CN117617351A - Preparation method of cottonseed meal and humic acid fermentation compound - Google Patents
Preparation method of cottonseed meal and humic acid fermentation compound Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Fodder In General (AREA)
Abstract
The invention belongs to the field of fermented feed, and in particular relates to a preparation method of a cottonseed meal and humic acid fermentation compound, which comprises the step of fermenting the cottonseed meal and the humic acid in the presence of a compound enzyme and a compound bacteria preparation. According to the invention, through more refined screening of the composite fermentation inoculant and the composite enzyme preparation, repeated application of the same type of microorganisms and the same type of biochemical enzyme preparation is avoided, so that the competing relationship among the same type of organisms is avoided, the synergistic effect of the composite fermentation inoculant and the composite enzyme preparation is effectively exerted, the degradation rate of free gossypol can be improved, and the content of acid soluble protein in a fermentation product is increased. In addition, the fermentation produces a substrate containing a large amount of amino acids and small peptides, and the amino acids and the small peptides in the substrate can carry out complexation reaction with humic acid to play a synergistic effect.
Description
Technical Field
The invention relates to the field of fermented feed, in particular to a preparation method of a cottonseed meal and humic acid fermentation compound.
Background
The soybean meal reduction substitution requires to increase the development of unconventional protein feed resources, fully utilizes the miscellaneous meal such as cottonseed meal, rapeseed meal, sunflower seed meal and the like, and reduces the use of the soybean meal in the feed.
The content of the crude protein in the cotton meal is 38% -50%, and the cotton meal is an ideal plant protein raw material for replacing soybean meal. The annual cotton seed cake produced in China is over 600 ten thousand tons, and the defect of plant protein resources in China can be relieved to a certain extent by reasonably developing and utilizing the cotton seed cake.
The screening of strains with good effect on fermented cottonseed meal is researched by screening of strains with good effect on fermented cottonseed meal and researching of fermentation activity products of the strains under the conditions of composite fermentation process (Xiacheng, nanjing agricultural university's Shuoshi) and finally, the combination of rhizopus oryzae, candida olivaceus and klebsiella is determined as a preferred fermentation strain, so that the content of gossypol in the fermentation products can be reduced, and the content of crude protein in the fermentation products can be increased.
Research on improving quality of cottonseed meal by solid state fermentation (Fengyu, et al, aquatic biology report) research on improving quality of cottonseed meal by solid state fermentation, and found that degradation rate of free gossypol, phytic acid and the like in cottonseed meal can be improved by adopting Saccharomyces cerevisiae (Saccharomyces cerevisiae) and lactobacillus plantarum (Lactobacillus plantarum) to be matched with bacillus subtilis and adding phytase and cellulase solution into a substrate to perform mixed bacteria solid state fermentation.
The research on preparing low bitter peptide by multi-enzyme hydrolysis of cottonseed protein isolate (Liu Jun, etc., food industry technology) discloses that the hydrolysis degree is used as an index, the enzymatic hydrolysis is adopted to prepare the cottonseed bioactive peptide, the double-enzyme hydrolysis process is discussed on the basis of single-enzyme hydrolysis, the optimal double-enzyme combination of alkaline protease and trypsin is determined, the hydrolysis degree can reach 26.67%, and the peptide yield is 87.35%. The flavor enzyme is adopted to continue hydrolysis, so that the bitter taste is effectively reduced, and the peptide yield is 85.26%.
Humic acid is an organic substance formed and accumulated by a series of processes of decomposition and transformation of plant remains and microorganisms and geochemistry. Humic acid as feed additive can reduce diarrhea rate and death rate of piglets, effectively improve immunity of livestock and poultry, reduce antibiotic consumption, promote growth, improve feed conversion rate, improve feeding environment, improve quality of livestock and poultry products, and is environment-friendly, and is one of the technical measures of current drug feed additives and antibiotic substitutes.
In the prior art, a composite fermentation microbial agent and a composite fermentation enzyme preparation are commonly adopted in the fermentation degradation process of cottonseed meal, but more microorganisms of the same type, biochemical enzyme preparations of the same type and the like exist in the existing composite fermentation microbial agent and composite enzyme preparation, and a certain competing relationship exists between the microorganisms and the biochemical enzyme preparations, so that the exertion of the actions of the composite microbial agent and the composite enzyme preparation can be influenced, and therefore, the objective requirement for further optimization of the composite fermentation microbial agent and the composite enzyme preparation exists. In addition, the active groups of untreated humic acid are often blocked by impurities, which affects the efficacy.
