Method for producing single-cell protein feed through synergy of enzyme and bacteria

Abstract

The invention relates to a method for producing a single-cell protein feed through synergy of enzymes and bacteria. The method comprises the following steps: adding a crushed straw raw material into compound cellulase compounded by endoglucanase, exonuclease and beta-glucosidase for enzymolysis; adding aspergillus oryzae and trichoderma longibrachiatum into the raw materials subjected to enzymolysis for fermentation; and then adding candida utilis for continuous fermentation to obtain the single-cell protein feed. The method has the advantages of short production time, high protein yield of the single-cell protein feed, low condition requirements in the production process, low production cost and the like.

Description

Technical Field

[0001] The invention relates to the technical field of feed production, and in particular to a method for producing single-cell protein feed in a coordinated manner with enzymes and bacteria. Background Art

[0002] Single-cell protein, also known as biological bacterial protein or microbial protein, refers to the bacterial protein produced by single-cell microorganisms such as yeast, fungi, molds, and non-pathogenic bacteria. By using this principle and the fermentation of microorganisms, various waste materials in industry and agriculture, such as straw, distiller's grains, sugar residues, wood, and waste products from the paper industry, are converted into single-cell protein feed, also known as microbial protein feed.

[0003] Single-cell protein feed can be used to feed all kinds of poultry and livestock. It not only has high protein content, but also contains fat, carbohydrates, nucleic acids, vitamins and inorganic salts, as well as various amino acids necessary for animal bodies, especially lysine, methionine and tryptophan that are lacking in plant feed. Its biological value is much better than that of plant protein feed. It also has the advantages of fast growth, high efficiency, easy cultivation and easy collection.

[0004] The types of microorganisms commonly used to prepare single-cell protein feeds are: bacteria, fungi (yeast, mold, basidiomycetes) and microalgae. The main bacteria are lactic acid bacteria, fecal streptococci, bifidobacteria, photosynthetic bacteria, bacillus, cellulose decomposing bacteria, etc.; the main yeasts are brewer's yeast, Candida utilis, Candida tropicalis, Candida lipolytica, etc.; the main molds are Rhizopus, Aspergillus, Penicillium and Trichoderma, etc.; the main basidiomycetes are Microcystis microdentata and Willow Leaf Parasol, and there are a large number of edible fungi such as Cibotium chinense and Auricularia auricula; the main microalgae are Spirulina and Chlorella, etc.

[0005] Although some technologies for the production and preparation of single-cell protein feed are recorded in existing literature, the methods in the existing technology have disadvantages such as long production time, high production cost, complex process and high cost. Therefore, there is still room for improvement in production efficiency, cost control, protein yield and other indicators. Summary of the invention

[0006] The purpose of the present invention is to provide a method for the coordinated production of single-cell protein feed by enzyme and bacteria, which has the advantages of short production time, high single-cell protein feed protein output rate, low production process condition requirements, low production cost, etc.

[0007] To achieve the above object, the technical solution of the present invention is as follows: A method for producing single-cell protein feed in collaboration with enzymes and bacteria, characterized in that: a composite cellulase composed of endoglucanase, exoglucanase and beta-glucosidase is added to the crushed straw raw material for enzymolysis; Aspergillus oryzae and Trichoderma longibrachiatum are added to the raw material after enzymolysis for fermentation; and then Candida utilis is added to continue fermentation for 24-48 hours to obtain the single-cell protein feed.

[0008] The ratio of endoglucanase, exoglucanase and beta-glucosidase in the composite cellulase is 1:1:1, and the added amount is 1 / 500 to 1 / 1500 relative to the dry matter of the straw.

[0009] The ratio of Aspergillus oryzae to Trichoderma longibotium is 1:1, and the added amount is 10-20% of the weight of the straw.

[0010] The added amount of the Candida utilis is 10-20% of the weight of the straw.

