A composite microbial preparation, a method for fermenting oil tea dregs and application thereof

Abstract

The application provides a kind of composite microbial preparation, the method and application of fermented oil tea dregs, and belongs to the field of microbial technology.The composite microbial preparation is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus in the ratio of 1:1:1-2:1:1:1 according to the number of viable bacteria.The composite microbial preparation provided by the application can not only reduce the content of tea saponin in oil tea dregs, but also increase the content of protein and vitamin in oil tea dregs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a compound microbial preparation, a method for fermenting oil tea dregs and application thereof. BACKGROUND

[0002] Camellia oleifera is a plant of Theaceae, and the main product of Camellia oleifera is tea oil. Tea oil is a healthy plant oil with high nutritional value, and the unsaturated fatty acid accounts for 90.2%, and the saturated fatty acid accounts for only 9.3%, and it is also rich in fat-soluble vitamins A, E and K. The various functional components in tea oil have obvious effects on maintaining the function of cardiovascular system, improving human immunity, reducing cholesterol and preventing and treating hypertension.

[0003] At present, the deep processing and comprehensive utilization technology of oil tea dregs is insufficient, which restricts the rapid development of the deep processing industry of oil tea. Oil tea dregs, also known as tea cake, tea dregs and dry cake, is the cake left after the oil tea seeds are pressed to extract tea oil, and its yield is about three times that of tea oil. However, due to the bitter taste of oil tea dregs, the content of tea saponin and crude fiber and other anti-nutritional substances, at present, it is basically used as fertilizer, fuel, pond cleaning agent or even abandoned, which causes great waste of resources. Tea saponin has pungent and bitter taste, and has strong irritation to nasal mucosa, which affects the palatability of feed and livestock refuses to eat. Moreover, the content of toxic components contained therein is high, which has great influence on animals, and cannot be directly used as feed. In addition, it is particularly sensitive to the toxicity of fish, and especially cannot be directly used as fish feed. This seriously restricts the application of oil tea cake in the feed industry.

[0004] Tea saponin can destroy animal red blood cells, produce hemolysis phenomenon, but it only produces hemolysis on red blood cells, and has no effect on white blood cells. The premise of hemolysis of tea saponin is that it must act on blood. It has been found that the daily methane production of lambs is reduced by 27.7% when 3g / d of tea saponin is added to the feed of the lambs; the addition of tea saponin can significantly reduce the methane production and the gene expression of mcrA in rumen fermentation, and will not adversely affect the function of rumen. Therefore, the addition of an appropriate amount of tea saponin in the feed of ruminants can inhibit the production of methane, reduce the harm of methane to the ecological environment, and enhance the production performance of animals. The addition of 500mg / kg of tea saponin in the diet of broilers can significantly increase the average daily gain of broilers, significantly reduce the feed to weight ratio and the drip loss of chicken meat, and significantly improve the pH value of chicken meat. This proves that a small amount of tea saponin can promote the growth of animals. In terms of taste, tannin tastes bitter and astringent, has astringency, affects the taste of feed, and reduces the feed intake of animals. Therefore, the oil tea cake meal needs to be detoxified before being used as feed to feed animals. Most of the oil tea cake fermentation on the market needs to use mold, which is easy to produce toxic metabolites-fungal toxins during fermentation, and needs to be treated by removing saponin before fermentation, which has many operation steps and consumes a lot of energy, resulting in the limited use of oil tea cake for fermentation, and currently there are few studies on the production of poultry and livestock feed from oil tea cake. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a composite microbial preparation which can be used for fermenting oil tea cake, can effectively reduce the content of tea saponin and cellulose in the oil tea cake, and can increase the content of protein and vitamins in the oil tea cake, so that the oil tea cake can be used as an excellent protein resource for feed.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0007] The composite microbial preparation is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus in a ratio of 1:1:1-2:1:1:1 in terms of the number of viable bacteria.

[0008] Preferably, the composite microbial preparation is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus in a ratio of 1:1:1:1:1:1 in terms of the number of viable bacteria.

