Medium and fermentation method for the fermentative production of lactulose

Abstract

The present application belongs to the technical field of microbial fermentation, and specifically provides a culture medium for fermenting lactulose, which comprises 30-40% of corn enzymatic solution, 5-20% of bran enzymatic solution, 30-40% of chicken enzymatic solution, 8-16% of flying water chrysopsis enzymatic solution, and the balance of nutritional supplements in percentage by mass. The culture medium for fermenting lactulose provided by the present application has simple manufacturing process and low cost. By culturing Kluyveromyces marxianus in the culture medium, lactulose can be fermented, the fermentation process is simple and controllable, the production cost is low, and the culture medium is suitable for application in animal breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a culture medium and fermentation method for the fermentation production of lactobiose. Background Technology

[0002] In recent years, my country's livestock and feed industries have been actively developing safe and efficient new feed additives, such as acidifiers, enzyme preparations, microecological preparations, and oligosaccharides. Among them, oligosaccharides can enhance animal immunity, reduce morbidity, and increase the number and activity of beneficial bacteria in the animal's intestines.

[0003] Lactobiose, an important component of oligosaccharides, is also an important substrate for Bifidobacteria in humans and animals to maintain their life activities. Only a small amount is needed to promote the colonization and proliferation of Bifidobacteria in the intestines of infants. However, lactobiose is usually found in the milk of lactating animals and is difficult to use in animal husbandry. Summary of the Invention

[0004] The purpose of this invention is to overcome the problem that the production of lactobiose in the prior art is low, making it difficult to use and apply in animal husbandry.

[0005] Therefore, the present invention provides a culture medium for fermentation production of lactobiose, comprising, by weight percentage, 30-40% corn hydrolysate, 5-20% wheat bran hydrolysate, 30-40% chicken hydrolysate, 8-16% water thistle hydrolysate, and the remainder being a nutritional supplement.

[0006] Specifically, the preparation method of the above-mentioned corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20-50%, add 0.1-0.5% α-amylase and 0.1-0.3% glucanase to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 5-6 to obtain the corn enzymatic hydrolysate.

[0007] Specifically, the preparation method of the above-mentioned bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20-50%, add 0.8-2% phosphoric acid to it, soak for 8 hours, add 0.1-0.6% cellulase, and enzymatically hydrolyze for 8-14 hours at 30-60℃ and pH 4-6 to obtain the bran enzymatic hydrolysate.

[0008] Specifically, the preparation method of the above-mentioned chicken enzymatic hydrolysate is as follows: prepare a chicken powder solution with a mass concentration of 20-50%, add 0.05-0.15% papain and 0.01-0.06% proteinase K to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 5-6 to obtain the chicken enzymatic hydrolysate.

[0009] Specifically, the preparation method of the above-mentioned water thistle enzymatic hydrolysate is as follows: Prepare a water thistle meal powder solution with a mass concentration of 20-50%, add 0.1-0.5% mannanase, 0.1-0.5% xylanase, and 0.1-0.8% galactosidase to the solution, and then enzymatically hydrolyze the solution at 30-60℃ and pH 4.5-5.5 for 8-14 hours to obtain the water thistle enzymatic hydrolysate. This invention also provides a method for producing lactobiose by fermentation of Kluyveromyces martensii, comprising the following steps:

[0010] (1) Prepare the culture medium described above for the fermentation production of lactobiose;

[0011] (2) Inoculate the Kluyveromyces martensii into the culture medium of step (1) for liquid fermentation;

[0012] (3) After fermentation, the fermentation liquid is inoculated into a mixture of soybean meal, corn flour and wheat bran for solid-state fermentation to obtain a fermentation product containing lactobiose.

[0013] Specifically, in step (2) above, 20 billion CFU / g of Kluyveromyces macrocarpa powder is inoculated into the culture medium of step (1) at an addition rate of 0.5-2% for liquid fermentation.

[0014] Specifically, in step (2) above, the liquid fermentation temperature is 25-35℃ and the fermentation time is 16-24h.

[0015] Specifically, in step (3) above, the mass ratio of soybean meal, corn flour and wheat bran is 3:2:5.

