阅读说明：本技术 一种茶油饼粕益生菌猪饲料的制备方法 (Preparation method of tea oil cake meal probiotic pig feed ) 是由 张涛 李逢 陈勇 李艳 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种茶油饼粕益生菌猪饲料的制备方法,包括以下步骤：1)茶油饼粕预处理；2)黑曲霉、枯草芽孢杆菌的活化、扩大培养,制备菌悬液；3)制备含茶油饼粕的固态发酵培养基；4)混合固态发酵：将黑曲霉和枯草芽孢杆菌接种至含茶油饼粕的固态发酵培养基进行发酵降解茶皂素,得到发酵茶油饼粕；5)猪粪便中筛选益生菌；6)制备含发酵茶油饼粕的益生菌固态培养基；7)益生菌固态发酵：将筛选的益生菌接种至益生菌固体培养基发酵培养；8)真空冷冻干燥,得到益生菌猪饲料。利用黑曲霉和枯草芽孢杆菌固态发酵脱脂后的茶油饼粕,并用地衣芽孢杆菌作为益生菌固态发酵发酵茶油饼粕,降解茶油饼粕中的茶皂素,提高猪的生长性能。(The invention discloses a preparation method of tea oil cake meal probiotic pig feed, which comprises the following steps: 1) pretreating tea oil cake; 2) activating and carrying out amplification culture on aspergillus niger and bacillus subtilis to prepare bacterial suspension; 3) preparing a solid fermentation culture medium containing tea oil cake; 4) mixed solid state fermentation: inoculating aspergillus niger and bacillus subtilis to a solid-state fermentation culture medium containing tea oil cake meal to ferment and degrade tea saponin to obtain fermented tea oil cake meal; 5) screening probiotics from pig manure; 6) preparing a probiotic solid culture medium containing fermented tea oil cake meal; 7) solid-state fermentation of probiotics: inoculating the screened probiotics to a probiotic solid culture medium for fermentation culture; 8) and (5) carrying out vacuum freeze drying to obtain the probiotic pig feed. Aspergillus niger and bacillus subtilis are used for solid-state fermentation of the degreased tea oil cake, and bacillus licheniformis is used as probiotics for solid-state fermentation of the tea oil cake to degrade tea saponin in the tea oil cake and improve the growth performance of pigs.)

1. A preparation method of tea oil cake meal probiotic pig feed is characterized by comprising the following steps: the method comprises the following steps: 1) pretreating tea oil cake; 2) activating and carrying out amplification culture on aspergillus niger and bacillus subtilis to prepare aspergillus niger suspension and bacillus subtilis suspension; 3) preparing a solid fermentation culture medium containing tea oil cake; 4) mixed solid state fermentation: inoculating aspergillus niger and bacillus subtilis to a solid-state fermentation culture medium containing tea oil cake meal to ferment and degrade tea saponin to obtain fermented tea oil cake meal; 5) screening probiotics from pig manure, and preparing seed liquid and bacterial suspension of the probiotics; 6) preparing a probiotic solid culture medium containing fermented tea oil cake meal; 7) solid-state fermentation of probiotics: inoculating the screened probiotics to a probiotic solid culture medium containing fermented tea oil cake meal for fermentation culture; 8) and (5) carrying out vacuum freeze drying to obtain the probiotic pig feed.

2. The preparation method of the tea oil cake meal probiotic pig feed according to claim 1, characterized by comprising the following steps: the specific operation steps of the step 1) are as follows: drying and crushing the tea oil cake dregs, and degreasing the crushed dregs with petroleum ether for later use.

3. The preparation method of the tea oil cake meal probiotic pig feed according to claim 1, characterized by comprising the following steps: the specific operation steps of the Aspergillus niger amplification culture in the step 2) are as follows: inoculating the activated aspergillus niger strains into an aspergillus niger liquid seed culture medium, and carrying out constant-temperature shaking table culture for later use; the specific operation steps of the bacillus subtilis expanded culture are as follows: inoculating the activated bacillus subtilis strain into a bacillus subtilis liquid seed culture medium, and carrying out constant-temperature shaking table culture for later use.

4. The preparation method of the tea oil cake meal probiotic pig feed according to claim 1, characterized by comprising the following steps: the specific operation steps of the step 3) are as follows: 100g of tea oil cake meal pretreated in the step 1) and KH₂PO₄ 1g、MgSO₄·7H₂O 0.5g、（NH₄）₂SO₄1g, stirring uniformly, and sterilizing at 121 ℃ for 30 min.

5. The preparation method of the tea oil cake meal probiotic pig feed according to claim 1, characterized by comprising the following steps: the specific operation steps of the step 4) are as follows: the total inoculation amount of aspergillus niger and bacillus subtilis is 8-14%, and the mixing ratio of bacillus subtilis suspension to aspergillus niger suspension is 2:1-1:2 inoculating the mixture into a solid fermentation culture medium containing tea oil cake dregs for fermentation culture.

