Complex enzyme preparation for reducing fiber content and increasing oligosaccharide content of sunflower seed meal

Abstract

The invention relates to the field of feed, in particular to a compound enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content. Based on 50 parts by weight of raw materials, the feed additive comprises 3-5 parts of xylanase, 3-5 parts of acidic cellulase, 3-5 parts of laccase, 3-5 parts of feruloyl esterase, 3-5 parts of saccharifying enzyme, 6-8 parts of alkaline protease and the balance of a carrier. The compound enzyme preparation provided by the invention can more efficiently degrade crude fibers in sunflower seed meal, and the degradation rate of the crude fibers exceeds 40% and is greatly higher than that of the conventional fermentation method and enzymolysis method.

Description

Technical Field

[0001] The present invention relates to the field of feed, and in particular to a composite enzyme preparation capable of reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content. Background Art

[0002] Sunflower seed meal is the material remaining after the oil is extracted from sunflower seeds through mechanical pressing or solvent extraction. It is rich in 29% to 43% high-quality plant protein and has a relatively balanced amino acid composition. The protein types are mainly globulin, albumin and gluten, and the content of indigestible alcohol-soluble protein is less than 5%.

[0003] Sunflower meal, as a protein feed, has many limiting factors, among which high crude fiber content is the primary factor limiting its use in feed. When the fiber content in the feed is too high, the animal's digestibility will decrease significantly, thereby affecting its absorption and utilization of other nutrients. Studies have found that adding 15% sunflower meal to the feed significantly reduces the daily weight gain of yellow-feathered broilers between 51 and 70 days old, reduces feed conversion efficiency, and increases feed consumption to weight gain ratio. Furthermore, as the amount of sunflower meal increases and the crude fiber content rises, feed conversion efficiency tends to decline further.

[0004] To improve the feeding effect of sunflower meal, fermentation is used to reduce the crude fiber content of sunflower meal and increase the utilization rate of nutrients. CN 114027435 A uses Bacillus subtilis, Saccharomyces cerevisiae, and Trichoderma longifolia to ferment and pretreat sunflower meal. The fermentation can degrade anti-nutritional factors such as crude fiber, chlorogenic acid, and phytic acid in the sunflower meal by 30% to 40%, with the crude fiber degradation rate reaching 25% to 30%. At the same time, the crude protein content can be increased by 20% to 25%, and the small molecule protein content can be increased by approximately 17% to 20%.

[0005] In addition to fermentation, the utilization rate of sunflower seed meal can be improved by adding complex enzymes to degrade anti-nutritional factors in sunflower seed meal. For example, CN 115340988 B provides a complex enzyme preparation including four single enzymes: phytase, neutral protease, xylanase, and cellulase, which can effectively improve the utilization rate of sunflower seed meal and the use ratio and feeding effect of enzymatically hydrolyzed sunflower meal in grass carp feed. However, the lignin in sunflower seed meal forms a cross-linked structure with cellulose and hemicellulose. In the absence of effective technical means to remove lignin, the high content of lignin hinders the removal of the other two components. Therefore, ordinary enzymatic hydrolysis and fermentation methods cannot effectively reduce the crude fiber content in sunflower seed meal.

[0006] Enzymatic removal of anti-nutritional factors such as xylans and mannans can produce a certain proportion of xylooligosaccharides and mannan oligosaccharides. Oligosaccharides, as prebiotics, can enhance the immunity of farmed animals, improve the balance of intestinal microbial flora, promote digestion and absorption, increase feed conversion rate, reduce the incidence of disease, and improve meat quality and growth rate.

[0007] In summary, there is currently no composite enzyme preparation that can effectively degrade the crude fiber content in sunflower meal (removal rate of more than 40%) and increase the oligosaccharide content. The present invention provides a composite enzyme preparation that can significantly degrade the crude fiber in sunflower meal and effectively increase the oligosaccharide content in enzymatically hydrolyzed sunflower meal. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a composite enzyme preparation which can significantly reduce the crude fiber content in sunflower meal and simultaneously increase the oligosaccharide content.

