Trichoderma harzianum pretreatment-based method for producing protein feed through white spirit vinasse solid-state fermentation

Abstract

The invention relates to the technical field of microbial fermentation feeds, in particular to a method for producing a protein feed through solid-state fermentation of distiller's grains based on Trichoderma harzianum pretreatment. According to the method, the vinasse is pretreated by the crude enzyme prepared from trichoderma harzianum, and the formula and fermentation conditions of an enzyme production culture medium are optimized, so that the filter paper enzyme activity of cellulase is remarkably improved. By adding the surfactant into a fermentation system and optimizing the variety of the surfactant, the yield of the fermented monosaccharide is improved, and the problem of musty taste in a traditional fermented product is solved while the degradation efficiency of lignocellulose is improved. Furthermore, a synchronous saccharification and fermentation process is adopted, vinasse is fermented through compounded saccharomycetes and crude enzyme liquid, the trichoderma harzianum fermented protein feed is prepared and obtained, digestion and absorption of animals are easier, lactic acid fermentation is conducted on the trichoderma harzianum fermented protein feed again through lactobacillus fermentum, the flavor of the feed is improved, the bitter taste of the feed is reduced, and the sour taste is improved. The method realizes efficient utilization of vinasse resources.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermented feed, and in particular to a method for producing protein feed based on solid-state fermentation of distiller's grains pretreated with Trichoderma harzianum. Background Art

[0002] Traditional feed protein sources, such as soybeans and fish meal, face problems such as unstable supply and drastic price fluctuations, making it difficult to meet the growing market demand for feed protein at this stage. Therefore, seeking alternative feed protein resources has become an important issue that needs to be urgently addressed in the current agricultural and food industry. As an industrial by-product rich in protein, fiber and various nutrients, white wine lees has huge potential for development and utilization. However, its high content of lignocellulose (rice husk accounts for more than 2 / 3) and low fermentable sugar content make its direct utilization face huge challenges; at the same time, its complex chemical composition and low digestibility make it subject to many restrictions when used directly as feed.

[0003] In recent years, biotechnology has shown great potential in resource recycling and waste treatment. However, traditional treatment methods have the following defects: (1) Limitations of Aspergillus niger fermentation process: The existing technology uses Aspergillus niger as a cellulose-degrading bacterium. Although it can improve enzyme activity, the fermentation product often has thick black smoke and musty smell, which seriously affects the palatability of feed and limits its application in the feed field; (2) High pretreatment cost: Relying on commercial enzymes for lignocellulose degradation is not only economical, but also the compatibility of exogenous enzymes with substrates is insufficient, making it difficult to achieve efficient degradation. Trichoderma harzianum is a fungus widely used in the field of biodegradation and biocontrol. The crude enzyme system it produces has fiber degradation ability.

[0004] In view of this, the development of a low-cost distiller's grains resource technology that can efficiently degrade lignocellulose and improve feed flavor has become an urgent need in the current industry. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for producing protein feed based on solid-state fermentation of white distiller's grains pretreated with Trichoderma harzianum, in view of the shortcomings of the prior art distiller's grains feed, such as low protein content, high fiber content and high cost of commercial cellulase.

[0006] The invention aims at the common problems of black smoke and moldy smell during fermentation of Aspergillus niger, based on the design concept of efficiently degrading lees lignocellulose while reducing the moldy smell after fermentation, and selects Trichoderma harzianum fungus as the fermentation enzyme production strain.

[0007] To solve the above technical problems, the present invention discloses a method for producing protein feed by solid-state fermentation of distillers' grains based on Trichoderma harzianum pretreatment, comprising the following steps:

[0008] (1) Inoculate the spore suspension of Trichoderma harzianum into an enzyme-producing medium for solid-state fermentation to obtain a fermented solid;

[0009] (2) Extract the crude enzyme solution of cellulase from the fermented solid, and pre-incubate the crude enzyme solution with a surfactant to obtain a treated crude enzyme solution;

[0010] (3) Add the treated crude enzyme solution to the distillers' grains raw material, and simultaneously inoculate the seed solution of yeast into the distillers' grains raw material added with the treated crude enzyme solution for synchronous saccharification fermentation to prepare Trichoderma harzianum fermented protein feed.

[0011] Among them, in step (1), the enzyme-producing medium includes a solid part, a liquid part and an additive;

[0012] Specifically, the solid-liquid ratio of the solid part to the liquid part is 1 g: 0.5 - 1.5 mL; preferably, the solid-liquid ratio is 1:1.

[0013] Among them, the solid part includes any one or a combination of several of corn straw, distillers' grains sieved rice husk and wheat bran; the mass ratio of corn straw, distillers' grains sieved rice husk and wheat bran is 0 - 6: 0 - 10: 0 - 2, and the three are not all 0 at the same time.

[0014] Preferably, the solid part includes corn straw, distillers' grains sieved rice husk and wheat bran; the mass ratio of corn straw, distillers' grains sieved rice husk and wheat bran is 4: 4: 2.

[0015] Among them, the formula of the part is as follows: ammonium sulfate 1 - 2 g / L, magnesium sulfate 2 - 5 g / L, potassium dihydrogen phosphate 2 - 8 g / L.

[0016] In some embodiments of the present invention, the formula of the liquid part is as follows: ammonium sulfate 2 g / L, magnesium sulfate 5 g / L, potassium dihydrogen phosphate 8 g / L.

