Complex enzyme preparation for fermenting rice bran and feed rich in rice bran, application of complex enzyme preparation and preparation of fermented feed

Abstract

The invention relates to the field of biological fermentation feed, and particularly discloses a compound enzyme preparation for fermenting rice bran and feed rich in rice bran, application and preparation of the fermented feed. The complex enzyme preparation is composed of cellulase, beta-mannase and beta-glucosidase, rice bran and biomass materials rich in the rice bran are fermented through bacteria and enzymes in a synergistic mode, antinutritional factors of the rice bran or biomass rich in the rice bran are reduced, the nutritional value of the rice bran or fermented feed rich in the biomass rich in the rice bran is increased, and resource waste is reduced. The fermented rice bran feed and the preparation method thereof have a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of bio-fermented feed of ground-source agricultural and sideline products, and in particular to a method for preparing fermented rice bran and feed rich in rice bran by utilizing bacterial enzyme synergistic fermentation technology and application thereof. Background Art

[0002] Rice bran (current national standard rice bran) is the main by-product of rice processing, mainly composed of pericarp, seed coat, endosperm, endosperm and embryo, accounting for about 5-7% of the mass of rice. The annual output of rice bran in the world is about 700 million tons, and my country's annual output of rice bran exceeds 18 million tons, ranking first in the world. Rice bran is rich in nutrients, with a protein content of 11.5-17.2%, oil and fat 12.8-22.6%, and rich in plant active ingredients such as phenolic substances. It is a "god-given nutrient source". Rice bran protein is mainly composed of albumin (37%), globulin (36%), alcohol-soluble protein (5%) and glutenin (22%). It is a recognized high-quality plant protein. Its essential amino acid composition is balanced and reasonable, close to the FAO / WHO recommended model. Among them, the lysine content is high, which is unmatched by other plant proteins, and the nutritional value is very considerable.

[0003] However, the oil components of rice bran are mainly unsaturated fatty acids such as palmitic acid (17%), oleic acid (40%) and linoleic acid (34%), which are very easy to oxidize and deteriorate. Therefore, the shelf life of fresh rice bran is very short, and the storage period in summer is generally 3-5 days. In order to extend the shelf life, large rice mills generally process rice bran into rice bran meal or defatted rice bran after oil extraction. The shelf life of rice bran meal or defatted rice bran can be extended to about half a year. However, the anti-nutritional factors such as cellulose and mannan in rice bran meal are too high, which will interfere with the utilization of nutrients in the diet, so that most of them are used in low-grade feed. The rice bran oil produced by its processing has a high content of unsaturated fatty acids and has high requirements for the production process and technology. It is rarely used for food, but is mostly used to make industrial raw materials such as feed, soap, hardened oil, glycerin, stearic acid, oleic acid, paint resin, etc. Studies have shown that the use of microbial fermentation can reduce the anti-nutritional effects of cellulose in rice bran meal or defatted rice bran, and improve the utilization efficiency of nutrients such as protein, dietary fiber, and phenolic substances (CN109221920A, CN108850549A, CN106666077A, CN105581240A), but most of the documents disclosed are the treatment of rice bran meal or defatted rice bran by probiotics, and do not involve fresh rice bran. The loss of nutritional value caused by the degreasing process of fresh rice bran or the process of oil extraction into rice bran meal has been seriously ignored.

[0004] my country is rich in rice bran resources and biomass rich in rice bran, such as distiller's grains. It is imperative to adopt appropriate processing methods and techniques to fully utilize the nutritional value of rice bran and biomass rich in rice bran, extend the shelf life of rice bran and biomass rich in rice bran, and effectively reduce resource waste. Summary of the invention

[0005] In order to solve the above problems, the present invention develops a composite enzyme preparation specifically for rice bran and biomass materials rich in rice bran, which reduces anti-nutritional factors while increasing protein content, thereby improving the nutritional value of rice bran and biomass materials rich in rice bran and reducing resource waste.

[0006] The invention provides a composite enzyme preparation specially used for rice bran and biomass materials rich in rice bran. The composite enzyme preparation consists of cellulase, beta-mannanase and beta-glucosidase.

[0007] Preferably, the enzyme activity ratio of cellulase, β-mannanase and β-glucosidase is 8.5:4:1, and 0.4-0.9‰ is added to the rice bran mixed raw material in U units.