[0009] Another purpose of the present application is to provide the use of the composite microbial preparation in the fermentation of oil tea cake.

[0010] Preferably, the composite microbial preparation can reduce the content of tea saponin and cellulose in the oil tea cake.

[0011] Another object of the present application is to provide a method for fermenting oil tea cake using the composite microbial preparation, comprising the following steps: adding cellulase to the crushed oil tea cake to obtain oil tea cake hydrolysate; adding molasses to the oil tea cake hydrolysate, and then inoculating the composite microbial preparation to ferment.

[0012] Preferably, the crushed oil tea cake is sieved through a 20-50 mesh sieve.

[0013] Preferably, the amount of cellulase added is 10-2000 U / g of oil tea cake, the amount of the composite microbial preparation inoculated is 0.05%-1% of the mass of the oil tea cake hydrolysate, and the amount of molasses added is 5%-30% of the mass of the oil tea cake hydrolysate.

[0014] Preferably, the temperature for enzyme hydrolysis is 37-60℃, and the time for enzyme hydrolysis is 2-24 h.

[0015] Preferably, the temperature for fermentation is 28-40℃, and the time for fermentation is 2-10 d.

[0016] Another object of the present application is to provide an oil tea cake feed prepared by the method for fermenting oil tea cake using the composite microbial preparation.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a composite microbial preparation. The composite microbial preparation provided by the present application can ferment oil tea cake, reduce the content of tea saponin and crude fiber in the oil tea cake, increase the content of protein and vitamins in the oil tea cake, cause little loss to the fermented material, require low equipment, and does not need to add any reagent during the microbial fermentation process. The fermentation product can be directly used without subsequent treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 For single-strain fermentation, A is the raw material; B is Lactococcus fermentation; C is Lactobacillus acidophilus fermentation; D is Bacillus subtilis fermentation; E is Bacillus subtilis fermentation; F is Saccharomyces cerevisiae CICC 33068 fermentation; G is Lactobacillus plantarum fermentation; H is Bacillus coagulans fermentation; and I is Saccharomyces cerevisiae CICC 32236 fermentation.

[0020] Figure 2 The tea saponin content in the tea cake after single-strain fermentation accounts for

[0021] Figure 3 The crude fiber content in the tea cake after single-strain fermentation accounts for

[0022] Figure 4The fermentation effects of test example 2 are as follows: A is only adding cellulase; B is only adding lactobacillus plantarum; C is adding cellulase first and then adding lactobacillus plantarum after 8 hours; D is processing oil tea dregs by the method of example 3; and G is processing oil tea dregs by the method of example 4.

[0023] Figure 5 The crude fiber contents of oil tea dregs after fermentation of each group of test example 2 are as follows: ***p<0.001.

[0024] Figure 6 The tea saponin contents of oil tea dregs after fermentation of each group of test example 2 are as follows: ***p<0.001.

[0025] Figure 7 The crude protein contents of oil tea dregs after fermentation of each group of test example 2 are as follows: ***p<0.001.

[0026] Figure 8 The acid-soluble protein contents of oil tea dregs after fermentation of each group of test example 2 are as follows: ***p<0.001.

[0027] Figure 9 The pH of oil tea dregs after fermentation of each group of test example 2 is as follows.

[0028] Figure 10 The crude fiber contents of oil tea dregs after fermentation of different combinations of strains in test example 3 are as follows.

[0029] Figure 11 The fermentation flowchart of group E in test example 3 for 7 days is as follows. DETAILED DESCRIPTION

[0030] The present application provides a composite microbial preparation, which is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, lactobacillus plantarum, bacillus subtilis, bacillus coagulans and lactobacillus acidophilus in a ratio of 1:1:1 to 2:1:1:1 in terms of the number of viable bacteria, and preferably in a ratio of 1:1:1:1:1:1 in terms of the number of viable bacteria.

[0031] Saccharomyces cerevisiae CICC 33068 in the present application is a strain disclosed in patent CN 106614572A and has been preserved in China Industrial Microbial Culture Collection Center (CICC) (address: No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological preservation number is CICC 33068.

