CN111690568A - Bacillus subtilis and application thereof in fermentation treatment of detoxication of flaxseed cake - Google Patents

Bacillus subtilis and application thereof in fermentation treatment of detoxication of flaxseed cake Download PDF

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CN111690568A
CN111690568A CN202010663114.XA CN202010663114A CN111690568A CN 111690568 A CN111690568 A CN 111690568A CN 202010663114 A CN202010663114 A CN 202010663114A CN 111690568 A CN111690568 A CN 111690568A
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bacillus subtilis
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蔡辉益
郭宝珠
刘国华
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Feed Research Institute of Chinese Academy of Agricultural Sciences
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Abstract

The invention discloses a bacillus subtilis BS, the preservation registration number of which is CGMCC No.18229, and the Bacillus subtilis BS is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the address is as follows: china, Beijing, institute of microbiology, China academy of sciences, No. 3 Xilu-1 of the Chaoyang district, Beijing city, zip code: 100101, preservation time of 2019, 7 months and 15 days. The invention also discloses a culture of the bacillus subtilis BS and a bacterial suspension of the bacillus subtilis BS. The invention also discloses a product, and the active ingredient of the product is bacillus subtilis BS, a culture of the bacillus subtilis BS or a bacterial suspension of the bacillus subtilis BS. The invention also discloses application of the bacillus subtilis BS, a culture of the bacillus subtilis BS or a bacterial suspension of the bacillus subtilis BS, wherein the application comprises the following steps: a) degrading cyanogenic glucoside; or b) degrading macromolecular protein in the linseed cake; or c) producing the polypeptide.

Description

Bacillus subtilis and application thereof in fermentation treatment of detoxication of flaxseed cake
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis and application of the bacillus subtilis in fermentation treatment of detoxication of flaxseed cakes.
Background
Linum usitatissimum L, also called flax, belongs to the family Linaceae and genus Linum. Flax is one of the oldest fiber crops in the world, and is widely divided into 3 types: the seeds of the oil flax, the fiber flax and the oil-fiber flax can be used for oil extraction, and become one of ten oil crops in the world. According to statistics, the annual yield of the global flaxseeds in 2018 is about 260 ten thousand tons, and the 7 th position of the world total oil yield is located. The oil flax is mainly produced in Canada, China, Argentina and the United states.
The oil flax is one of the important oil crops in China, is an important economic crop in China, is also a main oil crop planted in the arctic-alpine regions in North China, northwest China and northeast China, has the area and total yield second to Canada and is the second place in the world. China's linseed is mainly used for extracting oil, and occupies the 4 th position of the total oil production in China. China begins to plant in 2 century before the era, the initial flaxseeds are mainly used as medicines, and the seeds are not used for oil extraction until 16 century.
Flax seeds, also known as flaxseed, are seeds of flax of an annual or perennial herbaceous plant of the flax family, genus linum. Flaxseed consists of a hull and a kernel, the main components of which are oil and protein. Flaxseed contains 34% of oil, and flaxseed protein mainly comprises albumin and globulin, and is high-quality plant protein. The amino acid composition is similar to that of soybean, and lysine and methionine are deficient and rich in tryptophan. Flax seeds are rich in a variety of functional active ingredients such as alpha-linolenic acid, lignans, plant gums, flax diglycoside, phenolic acids, flavonoids, etc., many of which have been shown to have anti-cancer activity.
Linseed oil is the extracted oil of flaxseed. Oil production processes are generally divided into pressing and leaching processes. The by-product of the squeezing method is flax cake, and the by-product of the leaching method is flax meal.
The squeezing method is also called as physical squeezing method, namely, oil in the oil is squeezed out by external force, other chemical solvents and the like are not used, and the residue of organic solvents in the oil is avoided. The production process of squeezing keeps polyunsaturated fatty acid, protein, dietary fiber, vitamins, trace elements and other nutrient components beneficial to human body to the maximum extent, and is more suitable for special supplement and human body health care. The squeezing method is divided into a cold squeezing method and a hot squeezing method. The hot pressing method is that oil is produced by frying or steaming oil crops with high temperature fire and then physically pressing, and compared with the traditional pressing process, the hot pressing method has higher oil yield. The cold pressing method is that the vegetable oil is pressed by the huge pressure of physical machinery under the condition of low temperature, and the traditional high-temperature frying or steaming process is not carried out, so the oil is still distributed in the undenatured protein cells and contains very abundant inherent components of the flaxseeds.
The oil produced by the leaching process is also called leaching oil, namely the oil is leached by an organic solvent. The leaching method is an oil preparation process which adopts solvent oil to soak the oil raw material and then carries out high-temperature extraction to extract the oil.
The byproduct of linseed oil extraction is linseed cake meal. The flaxseed cake is rich in protein, alpha-linolenic acid and the like, has 28-36% of crude protein content, and can be used as a plant protein feed raw material. In addition, the flaxseed cake is also rich in various biologically functional active ingredients, such as alpha-linolenic acid, lignans and the like, so the flaxseed cake has higher feeding potential. With the rapid development of animal husbandry in China and the popularization of corn and soybean meal type daily rations, the requirement of the feed industry for soybean meal is increasing, the supply of protein feed raw materials is tense day by day, and the stability of the breeding industry is seriously influenced by the fluctuation of the price of the soybean meal in China. Therefore, the introduction of the mixed meal such as flaxseed meal, rapeseed meal, palm meal and the like can relieve the supply-demand contradiction of the soybean meal to a certain extent and promote the sustainable development of the livestock breeding industry with Chinese characteristics.
However, flaxseed meal also contains many toxic substances and anti-nutritional factors, such as flaxseed gum, phytic acid, allergens, cyanogenic glycosides, trypsin inhibitor, anti-VB6Factors and the like, particularly the toxicity of cyanogenic glycosides therein, greatly limit the amount of flaxseed meal used in an animal ration.
Research shows that cyanogenic glucoside is a main anti-nutritional factor in flaxseed cakes. Cyanogenic glycosides (hereinafter abbreviated as CGs), also known as cyanogenic glycosides and cyanohydrin glycosides, are glycosides formed by condensation of a hydroxyl group of a cyanohydrin derivative and D-glucose. Cyanogenic glycosides mainly exist in the hulls and kernels of flaxseeds, the cyanogenic glycosides in flaxseeds mainly include diglycoside and monoglycoside, the diglycoside is beta-gentiobiose acetone cyanohydrin and beta-gentiobiose methyl ethyl ketone cyanohydrin, the monoglycoside is linosid and picroside, the diglycoside content is high and is respectively 0.17% and 0.19%, and the monoglycoside content is low.