[0008] The present invention also provides a method for preparing a fermented feed, which is achieved by fermenting rice bran and a biomass material rich in rice bran with bacterial enzymes, and comprises the following steps:

[0009] (1) preparing feed raw materials: according to the mass fraction, 60-95% of rice bran, 5-20% of corn, 0-10% of bran, and 0-10% of soybean meal are mixed evenly, and molasses is added according to 1-2.5% of the dry matter of the materials;

[0010] (2) mixing the activated composite bacteria and composite enzyme preparation into the raw materials of step (1) in a certain proportion, and adding water to a water content of 35% to 38%;

[0011] (3) The mixture is placed in a packaging bag with a one-way breathing valve, heat-sealed, and placed in a fermentation room at a constant temperature of 30°C for fermentation.

[0012] The composite bacterial strain in step (2) is: silage bacterial agent, the number of bacteria after activation is not less than 1.0x10 6 -1.0x10 7 cfu / g were inoculated into the rice bran mixed raw material.

[0013] The composite enzyme preparation in step (2) is composed of cellulase, β-mannanase and β-glucosidase.

[0014] Preferably, the enzyme activity ratio of cellulase, β-mannanase and β-glucosidase is 8-9:2-5:1, preferably 8.5:4:1, and is added to the rice bran mixed raw material at 0.4-0.9‰ in U units.

[0015] Preferably, step (3) is left to ferment in a fermentation room in a ventilated and cool place, stacked flat, and can be used after fermenting for 3-7 days and having a sour aroma.

[0016] The present invention also provides rice bran feed prepared by the fermented feed preparation method.

[0017] The invention also provides application of rice bran feed for feeding pigs, cattle and the like.

[0018] Rice bran and biomass materials rich in rice bran contain anti-nutritional factors such as cellulose and mannan, which in feed will affect the digestion and utilization of other nutrients in the diet. Enzymatic hydrolysis of cellulose and mannan into usable monosaccharide forms is an important means to improve the digestibility and utilization of fermented feed.

[0019] Cellulose hydrolysis is the result of the synergistic action of multiple enzyme systems, among which β-glucosidase is an important component of the cellulase system, which is responsible for hydrolyzing the cellooligosaccharides and cellobiose released by the synergistic action of cellulose endo- and exo-enzymes into glucose. Therefore, by increasing the activity of β-glucosidase, not only can the overall enzyme activity of cellulase be effectively improved, but also the inhibitory effect of cellooligosaccharides and cellobiose on cellulose endo- and exo-enzymes can be reduced by degrading cellooligosaccharides and cellobiose, thereby improving the hydrolysis efficiency of the cellulase system. The cellulase system produced by Trichoderma reesei is insufficient in β-glucosidase, so it is necessary to compound β-glucosidase to balance the cellulase system.

[0020] Preferably, the cellulase comes from the Trichoderma reesei strain A2H capable of producing cellulase with enhanced activity, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number: CGMCC No.21470, the deposit time: March 17, 2021, and the classification name is Trichodema reesei: the address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] Preferably, β-mannanase: a Pichia strain with high mannanase production, the β-mannanase is from Pichia pastoris strain C1, deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCC NO.24324, the deposit date is January 17, 2022, and the classification name is: Pichia pastories: the address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] Preferably, the β-glucosidase is from Aspergillus niger, deposited in the General Microbiology Center of China Microorganism Culture Collection, with the deposit number: CGMCC No.22465, the deposit time: July 5, 2021, the classification name: Aspergillus niger, and the address of the depository unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] Rice bran and biomass materials rich in rice bran contain anti-nutritional factors such as cellulose and mannan, which seriously affect the digestion and utilization of nutrients in the diet. By conducting enzymatic hydrolysis experiments with cellulase: β-mannanase: β-glucosidase in the optimal ratio, the anti-nutritional factors of rice bran and biomass materials rich in rice bran are converted into monosaccharide forms to the greatest extent, which is conducive to the use of anaerobic bacteria to synthesize lactic acid. Microbial protein has the advantages of fast absorption, low energy consumption, high efficiency, and carrier saturation. At the same time, microbial protein can also give the product special biological activity, such as growth promotion, immune regulation, antibacterial, antiviral, lactation, antioxidant, appetite stimulation, mineral absorption and anti-tumor functions. Unsaturated fatty acids are degraded into short-chain saturated fatty acids, which are beneficial to animal absorption and utilization, and also extend the shelf life of fresh rice bran. The production of probiotic metabolites such as lactic acid and total acid improves the palatability of fermented rice bran feed, and the sour aroma has a strong appetizing effect. The production of acidic metabolites significantly reduces the pH value of fermented rice bran feed, which can inhibit the reproduction of harmful bacteria and spoilage bacteria. At the same time, the large amount of active probiotics contained in fermented rice bran will competitively inhibit the damage to the quality of rice bran by harmful bacteria and spoilage bacteria. The breathing bag is also an anaerobic environment, which prevents the rice bran from coming into contact with oxygen and undergoing redox reactions and becoming corrupt. In summary, the shelf life of rice bran can be effectively extended to more than one year.