[0032] Saccharomyces cerevisiae CICC 32236 is the strain disclosed in the literature "Preparation of microbial protein feed from corn bran by multi-strain solid-state fermentation" (Gao J, Yan YP, Li YL, et al. Journal of Biology, 2023, 40(03): 91-95.), and has been preserved in the China Industrial Microbial Culture Collection Center (CICC) (Address: No. 6, Building 6, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological preservation number is CICC 32236.

[0033] Lactobacillus plantarum is the strain disclosed in the literature "Construction of walnut protein / tea polyphenol / alginate complex for enhancing heat and gastrointestinal tolerance of lactic acid bacteria" (Li Y, Liu G, Liao N, et al. Food Hydrocolloids, 2024, 149: 109523.), and has been preserved in the China Center for Type Culture Collection (Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), and its biological preservation number is CCTCC AB 206133.

[0034] Bacillus coagulans is the strain disclosed in the patent CN113511936A, and has been preserved in the China Industrial Microbial Culture Collection Center (Address: No. 6, Building 6, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological preservation number is CICC 23843 。

[0035] Bacillus subtilis is the strain disclosed in the literature "Physiological and biochemical characteristics and detection and identification of Bacillus atrophaeus" (Ma D, Gong HY, Li MM, et al. Anhui Journal of Agricultural Sciences, 2014, 20(07): 33-35.), and has been preserved in the China Industrial Microbial Culture Collection Center (Address: No. 6, Building 6, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological preservation number is CICC 10275.

[0036] Lactobacillus acidophilus is the strain disclosed in the literature "Effect of fermentation by Lactobacillus acidophilus CH-2 on the enzymatic browning of pear juice" (Li X, Gao J, Simal-Gandara J, et al. Lwt, 2021, 147: 111489.), and has been preserved in the China Industrial Microbial Culture Collection Center (Address: No. 6, Building 6, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological preservation number is CICC 22162.

[0037] In the present application, the strains in the complex microbial preparation can be obtained through ordinary commercial channels, and the viable bacterial count in the complex microbial preparation is 1×10 10 ~ 1×10 12 CFU / g.

[0038] The present application also provides the application of the complex microbial preparation in fermented oil tea dregs. The complex microbial preparation provided by the present application can effectively reduce the content of tea saponin and cellulose in oil tea dregs and also can increase the content of protein and vitamin in oil tea dregs during the fermentation process of oil tea dregs.

[0039] The present application also provides a method for fermenting oil tea dregs by using the complex microbial preparation, which comprises the following steps: crushing oil tea dregs and adding cellulase to obtain oil tea dregs hydrolysate; adding molasses to the oil tea dregs hydrolysate and then inoculating the complex microbial preparation for fermentation.

[0040] In the method for fermenting oil tea dregs, the oil tea dregs are preferably crushed and then sieved through a 20-50 mesh sieve, and more preferably crushed and then sieved through a 30-40 mesh sieve; the cellulase is added in an amount of 10-2000 U / g of oil tea dregs, preferably 30-500 U / g of oil tea dregs, and more preferably 50-100 U / g of oil tea dregs; the molasses is added in an amount of 5%-30% of the mass of the oil tea dregs hydrolysate, preferably 10%-20%; and the complex microbial preparation is inoculated in an amount of 0.05%-1% of the mass of the oil tea dregs hydrolysate, preferably 0.1%-0.5%, wherein the inoculated mass of the complex microbial preparation refers to the dry weight of the microbial cells.

[0041] In the present application, the enzymolysis is performed under the following conditions: the enzymolysis temperature is 37-60℃, preferably 45-55℃, and the enzymolysis time is 2-24h, preferably 8-12h.

[0042] In the present application, after inoculating the complex microbial preparation, the water content of the fermentation system is adjusted and then fermentation is performed, preferably the water content is adjusted to 10%-60%, and more preferably the water content is adjusted to 30%-50%; and the fermentation is performed under the following conditions: the fermentation temperature is 28-40℃, preferably 32-37℃, and the fermentation time is 2-10d, preferably 3-7d.