When cold pressing is performed at low temperature, the linarin in the flaxseed remains intact in the cake.
Cyanogenic glycosides do not exhibit toxicity by themselves. Cyanogenic glucoside is hydrolyzed by hydrolase coexisting in plants to generate hydrocyanic acid (HCN) to cause animal poisoning. However, under normal conditions, cyanogenic glycoside and enzyme exist in different parts of the plant and cannot be contacted, so that hydrocyanic acid is not released. The reaction of cyanogenic glycoside to produce hydrocyanic acid is effected by two enzymes in combination. Cyanogenic glucoside is firstly decomposed under the action of beta-glucosidase to generate cyanohydrin and sugar, and the cyanohydrin is unstable and is naturally decomposed into corresponding ketone, aldehyde compounds and hydrocyanic acid. The degradation reaction can be accelerated by a cyanohydrin catabolic enzyme.
The main toxic effect of hydrocyanic acid is that after it is absorbed, it enters the cells of the tissue along with the circulation of blood and passes through the cell membrane into the mitochondria, the cyanide ion (CN)-) Ferric iron (Fe) capable of rapidly reacting with oxidative cytochrome oxidase3+) Binding to form a very stable ferricytochrome oxidase enzyme that is not converted to a enzyme having ferrous iron (Fe)2+) The reduced cytochrome oxidase in the method causes the cytochrome oxidase to lose the functions of transferring electrons and activating molecular oxygen, so that histiocytes can not utilize oxygen to form 'intracellular asphyxia', thereby causing toxic hypoxia of cells. Acute toxic symptoms of cyanogenic glucoside include heart rhythm disorder and muscleMeat paralysis and respiratory distress. The minimum lethal oral dose of the hydrocyanic acid is 0.5-3.5 mg/kg body weight. HCN taken orally in the form of cyanogenic glucoside has a minimum lethal dose of 2mg per kg body weight for cattle and sheep. The allowable content of cyanide in the feed is not higher than 50mg/kg in national feed hygiene standards.
The animal's daily ration with cyanogenic glycosides in amounts higher than the tolerance level of the animal can cause acute poisoning or death of the animal. The content of cyanogenic glucoside in daily ration is not higher than tolerance of animals, and can cause chronic poisoning of animals, such as thyroid enlargement, feed intake reduction, slow growth, optic nerve damage, etc. The mechanism of chronic poisoning is due to CN-In the animal body, the thiocyanate is catalyzed by rhodanese and converted into thiocyanate (1/200 with cyanide toxicity). Thiocyano group (CN) thereof-) Due to the addition of iodide ion (I)-) Has similar molecular volume and charge, and can be used for synthesizing I in thyroid acinar cell polyiodide process-Competition, thus reducing the concentration of iodine by thyroid acinar cells, resulting in the reduction of synthesis and secretion of thyroid hormone of the body. According to the regulation mechanism of the thyroid function regulating system (hypothalamus-pituitary gland-thyroid gland axis), when the concentration of thyroid hormone in blood decreases, the pituitary gland secretes a large amount of Thyroid Stimulating Hormone (TSH) by the action of negative feedback. TSH acts on the thyroid gland continuously, so that thyroid acinar cells show proliferative changes to form goiter. Flax cake poisoning is generally a chronic process of hydrocyanic acid poisoning, but symptoms gradually worsen.
Related researchers at home and abroad carry out extensive and intensive research on the detoxification method and the way of the cyanogenic glucoside of flaxseed, and a plurality of research results are obtained. The conventional methods for detoxifying flaxseed and its cake mainly include water boiling, warm heat treatment, acid treatment-wet heat treatment and dry heat treatment, and new methods developed in recent years, such as extrusion, microwave, autoclaving, microbiological and solvent methods. Although various detoxification methods can obviously reduce the content of cyanogenic glucoside in flaxseeds, the methods have defects of different degrees, and the detoxification rate is not high enough, equipment is not perfect, the cost is high, or secondary pollution is caused, the methods are difficult to apply in the actual production, most processing techniques can influence the nutrient content in the flaxseeds, and the nutritional value is reduced. Therefore, there is a need to continuously find a detoxification method with low cost, high efficiency and simple process equipment, and meanwhile, the method focuses on enhancing the nutritional value of flaxseeds and improving the utilization rate of nutrient substances.
The cyanogenic glucoside in the flaxseed cake is removed by adopting a microbial fermentation method, because microorganisms can generate beta-glucosidase through self metabolism, and the beta-glucosidase can degrade cyanogenic glucoside. The microbial fermentation method can overcome many defects in the traditional method, not only can effectively remove the cyanogenic glucoside as an anti-nutritional factor in the flaxseed cake, but also can improve the nutritional value of the flaxseed cake, and has better application prospect in the aspect of improving the feeding value of the flaxseed cake. Therefore, the anti-nutritional factors in the flaxseed cake meal are removed through microbial fermentation, so that the unique ground source type feed resources in China can be fully utilized, the contradiction between supply and demand of the soybean meal can be relieved, and the sustainable development of the characteristic livestock breeding industry in China is promoted. The detoxification is carried out by adopting a microbial fermentation method, and the method has the advantages of mild conditions, safety, high efficiency and lower cost. Therefore, the microbial fermentation detoxification is an environment-friendly and efficient method.
In the test, the plum oriole and the like (2013) detoxicate the flax cake by adopting a microbial fermentation method, and the optimal fermentation conditions of the microbes are determined as follows: saccharomyces cerevisiae CICC31077, strain inoculation amount of 3%, water content of 50%, fermentation temperature and fermentation time of 28 deg.C and 72 hr respectively, under which the cyanogenic glycoside removing rate can reach 76.91% (plum oriole, flax cake microbe detoxification process, food and fermentation industry, No. 39, No. 3: 111-.
The recombinant Pichia pastoris is used for fermenting the linseed, such as the von Aijuan, and the removal rate of HCN reaches more than 97.0 percent under the optimal fermentation condition (the von Aijuan and the like, the optimization of the detoxification process of the recombinant Pichia pastoris GS115-Ch-Glu fermented linseed, the food industry, No. 36, No. 2: 105-.
