Method for fermenting corn bran through single strain and application

Abstract

The invention discloses a method for fermenting corn bran through a single strain and application, and belongs to the technical field of fermentation engineering. According to the invention, lactic acid bacteria are used as a fermentation strain, alkaline protease is used as enzymolysis protease, cellulase is added for synergistic fermentation, and the fermented corn bran which can be used as a feed is prepared through a solid-state fermentation process; according to the fermented corn bran prepared by the method disclosed by the invention, the contents of small peptides and organic acids in the corn bran can be well increased, the contents of neutral detergent fibers and acid detergent fibers in the feed are reduced, and the flavor and palatability of the feed are improved.

Description

Technical Field

[0001] The present invention relates to a method for fermenting corn bran with a single strain and its application, belonging to the technical field of fermentation engineering. Background Art

[0002] In recent years, the livestock and feed industries in China have developed rapidly. Due to the high price of soybean meal raw materials, the livestock and feed industries have gradually started to pay attention to unconventional feed raw materials with lower prices and rich nutritional content to reduce feed costs. Corn is one of the main food crops in China, and the corn output in 2024 was 290 million tons.

[0003] Corn bran is a by-product after removing starch, germ, and zein during the production of corn starch or alcohol, accounting for 14-20% of the total raw material. Its chemical composition is rich, with glucose accounting for about 32%, starch accounting for about 10-20%, crude protein accounting for about 11.8%, and crude fat content accounting for about 6%. Corn bran is rich in various bioactive substances such as dietary fiber, corn yellow pigment, flavonoids, and polysaccharides, and has high development potential. However, there are many limitations when corn bran is directly used as feed. Its high fiber content leads to poor palatability, and it contains anti-nutritional factors such as non-starch polysaccharides, phytic acid, and mycotoxins, resulting in low utilization rate.

[0004] Existing technologies for fermenting corn bran have problems such as using multiple strains, long fermentation time, and insufficient nutrient content. And there is currently no report on the single-strain fermentation of corn bran by lactic acid bacteria in China.

[0005] Therefore, in order to improve the feeding value of corn bran, increase the utilization rate of corn bran, and expand the application of lactic acid bacteria in corn bran, providing a preparation method for corn bran feed has broad application prospects and practical significance. Summary of the Invention

[0006] To solve the above problems, the present invention provides a preparation method for corn bran feed, which includes: using corn bran as the fermentation raw material and fermenting it with Lactobacillus paracasei ZB-1 and protease; the taxonomic name of Lactobacillus paracasei ZB-1 is Lactobacillus paracasei DY2, which was deposited at the China Center for Type Culture Collection on June 5, 2017, and the deposit number is CCTCC NO: M2017303. The first object of the present invention is to provide a method for fermenting corn bran with a single strain, including the steps:

[0007] (1) Pretreatment of corn bran

[0008] After washing the corn bran, add cellulase and enzymatically hydrolyze it to obtain enzymatically hydrolyzed corn bran;

[0009] (2) Fermentation of corn bran

[0010] Lactobacillus paracasei ZB-1 was inoculated into corn cob raw materials, and alkaline protease and molasses were added, followed by fermentation to obtain single-strain fermented corn cob;

[0011] Among them, the taxonomic nomenclature of Lactobacillus paracasei ZB-1 is Lactobacillus paracasei DY2, which was deposited in the China Center for Type Culture Collection on June 5, 2017, with the deposit number CCTCC NO: M2017303.

[0012] In one embodiment, 0.5-2.5% w / w of sodium bicarbonate is added in step (1) or step (2);

[0013] Optionally, the addition amount of sodium bicarbonate is 1.5-2.5% w / w;

[0014] Optionally, the addition amount of sodium bicarbonate is 2% w / w;

[0015] Optionally, 2% w / w of sodium bicarbonate is added in step (2).

[0016] In one embodiment, the addition amount of cellulose in step (1) is 2500-5000 U / g of substrate.

[0017] In one embodiment, the enzyme activity of cellulose in step (1) is 50 U / mg of enzyme, and the addition amount is 5-10% w / w.

[0018] In one embodiment, the enzymatic hydrolysis conditions in step (1) are enzymatic hydrolysis at 55-60 °C for 24-30 h.

