Soybean fermentation bacillus and application thereof

Abstract

The invention provides a soybean fermentation bacillus and application thereof, the strain number is ASD02, the strain is preserved in China General Microbiological Culture Collection Center on May 15, 2019, called CGMCC for short, and the address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the postal code is 100101, and the preservation number is CGMCC No.17806; the Lactobacillus plantarum strain has a Latin literature name of Lactobacillus plantarum, and the Lactobacillus plantarum strain is Lactobacillus plantarum. Propagation of the strain ASD02 is carried out by using protein and fiber in bean sewage, and the produced acidic lipase can directly decompose grease in the bean sewage into fatty acid under a controlled condition, so that harmless treatment of the bean sewage is realized; and the obtained enzymatic hydrolysate can be directly used as an anaerobic biogas fermentation raw material, so that the continuous degree of bean sewage treatment is improved, and the resource utilization rate of bean sewage is further improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of Bacillus fermentum and its applications. Background Technology

[0002] Soybeans, mung beans, peas, and other legumes contain a large amount of oil, with oil content ranging from 4% to 24% depending on the variety. Legume processing is a water-intensive industry; during the production of legume starch, plant protein, and other products, a large amount of unused oil remains and is discharged with the wastewater. Taking pea vermicelli production as an example, the production process mainly includes peeling, washing, soaking, grinding, thickening, extruding and cooking, cooling and freezing, thawing, and drying of peas. Except for the thickening and freezing processes, the remaining steps consume a large amount of water. This water, after the impurity content increases, is collected and treated. The collected pea wastewater contains a large amount of organic pollutants, such as protein, starch, and suspended solids, as well as nutrient pollutants, such as nitrogen and phosphorus compounds, and oily compounds. The presence of these pollutants results in a pH of 3.0-5.0 and an oil content of 5-10 g / L in the pea wastewater. Among these, oily compounds can exacerbate wastewater turbidity and increase the difficulty of treating pea wastewater.

[0003] Biogas is a clean energy source obtained through anaerobic fermentation of manure, straw, sewage sludge, and other raw materials by microorganisms. Currently, commonly used raw materials for anaerobic biogas fermentation mainly include valuable waste such as livestock and poultry manure, wastewater, and straw. These raw materials contain certain sugars, cellulose, and proteins, which can be utilized by anaerobic microorganisms to produce biogas. However, there are still some problems in the practical application of these raw materials. For example, when using soybean wastewater for anaerobic fermentation to produce biogas, the oil in the wastewater coats the surface of the microorganisms, blocking their contact with nutrients and reducing their efficiency. Furthermore, excessive oil can cause microbial toxicity. Therefore, reducing the oil content in soybean wastewater is crucial when using it for biogas fermentation.

[0004] Currently, biological methods for treating legume wastewater mainly target proteins, sugars, and cellulose, failing to effectively remove oils. Furthermore, the high protein and sugar content slows down microbial metabolism, and the decomposition of proteins gradually lowers the pH of the liquid, hindering microbial proliferation and metabolism. Therefore, providing a strain that can adapt to fluctuations in pH and organic matter content in legume wastewater is of great significance for its treatment. Summary of the Invention

[0005] In view of this, the present invention provides a strain of *Bacillus fermentum* and its application. This strain, designated ASD02, was deposited on May 15, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 17806; its Latin scientific name is *Lactobacillus plantarum*. Bacillus glycinifermentans The strain ASD02, using legume wastewater as raw material, can utilize the protein and fiber in the wastewater as nitrogen and carbon sources for propagation, and produce acidic lipase, pectinase, and cellulase. Under controlled enzymatic hydrolysis conditions, it can directly decompose the oil in legume wastewater into fatty acids and the cellulose into monosaccharides, thus achieving the harmless treatment of legume wastewater. Furthermore, the obtained enzymatic hydrolysate can be directly used as a raw material for anaerobic biogas fermentation, improving the continuity of legume wastewater treatment and thereby increasing the resource utilization rate of legume wastewater.

[0006] The technical solution of the present invention is as follows: A strain of *Bacillus plantarum*, with accession number ASD02, was deposited on May 15, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 17806; its Latin name is *Lactobacillus plantarum*. Bacillus glycinifermentans .

[0007] Preferably, strain ASD02 was screened from straw anaerobic fermentation biogas slurry.

[0008] Preferably, the culture conditions for strain ASD02 are: LB medium, cultured at 37-42℃.

[0009] Preferably, when using liquid LB medium, the culture is carried out on a shaker at 200 rpm; when using slant or plate culture, the culture is carried out statically in an incubator.