6. The preparation method of the tea oil cake meal probiotic pig feed according to claim 5, characterized by comprising the following steps: the conditions of fermentation culture in the step 4) are as follows: the fermentation time is 3.5-5.5 days, the fermentation temperature is 28-35 ℃, and the fermentation is carried out once every 12 hours.

7. The preparation method of the tea oil cake meal probiotic pig feed according to claim 1, characterized by comprising the following steps: the probiotics screened in the step 5) are Bacillus licheniformis CFC-11 and MR-1.

8. The preparation method of the tea oil cake meal probiotic pig feed according to claim 1, characterized by comprising the following steps: the specific operation steps of the step 6) are as follows: sterilizing 20g of the fermented tea oil cake prepared in the step 4), 1-5g of glucose, 0.5-0.7g of ammonium sulfate, 0.15-0.25g of disodium hydrogen phosphate and 18ml of deionized water at the pH value of 7.0 for 20min at 121 ℃.

9. The preparation method of the tea oil cake meal probiotic pig feed according to claim 7, characterized by comprising the following steps: the specific operation steps of the step 7) are as follows: the total inoculation amount of the Bacillus licheniformis CFC-11 and MR-1 is 3-4%, and the bacterial suspension mixing ratio of the Bacillus licheniformis CFC-11 and MR-1 is 1: 3-3: 1 inoculating the mixture into a probiotic solid culture medium containing fermented tea oil cake dregs for fermentation culture.

10. The preparation method of the tea oil cake meal probiotic pig feed according to claim 9, characterized by comprising the following steps: the conditions of fermentation culture in the step 7) are as follows: the fermentation time is 28-35 deg.C, 60-84 hr, and turning over once every 12 hr.

Technical Field

The invention relates to the technical field of microbial engineering, and particularly relates to a preparation method of tea oil cake meal probiotic pig feed.

Background

The tea oil cake dregs are the cake dregs left after the tea oil is extracted from the camellia seeds, and belong to the processing byproducts of the tea oil industry. The tea oil cake contains 30-55% of polysaccharide, 10-15% of tea saponin, and part of crude protein, crude fiber, crude fat, amino acid and trace elements, so that the tea oil cake is fully and reasonably utilized, great economic benefit can be generated, and the development of the tea oil industry can be promoted.

The polysaccharide in the tea oil cake has certain activity, has the effects of resisting tumor, aging, oxidation and the like, and can be used as a biological reaction regulator to increase in-vivo and in-vitro immune response. The direct discarding of the crude protein and crude fiber in the tea oil cake is a waste of seeds, and in addition, the tea oil cake contains rich trace elements such as calcium, magnesium, iron and the like and can be used as raw materials of feeds, so that the functional feeds can be developed in an oriented manner.

The tea oil cake contains 10-15% of tea saponin, which is a pentacyclic triterpenoid saponin compound consisting of aglycone, sugar and organic acid. The tea saponin is a natural surfactant with good performance, and has antibacterial, antiinflammatory, alcohol inhibiting, snail killing, and hormone production stimulating effects. However, tea saponin can irritate nasal mucosa due to its pungent, spicy and bitter taste, contains antibiotic growth factors such as tannin and phytic acid, affects feed palatability, and excessive tea saponin has hemolytic effect, but a small amount of tea saponin feed additive can regulate blood pressure, tranquilize mind and enhance immunity. Researches show that the feed containing 1% of tea saponin can be directly used for animal production, not only does not influence the palatability of animals, but also can improve the quality of livestock and poultry products, improve the digestibility of the feed and enhance the immunity and disease resistance of animal organisms. Therefore, the degradation of tea saponin in the tea oil cake meal becomes a great difficulty in feed production.

CN201110231794.9 discloses a method for preparing an oilseed residue protein feed by adopting low-moisture solid state fermentation, which comprises the following steps: 1) preparation of a microbial inoculum: according to the reaction of sodium alginate solution and CaCl₂The volume ratio of the solution is = 1: 2-1: 10, the mixed solution (bacterial solution + sodium alginate solution) is injected into CaCl by a syringe₂In the solution, washing the solution for three times by using sterile normal saline after solidification to obtain a microcapsule microbial inoculum; 2) mixing and fermenting cake base materials: uniformly stirring and mixing the microcapsule microbial inoculum, the oilseed residue and water, and standing and fermenting at room temperature for 24-96 hours to obtain a fermentation product; 3) one of the following 2 methods is adopted: drying: after the fermentation is finished, drying the fermentation product to obtain the oil cake protein feed; secondly, directly vacuum packaging the fermentation product after fermentation to obtain the oil cake protein feed. When the method is used for preparing the feed, how to effectively degrade the tea saponin in the cake and the content of the tea saponin in the prepared feed are not involved, and the tea saponin in the prepared feed can influence the taste of the feed and is not beneficial to digestion and absorption of animals; in addition, in the preparation process of the feed, sodium alginate, calcium chloride and other reagents are required to be added to prepare the microcapsule microbial inoculum, so that the operation is complex, the feed preparation cost is improved, and the feed preparation efficiency is reduced.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: overcomes the defects of the prior art, and provides a preparation method of tea oil cake meal probiotic pig feed which has low preparation cost and can greatly reduce the content of tea saponin.

