Compound microbial preparation, method for fermenting oil tea meal and application of compound microbial preparation

Abstract

The invention provides a compound microbial preparation, a method for fermenting oil-tea meal and application of the compound microbial preparation, and belongs to the technical field of microorganisms. The compound microbial preparation is prepared from saccharomyces cerevisiae CICC 33068, saccharomyces cerevisiae CICC 32236, lactobacillus plantarum, bacillus subtilis, bacillus coagulans and lactobacillus acidophilus according to the viable count ratio of 1: 1: 1 to 2: 1: 1: 1. The compound microbial preparation provided by the invention is used for fermenting the oil-tea meal, so that the content of tea saponin in the oil-tea meal can be reduced, and the contents of protein and vitamins in the oil-tea meal can also be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a composite microbial preparation, a method for fermenting tea oil cake and applications thereof. Background Art

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

[0003] At present, the deep processing and comprehensive utilization technology of camellia oleifera cake is insufficient, which restricts the rapid development of the deep processing industry of camellia oleifera cake. Camellia oleifera cake, also known as tea cake, tea cake, and dried cake, is the cake left after the tea oil is extracted from the camellia oleifera seeds. Its output is about three times that of tea oil. However, because the camellia oleifera cake tastes bitter and contains anti-nutritional substances such as tea saponin and crude fiber, it is currently basically used as a fertilizer, fuel, pond cleaner, or even abandoned, resulting in a huge waste of resources. Tea saponin tastes spicy and bitter, has a strong irritation to the nasal mucosa, affects the palatability of feed, and livestock refuse to eat. Moreover, the toxic components contained in it are relatively high, which has a greater impact on animals and cannot be used directly as feed. In addition, it is particularly sensitive to toxic effects on fish, especially cannot be used directly as fish feed. This seriously restricts the application of camellia oleifera cake in the feed industry.

[0004] Tea saponin can destroy animal red blood cells and produce hemolysis, but it only produces hemolysis on red blood cells and has no effect on white blood cells. The premise for tea saponin to have a hemolytic effect is that it must react with blood. Studies have found that adding 3g / d of tea saponin to lamb feed can reduce the daily methane production of lambs by 27.7%; adding tea saponin can significantly reduce the production of methane and the gene expression of mcrA in rumen fermentation, and will not have an adverse effect on rumen function. Therefore, adding an appropriate amount of tea saponin to ruminant feed can inhibit the production of methane, reduce the harm of methane to the ecological environment, and enhance the production performance of animals. Adding 500mg / kg of tea saponin to broiler diets significantly increases the average daily weight gain of broilers, significantly reduces the feed-to-weight ratio and drip loss of chicken, and significantly increases the pH value of chicken. This proves that a small amount of tea saponin has a certain promoting effect on animal growth. In terms of taste, tannin tastes bitter and astringent, which affects the taste of feed and reduces the feed intake of animals. Therefore, the tea-oil cake needs to be detoxified before it can be used as feed for animals. Most of the tea-oil cake fermentation on the market requires the use of mold, which is easy to produce toxic metabolites - mycotoxins during the fermentation process, and it needs to be desoaped before fermentation. The operation steps are many and energy-intensive, which limits the fermentation use of tea-oil cake. At present, there are few studies on the production of poultry and livestock feed from tea-oil cake. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a composite microbial preparation that can be used for fermenting camellia oleifera meal, which can effectively reduce the saponin content and cellulose content of the camellia oleifera meal, and increase the protein and vitamin content in the camellia oleifera meal, thereby making the camellia oleifera meal an excellent feed protein resource.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A composite microbial preparation is prepared by using saccharomyces cerevisiae CICC 33068, saccharomyces cerevisiae CICC 32236, lactobacillus plantarum, bacillus subtilis, bacillus coagulans and lactobacillus acidophilus in a live bacteria quantity ratio of 1:1:1 to 2:1:1:1.

[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 live bacteria count ratio of 1:1:1:1:1:1:1.

[0009] Another object of the present invention is to provide the use of the composite microbial preparation in fermenting tea oil meal.

[0010] Preferably, the composite microbial preparation reduces the saponin content and cellulose content of tea oil seed cake.