[0010] Preferably, the LB culture composition is: 10g tryptone, 5g yeast extract, 10g sodium chloride, pH adjusted to 7.0 with NaOH solution, and volume brought to 1L.

[0011] Preferably, strain ASD02 has high salt tolerance, with a maximum salt tolerance of 10.0 g / L; low pH tolerance, with a minimum pH tolerance of 3.0; and can produce thermostable acidic lipase, cellulase and pectinase.

[0012] Preferably, the optimal reaction temperature for acid lipase is 30-80℃, the pH is 3.0-8.5, and the addition amount is 200-220 U / g.

[0013] The aforementioned application of Bacillus fermentum in soybeans specifically refers to the application of strain ASD02 in soybean wastewater treatment.

[0014] Preferably, the above application is specifically the application of strain ASD02 in the preparation of anaerobic biogas fermentation feedstock from legume wastewater.

[0015] Preferably, the bean wastewater is either non-fermented bean product wastewater or fermented bean product wastewater.

[0016] Preferably, the above application follows the following process: Step 1: Prepare seed solution Prepare liquid LB medium, inoculate strain ASD02 into the medium, and culture in a shaker at 37-42℃ and 200rpm for 24-36 hours to obtain seed culture; Step 2: Prepare the culture medium Adjust the protein content in the soybean wastewater to 15-20 g / L, starch content to 5-7 g / L, and sodium chloride content to 5-10 g / L. Adjust the pH to 6.0, sterilize, and obtain the culture medium for later use. Step 3, Fermentation Inoculate the seed culture into the culture medium at an inoculation rate of 3-5% (v / v), control the temperature at 40±1℃, ferment at a speed of 200-300 rpm for 36-48 hours, and sterilize at 50℃ to obtain the fermentation broth. Step 4: Prepare anaerobic biogas fermentation raw materials The fermentation broth is concentrated to a concentration of 200-220 U / g of acidic lipase. The temperature of the fermentation broth is controlled at 30-80℃ and the pH at 3.0-8.5. Enzymatic hydrolysis is carried out for 8-10 hours to obtain anaerobic biogas fermentation feedstock.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a strain of *Bacillus fermentum*, designated ASD02, which was deposited on May 15, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 17806; its Latin name is *Lactobacillus plantarum*. Bacillus glycinifermentans This strain, ASD02, has the advantages of being tolerant to high salt and low pH, making it suitable for use in soybean wastewater.

[0018] The strain ASD02 provided by this invention has metabolites including acidic lipase, cellulase and pectinase; among which, acidic lipase has the advantage of high temperature resistance and high activity in the pH range of 3.0-8.5; the wide pH range allows it to adapt to the low pH environment of the fermentation broth, ensuring that the acidic lipase can efficiently decompose oils and guarantee the decomposition effect.

[0019] Using strain ASD02 of the present invention for the treatment of soybean wastewater reduces the use of chemical agents, simplifies the treatment process, and lowers the cost of harmless treatment of soybean processing wastewater. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0021] In the following embodiments and comparative examples of the present invention, the bean wastewater used is non-fermented bean product wastewater, specifically pea wastewater, with a protein content of 73.5 g / L, a starch content of 10.3 g / L, a salt content (calculated as sodium chloride) of 8.5 g / L, and an oil content of 8.7 g / L.

[0022] Example 1: Isolation and Identification of Strains 1. Strains screening and purification (1) Sample preparation ① Take the biogas slurry from a well-functioning straw anaerobic biogas fermentation device in Yantai City, Shandong Province, add 100ml of sterile water, homogenize, and obtain 10 -1 suspension; ② Perform a 10-fold serial dilution on the solution from step ①, resulting in 10... -2 10 -3 10 -4 10 -5 10 -6 10 -7 ,stand-by; (2) Preparation of culture medium Solid LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, pH adjusted to 7.0 with NaOH solution, 15g agar, bring the volume to 1L, sterilize at 115℃ for 30min, and let cool before use; (3) Plate culture and selection of single colonies: The gradient solutions prepared in step ② were inoculated onto the solid LB medium in step (2) using the spread method, and then statically cultured at 40±0.1℃ for 36 hours. Single colonies were then selected. (4) Separation and purification The selected single colonies were inoculated onto the solid LB medium in step (2) using the streak method and cultured statically at 40±0.1℃ for 30 hours. Single colonies were then picked and stored in glycerol tubes at -80℃.