The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of tea oil cake meal probiotic pig feed comprises the following steps: 1) pretreating tea oil cake; 2) activating and carrying out amplification culture on aspergillus niger and bacillus subtilis to prepare aspergillus niger suspension and bacillus subtilis suspension; 3) preparing a solid fermentation culture medium containing tea oil cake; 4) mixed solid state fermentation: inoculating aspergillus niger and bacillus subtilis to a solid-state fermentation culture medium containing tea oil cake meal to ferment and degrade tea saponin to obtain fermented tea oil cake meal; 5) screening probiotics from pig manure, and preparing seed liquid and bacterial suspension of the probiotics; 6) preparing a probiotic solid culture medium containing fermented tea oil cake meal; 7) solid-state fermentation of probiotics: inoculating the screened probiotics to a probiotic solid culture medium containing fermented tea oil cake meal for fermentation culture; 8) and (5) carrying out vacuum freeze drying to obtain the probiotic pig feed.

Further, the specific operation steps of step 1) are as follows: drying and crushing the tea oil cake dregs, and degreasing the crushed dregs with petroleum ether for later use.

Further, the drying temperature is 65 ℃, the drying time is 24 hours, and the tea oil cake meal is crushed and then sieved by a 40-mesh sieve.

Further, the specific operation steps of the aspergillus niger amplification culture in the step 2) are as follows: inoculating the activated aspergillus niger strains into an aspergillus niger liquid seed culture medium, and carrying out constant-temperature shaking table culture for later use; the specific operation steps of the bacillus subtilis expanded culture are as follows: inoculating the activated bacillus subtilis strain into a bacillus subtilis liquid seed culture medium, and carrying out constant-temperature shaking table culture for later use.

Further, the Aspergillus niger liquid seed culture medium is composed of a potato culture medium (PDA), and the preparation steps are as follows: 200g of potato, 20g of glucose, 20g of agar and 1L of distilled water, and sterilizing at 121 ℃ for 30 min.

Further, the preparation method of the bacillus subtilis liquid seed culture medium comprises the following steps: 10g of peptone, 3g of beef extract, 5g of NaCl, 20g of agar, pH 7.0-7.2 and 1L of distilled water, and sterilizing at 121 ℃ for 30 min.

Further, when the Aspergillus niger in the step 2) is subjected to amplification culture, the culture temperature is 30 ℃, and the culture time is 3 d; when the bacillus subtilis is subjected to amplification culture, the culture temperature is 30 ℃, and the culture time is 2 d.

Further, the preparation steps of the aspergillus niger suspension and the bacillus subtilis suspension in the step 2) are as follows: after the amplification culture is finished, respectively centrifuging the bacterial liquid after the amplification culture of aspergillus niger and the bacterial liquid after the amplification culture of bacillus subtilis for 15min under the condition of 4000r/min, removing the supernatant, adding 10ml of 0.9% physiological saline, uniformly mixing by vortex oscillation, centrifuging the mixed liquid for 5min under the condition of 4000r/min, removing the supernatant, adding 0.9% physiological saline, repeatedly cleaning for 4 times, finally supplementing and uniformly mixing by the physiological saline, taking the physiological saline as a blank control, regulating the absorbance value of the bacterial liquid concentration by the physiological saline at the wavelength of 600nm, controlling the absorbance value at 0.8, and preparing aspergillus niger suspension and bacillus subtilis suspension.

Further, the specific operation steps of step 3) are as follows: 100g of tea oil cake meal pretreated in the step 1) and KH₂PO₄ 1g、MgSO₄·7H₂O 0.5g、（NH₄）₂SO₄1g, uniformly stirring, and sterilizing at 121 ℃ for 30min, wherein the water content of the tea oil cake is 40-60%.

Preferably, the tea oil cake has a water content of 50%.

Further, the specific operation steps of step 4) are as follows: the total inoculation amount of aspergillus niger and bacillus subtilis is 8-14%, and the mixing ratio of bacillus subtilis suspension to aspergillus niger suspension is 2:1-1:2 inoculating the mixture into a solid fermentation culture medium containing tea oil cake dregs for fermentation culture.

Preferably, the total inoculation amount of the aspergillus niger and the bacillus subtilis is 12%, and the mixing ratio of the bacillus subtilis suspension to the aspergillus niger suspension is 1: 1.

further, the conditions of the fermentation culture in the step 4) are as follows: the fermentation time is 3.5-5.5 days, the fermentation temperature is 28-35 ℃, and the fermentation is carried out once every 12 hours.

Preferably, the conditions of the fermentation culture in the step 4) are as follows: the fermentation time was 4 days and the fermentation temperature was 30 ℃.