Disclosure of Invention
The invention aims to provide bacillus subtilis BS and application thereof in detoxification of flaxseed cakes.
The Bacillus subtilis strain BS provided by the invention is separated from sheep rumen fluid collected from a pilot-scale base of Chinese academy of agricultural sciences. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu-Beijing university of Tokyo, Chaoyang district, China academy of sciences) within 15.07.15.2019, and the preservation registration number is CGMCC No. 18229. Bacillus subtilis (CGMCC No.18229, hereinafter abbreviated as Bacillus subtilis BS).
The present invention also provides a culture of bacillus subtilis BS.
The invention also provides a bacterial suspension of the bacillus subtilis BS. The bacterial suspension can be specifically obtained by resuspending the bacteria with sterile physiological saline.
The invention also provides a product, the active ingredient of which is the bacillus subtilis BS, the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS.
The invention also provides a method for degrading cyanogenic glucoside, which comprises the following steps: and (3) fermenting the substance containing cyanogenic glucoside by using the bacillus subtilis BS, the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS to realize the degradation of the cyanogenic glucoside.
The invention also provides a method for degrading the cyanogenic glucoside of the flaxseed cake, which comprises the following steps: and fermenting the linseed cake by using the bacillus subtilis BS, the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS to degrade the cyanogenic glucoside.
The invention also provides a method for degrading the macromolecular protein of the flaxseed cake, which comprises the following steps: and mixing the bacillus subtilis BS, the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS and the linseed cake for fermentation to degrade macromolecular proteins of the linseed cake.
The invention also provides a method for improving the polypeptide content in the flaxseed cake, which comprises the following steps: the method comprises the following steps of mixing and fermenting the bacillus subtilis BS, the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS and the flaxseed cake to improve the polypeptide content of the flaxseed cake.
The invention also provides a method for improving the nutritive value of the flaxseed cake, which comprises the following steps: the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS is mixed with the flaxseed cake for fermentation, so that the anti-nutritional factor, namely cyanogenic glucoside in the flaxseed cake is reduced, the indigestible macromolecular protein in the flaxseed cake is decomposed, and the polypeptide content of the flaxseed cake is improved, thereby improving the nutritional value of the flaxseed cake.
The invention also provides the application of the bacillus subtilis BS, the culture of the bacillus subtilis BS or the bacterial suspension of the bacillus subtilis BS, and the application comprises the following steps: a) degrading cyanogenic glucoside; or b) degrading macromolecular protein in the linseed cake; or c) producing the polypeptide.
The invention also provides application of the bacillus subtilis BS, a culture of the bacillus subtilis BS or bacterial suspension of the bacillus subtilis BS in preparation of fermented flaxseed cakes.
The invention also provides application of the bacillus subtilis BS, a culture of the bacillus subtilis BS or a bacterial suspension of the bacillus subtilis BS in the detoxification of flaxseed cakes. The detoxication of the flaxseed cake can be specifically degradation of cyanogenic glucoside, a main anti-nutritional factor in the flaxseed cake.
The invention also provides application of the bacillus subtilis BS, a culture of the bacillus subtilis BS or a bacterial suspension of the bacillus subtilis BS in improving the content of the linseed cake polypeptide.
The Bacillus subtilis BS, the culture of Bacillus subtilis BS or the bacterial suspension of Bacillus subtilis BS according to the invention can be used for degrading substances containing cyanogenic glycosides, such as flaxseed cakes. The flaxseed cake can be prepared by various methods. Because the low-temperature cold-pressed flaxseed cake furthest retains cyanogenic glucoside, and the toxicity of animals is particularly high, the method for fermenting the flaxseed cake can effectively remove the cyanogenic glucoside in the flaxseed cake.
The bacillus subtilis BS provided by the invention has the advantages of simple culture method, high growth speed, high temperature resistance and high safety. The bacillus subtilis provided by the invention can be applied to the following aspects: (1) because of having stronger cyanogenic glucoside degradation capability, the flax seed cake can be detoxified; (2) the content of the polypeptide in the flaxseed cake can be obviously improved, so that the method can be used for preparing fermented flaxseed cakes or fermented feed; (3) the strain has the capability of inhibiting harmful bacteria, so the strain can be used as a feed additive, promotes the digestion and absorption of animals to nutrient substances, inhibits the propagation of pathogenic bacteria in the gastrointestinal tract of the animals, and promotes the intestinal microecological balance.
Specifically, the present invention is as follows:
1. the bacillus subtilis BS has the preservation registration number of CGMCC No.18229, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and has the address: china, Beijing, institute of microbiology, China academy of sciences, No. 3 Xilu-1 of the Chaoyang district, Beijing city, zip code: 100101, preservation time of 2019, 7 months and 15 days.
2. A culture of the Bacillus subtilis BS of item 1.
3. A suspension of the Bacillus subtilis BS of item 1.
4. A product whose active ingredients comprise: the Bacillus subtilis BS of item 1, the culture of item 2, or the bacterial suspension of item 3.
5. A method of degrading cyanogenic glycosides comprising the steps of: mixing the Bacillus subtilis BS described in item 1, the culture described in item 2, or the suspension described in item 3 with a substance containing cyanogenic glycosides for fermentation.
6. A method for reducing cyanogenic glucoside in flaxseed cake comprises the following steps: and (3) mixing the bacillus subtilis BS of item 1, the culture of item 2 or the bacterial suspension of item 3 with a flaxseed cake for fermentation.
7. A method for degrading macromolecular protein of flaxseed cake and increasing polypeptide content in flaxseed cake comprises the following steps: and (3) mixing the bacillus subtilis BS of item 1, the culture of item 2 or the bacterial suspension of item 3 with a flaxseed cake for fermentation.
8. A method for improving the nutritive value of flaxseed cake comprises the following steps: and (3) mixing the bacillus subtilis BS of item 1, the culture of item 2 or the bacterial suspension of item 3 with a flaxseed cake for fermentation.
9. Use of the Bacillus subtilis BS of item 1, the culture of item 2, or the suspension of item 3, as follows: a) degrading cyanogenic glucoside; or b) degrading macromolecular protein in the linseed cake; or c) producing the polypeptide.