[0023] 2. Identification The single colonies preserved in glycerol tubes were identified, and their 16S rDNA sequences are shown in SEQ ID NO:1, as follows: SEQ ID NO:1 Based on 16S rDNA full-length sequencing and physiological and biochemical tests, it was identified as Bacillus fermentum of soybean (Bacillus fermentum). Bacillusglycinifermentans The strain, designated ASD02, was deposited on May 15, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 17806. Its Latin scientific name is *Lactobacillus plantarum*. Bacillus glycinifermentans .

[0024] Example 2: Tolerance test of strain ASD02 (1) Salt tolerance The strain ASD02 preserved in Example 1 was inoculated in equal amounts into 10 ml liquid LB medium tubes with Durham tubes containing different salt concentrations of sodium chloride (5.0 g / L, 8.0 g / L, 10.0 g / L, 11.0 g / L, and 12.0 g / L). The inoculation volume was 1 μl, scraped from one loop. The tubes were then cultured at 40 °C with shaking for 3 days. The growth status was observed and recorded. The results are shown in Table 1. Table 1. Salt tolerance results of strain ASD02

[0025] (2) pH tolerance The strain ASD02 preserved in Example 1 was inoculated in equal amounts into 10 ml liquid LB medium tubes with Durham tubes containing different pH values: 2.5, 3.0, 5.0, 6.0, and 7.0. The inoculation amount was 1 μl, with one loop scraped off. The tubes were then cultured at 40°C with shaking for 3 days. The growth status was observed and recorded. The results are shown in Table 2. Table 2 pH tolerance results of strain ASD02

[0026] As can be seen from Tables 1 and 2, the strain ASD02 provided by this invention has good salt tolerance and pH tolerance. When strain ASD02 ferments protein as a raw material, the amino acid content in the fermentation broth increases, causing the pH of the fermentation broth to decrease. However, the good pH tolerance of strain ASD02 of this invention enables it to maintain high activity continuously, thereby effectively utilizing the protein and starch in soybean wastewater and achieving pollution-free treatment of soybean wastewater.

[0027] Example 3 The application of strain ASD02 in the preparation of anaerobic biogas fermentation feedstock from pea wastewater is as follows: Step 1: Prepare seed solution Prepare liquid LB medium, inoculate strain ASD02 into the medium, and culture in a shaker at 40℃ and 200rpm for 30 hours to obtain seed culture; The LB culture composition was: 10g tryptone, 5g yeast extract, 10g sodium chloride, pH adjusted to 7.0 with NaOH solution, and volume brought to 1L. Step 2: Prepare the culture medium The protein content, starch content, and sodium chloride content in the pea wastewater were adjusted to 18 g / L, 6 g / L, and 8 g / L, respectively. The pH was adjusted to 6.0, and the culture medium was sterilized and then prepared for use. Step 3, Fermentation The seed culture was inoculated into the culture medium at an inoculation rate of 4% (v / v), the temperature was controlled at 40℃, fermented at 250 rpm for 40 hours, and then sterilized at 50℃ to obtain the fermentation broth; the acid lipase activity in the fermentation broth was 58.7 U / g. Step 4: Prepare anaerobic biogas fermentation raw materials The fermentation broth was concentrated, and the acidic lipase activity in the concentrate was 202.8 U / g. 245 U / g of acidic protease (purchased from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.) and 325 U / g of α-amylase (obtained from Aspergillus niger cultured on acid-resistant α-amylase medium) were added to the concentrate. The temperature of the fermentation broth was controlled at 70℃ and the pH at 5.5. Enzymatic hydrolysis was carried out for 8.5 hours to obtain the anaerobic biogas fermentation feedstock. The anaerobic biogas fermentation feedstock contains 61.6 g / L of amino acids, 7.7 g / L of monosaccharides, and 0.13 g / L of oil.

[0028] Comparative Example 1 The difference from Example 3 is that commercially available Bacillus subtilis is used, and the process is as follows: Step 1: Prepare seed solution Liquid LB medium was prepared, and Bacillus subtilis was inoculated into the medium and cultured in a shaker at 35°C and 200 rpm for 36 hours to obtain seed culture. The LB culture composition is the same as in Example 3; Step 2: Prepare the culture medium Same as Example 3; Step 3, Fermentation The seed culture was inoculated into the culture medium at an inoculation rate of 4% (v / v), the temperature was controlled at 35℃, fermented at 250 rpm for 60 hours, and then sterilized at 50℃ to obtain the fermentation broth; the acid lipase activity in the fermentation broth was 18.5 U / g.