Disclosure of Invention
The inventor discovers that the biological activity of a fermentation product can be greatly improved by fermenting cotton pulp and humic acid together and carrying out more refined screening combination on a composite fermentation microbial inoculum and a composite enzyme preparation, and the cotton pulp fermentation product and the humic acid together play a synergistic effect. Based on this, the present invention has been completed.
Therefore, the invention provides a high-efficiency microbial fermentation method for treating cotton pulp and humic acid, which adopts composite protease and composite bacterial strain to ferment and enzymolyze the cotton pulp to produce a substrate containing a large amount of amino acids and small peptides. Meanwhile, amino acid and small peptide in the substrates can be subjected to complexation reaction with humic acid to obtain a fermented cotton pulp and humic acid compound which is used in the fields of feed additives and the like.
The invention relates to a preparation method of a cottonseed meal and humic acid fermentation compound, which comprises the following steps:
step 1) crushing: crushing cotton seed meal by a crusher, wherein the rotating speed is 20000-24000 rpm, and the time is 30-60 seconds;
step 2) adding humic acid: mixing the crushed cotton meal powder and humic acid powder according to the weight ratio of 50-90:10-50, and uniformly stirring to obtain a fermentation material;
step 3) curing: sterilizing the fermented material under high pressure at 115-125deg.C and 1-2mpa, and aging for 1-2 hr;
step 4) humidity adjustment: cooling to room temperature after curing, and uniformly mixing the fermentation material and water according to the proportion of 1:0.6-1.0;
step 5) inoculation: mixing the complex enzyme and the complex bacterial preparation in a weight ratio of 1:2-5 at a temperature below 40 ℃, inoculating the complex enzyme and the complex bacterial preparation into a fermentation material according to an inoculum size of 2-5% by weight, and uniformly stirring;
step 6) fermentation front section: carrying out aerobic fermentation on the inoculated material, controlling the temperature to be 32+/-1 ℃, keeping the humidity to be more than or equal to 90%, inputting sterile air for 24-48 hours, and keeping the tank pressure to be 0.3-0.6mpa;
step 7) fermentation post-stage: after the air input is closed, stirring is carried out uniformly, the temperature is controlled to be 32+/-1 ℃, and the fermentation time is 24-48 hours;
step 8) buffer fermentation: after being stirred uniformly, the temperature is regulated to 30+/-1 ℃, the humidity is kept at 80-90%, and the fermentation time is 12-24 hours;
step 9) drying: and (3) drying the fermented product at 50-70 ℃ until the water content is below 12%. To obtain a fermentation complex.
In one embodiment of the invention, wherein:
the complex enzyme is a complex enzyme containing alkaline protease and cellulase, wherein the weight ratio of the alkaline protease to the cellulase is 2-4:1, preferably 2-3:1, for example 2:1, 2.5:1 and 3:1, and more preferably 2:1.
The composite bacterial preparation is a composite bacterial preparation containing rhizopus oryzae and candida olivaceus, wherein the weight ratio of rhizopus oryzae to candida olivaceus is 5-8:1, preferably 6-8:1, for example 6:1, 6.5:1, 7:1, 7.5:1, 8:1, more preferably 7:1.
In the preparation method of the invention, wherein:
in step 2), the weight ratio of the cotton seed meal to the humic acid powder is preferably 60-80:20-40, more preferably 70:30.
In step 3), the curing temperature is preferably 118-122 ℃, more preferably 121 ℃; the pressure is preferably 1.5-2mpa; the curing time is preferably 1 hour.
In step 4), the ratio of fermentation mass to water is preferably in the range of 1:0.7 to 0.9, for example 1:0.7, 1:0.8, 1:0.9, more preferably 1:0.9.
In step 5), the preferred inoculation temperature is 25-38deg.C, more preferably 28-34 deg.C, such as 28deg.C, 29 deg.C, 30 deg.C, 31 deg.C, 32 deg.C, 33 deg.C, 34 deg.C; the weight ratio of the complex enzyme to the complex bacterial preparation is preferably 1:2-4, more preferably 1:3. Preferably, the inoculum size is 2.5-4% by weight, more preferably 3-4%, for example 3%, 3.5%, 4% by weight, of the fermentation material.