[0011] The following steps are also included before and after the enzymatic hydrolysis and fermentation steps: A. Chopping: Chop the straw into pieces of 2-4 cm; B. Add bacteria to store: Add lactic acid bacteria to the raw materials and mix them evenly before storing them anaerobically; add lactic acid bacteria for early fermentation. Lactic acid bacteria are common bacteria in the intestines of most animals. Lactic acid bacteria can regulate the intestines and form a protective film, thereby reducing intestinal diseases. After adding lactic acid bacteria to the raw materials, the pH value can also be lowered, and miscellaneous bacteria cannot grow, thus avoiding the phenomenon of mold and corruption of the raw materials during storage.

[0012] C. Crushing: crush the raw materials into 5 mesh particles; D. Bacteria culture: Aspergillus oryzae, Trichoderma longibotium and Candida utilis were cultured; E. Enzymatic hydrolysis: adding composite cellulase, including endoglucanase, exoglucanase and β-glucosidase, to the raw material obtained in step D to perform enzymatic hydrolysis of cellulose; the composite cellulase converts cellulose, lignin and hemicellulose in the straw into glucose; F. Primary fermentation: adding Aspergillus oryzae and Trichoderma longibrachiatum to the raw material after enzymatic hydrolysis in step E and mixing them for fermentation; the mixed strains utilize their synergistic effect to further enhance the fermentation and decomposition of cellulose, lignin and hemicellulose in the straw; G. Secondary fermentation: Add Candida utilis for further fermentation; Candida utilis uses the glucose produced by enzymatic hydrolysis and primary fermentation to grow rapidly and produce a large amount of protein H. Drying: Dry the raw materials to make their moisture content less than 10%; I. Product inspection and packaging.

[0013] The enzymolysis time is 7-12h.

[0014] The pH value of the enzymatic hydrolysis is 4.5-5.5.

[0015] The temperature of the enzymatic hydrolysis is 45-55°C.

[0016] The time of the primary fermentation is 2-8 days.

[0017] The secondary fermentation time is 24-72 hours.

[0018] The strain culture in step D adopts three-stage culture, and the culture medium is YPD. YPD culture medium is also called yeast extract peptone glucose culture medium. The three-stage culture can play a role in expansion and provide a continuous supply of high-quality strains for production.

[0019] After the chopping in step A, the broken materials can be added with lactic acid bacteria and then packaged and baled for anaerobic storage. The advantages of anaerobic storage by packaging and baling are that the raw materials are stacked and placed to save storage space; the internal environment of the bale is anoxic, which is more conducive to the growth and fermentation of lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flowchart of the preparation method; Figure 2 This is the relationship curve between crude fiber degradation rate and temperature during the enzymatic hydrolysis stage; Figure 3 This is the relationship curve between crude fiber degradation rate and pH value during the enzymatic hydrolysis stage; Figure 4 This is the relationship curve between crude fiber degradation rate and time during the enzymatic hydrolysis stage. Advantages of the invention

[0022] 1. The single-cell protein feed production method of the present invention has a short production time. The entire production process takes less than one week, which greatly shortens the feed production cycle and improves production efficiency.

[0023] 2. The single-cell protein feed of the present invention can be produced using straw with a high fiber content as a raw material, and the protein content of the final feed is rapidly and substantially increased, which has great nutritional value for livestock and greatly improves the quality of the feed.

[0024] 3. The composite enzyme preparation of the present invention has a good digestive effect on cellulose: (1) endoglucanase, also known as EG enzyme or Cx enzyme; (2) exoglucanase, also known as cellobiohydrolase CBH or C1 enzyme; (3) β-glucosidase, referred to as BG.