[0043] The present application also provides an oil tea dregs feed prepared by the method for fermenting oil tea dregs by using the complex microbial preparation. The oil tea dregs feed provided by the present application has obvious fermentation aroma, the oil tea dregs becomes sour after fermentation, which greatly improves the palatability, the content of tea saponin and cellulose in the oil tea dregs feed is low, the content of protein and vitamin is high, and a new idea is provided for the subsequent utilization of oil tea dregs.

[0044] In the specific embodiments of the present application, the inoculum is activated in advance, wherein the Saccharomyces strain is cultured in YPD medium (yeast powder: 10 g / L, peptone: 2 g / L, glucose: 2 g / L) at 28 degrees in a shaker at 220 rpm for 2 days; the Bacillus strain is cultured in basic Bacillus medium (glucose: 10.0 g / L, calcium phosphate: 5.0 g / L, ammonium sulfate: 0.5 g / L, potassium chloride: 0.2 g / L, magnesium sulfate heptahydrate: 0.1 g / L, manganese sulfate: 0.0001 g / L, ferrous sulfate: 0.0001 g / L, yeast extract: 0.5 g / L) at 37 degrees in a shaker at 220 rpm for 2 days; the Lactobacillus strain is cultured in MRS medium (peptone: 10.0 g / L, beef extract powder: 5.0 g / L, yeast extract: 4.0 g / L, glucose: 20.0 g / L, Tween-80: 1.0 mL / L, potassium phosphate dibasic: 2.0 g / L, sodium acetate: 5.0 g / L, triammonium citrate: 2.0 g / L, magnesium sulfate heptahydrate: 0.2 g / L, manganese sulfate tetrahydrate: 0.05 g / L) at 37 degrees in a shaker at 220 rpm for 2 days.

[0045] In the specific embodiments of the present application, the oil tea cake is from Guangdong Baohua Agricultural Technology Co., Ltd.

[0046] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0047] Example 1

[0048] A composite microbial preparation, which is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus in a ratio of 1:1:1:1:1:1 by the number of viable bacteria.

[0049] Example 2

[0050] A composite microbial preparation, which is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus preferably in a ratio of 1:1:2:1:1:1 by the number of viable bacteria.

[0051] Example 3

[0052] A method for fermenting oil tea cake by using the composite microbial preparation, the steps are as follows:

[0053] Camellia meal was crushed and passed through a 40-mesh sieve, cellulase was added at a dosage of 50 U / g of Camellia meal, and enzymolysis was performed at 50°C for 8 h. After the enzymolysis was completed, Camellia meal enzymolysis products were obtained. Molasses was added to the Camellia meal enzymolysis products, the mass of the molasses being 20% of the mass of the Camellia meal enzymolysis products, then the composite microbial preparation in Example 1 was inoculated, the mass of the composite microbial preparation being 0.1% of the mass of the Camellia meal enzymolysis products (the number of viable bacteria being 1×10 10 ~ 1×10 12 CFU / g), the water content of the fermentation system was adjusted to 50%, and fermentation was performed at 37°C for 3 d.

[0054] Example 4

[0055] A method for fermenting Camellia meal using the composite microbial preparation, the difference between this embodiment and Example 3 being that fermentation was performed for 7 d.

[0056] Example 5

[0057] A method for fermenting Camellia meal using the composite microbial preparation, the difference between this embodiment and Example 3 being that the composite microbial preparation in Example 2 was inoculated for fermentation.

[0058] Example 6

[0059] A method for fermenting Camellia meal using the composite microbial preparation, the difference between this embodiment and Example 5 being that fermentation was performed for 7 d.

