Kluyveromyces marxianus hydrolysate for feed as well as preparation method and application of Kluyveromyces marxianus hydrolysate

Abstract

The invention belongs to the technical field of feed, and particularly relates to a kluyveromyces marxianus hydrolysate for feed as well as a preparation method and application of the kluyveromyces marxianus hydrolysate. Wherein the feed kluyveromyces marxianus hydrolysate comprises the following components in percentage by total mass of the feed kluyveromyces marxianus hydrolysate: more than or equal to 50% of crude protein and more than or equal to 32.5% of acid-soluble protein; wherein the mass of the acid-soluble protein accounts for more than or equal to 65% of the mass of the crude protein. The content of acid-soluble protein is higher than that of crude protein, so that the digestion and utilization rate of the raw materials can be increased by increasing the content of the acid-soluble protein, and the growth performance of animals is improved. The Kluyveromyces marxianus hydrolysate also contains rich nutrition and bioactive substances, such as mannan oligosaccharide, beta-glucan, amino acid, small peptide and nucleic acid, and can improve the intestinal health of animals, enhance the immunity of the animals and improve the utilization rate of the feed. When the feed additive is added into feed, the growth performance of piglets can be remarkably improved, meanwhile, the diarrhea phenomenon of the piglets can be remarkably improved, and the feed additive has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of feed, and in particular relates to a feed Kluyveromyces marxianus hydrolyzate and a preparation method and application thereof. Background Art

[0002] With the increasing material needs of the people, the demand for protein resources such as meat, eggs and milk is increasing. The supply of feed protein is difficult to meet the needs of the livestock and poultry farming industry, and has gradually become one of the bottlenecks restricting the development of the industry. Traditional feed protein, such as animal-derived protein raw materials such as plasma protein powder, fish meal and meat and bone meal, has high prices and continuous fluctuations. At the same time, animal-derived protein raw materials have biosafety risks; while plant-based protein raw materials such as soybean meal have disadvantages such as low digestibility and utilization and anti-nutritional factors. Therefore, it is urgent to develop new protein feed resources.

[0003] Future food sources should provide richer nutrition with the least resources in terms of land, time and production costs. Microbial protein provides a promising solution because it has many advantages such as producing more protein sources with fewer resources, high manufacturing efficiency and low carbon dioxide emissions. Its products are rich in nutrients such as high protein, amino acids, vitamins and minerals. Therefore, how to prepare feed using microbial cells as protein raw materials is of great research significance. Summary of the invention

[0004] Technical problems solved by the present invention: In the prior art, the products and application technology solutions of yeast strains in the field of feed breeding are mainly concentrated in live bacteria preparation type and solid fermentation type. The live bacteria preparation type is to add live yeast directly to the feed; the solid fermentation type is to add yeast to the solid culture medium as a fermentation agent to prepare feed; the single-cell direction of yeast for feeding is relatively less studied. Moreover, the content of acid-soluble protein in crude protein in the feed in the prior art is relatively low.

[0005] In view of the above technical problems, the present invention provides a feed Kluyveromyces marxianus hydrolyzate and a preparation method and application thereof.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a feed Kluyveromyces marxianus hydrolysate, which, based on the total mass of the feed Kluyveromyces marxianus hydrolysate, comprises a crude protein content of ≥50% and an acid-soluble protein content of ≥32.5%; and the mass of the acid-soluble protein accounts for a percentage of ≥65% of the crude protein mass.

[0008] Preferably, the crude protein content is 50% to 60%; more preferably, the crude protein content is 51% to 60%; further preferably, the crude protein content is 54% to 60%.

[0009] And / or, preferably, the content of acid-soluble protein is 35% to 50%; more preferably, the content of acid-soluble protein is 40% to 50%; further preferably, the content of acid-soluble protein is 45% to 50%.

[0010] And / or, preferably, the mass percentage of acid-soluble protein to crude protein is 65% to 90%; more preferably, the mass percentage of acid-soluble protein to crude protein is 70% to 90%; further preferably, the mass percentage of acid-soluble protein to crude protein is 75% to 90%; further preferably, the mass percentage of acid-soluble protein to crude protein is 80% to 90%.

