Preparation method of instant and ready-to-use bacillus pasteurii dry powder culture medium

Abstract

A preparation method of a ready-to-use bacillus pasteurii dry powder culture medium can realize large-scale production and convenient application of bacillus pasteurii, and is characterized by comprising the following steps: a, weighing 25-35 parts of bran and 25-30 parts of soybean meal, and mixing the bran and the soybean meal to obtain a dry mixture 1; 150-175 parts of purified water and 0.25 part of sodium bicarbonate are added into the dry mixture 1, the mixture is stirred and then left to stand for 1.5 h at the temperature of 45-50 DEG C, a reaction base material 2 is prepared, and bran and soybean meal in the dry mixture 1 are not sterilized; b, adding 1 part of protease powder (containing 0.91-0.97 part of glucose) into the reaction base material 2, reacting at the temperature of 45-50 DEG C for 12 hours, covering the reaction base material 2 with a cover surface net without turning over and stirring during the reaction base material 2, then filtering the reaction base material 2, and collecting filtrate as a reaction base solution 3; c, the reaction base solution 3 is subjected to cooking, concentration and spray drying in sequence, and powder 4 is prepared; and d, adding 5 parts of sodium bicarbonate and 20 parts of urea into the powder 4 to prepare the bacillus pasteurii dry powder culture medium.

Description

Technical Field

[0001] The invention relates to a method for preparing an instant Bacillus pasteurianus dry powder culture medium, belonging to the field of biotechnology. Background Art

[0002] MICP (microbial induced calcium carbonate precipitation) technology is an emerging reinforcement technology that uses microbial metabolic activities (including urea decomposition, photosynthesis, denitrification, sulfate reduction, etc.) to generate cementitious calcium carbonate to produce intergranular bonding and pore filling effects to improve the strength of the body to be reinforced. It can be used in engineering fields such as foundation treatment, slope reinforcement, windbreak and sand fixation, and concrete crack repair. At present, MICP technology has made gratifying progress in both indoor tests and field applications, but it still faces many challenges, such as adaptability to different application conditions, possible by-products, and instability in large-scale engineering applications, especially the problem of higher costs than chemical and traditional methods. Summary of the invention

[0003] Technical issues: Bacillus pasteurii is an engineered bacterium widely used in the application of MICP technology. It has the advantages of strong environmental adaptability and high urease activity. The secreted urease can decompose urea into carbonate and ammonia. The generated carbonate will react with extracellular calcium ions to generate gelling calcium carbonate. At present, yeast extract, beef extract, trypsin, etc. are mostly used as the main nutrient components for the enrichment culture of Bacillus pasteurii. Although the concentration of live bacteria cultured in this way can meet the requirements of industrial production, the culture cost is high. The cost of each ton of liquid culture medium is more than 1,500 yuan, which seriously restricts the promotion and application of MICP technology. Finding an economical, cheap and widely available culture medium raw material and developing a Bacillus pasteurii culture medium with this raw material as the main component is of great practical significance for realizing the industrial production of Bacillus pasteurii and promoting the widespread application of MICP technology.