[0016] Specifically, in step (3) above, the fermentation liquid is inoculated into the mixture at a ratio of 10-30% for solid-state fermentation, the fermentation moisture content is 35-50%, the fermentation temperature is 20-40℃, and the fermentation time is 3-5 days.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] The culture medium for fermenting lactobiose production provided by this invention has a simple and low-cost preparation process. Lactobiose can be produced by culturing *Kluyveromyces martensii* within this medium. The fermentation process is simple and controllable, with low production costs, making it suitable for animal husbandry applications. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0020] This invention provides a culture medium for the fermentation production of lactobiose, comprising, by weight percentage, 30-40% corn hydrolysate, 5-20% wheat bran hydrolysate, 30-40% chicken protein hydrolysate, 8-16% water thistle hydrolysate, with the remainder being a nutritional supplement, preferably corn steep liquor powder.

[0021] The preparation method of corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20-50%, add 0.1-0.5% α-amylase and 0.1-0.3% glucanase to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 5-6 to obtain corn enzymatic hydrolysate.

[0022] The preparation method of bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20-50%, add 0.8-2% phosphoric acid, soak for 8 hours, add 0.1-0.6% cellulase, and enzymatically hydrolyze for 8-14 hours at 30-60℃ and pH 4-6 to obtain bran enzymatic hydrolysate.

[0023] The preparation method of chicken enzymatic hydrolysate is as follows: prepare a chicken powder solution with a mass concentration of 20-50%, add 0.05-0.15% papain and 0.01-0.06% proteinase K to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 5-6 to obtain chicken enzymatic hydrolysate.

[0024] The preparation method of water thistle enzymatic hydrolysate is as follows: prepare a water thistle meal powder solution with a mass concentration of 20-50%, add 0.1-0.5% mannanase, 0.1-0.5% xylanase and 0.1-0.8% galactosidase to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 4.5-5.5 to obtain water thistle enzymatic hydrolysate.

[0025] This invention also provides a method for producing lactobiose by fermentation of Kluyveromyces martensii, comprising the following steps:

[0026] (1) Prepare the culture medium described above for the fermentation production of lactobiose;

[0027] (2) Inoculate 20 billion CFU / g of Kluyveromyces macrocarpa powder into the culture medium of step (1) at an addition rate of 0.5-2% for liquid fermentation at a fermentation temperature of 25-35℃ and a fermentation time of 16-24h.

[0028] (3) After fermentation, the fermentation liquid is inoculated into a mixture of soybean meal, corn flour and wheat bran for solid-state fermentation. The fermentation moisture content is 35-50%, the fermentation temperature is 20-40℃, and the fermentation time is 3-5 days to obtain a fermentation product containing lactobiose. The mass ratio of soybean meal, corn flour and wheat bran is 3:2:5.

[0029] The effects of the culture medium and fermentation method for producing lactobiose according to the present invention will be studied through specific embodiments below.

[0030] Example 1:

[0031] This embodiment provides a culture medium for the fermentation production of lactobiose, comprising, by weight percentage, 30% corn hydrolysate, 20% wheat bran hydrolysate, 40% chicken protein hydrolysate, 8% water thistle hydrolysate, and the remainder being corn steep liquor powder.

[0032] The preparation method of corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20%, add 0.1% α-amylase and 0.2% glucanase to it, and then enzymatically hydrolyze it for 12 hours at 40℃ and pH 5.5 to obtain corn enzymatic hydrolysate.

[0033] The preparation method of bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20%, add 0.8% phosphoric acid to it, soak for 8 hours, add 0.5% cellulase, and enzymatically hydrolyze for 12 hours at 50℃ and pH 4.5 to obtain bran enzymatic hydrolysate.

[0034] The preparation method of chicken enzymatic hydrolysate is as follows: prepare a chicken powder solution with a mass concentration of 20%, add 0.09% papain and 0.01% proteinase K to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.5 to obtain chicken enzymatic hydrolysate.

[0035] The preparation method of water thistle enzymatic hydrolysate is as follows: prepare a water thistle meal powder solution with a mass concentration of 20%, add 0.2% mannanase, 0.1% xylanase and 0.4% galactosidase to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.0 to obtain water thistle enzymatic hydrolysate.