[0009] To solve the above technical problems, the present invention provides a complex enzyme preparation, based on 50 parts by weight of raw materials, the complex enzyme preparation includes 3-5 parts of xylanase, 3-5 parts of acid cellulase, 3-5 parts of laccase, 3-5 parts of ferulic acid esterase, 3-5 parts of saccharifying enzyme, 6-8 parts of alkaline protease, and the balance carrier.

[0010] The complex enzyme preparation according to the present invention comprises, based on 50 parts by weight of raw materials, 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 3-5 parts of ferulic acid esterase, 3-5 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier.

[0011] According to the complex enzyme preparation of the present invention, based on 50 parts by weight of raw materials, 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance is carrier.

[0012] According to the complex enzyme preparation of the present invention, the ratio of the ferulic acid esterase activity (U) to the xylanase activity (U) is 1:500.

[0013] According to the complex enzyme preparation of the present invention, the ratio of the laccase activity (U) to the saccharifying enzyme activity (U) is 1:5.

[0014] According to the composite enzyme preparation of the present invention, the enzymatic activity of the xylanase is 50,000 u / g, the enzymatic activity of the acid cellulase is 1,000 u / g, the enzymatic activity of the laccase is 2,000 u / g, the enzymatic activity of the ferulic acid esterase is 100 u / g, the enzymatic activity of the saccharifying enzyme is 10,000 u / g, and the enzymatic activity of the alkaline protease is 50,000 u / g.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention significantly improves the degradation rate of lignin and further improves the degradation efficiency of crude fiber by adding xylanase, ferulic acid esterase and acid cellulase through the synergistic effect of compounding laccase and saccharifying enzyme. According to the technical solution of the present invention, compared with a single laccase, the laccase and saccharifying enzyme are compounded according to the preferred ratio, and the lignin degradation rate is increased by 1.71 times, and the crude fiber degradation rate is increased by 52.95%; for the laccase + saccharifying enzyme group and the laccase group with the same total number of parts, the laccase and saccharifying enzyme are compounded according to the preferred ratio, and the lignin degradation rate is increased by 1.51 times, and the crude fiber degradation rate is increased by 51.52%; compared with a single saccharifying enzyme, the laccase and saccharifying enzyme are compounded according to the preferred ratio, and the lignin degradation rate is increased by 1.51 times, and the crude fiber degradation rate is increased by 45.52%; for the laccase + saccharifying enzyme group and the saccharifying enzyme group with the same total number of parts, the laccase and saccharifying enzyme are compounded according to the preferred ratio, and the lignin degradation rate is increased by 1.35 times, and the crude fiber degradation rate is increased by 44.69%. The composite enzyme preparation provided by the present invention can more efficiently degrade crude fiber in sunflower seed meal, wherein the crude fiber degradation rate exceeds 50%, which is significantly higher than the current fermentation method and enzymatic hydrolysis method.

[0017] 2. The present invention makes full use of the synergistic effect of feruloyl esterase and xylanase when adding acid cellulase, laccase and saccharifying enzyme, and compounding feruloyl esterase and xylanase according to the preferred ratio can greatly increase the production of oligosaccharides. Compared with the single xylanase group, the oligosaccharide production rate of feruloyl esterase and xylanase compounded according to the preferred ratio of this application is increased by 51.61%; compared with the single saccharifying enzyme, the oligosaccharide production rate of feruloyl esterase and xylanase compounded according to the preferred ratio of this application is increased by 64.03%. The composite enzyme provided by the present invention can effectively increase the oligosaccharide content in sunflower seed meal, which is beneficial to improving the health level of livestock and poultry after feeding sunflower seed meal. DETAILED DESCRIPTION

[0018] In the following embodiments:

[0019] 1. Enzyme source and sample preparation: xylanase 50000u / g, acid cellulase 1000u / g, laccase 2000u / g, ferulic acid esterase 100u / g, saccharifying enzyme 10000u / g, alkaline protease 50000u / g;

[0020] 2. Preparation of enzymatic buffer and enzymatic hydrolysis method:

[0021] Enzyme hydrolysis buffer: Weigh 6.02 g of disodium hydrogen phosphate and 0.5 g of sodium dihydrogen phosphate, add 900 mL of deionized water, stir evenly, adjust the pH to 7.5 with 1 mol / L hydrochloric acid solution or 0.5 mol / L sodium hydroxide solution, and make up to 1000 mL.