[0029] Example 4 The application of strain ASD02 in the preparation of anaerobic biogas fermentation feedstock from pea wastewater is as follows: Step 1: Prepare seed solution Liquid LB medium was prepared, and strain ASD02 was inoculated into the medium and cultured in a shaker at 37°C and 200 rpm for 24 hours to obtain the seed culture. Step 2: Prepare the culture medium The protein content, starch content, and sodium chloride content in the soybean wastewater were adjusted to 15 g / L, 5 g / L, and the pH was adjusted to 6.0. After sterilization, the culture medium was obtained and ready for use. Step 3, Fermentation The seed culture was inoculated into the culture medium at an inoculation rate of 3% (v / v), the temperature was controlled at 40℃, fermented at 200 rpm for 48 hours, and then sterilized at 50℃ to obtain the fermentation broth; the acid lipase activity in the fermentation broth was 56.9 U / g. Step 4: Prepare anaerobic biogas fermentation raw materials The fermentation broth was concentrated, and the acidic lipase activity in the concentrate was 206.5 U / g. Acidic protease 245 U / g (purchased from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.) and α-amylase 325 U / g (obtained from Aspergillus niger cultured on acid-resistant α-amylase medium) were added to the concentrate. The temperature of the fermentation broth was controlled at 50℃ and the pH at 4.5. Enzymatic hydrolysis was carried out for 10 hours to obtain anaerobic biogas fermentation feedstock.

[0030] The anaerobic biogas fermentation feedstock contains 60.8 g / L of amino acids, 7.3 g / L of monosaccharides, and 0.17 g / L of oil.

[0031] Example 5 The application of strain ASD02 in the preparation of anaerobic biogas fermentation feedstock from pea wastewater is as follows: Step 1: Prepare seed solution Liquid LB medium was prepared, and strain ASD02 was inoculated into the medium and cultured in a shaker at 42℃ and 200rpm for 36 hours to obtain the seed culture. Step 2: Prepare the culture medium The protein content, starch content, and sodium chloride content in the soybean wastewater were adjusted to 20 g / L, 7 g / L, and 10 g / L, respectively. The pH was adjusted to 6.0, and the culture medium was sterilized and then prepared for use. Step 3, Fermentation The seed culture was inoculated into the culture medium at an inoculation rate of 5% (v / v), the temperature was controlled at 40℃, fermented at 300 rpm for 36 hours, and then sterilized at 50℃ to obtain the fermentation broth; the acid lipase activity in the fermentation broth was 55.9 U / g. Step 4: Prepare anaerobic biogas fermentation raw materials The fermentation broth was concentrated, and the acidic lipase activity in the concentrate was 203.8 U / g. 245 U / g of acidic protease (purchased from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.) and 325 U / g of α-amylase (obtained from Aspergillus niger cultured on acid-resistant α-amylase medium) were added to the concentrate. The temperature of the fermentation broth was controlled at 65℃ and the pH at 5.5. Enzymatic hydrolysis was carried out for 8 hours to obtain the anaerobic biogas fermentation feedstock.

[0032] The anaerobic biogas fermentation feedstock contains 59.8 g / L of amino acids, 7.0 g / L of monosaccharides, and 0.21 g / L of oil.

[0033] Example 6 The application of strain ASD02 in the preparation of anaerobic biogas fermentation feedstock from pea wastewater is as follows: Step 1: Prepare seed solution Liquid LB medium was prepared, and strain ASD02 was inoculated into the medium and cultured in a shaker at 40℃ and 200rpm for 36 hours to obtain the seed culture. Step 2: Prepare the culture medium The protein content, starch content, and sodium chloride content in the soybean wastewater were adjusted to 17 g / L, 5 g / L, and 6 g / L, respectively. The pH was adjusted to 6.0, and the culture medium was sterilized and then prepared for use. Step 3, Fermentation The seed culture was inoculated into the culture medium at an inoculation rate of 4% (v / v), the temperature was controlled at 40℃, fermented at 250 rpm for 40 hours, and then sterilized at 50℃ to obtain the fermentation broth; the acid lipase activity in the fermentation broth was 57.7 U / g. Step 4: Prepare anaerobic biogas fermentation raw materials The fermentation broth was concentrated, and the acidic lipase activity in the concentrate was 207.9 U / g. 245 U / g of acidic protease (purchased from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.) and 325 U / g of α-amylase (obtained from Aspergillus niger cultured on acid-resistant α-amylase medium) were added to the concentrate. The temperature of the fermentation broth was controlled at 30℃ and the pH at 3.0. Enzymatic hydrolysis was carried out for 10 hours to obtain the anaerobic biogas fermentation feedstock. The anaerobic biogas fermentation feedstock contains 60.8 g / L of amino acids, 7.4 g / L of monosaccharides, and 0.19 g / L of oil.