[0004] Technical solution: Bran and soybean meal are waste materials in the processing of agricultural products, and corn flour is directly crushed from corn. Soybean meal is rich in protein and contains a small amount of small peptides and amino acids. Bran and corn contain appropriate amounts of protein, starch, and a small amount of reducing sugar. The Chinese patent 2018105977073 applied by the applicant extracts amino acids, small peptides, reducing sugars and other nutrients from bran, soybean meal and corn flour for fermentation and culture of Bacillus pasteurianus, which effectively reduces the culture cost of Bacillus pasteurianus and provides a new idea for the development of cheap culture medium for Bacillus pasteurianus. However, directly dissolving bran, soybean meal and corn flour in water for steaming (high temperature sterilization) cannot obtain sufficient small peptides, amino acids and reducing sugars to provide nitrogen sources and nitrogen sources for rapid bacterial proliferation. The efficiency of culturing bacteria in this way is low, the portability is poor, it cannot be dissolved and used immediately, and the application scenarios are limited. The protein in soybean meal, bran and corn can be decomposed into small peptides or amino acids by protease, while the amylase contained in bran and corn can enzymolyze the starch contained therein into reducing sugar. Since bran and corn flour are both used to provide carbon sources for bacterial growth, their functional positioning overlaps. Although the starch content in bran is lower than that in corn, its purchase price is only half of that in corn. Therefore, enzymatic hydrolysis of bran and protein and starch in soybean meal for bacterial culture is more cost-effective than enzymatic hydrolysis of bran, corn, and protein and starch in soybean meal for bacterial culture. Soaking soybean meal and bran for a specific time under specific temperature and pH conditions can fully dissolve soluble protein, a small amount of amino acids, small peptides, reducing sugars, starch, and amylase from soybean meal and bran. In this process, free starch is further hydrolyzed to generate reducing sugars under the catalytic action of amylase, and the native bacteria of soybean meal and bran (Bacillus subtilis, lactic acid bacteria, etc.) begin to grow slowly. Then, protease powder is added to the soybean meal and bran soaking liquid. As the protein therein is decomposed into small peptides or amino acids, the nitrogen source content in the soaking liquid increases accordingly. The glucose carried in the protease powder supplements the carbon source required for bacterial growth. The relatively abundant carbon and nitrogen sources in the soybean meal and bran soaking liquid promote the rapid proliferation of the native bacteria therein and secrete a large amount of proteases, cellulases, and amylases. The combined action of the proteases secreted by bacteria and the exogenous enzymes further promotes the process of protein enzymatic hydrolysis. The degradation of cellulose in the bran by cellulase accelerates the dissolution of starch, and the large amount of dissolved starch is continuously converted into reducing sugars that can be used by bacteria by amylase. The above series of reactions produce a considerable amount of small peptides, amino acids, and reducing sugars in the soybean meal and bran soaking liquid. Extracting the products of the above reactions and adding sodium bicarbonate (for providing inorganic salts and adjusting the pH of the culture medium) and urea to prepare an instant dry powder culture medium is conducive to achieving low-cost cultivation of Bacillus pasteurianus, large-scale production, and convenient application, thereby promoting the large-scale promotion of MICP technology. Based on this, the present invention provides a method for preparing an instant dry powder culture medium for Bacillus pasteurianus based on bran and soybean meal as the main raw materials, characterized in that the method steps are as follows: a. Weigh 25-35 parts of bran and 25-30 parts of soybean meal respectively and mix the two to obtain a dry blend 1, add 150-175 parts of purified water and 0.25 parts of sodium bicarbonate to the dry blend 1, stir and place at 45-50 ° C for 1.5 hours to obtain a reaction base 2, wherein the bran and soybean meal in the dry blend 1 are not sterilized; b. Add 1 part of protease powder (containing 0.91-0.97 parts of glucose) to the reaction base 2 and place it at a temperature of 45-50 ° C for 12 hours, during which the reaction base 2 is covered with a screen and is not stirred, and then the reaction base 2 is filtered and the filtrate is collected as the reaction base liquid 3; c. The reaction base liquid 3 was successively subjected to cooking, concentration, and spray drying to obtain a powder 4. The temperature of the reaction base liquid 3 was controlled at 85°C and the cooking time was 20min; d. Add 5 parts of sodium bicarbonate and 20 parts of urea to powder 4 to obtain Bacillus pasteurianus dry powder culture medium.

[0005] Beneficial effects of the present invention: (1) The present invention uses bran and soybean meal, which are widely available, cheap and easily available, as raw materials to prepare the culture medium. Under the premise of ensuring good bacterial growth indicators and working performance, the culture cost of Bacillus pasteurianus is greatly reduced. Compared with industrial culture medium, the culture cost can be reduced by up to 90%; (2) Nutrients in bran and soybean meal are extracted through a series of biochemical reactions and made into powders, thereby solving the problem of low raw material utilization rate and inability to dissolve and use immediately after bacterial fermentation, which limits the application scenarios; (3) The filter residue left after filtering the reaction liquid contains rich protein and low anti-nutritional factor content, which can be eaten by livestock, realizing the comprehensive resource utilization of agricultural and forestry wastes and avoiding the pollution of the environment caused by discarded filter residues. DETAILED DESCRIPTION

[0006] The technical solution of the present invention is described below in conjunction with specific embodiments. The following is a preferred embodiment of the present invention, which can be used to explain and support the present invention, but does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the following embodiments that do not deviate from the content of the technical solution of the present invention and are based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

