升培养矿化微生物所用培养基成分包括牛肉膏2～5g，蛋白胨3～8g。
  3. 根据权利要求1所述的矿化微生物粉剂制备方法，其特征在于，步骤(1)中，高速离心是指：4000～6000r/min离心5～15min。
  4. 根据权利要求1所述的矿化微生物粉剂制备方法，其特征在于，步骤(2)中，所述冷冻保护剂为甘油、葡萄糖、L–半胱氨酸盐、脱脂奶粉、吐温-80、谷氨酸钠的一种或几种复配。
  5. 根据权利要求1所述的矿化微生物粉剂制备方法，其特征在于，步骤(2)中，浓缩菌液与保护剂混合要求为：保护剂pH值优选为7～9，保护剂与菌液的混合比例优选为2:3～3:2，混合平衡时间优选为50～70min。
  6. 根据权利要求5所述的矿化微生物粉剂制备方法，其特征在于，保护剂pH使用乙酸和碳酸氢钠调节。
  7. 根据权利要求1所述的矿化微生物粉剂制备方法，其特征在于，步骤(2)中，所述预冻冻干厚度为3～8mm，使用超低温冰箱预冻2～3h。
  8. 根据权利要求1所述的矿化微生物粉剂制备方法，其特征在于，步骤(3)中，储存方法为低温储存，温度为-68～4℃，温度越低，粉剂越稳定。
PCT/CN2021/113383 2020-12-31 2021-08-19 矿化微生物粉剂制备方法 WO2022142381A1 (zh)

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