Stevia rebaudiana waste residue fermentation product compound detoxified cottonseed kernel feed and preparation method thereof
Technical Field
The invention relates to a stevia rebaudiana waste residue fermentation product compound detoxified cottonseed kernel feed and a preparation method thereof, belonging to the field of feed processing.
Background
With the development of animal husbandry, the demand of high-protein and high-energy feed is increasing, but because the crude fiber content of stevia rebaudiana residue is high, the palatability is poor, the cottonseed is rich in toxic substance free gossypol, and the utilization of the two in the field of feed is severely limited. Therefore, it is important to develop a method for utilizing the stevia rebaudiana residue and the toxic cotton seed kernel while containing high-energy and high-protein and low-production cost feed. In addition, the gossypol content and fat content of the cottonseed are much higher than those of the cottonseed meal, so that the gossypol removal of the cottonseed is more difficult than that of the cottonseed meal.
Disclosure of Invention
The invention aims to solve the technical problem that cottonseed is difficult to remove gossypol from cottonseed, and provides a detoxified cottonseed feed compounded with stevia rebaudiana residue fermentation products prepared from stevia rebaudiana residue and cottonseed as raw materials and a preparation method thereof.
Technical scheme
A preparation method of a stevia rebaudiana waste residue fermentation product compound detoxified cottonseed kernel feed comprises the following steps:
(1) pretreatment:
soaking stevia rebaudiana residue in alkali liquor for more than 24h, and then performing high-temperature steam treatment; the high-temperature steam treatment temperature is above 120 ℃; preferably 121 ℃; the treatment time is more than 20min, preferably 20 min;
sterilizing the semen gossypii in high pressure steam sterilizing pot at 120 deg.C for more than 20 min; sterilizing at 120 deg.C for 20 min;
(2) fermenting and detoxifying the cottonseed kernels:
taking the pretreated cotton kernels as a fermentation substrate;
adding the monascus seed liquid into a fermentation substrate according to the inoculation amount of 5%, and performing aerobic fermentation for 120h at the temperature of 32 ℃;
(3) fermentation of stevia rebaudiana residue
Inoculating the Trichoderma viride seed solution into stevia rebaudiana residue according to the inoculation amount of 10%, fermenting at 28 deg.C for 25 days;
(4) respectively drying the fermentation products obtained in the steps (2) and (3); then according to the following steps of 8: 2 by mass ratio.
In the preparation method, preferably, the mass concentration of the alkali liquor is 10%; the drying means drying under the temperature condition of less than or equal to 65 ℃ until the moisture is less than or equal to 15 wt%.
In the preparation method, the mixture obtained in the step (4) is the detoxified cottonseed kernel feed compounded with the fermentation product of the stevia rebaudiana waste residue; the mixture may also be further processed into various shapes.
According to the preparation method, after the stevia rebaudiana residue is fermented by the trichoderma viride, the cellulose and protein contents are obviously changed after fermentation for 10 days, the cellulose content is basically not changed after fermentation for 25 days, the protein content is up to 12.57% after fermentation, and the cellulose is degraded by about 18%.
According to the preparation method, after the cotton kernels are detoxified by fermentation of monascus, the degradation rate of free gossypol reaches 98.9% (from 7812.6mg/kg to about 80 mg/kg), the content of crude protein is increased from 43.76% to 48.3%, the content of phytic acid is reduced by 46.9%, the content of crude fat is increased from 34.29% to 35.16%, and macromolecular protein is changed into micromolecular protein.
The invention is characterized in that monascus is used as the cottonseed detoxification zymocyte. Although there have been reports in the prior art of fermentative degradation of gossypol; however, the prior art is directed to cottonseed meal; the invention aims at the cotton seed kernel, and the content of gossypol and fat in the cotton seed kernel is obviously higher than that of the cotton seed meal. It is well known to those skilled in the art that gossypol at high concentrations acts as an inhibitor of the fermentation degradation of gossypol strains. Therefore, when the technology for degrading the gossypol in the cottonseed meal is adopted to ferment and degrade the gossypol in the cottonseed, the degradation effect is inevitably reduced. For example, the degradation rate of the cottonseed by adopting candida tropicalis, endoplasmic reticulum and lactobacillus plantarum in a mass ratio of 3:2:1 as fermentation degradation bacteria can only reach 91%, and is obviously lower than the 94% degradation rate of the cottonseed when the cottonseed is degraded. However, the inventor finds that in the experimental process, the monascus is used as the degrading bacteria to degrade the cotton kernels, so that the degrading rate is high, and the operation is simple.