[0011] Another object of the present invention is to provide a method for fermenting tea oil meal using the composite microbial preparation, comprising the following steps: adding cellulase to the tea oil meal after crushing the tea oil meal for enzymolysis to obtain tea oil meal hydrolysate; adding molasses to the tea oil meal hydrolysate, and then inoculating the composite microbial preparation for fermentation.

[0012] Preferably, the tea oil meal is crushed and passed through a 20-50 mesh sieve.

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

[0014] Preferably, the enzymatic hydrolysis temperature is 37-60° C., and the enzymatic hydrolysis time is 2-24 h.

[0015] Preferably, the fermentation temperature is 28-40° C., and the fermentation time is 2-10 days.

[0016] Another object of the present invention is to provide a tea-leaf meal feed, which is prepared by the method of fermenting tea-leaf meal using a composite microbial preparation.

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

[0018] The present invention provides a composite microbial preparation. The composite microbial preparation provided by the present invention ferments tea oil cake, which can not only reduce the content of tea saponin and crude fiber in tea oil cake, but also increase the content of protein and vitamin in tea oil cake. The loss of fermented substances during the fermentation process is small, the equipment requirements are low, and no reagents need to be added during the microbial fermentation process, and the fermentation product can be directly used without subsequent treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a single strain fermentation, wherein 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; I is Saccharomyces cerevisiae CICC 32236 fermentation;

[0020] Figure 2 The percentage of tea saponin content in tea cake after fermentation by a single strain;

[0021] Figure 3 The percentage of crude fiber content in tea cake after fermentation with a single strain, **p<0.01;

[0022] Figure 4This is the fermentation effect of Experiment 2, wherein A is the addition of cellulase only; B is the addition of Lactobacillus plantarum only; C is the addition of cellulase for 8 hours before adding Lactobacillus plantarum for fermentation; D is the treatment of camellia oleifera meal by the method of Example 3; G is the treatment of camellia oleifera meal by the method of Example 4;

[0023] Figure 5 The crude fiber content of tea oil meal in each group after fermentation in Experimental Example 2, ***p<0.001;

[0024] Figure 6 The tea saponin content in tea oil cake after fermentation in each group of Experimental Example 2, ***p<0.001;

[0025] Figure 7 The crude protein content in tea oil meal after fermentation in each group of Experimental Example 2, ***p<0.001;

[0026] Figure 8 The acid-soluble protein content in tea oil meal after fermentation in each group of Experimental Example 2, ***p<0.001;

[0027] Fig. 9 The pH of tea oil meal after fermentation in each group of Experimental Example 2;

[0028] Fig.10 The crude fiber content after fermentation of different combinations of multiple strains in Experimental Example 3;

[0029] Fig.11 This is the fermentation flow chart of Group E in Experimental Example 3 for 7 days. DETAILED DESCRIPTION

[0030] The invention provides a composite microbial preparation. The composite microbial preparation is composed of saccharomyces cerevisiae CICC33068, saccharomyces cerevisiae CICC 32236, lactobacillus plantarum, bacillus subtilis, bacillus coagulans and lactobacillus acidophilus in a live bacteria ratio of 1:1:1 to 2:1:1:1; preferably, the live bacteria ratio is 1:1:1:1:1:1:1.

[0031] The brewer's yeast CICC 33068 in the present invention is a strain disclosed in patent CN 106614572A, and has been deposited in the China Industrial Microbiological Culture Collection Center (CICC) (address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological deposit number is CICC 33068.

[0032] Saccharomyces cerevisiae CICC 32236 is a strain disclosed in the document "Preparation of bacterial protein feed by solid-state fermentation of corn husks with multiple strains" (Gao Junjie, Yan Yaping, Li Yongli, et al. Journal of Biology, 2023, 40(03): 91-95.), and has been deposited in the China Industrial Microbiological Culture Collection Center (CICC) (address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological deposit number is CICC 32236.

[0033] Lactobacillus plantarum is a strain disclosed in the document "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 deposited 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 CCTCCAB 206133.