[0017] Among them, the additive is Tween-80, and the addition amount is 0.005 - 0.015 mL / g, based on the solid part of the enzyme-producing medium.

[0018] Preferably, the addition amount of Tween-80 is 0.15 mL / g.

[0019] Among them, the moisture content of the enzyme-producing medium is 40 - 60 wt%, and the preferred moisture content is 53 wt%.

[0020] Among them, in step (1), for the inoculation, the inoculation amount is 4×10 6 ~1×10 7 spores / g, calculated based on the solid part of the enzyme-producing medium.

[0021] Preferably, for the inoculation, the inoculation amount is 4×10 6 spores / g.

[0022] Among them, in step (1), for the solid-state fermentation, the fermentation conditions are: the initial pH value is natural, and it is cultured at 25 - 30°C for 3 - 5 days.

[0023] Preferably, the fermentation conditions are: the initial pH value is natural, and it is cultured at 30°C for 3 days.

[0024] Among them, in step (2), the surfactant includes any one or a combination of several of Tween-20, Tween-80, PEG4000, and PEG6000; the preferred surfactant is PEG4000.

[0025] Specifically, the addition amount of the surfactant is 0.01 - 0.05 g of surfactant added to every 1 mL of crude enzyme solution.

[0026] In some embodiments of the present invention, the addition amount of the surfactant is 0.01 g of surfactant added to every 1 mL of crude enzyme solution.

[0027] Among them, in step (2), for the pre-incubation treatment, the treatment conditions are: incubate at 0 - 4°C for 12 h.

[0028] In some embodiments of the present invention, for the pre-incubation treatment, the treatment conditions are: incubate at 4°C for 12 h.

[0029] Among them, in step (3), the crude enzyme solution for extracting cellulase from the fermentation solid is extracted using 0.1 M acetic acid - sodium acetate buffer.

[0030] Among them, in step (3), the distiller's grains raw material includes crushed distiller's grains and uncrushed distiller's grains, and the mass ratio of the crushed distiller's grains to the uncrushed distiller's grains is 0 - 10:0 - 10, and the two are not both 0 at the same time; preferably, the mass ratio of the crushed distiller's grains to the uncrushed distiller's grains is 8:2.

[0031] Specifically, the distiller's grains raw material includes urea, and the addition amount of the urea, calculated based on the dry weight of the distiller's grains raw material, is 2 - 5 g of urea added to every 100 g of distiller's grains raw material.

[0032] Specifically, the crushed distiller's grains are crushed distiller's grains with a mesh size of 40.

[0033] Specifically, the distiller's grains are white spirit distiller's grains, donated by Luzhou Laojiao.

[0034] Among them, in step (3), the addition amount of the treated crude enzyme solution is such that the moisture content of the distillers grains raw material is 55-70 wt%; preferably, the addition amount is such that the moisture content of the distillers grains raw material is 60 wt%.

[0035] Among them, in step (3), the yeast is Candida utilis, Saccharomyces cerevisiae, and Rhodotorula benthica.

[0036] Specifically, the inoculation volume ratio of Candida utilis, Saccharomyces cerevisiae, and Rhodotorula benthica is 1:1:1. The inoculation amount of the seed liquid of the three yeasts is based on the dry weight of the distillers grains raw material. For every 100 g of the distillers grains raw material, 2-5 mL of the seed liquid of Candida utilis, the seed liquid of Saccharomyces cerevisiae, and the seed liquid of Rhodotorula benthica are added respectively.

[0037] In some embodiments of the present invention, the inoculation amount of the seed liquid of the three yeasts is based on the dry weight of the distillers grains raw material. For every 100 g of the distillers grains raw material, 2 mL of different yeast seed liquids are added respectively.

[0038] Among them, in step (3), the conditions for the simultaneous saccharification and fermentation are as follows: simultaneous saccharification and fermentation are carried out at 25-30 °C for 7-11 days.

[0039] In some embodiments of the present invention, the conditions for the simultaneous saccharification and fermentation are as follows: simultaneous saccharification and fermentation are carried out at 30 °C for 11 days.

[0040] Among them, in step (3), the Trichoderma harzianum fermented protein feed is dried and pulverized, the pH is adjusted to 5-6, and the moisture content is adjusted to 50-60 wt% to obtain a fermented material. The seed liquid of Limosilactobacillus fermentum is inoculated into the fermented material, and glucose is added for lactic acid fermentation to prepare a lactic acid fermented feed.

[0041] Specifically, for the drying and pulverizing, the drying temperature is 105 °C, and the pulverizing particle size is 40 mesh.

[0042] Specifically, for the seed liquid of Limosilactobacillus fermentum, based on the dry weight of the fermentation material, 2 - 5 mL of the seed liquid of Limosilactobacillus fermentum is added to every 100 g of the fermentation material.

[0043] In some embodiments of the present invention, based on the dry weight of the fermentation material, 2 mL of the seed liquid of Limosilactobacillus fermentum is added to every 100 g of the fermentation material.

[0044] Specifically, for the glucose, based on the dry weight of the fermentation material, 2 - 5 mL of glucose is added to every 100 g of the fermentation material.

[0045] In some embodiments of the present invention, based on the dry weight of the fermentation material, 2 mL of glucose is added to every 100 g of the fermentation material.

[0046] Specifically, for the lactic acid fermentation, the conditions are as follows: ferment at 30°C - 37°C for 15 - 45 days under vacuum conditions.