[0036] This embodiment also provides a method for producing lactobiose by fermentation of Kluyveromyces martensii, including the following steps:

[0037] (1) Prepare the culture medium described above for the fermentation production of lactobiose;

[0038] (2) Inoculate 20 billion CFU / g of Kluyveromyces macrocarpa powder into the culture medium of step (1) at an addition rate of 0.5% for liquid fermentation at a fermentation temperature of 30°C and a fermentation time of 16 hours.

[0039] (3) After fermentation, the fermentation liquid was inoculated into a mixture of soybean meal, corn flour and wheat bran at a ratio of 10% for solid-state fermentation. The fermentation moisture content was 45%, the fermentation temperature was 30℃, and the fermentation time was 3 days to obtain a fermentation product containing lactobiose. The mass ratio of soybean meal, corn flour and wheat bran was 3:2:5.

[0040] The obtained fermentation product was analyzed by high performance liquid chromatography, and the lactobiose content in the product was 1%.

[0041] Example 2:

[0042] This embodiment provides a culture medium for the fermentation production of lactobiose, comprising, by weight percentage, 40% corn hydrolysate, 5% wheat bran hydrolysate, 30% chicken protein hydrolysate, 16% water thistle hydrolysate, and the remainder being corn steep liquor powder.

[0043] The preparation method of corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20%, add 0.1% α-amylase and 0.2% glucanase to it, and then enzymatically hydrolyze it for 12 hours at 40℃ and pH 5.5 to obtain corn enzymatic hydrolysate.

[0044] The preparation method of bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20%, add 0.8% phosphoric acid to it, soak for 8 hours, add 0.5% cellulase, and enzymatically hydrolyze for 12 hours at 50℃ and pH 4.5 to obtain bran enzymatic hydrolysate.

[0045] The preparation method of chicken enzymatic hydrolysate is as follows: prepare a chicken powder solution with a mass concentration of 20%, add 0.09% papain and 0.01% proteinase K to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.5 to obtain chicken enzymatic hydrolysate.

[0046] The preparation method of water thistle enzymatic hydrolysate is as follows: prepare a water thistle meal powder solution with a mass concentration of 20%, add 0.2% mannanase, 0.1% xylanase and 0.4% galactosidase to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.0 to obtain water thistle enzymatic hydrolysate.

[0047] This embodiment also provides a method for producing lactobiose by fermentation of Kluyveromyces martensii, including the following steps:

[0048] (1) Prepare the culture medium described above for the fermentation production of lactobiose;

[0049] (2) 20 billion CFU / g of Kluyveromyces macrocarpa powder was added to the culture medium in step (1) at a rate of 2% for liquid fermentation at a fermentation temperature of 30°C and a fermentation time of 24 hours.

[0050] (3) After fermentation, the fermentation liquid was inoculated into a mixture of soybean meal, corn flour and wheat bran at a ratio of 30% for solid-state fermentation. The fermentation moisture content was 45%, the fermentation temperature was 30℃, and the fermentation time was 3 days to obtain a fermentation product containing lactobiose. The mass ratio of soybean meal, corn flour and wheat bran was 3:2:5.

[0051] The obtained fermentation product was analyzed by high performance liquid chromatography, and the lactobiose content in the product was 5%.

[0052] Example 3:

[0053] This embodiment provides a culture medium for the fermentation production of lactobiose, comprising, by weight percentage, 35% corn hydrolysate, 10% wheat bran hydrolysate, 35% chicken protein hydrolysate, 16% water thistle hydrolysate, and the remainder being corn steep liquor powder.

[0054] The preparation method of corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20%, add 0.1% α-amylase and 0.2% glucanase to it, and then enzymatically hydrolyze it for 12 hours at 40℃ and pH 5.5 to obtain corn enzymatic hydrolysate.

[0055] The preparation method of bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20%, add 0.8% phosphoric acid to it, soak for 8 hours, add 0.5% cellulase, and enzymatically hydrolyze for 12 hours at 50℃ and pH 4.5 to obtain bran enzymatic hydrolysate.

[0056] The preparation method of chicken enzymatic hydrolysate is as follows: prepare a chicken powder solution with a mass concentration of 20%, add 0.09% papain and 0.01% proteinase K to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.5 to obtain chicken enzymatic hydrolysate.