[0022] Enzymatic hydrolysis method: crush the sunflower meal and pass it through a 40-mesh sieve. Accurately weigh 100 g of sunflower meal sample and place it in an enzymatic hydrolysis reaction vessel. Weigh 0.8 g of the corresponding component complex enzyme preparation and dissolve it in 200 ml of buffer solution. Pour the enzyme-containing buffer solution into the enzymatic hydrolysis reaction vessel and mix evenly. Seal the container with sealing film and perform enzymatic hydrolysis at 60 °C for 12 h. After the enzymatic hydrolysis is completed, dry it in an oven at 65 °C for testing.

[0023] 3. Measurement indicators and specific methods:

[0024] Lignin determination method: Acid hydrolysis method is used. Weigh a certain amount of sunflower meal sample (accurate to 0.0001g) and place it in a stoppered conical flask. Add 72% sulfuric acid and stir the reaction at 25°C for 2.5 hours. Then dilute the reaction solution to 3% sulfuric acid, heat and hydrolyze in a boiling water bath for 1.5 hours, filter to obtain the residue, and wash the residue with hot water until neutral. After drying the residue, weigh it and calculate the lignin content. The lignin content calculation formula is: Lignin content (%) = (m1 / m0) × 100%, where m1 is the residue mass (i.e., lignin mass) and m0 is the sample mass.

[0025] Crude fiber determination method: Crude fiber was determined using an ANKOMA 2000i fiber determination instrument according to the analytical method for determining crude fiber CF using the filter bag technique in the instruction manual.

[0026] Oligosaccharide determination method: The oligosaccharide extraction, separation, and purification of sunflower seed meal were carried out using the method of invention patent CN102086217A; quantitative determination was performed using a Shimadzu LC-64 liquid chromatograph by HPLC, and the specific steps were carried out according to the instructions.

[0027] Example 1

[0028] A composite enzyme preparation is prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier.

[0029] Example 2

[0030] A composite enzyme preparation is prepared, based on 50 parts by weight of raw materials, including 5 parts of xylanase, 5 parts of acid cellulase, 5 parts of laccase, 5 parts of ferulic acid esterase, 5 parts of saccharifying enzyme, 8 parts of alkaline protease, and the balance of carrier.

[0031] Example 3

[0032] A composite enzyme preparation is prepared, based on 50 parts by weight of raw materials, comprising 3 parts of active xylanase, 3 parts of acid cellulase, 3 parts of laccase, 3 parts of ferulic acid esterase, 3 parts of saccharifying enzyme, 6 parts of alkaline protease, and the balance being a carrier.

[0033] Comparative Example 1

[0034] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance being a carrier. Compared with Example 1, no xylanase was compounded.

[0035] Comparative Example 2

[0036] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, no acid cellulase was compounded.

[0037] Comparative Example 3

[0038] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, laccase was not compounded.

[0039] Comparative Example 4

[0040] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance being a carrier. Compared with Example 1, no ferulic acid esterase was compounded.

[0041] Comparative Example 5

[0042] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 7 parts of alkaline protease, and the balance being a carrier. Compared with Example 1, no saccharifying enzyme was compounded.

[0043] Comparative Example 6

[0044] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, and the balance being a carrier. Compared with Example 1, alkaline protease was not compounded.

[0045] Comparative Example 7

[0046] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 5 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of feruloyl esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, the ratio of feruloyl esterase activity (U) to xylanase activity (U) was lower than 1:500.

[0047] Comparative Example 8

[0048] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 5 parts of feruloyl esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, the ratio of feruloyl esterase activity (U) to xylanase activity (U) was higher than 1:500.