[0025] Composite cellulase plays a good synergistic role: endoglucanase first acts on the amorphous region of microcellulose, randomly hydrolyzes β-1,4-glycosidic bonds, and produces a large number of small molecular celluloses with non-reducing ends. Exoglucanase hydrolyzes β-1,4-glycosidic bonds from these non-reducing ends in turn to generate cellobiose and other low molecular cellodextrins, which are hydrolyzed into glucose molecules under the action of β-glucosidase. This synergistic effect is ubiquitous. In addition to the above synergistic effect, it can also occur between endonucleases, between exonucleases, and even between endonucleases and exonucleases from different bacterial sources. The synergistic effect is proportional to the crystallinity of the enzymatic substrate.

[0026] Cellulase preferentially acts on the amorphous region of cellulose and has a certain degree of degradation on crystalline cellulose, but it is difficult. Through research, EG and CBH can cause the dispersion and defibrillation of cellulose, disrupting the crystalline structure of cellulose and causing denaturation. Cellulase penetrates into the interface of cellulose molecules, increasing the pressure of the pore wall, cavity wall and microcrack wall, and water molecules intervene to destroy the hydrogen bonds between cellulose molecules and produce partially soluble microcrystals. The mechanism of action of a single component in cellulase is similar to that of lysozyme, following a double displacement mechanism. Implementation Example

[0027] A method for producing single-cell protein feed in collaboration with enzymes and bacteria, characterized in that: a composite cellulase composed of endoglucanase, exoglucanase and cellobiase is added to crushed straw raw materials for enzymolysis; Aspergillus oryzae and Trichoderma longibrachiatum are added to the raw materials after enzymolysis for fermentation; and then Candida utilis is added to continue fermentation for 24-48 hours to obtain the single-cell protein feed.

[0028] The ratio of endoglucanase, exoglucanase and cellobiase in the composite cellulase is 1:1:1, and the added amount is 1 / 1000 relative to the dry matter of the straw.

[0029] The ratio of Aspergillus oryzae to Trichoderma longibrachiatum is 1:1, and the added amount is 20% of the weight of the straw.

[0030] The added amount of the Candida utilis is 20% of the weight of the straw.

[0031] The following steps are also included before and after the enzymatic hydrolysis and fermentation steps: A. Chopping: Chop the straw into pieces of 2-4 cm; B. Add bacteria to store: Add lactic acid bacteria to the raw materials and mix evenly, then store anaerobically; C. Crushing: crush the raw materials into 5 mesh particles; D. Bacteria culture: Aspergillus oryzae, Trichoderma longibotium and Candida utilis were cultured; E. Enzymatic hydrolysis: adding composite cellulase, including endoglucanase, exoglucanase and cellobiase, to the raw material obtained in step D to perform enzymatic hydrolysis of cellulose; F. Primary fermentation: adding Aspergillus oryzae and Trichoderma longibrachiatum to the raw material after enzymolysis in step E for fermentation; G. Secondary fermentation: adding Candida utilis for further fermentation; H. Drying: Dry the raw materials to make their moisture content less than 10%; I. Product inspection and packaging.

[0032] The enzymatic hydrolysis time is 12h.

[0033] The pH value of the enzymatic hydrolysis is 5.

[0034] The temperature of the enzymatic hydrolysis is 50°C.

[0035] The time of the primary fermentation is 2 days.

[0036] The secondary fermentation time is 24 hours.

[0037] Application examples: Initial nutrient content of corn stover (dry matter basis) %

[0038] Enzymatic hydrolysis test: A. Detect the moisture content of corn stalks, weigh the corn stalks after yellowing (converted to 50g dry matter), add complex enzyme in an amount of 1 / 1000 of the dry matter of the raw material, and the total enzyme amount is 0.05g.

[0039] B. Place the raw materials into a steam sterilizer for sterilization (121°C, 30 min).

[0040] C. After sterilization is completed and the mixture is cooled, 20% sodium hydroxide solution is added to adjust the pH, and compound enzyme is added for enzymolysis.

[0041] D. After the enzymatic hydrolysis is completed, the raw materials are placed in an electric constant temperature forced air drying oven for drying (105°C, 6h).

[0042] E. Crush the dried raw materials and pass through a 40-mesh sieve.