[0057] The preparation method of water thistle enzymatic hydrolysate is as follows: prepare a water thistle meal powder solution with a mass concentration of 20%, add 0.2% mannanase, 0.1% xylanase and 0.4% galactosidase to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.0 to obtain water thistle enzymatic hydrolysate.

[0058] This embodiment also provides a method for producing lactobiose by fermentation of Kluyveromyces martensii, including the following steps:

[0059] (1) Prepare the culture medium described above for the fermentation production of lactobiose;

[0060] (2) Inoculate 20 billion CFU / g of Kluyveromyces macrocarpa powder into the culture medium of step (1) at an addition rate of 1% for liquid fermentation at a fermentation temperature of 30°C and a fermentation time of 20 h.

[0061] (3) After fermentation, the fermentation liquid was inoculated into a mixture of soybean meal, corn flour and wheat bran at a ratio of 25% for solid-state fermentation. The fermentation moisture content was 45%, the fermentation temperature was 30℃, and the fermentation time was 3 days to obtain a fermentation product containing lactobiose. The mass ratio of soybean meal, corn flour and wheat bran was 3:2:5.

[0062] The obtained fermentation product was analyzed by high performance liquid chromatography, and the lactobiose content in the product was 3%.

[0063] Comparative Example 1:

[0064] This comparative example provides a culture medium comprising, by weight percentage, 40% corn hydrolysate, 30% wheat bran hydrolysate, 20% water thistle hydrolysate, and the remainder corn steep liquor powder.

[0065] The preparation method of corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20%, add 0.1% α-amylase and 0.2% glucanase to it, and then enzymatically hydrolyze it for 12 hours at 40℃ and pH 5.5 to obtain corn enzymatic hydrolysate.

[0066] The preparation method of bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20%, add 0.8% phosphoric acid to it, soak for 8 hours, add 0.5% cellulase, and enzymatically hydrolyze for 12 hours at 50℃ and pH 4.5 to obtain bran enzymatic hydrolysate.

[0067] The preparation method of water thistle enzymatic hydrolysate is as follows: prepare a water thistle meal powder solution with a mass concentration of 20%, add 0.2% mannanase, 0.1% xylanase and 0.4% galactosidase to it, and then enzymatically hydrolyze it for 12 hours at 50℃ and pH 5.0 to obtain water thistle enzymatic hydrolysate.

[0068] This comparative example also provides a method for fermenting with *Kluyveromyces martensii*, comprising the following steps:

[0069] (1) Prepare the above culture medium;

[0070] (2) Inoculate 20 billion CFU / g of Kluyveromyces macrocarpa powder into the culture medium of step (1) at an addition rate of 1% for liquid fermentation at a fermentation temperature of 30°C and a fermentation time of 20 h.

[0071] (3) After fermentation, the fermentation liquid was inoculated into a mixture of soybean meal, corn flour and wheat bran at a ratio of 30% for solid-state fermentation. The fermentation moisture content was 45%, the fermentation temperature was 30℃, and the fermentation time was 3 days to obtain a fermentation product containing lactobiose. The mass ratio of soybean meal, corn flour and wheat bran was 3:2:5.

[0072] The obtained fermentation product was analyzed by high performance liquid chromatography, and the lactobiose content in the product was 0.5%.

[0073] Comparative Example 2:

[0074] 20 billion CFU / g of Kluyveromyces macrocephalae yeast powder was dissolved in water to prepare a 2% yeast solution. This solution was then inoculated into a mixture of soybean meal, corn flour, and wheat bran at a ratio of 50% for solid-state fermentation. The fermentation moisture content was 45%, the temperature was 30°C, and the fermentation time was 3 days. The mass ratio of soybean meal:corn flour:wheat bran was 3:2:5. After fermentation, high-performance liquid chromatography analysis was performed, and lactobiose was not detected in the fermentation product.

[0075] Comparative Example 3:

[0076] This comparative example provides a culture medium comprising, by weight percentage, 35% corn flour solution, 10% wheat bran suspension, 35% chicken meal solution, 16% water thistle meal powder solution, and the remainder being corn steep liquor powder.