Further, the probiotics screened in the step 5) are bacillus licheniformis CFC-11 and MR-1, and have antagonistic action on staphylococcus aureus, escherichia coli, salmonella enterica and enterobacter aerogenes.

Further, the specific operation steps of step 6) are as follows: sterilizing 20g of the fermented tea oil cake prepared in the step 4), 1-5g of glucose, 0.5-0.7g of ammonium sulfate, 0.15-0.25g of disodium hydrogen phosphate and 18ml of deionized water at the pH value of 7.0 for 20min at 121 ℃.

Preferably, the glucose is 3g, the ammonium sulfate is 0.5g, and the disodium hydrogen phosphate is 0.25 g.

Further, the specific operation steps of step 7) are as follows: the total inoculation amount of the Bacillus licheniformis CFC-11 and MR-1 is 3-4%, and the bacterial suspension mixing ratio of the Bacillus licheniformis CFC-11 and MR-1 is 1: 3-3: 1 inoculating the mixture into a probiotic solid culture medium containing fermented tea oil cake dregs for fermentation culture.

Preferably, the total inoculation amount of the bacillus licheniformis CFC-11 and MR-1 is 3.5 percent, and the mixing ratio of the bacterial suspensions of the bacillus licheniformis CFC-11 and MR-1 is 1: 1.

further, the conditions of the fermentation culture in the step 7) are as follows: the fermentation time is 28-35 deg.C, 60-84 hr, and turning over once every 12 hr.

Preferably, the conditions of the fermentation culture in the step 7) are as follows: the fermentation time is 30 ℃ and 70 h.

Further, the preparation steps of the bacterial suspension of the bacillus licheniformis CFC-11 and MR-1 are as follows: a) preparing a seed solution: respectively picking single colonies of the bacillus licheniformis CFC-11 and MR-1 in an NA liquid culture medium, and culturing at the temperature of 35 ℃, the rotating speed of 180r/min and the culturing time of 14 h; b) respectively centrifuging the bacillus licheniformis CFC-11 seed liquid and the bacillus licheniformis MR-1 seed liquid prepared in the step a) for 15min under the condition of 4000r/min, removing supernatant, adding 10ml of 0.9% physiological saline, uniformly mixing by vortex oscillation, centrifuging the mixed liquid for 5min under the condition of 4000r/min, removing supernatant, adding 0.9% physiological saline, repeatedly cleaning for 4 times, finally supplementing and uniformly mixing by physiological saline, taking the physiological saline as a blank control, regulating the absorbance value of the concentration of the bacterial liquid at the wavelength of 600nm by the physiological saline, and controlling the absorbance value at 0.8 to prepare bacterial suspensions of the bacillus CFC-11 and the bacillus licheniformis MR-1.

Further, the specific operation steps of step 8) are as follows: and (3) carrying out vacuum drying treatment on the fermentation product obtained after fermentation culture in the step 7) to obtain the probiotic feed.

The preparation method of the tea oil cake meal probiotic pig feed has the beneficial effects that:

(1) according to the invention, the tea saponin in the tea oil cake pulp can be greatly degraded by using the tea oil cake pulp after the aspergillus niger and the bacillus subtilis are subjected to solid state fermentation and degreasing, so that the content of the tea saponin is controlled within 0.5%, the tea saponin can be directly used for feed, the palatability of the pig feed is improved, the digestion and absorption of the feed are facilitated, and the growth performance of the pig is improved;

(2) the invention adopts the Bacillus licheniformis CFC-11 and MR-1 separated from the pig manure as probiotics to carry out solid state fermentation to obtain the fermented tea oil cake meal after the fermentation of Aspergillus niger and Bacillus subtilis, which has the function of bacteriostasis, can adjust the micro-ecological imbalance in a host body and keep the balance of microbial colonies; in the solid fermentation process of the probiotics, enzymes such as protease, cellulase and the like can be generated, macromolecular nutrient substances are decomposed into micromolecular nutrient substances, and the absorbability of the feed is improved;

(3) the preparation method disclosed by the invention is simple, strong in operability, economical and environment-friendly, effectively improves the utilization value of the tea oil cake and promotes the rapid development of the tea oil industry.

Drawings

FIG. 1 is a flow chart of a preparation process of the tea oil cake meal probiotic pig feed;

FIG. 2-is a graph showing the effect of different total inoculum sizes of Aspergillus niger and Bacillus subtilis on the rate of soap removal;

FIG. 3-is a graph showing the effect of different mixing ratios of Bacillus subtilis and Aspergillus niger on the soap removal rate;

FIG. 4-is a graph showing the effect of different total inoculum sizes of Bacillus licheniformis CFC-11 and MR-1 on probiotic bacteria;

FIG. 5-Effect of different mixing ratios of Bacillus licheniformis CFC-11 and MR-1 on probiotic bacteria;

FIG. 6 shows the effect of different contents of fermented tea seed cake on probiotics in solid culture medium of probiotics.

Detailed Description

The invention is further illustrated with reference to the following figures and examples, which are not intended to limit the scope of the invention in any way.

