CN113817635A - Method for culturing bacillus by using soybean whey wastewater - Google Patents
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Abstract
The invention belongs to the technical field of agricultural biology, and relates to application of soybean whey wastewater in fermentation production of bacillus and a method for producing bacillus subtilis by fermentation of the soybean whey wastewater, which solve the problem of the soybean whey wastewater, realize zero discharge of sewage, and provide a bacillus subtilis fermentation broth with low cost and high viable count. The invention firstly proposes to directly utilize the soybean whey wastewater to ferment and produce the bacillus, the production process is simple, the soybean whey wastewater does not need to be treated by centrifugation, concentration and the like, the production cost is reduced, the problem of the soybean whey wastewater is solved, the zero discharge of sewage is realized, and the water resource is recycled.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a method for producing bacillus by fermenting soybean whey wastewater.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The bacillus is aerobic or facultative anaerobic, gram-positive and spore-producing bacteria, has no pathogenicity to plants, human beings and animals, wide application range, good safety and excellent bacteriostasis and stress resistance. At present, bacillus subtilis, bacillus licheniformis, bacillus coagulans and other strains in bacillus are widely applied to the fields of agricultural plant protection, animal husbandry and breeding industry, environmental protection three-waste treatment, animal and plant disease control, food, biological medicine and the like. Currently, water is mainly used as a solvent in industry, and a certain amount of carbon source, nitrogen source and inorganic salt are added to produce bacillus through fermentation.
It is reported that a large amount of soybean whey wastewater is generated in the processing process of soybean protein isolate and bean products, and the main components of the wastewater are small molecular water-soluble protein, cane sugar and inorganic salt, and in addition, the wastewater also contains a small amount of high value-added substances with special health care functions on human bodies, such as soybean oligosaccharide, amylase, cytochrome, isoflavone compounds, soyasaponin, phytic acid and the like. If the waste water is treated only as waste water, the high-concentration organic substances contained in the waste water can cause great resource waste and environmental pollution, and the problems of effective utilization, resource saving and income improvement are solved at present.
In the prior art, the patent conditions related to the treatment of soybean whey wastewater are as follows: "a method for preparing soybean whey polypeptide with liver protection and antioxidation" (CN201210241261.3) "and" a method for preparing high-purity soybean oligosaccharide from soybean whey wastewater "(CN 201210192793.2), but these patents relate to the utilization and development of a certain component in the soybean whey wastewater, and are not beneficial to the treatment of a large amount of polluted wastewater. The patent "a method for producing yeast cells by fermenting clear bean water" (CN201310227567.8), relates to fermenting and culturing yeast by using soybean whey wastewater to obtain single cell protein, obviously reduces COD (chemical oxygen demand) in the wastewater, and greatly solves the problems of resource waste and environmental pollution, wherein the COD removal rate reaches 40%; the patent "application of soybean whey wastewater in preparing high-activity lactobacillus preparation" (CN201710799714.7), relates to the application of soybean whey wastewater in preparing active lactobacillus to obtain high-activity lactobacillus, the COD of the wastewater obtained in the process of the process is below 1000mg/L, and the treatment cost is greatly reduced; however, the prior art and the patent are more important than the improvement of a sewage treatment mode and the application of a new technology, the treatment process is more complex, the cost is high, the benefit is low, and the industrial utilization is difficult to enter. Therefore, how to treat the soybean whey wastewater efficiently needs to be further solved and perfected.
Disclosure of Invention
In order to overcome the problems, the invention provides the application of the soybean whey wastewater in the fermentation production of the bacillus and the method for producing the bacillus subtilis by fermenting the soybean whey wastewater, solves the problem of the soybean whey wastewater, realizes zero discharge of sewage, and can provide the bacillus subtilis fermentation liquor with low cost and high viable count.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for culturing bacillus using soybean whey wastewater, comprising:
preparing a culture medium by taking soybean whey wastewater as a raw material;
inoculating bacillus into the culture medium, and fermenting and culturing to obtain the bacillus subtilis.
