Method for preparing feruloyl oligosaccharide additive by mixed fermentation

Abstract

The present application relates to the technical field of biology, and particularly relates to a method for preparing feruloyl oligosaccharide additive by mixed bacteria fermentation. The method for hydrolyzing and extracting FOs from glycosidic bond in corn bran by using hydrothermal pretreatment and enzyme hydrolysis combination method or ionic liquid pretreatment and enzyme hydrolysis combination method has high cost and low yield. In view of the above problems, the present application provides a method for preparing feruloyl oligosaccharide additive by mixed bacteria fermentation. The method uses aspergillus oryzae, lactobacillus and saccharomyces cerevisiae as fermentation strains, and corn bran, bean dregs, sterile water and readily available carbon source as fermentation raw materials, so that the content of FOs in the obtained fermentation product is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing a ferulic acid oligosaccharide additive through mixed-culture fermentation. Background Technology

[0002] Ferulic acid oligosaccharides (FOs) are natural antioxidants formed by ester bonds between the carboxyl groups of ferulic acid and the hydroxyl groups of oligosaccharides. FOs possess the physiological functions of both ferulic acid and oligosaccharides, such as antioxidant activity, immune modulation, and inhibition of non-enzymatic glycosylation of proteins. They have been widely used in the development of functional foods in recent years.

[0003] FOS (Ferrous Oxide) preparation typically uses barley endosperm, barley straw, corn sprouts, corn bran, wheat bran, and sugarcane bagasse as raw materials. my country ranks second in the world in corn production, reaching 277 million tons in 2022, of which approximately 25% is used for deep processing into corn starch, ethanol, etc. Corn bran is a byproduct of corn starch production and is abundant. Studies have shown that corn bran contains 13%-18% arabinoxylan and about 3% ferulic acid.

[0004] Hydrothermal pretreatment combined with enzymatic hydrolysis, ionic liquid pretreatment combined with enzymatic hydrolysis, and microbial fermentation can all hydrolyze glycosidic bonds in corn bran to extract FOs. Compared with other methods, microbial fermentation of corn bran to produce FOs is lower in cost, safer, and can also increase the content of peptides and other active substances in the product through the action of various hydrolytic enzymes secreted by microorganisms, further increasing the added value of corn bran.

[0005] Dried soybean residue contains about 28% crude protein and about 60% dietary fiber. It can provide nitrogen and carbon sources for the growth and metabolism of microorganisms, and also provide raw materials for the synthesis of cell hydrolytic enzymes by microorganisms, which can increase the release of ferulic oligosaccharides.

[0006] Studies have shown that adding readily available carbon sources such as glucose mother liquor, molasses, and glucose to certain microbial fermentation systems can promote the production of oligosaccharides by yeast, β-Ffase enzyme by Aspergillus niger, and carotenoids by Zygophyllum demersum fermentation of wheat bran, while also increasing the yeast cell count. However, this invention has found that, for the microbial fermentation raw materials and composite strain system of this invention, only readily available carbon source such as glucose mother liquor can significantly increase the yield of ferulic acid oligosaccharides. Other readily available carbon sources, such as molasses, not only fail to increase the yield of ferulic acid oligosaccharides but also reduce it. Summary of the Invention

[0007] To address the problems existing in the prior art, the technical problem to be solved by the present invention is: the yield of FOs extracted by hydrolysis of glycosidic bonds in corn bran using a combination of hydrothermal pretreatment and enzymatic hydrolysis or ionic liquid pretreatment and enzymatic hydrolysis is low.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation, comprising a fermentation culture medium and a mixed-culture seed liquid; The fermentation medium includes corn bran, soybean residue, sterile water, and a readily available carbon source. The mass ratio of corn bran to soybean residue is (2-10):(0-8). The water content in the fermentation medium is 30-70% by mass. The amount of readily available carbon source added is 0.02-0.05% of the total mass of the fermentation medium.