[0060] Example 7

[0061] A method for fermenting Camellia meal using the composite microbial preparation, the steps being as follows:

[0062] Camellia meal was crushed and passed through a 50-mesh sieve, cellulase was added at a dosage of 200 U / g of Camellia meal, and enzymolysis was performed at 40°C for 12 h. After the enzymolysis was completed, Camellia meal enzymolysis products were obtained. Molasses was added to the Camellia meal enzymolysis products, the mass of the molasses being 10% of the mass of the Camellia meal enzymolysis products, then the composite microbial preparation in Example 1 was inoculated, the mass of the composite microbial preparation being 0.2% of the mass of the Camellia meal enzymolysis products (the number of viable bacteria being 1×10 10 ~ 1×10 12 CFU / g), the water content of the fermentation system was adjusted to 30%, and fermentation was performed at 30°C for 7 d.

[0063] Example 8

[0064] A method for fermenting Camellia meal using the composite microbial preparation, the steps being as follows:

[0065] Camellia oleifera meal was pulverized and passed through a 50-mesh sieve. Cellulase was added at a rate of 500 U / g of camellia oleifera meal, and enzymatic hydrolysis was carried out at 45°C for 5 hours. After hydrolysis, camellia oleifera meal hydrolysate was obtained. Molasses was added to the camellia oleifera meal hydrolysate at a mass of 25% of the hydrolysate mass. Then, the compound microbial preparation from Example 2 was inoculated at a mass of 0.6% of the camellia oleifera meal hydrolysate mass (viable count of 1×10⁻⁶). 10 ~1×10 12 The water content of the fermentation system was adjusted to 40% (CFU / g) and then fermented at 35℃ for 3 days.

[0066] Comparative Example 1

[0067] A method for fermenting camellia seed cake using the aforementioned compound microbial preparation differs from Example 3 in that the inoculated compound microbial preparation consists of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, and Lactobacillus plantarum in a live bacteria ratio of 1:1:1.

[0068] Comparative Example 2

[0069] A method for fermenting camellia seed cake using the aforementioned compound microbial preparation differs from Example 3 in that the inoculated compound microbial preparation is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, and Bacillus subtilis in a live cell ratio of 1:1:1:1.

[0070] Comparative Example 3

[0071] A method for fermenting camellia seed cake using the aforementioned compound microbial preparation differs from Example 3 in that the inoculated compound microbial preparation is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, and Bacillus coagulans in a live cell ratio of 1:1:1:1:1.

[0072] Experimental Example 1

[0073] 1. The effect of single-strain fermentation of camellia seed meal.

[0074] The following bacterial strains were used for fermentation of camellia seed cake:

[0075] A: The camellia seed cake was crushed and passed through a 40-mesh sieve, but no inoculum was inoculated.

[0076] B: After crushing the camellia seed cake, pass it through a 40-mesh sieve, add molasses at a rate of 10% (w / w), inoculate with Lactococcus lactis (CICC6246) (100 billion CFU / g) at a rate of 0.1% (w / w), adjust the moisture content to 50%, and ferment at 37℃ for 3 days.

[0077] C: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Lactobacillus acidophilus (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d;

[0078] D: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Bacillus subtilis (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d;

[0079] E: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Bacillus subtilis (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d;

[0080] F: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Saccharomyces cerevisiae CICC33068 (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d; recorded as Saccharomyces cerevisiae 1;

[0081] G: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Lactobacillus plantarum (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d;

[0082] H: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Bacillus coagulans (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d;

[0083] I: Camellia oleifera cake was crushed and passed through a 40-mesh sieve, added molasses, the addition amount was 10% (w / w), inoculated with Saccharomyces cerevisiae CICC32236 (viable bacteria number 100 billion CFU / g), the inoculation amount was 0.1% (w / w), adjusted the moisture content to 50%, fermented at 37℃ for 3d; recorded as Saccharomyces cerevisiae 2.

[0084] Each treatment was repeated 3 times, and the contents of tea saponin and crude fiber in the fermented products were detected after fermentation, and the average value of 3 repeated tests was taken.