[0019] In one embodiment, the inoculation amount of Lactobacillus paracasei ZB-1 in step (2) is 10 7 ~10 8 CFU / mL of corn cob raw materials.

[0020] In one embodiment, the cell concentration of Lactobacillus paracasei ZB-1 in step (2) is 1×10 9 ~5×10 9 CFU / mL, and the inoculation amount is 1-5% v / v.

[0021] In one embodiment, the mass fraction of water during fermentation in step (2) is 40-55% w / w;

[0022] Optionally, the mass fraction of water during fermentation is 45-55% w / w.

[0023] In one embodiment, the addition amount of alkaline protease in step (2) is 1000-2000 U / g of corn cob, and the addition amount of molasses is 1-5 w / w.

[0024] In one embodiment, the fermentation time in step (2) is 24 - 48 h at 30 - 37 °C.

[0025] The second object of the present invention is to provide a single-strain fermented corn bran prepared by any of the above methods.

[0026] The third object of the present invention is to provide the application of any of the above methods or the above single-strain fermented corn bran in the preparation of feed.

[0027] The fourth object of the present invention is to provide a method for simultaneously increasing the contents of small peptides and organic acids in fermented corn bran and decreasing the contents of neutral detergent fiber and acid detergent fiber. The method uses Lactobacillus paracasei ZB-1 to ferment corn bran, and the steps are as follows:

[0028] (1) Pretreatment of corn bran

[0029] After washing the corn bran with water, add cellulase and enzymatically hydrolyze to obtain enzymatically hydrolyzed corn bran;

[0030] (2) Fermentation of corn bran

[0031] Inoculate Lactobacillus paracasei ZB-1 into the corn bran raw material, add alkaline protease and molasses, and ferment to obtain corn bran with increased contents of small peptides and organic acids and decreased contents of neutral detergent fiber and acid detergent fiber;

[0032] Among them, the taxonomic nomenclature of Lactobacillus paracasei ZB-1 is Lactobacillus paracasei DY2, which was deposited in the China Center for Type Culture Collection on June 5, 2017, and the deposit number is CCTCC NO: M2017303.

[0033] In one embodiment, 0.5 - 2.5% w / w of sodium bicarbonate is added in step (1) or step (2);

[0034] Optionally, the addition amount of sodium bicarbonate is 1.5 - 2.5% w / w;

[0035] Optionally, the addition amount of sodium bicarbonate is 2% w / w;

[0036] Optionally, 2% w / w of sodium bicarbonate is added in step (2).

[0037] In one embodiment, the addition amount of cellulose in step (1) is 2500 - 5000 U / g of substrate.

[0038] In one embodiment, the enzyme activity of cellulose in step (1) is 50 U / mg of enzyme, and the addition amount is 5 - 10% w / w.

[0039] In one embodiment, the enzymatic hydrolysis conditions in step (1) are enzymatic hydrolysis at 55 - 60°C for 24 - 30 h.

[0040] In one embodiment, the inoculation amount of Lactobacillus paracasei ZB-1 in step (2) is 10 7 ~10 8 CFU / mL corn bran raw material.

[0041] In one embodiment, the cell concentration of Lactobacillus paracasei ZB-1 in step (2) is 1×10 9 ~5×10 9 CFU / mL, and the inoculation amount is 1 - 5% v / v.

[0042] In one embodiment, the mass fraction of water during fermentation in step (2) is 40 - 55% w / w;

[0043] Optionally, the mass fraction of water during fermentation is 45 - 55% w / w.

[0044] In one embodiment, the addition amount of alkaline protease in step (2) is 1000 - 2000 U / g of corn bran, and the addition amount of molasses is 1 - 5 w / w.

[0045] In one embodiment, the fermentation time in step (2) is fermentation at 30 - 37°C for 24 - 48 h.

[0046] Advantages of the present invention

[0047] The Lactobacillus paracasei ZB-1 provided by the present invention ferments corn bran at 37°C, which can effectively increase the contents of small peptides and organic acids in corn bran, and can also reduce the contents of NDF and ADF.