The invention firstly proposes to directly utilize the soybean whey wastewater to ferment and produce the bacillus, the production process is simple, the soybean whey wastewater does not need to be treated by centrifugation, concentration and the like, the production cost is reduced, the problem of the soybean whey wastewater is solved, the zero discharge of sewage is realized, and the water resource is recycled.
The research shows that: although the prior art has the technology of utilizing the soybean whey wastewater to culture the strains, the method is limited by the factors of complex wastewater source, nutrient composition of a culture medium, fermentation conditions of the strains and the like, and the fermentation level is lower. Therefore, the invention takes the soybean whey wastewater with specific sources as the main raw material, and discovers the following components by compounding, process optimization and strain screening: the bacillus is directly produced by fermenting the soybean whey wastewater, the fermentation level is high, and the industrialization prospect is good.
In a second aspect of the invention, there is provided a bacillus cultured by any of the above methods.
The invention has the advantages of short fermentation period of about 24-48 hours, high fermentation speed, saving a large amount of manpower and material resources, low requirement on operation level, large bacillus subtilis bacterial quantity, good spore formation and industrial development value.
In a third aspect of the invention, the application of the soybean whey wastewater in the fermentation production of bacillus is provided.
The final fermentation level of the fermentation technology provided by the invention is more than 100 hundred million/ml, and the industrial production level of bacillus is achieved.
The fermentation technology provided by the invention can finally produce the polyglutamic acid by fermentation in an amount of 20 g/L.
The invention has the beneficial effects that:
(1) the invention firstly proposes to directly utilize the soybean whey wastewater to ferment and produce the bacillus, the production process is simple, the soybean whey wastewater does not need to be treated by centrifugation, concentration and the like, the production cost is reduced, the problem of the soybean whey wastewater is solved, the zero discharge of sewage is realized, and the water resource is recycled.
(2) The final fermentation level of the fermentation technology provided by the invention is more than 100 hundred million/ml, and the industrial production level of bacillus is achieved.
(3) The fermentation technology provided by the invention can finally produce the polyglutamic acid by fermentation in an amount of 20 g/L.
(4) The invention has the advantages of short fermentation period of about 24-48 hours, high fermentation speed, saving a large amount of manpower and material resources, low requirement on operation level, large bacillus subtilis bacterial quantity, good spore formation and industrial development value.
(5) The production method is simple, convenient to operate, high in practicability and easy to popularize.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention aims to provide application of soybean whey wastewater in fermentation production of bacillus.
The soybean whey wastewater in the invention is especially liquid left after defatted soybeans are subjected to alkali extraction and acid precipitation processes and protein is removed by centrifugation.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of soybean whey wastewater in fermentation production of bacillus.
The specific species of bacillus is not particularly limited in this application and in some embodiments, the bacillus includes but is not limited to bacillus subtilis, bacillus licheniformis, bacillus coagulans, bacillus lentus, bacillus megaterium, bacillus mucilaginosus, or bacillus thuringiensis for better development value.