[0009] The compound microbial seed liquid includes Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid, with a volume ratio of Aspergillus oryzae seed liquid to Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid of 1:1:1. The Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid were obtained by seed propagation of Aspergillus oryzae, Lactobacillus, and Saccharomyces cerevisiae, respectively. The inoculation amount of the compound bacterial seed liquid on the fermentation medium is 4-12%, where "%" refers to the volume of seed liquid contained in 100g of medium. The method includes the following steps: (1) At room temperature, corn bran, soybean residue, fast-acting carbon source and sterile water are mixed evenly to obtain fermentation culture medium; (2) Add the compound bacterial seed liquid to the fermentation medium and carry out fermentation by solid-state fermentation to obtain the fermentation product; (3) Place the fermentation product in a 60℃ oven and dry for 4 hours. Crush the dried fermentation product and place it in an Erlenmeyer flask. Add distilled water at a material-to-water ratio of 1:100 and extract by shaking at 25℃ and 150-200r / min for 20-30 minutes. After extraction, centrifuge the product. After centrifugation, take the supernatant to obtain the additive containing ferulic acid oligosaccharides.

[0010] Specifically, the method for propagating Aspergillus oryzae seeds includes the following steps: (1) Slant culture of strain A single Aspergillus oryzae was picked and inoculated onto an agar slant culture medium and cultured at 28-30℃ for 3-5 days to obtain primary seed culture. The agar slant culture medium was PDA solid medium. The PDA solid culture medium is obtained by dissolving a mixture of 200g potato, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 120-121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 28-30℃ for 24 hours to obtain Aspergillus oryzae seed liquid. The seed culture medium is a mixture of 200g potato and 20g glucose, dissolved in sterile water and brought to a volume of 1000mL, and sterilized at 121℃ for 30min.

[0011] Specifically, the method for propagating Lactobacillus seeds includes the following steps: (1) Slant culture of strain Single Lactobacillus bacteria were picked and inoculated onto slant culture medium and cultured at 28-30℃ for 1-3 days to obtain primary seed culture. The slant culture medium was MRS solid medium. The MRS solid culture medium is prepared by adding sterile water to a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and 20g agar to a volumetric flask and adjusting the volume to 1-2mm below the mark. Then, the pH of the solution is adjusted to 6.2-6.6 with 0.5mol / L sodium hydroxide aqueous solution, and then sterile water is added to bring the volume to 1000mL. After shaking well, the solution is sterilized at 120-121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into a seed culture medium with an inoculation amount of one loopful of bacteria. The culture is carried out at 28-30℃ to obtain a Lactobacillus seed solution with an OD value of 0.55-0.60. The seed culture medium is a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, and 10g beef extract. The mixture is dissolved in sterile water and brought to a final volume of 1-2mm below the final volume mark on the volumetric flask. The pH of the solution is then adjusted to 6.2-6.6 with 0.5mol / L sodium hydroxide aqueous solution. Sterile water is then added to bring the final volume to 1000mL. The solution is shaken well and then sterilized at 120-121℃ for 30min.

[0012] Specifically, the method for propagating the brewer's yeast seed includes the following steps: (1) Slant culture of strain Single cells of Saccharomyces cerevisiae were inoculated onto slant culture medium and cultured at 28-30℃ for 1-3 days to obtain primary seed culture. The slant culture medium was YPD solid medium. The YPD solid culture medium is obtained by dissolving a mixture of 20g tryptone, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 120-121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 27-30℃ to obtain a Saccharomyces cerevisiae seed liquid with an OD value of 0.55-0.60. The seed culture medium is obtained by dissolving a mixture of 20g tryptone and 20g glucose in sterile water to a final volume of 1000mL and sterilizing it at 120-121℃ for 30min.

[0013] Specifically, the fermentation temperature is 26-32℃, and the fermentation time is 2-6 days.

[0014] Specifically, the mass ratio of corn bran to soybean residue is 8:2.

[0015] Specifically, the fast-acting carbon source includes at least one of glucose mother liquor, molasses, and glucose.

[0016] Specifically, the obtained feruloyl oligosaccharide additives can be used as additives in food and animal feed.