The invention has the following unexpected findings in the research and experiment process: a small amount of stevia rebaudiana residue is added in the detoxification process of the cotton seeds, so that the degradation rate of the gossypol can be further improved.
Therefore, it is preferable that the stevia residue after pretreatment and the cotton seed kernel after pretreatment are mixed at a ratio of 0.2 to 0.5: 100 as fermentation substrate.
According to the preparation method, after the cottonseed kernels are fermented and detoxified, the degradation rate of free gossypol reaches 99.6% (from 7812.6mg/kg to about 35 mg/kg).
The invention also provides the detoxified cottonseed feed compounded with the stevia rebaudiana waste residue fermentation product prepared by the method.
The stevia waste residue fermentation product compounded detoxified cottonseed kernel feed is dark red solid particles doped with a little brown color, and is suitable for most livestock. According to the national standard GB-13078-91, the allowable amount of free gossypol in the feed is that the cottonseed meal of the raw material is less than or equal to 1200mg/kg, the mixed feed for growing and fattening pigs is less than or equal to 60mg/kg, the feed for laying hens is less than or equal to 20mg/kg, and the allowable amount of broiler chicks and growing chickens is less than or equal to 100 mg/kg.
Trichoderma viride (Trichoderma viride) used in the present inventionTrichoderma viride) Monascus purpureus went (Monascus purpureus Went) (ii) a Is a commonly used bacterium; are all available in the market.
The invention has the beneficial effects that: the degradation rate of gossypol in the cotton kernels is high; therefore, the feed has good palatability, high protein content, rich and balanced nutrition, is easy to digest and absorb by animals, and effectively improves the quality of the feed; the utilization value of the industrial waste stevia rebaudiana residue and rich protein and grease which are rich in cotton kernels can be fully exerted. The method has the advantages of obvious significance for improving the resource utilization rate and reducing the production cost of the feed industry, simple process, no pollution in the production process, low cost and simple equipment.
Drawings
FIG. 1 shows the protein size change before and after fermentation.
Detailed Description
Example 1
(1) Preparation of PDA Medium
Weighing 50g of peeled potatoes, slicing the peeled potatoes into a big beaker, adding about 100ml of water, putting the big beaker into a microwave oven, heating the big beaker to be well cooked (preventing the water from boiling out), then filtering the big beaker by 8 layers of gauze, collecting filtrate to another beaker, adding 2g of glucose (if solid PDA is prepared, adding 2g of agar), stirring the mixture evenly, then heating the mixture until the glucose is completely dissolved evenly, subpackaging the mixture, sterilizing the mixture at 121 ℃ for 20min, taking out the mixture after pressure reduction, and cooling the mixture for later use.
(2) Preparation of seed liquid
The trichoderma viride is subjected to slant culture for 24h by using a PDA culture medium, and then is transferred into a PDA liquid culture medium to be cultured for 24h to serve as seed liquid for later use.
(3) Pretreatment of stevia residue
Immersing stevia rebaudiana residue in 10% alkali liquor for 24h to loosen the cell wall of the stevia rebaudiana residue, and separating lignin from the wood cellulose so as to improve the enzymatic saccharification rate and yield of the cellulase.
(4) Sterilizing stevia rebaudiana residue at high temperature
Adjusting relative humidity of the alkali-treated stevia rebaudiana residue to about 70%, and sterilizing with high temperature steam for 20 min.
(5) Fermentation of
Inoculating the seed solution prepared in the step (2) into the stevia rebaudiana residue treated in the step (4) according to the inoculation amount of 10%, fermenting at the temperature of 28 ℃ for 10 days, wherein the cellulose content and the protein content are obviously changed, the cellulose content is basically not changed after fermenting for 25 days, and detecting the crude protein content before and after fermenting by using a Kjeldahl method; determining the content of neutral detergent fiber and acidic detergent fiber by a fiber bag method; detecting crude fat by gas chromatography; the measurement results are shown in table 1;
TABLE 1 Change before and after fermentation of stevia rebaudiana residue with Trichoderma viride
In Table 1, "%" indicates mass percent.