The specific culture method of Bacillus is also not particularly limited in the present application, and in some embodiments, the use of the soybean whey wastewater in the fermentation production of Bacillus comprises the following steps: 1) preparing a soybean whey wastewater culture medium: adding 0-10% of carbon source, 0-10% of nitrogen source, 0-10% of pH buffer salt, 0-10% of other nutrient components and a pH range of 4.0-9.0 into the soybean whey wastewater, placing at 105 ℃, and sterilizing for 20 minutes; 2) preparing a seed solution: inoculating the bacillus preserved on the inclined plane into a conical flask filled with a liquid culture medium for amplification culture, and culturing at the constant temperature of 37 ℃ for 8-16 hours at the rotating speed of 200 rpm; 3) and (3) fermentation in a tank: inoculating the seed solution into a sterilized fermentation tank for liquid fermentation when the OD value of the seed solution is more than 30, wherein the inoculation amount is 10 percent, the liquid loading amount is 70 percent, the seed solution is cultured at the constant temperature of 37 ℃ for 24-36 hours, the rotating speed is 300rpm at most, the ventilation ratio is 1.0vvm, and the pH value is controlled to be more than 6.5; in the process of fermentation tank culture, the pH is firstly reduced and then raised, the pH is controlled to be 6.0-7.5 within 12 hours, the pH is naturally raised after 12 hours, the culture is carried out until the OD value is not increased any more, the spore rate is more than or equal to 90%, and the fermentation is stopped. The experimental results show that: the bacillus is directly produced by fermenting the soybean whey wastewater, the production process is simple, and the thallus density order of the obtained bacillus fermentation liquor can reach 1010cfu/ml, reaches the industrial production level of bacillus and has better market prospect.
The soybean whey wastewater is a byproduct in the preparation process of the soybean protein isolate and is organic wastewater left after protein precipitation.
The invention adopts the soybean whey wastewater, a carbon source, a nitrogen source, necessary salts and nutrient substances to form a culture medium to support the growth of bacillus, and in order to achieve a better culture effect, the invention carries out a wide screening test to obtain a culture medium formula with higher commercial value. Thus, in some embodiments, the formulation of the medium comprises: na (Na)2HPO4Or K2HPO4，CaCO3Mixing with at least one of soluble starch, corn steep powder, corn flour, yeast extract powder, molasses, yeast powder, and ureaAnd/or at least one of glucose and monosodium glutamate. The research result shows that: the combination of the raw materials is easy to obtain, and can well support the growth of various bacillus, and in the practical application process, technicians can select corresponding components from the raw materials to culture according to the characteristics and production conditions of strains so as to obtain better industrial production conditions of the bacillus subtilis.
Further, the application further gives preferred solutions for several commonly used bacillus species, such as: in some embodiments, for Bacillus subtilis, the recommended preparation method using liquid medium is: adding corn starch powder or 4 percent of corn flour and K into the soybean whey wastewater2HPO43％、CaCO34 percent, adjusting the pH value to 7.5, placing at 105 ℃ or 110 ℃, and sterilizing for 20 minutes to obtain the bacillus subtilis preparation, wherein each percentage is the percentage of the raw materials corresponding to the volume of the soybean whey wastewater so as to better support the growth of the bacillus subtilis.
In yet other embodiments, the liquid medium may be selected for the culture of Bacillus amyloliquefaciens by: adding 3 percent of corn flour and Na into the soybean whey wastewater2HPO4 2％、CaCO31% and 2% of urea, adjusting the pH value to 7.2, placing at 110 ℃, and sterilizing for 20 minutes to obtain the product. More preferably, molasses may be used instead of corn flour as the culture medium for bacillus amyloliquefaciens.
Also, in some embodiments, for the production of bacillus licheniformis, the medium may be prepared by: adding yeast extract powder 1.5%, urea 2.5%, and K into soybean whey wastewater2HPO4 3.5％、CaCO32 percent and glucose 4 percent, adjusting the pH value to 7.5, placing at 110 ℃, and sterilizing for 20 minutes to obtain the yeast extract powder, which promotes the growth of the bacillus licheniformis by utilizing the characteristic that the yeast extract powder is rich in protein, amino acid, polypeptide, nucleotide, B vitamins and growth factors.
In other embodiments, the invention selects 3 percent of corn flour and 3 percent of yeast extract powder to better utilize soybean whey wastewater to ferment and produce the bacillus natto and the polyglutamic acid1.5%, urea 2.5%, K2HPO4 1.5％、CaCO31.5 percent, 4 percent of glucose and 5 percent of monosodium glutamate are taken as components of the culture medium, meanwhile, the pH value is adjusted to 7.2, the culture medium is placed at 110 ℃ and sterilized for 20 minutes to obtain a qualified culture medium, and the experimental result shows that: the viable count of the bacillus natto fermentation liquor and the yield of the polyglutamic acid reach higher levels.