[0047] In some embodiments of the present invention, the lactic acid fermentation is anaerobic fermentation, and the conditions are as follows: ferment at 37°C for 45 days under vacuum conditions.

[0048] Furthermore, the flavors of different protein feeds were compared. The order of the bitterness intensity of the four feeds is: Trichoderma harzianum fermented protein feed > Aspergillus niger fermented protein feed > lactic acid fermented feed > distiller's grains raw material. Therefore, through the lactic acid bacteria fermentation treatment, the flavor characteristics of the Trichoderma harzianum fermented protein feed have been significantly improved. Specifically, the bitterness in the feed has decreased, while the sourness has increased, and the overall flavor has become more harmonious, enhancing the palatability.

[0049] Furthermore, the in vitro digestion verification experiment of rumen fluid was used to verify the effectiveness and safety of different protein feeds. It was found that the Trichoderma harzianum fermented protein feed is more conducive to digestion and easier for animals to absorb.

[0050] Beneficial effects:

[0051] (1) The present invention uses the by - product of grain brewing, distiller's grains, as the raw material to produce feed, realizing the efficient utilization of resources, reducing resource waste, and at the same time reducing environmental pollution, with significant environmental protection benefits.

[0052] (2) The present invention adopts the cellulase hydrolysis process to treat distiller's grains, replaces the use of commercial enzymes through microbial pretreatment, effectively reduces the production cost, and improves the economic efficiency of the process.

[0053] (3) The crude enzyme solution prepared from Trichoderma harzianum is used in the present invention to pretreat distillers' grains, and combined with the optimization of the enzyme-producing medium formula and fermentation conditions, the filter paper activity of cellulase is significantly increased by 23.7%. Further, by adding surfactants to the fermentation system and optimizing the types of surfactants, the yield of fermentable monosaccharides is increased by 10.75%. Finally, while improving the degradation efficiency of lignocellulose, the problem of musty smell in traditional fermentation products is solved.

[0054] (4) The present invention adopts a simultaneous saccharification and fermentation process, and uses a compound yeast to carry out simultaneous saccharification and fermentation on the distillers' grains obtained by the synergistic action of Trichoderma harzianum, the enzyme-producing medium formula, the crude enzyme solution, the fermentation conditions and the surfactant, and a Trichoderma harzianum fermented protein feed is prepared. This feed is more conducive to digestion and easier for animals to absorb.

[0055] (5) The present invention further introduces a lactic acid fermentation link in the production process of the protein feed. By further carrying out lactic acid fermentation on the Trichoderma harzianum fermented protein feed, the pH value of the product is reduced, thereby extending the storage period. At the same time, the fermentation process improves the flavor of the feed, reduces the bitterness of the feed, and enhances the sour taste, making it more suitable for animal consumption. Description of the Drawings

[0056] The following further specifically describes the present invention with reference to the drawings, and the above and / or other advantages of the present invention will become clearer.

[0057] Figure 1 Results of the filter paper activity of cellulase produced by the enzyme-producing medium containing different contents of rice husk.

[0058] Figure 2 Results of the reducing sugar and sugar content in the enzymatic hydrolysis reaction with different surfactants added. Among them, (a) is the result of reducing sugar, and (b) is the result of sugar content.

[0059] Figure 3 Results of the determination of the true protein content of the Trichoderma harzianum fermented protein feed.

[0060] Figure 4 Results of the principal component analysis (PCA) of the flavor detection of 4 kinds of feeds (lactic acid fermentation feed, Trichoderma harzianum fermented protein feed, Aspergillus niger fermented protein feed, distillers' grains raw material) by using electronic nose technology.

[0061] Figure 5 Results of the electronic tongue signals of the flavor detection of 4 kinds of feeds (lactic acid fermentation feed, Trichoderma harzianum fermented protein feed, Aspergillus niger fermented protein feed, distillers' grains raw material) by using electronic tongue technology.

[0062] Figure 6Results of dry matter degradation rate and crude protein degradation rate of different feeds. Detailed implementation manners

[0063] The following further specifically describes the present invention in conjunction with the detailed implementation manners, and the above and / or other advantages of the present invention will become clearer.

[0064] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0065] In the following examples, the Trichoderma harzianum has been disclosed in the master's degree thesis of Zhengzhou University, "System Construction and Mechanism Research on Enzyme-Fungi Co-Fermentation for Producing Distillers' Grains Protein Feed"; the Aspergillus niger, Candida utilis, Saccharomyces cerevisiae, Rhodotorula benthica and Limosilactobacillus fermentum were all gifted by the National Biochemical Engineering Technology Research Center of Nanjing Tech University.

[0066] In the following examples, the formula of the PDA medium is: 200 g / L of potato, 20 g / L of glucose, 20 g / L of agar; the formula of the YPD liquid medium is: 10 g / L of yeast powder, 20 g / L of peptone, 20 g / L of glucose; the formula of the MRS liquid medium is: 10 g / L of peptone, 10 g / L of beef extract, 5 g / L of yeast powder, 5 g / L of glucose, 20 g / L of calcium carbonate, 1 g / L of Tween-80, 2 g / L of dipotassium hydrogen phosphate, 5 g / L of sodium acetate, 2 g / L of diammonium citrate, 0.2 g / L of magnesium sulfate heptahydrate, 0.05 g / L of manganese sulfate monohydrate.