[0043] F. The crude fiber, neutral detergent fiber and acid detergent fiber contents of the treated raw materials are determined using a near infrared detector.

[0044] G. Take some samples manually to determine the contents of crude fiber, neutral detergent fiber and acid detergent fiber for inspection.

[0045] 1. Effect of enzymatic hydrolysis temperature on crude fiber degradation rate Depend on Figure 2 It can be seen that when the enzymatic hydrolysis temperature is between 30℃ and 50℃, the crude fiber degradation rate increases with the increase of the enzymatic hydrolysis temperature; when the enzymatic hydrolysis temperature is between 45℃ and 50℃, there is a higher crude fiber degradation rate; when the enzymatic hydrolysis temperature is greater than 50℃, the crude fiber degradation rate shows a downward trend. Therefore, the optimal enzymatic hydrolysis temperature for the composite enzyme to hydrolyze corn straw is 50℃.

[0046] 2. Effect of pH on crude fiber degradation rate Depend on Figure 3 It can be seen that pH has a significant effect on the crude fiber degradation rate. When the pH is 5.0, the crude fiber degradation rate is the highest. When the pH is 4.4-5.0, the crude fiber degradation rate gradually increases; when the pH is 5.0-5.6, the crude fiber degradation rate gradually decreases. Therefore, the optimal pH for the enzyme to hydrolyze corn straw is 5.0.

[0047] 3. Effect of enzymatic hydrolysis time on crude fiber degradation rate Depend on Figure 4 It can be seen that, overall, the crude fiber degradation rate tends to increase with the extension of enzymatic hydrolysis time. When the enzymatic hydrolysis time is 7h-12h, the crude fiber degradation rate does not change much, only increasing by about 1%, while when the enzymatic hydrolysis time is 6h-7h, the crude fiber degradation rate changes greatly, increasing by about 1%.

[0048] Orthogonal test: Table 1: Orthogonal test results

[0049] Range analysis of crude fiber degradation rates under different treatments Table 2: Analysis of the extreme difference in crude fiber degradation rate:

[0050]

[0051] Note: K1, K2, K3 represent the sum of the observations of the same factor at different levels, k1, k2, k3 represent the average of the observations of the same factor at different levels, R represents the range.

[0052] As can be seen from Table 2, different factors and levels have different effects on crude fiber degradation rate. From the perspective of the range value R, the range of the enzymatic hydrolysis time (B) factor is the largest, which is 0.99, which is greater than the other two factors. The larger the range, the greater the impact of the level change of the factor on the test results. Therefore, it is shown that the enzymatic hydrolysis time has the greatest influence on crude fiber degradation and is the most important factor affecting crude fiber degradation rate, followed by pH, and then enzymatic hydrolysis temperature. From the average value of the observed values ​​of different factors at different levels, the highest crude fiber degradation rate is the third level of the enzymatic hydrolysis time (B) factor, that is, 12h is the best; the best pH (C) factor is the second level, that is, 5.0, the highest crude fiber degradation rate; the best enzymatic hydrolysis temperature (A) factor is the third level, that is, 50℃.

[0053] Effects of different factors and treatment levels on crude fiber degradation rate Table 3: ANOVA of crude fiber degradation rate under different factors and levels

[0054] From the variance analysis results in Table 3, it can be seen that enzymatic hydrolysis temperature, enzymatic hydrolysis time and pH have significant effects on crude fiber degradation rate, among which: enzymatic hydrolysis time has the greatest effect, followed by pH, and enzymatic hydrolysis temperature has the least effect. Among the three different levels of enzymatic hydrolysis conditions of enzymatic hydrolysis temperature, the best enzymatic hydrolysis temperature is 50℃; among the three different levels of enzymatic hydrolysis conditions of enzymatic hydrolysis time, the best enzymatic hydrolysis time is 12h; among the three different levels of enzymatic hydrolysis conditions of pH, the best pH is 5.0. Therefore, the selection of the optimal enzymatic hydrolysis temperature, enzymatic hydrolysis time and pH combination can significantly improve the crude fiber degradation rate. A3B3C2 is the best experimental combination, that is, the enzymatic hydrolysis condition combination of 50℃ enzymatic hydrolysis temperature, 12h enzymatic hydrolysis time and pH value of 5.0.