[0048] Specifically, the content of small peptides in corn bran fermented by Lactobacillus paracasei ZB-1 is increased from 17.4 mg / g to 50.2 mg / g, the content of organic acids is increased from 0.62% w / w to 3.23%, the content of neutral detergent fiber NDF is reduced from 74.67% to 63.72%, and the content of acid detergent fiber ADF is reduced from 19.01% to 15.82%. The flavor and palatability of the feed are significantly improved.

[0049] Biological material preservation

[0050] Lactobacillus paracasei ZB-1, taxonomically named Lactobacillus paracasei DY2, was deposited at the China Center for Type Culture Collection on June 5, 2017, with the deposit number CCTCC NO: M2017303, and the deposit address is Wuhan University, Wuhan, China.

[0051] Lactobacillus paracasei ZB-1, with the preservation number of CCTCC NO: M2017303, was disclosed in Patent CN118680234A on September 24, 2024. Description of the Drawings

[0052] Figure 1 Fermentation results of different lactic acid bacteria;

[0053] Figure 2 Results of non-pretreatment and different alkali pretreatments of corn bran;

[0054] Figure 3 Effects of different alkali addition times on fermented small peptides, organic acids, NDF, and ADF;

[0055] Figure 4 Effects of different sodium bicarbonate addition amounts on fermented small peptides, organic acids, NDF, and ADF;

[0056] Figure 5 Effects of different fermentation water contents on fermented small peptides, organic acids, NDF, and ADF. Detailed Embodiments

[0057] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0058] Raw materials used in the embodiments:

[0059] Lactobacillus paracasei DY2, named ZB-1 in the embodiment, with the preservation number of CCTCC NO: M2017303;

[0060] Lactobacillus plantarum DY1, named ZB-2 in the embodiment, preserved in the National Engineering Laboratory of Cereal Fermentation Technology and Technology, Jiangnan University;

[0061] Lactobacillus rhamnosus DY4, named ZB-5 in the embodiment, with the preservation number of CCTCC NO: M2017279;

[0062] Lactobacillus paracasei ZB-3, Lactobacillus paracasei ZB-4; Pediococcus acidilactici ZB-6; Lactobacillus plantarum ZB-7; Lactobacillus plantarum ZB-8; Lactobacillus rhamnosus ZB-9 are all preserved in the laboratory.

[0063] Corn bran was purchased from Fufeng Group Co., Ltd.;

[0064] Cellulase was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., with an enzyme activity of 50 U / mg.

[0065] Testing methods:

[0066] 1. Detection of small peptide content:

[0067] Refer to the paper "Fang Le, Ge Xiangyang, Tang Jiangwu, et al. Research on the preparation of small peptides for feed by the synergistic treatment of bacteria and enzymes from soybean meal [J]. China Feed, 2011(05): 17-20. DOI: 10.3969 / j.issn.1004-3314.2011.05.006".

[0068] 2. Organic acid determination method

[0069] Refer to GB / T 5009.157-2016.

[0070] 3. Determination of neutral detergent fiber and acid detergent fiber

[0071] Refer to the paper "Yuan Cuilin, Zhu Yajun, Lin Yingting, et al. Comparison of different determination methods of neutral detergent fiber and acid detergent fiber in feed [J]. Cereal & Feed Industry, 2014(4): 4. DOI: 10.7631 / j.issn.1003-6202.2014.04.017.".

[0072] Example 1: A method for single-strain fermentation of corn bran

[0073] 1. Strain activation

[0074] Lactobacillus paracasei ZB-1 was inoculated into MRS medium at an inoculation amount of 2% v / v and activated at 37 °C for 12 h, repeated 2-3 times, to obtain the starting strain bacterial liquid, and the bacterial concentration was adjusted to 1×10 9 CFU / mL.

[0075] 2. A method for single-strain fermentation of corn bran, including the steps:

[0076] (1) Pretreatment of corn bran

[0077] 100 g of corn bran was added with 6% w / w of cellulase, 2% w / w of NaHCO3, and a moisture mass fraction of 25%, and enzymatically hydrolyzed at 60 °C for 24 h to obtain enzymatically hydrolyzed corn bran;

[0078] (2) Fermentation of corn bran

[0079] The starting strain bacterial liquid was inoculated into the corn bran raw material at an inoculation amount of 5% v / v, 1350 U / g of alkaline protease of corn bran, 3% w / w of molasses, and a moisture mass fraction of 45% w / w, and fermented at 37 °C for 48 h to obtain fermented corn bran.