In step 6), the fermentation time is preferably 24 to 36 hours, more preferably 24 hours. Preferably, the can pressure is maintained between 0.4 and 0.5mpa.
In step 7), the fermentation time is preferably 24 to 36 hours, more preferably 24 hours.
In step 8), the fermentation time is preferably 12 hours.
In step 9), it is preferable to dry at 60℃to a water content of 10% or less.
Another aspect of the invention relates to a fermented complex of cottonseed meal and humic acid obtained by the above process. The steps for preparing the cotton seed meal and humic acid fermentation compound and the preferred steps are as described above.
In a further aspect the invention relates to the use of the fermented complex of cottonseed meal and humic acid obtained by the above-described process for feed additives in an amount of 0.1-10% by weight, preferably in an amount of 0.5-5% by weight, more preferably in an amount of 1-4% by weight, such as 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% by weight, in a feed.
The cotton seed meal and humic acid fermentation compound can be widely added into feeds for laying hens, broilers, piglets and fattening pigs, so that the laying rate of the laying hens is effectively improved, the feed-egg ratio of the laying hens is reduced, the average daily gain of the broilers is increased, the feed-meat ratio of the broilers is reduced, the average daily gain of the piglets is increased, and the diarrhea rate and the death rate of the piglets are reduced.
The beneficial effects are that:
in the preparation process of the cotton seed meal and humic acid fermentation compound, the composite fermentation microbial inoculum and the composite enzyme preparation are subjected to finer screening, so that repeated application of the same type of microorganisms (such as same genus microorganisms) and the same type of biochemical enzyme preparation is avoided, the competing relation among the same type of organisms is further avoided, and the synergistic effect of the composite fermentation microbial inoculum and the composite enzyme preparation is effectively exerted.
The cotton dreg fermentation product and the humic acid are fermented together, wherein the fermentation produces substrates containing a large amount of amino acids and small peptides, the amino acids and the small peptides in the substrates can carry out complexation reaction with the humic acid to play a synergistic effect, and meanwhile, the active groups of the untreated humic acid are prevented from being often blocked by impurities to influence the efficacy.
Detailed Description
The following examples are provided to further illustrate the invention and are not to be construed as limiting the invention in any way.
The raw materials used in the following examples of the present invention are all commercially available.
In the present invention, unless otherwise specified, the proportions are weight ratios, and the percentages are weight percentages.
In the following examples of the present invention, rhizopus oryzae (strain number: CICC 41158Rhizopus oryzae) was purchased from China center for type culture Collection of microorganisms; candida olive (strain number: CICC 31860,Candida oleophila) was purchased from China center for Industrial microorganism culture collection management. Alkaline protease (50000U/g) was purchased from Nanning east Henghua Biotechnology Co., ltd; cellulases (10000U/g) were purchased from Nanning east Henghua Biotechnology Co., ltd.
1. Complex bacterium and complex enzyme screening
1. And (3) expanding culture of strains:
inoculating rhizopus oryzae and candida albicans preserved in advance in a bevel into a potato liquid culture medium, and culturing for 24 hours at 30 ℃ under 150 rpm shaking to obtain primary seed liquid. Then inoculating the first-stage seed liquid into an conical flask containing potato liquid culture medium in an amount of 5%, culturing at 30deg.C for 24h under 150 rpm shaking, and placing in a refrigerator at 4deg.C for use. Wherein the number of living rhizopus oryzae is 3.42X10 8 CFU/mL, the viable count of Candida olive is 2.67×10 7 CFU/mL。
2. Complex bacterium and complex enzyme proportion screening
Crushing cotton seed meal for 45 seconds by using a crusher at the rotation speed of 20000 revolutions per minute, and then mixing the crushed cotton seed meal with humic acid powder according to the weight ratio of 70:30, and uniformly stirring. Thereafter, the resulting fermented mass was aged at 121℃under a pressure of 2mpa for 1 hour and autoclaved. And then cooling the material to room temperature, and uniformly mixing the material with sterilized water according to the proportion of 1:0.8.