[0017] The beneficial effects of this invention are: (1) When glucose mother liquor is added to the mixed-culture fermentation system of the present invention as a fast-acting carbon source, it can significantly increase the FO content of the obtained fermentation products compared with other fast-acting carbon sources (glucose, molasses, sucrose, xylan, etc.). S The content of glucose mother liquor is higher because, in addition to a large amount of glucose, it also contains a small amount of acid and other trace elements, such as vitamins and minerals, which provide necessary growth factors for microorganisms. Compared with nutrients composed of single components such as glucose, sucrose, and xylan, it is easier to promote cell growth. Molasses mainly contains sucrose, which needs to be degraded into monosaccharides in microbial fermentation before it can be further utilized. In addition, molasses contains about 300mg GAE / 100g of total phenols, which may have a certain antibacterial activity against the microorganisms in this invention. Therefore, its effect on promoting FOs production in this invention is not as good as that of glucose mother liquor. (2) The fermentation culture medium of the mixed-culture fermentation system of the present invention also contains soybean residue as a fermentation raw material. Compared with soybean meal, soybean residue has a higher content of cellulose and hemicellulose, containing about 60% dietary fiber, which provides abundant cellulose and hemicellulose substrates for the production of FOs, thereby helping to increase the FO content in the fermentation products. S The content of FO in the mixed fermentation system of the present invention; (3) The mixed fermentation system of the present invention uses specific fermentation strains, and the FO content in the fermentation product is high. S The content is higher. Detailed Implementation

[0018] The glucose mother liquor in the following embodiments of the present invention has a total sugar content of about 85%-89%, of which glucose accounts for about 50%, and contains about 11%-12% ash. It was purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.

[0019] The molasses in the following embodiments of the present invention contains approximately 50% sucrose, and the other 50% mainly contains potassium, sodium, a small amount of betaine, fructooligosaccharides, etc., and was purchased from Yian Dongfang Ruixue Sugar Industry Co., Ltd.

[0020] The glucose used in the following embodiments of the present invention was purchased from Tianjin Kaitong Chemical Reagent Co., Ltd., product number 50-99-7.

[0021] The lactose used in the following embodiments of the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number 5989-81-1.

[0022] The xylan used in the following embodiments of the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with product number 9014-63-5.

[0023] The sucrose used in the following embodiments of the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number 57-50-1.

[0024] The Aspergillus oryzae used in the following embodiments of the present invention is strain YY-21 with accession number CGMCC No. 6381. The depository unit is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 19, 2012. The classification name is Aspergillus oryzae.

[0025] The Lactobacillus strain YY-2 with accession number CGMCC No. 13110 in the following embodiments of the present invention is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 13, 2016. Its classification and name are Lactobacillus sp.

[0026] The YY-4 strain of *Saccharomyces cerevisiae* with accession number CGMCC No. 21353 in the following embodiments of the present invention is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 11, 2020. Its classification and name are *Saccharomyces cerevisiae*.

[0027] Example 1

[0028] A method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation is as follows: (1) At room temperature, corn bran, soybean residue, fast-acting carbon source and sterile water are mixed evenly to obtain fermentation culture medium; (2) The compound bacterial seed liquid is added to the fermentation medium. The inoculation amount of the compound bacterial seed liquid on the fermentation medium is 10%. Solid fermentation is carried out. The fermentation temperature is 32℃ and the fermentation time is 4 days to obtain the fermentation product. (3) Place the fermentation product in a 60℃ oven for 4 hours, crush the dried fermentation product and place it in an Erlenmeyer flask, add distilled water at a material-to-water ratio of 1:100, and shake and extract at 25℃ and 150r / min for 20 minutes. After extraction, centrifuge the product and take the supernatant to obtain the additive containing ferulic acid oligosaccharide.

[0029] The fermentation medium consists of corn bran, soybean residue, sterile water, and glucose mother liquor. The mass ratio of corn bran to soybean residue is 8:2. The water content in the fermentation medium is 60% by mass. The amount of glucose mother liquor added is 0.02% of the total mass of the fermentation medium.

[0030] The compound bacterial seed liquid is composed of Aspergillus oryzae seed liquid, Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid, with a volume ratio of Aspergillus oryzae seed liquid to Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid of 1:1:1. The Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid were obtained by seed propagation of Aspergillus oryzae, Lactobacillus, and Saccharomyces cerevisiae, respectively. Specifically, the method for propagating Aspergillus oryzae seeds is as follows: (1) Slant culture of strain A single Aspergillus oryzae was picked and inoculated onto an agar slant culture medium and cultured at 28°C for 3 days to obtain primary seed culture. The agar slant culture medium was PDA solid medium. The PDA solid culture medium was prepared by dissolving a mixture of 200g potato, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 28°C for 24 hours to obtain Aspergillus oryzae seed liquid. The seed culture medium is a mixture of 200g potato and 20g glucose, which is dissolved in sterile water and brought to a volume of 1000mL. The mixture is then sterilized at 120°C for 30 minutes.