[0034] Bacillus coagulans is a strain disclosed in patent CN113511936A and has been deposited in the China Industrial Microbiological Culture Collection Center (address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological deposit number is CICC23843 。

[0035] Bacillus subtilis is a strain disclosed in the document "Physiological and biochemical characteristics and detection and identification of Bacillus atrophaeus" (Ma Dinghong, Gong Haiyan, Li Mengmeng, et al. Anhui Agricultural Science Bulletin, 2014, 20(07): 33-35.), and has been deposited in the China Industrial Microbiological Culture Collection Center (address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological deposit number is CICC 10275.

[0036] The strain of Lactobacillus acidophilus disclosed in the literature "Effect offermentation 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.) has been deposited in the China Industrial Microbiological Culture Collection Center (address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing), and its biological deposit number is CICC 22162.

[0037] In the present invention, the strains in the composite microbial preparation can be obtained through common commercial channels, and the number of viable bacteria in the composite microbial preparation is 1×10 10 ~1×10 12 CFU / g.

[0038] The present invention also provides the use of the composite microbial preparation in the fermentation of tea oil cake. The composite microbial preparation provided by the present invention can effectively reduce the content of tea saponin and cellulose in tea oil cake during the tea oil cake fermentation process, and can also increase the content of protein and vitamins in tea oil cake.

[0039] The present invention also provides a method for fermenting camellia oleifera meal using the composite microbial preparation, comprising the following steps: adding cellulase to crush the camellia oleifera meal for enzymolysis to obtain camellia oleifera meal hydrolysate; adding molasses to the camellia oleifera meal hydrolysate, and then inoculating the composite microbial preparation for fermentation.

[0040] In the method for fermenting camellia oleifera meal of the present invention, the camellia oleifera meal is preferably sieved through a 20-50 mesh sieve after being crushed, and more preferably sieved through a 30-40 mesh sieve after being crushed; the amount of cellulase added is 10-2000U / g camellia oleifera meal, preferably 30-500U / g camellia oleifera meal, and more preferably 50-100U / g camellia oleifera meal; the mass of molasses added is 5%-30% of the mass of the camellia oleifera meal enzymatic hydrolysate, and the preferred amount is 10%-20%; the mass of the composite microbial preparation inoculated is 0.05%-1% of the mass of the camellia oleifera meal enzymatic hydrolysate, and the preferred inoculation amount is 0.1%-0.5%. The mass of the composite microecological preparation inoculated in the present invention refers to the dry weight of the microbial cells.

[0041] In the present invention, the conditions for enzymolysis are: the enzymolysis temperature is 37 to 60° C., preferably 45 to 55° C., and the enzymolysis time is 2 to 24 hours, preferably 8 to 12 hours.

[0042] In the present invention, after inoculating the composite microbial preparation, the water content of the fermentation system is adjusted before fermentation, preferably the water content is adjusted to 10% to 60%, more preferably the water content is adjusted to 30% to 50%; the fermentation conditions are: the fermentation temperature is 28 to 40°C, preferably 32 to 37°C, and the fermentation time is 2 to 10 days, preferably 3 to 7 days.

[0043] The present invention also provides a tea-seed meal feed, which is prepared by the method of fermenting tea-seed meal with the composite microbial preparation. The tea-seed meal feed provided by the present invention has an obvious fermentation aroma, and the tea-seed meal becomes sour after fermentation, which greatly improves the palatability. In addition, the tea saponin and cellulose content in the tea-seed meal feed is low, and the protein and vitamin content is high, which provides a new idea for the subsequent utilization of tea-seed meal.

[0044] In a specific embodiment of the present invention, the bacterial agent is activated before inoculation, wherein the yeast strain uses YPD medium (yeast powder: 10 g / L, peptone: 2 g / L, glucose: 2 g / L) and is cultured in a 28 degree shaker at 220 rpm for 2 days; the Bacillus strain uses 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), cultured in a 37-degree shaker at 220 rpm for 2 days; Lactobacillus strains used 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 dihydrogen phosphate: 2.0 g / L, sodium acetate: 5.0 g / L, ammonium citrate: 2.0 g / L, magnesium sulfate heptahydrate: 0.2 g / L, manganese sulfate tetrahydrate: 0.05 g / L), cultured in a 37-degree shaker at 220 rpm for 2 days.

[0045] In a specific embodiment of the present invention, the tea seed meal comes from Guangdong Baohua Agricultural Science and Technology Co., Ltd.