Example 1

A preparation method of tea oil cake meal probiotic pig feed comprises the following steps:

1) tea oil cake meal pretreatment: drying the tea seed cake at 65 ℃ for 24h, crushing and sieving by a 40-mesh sieve, and degreasing by using petroleum ether for later use;

2) and (3) amplification culture of aspergillus niger: inoculating the activated aspergillus niger strains into a 250ml triangular flask filled with 100ml of aspergillus niger liquid seed culture medium, and carrying out shake culture at the constant temperature of 30 ℃ for 3 d; the Aspergillus niger liquid seed culture medium comprises a potato culture medium (PDA) and is prepared by the following steps: 200g of potato, 20g of glucose, 20g of agar and 1L of distilled water, and sterilizing at 121 ℃ for 30 min;

3) b, expanding culture of the bacillus subtilis: inoculating the activated bacillus subtilis strain into a 250ml triangular flask filled with 100ml bacillus subtilis liquid seed culture medium, and performing shake culture at the constant temperature of 30 ℃ for 2 d; the preparation method of the bacillus subtilis liquid seed culture medium comprises the following steps: 10g of peptone, 3g of beef extract, 5g of NaCl, 20g of agar, pH 7.0-7.2 and 1L of distilled water, and sterilizing at 121 ℃ for 30 min;

4) preparation of aspergillus niger suspension: after the amplification culture of the aspergillus niger is finished, centrifuging the bacterial liquid for 15min under the condition of 4000r/min, removing supernatant, adding 10ml of 0.9% physiological saline, uniformly mixing by vortex oscillation, centrifuging the mixed liquid for 5min under the condition of 4000r/min, removing supernatant, adding 0.9% physiological saline, repeatedly cleaning for 4 times, finally supplementing and uniformly mixing with physiological saline, taking the physiological saline as a blank control, regulating the absorbance value of the concentration of the bacterial liquid at the wavelength of 600nm by using the physiological saline, and controlling the absorbance value at 0.8 to prepare aspergillus niger suspension;

5) b, preparing a bacillus subtilis suspension: after the bacillus subtilis is subjected to enlarged culture, centrifuging the bacterial liquid for 15min under the condition of 4000r/min, removing supernatant, adding 10ml of 0.9% physiological saline, uniformly mixing by vortex oscillation, centrifuging the mixed liquid for 5min under the condition of 4000r/min, removing supernatant, adding 0.9% physiological saline, repeatedly cleaning for 4 times, finally supplementing and uniformly mixing with physiological saline, taking the physiological saline as a blank control, regulating the absorbance value of the concentration of the bacterial liquid at the wavelength of 600nm by using the physiological saline, and controlling the absorbance value at 0.8 to prepare bacillus subtilis suspension; (ii) a

6) Preparing a solid fermentation culture medium containing tea oil cake meal: 100g of tea oil cake meal pretreated in the step 1) and KH₂PO₄ 1g、MgSO₄·7H₂O 0.5g、（NH₄）₂SO₄1g, sterilizing at 121 ℃ for 30min after uniformly stirring, and controlling the water content of the tea oil cake to be 50%;

7) mixed solid state fermentation: taking the total inoculation amount of aspergillus niger and bacillus subtilis as 12%, wherein the mixing ratio of aspergillus niger suspension to bacillus subtilis suspension is 1: 1: inoculating the mixture to a solid state fermentation culture medium containing tea oil cake dregs, fermenting for 4d at the fermentation temperature of 30 ℃, and turning over once every 12h to obtain fermented tea oil cake dregs;

8) screening probiotics in pig manure: bacillus licheniformis CFC-11 and MR-1 are separated from pig manure and have antagonistic action on staphylococcus aureus, escherichia coli, salmonella enterica and enterobacter aerogenes;

9) preparation of seed solutions of Bacillus licheniformis CFC-11 and MR-1: respectively selecting single colonies of the bacillus licheniformis CFC-11 and MR-1, and putting the single colonies into a NA liquid culture medium containing 100ml, wherein the culture temperature is 35 ℃, the rotation speed is 180r/min, and the culture time is 14 h; the preparation steps of the NA liquid culture medium are as follows: 3g of beef extract, 10g of peptone, 5g of sodium chloride, 2g of agar and 1L of deionized water, adjusting the pH value to 6.8-7.0, and sterilizing at 121 ℃ for 20 min;

10) preparation of bacterial suspension of Bacillus licheniformis CFC-11 and MR-1: respectively centrifuging the bacillus licheniformis CFC-11 seed liquid and the bacillus licheniformis MR-1 seed liquid prepared in the step 9) for 15min under the condition of 4000r/min, removing supernatant, adding 10ml of 0.9% physiological saline, uniformly mixing by vortex oscillation, centrifuging the mixed liquid for 5min under the condition of 4000r/min, removing supernatant, adding 0.9% physiological saline, repeatedly cleaning for 4 times, finally supplementing and uniformly mixing by using the physiological saline, using the physiological saline as a blank control, regulating the absorbance value of the concentration of the bacteria liquid at the position of 600nm of wavelength by using the physiological saline, controlling the absorbance value at 0.8, and preparing the bacteria suspension of the bacillus licheniformis CFC-11 and the bacillus licheniformis MR-1;