Then, according to the 3-factor 3-level orthogonal test, fermentation effects at different ratios were analyzed by taking the weight ratio of the complex enzyme to the complex microbial inoculum, the weight ratio of alkaline protease and cellulase in the complex enzyme, and the weight ratio of rhizopus oryzae and candida olivaceus in the complex microbial inoculum as investigation factors (the orthogonal test design is shown in table 1). Specifically, 1000g of fermentation material after pretreatment is taken for each treatment, and composite protease and composite bacterial strain are inoculated at 30 ℃, wherein the total inoculation amount of the composite protease and the composite bacterial strain is 5% of the total fermentation material. Then, the inoculated material is subjected to aerobic fermentation, sterile air is input, the tank pressure is kept at 0.5mpa, the temperature is controlled at 32 ℃, the humidity is kept at 90%, and the fermentation is carried out for 24 hours. And stopping inputting air, uniformly stirring, controlling the temperature to be 32 ℃, and continuing fermentation for 24 hours. Then, the mixture was stirred uniformly, the temperature was adjusted to 30℃and the humidity was kept at 85%, and the mixture was fermented for 12 hours. And finally, drying the fermented product at 60 ℃ until the water content is about 8%, thus obtaining the fermentation compound.
The free gossypol content and acid soluble protein content of the fermentation complex obtained by each treatment were measured, and the fermentation effect of each treatment was evaluated. Wherein the free gossypol content is determined by the method described in GB/T13086-2020 and the acid soluble protein content is determined by the method described in NY/T3801-2020. The specific measurement results are shown in table 2 below.
TABLE 1 orthogonal test design
TABLE 2 fermentation effects of each treatment
* The control was unfermented feedstock.
As shown in the above results, the alkaline protease in the complex enzyme treated in T1.4 is 2:1, the rhizopus oryzae is 7:1, the degradation rate of gossypol is maximum when the complex enzyme is 1:3, and the acid soluble protein content in the fermentation product is increased to 115.63%, so that the complex enzyme and the complex microbial inoculum are the most preferable combined fermentation microbial enzyme complex. The degradation rate of other treatments on gossypol is also obvious, and the content of acid soluble protein can be obviously improved, but the treatment fermentation effect of T1.3 is relatively poor.
2. Screening of optimal fermentation conditions
Screening fermentation conditions by adopting the screened complex enzyme and complex microbial inoculum (wherein the complex enzyme is complex microbial inoculum=1:3, and the alkaline protease is cellulase=2:1, and the rhizopus oryzae is candida olivaceus=7:1) in the complex microbial inoculum, and designing a 3-factor 3-level orthogonal test by taking the feed-water ratio, the inoculation amount and the aerobic fermentation time as investigation factors (the orthogonal test design is shown in table 3).
The specific fermentation method comprises the following steps: crushing cotton seed meal for 45 seconds by using a crusher at the rotation speed of 20000 revolutions per minute, and then mixing the crushed cotton seed meal with humic acid powder according to the weight ratio of 70:30, and uniformly stirring. Thereafter, the resulting fermented mass was aged at 121℃under a pressure of 2mpa for 1 hour and autoclaved. And then cooling the materials to room temperature, and uniformly mixing the materials with sterilized water according to a set proportion. 1000g of the pretreated fermentation material is taken, and the composite protease and the composite strain with the proportion are inoculated at 30 ℃ according to the set inoculation amount (the total inoculation amount is calculated as the percentage of the total fermentation material). Then, the inoculated material is subjected to aerobic fermentation, sterile air is input, the tank pressure is kept at 0.5mpa, the temperature is controlled at 32 ℃, the humidity is kept at 90%, and the fermentation is carried out for a set time. And stopping inputting air, uniformly stirring, controlling the temperature to be 32 ℃, and continuing fermentation for 24 hours. Then, the mixture was stirred uniformly, the temperature was adjusted to 30℃and the humidity was kept at 85%, and the mixture was fermented for 12 hours. And finally, drying the fermented product at 60 ℃ until the water content is about 8%, thus obtaining the fermentation compound.
The free gossypol content and acid soluble protein content of the fermentation complex obtained by each treatment were measured, and the fermentation effect of each treatment was evaluated, and the results are shown in Table 4 below.
TABLE 3 orthogonal test design
TABLE 4 fermentation effects of each treatment
* The control was unfermented feedstock.
As shown in the above results, the degradation rate of the treated T2.8 gossypol was 92.19%, the improvement rate of the acid soluble protein content in the fermentation product was 191.07%, and it was preferable in the present invention, that is, the feed water ratio was 1:0.9, the total inoculum size was 4% and the aerobic fermentation time was 24 hours. The degradation rate of other treatments on gossypol and the improvement rate of acid soluble protein are also obvious, but the treatment fermentation effect of T2.3 is relatively poor.