10. Use of the Bacillus subtilis BS of item 1, the culture of item 2, or the suspension of item 3 in the preparation of a fermented flaxseed cake.
Drawings
FIG. 1 is a photograph of a colony of Bacillus subtilis BS.
FIG. 2 is a microscope picture of Bacillus subtilis BS.
FIG. 3 is a growth curve of Bacillus subtilis BS.
FIG. 4 shows the effect of time on Bacillus subtilis BS degradation of flaxseed cake cyanogenic glycosides.
FIG. 5 shows the effect of temperature on the degradation of cyanogenic glycosides in flaxseed cake by Bacillus subtilis BS.
FIG. 6 shows the effect of feed water ratio on Bacillus subtilis BS degradation of cyanogenic glycosides in flaxseed cake.
FIG. 7 shows the effect of inoculum size on the degradation of cyanogenic glycosides in flaxseed cake by Bacillus subtilis BS.
FIG. 8 shows the cyanogenic glycoside content of linseed cake treated by Bacillus subtilis BS fermentation in the optimal process.
FIG. 9 shows the content of the linseed cake polypeptide fermented by the Bacillus subtilis BS in the optimal process.
Biological material preservation information
Bacillus subtilis (Bacillus subtilis) strain BS with the preservation registration number of CGMCC No.18229, preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, and the address: china, Beijing, institute of microbiology, China academy of sciences, No. 3 Xilu-1 of the Chaoyang district, Beijing city, zip code: 100101, preservation time of 2019, 7 months and 15 days.
Detailed Description
The following examples are given to facilitate a better understanding of the present invention, but the present invention is not limited to these examples. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged. In the following examples, the water used was deionized water. In the following examples, the used flaxseed cakes were cold-pressed flaxseed cakes obtained from Mongolian aroma biotechnology, Inc., Chonghaote, unless otherwise specified.
Example 1 isolation, purification and characterization of Bacillus subtilis
1.1 isolation and purification of Bacillus subtilis
1.1.1 test materials
The linseed cake is purchased from Mongolian Guxiang Biotech, Inc., called Haoyote.
The rumen fluid was collected from the pilot plant base of Chinese academy of agricultural sciences and stored at 4 ℃.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
1.1.2 culture Medium
Bacterial screening solid medium (esculin-LB solid medium): 0.5g of yeast extract, 1.5g of agar powder, 1g of peptone, 1g of sodium chloride, 0.05g of esculin, 0.01g of ferric citrate and 100mL of distilled water, and sterilizing at 121 ℃ for 20 min.
Bacterial basal liquid medium (LB liquid medium): yeast extract 0.5g, peptone 1g, sodium chloride 1g, distilled water 100mL, 121 degrees C sterilization for 20 min.
LB solid medium: 0.5g of yeast extract, 1.5g of agar powder, 1g of peptone, 1g of sodium chloride and 100mL of distilled water, and sterilizing at 121 ℃ for 20 min.
1.1.3 test methods
1.1.3.1 isolation of Cyanogenic Glycoside (CGs) degrading bacteria
In this experiment, a solid medium containing iron esculin citrate was used, and a target strain was isolated by a dilution-plating method. The principle is as follows: the degradation of cyanogenic glycosides requires the involvement of the key enzyme β -glucosidase. Esculin can be decomposed by beta-glucosidase to produce escin, and escin reacts with iron ions to form black compounds, and strains producing beta-glucosidase can be screened according to the condition of producing black circles.
Taking 2 250mL conical flasks which are respectively marked as A1 and A2, respectively adding 10g of flaxseed cake powder and 10mL of sterile distilled water into each conical flask, uniformly mixing the sheep rumen fluid stored at 4 ℃, respectively adding 5mL of sheep rumen fluid into the conical flasks, uniformly stirring, sealing the mouth of each conical flask by a membrane, sealing the mouth of each conical flask by the membrane, placing the A1 conical flasks and the A2 conical flasks in an incubator at 37 ℃, and culturing for 72 hours to enrich strains capable of utilizing flaxseed cakes. After 72h of incubation, 10g of the culture in the A1 and A2 Erlenmeyer flasks were placed in another 2 250mL Erlenmeyer flasks, 100mL of sterile distilled water and several sterile glass beads were added, and the mixture was shaken for 30min on a shaker at 200 r/min. Then all the conical flasks are taken out, kept stand for 10min, and after supernatant liquid appears, 1mL of supernatant liquid is taken according to the proportion of 10-1、10-2、10-3、10-4And (5) diluting by times. Respectively coating the supernatant diluent of A1 and A2 on esculin-LB solid medium, and culturing at 37 deg.C for 72 h. After culturing for 72h, colonies with the periphery blackened are picked, streaked and separated, and streaking separation and purification are repeated according to the method until single colonies appear.
1.1.3.2 Primary screening for Cyanogenic Glucoside (CGs) degrading bacteria
The strains which are purified and have the discoloring circles are respectively inoculated in 10mL LB liquid culture medium, after 24h of culture, shaking up, respectively taking 10 microliter of culture liquid to drop in the center of the corresponding esculin solid culture medium, after 48h of culture, respectively measuring the sizes of the discoloring circles (d1) and the colony diameter (d2) by using a graduated scale. Three replicates were set up per strain. And screening 3 strains with larger d1/d2 ratio results by taking the result of d1/d2 as an index and preserving the strains.
1.1.3.3 rescreening of cyanogenic glucoside degrading bacteria
After the 3 strains obtained by primary screening are subjected to expanded culture, 3mL of bacterial liquid is respectively taken to be put into a 300mL beaker, 21mL of sterile distilled water is added to be mixed uniformly, then 30g of flaxseed cake powder is added to be stirred uniformly, a bottle mouth sealing film is used for sealing, and the mixture is placed in an incubator at 37 ℃ for culture for 72 hours. Each process set 3 replicates. And after the fermentation is finished, airing the fermented material in a natural state. And (3) determining the content of the cyanogenic glucoside in each test sample, and selecting strains with good cyanogenic glucoside degradation effect for identification.
1.1.4 results and analysis
1.1.4.1 data statistics and analysis
The test data is used for establishing a database by Excel, a one-factor variance analysis method in SPSS19.0 software is used for analyzing, the test results are all expressed in the form of mean value plus or minus standard error, and the difference is obvious when P < 0.05.