[0024] When the optimal enzyme system is added to rice bran or rice bran-rich biomass materials for fermentation feed preparation, the monosaccharide content is the highest after enzymatic hydrolysis, and the anaerobic bacteria concentration is greater than or equal to 1.0x10 8cfu / g. In addition to improving the digestive system, probiotics can also improve the intestinal microecology, allowing nutrients to be quickly absorbed and comprehensively improving animal health. According to relevant analysis, probiotics can not only reduce the number of E. coli in the intestines of livestock and poultry to a certain extent, but also increase the number of lactobacilli in the intestines, significantly reduce the feed-to-weight ratio, increase daily weight gain, increase the level of lactic acid bacteria in their feces, significantly reduce the content of Salmonella and E. coli, and can significantly improve the growth performance of fattening pigs and dairy cows.

[0025] The beneficial effects of the present invention are as follows:

[0026] The present invention reduces the anti-nutritional factors of rice bran or rice bran-rich biomass fermented feed by developing an enzyme preparation suitable for rice bran or rice bran-rich biomass fermented feed, improves the nutritional value of rice bran or rice bran-rich biomass fermented feed, and reduces resource waste. The pH, crude fiber content, and unsaturated fatty acid content of the fermented rice bran feed obtained after fermentation treatment are significantly reduced, the crude fiber content is reduced by 15.22%, and the unsaturated fatty acid content is reduced by 51.58%; while the crude protein content, lactic acid content, and total acid content are significantly increased, and the microbial cell protein content is increased by 39.82%. After adding the fermented rice bran feed prepared by the present invention to the feed of mammals, compared with the feed without adding the fermented rice bran feed, the body weight, average daily weight gain, and average daily feed intake of the mammals are significantly increased, and the milk production and the milk protein and milk fat content in the milk are also significantly increased. The fermented rice bran feed of the present invention and the preparation method thereof have broad application prospects. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] The following examples involve the following indicator determination methods:

[0029] The crude protein content shall refer to GB / T 6432-2018, the acid-soluble protein content shall refer to GB / T 22492-2008, the crude fiber content shall refer to GB / T 6434-2006, the total acid content shall refer to GB / T 12456-2008, the unsaturated fatty acid content shall refer to GB / T5009.168-2016, the pH test shall refer to GB 5009.237-2016 and the lactic acid content shall refer to the biosensor enzyme membrane method.

[0030] In the following examples, the raw materials are derived from:

[0031] The fermentation bacteria were purchased from commercial silage agents.

[0032] The cellulase used in the embodiment is from the Trichoderma reesei strain A2H capable of producing cellulase with improved activity, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.21470, the deposit time: March 17, 2021, and the deposit unit address: Institute of Microbiology, Chinese Academy of Sciences.

[0033] β-mannanase: a Pichia pastoris strain with high mannanase production, the β-mannanase is from the Pichia pastoris strain C1, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number: CGMCCNO.24324.

[0034] The β-glucosidase comes from Aspergillus niger, and is characterized in that it is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.22465, and the deposit time is: July 5, 2021.

[0035] Example 1: Selection of enzyme addition amount

[0036] The compound ratio of cellulase and β-glucosidase has been disclosed in the previous patent (high-yield β-glucosidase Aspergillus niger strain and application, patent application number: ZL202110967072.3), so the compound ratio of cellulase and β-glucosidase is preferably 8.5:1.