[0007] Example 1

[0008] In this example, the instant dry powder culture medium is prepared by using the present invention, and the prepared dry powder culture medium is used for culturing Bacillus pasteurianus. The specific steps and implementation effects are as follows: a. Weigh 25 parts of bran and 25 parts of soybean meal respectively and mix the two to obtain a dry blend 1, then add 150 parts of purified water and 0.25 parts of sodium bicarbonate to the dry blend 1, stir and place at 45 ° C for 1.5 hours to obtain a reaction base 2, wherein the bran and soybean meal in the dry blend 1 are not sterilized; b. Add 1 part of protease powder to the reaction base 2 and place it at a temperature of 45 ° C for 12 hours, during which the reaction base 2 is covered with a screen and is not stirred, and then the reaction base 2 is filtered by coarse filtration and fine filtration in two steps and the filtrate is collected as the reaction base liquid 3. The protease added to the reaction base 2 is extracted and produced by fermentation of Bacillus subtilis, and 1 part of protease powder contains 0.91 parts of glucose; c. The reaction base liquid 3 was successively subjected to cooking, concentration, and spray drying to obtain a powder 4. The temperature of the reaction base liquid 3 was controlled at 85°C and the cooking time was 20min; d. Add 5 parts of sodium bicarbonate and 20 parts of urea to powder 4 to obtain Bacillus pasteurianus dry powder culture medium. The prepared Bacillus pasteurian powder culture medium was dissolved in 1000 parts of purified water to prepare 1L culture solution. 200mL of the culture solution was placed in a 500mL conical flask and inoculated with Bacillus pasteurianus. The culture was placed in a constant temperature shaking culture at 30℃ for 48h, and the OD of the culture solution was measured. 600 The urease activity of the bacteria was 10.5 mmol / min / L. Under the same test conditions, the OD value of the bacterial solution obtained by culturing with the industrial culture medium (containing 5 g / L soy peptone, 15 g / L tryptone, 5 g / L sodium chloride, and 20 g / L urea) prepared with the formula recommended by the bacterial culture collection center was 3.86. 600 The dry powder culture medium prepared in the present invention can be dissolved in water to prepare 1L of culture solution at a cost of about 0.15 yuan, while the cost of preparing 1L of industrial culture medium according to the formula recommended by the preservation center is about 1.50 yuan. It can be seen that the dry powder culture medium prepared by the present invention can be used immediately after dissolving, and the cost of culturing Bacillus pasteurianus using the dry powder culture medium is low and the effect is good.

[0009] Example 2

[0010] In this example, the instant dry powder culture medium is prepared by using the present invention, and the prepared dry powder culture medium is used to cultivate Bacillus pasteurianus for sand solidification. The specific steps and implementation effects are as follows: a. Weigh 30 parts of bran and 30 parts of soybean meal respectively and mix the two to obtain a dry blend 1, then add 170 parts of purified water and 0.25 parts of sodium bicarbonate to the dry blend 1, stir and place at 47 ° C for 1.5 hours to obtain a reaction base 2, wherein the bran and soybean meal in the dry blend 1 are not sterilized; b. Add 1 part of protease powder to the reaction base 2 and place it at 47 ° C for 12 hours, during which the reaction base 2 is covered with a screen and is not stirred, and then the reaction base 2 is filtered by coarse filtration and fine filtration in two steps and the filtrate is collected as the reaction base liquid 3. The protease added to the reaction base 2 is extracted and produced by fermentation of Bacillus subtilis, and 1 part of protease powder contains 0.95 parts of glucose; c. The reaction base liquid 3 was successively subjected to cooking, concentration, and spray drying to obtain a powder 4. The temperature of the reaction base liquid 3 was controlled at 85°C and the cooking time was 20min; d. Add 5 parts of sodium bicarbonate and 20 parts of urea to powder 4 to obtain Bacillus pasteurianus dry powder culture medium. The prepared Bacillus pasteurian powder culture medium was dissolved in 1000 parts of purified water to prepare 1L culture solution. 200mL of the culture solution was placed in a 500mL conical flask and inoculated with Bacillus pasteurianus. The culture was placed in a constant temperature shaking culture at 30℃ for 48h, and the OD of the culture solution was measured. 600 The urease activity of the bacteria was 11.7 mmol / min / L, and then the bacterial solution was injected into the loose sand. After an interval of 12 hours, a cementing solution composed of 1 mol / L urea and calcium chloride in an equal molar ratio was injected. A total of 1 bacterial solution and 4 cementing solutions were injected to solidify the sand. The unconfined compressive strength of the solidified sand was 378 kPa. Under the same test conditions, the OD of the bacterial solution obtained by culturing with the industrial culture medium (containing 5 g / L soy peptone, 15 g / L tryptone, 5 g / L sodium chloride, and 20 g / L urea) prepared with the formula recommended by the strain collection center was 0. 600 The urease activity of the bacteria is 2.75, and the unconfined compressive strength of the sand solidified by the bacterial liquid is 232 kPa. The cost of preparing 1L of culture solution by dissolving the dry powder culture medium in water is about 0.17 yuan, while the cost of preparing 1L of industrial culture medium according to the formula recommended by the preservation center is about 1.50 yuan. It can be seen that the dry powder culture medium prepared by the present invention can be dissolved and used immediately, and the cost of culturing Bacillus pasteurianus and using it for sand solidification is low and the effect is good.