11) preparing a probiotic solid culture medium containing fermented tea oil cake meal: 20g of fermented tea oil cake meal, 3g of glucose, 0.5g of ammonium sulfate, 0.25g of disodium hydrogen phosphate and 18ml of deionized water, wherein the pH value is 7.0, and the fermented tea oil cake meal is sterilized at 121 ℃ for 20 min;

12) solid-state fermentation of probiotics: the total inoculation amount of the Bacillus licheniformis CFC-11 and MR-1 is 3.5 percent, and the bacterial suspension mixing ratio of the Bacillus licheniformis CFC-11 and MR-1 is 1:1 inoculating the mixture into a probiotic solid culture medium containing fermented tea oil cake meal for fermentation culture, wherein the culture conditions are as follows: 1:1 of bacterial suspension, 3.5 percent of inoculation amount, 30 ℃ of culture temperature and 70 hours of culture time; during the research on the degradation of tea saponin in the oil tea cake, the screening of probiotics for feed and the optimization of fermentation conditions, the bacillus licheniformis CFC-11 and MR-1 are obtained by separating and identifying fresh piglet manure, and the biological characteristics of the bacillus licheniformis CFC-11 and MR-1 are subjected to relevant experiments, so that the bacillus licheniformis CFC-1 and the bacillus licheniformis CFC-1 both have certain tolerance to NaCl and bile salt, can produce protease and cellulase and have certain tolerance to high temperature;

13) vacuum freeze drying: and (3) drying the fermented product obtained after the fermentation culture in the step 12) in vacuum to obtain the probiotic pig feed.

The flow chart of the pig feed preparation process in this example 1 is shown in fig. 1, and in this example 1, the content of tea saponin in the raw material tea oil cake meal is 13.8%, the content of tea saponin in the pig feed prepared by the present invention is 0.49%, and the number of bacillus licheniformis is 2.0 × 10⁹CFU/g shows that the method for preparing the pig feed can effectively reduce the content of the tea saponin, so that the content of the tea saponin in the pig feed is kept at a lower level, and the prepared pig feed contains a large amount of probiotics (bacillus licheniformis) which can effectively promote the digestion and absorption of the pig feed and improve the growth performance of pigs.

Comparative example 1

This comparative example differs from example 1 in that: and 8) -12), namely, aspergillus niger and bacillus subtilis are adopted to carry out mixed solid state fermentation on the tea oil cake meal, and then the fermentation product is directly subjected to vacuum freeze drying to obtain the pig feed, and the content of tea saponin, the content of probiotics and the influence of the tea saponin content and the probiotics content on growth indexes such as the growth performance and the survival rate of piglets in the feed are researched, and the results are shown in table 1.

Comparative example 2

This comparative example differs from example 1 in that: the steps 2) -5) and 7) are not included, namely the bacillus licheniformis CFC-11 and MR-1 are adopted to carry out probiotic solid state fermentation on the tea oil cake meal, the fermentation product is directly subjected to vacuum freeze drying to obtain the pig feed, the tea saponin content, the probiotic content and the influence of the tea saponin content and the probiotic content on the growth indexes such as the growth performance, the survival rate and the like of the piglets in the feed are researched, and the results are shown in the table 1.

Comparative example 3

This comparative example differs from example 1 in that: only Aspergillus niger is adopted to carry out single solid state fermentation on tea oil cake pulp, then Bacillus licheniformis CFC-11 and MR-1 are adopted to carry out probiotic solid state fermentation on the fermented tea oil cake pulp fermented by the Aspergillus niger, and the tea saponin content, the probiotic content and the influence of the probiotic content on the growth indexes such as the growth performance, the survival rate and the like of piglets contained in the feed are researched, and the results are shown in Table 1.

Comparative example 4

This comparative example differs from example 1 in that: the tea oil cake pulp is subjected to single solid-state fermentation only by using bacillus subtilis, then the fermented tea oil cake pulp fermented by the bacillus subtilis is subjected to probiotic solid-state fermentation by using bacillus licheniformis CFC-11 and MR-1, and the tea saponin content, the probiotic content and the influence of the probiotic content on growth indexes such as the growth performance, the survival rate and the like of piglets contained in the feed are researched, and the results are shown in Table 1.