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

[0047] Example 1

[0048] A composite microbial preparation is prepared by using saccharomyces cerevisiae CICC 33068, saccharomyces cerevisiae CICC 32236, lactobacillus plantarum, bacillus subtilis, bacillus coagulans and lactobacillus acidophilus in a live bacteria ratio of 1:1:1:1:1:1:1.

[0049] Example 2

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

[0051] Example 3

[0052] A method for fermenting tea oil meal using the composite microbial preparation comprises the following steps:

[0053] The tea-seed meal was crushed and passed through a 40-mesh sieve. Cellulase was added in an amount of 50 U / g tea-seed meal. Enzymolysis was performed at 50° C. for 8 h. After the enzymolysis was completed, tea-seed meal hydrolysate was obtained. Molasses was added to the tea-seed meal hydrolysate. The mass of the molasses added was 20% of the mass of the tea-seed meal hydrolysate. Then, the composite microbial preparation in Example 1 was inoculated. The mass of the composite microbial preparation inoculated was 0.1% of the mass of the tea-seed meal hydrolysate (the number of viable bacteria was 1×10 10 ~1×10 12 CFU / g), and the water content of the fermentation system was adjusted to 50% and then fermented at 37°C for 3 days.

[0054] Example 4

[0055] A method for fermenting tea oil cake using the composite microbial preparation. The difference between this embodiment and embodiment 3 is that the fermentation is performed for 7 days.

[0056] Example 5

[0057] A method for fermenting tea oil cake using the composite microbial preparation. The difference between this embodiment and embodiment 3 is that the composite microbial preparation in embodiment 2 is inoculated for fermentation.

[0058] Example 6

[0059] A method for fermenting tea oil cake using the composite microbial preparation. The difference between this embodiment and embodiment 5 is that the fermentation is performed for 7 days.

[0060] Example 7

[0061] A method for fermenting tea oil meal using the composite microbial preparation comprises the following steps:

[0062] The tea-seed meal was crushed and passed through a 50-mesh sieve. Cellulase was added in an amount of 200 U / g tea-seed meal. Enzymolysis was performed at 40° C. for 12 h. After the enzymolysis was completed, tea-seed meal hydrolysate was obtained. Molasses was added to the tea-seed meal hydrolysate. The mass of the molasses added was 10% of the mass of the tea-seed meal hydrolysate. Then, the composite microbial preparation in Example 1 was inoculated. The mass of the composite microbial preparation inoculated was 0.2% of the mass of the tea-seed meal hydrolysate (the number of viable bacteria was 1×10 10 ~1×10 12 CFU / g), and the water content of the fermentation system was adjusted to 30% and then fermented at 30°C for 7 days.

[0063] Example 8

[0064] A method for fermenting tea oil meal using the composite microbial preparation comprises the following steps:

[0065] The tea-seed meal was crushed and passed through a 50-mesh sieve. Cellulase was added in an amount of 500 U / g tea-seed meal. Enzymolysis was performed at 45° C. for 5 h. After the enzymolysis was completed, tea-seed meal hydrolysate was obtained. Molasses was added to the tea-seed meal hydrolysate. The mass of the molasses added was 25% of the mass of the tea-seed meal hydrolysate. Then, the composite microbial preparation in Example 2 was inoculated. The mass of the composite microbial preparation inoculated was 0.6% of the mass of the tea-seed meal hydrolysate (the number of viable bacteria was 1×10 10 ~1×10 12 CFU / g), and the water content of the fermentation system was adjusted to 40% and then fermented at 35°C for 3 days.

[0066] Comparative Example 1

[0067] A method for fermenting tea oil cake using the composite microbial preparation is different from Example 3 in that the inoculated composite microbial preparation is composed 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 tea oil cake using the composite microbial preparation, which differs from Example 3 in that the inoculated composite microbial preparation is composed of saccharomyces cerevisiae CICC 33068, saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum and Bacillus subtilis in a live bacteria ratio of 1:1:1:1.