[0085] 1.1, tea saponin detection method

[0086] The content of tea saponin was determined by vanillin sulfuric acid method. The tea saponin standard was accurately weighed, and 1 mg / mL standard solution was prepared with 80% ethanol solution. 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL and 10 mL of the standard solution were respectively taken into a test tube with a stopper, and 0.5 mL of 8% vanillin ethanol solution and 4 mL of 77% sulfuric acid solution were added to make up to 0.5 mL under ice water bath. After mixing, the mixture was reacted at 60°C water bath for 20 min, and then cooled in ice water bath for 10 min. After taking out for 10 min, the absorbance was measured at 545 nm, and the standard curve of tea saponin was drawn.

[0087] 0.1 g of fermented and dried tea cake sample was weighed into a 10 mL centrifuge tube, 5 mL of 80% ethanol was added, and the mixture was shaken for 30 min and centrifuged at 4000 rpm for 10 min. 0.5 mL of supernatant was taken, 0.5 mL of 8% vanillin ethanol solution was added under ice water bath, and 4 mL of 77% sulfuric acid solution was added. After mixing, the mixture was reacted at 60°C water bath for 20 min, and then cooled in ice water bath for 10 min. After taking out for 10 min, the absorbance was measured at 545 nm.

[0088] 1.2, Crude fiber detection method

[0089] The content of crude fiber in feed was determined according to GB / T 6434-2006.

[0090] 1.3, The results are shown in Figures 1-3 .

[0091] As shown in Figure 1 , the raw material is a light yellow powder with a strong traditional Chinese medicine and lignin flavor. After fermentation, the color deepens, there is a fermented sour taste, the PH decreases, and the palatability of tea cake is improved. After 3 days of fermentation, the contents of crude fiber and tea saponin in fermented tea cake were detected, respectively. As shown in Figure 2 , after 3 days of fermentation, each strain can degrade part of tea saponin, among which Lactobacillus plantarum and Saccharomyces cerevisiae 1 have the best tea saponin degradation ability, as shown in Figure 3 , Bacillus coagulans and Lactobacillus plantarum have certain crude fiber degradation ability.

[0092] 2, Fermentation effect of Lactobacillus plantarum

[0093] Orthogonal analysis method (L164 ^ 4) was used to explore the optimal conditions for Lactobacillus plantarum to degrade crude fiber. After fermentation, the final crude fiber content of tea cake was detected. The test conditions and detection results are shown in Tables 1 and 2.

[0094] Table 1 Lactobacillus plantarum L16 orthogonal experiment table

[0095]

[0096] Table 2 Comprehensive score range analysis of L. plantarum fermented oil tea cake

[0097] Inoculum Fermentation time h Temperature °C Molasses concentration % K1 1.2451 1.3244 1.2962 1.3851 K2 1.3023 1.2760 1.3792 1.3675 K3 1.3011 1.2722 1.2172 1.2872 K4 1.3717 1.3475 1.3275 1.1803 k1 0.3113 0.3311 0.3241 0.3463 k2 0.3256 0.3190 0.3448 0.3419 k3 0.3253 0.3181 0.3043 0.3218 k4 0.3429 0.3369 0.3319 0.2951 R 0.0317 0.0188 0.0405 0.0512

[0098] R analysis results: molasses concentration > temperature > inoculum > fermentation time, the optimal fermentation conditions of L. plantarum are: inoculum 0.1%, fermentation time 48 h, temperature 37℃, and molasses concentration 20%.

[0099] 3. Enzymolysis effect of cellulase.

[0100] L16 orthogonal analysis method (L164 ^ 4) Optimal conditions for cellulase to degrade crude fiber, the oil tea cake was crushed and sieved, indicating that the oil tea cake powder was sieved through a 40 mesh sieve, and the unsieved indicates that the oil tea cake powder was directly subjected to enzymolysis, and the final crude fiber content of the tea cake was detected after the enzymolysis, and the test conditions and detection results are shown in Tables 3 and 4.