And (4) drying the fermentation product obtained in the step (5) in an oven at the temperature of less than or equal to 65 ℃ until the water content is less than or equal to 15% for later use.
Example 2
(1) Preparation of PDA Medium
Weighing 50g of peeled potatoes, slicing the peeled potatoes into a big beaker, adding about 100ml of water, putting the big beaker into a microwave oven, heating the big beaker to be well cooked (preventing the water from boiling out), then filtering the big beaker by 8 layers of gauze, collecting filtrate to another beaker, adding 2g of glucose (if solid PDA is prepared, adding 2g of agar), stirring the mixture evenly, then heating the mixture till the mixture is completely dissolved evenly, subpackaging the mixture, sterilizing the mixture at 121 ℃ for 20min, taking out the mixture after pressure reduction, and cooling the mixture for later use.
(1) Corn flour culture medium preparation
Corn flour culture medium: 2% of corn flour, 0.3% of peptone, 0.1% of dipotassium phosphate, 0.05% of potassium chloride, 0.05% of magnesium sulfate and the balance of water; "%" is mass percent.
(2) Slant inoculation
Inoculating monascus strains to a 2% solid PDA culture medium in a streaking manner, and culturing for 48h at the temperature of 30 ℃;
(3) preparation of seed liquid
Inoculating monascus activated by a slant, and culturing the monascus in a corn flour culture medium at 32 ℃ for 48 hours for later use.
(4) Pretreatment of cotton seed kernels
The cotton seed kernel is sterilized in a high temperature high pressure steam sterilizing pot at 120 deg.C for 20min before fermentation to achieve the purpose of sterilization.
(5) Fermentation of
Inoculating the seed liquid prepared in the step (3) into the cotton kernels treated in the step (4) according to the inoculation amount of 10%, and performing aerobic fermentation for 120h at 32 ℃. Detecting the content of free gossypol by adopting a phloroglucinol method; detecting the content of phytic acid by a ferric chloride titration method; detecting the content of crude protein before and after fermentation by a Kjeldahl method; and (4) measuring the crude fat by gas chromatography. The measurement results are shown in table 2:
TABLE 2 Change of Cotton seed kernels before and after fermentation with Monascus purpureus
In Table 2, "%" indicates mass percent.
Analyzing the change of protein molecular weight before and after fermentation by Tricine-SDS-PAGE; the results are shown in FIG. 1.
Drying the fermentation product obtained in the step (5) in an oven at the temperature of less than or equal to 65 ℃ until the water content is less than or equal to 15% for later use;
example 3
Uniformly mixing the stevia rebaudiana residue fermentation product prepared in example 1 and the detoxified cottonseed fermentation product according to the ratio of 2:8, and preparing into granules with the diameter of 1.5-2.5 mm and the length of 4-7 mm by using a granulator; the detoxified cottonseed kernel feed compounded with the fermentation product of the stevia rebaudiana waste residue is obtained. The quality evaluation of the feed is shown in table 3:
TABLE 3 evaluation of the quality of the finished feed
In Table 3, "%" indicates mass percent.
Example 4
The stevia rebaudiana residue after pretreatment of example 1 and the cotton seed kernel after pretreatment of example 2 were mixed at a ratio of 0.2-0.5: 100 as fermentation substrate; inoculating the seed liquid prepared in the step (3) into a fermentation substrate according to the inoculation amount of 10%, and carrying out aerobic fermentation for 120h at 32 ℃. The free gossypol content is detected by adopting a phloroglucinol method, the gossypol content is less than or equal to 35mg/kg, and is obviously lower than the gossypol content in the fermentation product of the embodiment 3.
Comparative example 1
Candida tropicalis, endoplasmic reticulum and lactobacillus plantarum in a mass ratio of 3:2:1 are used as fermentation degrading bacteria; the cotton seed kernels treated in the step (4) of the example 2 are used as fermentation substrates. Respectively carrying out amplification culture on the zymophyte to obtain seed liquid, inoculating the seed liquid into a fermentation substrate according to the inoculation amount of 10%, and carrying out aerobic fermentation for 120h at 25 ℃. Detecting the content of free gossypol by adopting a phloroglucinol method; the gossypol content is more than or equal to 700mg/kg, which is obviously higher than that in the fermentation product of the embodiment 3.