[0080] Example 2: A method for single-strain fermentation of corn bran

[0081] 1. Strain activation

[0082] Lactobacillus paracasei ZB-1 was inoculated into MRS medium at an inoculation amount of 2% v / v and activated at 37 °C for 12 h, repeated 2-3 times to obtain the starting strain bacterial solution, and the bacterial concentration was adjusted to 1×10 9 CFU / mL.

[0083] 2. A method for fermenting corn bran with a single strain, comprising the steps of:

[0084] (1) Pretreatment of corn bran

[0085] 100 g of corn bran was added with 6% w / w of cellulase, the moisture mass fraction was 25%, and enzymolysis was carried out at 60 °C for 24 h to obtain enzymolyzed corn bran;

[0086] (2) Fermentation of corn bran

[0087] The starting strain bacterial solution was inoculated into the corn bran raw material at an inoculation amount of 5% v / v, 2% w / w NaHCO3, 1350 U / g alkaline protease, 3% w / w molasses were added, the moisture mass fraction was 50% w / w, and fermentation was carried out at 37 °C for 48 h to obtain fermented corn bran. Comparative Example 1: Using other strains to ferment corn bran

[0088] On the basis of Example 1, Lactobacillus paracasei ZB-1 was replaced with Lactobacillus plantarum ZB-2, Lactobacillus paracasei ZB-3, Lactobacillus paracasei ZB-4, Lactobacillus rhamnosus ZB-5, Pediococcus acidilactici ZB-6, Lactobacillus plantarum ZB-7, Lactobacillus plantarum ZB-8, Lactobacillus rhamnosus ZB-9 respectively, and the remaining steps were the same as those in Example 1 to prepare fermented corn bran.

[0089] Comparative Example 2: Without pretreatment

[0090] On the basis of Example 1, step (1) was omitted, and the remaining steps were the same as those in Example 1 to prepare fermented corn bran (control group).

[0091] Taking the corn bran raw material, the raw material after washing the corn bran (Y1), and the fermented product after washing the corn bran (Y2) as controls, the fermentation conditions were the same as those in Example 1.

[0092] Comparative Example 3: Using sodium hydroxide pretreatment

[0093] On the basis of Example 1, NaHCO3 was replaced with NaOH, and the remaining steps were the same as those in Example 1 to prepare fermented corn bran (Y6).

[0094] Taking the raw material after treating corn bran with NaHCO3 (Y3), the fermented product after treating corn bran with NaHCO3 (i.e., Example 1, Y4), and the raw material after treating corn bran with NaOH (Y5) as controls, the fermentation conditions were the same as those in Example 1.

[0095] Example 3: Optimization of Fermentation Conditions

[0096] (1) Addition amount of NaHCO3

[0097] On the basis of Example 1, the addition amounts of NaHCO3 in step (1) were changed to 0.5%, 1.0%, 1.5%, and 2.5% respectively, and the remaining steps were the same as those in Example 1 to prepare fermented corn bran.

[0098] (2) Moisture content of corn bran fermentation

[0099] On the basis of Example 1, the mass fraction of moisture in step (2) was changed to 40%, 50%, and 55%, and the remaining steps were the same as those in Example 1 to prepare fermented corn bran.

[0100] Example 4: Detection of Peptide and Organic Acid Contents

[0101] Take the fermented corn bran prepared in Examples 1 to 3 and Comparative Examples 1 to 3 to detect their organic acid and small peptide contents. The results are shown in Table 1, Figures 1 to 5 as shown.

[0102] Table 1 Organic Acid and Small Peptide Contents

[0103]

[0104] Note: " / " indicates not detected.

[0105] Comparing Example 1 and Comparative Example 1, it can be seen that using other lactic acid bacteria strains for fermentation has a poor effect, and they are all inferior to Lactobacillus paracasei ZB-1. The results are as Figure 1 shown; among them, ZB-1 is significantly superior to Lactobacillus plantarum, other species of Lactobacillus paracasei, Pediococcus acidilactici, and Lactobacillus rhamnosus.