1.1.4.2 preliminary screening results
Using moroxydine as a flora source, 8 strains with black circles were isolated from the LB solid medium by a dilution plating method using LB solid medium containing esculin ferric citrate, and table 1 shows the measurement values of the black circle diameter (d1) and the colony diameter (d2) of the 8 target strains and the calculation results of d1/d 2. Among the 8 target strains, 3 strains (L1, L3, L4) had a d1/d2 result of more than 1.5.
TABLE 1 ratio of diameter of discoloring ring (d1) to diameter of colony (d2) of 48h cultured by target strain
Figure BDA0002579348870000081
1.1.4.3 rescreening results
The content of Cyanogenic Glycosides (CGs) in the fermented flaxseed cake is used as an index, and the primary screened 3 strains are re-screened, so that the result is shown in Table 2, the detoxification effect of the strain L4 is best and is remarkably higher than the detoxification effects of the strain L1 and the strain L3 (P is less than 0.05), and therefore the strain L4 can be used as a target strain for fermenting the flaxseed cake.
TABLE 2 detoxification effect of fermentation treatment of flaxseed cake with different purpose strains
Figure BDA0002579348870000091
Note: the data in the same column are marked with lower case letters, indicating significant differences (P < 0.05).
1.2 identification of the species
1.2.1 morphological Observation
The screened strains are preliminarily identified according to a manual for identifying a common bacteria system, and the color, the shape, the size, the roughness, the edge and the like of a single colony are observed after the target strains are cultured for 48 hours. FIG. 1 is a photograph of a colony of strain L4. After the strain L4 obtained by rescreening is cultured on a separation plate for 48 hours, the strain L4 can be observed to form a round colony, the edge is irregular, and the surface is dry and rough.
FIG. 2 is a microscopic morphological picture of strain L4. The bacterial cells of L4 were rod-shaped and gram-positive under the microscope.
1.2.2 molecular biological identification
The target bacteria were entrusted to the biological medicine science and technology limited of Mei Jisang Beijing to identify 16S rDNA strains. The 16S rDNA of the L4 strain was amplified, and the purified PCR product was sequenced.
The 16S rDNA strain identification result shows that: the strain L4 belongs to the genus Bacillus, and has 100% similarity with Bacillus subtilis strain (KY652941.1Bacillus subtilis strain). And determining that the L4 is the Bacillus subtilis and named as Bacillus subtilis strain BS by integrating the results of morphological identification and molecular biological identification.
The Bacillus subtilis strain BS is preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 Hospital No.1 of Beijing, Naja Kogyo, China academy of sciences) in 15.07.2019, and the preservation registration number is CGMCC No. 18229. Bacillus subtilis (CGMCC) No.18229, herein called Bacillus subtilis BS.
Example 2 study on function of Bacillus subtilis BS for fermenting flaxseed cake to degrade cyanogenic glucoside
2.1 Bacillus subtilis BS growth Curve determination
Streaking the separated Bacillus subtilis BS plate, selecting single colony on the plate, placing in 10mL LB liquid culture medium, and placing at 37 deg.CPerforming shake culture on a 200r/min shaking table for 24h to prepare a seed solution, inoculating the seed solution into 250mL of liquid LB culture medium according to the inoculation amount of 1%, placing the seed solution into a shaking table for shake culture at 37 ℃ and 200r/min for 36h, sampling every 2h, measuring the Optical Density (OD) value at 600nm of an ultraviolet visible spectrophotometer, and measuring the OD value by using the OD600The value represents the amount of growth of the cells and the OD is plotted600Graph with time.
FIG. 3 is a growth curve of Bacillus subtilis BS. As shown in FIG. 3, Bacillus subtilis BS goes through a short slow-growth phase, is in a logarithmic growth phase for 2h to 24h, reaches a maximum growth concentration for 24h, and then enters a plateau phase. Therefore, the bacterial liquid cultured for 24h was selected as the seed liquid for fermentation in the subsequent fermentation test.
2.2 measurement of indices and methods
The content of cyanogenic glucoside is determined by referring to a colorimetric method in GB/T13084-2006.
The results of all the above measurements are based on oven dry mass.
2.3 data statistics and analysis
The test data is used for establishing a database by Excel, a one-factor variance analysis method in SPSS19.0 software is used for analyzing, the test results are all expressed in the form of mean value plus or minus standard error, and the difference is obvious when P is less than 0.05.
2.4 Single-factor test of influence of time, temperature, material-water ratio and inoculation amount on degradation of cyanogenic glucoside by fermenting flaxseed cakes with Bacillus subtilis BS
And (3) respectively researching the influence of fermentation time, fermentation temperature, material-water ratio and inoculation amount on the fermentation effect of the target strain by adopting a variable control method. The content of cyanogenic glucoside of the fermented flaxseed cake is taken as an evaluation index. Different single factor levels were set, with three replicates per treatment set.
2.4.1 preparation of bacterial suspensions
(1) Single colonies on the plate are picked up and placed in 10mL LB liquid medium and shake-cultured for 24h at 37 ℃ and 200r/min by a shaking table to prepare seed liquid.
(2) Inoculating the seed solution obtained in the step (1) into 250mL of LB liquid culture medium according to the inoculation amount of 1%, placing the seed solution into a shaker at 37 ℃ and 200r/min for shaking culture for 24h, centrifuging the seed solution at 4000rpm for 10min, collecting thalli, and washing the thalli for 3 times by using sterile normal saline.
(3) Taking the thallus obtained in the step (2), and re-suspending the thallus by using 250mL of sterile physiological saline to prepare a bacterial suspension, wherein the bacterial concentration in the bacterial suspension is 1.0 × 1010～2.0×1010cfu/mL。
2.4.2 Effect of fermentation time on degradation of cyanogenic glucoside by fermenting and treating flaxseed cake with Bacillus subtilis BS
Taking the bacterial suspension obtained in the step 2.4.1, keeping the inoculum size at 10%, the ratio of material to water at 1: 0.8, keeping the temperature at 37 ℃ unchanged, and setting the fermentation time to 24h, 36h, 48h, 60h and 72h respectively. Fermenting according to the parameters, drying and crushing in the air in a natural state after fermentation is finished, and measuring the content of cyanogenic glucoside.