[0049] Comparative Example 9

[0050] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 5 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance being a carrier. Compared with Example 1, the ratio of laccase activity (U) to saccharifying enzyme activity (U) was higher than 1:5.

[0051] Comparative Example 10

[0052] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 5 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, the ratio of laccase activity (U) to saccharifying enzyme activity (U) was lower than 1:5.

[0053] Comparative Example 11

[0054] The blank control group, based on 50 parts by weight of the raw materials, did not contain any enzyme preparation, but was entirely composed of carriers. Compared with Example 1, there was no enzyme preparation.

[0055] Comparative Example 12

[0056] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of ferulic acid esterase, 8 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, laccase was not compounded, but the number of saccharifying enzymes added was increased, keeping the total number of laccase and saccharifying enzymes added consistent.

[0057] Comparative Example 13

[0058] A composite enzyme preparation was prepared, based on 50 parts by weight of the raw materials, including 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of ferulic acid esterase, 8 parts of laccase, 7 parts of alkaline protease, and the balance of carrier. Compared with Example 1, no saccharifying enzyme was compounded, but the number of laccase added was increased, keeping the total number of laccase and saccharifying enzyme added consistent.

[0059] Since the enzymes related to lignin degradation in the present invention are laccase and saccharifying enzyme, the detection of lignin only involves the examples or comparative examples related to the addition levels of laccase and saccharifying enzyme, namely, Examples 1 to 3, Comparative Example 3, Comparative Example 5, and Comparative Examples 9 to 13. According to the experimental method described in the above test, the changes in lignin content in Example 1, Comparative Example 3, and Comparative Example 5 were measured, and the results are shown in Table 1 below.

[0060] Table 1 Effects of different complex enzymes on the lignin content of sunflower seed meal (dry matter basis)

[0061]

[0062] Compared with Example 1, Comparative Example 3 did not contain laccase; Comparative Example 12 did not contain laccase, and the number of saccharifying enzymes added was the sum of the number of laccases and saccharifying enzymes added in Example 1; Comparative Example 5 did not contain saccharifying enzymes, and Comparative Example 13 did not contain saccharifying enzymes, and the number of laccases added was the sum of the number of laccases and saccharifying enzymes added in Example 1. The test results showed that the lignin content in Example 1 was significantly lower than that in Comparative Example 3, Comparative Example 5, Comparative Example 12, and Comparative Example 13, confirming that the addition of laccase and saccharifying enzymes in the ratio provided by the present invention can synergistically improve the degradation rate of lignin.

[0063] According to the experimental method described in the above test, the changes in crude fiber and oligosaccharide content of Examples 1 to 3 and Comparative Examples 1 to 11 were measured. The results are shown in Table 2 below.

[0064] Table 2 Effects of different complex enzymes on crude fiber and oligosaccharide content of sunflower seed meal (dry matter basis)

[0065]

[0066]

[0067] Compared with Example 1, the ratio of feruloyl esterase activity (U) to xylanase activity (U) in Comparative Example 7 was lower than 1:500; compared with Example 1, the ratio of feruloyl esterase activity (U) to xylanase activity (U) in Comparative Example 8 was higher than 1:500. Compared with Example 1, the ratio of laccase activity (U) to saccharifying enzyme activity (U) in Comparative Example 9 was higher than 1:5; compared with Example 1, the ratio of laccase activity (U) to saccharifying enzyme activity (U) in Comparative Example 10 was lower than 1:5. The results showed that the crude fiber content of Examples 1 to 3 and Comparative Examples 7 to 10 was significantly lower than that of the other comparative example groups, confirming that the composite enzyme composed of the enzyme spectrum of the present invention can significantly reduce the crude fiber content.

[0068] The crude fiber content of Examples 1 to 3 is significantly lower than that of Comparative Examples 1 to 13, which confirms that the addition of xylanase and ferulic acid esterase, as well as laccase and saccharifying enzyme in the ratio given by the present invention can significantly improve the degradation rate of crude fiber.