TABLE 1 feed in example 1 and comparative examples 1-4 contains tea saponin content, probiotic content and its effect on piglet growth index

As can be seen from the above table, the average body weight of the piglets fed by the feed prepared by the method of example 1 is higher than that of the piglets fed by the feeds prepared by the comparative examples 1 and 2, and the weight gain per day is obviously less than that of the feed prepared by the comparative example 1 although the daily feed intake is not obviously different from that of the feed prepared by the example 1, because the tea saponin content of the feed prepared by the comparative example 2 is increased, the palatability is poor, and the soaping is a necessary process step; the probiotic content of comparative example 1 is significantly less than that of example 1 and the piglets of comparative example 1 gained weight less than example 1, indicating that the probiotic feed of the invention contributes to the growth of piglets. Therefore, the invention highlights the advantages that the tea oil seed meal fermented by the aspergillus niger and the bacillus subtilis is used as the solid culture medium, and the bacillus licheniformis is used for preparing the pig feed. Compared with the example 1, the daily feed intake, daily average weight gain, probiotics and tea saponin content of the piglets of the comparative examples 3 and 4 are all lower than that of the piglet of the example 1, especially the tea saponin content is obviously lower than that of the piglet of the example 1, the parts 3 and 4 adopt the aspergillus niger and the bacillus subtilis to ferment the tea oil seed meal to prepare the pig feed, and the part 1 adopts the aspergillus niger and the bacillus subtilis to ferment the tea oil seed meal in a mixed way to prepare the tea oil seed meal, which shows that the pig feed prepared by fermenting the mixed strain of the aspergillus niger and the bacillus subtilis can obviously improve the growth performance of the piglets compared with the pig feed prepared by fermenting the tea oil seed meal by adopting any one of the aspergillus niger and the bacillus subtilis.

Experimental example 1

The experimental example mainly researches the enzyme production effect of the bacillus licheniformis, and comprises the following steps:

1) inoculating the bacterial suspension of the bacillus licheniformis into a culture medium which produces protease and cellulase, wherein the fermentation conditions are as follows:

the ratio of CFC-11 to MR-1 of the bacillus licheniformis to the total culture medium is 1:1, the inoculation amount is 2 percent, the fermentation temperature is 35 ℃, the rotation speed is 180r/min, and the culture time is 48 hours.

2) Protease-producing culture medium: 5g of peptone, 7g of yeast extract, 5g of sodium chloride and 1L of deionized water, and sterilizing at the temperature of 7.0-7.2,121 ℃ for 20 min.

3) A cellulase production culture medium: 10g of bran, 10g of peptone, 5g of sodium chloride, 5g of yeast extract powder and 1L of deionized water, and sterilizing at 121 ℃ for 20 min.

4) Determination of protease: and (4) centrifuging the product after the fermentation is finished in a refrigerated centrifuge of 5000r/min for 8min, wherein the supernatant is the required crude enzyme liquid. The activity of the protease was determined by the Folin-phenol method.

5) Determination of cellulase: and (4) centrifuging the product after the fermentation is finished in a refrigerated centrifuge of 5000r/min for 8min, wherein the supernatant is the required crude enzyme liquid. The activity of the cellulase is determined by using a DNS reagent method.

The experimental result shows that when the strain is under the condition, the protease activity can reach 63.2U/mL, and the cellulase activity is 18U/mL.

Experimental example 2

The experimental case mainly researches the components of the probiotic solid culture medium, and comprises the following steps:

a. determination of optimal carbon source: the bacterial suspension (the absorbance value is 0.8) of the CFC-11 and MR-1 of the bacillus licheniformis is uniformly shaken every 12h at the fermentation temperature of 35 ℃ for 48h according to the inoculation amount of 2 percent, the ratio of the bacterial liquid to the bacterial liquid is 1:1, 2g of glucose, sucrose, starch, maltose and glycerol are respectively added, and the number of spores after fermentation of culture media with different carbon sources is researched. From the experimental results, it was found that when glucose was used as the carbon source, the influence on the number of spores was the greatest, and was 5.8 x 10⁸CFU/g。

b. Determination of optimal nitrogen source: the bacterial suspension (the absorbance value is 0.8) of the CFC-11 and MR-1 of the bacillus licheniformis is uniformly shaken every 12h at the fermentation temperature of 35 ℃ for 48h according to the inoculation amount of 2 percent, the ratio of the bacterial liquid to the bacterial liquid is 1:1, 0.5g of peptone, yeast extract, corn flour, ammonium nitrate, ammonium sulfate and urea are respectively added during the fermentation for research on the number of spores after fermentation of different nitrogen source culture media is finished. From the experimental results, it can be found that the above 6 nitrogen sources can promote the generation of spores, but the promotion effect of ammonium sulfate is the largest, and can reach 6.2 x 10⁸CFU/g。

c. Determination of the optimal inorganic salts: the bacterial suspension (the absorbance value is 0.8) of the bacillus licheniformis CFC-11 and MR-1 is uniformly shaken every 12 hours at the fermentation temperature of 35 ℃ for 48 hours according to the inoculum concentration of 2 percent, the ratio of the bacterial liquid to the bacterial liquid is 1:1, 0.15g of calcium chloride, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and ferrous sulfate are respectively added, and the number of spores after fermentation of different inorganic salt culture media is researched. From the results of the experiments, it was found that when disodium hydrogenphosphate was added as an inorganic salt,the number of spores capable of promoting solid state fermentation reached the maximum value in the experiment, which was 5.1 x 10⁸CFU/g

The experiment shows that during mixed fermentation, the most suitable carbon source is glucose, the most suitable nitrogen source is ammonium sulfate, and the most suitable inorganic salt is disodium hydrogen phosphate.