[0067] In the following examples, the corn straw, distillers' grains sieved rice husk, and wheat bran need to be dried at 105 °C and then crushed to a particle size of 40 mesh.

[0068] Example 1: Directed domestication of the enzyme production specificity of Trichoderma harzianum

[0069] In this study, the rice husk sieved from distillers' grains was used to direct the domestication of the enzyme production specificity of Trichoderma harzianum, so that the cellulase produced by Trichoderma harzianum showed specificity for the enzymatic hydrolysis of distillers' grains and enhanced its decomposition ability.

[0070] 1. Inoculate Trichoderma harzianum into PDA medium and culture it at a constant temperature of 30 °C for 5 days. Then, use 0.05% (v / v) Tween-80 solution to elute the spores on the surface of the plate. After counting with a hemocytometer, adjust the spore concentration of the spore suspension of Trichoderma harzianum to 4×10 7 spores / mL.

[0071] 2. Inoculate the spore suspension of Trichoderma harzianum into different enzyme-producing media at an inoculum amount of 4×10 6 spores / g (calculated based on the mass of the solid part), and culture it at 30 °C for 3 days for solid-state fermentation to produce cellulase.

[0072] (1) Basic enzyme-producing medium (i.e., rice husk content 0%): It includes a solid part and a liquid part, and the solid-liquid ratio is 1:1 (i.e., the water content is 50 wt%). The solid part is corn straw and wheat bran with a mass ratio of 4:1, and the liquid part is a nutrient solution composed of 2 g / L of ammonium sulfate, 5 g / L of magnesium sulfate, and 8 g / L of potassium dihydrogen phosphate (initial pH value is natural).

[0073] Based on 10 g of the solid part, that is, the enzyme-producing system is 10 g of the solid part (8 g of corn straw powder, 2 g of wheat bran), 10 mL of the nutrient solution, and 1 mL of the spore suspension of Trichoderma harzianum.

[0074] (2) Enzyme-producing medium containing 20% rice husk content: Replace 20% of the total mass of the solid part of the basic enzyme-producing medium with rice husk screened from distiller's grains. At this time, the solid part is corn straw, rice husk screened from distiller's grains, and wheat bran with a mass ratio of 6:2:2.

[0075] Based on 10 g of the solid part, that is, the enzyme-producing system is 10 g of the solid part (6 g of corn straw, 2 g of rice husk screened from distiller's grains, 2 g of wheat bran), 10 mL of the nutrient solution, and 1 mL of the spore suspension of Trichoderma harzianum.

[0076] (3) Enzyme-producing medium containing 40% rice husk content: Replace 40% of the total mass of the solid part of the basic enzyme-producing medium with rice husk screened from distiller's grains. At this time, the solid part is corn straw, rice husk screened from distiller's grains, and wheat bran with a mass ratio of 4:4:2.

[0077] Based on 10 g of the solid part, that is, the enzyme-producing system is 10 g of the solid part (4 g of corn straw, 4 g of rice husk screened from distiller's grains, 2 g of wheat bran), 10 mL of the nutrient solution, and 1 mL of the spore suspension of Trichoderma harzianum.

[0078] (4) Enzyme-producing medium containing 60% rice husk content: Replace 60% of the total mass of the solid part of the basic enzyme-producing medium with rice husk screened from distiller's grains. At this time, the solid part is corn straw, rice husk screened from distiller's grains, and wheat bran with a mass ratio of 2:6:2.

[0079] Based on 10 g of the solid part, that is, the enzyme-producing system is 10 g of the solid part (2 g of corn straw, 6 g of rice husk sieved from distiller's grains, 2 g of wheat bran), 10 mL of nutrient solution, and 1 mL of Trichoderma harzianum spore suspension.

[0080] (5) Enzyme-producing medium containing 80% rice husk content: Replace 80% of the total mass of the solid part of the basic enzyme-producing medium with rice husk sieved from distiller's grains. At this time, the solid part is corn straw, rice husk sieved from distiller's grains, and wheat bran with a mass ratio of 0:8:2.

[0081] Based on 10 g of the solid part, that is, the enzyme-producing system is 10 g of the solid part (8 g of rice husk sieved from distiller's grains, 2 g of wheat bran), 10 mL of nutrient solution, and 1 mL of Trichoderma harzianum spore suspension.

[0082] (6) Enzyme-producing medium containing 100% rice husk content: Replace all of the solid part in the basic enzyme-producing medium with rice husk sieved from distiller's grains. At this time, the solid part is rice husk sieved from distiller's grains.

[0083] Based on 10 g of the solid part, that is, the enzyme-producing system is 10 g of the solid part (10 g of rice husk sieved from distiller's grains), 10 mL of nutrient solution, and 1 mL of Trichoderma harzianum spore suspension.

[0084] By comparing the filter paper enzyme activity of cellulase under different rice husk ratios, the optimal rice husk ratio was screened to obtain the optimal enzyme-producing medium. The results are as Figure 1 shown. The filter paper enzyme activity first increased and then decreased with the increase in rice husk content. When the rice husk content was 40%, the filter paper enzyme activity of cellulase reached the maximum value of 1.14 U / g. Therefore, the rice husk content of the enzyme-producing medium in subsequent experiments was determined to be 40%.