[0070] Comparative Example 3

[0071] A method for fermenting tea oil cake using the composite microbial preparation, which differs from Example 3 in that: the inoculated composite microbial preparation is composed of saccharomyces cerevisiae CICC 33068, saccharomyces cerevisiae CICC 32236, Lactobacillus plantarum, Bacillus subtilis and Bacillus coagulans in a live bacteria ratio of 1:1:1:1:1.

[0072] Test Example 1

[0073] 1. The effect of single strain fermentation on tea oil meal.

[0074] The following strains were used for tea seed meal fermentation:

[0075] A: Camellia oleifera meal was crushed and passed through a 40-mesh sieve without inoculation of bacteria;

[0076] B: After the camellia oleifera meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and lactococci (CICC6246) (viable count 100 billion CFU / g) were inoculated at a rate of 0.1% (w / w), the moisture content was adjusted to 50%, and fermented at 37°C for 3 days;

[0077] C: After the camellia oleifera meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and Lactobacillus acidophilus (live bacteria count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w), the moisture content was adjusted to 50%, and fermented at 37°C for 3 days;

[0078] D: After the camellia oleifera meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and Bacillus subtilis (viable count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w), the moisture content was adjusted to 50%, and fermented at 37°C for 3 days;

[0079] E: After the camellia oleifera meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and Bacillus subtilis (viable count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w), the moisture content was adjusted to 50%, and fermented at 37°C for 3 days;

[0080] F: After the tea seed meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and Saccharomyces cerevisiae CICC33068 (viable count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w). The water content was adjusted to 50%, and fermented at 37°C for 3 days; recorded as Saccharomyces cerevisiae 1;

[0081] G: After the camellia oleifera meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and Lactobacillus plantarum (viable count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w). The moisture content was adjusted to 50%, and fermented at 37°C for 3 days;

[0082] H: After the camellia oleifera meal was crushed and passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and Bacillus coagulans (viable count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w), the moisture content was adjusted to 50%, and fermented at 37°C for 3 days;

[0083] I: After the camellia oleifera meal was crushed, it was passed through a 40-mesh sieve, molasses was added at a rate of 10% (w / w), and saccharomyces cerevisiae CICC32236 (viable count 100 billion CFU / g) was inoculated at a rate of 0.1% (w / w). The moisture content was adjusted to 50%, and fermented at 37°C for 3 days; recorded as saccharomyces cerevisiae 2.

[0084] Each treatment was repeated three times. After fermentation, the tea saponin and crude fiber content of the fermentation product were detected, and the result was taken as the average of the three repeated tests.

[0085] 1.1. Tea saponin detection method

[0086] The tea saponin content was determined by the vanillin sulfuric acid method. Accurately weigh the tea saponin standard, prepare a 1 mg / mL standard solution with 80% ethanol solution, pipette 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL into 10 mL stoppered test tubes, and add 80% ethanol to 0.5 mL, add 0.5 mL of 8% vanillin ethanol solution in an ice water bath, then add 4 mL of 77% sulfuric acid solution, mix well, react in a 60°C water bath for 20 minutes, then cool in an ice water bath for 10 minutes, take out 10 minutes later, measure the absorbance at 545 nm, and draw a standard curve for tea saponin.

[0087] Weigh 0.1g of fermented and dried tea meal sample into a 10mL centrifuge tube, add 5mL of 80% ethanol, shake for 30min, centrifuge at 4000rpm for 10min, take 0.5mL of supernatant, add .5mL of 8% vanillin ethanol solution in an ice-water bath, then add 4mL of 77% sulfuric acid solution, mix well, react in a 60℃ water bath for 20min, cool in an ice-water bath for 10min, take out 10min later, and measure the absorbance at 545nm.

[0088] 1.2. Crude fiber detection method

[0089] The method for determining crude fiber content in feed shall refer to GB / T 6434-2006.

[0090] 1.3. The results are as follows Figure 1 to Figure 3 shown.

[0091] like Figure 1 As shown in the figure, the raw material is a light yellow powder with a strong taste of Chinese patent medicine and lignin. After fermentation, the color deepens and there is a sour taste after fermentation. The pH drops, which improves the palatability of the tea meal. After 3 days of fermentation, the crude fiber and tea saponin content in the fermented tea meal are tested. Figure 2 As shown in the figure, after 3 days of fermentation, each strain can degrade some tea saponins, among which Lactobacillus plantarum and Saccharomyces cerevisiae 1 have the best tea saponin degradation ability. Figure 3 The Bacillus coagulans and Lactobacillus plantarum strains shown have certain crude fiber degradation capabilities.