Experimental example 3

The experimental example mainly researches the influence of the total inoculation amounts of different aspergillus niger and bacillus subtilis on the soap removing rate, wherein the soap removing rate = the content of tea saponin in raw tea oil cake/the content of tea saponin in pig feed multiplied by 100%, and comprises the following steps: the mixing ratio of the bacterial suspension is 1:1, the water content of the tea seed cake is 50%, the fermentation time is 4d, the fermentation temperature is 30 ℃, the tea seed cake is turned over once every 12h, and the influence of different total inoculum sizes of 4%, 6%, 8%, 10%, 12%, 14% and 16% on the soap removal rate is researched.

As can be seen from the experimental result figure 2, when the inoculation amount is small, the concentration of the strain is too low, so that the fermentation is incomplete, the content of tea saponin in the pig feed is high, and the soap removal rate is not ideal; when the inoculation amount is increased to 8%, the soap stripping rate can reach 92.68%, and then the soap stripping rate continuously rises along with the increase of the inoculation amount. When the inoculation amount reaches 14%, the soap removal rate shows a descending trend. Therefore, in the optimization test, the inoculation amount is optimal at 8% -14%.

Experimental example 4

The experimental example mainly researches the influence of the mixing ratio of the bacillus subtilis suspension and the aspergillus niger suspension on the soap removal rate, and comprises the following steps: the tea seed cake is stirred once every 12 hours at the fermentation temperature of 30 ℃ and the total inoculation amount of 50% of the water content of the tea seed cake, the fermentation time of 4d and the fermentation temperature of 30 ℃, and the influence of the mixing ratio of the bacillus subtilis suspension to the aspergillus niger suspension on the content of tea saponin in the tea oil cake is researched, wherein the mixing ratio of the bacillus subtilis suspension to the aspergillus niger suspension is 4:1, 3:1, 2:1, 1:2, 1:3 and 1: 4.

As can be seen from the experimental results of fig. 3, as the mixing ratio increases, the soap removal rate increases and then decreases, when the mixing ratio is 4:1, 3:1, the soap removal rate is less than 90%, when the mixing ratio is 2:1-1: the soap removing rate can reach 93-94% in case of 2, and 90.31% and 89.3% in case of 1:3 and 1:4 respectively. The synergistic soap removing effect between the two reaches the best state when the mixing ratio is 2:1-1:2, and the soap removing capability of the soap remover is stronger because the capability of growing and secreting enzyme systems is strongest.

Experimental example 5

The experimental example mainly researches the influence of the total inoculation amounts of different bacillus licheniformis CFC-11 and MR-1 on probiotics in pig feed, and comprises the following steps: the influence of the total inoculum size of 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0% on probiotics in pig feed is studied by mixing the suspension of Bacillus licheniformis CFC-11 and MR-1 at a mixing ratio of 1:1, at a culture temperature of 30 ℃, for a culture time of 70h, and turning over every 12 h.

As can be seen from FIG. 4, as the total inoculum size increased, the number of spores increased first and then decreased; when the inoculation amount is 3.5%, the number of spores reaches the maximum. According to the combination data, 3% -4% is selected as the optimal condition.

Experimental example 6

The experimental example mainly researches the influence of different bacillus licheniformis CFC-11 and MR-1 bacterial suspensions on probiotics in pig feed, and comprises the following steps:

the total inoculation amount of the Bacillus licheniformis CFC-11 and MR-1 is 3.5 percent, the culture temperature is 30 ℃, the culture time is 70 hours, and the Bacillus licheniformis CFC-11 and MR-1 are turned over once every 12 hours in the period, and the influence on the solid fermentation probiotic production when the different mixing ratios are 4:1, 3:1, 2:1, 1:2, 1:3 and 1:4 is researched.

As can be seen from FIG. 5, the spore number increases and then decreases with increasing ratio, and the ratio of CFC-11 and MR-1 suspensions of Bacillus licheniformis is selected as the experimental condition because the spore number is higher at 1: 3-3: 1.

Experimental example 7

The experimental example mainly researches the influence of the content of the fermented tea oil cake meal in the probiotic solid culture medium on the probiotics in the pig feed, and comprises the following steps: the probiotic solid culture medium comprises the following components: 3g of glucose, 0.5g of ammonium sulfate, 0.25g of disodium hydrogen phosphate and 18ml of deionized water, wherein the mold is cultured at the pH value of 7.0,121 ℃ for 20min, the inoculation amount of the mixed bacteria is 3.5 percent, and the ratio is 1: 1. The influence of different tea oil cake meal contents on probiotics in pig feed is researched. As can be seen from FIG. 6, the spore number was the greatest when the tea oil cake content was 20 g.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.