[0037] The β-mannanase expressed by Pichia pastoris was added to the above-mentioned cellulase + β-glucosidase mixed enzyme system, and the addition ratio was shown in Table 1 below. 1% (w / w) silage agent was added to rice bran as a substrate, and the above-mentioned β-mannanase, cellulase and β-glucosidase complex enzyme preparation was added, and anaerobic fermentation was performed in a breathing bag for 15 days. After the anaerobic fermentation was completed, the breathing bag was opened to measure the acidity, lactic acid content and pH changes of the fermented samples.

[0038] The results showed that when the ratio of cellulase:β-mannanase:β-glucosidase was 8.5:2-5:1, the relative amount of lactic acid was greater than 90%, and when the ratio of cellulase:β-mannanase:β-glucosidase was 8.5:4:1, the final rice bran fermented feed had the most obvious sour aroma and the highest lactic acid yield, indicating that when the ratio of cellulase:β-mannanase:β-glucosidase was the most appropriate (8.5:4:1), cellulose and mannan were most conducive to conversion into monosaccharides for anaerobic fermentation in silage to produce lactic acid. This was used for subsequent experiments.

[0039] Table 1. Relative amount of lactic acid under different ratios of cellulase, β-mannanase and β-glucosidase

[0040] Serial number Cellulase: β-mannanase: β-glucosidase Relative amount of lactic acid 1 8.5:1:1 84.3％ 2 8.5:2:1 89.9％ 3 8.5:3:1 92.3％ 4 8.5:4:1 100％ 5 8.5:5:1 94.7％

[0041] Example 2: Feed preparation

[0042] Weigh the silage agent, dissolve it in clean water at a ratio of 1:50 (weight ratio), activate it in a strain activation tank at 30°C for 12 to 24 hours, and use it as seed liquid for later use;

[0043] The raw materials were weighed according to the raw material formula: 80 kg rice bran, 10 kg corn flour, 5 kg bran, 5 kg soybean meal (large particles need to be crushed and passed through a 40-mesh sieve), and then a composite enzyme preparation consisting of cellulase, β-mannanase and β-glucosidase was added at 0.5‰ (weight ratio), wherein the addition ratio of cellulase: β-mannanase: β-glucosidase was 8.5:(1-5):1.

[0044] The above-mentioned complex enzyme preparation is premixed for 2-5 minutes; clean water, 2% molasses and seed liquid are added to the liquid preparation tank in proportion and mixed evenly for 0.5 minutes as bacterial liquid for standby use; the bacterial liquid is sprayed into the raw materials being stirred, and stirring is continued for 2-5 minutes after the bacterial liquid is added.

[0045] After discharging, put it into a packaging bag with a one-way breathing valve, weighing: 20kg / bag, and seal it.

[0046] Put it into storage and let it ferment, place it in a ventilated and cool place, lay it flat, and continue to ferment for 3-7 days. It can be used with a sour aroma. See Table 2. Under the most appropriate addition ratio of cellulase: β-mannanase: β-glucosidase (8.5:4:1), the crude protein content of the fermented product reached 18.2%, the acid-soluble protein content was 4.27%, the lactic acid content was 12.7g / L, the total acid content was 16.8g / L, and the crude fiber content was reduced to 8.58%. The nutritional value of the fermented feed was greatly improved by increasing the protein content and reducing the crude fiber content.

[0047] Table 2. Comparison of the content of various substances before and after fermentation (on a dry basis)

[0048]

[0049] Embodiment 3: feedstuff feeding

[0050] Feeding trial 1

[0051] Test location: a pig farm in Tianjin

[0052] Experimental plan: 40 fattening pigs with a body weight of about 18kg were selected and randomly divided into two groups, the experimental group and the control group, with 20 pigs in each group. The experimental period was 160 days, including a 10-day pre-trial period and a 150-day full-trial period. The control group was fed a basic complete diet, and the experimental group was fed a 10.0% bacterial enzyme-fermented rice bran feed added to the diet.