[0085] Example 2: Response surface optimization of Trichoderma harzianum solid-state fermentation for enzyme production

[0086] During the process of solid-state fermentation of molds for enzyme production, various factors have a significant impact on the enzyme production efficiency. In this example, the "Plackett-Burman (PB) design method" was used to systematically screen the influencing factors of Trichoderma harzianum solid-state fermentation for enzyme production, with the filter paper enzyme activity as the response value index. Specifically, through the comprehensive evaluation of 7 factors, namely fermentation time, fermentation temperature, initial pH of the enzyme-producing medium, moisture content of the enzyme-producing medium, inoculation amount of Trichoderma harzianum spore suspension, addition amount of Tween-80 added to the enzyme-producing system (calculated based on the mass of the solid part), and addition amount of soy peptone added to the enzyme-producing system (calculated based on the mass of the solid part), 3 factors that have the most significant impact on cellulase production were screened out, which are: moisture content of the enzyme-producing medium, fermentation time, and addition amount of Tween-80 added to the enzyme-producing medium.

[0087] Response surface Box-Behnken design was used to optimize the response surface of cellulase production by solid-state fermentation. Specifically, with the moisture content (X1), fermentation time (X2), and addition amount of Tween-80 (X3) as factor variables, three levels of low, medium, and high were selected for each factor, and the filter paper enzyme activity was selected as the response value. Experiments were carried out according to the 17 groups of experimental conditions designed by the response surface. To ensure the accuracy of the experiment, three parallel control experiments were carried out at the same time. The response surface design scheme is shown in Table 1.

[0088] Table 1 Response surface design scheme

[0089]

[0090] It can be seen from the filter paper enzyme activity data in Table 1 that as the fermentation time prolongs, the filter paper enzyme activity gradually decreases, which may be due to the exhaustion of nutrients in the enzyme-producing medium and the gradual loss of the activity of Trichoderma harzianum; when the moisture content increases from 40wt% to 60wt%, the filter paper enzyme activity shows a trend of first increasing and then decreasing, with a maximum value near the moisture content of 50wt%; as the addition amount of Tween-80 increases, the filter paper enzyme activity slightly decreases. The results show that the moisture content is the most important factor affecting enzyme production by solid-state fermentation.

[0091] The filter paper enzyme activity after fermentation was input and analyzed and fitted by software. The relationship between the filter paper enzyme activity and the moisture content, addition amount of Tween-80, and fermentation time is shown by the following formula.

[0092]

[0093] Among them, Y is the filter paper enzyme activity (U / g), and X1, X2, and X3 are the actual values of the moisture content (wt%), time (d), and addition amount of Tween-80 (mL / g), respectively. The ANOVA of the quadratic regression model shows that the F value is 31.23 and the P value is less than 0.0001, indicating that the model is significant. The P values of X2 and X3 are relatively low (<0.05), which are significant model terms and the model differences are significant.

[0094] According to the response surface optimization results, when the moisture content of the enzyme-producing medium is 53wt%, the fermentation time is 3 days, and Tween-80 is added to the enzyme-producing system with an addition amount of 0.15 mL / g, the predicted highest enzyme activity is 1.45 U / g. Under this condition, a verification experiment was carried out according to the enzyme production steps in Example 1, and the filter paper enzyme activity of cellulase was measured to be 1.41 U / g, which is close to the predicted value. Compared with the initial value of 1.14 U / g, it increased by 23.7%.

[0095] Example 3: Exploration of the conditions for the enzymatic hydrolysis of distillers' grains by crude enzyme solution

[0096] After the fermented solid containing cellulase optimized in Example 2 was extracted with 0.1 M acetic acid-sodium acetate buffer solution, a crude enzyme solution was obtained. After detection, the filter paper enzyme activity of the crude enzyme solution was 0.14 U / mL.

[0097] 1. Pre-incubate the crude enzyme solution with surfactants

[0098] In a 100 mL conical flask, 0.05 g of different surfactants (Tween-20, Tween-80, PEG4000, PEG6000) were respectively added to 5 mL of the crude enzyme solution and dissolved thoroughly by shaking. Subsequently, the conical flask was sealed with plastic wrap and incubated in a 4°C refrigerator for 12 h to obtain the crude enzyme solutions with different pre-incubation treatments.

[0099] 2. Enzymatic hydrolysis of the crude enzyme solution

[0100] Take a 50 mL centrifuge tube, add 1 g of crushed distiller's grains dried and ground to 40 mesh as the substrate, and add the crude enzyme solutions with different pre-incubation treatments to the centrifuge tube respectively. The addition amount of the crude enzyme solution was 5 mL to make the crude enzyme solution fully contact with the substrate. At this time, the enzyme loading of the crushed distiller's grains was 0.7 U / g. Subsequently, the centrifuge tube was placed in a 50°C constant temperature shaker, the rotation speed was adjusted to 200 rpm, and an enzymatic hydrolysis reaction was carried out for 72 h. The crude enzyme solution without surfactant treatment was used as the control.

[0101] The results are as Figure 2 shown. After adding surfactants, except for the Tween-80 group, the reducing sugar yields of the other experimental groups increased after enzymatic hydrolysis. The Tween-20 group, PEG4000 group, and PEG6000 group increased by 3.77%, 10.75%, and 3.33% respectively compared with the raw material group. The glucose yield after enzymatic hydrolysis only increased by 20.23% in the PEG4000 group compared with the raw material group, and no obvious differences were observed between the other experimental groups and the raw material group. Therefore, after the crude enzyme solution was pre-incubated with the surfactant PEG4000, its enzymatic hydrolysis effect on distiller's grains was enhanced.