FIG. 4 is a graph showing the effect of fermentation time on the degradation of linseed cake cyanogenic glycosides by Bacillus subtilis BS. As shown in figure 4, when the inoculation amount is 10%, the ratio of feed to water is 1: 0.8, and the temperature is 37 ℃, the content of cyanogenic glucoside in the fermented flaxseed cake gradually decreases and becomes stable along with the increase of the fermentation time, and the content of cyanogenic glucoside in the fermented flaxseed cake is the lowest at the time of 72 h. It is suggested that the level of fermentation time can be set between 48h and 72h in the orthogonal experiments.
2.4.3 Effect of fermentation temperature on degradation of cyanogenic glucoside by fermenting and treating flaxseed cake with Bacillus subtilis BS
Taking the bacterial suspension in the step (1), keeping the inoculum size at 10%, the material-water ratio at 1: 0.8, and the fermentation time at 72h, and setting the fermentation temperature at 31 ℃, 33 ℃, 35 ℃, 37 ℃ and 39 ℃ respectively. Fermenting according to the parameters, drying and crushing in the air in a natural state after fermentation is finished, and measuring the content of cyanogenic glucoside.
FIG. 5 is a graph showing the effect of fermentation temperature on the degradation of cyanogenic glycosides in flaxseed cake by Bacillus subtilis BS. As shown in figure 5, when the inoculation amount is 10%, the ratio of feed to water is 1: 0.8, and the time is 72 hours, the content of cyanogenic glucoside in the fermented flaxseed cake decreases and then becomes stable along with the increase of the fermentation temperature, the content of cyanogenic glucoside in the fermented flaxseed cake becomes stable between 37 ℃ and 39 ℃, and the content of cyanogenic glucoside in the fermented flaxseed cake reaches the lowest. It is suggested that the orthogonal assay allows setting the fermentation temperature level between 35 ℃ and 39 ℃.
2.4.4 Effect of feed water ratio on degradation of cyanogenic glucoside by fermenting and treating flaxseed cake with Bacillus subtilis BS
Taking the bacterial suspension in the step (1), keeping the inoculum size at 10%, the temperature at 37 ℃ and the time for 72 hours unchanged, wherein the material-water ratio is 1: 0.6, 1: 0.7, 1: 0.8, 1: 1 and 1: 1.2 respectively. Fermenting according to the parameters, drying and crushing in the air in a natural state after fermentation is finished, and measuring the content of cyanogenic glucoside.
FIG. 6 is a graph showing the effect of feed water ratio on the degradation of cyanogenic glycosides in flaxseed cake by Bacillus subtilis BS. As shown in FIG. 6, when the inoculation amount is 10%, the temperature is 37 ℃, and after fermentation is carried out for 72 hours, the content of cyanogenic glucoside in the fermented flaxseed cake has a trend of first decreasing and then increasing along with the increase of the feed-water ratio, which indicates that the level of the fermented feed-water ratio can be set between 1: 0.6 and 1: 0.8 in the orthogonal test.
2.4.5 Effect of inoculum size on degradation of cyanogenic glucoside by fermenting and treating flaxseed cake with Bacillus subtilis BS
Taking the bacterial suspension in the step (1), keeping the ratio of material to water of 1: 0.8, the temperature of 37 ℃ and the fermentation time of 72h unchanged, wherein the inoculation amount is 2%, 6%, 10%, 14% and 18% respectively. Fermenting according to the parameters, drying and crushing in the air in a natural state after fermentation is finished, and measuring the content of cyanogenic glucoside.
FIG. 7 shows the effect of the inoculum size on the degradation of cyanogenic glycosides in flaxseed cake by Bacillus subtilis BS. As shown in figure 7, when the feed-water ratio is 1: 0.8, the temperature is 37 ℃, and after fermentation is carried out for 72 hours, the content of cyanogenic glucoside in the fermented flaxseed cake tends to decrease firstly and then increase along with the increase of the inoculation amount. It is suggested that the level of fermentation inoculum can be set between 4% and 8% in orthogonal experiments.
Example 3 Process optimization for degradation of cyanogenic glycosides by fermenting flaxseed cakes with Bacillus subtilis BS
3.1 measurement index and method
The content of cyanogenic glucoside is determined by referring to a colorimetric method in GB/T13084-2006, the content of crude protein is determined by adopting a Dumas full-automatic azotometer, the content of crude fiber is determined by adopting a filter bag method, the content of crude fat is determined by adopting a Soxhlet extraction method, and the content of polypeptide is determined by referring to a determination method of GB/T22492-2008 soybean peptide powder.
The results of all the above measurements are based on oven dry mass.
3.2 data statistics and analysis
The test data was analyzed by Excel to create a database and by one-way anova in SPSS19.0 software. Except for the orthogonal test, the results of the other tests were expressed as mean ± sem, and the difference was significant as P < 0.05.
3.3 preparation of the bacterial suspension
(1) Single colonies on the plate are picked up and placed in 10mL LB liquid medium and shake-cultured for 24h at 37 ℃ and 200r/min by a shaking table to prepare seed liquid.
(2) Inoculating the seed solution obtained in the step (1) into 250mL of liquid LB culture medium according to the inoculation amount of 1%, placing the seed solution into a shaker at 37 ℃ and 200r/min for shaking culture for 24h, centrifuging the seed solution at 4000rpm for 10min, collecting thalli, and washing the thalli for 3 times by using sterile normal saline.
(3) Taking the thallus obtained in the step (2), and re-suspending the thallus by using 250mL of sterile physiological saline to prepare a bacterial suspension, wherein the bacterial concentration in the bacterial suspension is 1.0 × 1010～2.0×1010cfu/mL。
3.4 fermentation Process optimization
3.4.1 orthogonal test
On the basis of a single-factor test, the optimal process for fermenting the flaxseed cake by the target strain is determined. Taking the bacterial suspension obtained in the step 3.3, taking the content of cyanogenic glucoside after fermentation as an index, and adopting L9(34) The orthogonal test design optimizes four factors of fermentation time (A), fermentation temperature (B), material-water ratio (C) and inoculation amount (D), each factor is provided with three levels, an orthogonal optimization test of three levels and four factors is carried out, and a table 3 is an orthogonal test design factor level table.