[0053] Table 3. Effects of bacterial enzyme synergistic fermentation of rice bran on growth performance of growing and finishing pigs

[0054]

[0055] As shown in Table 3, when the test group was fed with fermented rice bran feed with the optimal ratio of cellulase:β-mannanase:β-glucosidase (8.5:4:1), the final weight (kg / head), average daily weight gain (kg / head / day) and average daily feed intake (kg / head / day) of the animals were significantly higher than those of the control group, while the feed-to-meat ratio was significantly lower than that of the control group. Compared with the control group, the average daily weight gain of the test group increased by 11.98%, the average daily feed intake increased by 5.86%, and the feed-to-weight ratio decreased by 4.38%, indicating that the optimal ratio of cellulase:β-mannanase:β-glucosidase (8.5:4:1) fermented rice bran feed has the best nutritional function.

[0056] Feeding experiment 2

[0057] Test location: a dairy farm in Hebei

[0058] Experimental plan: 20 Holstein cows with basically the same age, weight, parity and milk production were selected and randomly divided into an experimental group and a control group, with 10 cows in each group, and the experimental period was 30 days. The diet composition of the control group was concentrate supplement + silage + alfalfa hay, and the diet composition of the experimental group added 20% fermented feed to the concentrate supplement, and then the concentrate supplement was mixed with silage and alfalfa hay to form the experimental group diet. During the experiment, all the experimental cows in the two groups were raised in a standardized manner, fed 3 times a day, milked 2 times, and had free drinking water, and the management conditions were consistent. During the experiment, the milk production of each cow was recorded every day; samples were taken at the beginning and end of the experiment to detect the milk protein and milk fat content in the milk.

[0059] Table 4. Effects of different feeds on dairy cow production performance

[0060]

[0061] As shown in Table 4, when fermented rice bran feed was added with the optimal ratio of cellulase:β-mannanase:β-glucosidase (8.5:4:1), the yield of each dairy cow in the experimental group was 0.62kg higher than that in the control group, and the milk fat rate and milk protein increased by 0.07g / 100ml and 0.09g / 100ml respectively.

Claims

1. A composite enzyme preparation for fermenting rice bran or a biomass material rich in rice bran, characterized in that: The complex enzyme preparation consists of cellulase, beta-mannanase and beta-glucosidase.

2. The complex enzyme preparation according to claim 1, characterized in that The cellulase is from Trichoderma reesei strain A2H, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with the preservation number of CGMCC No.21470.

3. The complex enzyme preparation according to claim 1, characterized in that The β-mannanase is from Pichia pastoris strain C1 and is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number: CGMCC NO.24324.

4. The complex enzyme preparation according to claim 1, characterized in that The β-glucosidase is from Aspergillus niger, and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.22465.

5. The complex enzyme preparation according to claim 1, characterized in that The enzyme activity ratio of the cellulase, β-mannanase and β-glucosidase is 8-9:2-5:1, preferably 8.5:4:

1.

6. Use of the complex enzyme preparation according to any one of claims 1 to 5 in the fermentation of rice bran or biomass materials rich in rice bran.

7. A method for preparing fermented feed, characterized in that: The method is achieved by fermenting rice bran or a biomass material rich in rice bran with bacterial enzymes, and the method comprises the following steps: (1) Prepare feed raw materials: Mix 60-95% rice bran, 5-20% corn, 0-10% bran, and 0-10% soybean meal evenly according to mass fraction, and add molasses at 1-2.5% of the dry matter of the material; (2) Mixing the activated composite bacteria with the composite enzyme preparation according to any one of claims 1 to 5, adding the mixture to the feed raw material of step (1), and adding water to a water content of 35% to 38%; (3) Put the product into a packaging bag with a one-way breathing valve, seal it, and place it in a constant temperature of 30°C for fermentation.

8. The preparation method according to claim 7, characterized in that: The composite bacterial strain in step (2) is a silage bacterial agent, and the bacterial count after activation is not less than 1.0x10 6 (e.g. 1.0x10 6 -1.0x10 7 cfu / g) was inoculated into the rice bran mixed raw material; the complex enzyme preparation was calculated in U units and added at 0.4-0.9‰ of the raw material.

9. The preparation method according to claim 7, characterized in that: The step (3) is to place the mixture in a ventilated and cool place for fermentation. After 3-7 days of fermentation, it can be used when it has a sour aroma.

10. The fermented feed prepared by the preparation method according to any one of claims 7 to 9.

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