In some examples, the fermentation is carried out in a fermentor, which is then transferred to a sterilized fermentor for liquid fermentation, with an inoculum size of 5% and a liquid loading of 35%, and incubated at 37 ℃ for 48 hours.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
In the following examples, the soybean whey wastewater is a liquid left after defatted soybeans of a certain factory are subjected to alkali extraction and acid precipitation processes and protein is removed by centrifugation.
The specific method comprises the following steps: mixing defatted soybean with distilled water at a ratio of 1: 10, regulating the pH value of the mixture to 7-9 by NaOH, fully stirring and leaching alkali-soluble soybean protein, performing centrifugal separation, adjusting the pH value of supernatant to 4.3-4.8 by using dilute HCl, precipitating protein, performing centrifugal separation to obtain soybean whey wastewater and precipitate, dissolving the precipitate in NaOH solution with the pH value of 7.0-8.0 again, and performing spray drying or freeze drying to obtain the soybean protein isolate.
In the following examples, the viable count of Bacillus subtilis fermentation broth was performed by serial dilution; the yield of polyglutamic acid was determined by ninhydrin colorimetry.
Example 1
The application of the soybean whey wastewater in fermentation production of bacillus subtilis comprises the following steps:
1) preparation of a culture medium: adding 4 percent of corn steep liquor powder and K into the soybean whey wastewater2HPO43％、CaCO34 percent, adjusting the pH value to 7.5, placing at 105 ℃ and sterilizing for 20 minutes; each percentage is the percentage of each raw material corresponding to the volume of the soybean whey wastewater.
2) Preparing a seed solution: inoculating the bacillus subtilis stored on the inclined plane into a conical flask filled with a liquid culture medium for amplification culture, inoculating 1-ring lawn, and culturing at a constant temperature of 37 ℃ for 12 hours at a rotation speed of 200 rpm;
3) and (3) fermentation in a tank: inoculating into a sterilized fermentation tank for liquid fermentation, wherein the inoculation amount is 5%, the liquid loading amount is 65%, culturing is carried out at the constant temperature of 37 ℃ for 32 hours, the rotating speed is 300rpm at most, the ventilation ratio is 1.0vvm, and the pH is controlled to be more than 6.5; cultured to OD600No increase, the spore rate is more than or equal to 90 percent, the fermentation is stopped, and the viable count of the obtained bacillus subtilis fermentation liquor is 1.8 multiplied by 1010cfu/ml。
Example 2
The application of the soybean whey wastewater in fermentation production of bacillus amyloliquefaciens comprises the following steps:
1) preparation of a culture medium: adding molasses 3% and Na into soybean whey wastewater2HPO44％、CaCO33 percent, adjusting the pH value to 7.2, placing at 105 ℃ and sterilizing for 20 minutes; each percentage is the percentage of each raw material corresponding to the volume of the soybean whey wastewater.
2) Preparing a seed solution: inoculating the bacillus subtilis stored on the inclined plane into a conical flask filled with a liquid culture medium for amplification culture, inoculating 1-ring lawn, and culturing at a constant temperature of 37 ℃ for 8 hours at a rotation speed of 200 rpm;
3) and (3) fermentation in a tank: inoculating into a sterilized fermentation tank for liquid fermentation, wherein the inoculation amount is 5%, the liquid loading amount is 65%, culturing is carried out at the constant temperature of 37 ℃ for 24 hours, the rotating speed is 300rpm at most, the ventilation ratio is 1.0vvm, and the pH is controlled to be more than 6.5; culturing until OD value is not increased any more, spore rate is greater than or equal to 90%, stopping fermentation, and viable count of the obtained Bacillus amyloliquefaciens fermentation liquor is 1.4 × 1010cfu/ml。
Example 3
The application of the soybean whey wastewater in the fermentation production of the bacillus licheniformis comprises the following steps:
1) preparation of a culture medium: adding yeast powder 1.5%, urea 2.5%, and K into soybean whey wastewater2HPO43.5％、CaCO36 percent, adjusting the pH value to 7.8, placing at 105 ℃ and sterilizing for 20 minutes; each percentage is the percentage of each raw material corresponding to the volume of the soybean whey wastewater.