[0102] Example 4: Simultaneous saccharification process for fermenting Trichoderma harzianum fermented protein feed and lactic acid re-fermentation of Trichoderma harzianum fermented protein feed

[0103] 1. Simultaneous saccharification process for fermenting Trichoderma harzianum fermented protein feed

[0104] After Candida utilis, Saccharomyces cerevisiae, and Rhodotorula benthica were activated, they were respectively inoculated into YPD liquid medium and cultured at 30°C and 200 rpm for 20 h to obtain their respective seed solutions.

[0105] The distiller's grains are dried and crushed into crushed distiller's grains with a mesh size of 40. The crushed distiller's grains and uncrushed distiller's grains are mixed at a mass ratio of 8:2 to obtain the original distiller's grains raw material. 10 g of the original distiller's grains raw material is added to a 250 mL conical flask, and then 2% (based on dry weight) urea (i.e., 0.2 g) is added to obtain the distiller's grains raw material. Then, it is autoclaved at 121 °C for 20 min. 15 mL of the crude enzyme solution pre-incubated in Example 3 is added to the autoclaved distiller's grains raw material to make the water content of the distiller's grains raw material 60 wt%. Then, the seed solutions of Candida utilis, Saccharomyces cerevisiae, and Rhodotorula marina are inoculated into the distiller's grains raw material with a water content of 60 wt% at an inoculation amount of 2% (based on dry weight) (i.e., 0.2 mL each). After mixing evenly, synchronous saccharification and fermentation are carried out at 30 °C for 11 days to obtain Trichoderma harzianum fermented protein feed.

[0106] After the fermentation is completed, the true protein content of the obtained Trichoderma harzianum fermented protein feed is measured. As Figure 3 shown, the true protein content increases from 10.71% before fermentation to 15.83% after fermentation, an increase of 47.8% compared with before fermentation.

[0107] 2. Lactic acid re-fermentation of protein feed

[0108] After activating Limosilactobacillus fermentum, it is inoculated into MRS liquid medium and cultured at 37 °C and 200 rpm for 24 h to obtain the seed solution of Limosilactobacillus fermentum.

[0109] The Trichoderma harzianum fermented protein feed is dried, crushed, and sieved through a 40-mesh sieve at 105 °C, the pH is adjusted to 5.6, and then autoclaved at 121 °C. A 250 mL conical flask is used as the fermentation system, the loading amount is 10 g, and 10 mL of sterile water is added to make the water content 50 wt%. Then, 2% (based on the dry weight of the material) of the seed solution of Limosilactobacillus fermentum is inoculated into it, and 2% (based on the dry weight of the material) of glucose is added, and fermentation is carried out at 37 °C for 45 days under vacuum conditions to obtain lactic acid fermented feed. During this period, samples are taken on the 0th, 3rd, 7th, 11th, 15th, and 45th days respectively, and sterile water is used instead of the seed solution of Limosilactobacillus fermentum as the control group to compare the fermentation effect.

[0110] It is found that as the lactic acid fermentation time prolongs, the pH value shows a downward trend. It shows an obvious three-stage change pattern: in the initial stage (0 - 3 d), it gradually decreases from 6.00 on the 0th day to 5.74, in the middle stage (3 - 11 d), there is a rapid decline stage, and in the later stage (11 - 45 d), it enters a stable slow decline period, and finally reaches an equilibrium value of 4.47.

[0111] Example 5: Flavor comparison of Trichoderma harzianum fermented protein feed

[0112] 1. Production of protein feed fermented by Aspergillus niger

[0113] The production method of protein feed fermented by Aspergillus niger is similar to that of protein feed fermented by Trichoderma harzianum. Only replace the Trichoderma harzianum strain in Examples 1 - 4 above with Aspergillus niger strain, and the rest remains unchanged. The specific operation is as follows: Use the optimal enzyme - producing medium obtained in Example 1, combined with the solid - state fermentation enzyme - producing conditions obtained by response surface optimization method in Example 2, that is, the moisture content is 53wt%, the fermentation time is 3 days, and the addition amount of Tween - 80 is 0.15 mL / g. Replace Trichoderma harzianum with Aspergillus niger therein and conduct solid - state fermentation to produce enzymes. Use the crude enzyme liquid extraction method and pre - incubation conditions obtained in Example 3, combined with the synchronous saccharification and protein production process in Example 4, to conduct solid - state fermentation to produce protein feed fermented by Aspergillus niger.

[0114] 2. Flavor detection

[0115] Use electronic nose and electronic tongue technologies to detect the flavors of 4 kinds of feeds (lactic acid - fermented feed, protein feed fermented by Trichoderma harzianum, protein feed fermented by Aspergillus niger, distiller's grains raw material). The results are as Figure 4 and Figure 5 shown. Figure 4 Principal component analysis (PCA) of electronic nose data shows that the cumulative contribution rate of principal component 1 and principal component 2 has exceeded 99%. This means that PCA can distinguish the odor differences between different samples, indicating that there are significant differences in odor among distiller's grains raw material, protein feed fermented by Aspergillus niger, protein feed fermented by Trichoderma harzianum, and lactic acid - fermented feed.