TABLE 3L9(34) Level meter for orthogonal test factors
Figure BDA0002579348870000121
3.4.2 results of orthogonal experiments
The orthogonal test is carried out on the basis of a single-factor test.
Table 4 is a visual analysis table of the results of the process optimization orthogonal test of the fermented flaxseed cake. The R value of the table 4 is directly analyzed according to the process optimization orthogonal test result of the fermented flaxseed cake, the fermentation temperature has the largest influence on the content of cyanogenic glucoside after fermentation of the flaxseed cake, the fermentation time is the next time, and the influence on the material-water ratio and the inoculation amount is small, namely B is more than A and more than D is more than C. The K value shows that the test treatment of A3B3C2D3 can reduce the cyanogenic glucoside content in the fermented flaxseed cake to the optimum level, namely the fermentation time is 72h, the fermentation temperature is 39 ℃, the material-water ratio is 1: 0.7, and the inoculation amount is 8%.
TABLE 4 fermentation of flaxseed cake process optimization orthogonal experiment design factor level table and visual analysis table of results
Figure BDA0002579348870000122
Figure BDA0002579348870000131
Table 5 is a table of analysis of variance of the process optimization orthogonal test of the fermented flaxseed cake. As shown in the table 5, the fermentation linseed cake process optimization orthogonal experiment analysis of variance table shows that the content of cyanogenic glucoside of the fermentation time level 3 is obviously lower than that of cyanogenic glucoside of the fermentation time levels 1 and 2 (P is less than 0.05); the content of cyanogenic glycosides at the fermentation temperature levels 2 and 3 is obviously lower than that of cyanogenic glycosides at the fermentation temperature level 1 (P is less than 0.05), the content of cyanogenic glycosides at the fermentation temperature level 3 is lower than that of cyanogenic glycosides at the fermentation temperature level 2, the difference is not significant statistically, but P is 0.051, which shows the trend that the difference is significant; the difference between the material-water ratios is not significant (P is more than 0.05); the difference between the various levels of inoculation was not significant (P > 0.05). According to the analysis results and the production practice, the optimal conditions for removing cyanogenic glucoside by fermenting the flaxseed cake with the bacillus subtilis BS are as follows: A3B3C2D1, namely fermentation time is 72h, fermentation temperature is 39 ℃, material-water ratio is 1: 0.7, and inoculation amount is 4%.
TABLE 5 analysis of variance table of results of process optimization orthogonal test of fermented flaxseed cake
Figure BDA0002579348870000132
Note: the data in the same column are marked with lower case letters, indicating significant differences (P < 0.05).
3.4.3 validation and amplification tests
And (4) carrying out a verification test according to the optimal fermentation condition determined by the orthogonal test, setting three times of repetition and measuring the content of the cyanogenic glucoside after fermentation.
The scale-up test was carried out according to the optimum fermentation conditions determined in the orthogonal test and scaling up the raw material to 30kg, grouped as follows,
and (3) fermentation group: the fermentation time is 72h, the fermentation temperature is 39 ℃, the material-water ratio is 1: 0.7, and the inoculum size is 4 percent (4 percent of strain culture solution);
control group: the fermentation time is 72h, the fermentation temperature is 39 ℃, the material-water ratio is 1: 0.7, the LB liquid culture medium is 4 percent, and the potassium sorbate is 1 percent;
untreated group: the flax cake raw material was not subjected to any treatment.
And (4) setting three times of treatment, naturally air-drying the treated sample, measuring the content of the CGs, and calculating the detoxification rate of the CGs. CGs detoxification rate (%) [ (content of untreated cyanogenic glycoside-content of cyanogenic glycoside after fermentation)/content of untreated cyanogenic glycoside ] × 100.
And analyzing the content of crude protein, polypeptide, crude fiber and crude fat of the flaxseed cakes of each treatment group in the expanded experiment.
3.4.4 validation and amplification test results
And (5) carrying out a verification test according to the optimal fermentation condition obtained by the orthogonal experiment. The result of the verification test shows that the content of cyanogenic glucoside is 33.42 +/-2.05 mg/kg, and the content (38.78 +/-0.20 mg/kg) of cyanogenic glucoside treated by A3B3C2D1 in the orthogonal test is not different significantly (P is more than 0.05).
The scale-up test was performed according to the optimal fermentation conditions obtained from the orthogonal experiments. Table 6 shows the results of the optimum process expansion test for fermented flax cakes. The results of the amplification experiment showed that the content of cyanogenic glycosides was 34.78. + -. 1.86mg/kg, which was not significantly different (P > 0.05) from the content of cyanogenic glycosides treated in the orthogonal experiment with A3B3C2D1 (38.78. + -. 0.20 mg/kg). As shown in the enlarged test result of Table 6, after the bacillus subtilis BS is fermented under the optimal conditions of 72 hours of fermentation time, 39 ℃, 1: 0.7 of material-water ratio and 4 percent of inoculation amount, the content of cyanogenic glucoside of the flaxseed cake is reduced to 34.78 +/-1.86 mg/kg from 584.47 +/-8.76 mg/kg of an untreated group, and the detoxification rate of CGs reaches 94.05 percent.
TABLE 6 optimal Process expansion test results for fermented flaxseed cake
Figure BDA0002579348870000141
3.4.5 Effect of fermentation treatment on nutritional value of Linum usitatissimum cake
Table 7 shows the effect of the fermentation treatment on the nutritional composition of flaxseed cake. The fermentation not only removes the main anti-nutritional factor CGs in the flaxseed cake, but also improves the nutritional value of the flaxseed cake. Wherein the content of Crude Protein (CP) is remarkably improved from 36.79 +/-0.37 percent to 39.43 +/-0.53 percent (P is less than 0.05); the polypeptide content is remarkably improved from 1.65 +/-0.06 percent to 4.41 +/-0.03 percent (P is less than 0.05); the content of crude fat is remarkably improved from 8.81 plus or minus 0.09 percent to 9.55 plus or minus 0.07 percent (P is less than 0.05); the crude fiber content varied insignificantly (P > 0.05).
TABLE 7 Effect of fermentation treatment on the nutritional composition of flaxseed cake
Figure BDA0002579348870000142
Note: the data in the same column are marked with lower case letters, indicating significant differences (P < 0.05).
Example 4, the bacillus subtilis BS fermentation treatment of the linseed cake cyanogenic glucoside and the polypeptide content and the detoxification rate of the cyanogenic glucoside under the optimal process parameters.