[0055] Fermentation experiment: 1. Bacteria culture Aspergillus oryzae, Trichoderma longifolia liquid culture The spores on the working plate were washed into 300 mL of YPD medium with 10 mL of sterile water and incubated at 28°C and 180 rpm until the bacteria became thick.

[0056] Candida utilis Use an inoculation loop to pick up a loop of bacteria on the slant, put it into 300 mL of YPD medium, and culture it at 28°C and 180 r / min for 18 h.

[0057] 2. Batching process Using crude corn stalks as fermentation raw materials:

[0058] (1) Weigh the coarse corn stalks, add water to adjust the moisture content to 60%, and sterilize them in a sterilizer at 121℃ for 30 minutes.

[0059] (2) After cooling, divide the mixture into 200g portions. Use a spray bottle to add 6g of complex cellulase solution to each portion and mix well. Add 40g of bacterial solution to 40g of sterilized bran. Then mix the bran and straw materials well. Place the mixture in a culture room and ferment at room temperature for 48h.

[0060] (3) Add 40 g of Candida utilis solution and continue culturing for 24 h.

[0061] (4) Dry the material at 120°C and then crush and test it.

[0062]

[0063] It can be seen that the production of crude protein by the composite mold is about 3 times higher than that of single Aspergillus oryzae or Trichoderma longibotium, and the increase in protein is significant.

Claims

1. A method for producing single-cell protein feed by enzyme-bacteria collaboration, characterized in that: The crushed straw raw material is added with a composite cellulase composed of glucan endonuclease, glucan exonuclease and β-glucosidase for enzymolysis; the raw material after enzymolysis is added with Aspergillus oryzae and Trichoderma longibrachiatum for fermentation; then Candida utilis is added to continue fermentation to obtain single-cell protein feed.

2. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The ratio of endoglucanase, exoglucanase and beta-glucosidase in the composite cellulase is 1:1:1, and the added amount is 1 / 500 to 1 / 1500 relative to the dry matter of the straw.

3. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The ratio of Aspergillus oryzae to Trichoderma longibotium is 1:1, and the added amount is 10-20% of the weight of the straw.

4. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The added amount of the Candida utilis is 10-20% of the weight of the straw.

5. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The following steps are also included before and after the enzymatic hydrolysis and fermentation steps: A. Chopping: Chop the straw into pieces of 2-4 cm; B. Add bacteria to store: Add lactic acid bacteria to the raw materials and mix evenly, then store anaerobically; C. Crushing: crush the raw materials into 5 mesh particles; D. Bacteria culture: Aspergillus oryzae, Trichoderma longibotium and Candida utilis were cultured; E. Enzymatic hydrolysis: adding composite cellulase, including endoglucanase, exoglucanase and β-glucosidase, to the raw material obtained in step D to perform enzymatic hydrolysis of cellulose; F. Primary fermentation: adding Aspergillus oryzae and Trichoderma longibrachiatum to the raw material after enzymolysis in step E for fermentation; G. Secondary fermentation: adding Candida utilis for further fermentation; H. Drying: Dry the raw materials to make their moisture content less than 10%; I. Product inspection and packaging.

6. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The enzymatic hydrolysis time is 7-12h.

7. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The pH value of the enzymatic hydrolysis is 4.5-5.

5.

8. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The temperature of the enzymatic hydrolysis is 45-55°C.

9. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The time of the primary fermentation is 2-8 days.

10. The method for producing single-cell protein feed by enzyme-bacteria collaboration according to claim 1, characterized in that: The secondary fermentation time is 24-72 hours.

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