[0116] Figure 5 shows the electronic tongue signals of distiller's grains raw material and three kinds of fermented feed samples. 8 main flavor substances are detected. There are no significant differences among the 4 samples in terms of saltiness, richness, umami, astringency, and aftertaste, but there are obvious differences in sourness and bitterness. The tasteless point of sourness is - 13. Below this value, there is no sourness, otherwise there is. Through fermentation, the sourness of protein feed fermented by Trichoderma harzianum and Aspergillus niger is lower than the tasteless value, indicating no sourness. The distiller's grains raw material has the lowest pH and shows the strongest sourness. The reduction of sourness may lead to an increase in bitterness. The tasteless point of bitterness is 0. The bitterness intensity order of the four feeds is: protein feed fermented by Trichoderma harzianum > protein feed fermented by Aspergillus niger > lactic acid - fermented feed > distiller's grains raw material. Therefore, after lactic acid bacteria fermentation treatment, the flavor characteristics of protein feed fermented by Trichoderma harzianum have been significantly improved. Specifically, the bitterness in the feed is reduced, while the sourness is increased, and the overall flavor becomes more harmonious, enhancing the palatability.

[0117] Example 6: In vitro rumen digestion verification experiment

[0118] Artificial rumen culture solution was prepared by collecting fresh rumen fluid, and an in vitro digestion simulation experiment was carried out. The following steps were taken to collect fresh rumen fluid: Fill a thermos flask with hot water, and introduce CO2 to expel air to ensure an anaerobic environment. Quickly pour out the hot water, take the residue from the bovine rumen, wrap it with gauze and squeeze it, quickly collect the rumen fluid and store it in a collection bottle. Immediately after the collection, the sample was taken back to the laboratory for processing. After the rumen fluid brought back to the laboratory was stirred and mixed evenly, it was filtered through four layers of gauze to remove large feed particles and residues. CO2 gas was continuously introduced into the filtered rumen fluid to maintain an anaerobic environment. After filtration, the rumen fluid was quickly mixed with Menke medium at a ratio of 1:2 to prepare a mixed artificial rumen culture solution.

[0119] Among them, the Menke medium is as follows:

[0120] (1) Trace element solution (solution A): 13.20 g of CaCl2·2H2O, 10.00 g of MnCl2·4H2O, 1.00 g of CoCl2·6H2O, 8.00 g of FeCl3·6H2O. Add deionized water to make up to 100 mL, and after volume fixation, store it in a refrigerator at 4°C.

[0121] (2) Buffer solution (solution B): 4.00 g of NH4HCO3, 35.00 g of NaHCO3. Add deionized water to make up to 1000 mL, and after volume fixation, store it in a refrigerator at 4°C.

[0122] (3) Macroelement solution (solution C): 9.45 g of Na2HPO4·12H2O, 6.20 g of KH2PO4, 0.60 g of MgSO4·7H2O. Add deionized water to make up to 1000 mL, and after volume fixation, store it in a refrigerator at 4°C.

[0123] (4) 0.1% Resazurin solution (solution D): Add 100 mg of resazurin to 100 mL of deionized water. After volume fixation, transfer it to a bottle, seal it with a rubber stopper and an aluminum cap, wrap it with aluminum foil on the outside for light protection, and store it in a refrigerator at 4°C. When in use, draw the solution with a syringe.

[0124] (5) Reducing agent solution (solution E): 625 mg of L-cysteine hydrochloride, 4.00 mL of 1 M NaOH solution, 625 mg of Na2S·9H2O, 95 mL of distilled water. The reducing agent solution needs to be prepared and used immediately, and added to the Menke medium when about to add rumen fluid.

[0125] Add the prepared solutions A, B, C, D and distilled water into the Erlenmeyer flask in sequence according to the proportion, and gently shake the Erlenmeyer flask to fully mix the solutions. Subsequently, place the Erlenmeyer flask in a constant temperature water bath (maintaining the temperature at 39 °C), and continuously introduce CO2 gas into the flask for about 1 h. After that, use a portable pH meter to measure the pH value of the culture medium. If the pH is lower than 6.8, adjust it by dropwise adding 2 mol / L NaOH solution until the pH reaches 6.8. After the adjustment is completed, add the reducing agent solution (Solution E) to remove the residual oxygen in the solution. When the color of the culture medium turns to light yellow, add the filtered rumen fluid.

[0126] In the experiment, 4 sampling points were set, and the fermentation times were 4 h, 14 h, 24 h and 48 h respectively. A blank control group was set at each sampling point. The blank control group was artificial rumen culture medium without adding substrates. The mixed artificial rumen culture medium in the experimental group was dispensed into 500 mL Erlenmeyer flasks, 200 mL was dispensed into each flask, and 1.5 g of substrate was added. The bottle mouth was sealed with an isobutyl rubber stopper and fixed with an aluminum cap. Subsequently, the Erlenmeyer flasks were placed in a 39 °C constant temperature oscillator for cultivation, and the oscillation frequency was set at 150 rpm.

[0127] After fermentation at each stage, quickly take out the sample from the water bath shaker and place it in ice water to terminate the fermentation. Filter the rumen fermentation fluid with four layers of gauze, wash it three times with pure water, and dry the filtered solid at 105 °C to constant weight for subsequent determination.

[0128] The in vitro dry matter digestibility (IVDMD) was calculated according to formula (1):

[0129]

[0130] In the formula:

[0131] IVDMD: in vitro dry matter digestibility (%);

[0132] m: the mass of the dry matter of the substrate after fermentation (g);

[0133] M: the mass of the dry matter before fermentation (g).