[0092] 2. The fermentation effect of Lactobacillus plantarum.

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

[0094] Table 1 Orthogonal experiment table of Lactobacillus plantarum L16

[0095]

[0096] Table 2 Range analysis of comprehensive scores of tea oil meal fermented by Lactobacillus plantarum

[0097] Inoculation volume Fermentation time Temperature ℃ 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 > inoculation amount > fermentation time. The optimal fermentation conditions for Lactobacillus plantarum are: inoculation amount 0.1%, fermentation time 48h, temperature 37℃, and added molasses concentration 20%.

[0099] 3. Enzymatic hydrolysis effect of cellulase.

[0100] Orthogonal analysis (L164 ^ 4) To explore the optimal conditions for cellulase degradation of crude fiber, the tea seed meal was crushed. Screening means that the tea seed meal powder was passed through a 40-mesh sieve, and not screening means that the tea seed meal powder was directly enzymatically hydrolyzed. After the enzymatic hydrolysis, the final crude fiber content of the tea seed meal was detected. The experimental conditions and test results are shown in Tables 3 and 4.

[0101] Table 3 Orthogonal experiment table of cellulase L16

[0102]

[0103]

[0104] Table 4 Analysis of the range of comprehensive scores of crude fiber in tea oil meal degraded by cellulase

[0105] Raw material status Concentration U / g Temperature ℃ Reaction time 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 > added enzyme concentration. The optimal conditions for cellulase hydrolysis are: raw material state: passed through a 40-mesh sieve; added enzyme concentration: 50U / g; temperature 50℃; reaction time: 8h.

[0107] According to the above results, the crude fiber content in tea meal was reduced to below 30% by degradation by Lactobacillus plantarum or cellulase, but the nutritional components in tea meal were insufficient.

[0108] Test Example 2

[0109] Fermentation effect of single strain and composite bacterial agents.

[0110] The following methods are used to process tea seed meal:

[0111] A: Compared with Example 3, only cellulose was added for enzymatic hydrolysis, and the composite microbial preparation was not inoculated;

[0112] B: Compared with Example 3, no cellulose was added for enzymatic hydrolysis, and only Lactobacillus plantarum was inoculated;

[0113] C: Compared with Example 3, fermentation was carried out by inoculating Lactobacillus plantarum;

[0114] D: The method of Example 3 is adopted for treatment;

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

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

[0117] 1. Crude protein determination

[0118] Determination by Kjeldahl method, refer 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. Weigh 0.1g of the sample, accurate to 1mg, and place it in a 10mL, stoppered conical flask. At 10℃~35℃, accurately add 5mL of trichloroacetic acid solution. After shaking, oscillate at 2500r / min for 30min. Centrifuge: Centrifuge at 4000r / min for 10min, and the supernatant is the sample extract. If there are floating objects in the upper solution, it needs to be filtered. The reagent extract uses the Kjeldahl method to detect the nitrogen content in the solution.

[0121] 3. pH detection

[0122] Take 0.1 g of sample, accurate to 1 mg, add 5 mL of distilled water, and shake to test the pH of the suspension.

[0123] The results are as follows Figures 4 to 9 shown.

[0124] like Figure 4 As shown, after fermentation, the color deepens and there is a sour taste after fermentation. Figure 5 , Figure 6 As shown in the figure, compared with the unfermented raw materials, the crude fiber content after fermentation was less than 30%, among which the crude fiber content of group B fermented with a single plant lactobacillus strain and group G fermented for 7 days was about 25%, and the tea saponin content of groups A, C and G dropped to less than 10%. Figure 7 and Figure 8 As shown in the figure, after 7 days of multi-strain combined fermentation, the crude protein content increased significantly, from 6% to 8% of the raw material to 15% to 18%, and the acid-soluble protein content increased from about 1% of the raw material to 2% to 3%. Fig. 9 As shown, the pH after fermentation according to the method provided by the present invention decreases significantly.