[0069] The crude fiber degradation rates of Examples 1 to 3 and Comparative Examples 7 to 10 were significantly higher than those of the other comparative example groups, which also confirmed that the composite enzyme composed of the enzyme spectrum of the present invention can significantly improve the crude fiber degradation rate.

[0070] The crude fiber degradation rates of Examples 1 to 3 were significantly higher than those of Comparative Examples 1 to 13, which also confirmed that the addition of xylanase and ferulic acid esterase, as well as laccase and saccharifying enzyme in the ratio given by the present invention can significantly improve the degradation rate of crude fiber.

[0071] The oligosaccharide content in Examples 1 to 3 and Comparative Examples 7 to 10 was significantly higher than that in the other comparative example groups, confirming that the complex enzyme constructed according to the enzyme spectrum of the present invention can significantly increase the oligosaccharide content of sunflower seed meal after enzymatic hydrolysis.

[0072] The oligosaccharide content of Examples 1 to 3 was significantly higher than that of Comparative Examples 1 to 13, confirming that the addition of xylanase and ferulic acid esterase, as well as laccase and saccharifying enzyme in the ratio given by the present invention can significantly increase the oligosaccharide content of sunflower seed meal after enzymatic hydrolysis.

[0073] The present invention discloses a composite enzyme preparation for enzymatic hydrolysis of sunflower seed meal, comprising xylanase, acid cellulase, laccase, feruloyl esterase, saccharifying enzyme, and alkaline protease. The ratio of the feruloyl esterase activity (u) to the xylanase activity (u) is 1:500; and the ratio of the laccase activity (u) to the saccharifying enzyme activity (u) is 1:5. The composite enzyme preparation provided by the present invention can significantly degrade the crude fiber content in sunflower meal. After enzymatic hydrolysis, the crude fiber content of the sunflower meal is reduced by more than 40%, and the crude fiber degradation effect is much higher than the current fermentation treatment and known enzymatic hydrolysis treatment. At the same time, the composite enzyme preparation provided by the present invention can significantly increase the oligosaccharide content in the enzymatic hydrolysis product by enzymatic hydrolysis of sunflower seed meal.

[0074] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. A composite enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content, characterized in that: Based on 50 parts by weight of the raw materials, the invention comprises 3-5 parts of xylanase, 3-5 parts of acid cellulase, 3-5 parts of laccase, 3-5 parts of ferulic acid esterase, 3-5 parts of saccharifying enzyme, 6-8 parts of alkaline protease and the balance of carrier.

2. The composite enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content according to claim 1, characterized in that: Based on 50 parts by weight of the raw materials, 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 3-5 parts of ferulic acid esterase, 3-5 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance is carrier.

3. The composite enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content according to claim 2, characterized in that: Based on 50 parts by weight of the raw materials, 4 parts of xylanase, 4 parts of acid cellulase, 4 parts of laccase, 4 parts of ferulic acid esterase, 4 parts of saccharifying enzyme, 7 parts of alkaline protease, and the balance is carrier.

4. The complex enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content according to any one of claims 1 to 3, characterized in that: The ratio of the ferulic acid esterase activity U to the xylanase activity U is 1:

500.

5. The complex enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content according to any one of claims 1 to 3, characterized in that: The ratio of the laccase activity U to the saccharifying enzyme activity U is 1:

5.

6. The composite enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content according to claim 1, characterized in that: The enzymatic activity of the xylanase is 50,000 u / g, the enzymatic activity of the acid cellulase is 1,000 u / g, the enzymatic activity of the laccase is 2,000 u / g, the enzymatic activity of the ferulic acid esterase is 100 u / g, the enzymatic activity of the saccharifying enzyme is 10,000 u / g, and the enzymatic activity of the alkaline protease is 50,000 u / g.

7. A method for degrading sunflower seed meal, characterized in that: The method comprises the step of degrading sunflower seed meal using the composite enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content according to claim 1, wherein the addition amount of the composite enzyme preparation for reducing the fiber content of sunflower seed meal and increasing the oligosaccharide content is 4 kg / t sunflower seed meal.

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