[0101] Table 3 L16 orthogonal experiment table of cellulase

[0102]

[0103]

[0104] Table 4 Comprehensive score range analysis of cellulase degradation of crude fiber in oil tea cake

[0105] State of raw material Concentration U / g Temperature °C Reaction time h K1 2.514 1.525 1.487 1.559 K2 3.637 1.553 1.560 1.435 K3 0.000 1.534 1.501 1.534 K4 0.000 1.539 1.604 1.622 k1 0.629 0.381 0.372 0.390 k2 0.909 0.388 0.390 0.359 k3 0.000 0.383 0.375 0.384 k4 0.000 0.385 0.401 0.406 R 0.281 0.007 0.026 0.047

[0106] R analysis results: raw material state > reaction time > temperature > enzyme concentration, the optimal conditions for cellulase to perform enzymolysis are: raw material state: sieved through a 40 mesh sieve; enzyme concentration: 50 U / g; temperature: 50℃; and reaction time: 8 h.

[0107] According to the above results, through the degradation of L. plantarum or cellulase, the crude fiber content in the tea cake is reduced to below 30%, but the nutritional ingredients in the tea cake are insufficient.

[0108] Test Example 2

[0109] Fermentation effect of single strain and complex microbial agent.

[0110] The oil tea cake was treated by the following methods:

[0111] A: compared with Example 3, only cellulose was added for enzymolysis, and no complex microbial preparation was inoculated;

[0112] B: compared with Example 3, no cellulose was added for enzymolysis, and only L. plantarum was inoculated;

[0113] C: Compared with Example 3, inoculate Lactobacillus plantarum for fermentation;

[0114] D: Take the method of Example 3 for processing;

[0115] G: Take the method of Example 4 for processing.

[0116] After the fermentation is completed, the tea saponin, crude fiber content, crude protein and acid-soluble protein content of the fermentation product are detected by the method described in Test Example 1, and the detection method of pH is as follows.

[0117] 1. Crude protein determination

[0118] Determined by Kjeldahl nitrogen determination, according to GB / T 5009.5-2003.

[0119] 2. Acid-soluble protein determination

[0120] The acid-soluble protein detection method refers to NYT3801-2020, and two tests are performed in parallel. 0.1 g of sample is weighed to 1 mg, and placed in a 10 mL stoppered flask. Under the condition of 10℃-35℃, 5 mL of trichloroacetic acid solution is accurately added. After shaking, oscillate at 2500 r / min for 30 min. Centrifuge: centrifuge at 4000 r / min for 10 min, and the supernatant is the sample extract. If there is floating material in the upper solution, it needs to be filtered. The reagent extract is detected by Kjeldahl nitrogen determination.

[0121] 3. pH detection

[0122] 0.1 g of sample is taken, and 5 mL of distilled water is added after shaking to detect the pH of the suspension.

[0123] The results are shown in Figures 4-9 .

[0124] As shown in Figure 4 , after fermentation, the color deepens, and there is a sour taste after fermentation. As shown in Figure 5 , Figure 6 , compared with the unfermented raw materials, the crude fiber content of the fermented materials is less than 30%, among which the crude fiber content of the single Lactobacillus plantarum strain fermentation of group B and the prolonged fermentation of 7 days of group G is about 25%, and the tea saponin content of groups A, C and G is reduced to below 10%. The crude protein and acid-soluble protein content is shown in Figure 7 and Figure 8 , after 7 days of multi-strain combined fermentation, the crude protein content increases significantly, from 6%-8% of the raw material to 15%-18%, and the acid-soluble protein content increases from about 1% of the raw material to 2%-3%. As shown in Figure 9 , the pH of the fermentation product provided by the method of the present application decreases significantly.

[0125] The method provided by this invention ferments camellia seed meal, which increases the protein content of the camellia seed meal and lowers the pH of the tea seed meal to about 3.5, thereby improving the palatability of the feed. The increased content of acid-soluble protein also indicates an increase in the content of small peptides and free amino acids in the tea seed meal, thus enhancing the nutrient absorption efficiency of the tea seed meal.

[0126] Experimental Example 3

[0127] Fermentation effects of single strains and compound inoculants.