[0077] Among them, the corn flour solution, bran suspension, chicken meal solution, and water thistle meal powder solution are the corresponding solutions in Example 2 with a mass concentration of 20% that have not been enzymatically hydrolyzed.

[0078] This comparative example also provides a method for fermenting with *Kluyveromyces martensii*, comprising the following steps:

[0079] (1) Prepare the above culture medium;

[0080] (2) 20 billion CFU / g of Kluyveromyces macrocarpa powder was added to the culture medium in step (1) at a rate of 2% for liquid fermentation at a fermentation temperature of 30°C and a fermentation time of 24 hours.

[0081] (3) After fermentation, the fermentation liquid was inoculated into a mixture of soybean meal, corn flour and wheat bran at a ratio of 30% for solid-state fermentation. The fermentation moisture content was 45%, the fermentation temperature was 30℃, and the fermentation time was 3 days to obtain a fermentation product containing lactobiose. The mass ratio of soybean meal, corn flour and wheat bran was 3:2:5.

[0082] The obtained fermentation product was analyzed by high performance liquid chromatography, and the lactobiose content in the product was 0.1%.

[0083] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A method for producing lactobiose by fermentation of Kluyveromyces martensii, characterized in that, Includes the following steps: (1) Prepare a culture medium for fermentation production of lactobiose. By mass percentage, the culture medium includes 30-40% corn hydrolysate, 5-20% wheat bran hydrolysate, 30-40% chicken hydrolysate, 8-16% water thistle hydrolysate, and the remainder is a nutrient supplement. The method for preparing the corn enzymatic hydrolysate is as follows: prepare a corn flour solution with a mass concentration of 20-50%, add 0.1-0.5% α-amylase and 0.1-0.3% glucanase to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 5-6 to obtain the corn enzymatic hydrolysate. The preparation method of the bran enzymatic hydrolysate is as follows: prepare a bran suspension with a mass concentration of 20-50%, add 0.8-2% phosphoric acid to it, soak for 8 hours, add 0.1-0.6% cellulase, and enzymatically hydrolyze for 8-14 hours at 30-60℃ and pH 4-6 to obtain the bran enzymatic hydrolysate; The preparation method of the chicken enzymatic hydrolysate is as follows: prepare a chicken powder solution with a mass concentration of 20-50%, add 0.05-0.15% papain and 0.01-0.06% proteinase K to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 5-6 to obtain the chicken enzymatic hydrolysate. The preparation method of the water thistle enzymatic hydrolysate is as follows: prepare a water thistle meal powder solution with a mass concentration of 20-50%, add 0.1-0.5% mannanase, 0.1-0.5% xylanase and 0.1-0.8% galactosidase to it, and then enzymatically hydrolyze it for 8-14 hours at 30-60℃ and pH 4.5-5.5 to obtain the water thistle enzymatic hydrolysate; The nutritional supplement is corn steep liquor powder; (2) Inoculate the Kluyveromyces martensii into the culture medium of step (1) for liquid fermentation; (3) After fermentation, the fermentation liquid is inoculated into a mixture of soybean meal, corn flour and wheat bran for solid-state fermentation to obtain a fermentation product containing lactobiose.

2. The method for producing lactobiose by fermentation of Kluyveromyces martensii as described in claim 1, characterized in that: In step (2), 20 billion CFU / g of Kluyveromyces macrocarpa powder is inoculated into the culture medium of step (1) at an addition rate of 0.5-2% for liquid fermentation.

3. The method for producing lactobiose by fermentation of Kluyveromyces martensii as described in claim 1, characterized in that: In step (2), the liquid fermentation temperature is 25-35℃ and the fermentation time is 16-24h.

4. The method for producing lactobiose by fermentation of Kluyveromyces martensii as described in claim 1, characterized in that: In step (3), the mass ratio of soybean meal, corn flour, and wheat bran is 3:2:

5.

5. The method for producing lactobiose by fermentation of Kluyveromyces martensii as described in claim 1, characterized in that: In step (3), the fermentation liquid is inoculated into the mixture at a ratio of 10-30% for solid-state fermentation, the fermentation moisture content is 35-50%, the fermentation temperature is 20-40℃, and the fermentation time is 3-5 days.