[0106] Comparing Example 1 and Comparative Example 2, it can be seen that the organic acids in the corn bran do not change without pretreatment, which is significantly lower than that in Example 1. The results are as Figure 2 shown.

[0107] Comparing Example 1 and Comparative Example 3, it can be seen that the fermentation effect of treating corn bran with NaOH is inferior to that with NaHCO3. The results are as Figure 2 shown; and NaHCO3 has no strong corrosiveness, reducing the operation risk, and has more advantages compared with NaOH.

[0108] Comparing Example 1 and Example 2, it can be seen that adding alkali during fermentation has a better effect than adding alkali during enzymatic hydrolysis, with higher organic acid production and lower NDF and ADF contents. The results are as Figure 3 shown.

[0109] Comparing Example 1 with Example 3, it can be seen that changing the addition amount of NaHCO3 and the mass fraction of fermentation moisture will affect the contents of small peptides and organic acids. The optimal addition amount of NaHCO3 is 2% w / w, and the optimal mass fraction of fermentation moisture is 50% w / w. The results are as Figure 4 , Figure 5 shown.

[0110] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for fermenting corn husks with a single bacterial strain, characterized in that: Includes steps: (1) Corn husk pretreatment After the corn husk is washed, cellulase is added to perform enzymolysis to obtain enzymolyzed corn husk; (2) Corn husk fermentation Lactobacillus paracasei ZB-1 was inoculated into corn husk raw material, alkaline protease and molasses were added, and fermentation was performed to obtain single-strain fermented corn husk; Among them, the taxonomic name of Lactobacillus paracasei ZB-1 is Lactobacillus paracasei DY2, which was deposited in the China Center for Type Culture Collection on June 5, 2017, with the deposit number of CCTCC NO:M2017303.

2. The method according to claim 1, characterized in that Adding 0.5-2.5% w / w sodium bicarbonate in step (1) or step (2); Optionally, the amount of sodium bicarbonate added is 1.5-2.5% w / w; Optionally, sodium bicarbonate is added in an amount of 2% w / w; Optionally, 2% w / w sodium bicarbonate is added in step (2).

3. The method according to claim 1, characterized in that The amount of cellulose added in step (1) is 2500-5000 U / g substrate.

4. The method according to claim 1, characterized in that: The enzymatic hydrolysis conditions in step (1) are 55-60° C. for 24-30 hours.

5. The method according to claim 1, characterized in that The moisture content during fermentation in step (2) is 40-55% w / w; Optionally, the moisture content during fermentation is 45-55% w / w; Optionally, the inoculum size of Lactobacillus paracasei ZB-1 is 10 7 ~10 8 CFU / mL corn husk raw material.

6. The method according to claim 1, characterized in that In step (2), the amount of alkaline protease added is 1000-2000 U / g corn husk, and the amount of molasses added is 1-5 w / w.

7. The method according to claim 1, characterized in that The fermentation time in step (2) is 24 to 48 hours at 30 to 37°C.

8. Corn bran fermented by a single strain prepared by the method according to any one of claims 1 to 7.

9. Use of the method according to any one of claims 1 to 7 or the single-strain fermented corn husk according to claim 8 in preparing feed.

10. A method for simultaneously increasing the content of small peptides and organic acids in fermented corn husks and reducing the content of neutral detergent fiber and acid detergent fiber, characterized in that: Ferment corn husks with Lactobacillus paracasei ZB-1, the steps are as follows: (1) Corn husk pretreatment After the corn husk is washed, cellulase is added to perform enzymolysis to obtain enzymolyzed corn husk; (2) Corn husk fermentation Lactobacillus paracasei ZB-1 is inoculated into corn husk raw material, alkaline protease and molasses are added, and fermentation is performed to obtain corn husk with increased content of small peptides and organic acids and reduced content of neutral detergent fiber and acid detergent fiber; Among them, the taxonomic name of Lactobacillus paracasei ZB-1 is Lactobacillus paracasei DY2, which was deposited in the China Center for Type Culture Collection on June 5, 2017, with the deposit number of CCTCC NO:M2017303.

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