[0031] Specifically, the method for propagating the Lactobacillus seed is as follows: (1) Slant culture of strain Single Lactobacillus bacteria were picked and inoculated onto an slant culture medium and cultured at 28°C for 1 day to obtain primary seed culture. The slant culture medium was MRS solid medium. The MRS solid culture medium is prepared by adding sterile water to a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and 20g agar to a volumetric flask and adjusting the volume to 1mm below the mark. Then, the pH of the solution is adjusted to 6.2 with 0.5mol / L sodium hydroxide aqueous solution, and then sterile water is added to bring the volume to 1000mL. After shaking well, the solution is sterilized at 121℃ for 30min. (2) The primary seeds obtained in step (1) were inoculated into the seed culture medium. The inoculation amount was one loopful of bacteria. The culture was carried out at 28°C to obtain a Lactobacillus seed solution with an OD value of 0.55. The seed culture medium was a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, and 10g beef extract. The mixture was dissolved in sterile water and the volume was adjusted to 1mm below the volume mark on the volumetric flask. Then, the pH of the solution was adjusted to 6.2 with 0.5mol / L sodium hydroxide aqueous solution. Then, sterile water was added to adjust the volume to 1000mL. After shaking, the solution was sterilized at 121°C for 30min.

[0032] Specifically, the method for propagating the brewer's yeast seed is as follows: (1) Slant culture of strain A single strain of Saccharomyces cerevisiae was inoculated onto an agar slant and cultured at 28°C for 1 day to obtain primary seed culture. The agar slant culture medium was YPD solid medium. The YPD solid culture medium is obtained by dissolving a mixture of 20g tryptone, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 27°C to obtain a yeast seed liquid with an OD value of 0.55-0.60. The seed culture medium is obtained by dissolving a mixture of 20g tryptone and 20g glucose in sterile water to a final volume of 1000mL and sterilizing at 121°C for 30min.

[0033] Example 2

[0034] A method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation is as follows: (1) At room temperature, corn bran, soybean residue, fast-acting carbon source and sterile water are mixed evenly to obtain fermentation culture medium; (2) The compound bacterial seed liquid is added to the fermentation medium. The inoculation amount of the compound bacterial seed liquid on the fermentation medium is 12%. Solid fermentation is carried out. The fermentation temperature is 28℃ and the fermentation time is 5 days to obtain the fermentation product. (3) Place the fermentation product in a 60℃ oven for 4 hours, crush the dried fermentation product and place it in an Erlenmeyer flask, add distilled water at a material-to-water ratio of 1:100, and shake and extract at 25℃ and 150r / min for 30 minutes. After extraction, centrifuge the product and take the supernatant to obtain the additive containing ferulic acid oligosaccharide.

[0035] The fermentation medium consists of corn bran, soybean residue, sterile water, and glucose mother liquor. The mass ratio of corn bran to soybean residue is 10:0. The water content in the fermentation medium is 70%. The amount of glucose mother liquor added is 0.03% of the total mass of the fermentation medium.

[0036] The compound bacterial seed liquid is composed of Aspergillus oryzae seed liquid, Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid, with a volume ratio of Aspergillus oryzae seed liquid to Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid of 1:1:1. The Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid were obtained by seed propagation of Aspergillus oryzae, Lactobacillus, and Saccharomyces cerevisiae, respectively. Specifically, the method for propagating Aspergillus oryzae seeds is as follows: (1) Slant culture of strain A single Aspergillus oryzae was picked and inoculated onto an agar slant culture medium and cultured at 29°C for 4 days to obtain primary seed culture. The agar slant culture medium was PDA solid medium. The PDA solid culture medium was prepared by dissolving a mixture of 200g potato, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 29°C for 24 hours to obtain Aspergillus oryzae seed liquid. The seed culture medium is a mixture of 200g potato and 20g glucose, which is dissolved in sterile water and brought to a volume of 1000mL. The mixture is then sterilized at 121°C for 30 minutes.