[0034] The prior art portion of this application will not be described in detail.

[0035] Application Examples The anaerobic biogas fermentation raw material provided in Example 3 is applied as follows: S1, take 100L of anaerobic biogas fermentation raw material obtained in Example 4, adjust the pH to 7.0 with sodium bicarbonate (NaHCO3), place it in a continuous stirred tank reactor (CSTR), sterilize and set aside; S2, using anaerobic digested sludge from a well-functioning straw anaerobic biogas fermentation device in Yantai City, Shandong Province as inoculum, inoculated at an inoculation rate of 25% (v / v), i.e., 25L of inoculum, and mixed thoroughly; S3, purge with nitrogen for 10 minutes to ensure oxygen concentration in the reactor is <0.1%, then seal; control the temperature in the continuous stirred tank reactor at 37℃ and the pH during the reaction process at 7.0, ferment for 15 days under stirring at 120 rpm, ensuring that the raw materials and inoculum are mixed evenly during the stirring process; The produced gas is collected through a water-sealed gas storage tank; Tests showed that the biogas content in the produced gas was 62.5%.

[0036] Although the present invention has been described in detail with reference to preferred embodiments, it is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the claims.

Claims

1. A strain of Bacillus fermentum soybean, characterized in that, This strain, with accession number ASD02, was deposited on May 15, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 17806; its Latin scientific name is *Lactobacillus plantarum*. Bacillus glyciniferan .

2. The Bacillus fermentum of soybean as described in claim 1, characterized in that, Straw strain ASD02 was screened from anaerobic fermentation biogas slurry from straw.

3. The soybean fermentation Bacillus as described in claim 1, characterized in that, The culture conditions for strain ASD02 were: LB medium, cultured at 37-42℃; When using liquid LB medium, culture on a shaker at 200 rpm; when using slant or plate culture, culture statically in an incubator.

4. The soybean fermentation Bacillus as described in claim 3, characterized in that, The LB culture composition was as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, pH adjusted to 7.0 with NaOH solution, and volume brought to 1L.

5. The soybean fermentation Bacillus as described in claim 1, characterized in that, Strain ASD02 exhibits high salt tolerance, with a maximum salt tolerance of 10.0 g / L; it also exhibits low pH tolerance, with a minimum pH tolerance of 3.0; strain ASD02 is capable of producing thermostable acidic lipase, cellulase, and pectinase.

6. The Bacillus fermentum of soybean as described in claim 5, characterized in that, The optimal reaction temperature for acid lipase is 30-80℃, the pH is 3.0-8.5, and the addition amount is 200-220 U / g.

7. The application of *Bacillus fermentum* as described in claim 1, characterized in that, Specifically, this involves the application of strain ASD02 in the treatment of legume wastewater.

8. The application of *Bacillus fermentum* as described in claim 7, characterized in that, The specific application is the use of strain ASD02 in preparing biogas fermentation feedstock from soybean wastewater.

9. The application of *Bacillus fermentum* as described in claim 8, characterized in that, The application in which the bean wastewater is defined as wastewater from non-fermented bean products and wastewater from fermented bean products.

10. The application of *Bacillus fermentum* as described in claim 7, characterized in that, The application process is as follows: Step 1: Prepare seed solution Prepare liquid LB medium, inoculate strain ASD02 into the medium, and culture in a shaker at 37-42℃ and 200rpm for 24-36 hours to obtain seed culture; Step 2: Prepare the culture medium Adjust the protein content in the soybean wastewater to 15-20 g / L, starch content to 5-7 g / L, and sodium chloride content to 5-10 g / L. Adjust the pH to 6.0, sterilize, and obtain the culture medium for later use. Step 3, Fermentation Inoculate the seed culture into the culture medium at an inoculation rate of 3-5% (v / v), control the temperature at 40±1℃, ferment at a speed of 200-300 rpm for 36-48 hours, and sterilize at 50℃ to obtain the fermentation broth. Step 4: Prepare biogas fermentation feedstock The fermentation broth is concentrated to a concentration of 200-220 U / g of acidic lipase. The temperature of the fermentation broth is controlled at 30-80℃ and the pH at 3.0-8.

5. Enzymatic hydrolysis is carried out for 8-10 hours to obtain anaerobic biogas fermentation feedstock.

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