2) Preparing a seed solution: inoculating the bacillus subtilis stored on the inclined plane into a conical flask filled with a liquid culture medium for amplification culture, inoculating 1-ring lawn, and culturing at a constant temperature of 37 ℃ for 8-16 hours at a rotation speed of 200 rpm;
3) and (3) fermentation in a tank: inoculating into a sterilized fermentation tank for liquid fermentation, wherein the inoculation amount is 5%, the liquid loading amount is 65%, culturing is carried out at the constant temperature of 37 ℃ for 36 hours, the rotating speed is 300rpm at most, the ventilation ratio is 1.0vvm, and the pH is controlled to be more than 6.5; culturing until OD value is not increased, spore rate is not less than 90%, stopping fermentation, and the viable count of the obtained Bacillus licheniformis fermentation liquor is 1.6 × 1010cfu/ml。
Example 4
The application of the soybean whey wastewater in the fermentation production of the bacillus natto comprises the following steps:
1) preparation of a culture medium: adding 3% of corn flour, 1.5% of yeast extract powder, 2.5% of urea and K into the soybean whey wastewater2HPO41.5 percent, CaCO 31.5 percent, glucose 4 percent and monosodium glutamate 5 percent, adjusting the pH value to 7.2, placing at 110 ℃, and sterilizing for 20 minutes; each percentage is the percentage of each raw material corresponding to the volume of the soybean whey wastewater.
2) Preparing a seed solution: inoculating the bacillus natto stored on the inclined plane into a conical flask filled with a liquid culture medium for amplification culture, inoculating 1-ring lawn, and culturing at a constant temperature of 37 ℃ for 8-16 hours at a rotation speed of 200 rpm;
3) and (3) fermentation in a tank: inoculating into a sterilized fermentation tank for liquid fermentation, wherein the inoculation amount is 5%, the liquid loading amount is 30%, the culture is carried out at the constant temperature of 37 ℃ for 48 hours, the rotating speed is 300rpm at most, the ventilation ratio is 1.0vvm, and the pH is controlled to be more than 6.5; culturing until OD value is not increased any more, spore rate is not less than 90%, stopping fermentation, and the viable count of the obtained Bacillus natto fermentation liquid is 1.3 × 1010The yield of the polyglutamic acid can reach 20g/L by cfu/ml.
Comparative example 1
(1) Activation culture: the bacillus preserved in the slant is inoculated into an activation medium for activation. The activation medium consisted of (in weight percent): 0.5-1.5% of soluble starch, 0-1.0% of polypeptone, 0-0.5% of sodium chloride, 0-0.3% of monopotassium phosphate, 0-0.3% of dipotassium hydrogen phosphate and the balance of yellow serous water, uniformly stirring and heating. After the components are dissolved, adjusting the pH value to 6.0-8.0 by using 1mol/L NaOH or HCl, and supplementing yellow slurry water to 1000 mL. Preparing yellow serofluid: centrifuging fresh yellow serofluid, and taking supernatant as a liquid culture medium raw material. Subpackaging the prepared culture medium into 250mL triangular bottles, wherein the packaging amount of each bottle is 50mL, sealing with a breathable sealing film, and sterilizing with high-pressure steam at 121 ℃ for 30 min. The activation culture conditions are as follows: shaking and culturing for 20-24 h at the temperature of 30-42 ℃ and the rotating speed of 100-250 rpm.