3. Feed additive test
Fermentation complexes obtained by treating T2.3, T2.8 as described above
The experiment was performed with 60 healthy piglets of 30 days of age, randomly divided into 3 groups of 20 piglets. 2 experimental groups were set, and the feeds to which the fermented complex obtained by the treatments T2.3 and T2.8 were added were fed, respectively, with an addition amount of 2%. 1 control group was given to feed without any fermentation complex. The pigs are managed according to the conventional method of a pig farm, and after being fed for 30 days, average daily gain and diarrhea rate of different treated piglets are observed. The results are shown in Table 5.
TABLE 5 piglet feeding effect
The results show that the average daily gain is obviously improved and the diarrhea rate is obviously reduced in the treatment of adding the T2.3 and T2.8 fermentation complex compared with the control treatment. Wherein, the treatment effect of the T2.8 fermentation compound is better.
The advantages of the present invention have been shown and described above. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, and that the foregoing embodiments and descriptions are merely illustrative of the invention, and that any changes and modifications may be made without departing from the spirit and scope of the invention as claimed.
Claims (8)
1. The preparation method of the cottonseed meal and humic acid fermentation compound is characterized by comprising the following steps:
step 1) crushing: crushing cotton seed meal by a crusher, wherein the rotating speed is 20000-24000 rpm, and the time is 30-60 seconds;
step 2) adding humic acid: mixing the crushed cotton meal powder and humic acid powder according to the weight ratio of 50-90:10-50, and uniformly stirring to obtain a fermentation material;
step 3) curing: sterilizing the fermented material under high pressure at 115-125deg.C and 1-2mpa, and aging for 1-2 hr;
step 4) humidity adjustment: cooling to room temperature after curing, and uniformly mixing the fermentation material and water according to the proportion of 1:0.6-1.0;
step 5) inoculation: mixing the complex enzyme and the complex bacterial preparation in a weight ratio of 1:2-5 at a temperature below 40 ℃, inoculating the complex enzyme and the complex bacterial preparation into a fermentation material according to an inoculum size of 2-5% by weight, and uniformly stirring;
step 6) fermentation front section: carrying out aerobic fermentation on the inoculated material, controlling the temperature to be 32+/-1 ℃, keeping the humidity to be more than or equal to 90%, inputting sterile air for 24-48 hours, and keeping the tank pressure to be 0.3-0.6mpa;
step 7) fermentation post-stage: after the air input is closed, stirring is carried out uniformly, the temperature is controlled to be 32+/-1 ℃, and the fermentation time is 24-48 hours;
step 8) buffer fermentation: after being stirred uniformly, the temperature is regulated to 30+/-1 ℃, the humidity is kept at 80-90%, and the fermentation time is 12-24 hours;
step 9) drying: and (3) drying the fermented product at 50-70 ℃ until the water content is below 12%, thus obtaining the fermentation compound.
2. The method of claim 1, wherein the step of determining the position of the substrate comprises,
the complex enzyme is complex enzyme containing alkaline protease and cellulase, wherein the weight ratio of the alkaline protease to the cellulase is 2-4:1;
the composite bacterial preparation is a composite bacterial preparation containing rhizopus oryzae and candida olivaceus, wherein the weight ratio of rhizopus oryzae to candida olivaceus is 5-8:1.
3. The method according to claim 1, characterized in that in step 2) the weight ratio of cotton meal to humic acid powder is preferably 60-80:20-40.
4. The method according to claim 1, characterized in that in step 3), the curing temperature is preferably 118-122 ℃; the pressure is preferably 1.5-2mpa; the curing time is preferably 1 hour.
5. The method according to claim 1, characterized in that in step 4) the ratio of fermentation mass to water is preferably 1:0.7-0.9.
6. The method according to claim 1, characterized in that in step 5), the preferred inoculation temperature is 25-38 ℃; the weight ratio of the complex enzyme to the complex bacteria preparation is preferably 1:2-4; preferably in an inoculum size of 2.5-4% by weight.
7. A fermented cotton seed meal and humic acid complex obtainable by a process according to any one of claims 1 to 6.
8. Use of the cottonseed meal and humic acid fermented complex prepared according to the method of any one of claims 1 to 6 for feed additives, characterized in that the amount added in the feed is 0.1 to 10%.
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