[0037] Specifically, the method for propagating the Lactobacillus seed is as follows: (1) Slant culture of strain Single Lactobacillus bacteria were picked and inoculated onto slant culture medium and cultured at 29°C for 2 days to obtain primary seed culture. The slant culture medium was MRS solid medium. The MRS solid culture medium is prepared by adding sterile water to a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and 20g agar to a volumetric flask. The volume is then adjusted to 1mm below the mark with sterile water. The pH of the solution is then adjusted to 6.3 with 0.5mol / L sodium hydroxide aqueous solution. Sterile water is then added to bring the volume to 1000mL. After shaking well, the solution is sterilized at 121℃ for 30min. (2) The primary seeds obtained in step (1) were inoculated into the seed culture medium. The inoculation amount was one loopful of bacteria. The culture was carried out at 29°C to obtain a Lactobacillus seed solution with an OD value of 0.58. The seed culture medium was a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, and 10g beef extract. The mixture was dissolved in sterile water and the volume was adjusted to 1mm below the volume mark on the volumetric flask. Then, the pH of the solution was adjusted to 6.3 with 0.5mol / L sodium hydroxide aqueous solution. Then, sterile water was added to adjust the volume to 1000mL. After shaking, the solution was sterilized at 121°C for 30min.

[0038] Specifically, the method for propagating the brewer's yeast seed is as follows: (1) Slant culture of strain Single cells of Saccharomyces cerevisiae were inoculated onto slant culture medium and cultured at 29°C for 2 days to obtain primary seed culture. The slant culture medium was YPD solid medium. The YPD solid culture medium is obtained by dissolving a mixture of 20g tryptone, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 28°C to obtain a yeast seed liquid with an OD value of 0.58. The seed culture medium is obtained by dissolving a mixture of 20g tryptone and 20g glucose in sterile water to a final volume of 1000mL and sterilizing at 121°C for 30min.

[0039] Example 3

[0040] A method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation is as follows: (1) At room temperature, corn bran, soybean residue, fast-acting carbon source and sterile water are mixed evenly to obtain fermentation culture medium; (2) The compound bacterial seed liquid is added to the fermentation medium. The inoculation amount of the compound bacterial seed liquid on the fermentation medium is 4%. Solid fermentation is carried out. The fermentation temperature is 30℃ and the fermentation time is 6 days to obtain the fermentation product. (3) Place the fermentation product in a 60℃ oven for 4 hours, crush the dried fermentation product and place it in an Erlenmeyer flask, add distilled water at a material-to-water ratio of 1:100, and extract by shaking at 25℃ and 200r / min for 30 minutes. After extraction, centrifuge the product and take the supernatant to obtain the additive containing ferulic acid oligosaccharide.

[0041] The fermentation medium consists of corn bran, soybean residue, sterile water, and glucose mother liquor. The mass ratio of corn bran to soybean residue is 6:4. The water content in the fermentation medium is 40%. The amount of glucose mother liquor added is 0.05% of the total mass of the fermentation medium.

[0042] The compound bacterial seed liquid is composed of Aspergillus oryzae seed liquid, Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid, with a volume ratio of Aspergillus oryzae seed liquid to Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid of 1:1:1. The Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid were obtained by seed propagation of Aspergillus oryzae, Lactobacillus, and Saccharomyces cerevisiae, respectively. Specifically, the method for propagating Aspergillus oryzae seeds is as follows: (1) Slant culture of strain A single Aspergillus oryzae was picked and inoculated onto an agar slant culture medium and cultured at 30°C for 5 days to obtain primary seed culture. The agar slant culture medium was PDA solid medium. The PDA solid culture medium was prepared by dissolving a mixture of 200g potato, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 30°C for 24 hours to obtain Aspergillus oryzae seed liquid. The seed culture medium is a mixture of 200g potato and 20g glucose, which is dissolved in sterile water and brought to a volume of 1000mL. The mixture is then sterilized at 121°C for 30 minutes.

[0043] Specifically, the method for propagating the Lactobacillus seed is as follows: (1) Slant culture of strain Single Lactobacillus bacteria were picked and inoculated onto an slant culture medium and cultured at 30°C for 3 days to obtain primary seed culture. The slant culture medium was MRS solid medium. The MRS solid culture medium is prepared by adding sterile water to a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and 20g agar to a volumetric flask and adjusting the volume to 2mm below the mark. Then, the pH of the solution is adjusted to 6.6 with 0.5mol / L sodium hydroxide aqueous solution, and then sterile water is added to bring the volume to 1000mL. After shaking well, the solution is sterilized at 121℃ for 30min. (2) The primary seeds obtained in step (1) were inoculated into a seed culture medium with an inoculation amount of one loopful of bacteria. The culture was carried out at 30°C to obtain a Lactobacillus seed solution with an OD value of 0.60. The seed culture medium was a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, and 10g beef extract. The mixture was dissolved in sterile water and brought to a volumetric flask 2mm below the volumetric flask mark. The pH of the solution was then adjusted to 6.6 with 0.5mol / L sodium hydroxide aqueous solution. Sterile water was then added to bring the volume to 1000mL. The solution was shaken well and then sterilized at 121°C for 30min.