[0125] The method provided by the present invention ferments tea oil meal, thereby increasing the protein content in the tea oil meal and reducing the pH of the tea meal to about 3.5, thereby improving the palatability of the feed; the increase in the content of acid-soluble protein also indicates an increase in the content of small peptides and free amino acids in the tea meal, thereby enhancing the nutrient absorption efficiency of the tea meal.

[0126] Test Example 3

[0127] Fermentation effect of single strain and composite bacterial agents.

[0128] The following methods are used to process tea seed meal:

[0129] Group A: 3 days: treated with the method of comparative example 1;

[0130] Group A: 7 days: adopt the method of comparative example 1 and ferment for 7 days;

[0131] Group B: 3 days: treated with the method of comparative example 2;

[0132] Group B: 7 days: adopt the method of comparative example 2 and ferment for 7 days;

[0133] Group C: 3 days: treated with the method of Example 5;

[0134] Group C: 7 days: treated with the method of Example 6;

[0135] Group D: 3 days: treated with the method of comparative example 3;

[0136] Group D: 7 days: adopt the method of comparative example 3 and ferment for 7 days;

[0137] Group E: 3 days: treated with the method of Example 3;

[0138] Group E: 7 days: treated using the method of Example 4.

[0139] After the fermentation was completed, the crude fiber content of the fermentation product was detected by the method described in Experimental Example 1. Figure 10-11 shown.

[0140] like Fig.10As shown, the crude fiber content in the tea meal after 3 and 7 days of fermentation of each group of composite strains was compared, and there was no significant difference in the crude fiber content after different fermentation times of each group of composite microbial agents. The crude fiber content of the oil tea meal was lower after treatment with the method provided by the present invention. The various probiotics in the composite microbial preparation of the present invention synergistically fermented with the cellulase enzyme, and without the previous desoap treatment of the tea meal, the tea saponin content was significantly reduced, the crude protein content reached about 18%, and the crude fiber was reduced to 20%. The problem of the need for additional desoap treatment before the fermentation of the tea meal was solved, which reduced the energy consumption during the fermentation process and provided a new idea for the subsequent utilization of the tea meal.

[0141] Fig.11 This is the fermentation flow chart of Group E of this embodiment for 7 days. After the camellia oleifera meal raw material is crushed, there are obvious fibrous filaments and obvious particles. After being treated with cellulase, the fibrous nature of the camellia oleifera meal is reduced. After being inoculated with the composite microbial preparation and fermented for 7 days, the color of the camellia oleifera meal powder deepens and it has an obvious wine aroma.

[0142] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite microbial preparation, characterized in that: 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 live bacteria quantity ratio of 1:1:1 to 2:1:1:

1.

2. The composite microbial preparation according to claim 1, characterized in that: 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 1:1:1:1:1:1:1 in terms of live bacteria count.

3. Use of the composite microbial preparation according to claim 1 or 2 in fermenting camellia oleifera meal.

4. The use according to claim 3, characterized in that: The composite microbial preparation reduces the tea saponin content and cellulose content of tea oil cake.

5. A method for fermenting tea oil cake using the composite microbial preparation according to claim 1 or 2, characterized in that: The steps include: After the tea-seed meal is crushed, cellulase is added for enzymolysis to obtain tea-seed meal hydrolysate; molasses is added to the tea-seed meal hydrolysate, and then the composite microbial preparation according to claim 1 or 2 is inoculated for fermentation.

6. The method according to claim 5, characterized in that The tea oil meal is crushed and passed through a 20-50 mesh sieve.

7. The method according to claim 5, characterized in that The added amount of cellulase is 10-2000U / g of camellia oleifera meal, the mass of the composite microbial preparation inoculated is 0.05%-1% of the mass of the camellia oleifera meal enzymolyte, and the mass of molasses added is 5%-30% of the mass of the camellia oleifera meal enzymolyte.

8. The method according to claim 5, characterized in that The temperature of enzymolysis is 37-60°C, and the time of enzymolysis is 2-24h.

9. The method according to claim 5, characterized in that The fermentation temperature is 28-40°C and the fermentation time is 2-10 days.

10. A tea seed meal feed, characterized in that: The product is prepared by the method described in any one of claims 5 to 9.

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