[0128] The following methods were used to process camellia seed cake:

[0129] Group A (3 days): The same method as in Comparative Ratio 1 was used for treatment;

[0130] Group A (7 days): Fermented for 7 days using the method of Comparative Example 1;

[0131] Group B, 3 days: The same method as in Comparative Ratio 2 was used for treatment;

[0132] Group B (7 days): Fermented for 7 days using the method of Comparative Example 2;

[0133] Group C (3 days): The treatment was carried out using the method described in Example 5;

[0134] Group C (7 days): The treatment was carried out using the method described in Example 6;

[0135] Group D (3 days): The treatment method of Comparative Ratio 3 was adopted;

[0136] Group D (7 days): Fermented for 7 days using the method of Comparative Example 3;

[0137] Group E, 3 days: The treatment was carried out using the method described in Example 3;

[0138] Group E (7 days): The treatment was carried out using the method described in Example 4.

[0139] After fermentation, the crude fiber content of the fermentation product was determined using the method described in Example 1. The results are as follows: Figures 10-11 As shown.

[0140] like Figure 10As shown, the crude fiber content in tea meal after 3 and 7 days of fermentation for each group of compound microbial strains was compared. There was no significant difference in crude fiber content after different fermentation times for each group of compound microbial agents. The method provided by this invention results in even lower crude fiber content in the tea meal. The various probiotics and cellulase enzymes in the compound microbial preparation of this invention synergistically ferment, significantly reducing tea saponin content, achieving a crude protein content of approximately 18%, and decreasing crude fiber to 20% without prior tea meal desapification treatment. This solves the problem of requiring additional desapification treatment before tea meal fermentation, reduces energy consumption during fermentation, and provides a new approach for the subsequent utilization of tea meal.

[0141] Figure 11 The following is a 7-day fermentation flowchart for Group E in this embodiment. After being crushed, the camellia oil meal raw material has obvious fibrous residue and obvious particles. After being treated with cellulase, the fibrous content of the camellia oil meal is reduced. After fermentation for 7 days with compound microbial preparation, the color of the camellia oil meal powder is darker and it has a distinct wine aroma.

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of compound microbial preparations in fermented camellia seed meal, characterized in that, The compound microbial preparation is composed of Saccharomyces cerevisiae CICC 33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus in a live cell ratio of 1:1:1 to 2:1:1:

1. The biopreservation number of *Lactobacillus plantarum* is CCTCAB 206133; the biopreservation number of *Bacillus subtilis* is CICC 10275; the biopreservation number of *Bacillus coagulans* is CICC 23843; and the biopreservation number of *Lactobacillus acidophilus* is CICC 22162.

2. The application according to claim 1, characterized in that, The compound microbial preparation is composed of Saccharomyces cerevisiae CICC33068, Saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans and Lactobacillus acidophilus in a ratio of 1:1:1:1:1:

1.

3. The application according to claim 1, characterized in that, The compound microbial preparation reduces the saponin and cellulose content of camellia oil meal.

4. A method for fermenting camellia seed cake, characterized in that, Includes the following steps: Camellia oleifera meal is crushed and then enzymatically hydrolyzed with cellulase to obtain camellia oleifera meal hydrolysate; molasses is added to the camellia oleifera meal hydrolysate, and then the compound microbial preparation described in claim 1 or 2 is inoculated for fermentation.

5. The method according to claim 4, characterized in that, The camellia oil meal was crushed and passed through a 20-50 mesh sieve.

6. The method according to claim 4, characterized in that, The amount of cellulase added is 10~2000U / g camellia seed cake, the mass of the compound microbial preparation inoculated is 0.05%~1% of the enzymatic hydrolysate of camellia seed cake, and the mass of molasses added is 5%~30% of the enzymatic hydrolysate of camellia seed cake.

7. The method according to claim 4, characterized in that, The enzymatic hydrolysis temperature is 37~60℃, and the enzymatic hydrolysis time is 2~24h.

8. The method according to claim 4, characterized in that, The fermentation temperature is 28~40℃, and the fermentation time is 2~10 days.

9. A camellia seed meal feed, characterized in that, It is prepared by the method described in any one of claims 4 to 8.

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