[0011] Example 3

[0012] In this example, the dry powder culture medium prepared by the present invention is also used to culture Bacillus pasteurianus and used for sand solidification. The specific steps and implementation effects are as follows: a. Weigh 35 parts of bran and 30 parts of soybean meal respectively and mix the two to obtain a dry blend 1, then add 175 parts of purified water and 0.25 parts of sodium bicarbonate to the dry blend 1, stir and place at 50 ° C for 1.5 hours to obtain a reaction base 2, wherein the bran and soybean meal in the dry blend 1 are not sterilized; b. Add 1 part of protease powder to the reaction base 2 and place it at a temperature of 50 ° C for 12 hours, during which the reaction base 2 is covered with a screen and is not stirred, and then the reaction base 2 is filtered by coarse filtration and fine filtration in two steps and the filtrate is collected as the reaction base liquid 3. The protease added to the reaction base 2 is extracted and produced by fermentation of Bacillus subtilis, and 1 part of protease powder contains 0.97 parts of glucose; c. The reaction base liquid 3 was successively subjected to cooking, concentration, and spray drying to obtain a powder 4. The temperature of the reaction base liquid 3 was controlled at 85°C and the cooking time was 20min; d. Add 5 parts of sodium bicarbonate and 20 parts of urea to powder 4 to obtain Bacillus pasteurianus dry powder culture medium. The prepared Bacillus pasteurian powder culture medium was dissolved in 1000 parts of purified water to prepare 1L culture solution. 200mL of the culture solution was placed in a 500mL conical flask and inoculated with Bacillus pasteurianus. The culture was placed in a constant temperature shaking culture at 30℃ for 48h, and the OD of the culture solution was measured. 600 The urease activity of the bacteria was 18.6 mmol / min / L, and then the bacterial solution was injected into the loose sand. After an interval of 12 hours, a cementing solution composed of 1 mol / L urea and calcium chloride in an equal molar ratio was injected. A total of 1 bacterial solution and 4 cementing solutions were injected to solidify the sand. The unconfined compressive strength of the solidified sand was 564 kPa. Under the same test conditions, the OD of the bacterial solution obtained by culturing with the industrial culture medium (containing 5 g / L soy peptone, 15 g / L tryptone, 5 g / L sodium chloride, and 20 g / L urea) prepared with the formula recommended by the strain collection center was 0. 600 The urease activity of the bacteria is 2.75, and the unconfined compressive strength of the sand solidified by the bacterial liquid is 232 kPa. The cost of preparing 1L of culture solution by dissolving the dry powder culture medium in water is about 0.21 yuan, while the cost of preparing 1L of industrial culture medium according to the formula recommended by the preservation center is about 1.50 yuan. It can be seen that the dry powder culture medium prepared by the present invention can be dissolved and used immediately, and the cost of culturing Bacillus pasteurianus and using it for sand solidification is low and the effect is good.

Claims

1. A method for preparing an instant Bacillus pasteurianus dry powder culture medium, characterized in that The steps of this method are as follows: a. Weigh 25-35 parts of bran and 25-30 parts of soybean meal respectively and mix the two to obtain a dry mix (1), then add 150-175 parts of purified water and 0.25 parts of sodium bicarbonate to the dry mix (1), stir, and then place at 45-50 ° C for 1.5 hours to obtain a reaction base (2), wherein the bran and soybean meal in the dry mix (1) are not sterilized; b. Add 1 part of protease powder (containing 0.91-0.97 parts of glucose) to the reaction base (2) and place it at a temperature of 45-50 ° C for 12 hours, during which the reaction base (2) is covered with a screen and is not stirred, and then the reaction base (2) is filtered and the filtrate is collected as the reaction base liquid (3); c. The reaction base liquid (3) is successively subjected to steaming, concentrating and spray drying to obtain a powder (4). The temperature of the reaction base liquid (3) is controlled at 85° C. and the steaming time is 20 min. d. Add 5 parts of sodium bicarbonate and 20 parts of urea to the powder (4) to obtain a Bacillus pasteurianus dry powder culture medium.