[0044] Specifically, the method for propagating the brewer's yeast seed is as follows: (1) Slant culture of strain Single cells of Saccharomyces cerevisiae were inoculated onto slant culture medium and cultured at 30°C for 3 days to obtain primary seed culture. The slant culture medium was YPD solid medium. The YPD solid culture medium is obtained by dissolving a mixture of 20g tryptone, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 30°C to obtain a yeast seed liquid with an OD value of 0.60. The seed culture medium is obtained by dissolving a mixture of 20g tryptone and 20g glucose in sterile water to a final volume of 1000mL and sterilizing at 121°C for 30min.

[0045] Comparative Example 1 is the same as Example 1, except that Comparative Example 1 uses the same mass of molasses instead of the glucose mother liquor in Example 1.

[0046] Comparative Example 2 is the same as Example 1, except that Comparative Example 2 uses the same mass of glucose instead of the glucose mother liquor in Example 1.

[0047] Comparative Example 3 is the same as Example 1, except that Comparative Example 3 uses the same mass of lactose instead of the glucose mother liquor in Example 1.

[0048] Comparative Example 4 is the same as Example 1, except that Comparative Example 4 uses the same mass of sucrose instead of the glucose mother liquor in Example 1.

[0049] Comparative Example 5 is the same as Example 1, except that Comparative Example 5 uses the same mass of xylan instead of the glucose mother liquor in Example 1.

[0050] Comparative Example 6 is the same as Example 1, except that soybean residue was not added to the fermentation medium of Comparative Example 6.

[0051] Comparative Example 7 is the same as Example 1, except that soybean meal is used instead of soybean residue in the fermentation culture medium of Comparative Example 7.

[0052] Comparative Example 8 is the same as Example 1, except that the Aspergillus oryzae preservation number used in Comparative Example 8 is CGMCC3.951.

[0053] Comparative Example 9 is the same as Example 1, except that the Lactobacillus accession number used in Comparative Example 9 is CGMCC 23998, the depositary institution is China General Microbiological Culture Collection Center, the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 29, 2021, and the classification name is Lactobacillus paracasei.

[0054] Comparative Example 10 is the same as Example 1, except that the Saccharomyces cerevisiae used in Comparative Example 10 has the accession number CGMCC 24697, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 18, 2022, the classification name is Saccharomyces cerevisiae, and the Latin scientific name is Saccharomyces cerevisiae.

[0055] Comparative Example 11 is the same as Example 1, except that Comparative Example 11 uses wheat bran of the same mass instead of corn bran in Example 1.

[0056] Performance testing The content of FOs and other components in the additives containing feruloyl oligosaccharides obtained in Examples 1-3 and Comparative Examples 1-11 of the present invention were determined, and the specific test results are shown in Table 1.

[0057] The method for testing FOs content is as follows: Table 1

[0058] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation, characterized in that, Including fermentation medium and compound bacterial seed liquid; The fermentation medium includes corn bran, soybean residue, sterile water, and a readily available carbon source. The mass ratio of corn bran to soybean residue is (2-10):(0-8). The water content in the fermentation medium is 30-70% by mass. The amount of readily available carbon source added is 0.02-0.05% of the total mass of the fermentation medium. The compound microbial seed liquid includes Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid, with a volume ratio of Aspergillus oryzae seed liquid to Lactobacillus seed liquid and Saccharomyces cerevisiae seed liquid of 1:1:

1. The Aspergillus oryzae seed liquid, Lactobacillus seed liquid, and Saccharomyces cerevisiae seed liquid were obtained by seed propagation of Aspergillus oryzae, Lactobacillus, and Saccharomyces cerevisiae, respectively. The Aspergillus oryzae strain mentioned is strain YY-21 with accession number CGMCC No. 6381; The lactobacillus mentioned is strain YY-2 of lactobacillus sp. with accession number CGMCC No.13110; The brewing yeast is Saccharomyces cerevisiae YY-4 strain with accession number CGMCC No.21353; The inoculum amount of the compound bacterial seed liquid in the fermentation medium is 4-12%; The method includes the following steps: (1) At room temperature, corn bran, soybean residue, fast-acting carbon source and sterile water are mixed evenly to obtain fermentation culture medium; The fast-acting carbon source is glucose mother liquor; (2) Add the compound bacterial seed liquid to the fermentation medium and carry out fermentation by solid-state fermentation to obtain the fermentation product; (3) Place the fermentation product in a 60℃ oven and dry for 4 hours. Crush the dried fermentation product and place it in an Erlenmeyer flask. Add distilled water at a material-to-water ratio of 1:100 and extract by shaking at 25℃ and 150-200r / min for 20-30 minutes. After extraction, centrifuge the product. After centrifugation, take the supernatant to obtain the additive containing ferulic acid oligosaccharides.

2. The method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation according to claim 1, characterized in that, The method for propagating Aspergillus oryzae seeds includes the following steps: (1) Slant culture of strain A single Aspergillus oryzae was picked and inoculated onto an agar slant culture medium and cultured at 28-30℃ for 3-5 days to obtain primary seed culture. The agar slant culture medium was PDA solid medium. The PDA solid culture medium is obtained by dissolving a mixture of 200 g potato, 20 g glucose and 20 g agar powder in sterile water and bringing the volume to 1000 mL, and then sterilizing it at 120-121℃ for 30 min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 28-30℃ for 24 h to obtain Aspergillus oryzae seed liquid. The seed culture medium is a mixture of 200 g potato and 20 g glucose, dissolved in sterile water and brought to a volume of 1000 mL. The mixture is then sterilized at 120-121℃ for 30 min.

3. The method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation according to claim 1, characterized in that, The method for propagating Lactobacillus seeds includes the following steps: (1) Slant culture of strain Single Lactobacillus bacteria were picked and inoculated onto slant culture medium and cultured at 28-30℃ for 1-3 days to obtain primary seed culture. The slant culture medium was MRS solid medium. The MRS solid culture medium is prepared by adding sterile water to a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and 20g agar to a volumetric flask and adjusting the volume to 1-2mm below the mark. Then, the pH of the solution is adjusted to 6.2-6.6 with 0.5mol / L sodium hydroxide aqueous solution, and then sterile water is added to bring the volume to 1000mL. After shaking well, the solution is sterilized at 120-121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into a seed culture medium with an inoculation amount of one loopful of bacteria. The culture is carried out at 28-30℃ to obtain a Lactobacillus seed solution with an OD value of 0.55-0.

60. The seed culture medium is a mixture of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, Tween 80, 2g diammonium hydrogen citrate, and 10g beef extract. The mixture is dissolved in sterile water and brought to a final volume of 1-2mm below the final volume mark on the volumetric flask. The pH of the solution is then adjusted to 6.2-6.6 with 0.5mol / L sodium hydroxide aqueous solution. Sterile water is then added to bring the final volume to 1000mL. The solution is shaken well and then sterilized at 120-121℃ for 30min.

4. The method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation according to claim 1, characterized in that, The method for propagating the brewer's yeast seed includes the following steps: (1) Slant culture of strain Single cells of Saccharomyces cerevisiae were inoculated onto slant culture medium and cultured at 28-30℃ for 1-3 days to obtain primary seed culture. The slant culture medium was YPD solid medium. The YPD solid culture medium is obtained by dissolving a mixture of 20g tryptone, 20g glucose and 20g agar powder in sterile water and bringing the volume to 1000mL, and then sterilizing it at 121℃ for 30min. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium. The inoculation amount is one loopful of bacteria. The culture is carried out at 27-30℃ to obtain a Saccharomyces cerevisiae seed liquid with an OD value of 0.55-0.

60. The seed culture medium is obtained by dissolving a mixture of 20g tryptone and 20g glucose in sterile water to a final volume of 1000mL and sterilizing it at 120-121℃ for 30min.

5. The method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation according to claim 1, characterized in that, In step (2) of the method, the fermentation temperature of the solid fermentation is 26-32℃ and the fermentation time is 2-6 days.

6. The method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation according to claim 1, characterized in that, The mass ratio of corn bran to soybean residue is 8:

2.

7. A method for preparing a ferulic acid oligosaccharide additive by mixed-culture fermentation according to any one of claims 1-6, characterized in that, The obtained feruloyl oligosaccharide additives can be used as food and animal feed additives.

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