(2) Fermentation culture: inoculating the activated bacillus liquid into a fermentation culture medium according to the inoculation amount (v/v) of 1-5%. The fermentation medium consists of the following raw materials in percentage by weight: 0.5-1.5% of soluble starch, 0-1.0% of polypeptone, 0-0.5% of sodium chloride, 0-0.3% of monopotassium phosphate, 0-0.3% of dipotassium phosphate, 0.05-0.15% of magnesium sulfate, 0.005-0.015% of calcium carbonate, 0.05-0.15% of manganese sulfate and yellow serous water as the balance, and the pH value is adjusted to be 6.0-8.0. The fermentation culture conditions are as follows: the culture temperature is 30-42 ℃, the rotating speed is 100-250 rpm, the inoculation amount is 1-5% (v/v), and the shaking culture is performed for 20-36 h.
The cell biomass of the bacillus cultured by the method reaches 1010More than one/mL.
As can be seen from the comparison between examples 1-4 and comparative example 1, the method for producing bacillus by directly fermenting soybean whey wastewater has simple production process, and the soybean whey wastewater does not need to be treated by centrifugation, concentration and the like, thereby not only reducing the production cost, but also solving the problem of the soybean whey wastewater, realizing zero discharge of sewage and recycling water resources. Meanwhile, the final fermentation level is more than 100 hundred million/ml, and the industrial production level of the bacillus is achieved. Especially for the fermentation production of the bacillus natto, the coproduction of the bacillus natto and the polyglutamic acid is realized.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. Application of soybean whey wastewater in culturing bacillus.
2. The use of claim 1, wherein the bacillus comprises: bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus lentus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus thuringiensis, and Bacillus natto.
3. Application of soybean whey wastewater in producing polyglutamic acid.
4. Application of soybean whey wastewater in culturing bacillus natto and co-producing polyglutamic acid.
5. A method for culturing bacillus by utilizing soybean whey wastewater is characterized by comprising the following steps:
preparing a culture medium by taking soybean whey wastewater as a raw material;
inoculating bacillus into the culture medium, and fermenting and culturing to obtain the bacillus subtilis.
6. The method for culturing Bacillus using soybean whey wastewater as claimed in claim 5, wherein the soybean whey wastewater is a liquid remaining after the defatted soybeans are subjected to alkali extraction and acid precipitation steps and then centrifuged to remove proteins.
7. The method for culturing bacillus using soybean whey wastewater as claimed in claim 5, wherein the culture medium further comprises: 0-10% of carbon source, 0-10% of nitrogen source, 0-10% of pH buffer salt, 0-10% of other nutrient components and pH range of 4.0-9.0.
8. The method for culturing bacillus using soybean whey wastewater as claimed in claim 5, wherein the formulation of the culture medium comprises: na (Na)2HPO4Or K2HPO4，CaCO3And at least one of soluble starch, corn flour, yeast extract powder, molasses, yeast powder and urea, and/or at least one of glucose and monosodium glutamate.
9. The method for culturing bacillus using soybean whey wastewater as claimed in claim 5, wherein the fermentation comprises: preparing seed liquid and fermenting in a tank; the fermentation time is 24-48 hours.
10. The method for culturing Bacillus using soybean whey wastewater as claimed in claim 7, wherein the culture medium is sterilized at 105 to 110 ℃.
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| CN114455694A (en) * | 2022-02-28 | 2022-05-10 | 青海洁神环境科技股份有限公司 | Method for quickly realizing biofilm formation starting of biological rotating disc |
| CN117969862A (en) * | 2024-04-02 | 2024-05-03 | 中国农业科学院农产品加工研究所 | Screening method and application of soybean protein as microbial culture nitrogen source |
| CN118256403A (en) * | 2024-05-30 | 2024-06-28 | 中国农业科学院农产品加工研究所 | Novel bacillus and application thereof in soybean protein extraction |
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