4.1 preparation of the bacterial suspension
(1) Single colonies on the plate are picked up and placed in 10mL LB liquid medium and subjected to shaking culture at 37 ℃ and 200r/min for 24h to prepare seed liquid.
(2) Inoculating the seed solution obtained in the step (1) into 250mL of liquid LB culture medium according to the inoculation amount of 1%, placing the seed solution into a shaker at 37 ℃ and 200r/min for shaking culture for 24h, centrifuging the seed solution at 4000rpm for 10min, collecting thalli, and washing the thalli for 3 times by using sterile normal saline.
(3) Taking the thallus obtained in the step (2), and re-suspending the thallus by using 250mL of sterile physiological saline to prepare a bacterial suspension, wherein the bacterial concentration in the bacterial suspension is 1.0 × 1010～2.0×1010cfu/mL。
4.2 optimum technological parameters of Bacillus subtilis BS fermentation treatment on the content and detoxification rate of linseed cake to cyanogenic glucoside
And (3) taking the bacterial suspension obtained in the step (4.1), fermenting and treating the flaxseed cake according to the optimal fermentation process determined in the embodiment 3, naturally drying and crushing the fermented material after fermentation, measuring the content of cyanogenic glucoside and calculating the detoxification rate of the cyanogenic glucoside.
The cyanogenic glucoside detoxification rate (%) - (content of cyanogenic glucoside in unfermented flaxseed cake-content of cyanogenic glucoside in fermented flaxseed cake)/content of cyanogenic glucoside in unfermented flaxseed cake ] × 100%
The result is shown in figure 8, the cyanogenic glucoside generated by the flax seed cake fermented by the bacillus subtilis BS is effectively removed, and the detoxification rate of the cyanogenic glucoside reaches 94.05 percent.
4.3 Effect of optimum Process parameters Bacillus subtilis BS fermentation on the polypeptide content
And (2) taking the bacterial suspension in the step (1), fermenting and treating the flaxseed cake according to the optimal fermentation process determined in the embodiment 3, naturally drying and crushing the fermented material after fermentation, and determining the polypeptide content.
The result is shown in figure 9, the content of the polypeptide is obviously improved when the linseed cake is fermented by the bacillus subtilis BS. The protease secreted by the bacillus subtilis BS converts macromolecular proteins into polypeptides which are easier to digest and absorb by animals, and improves the nutritional value of the flaxseed cake, so that the flaxseed cake can be better utilized by the animals.

Claims (10)

1. The bacillus subtilis BS has the preservation registration number of CGMCC No.18229, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and has the address: china, Beijing, institute of microbiology, China academy of sciences, No. 3 Xilu-1 of the Chaoyang district, Beijing city, zip code: 100101, preservation time of 2019, 7 months and 15 days.
2. A culture of Bacillus subtilis BS according to claim 1.
3. A suspension of the Bacillus subtilis BS of claim 1.
4. A product whose active ingredients comprise: the Bacillus subtilis BS of claim 1, the culture of claim 2, or the suspension of claim 3.
5. A method of degrading cyanogenic glycosides comprising the steps of: mixing the Bacillus subtilis BS of claim 1, the culture of claim 2 or the suspension of claim 3 with a substance containing cyanogenic glycosides for fermentation.
6. A method for reducing cyanogenic glucoside in flaxseed cake comprises the following steps: mixing the Bacillus subtilis BS of claim 1, the culture of claim 2 or the suspension of claim 3 with flaxseed cake for fermentation.
7. A method for degrading macromolecular protein of flaxseed cake and increasing polypeptide content in flaxseed cake comprises the following steps: mixing the Bacillus subtilis BS of claim 1, the culture of claim 2 or the suspension of claim 3 with flaxseed cake for fermentation.
8. A method for improving the nutritive value of flaxseed cake comprises the following steps: mixing the Bacillus subtilis BS of claim 1, the culture of claim 2 or the suspension of claim 3 with flaxseed cake for fermentation.
9. Use of the Bacillus subtilis BS of claim 1, the culture of claim 2 or the bacterial suspension of claim 3, as follows:
a) degrading cyanogenic glucoside; or
b) Degrading macromolecular protein in the flaxseed cake; or
c) Producing the polypeptide.
10. Use of the bacillus subtilis BS of claim 1, the culture of claim 2 or the suspension of claim 3 for the preparation of a fermented flaxseed cake.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725222A (en) * 2020-12-21 2021-04-30 齐鲁工业大学 Bacillus subtilis Q3 for producing complex enzyme, culture method and application
CN113080312A (en) * 2021-04-26 2021-07-09 中国农业科学院饲料研究所 Small peptide-rich bacterium-enzyme synergistic fermented chicken sausage and preparation method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080044501A1 (en) * 2006-08-16 2008-02-21 Genebiotech Co. Ltd. Fermented soybean products and methods of producing them

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080044501A1 (en) * 2006-08-16 2008-02-21 Genebiotech Co. Ltd. Fermented soybean products and methods of producing them

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
庞钰鑫 等: "市售川产水豆豉分离株中高产蛋白酶与β－葡萄糖苷酶菌株的菌属鉴定与产酶能力评价", 《四川大学学报（医学版）》 *
梅莺等: "亚麻饼粕微生物脱毒工艺", 《食品与发酵工业》 *
郭宝珠 等: "固态发酵亚麻籽饼工艺优化及其对1~21日龄肉鸡生长性能、免疫及抗氧化功能的影响", 《动物营养学报》 *
郭宝珠 等: "生氰糖苷脱毒菌株筛选及其发酵亚麻籽饼研究", 《饲料工业》 *
郭宝珠: "亚麻籽饼固态发酵工艺及其对肉鸡生产性能影响的研究", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725222A (en) * 2020-12-21 2021-04-30 齐鲁工业大学 Bacillus subtilis Q3 for producing complex enzyme, culture method and application
CN112725222B (en) * 2020-12-21 2023-01-31 齐鲁工业大学 Bacillus subtilis Q3 for producing complex enzyme, culture method and application
CN113080312A (en) * 2021-04-26 2021-07-09 中国农业科学院饲料研究所 Small peptide-rich bacterium-enzyme synergistic fermented chicken sausage and preparation method thereof

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