[0134] The crude protein content of the dried sample was determined by the Kjeldahl method, and the in vitro crude protein digestibility (IVCPD) was calculated by the following formula (2):

[0135]

[0136] In the formula:

[0137] IVCPD: Crude protein degradation rate (%);

[0138] CP 后 : Crude protein content after digestion (%);

[0139] CP 前 : Crude protein content before digestion (%).

[0140] By detecting the dry matter degradation rate and the crude protein degradation rate, feed that is easy to digest is speculated. The results are as Figure 6 shown. Trichoderma harzianum fermented protein feed performs excellently in terms of the dry matter degradation rate and the crude protein degradation rate, reaching 69.0% and 78.5% respectively. Compared with Aspergillus niger fermented protein feed, the dry matter degradation rate and the crude protein degradation rate of Trichoderma harzianum fermented protein feed are increased by 3.4% and 8.0% respectively. Therefore, Trichoderma harzianum fermented protein feed is more conducive to digestion and easier for animals to absorb.

[0141] The present invention provides an idea and method for a method of producing protein feed by solid-state fermentation of distillers' grains pretreated with Trichoderma harzianum. There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by existing technologies.

Claims

1. A method for producing protein feed by solid-state fermentation of distillers grains based on Trichoderma harzianum pretreatment, characterized in that, It includes the following steps: (1) Inoculate the spore suspension of Trichoderma harzianum into an enzyme-producing medium for solid-state fermentation to obtain a fermented solid; (2) Extract the crude enzyme solution of cellulase from the fermented solid, and perform pre-incubation treatment on the crude enzyme solution with a surfactant to obtain the treated crude enzyme solution; (3) Add the treated crude enzyme solution to the distiller's grains raw material, and at the same time inoculate the seed solution of yeast into the distiller's grains raw material added with the treated crude enzyme solution for synchronous saccharification fermentation to prepare Trichoderma harzianum fermented protein feed.

2. The method according to claim 1, characterized in that, In step (1), the enzyme-producing medium includes a solid part, a liquid part and an additive; Among them, the solid-liquid ratio of the solid part to the liquid part is 1 g: 0.5-1.5 mL; Among them, the solid part includes any one or a combination of several of corn straw, distiller's grains sieved rice husk and wheat bran; the mass ratio of corn straw, distiller's grains sieved rice husk and wheat bran is 0-6: 0-10: 0-2, and the three are not 0 at the same time; Among them, the formula of the liquid part is as follows: ammonium sulfate 1-2 g / L, magnesium sulfate 2-5 g / L, potassium dihydrogen phosphate 2-8 g / L; Among them, the additive is Tween-80, and the addition amount is 0.005-0.015 mL / g, based on the solid part of the enzyme-producing medium.

3. The method according to claim 2, characterized in that, In step (1), for the inoculation, the inoculation amount is 4×10 6 ~1×10 7 spores / g, based on the solid part of the enzyme-producing medium.

4. The method according to claim 1, wherein In step (1), for the solid-state fermentation, the fermentation conditions are: the initial pH value is natural, and it is cultured at 25-30 °C for 3-5 days.

5. The method according to claim 1, wherein In step (2), the surfactant includes any one or a combination of several of Tween-20, Tween-80, PEG4000, PEG6000; the addition amount of the surfactant is 0.01-0.05 g of surfactant added to each 1 mL of crude enzyme solution.

6. The method according to claim 1, wherein In step (2), for the pre-incubation treatment, the treatment conditions are: incubate at 0-4 °C for 12 h.

7. The method according to claim 1, characterized in that In step (3), the distiller's grains raw material includes crushed distiller's grains and uncrushed distiller's grains, and the mass ratio of crushed distiller's grains to uncrushed distiller's grains is 0-10: 0-10, and the two are not 0 at the same time; the addition amount of the treated crude enzyme solution is such that the moisture content of the distiller's grains raw material is 55-70 wt%.

8. The method according to claim 1, characterized in that In step (3), the yeasts are Candida utilis, Saccharomyces cerevisiae, and Rhodotorula benthica; the inoculation volume ratio of Candida utilis, Saccharomyces cerevisiae, and Rhodotorula benthica is 1:1:

1. The inoculation amount of the seed liquid of the three yeasts is based on the dry weight of the distiller's grains raw material. For every 100 g of the distiller's grains raw material, 2 - 5 mL of the seed liquid of Candida utilis, the seed liquid of Saccharomyces cerevisiae, and the seed liquid of Rhodotorula benthica are added respectively; for the simultaneous saccharification and fermentation, the conditions are: carry out simultaneous saccharification and fermentation at 25 - 30 °C for 7 - 11 days.

9. The method according to claim 1, wherein In step (3), dry and crush the Trichoderma harzianum fermented protein feed, adjust the pH to 5 - 6 and the moisture content to 50 - 60 wt% to obtain a fermented material. Inoculate the seed liquid of Limosilactobacillus fermentum into the fermented material, and add glucose to carry out lactic acid fermentation to prepare a lactic acid fermented feed.

10. The method according to claim 9, wherein For the seed liquid of Limosilactobacillus fermentum, the inoculation amount is based on the dry weight of the fermented material. For every 100 g of the fermented material, 2 - 5 mL of the seed liquid of Limosilactobacillus fermentum is added; for the glucose, based on the dry weight of the fermented material, 2 - 5 mL of glucose is added to every 100 g of the fermented material; for the lactic acid fermentation, the conditions are: ferment at 30 °C - 37 °C for 15 - 45 days under vacuum conditions.

Images