[0011] And / or, preferably, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises manno-oligosaccharides, and the content thereof is ≥10.0%; more preferably, the content of manno-oligosaccharides is 10% to 15%.

[0012] And / or, preferably, based on the total mass of the Kluyveromyces marxianus hydrolysate for feeding, the Kluyveromyces marxianus hydrolysate for feeding further comprises β-glucan, and its content is ≥10.0%; more preferably, the content of β-glucan is 10% to 20%; further preferably, the content of β-glucan is 15% to 20%.

[0013] And / or preferably, based on the dry weight of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate also includes RNA, and its content is ≥5.0%; more preferably, the RNA content is 5% to 10%; further preferably, the RNA content is 7% to 9%.

[0014] And / or preferably, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises free amino acids, the content of which is ≥10%; more preferably, the content of the free amino acids is 10% to 15%.

[0015] And / or, preferably, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate also includes hydrolyzed amino acids, and the content thereof is ≥45%; more preferably, the content of the hydrolyzed amino acids is 45% to 55%; further preferably, the content of the hydrolyzed amino acids is 47% to 52%.

[0016] And / or, preferably, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate also includes small peptides, and the content thereof is ≥15%; more preferably, the content of the small peptides is 20% to 37%; further preferably, the content of the small peptides is 29% to 37%; further preferably, the content of the small peptides is 31% to 36%.

[0017] And / or preferably, the feed Kluyveromyces marxianus hydrolyzate is prepared by using Kluyveromyces marxianus AMCC 30634, and the deposit number of Kluyveromyces marxianus AMCC 30634 is CCTCC NO:M 2023217.

[0018] In a second aspect, the present invention provides a method for preparing a hydrolyzate of Kluyveromyces marxianus for feeding, comprising the following steps:

[0019] Step 1: fermenting and culturing Kluyveromyces marxianus to obtain yeast milk;

[0020] Step 2: subjecting the fermented Kluyveromyces marxianus to a heat shock treatment to perform autolysis;

[0021] Step 3: adding a composite enzyme to the solution obtained in step 3 for enzymolysis; and

[0022] Step 4: The enzymatic hydrolysate obtained in step 3 is inactivated, concentrated and dried to obtain Kluyveromyces marxianus hydrolysate.

[0023] Preferably, in step 1, the fermentation culture comprises the following steps: inoculating Kluyveromyces marxianus into a seed tank for culture to obtain Kluyveromyces marxianus seed liquid; and then inoculating the Kluyveromyces marxianus seed liquid into a fermentation tank for culture;

[0024] More preferably, the volume percentage of the inoculation amount of the Kluyveromyces marxianus seed solution inoculated into the fermentation tank is 20% to 40%.

[0025] And / or more preferably, the fermentation conditions in the fermenter are: temperature 30-37°C, time 25-30h, ventilation volume 1-3vvm, and / or pH 4.5-6.5.

[0026] And / or more preferably, the culture medium of the fermentation tank includes corn syrup, wherein the mass percentage of corn syrup is 50% to 100%; preferably, the culture medium of the fermentation tank also includes molasses, wherein the mass percentage of molasses is 0% to 50%, and the sum of the mass percentages of molasses and corn syrup is 100%.

[0027] And / or, preferably, in step 1, the culturing process in the seed tank comprises: inoculating Kluyveromyces marxianus into a primary seed tank for culturing, to obtain a Kluyveromyces marxianus seed liquid obtained by culturing in the primary seed tank;

[0028] More preferably, the culture conditions in the primary seed tank are: temperature 30-37°C, time 24-30h, and / or ventilation volume 15-30m 3 / h; further preferably, the culture conditions in the primary seed tank are: temperature 30-32°C, and / or time 24-25h; and / or, more preferably, based on the total mass of the culture medium, the culture medium of the seed tank comprises 10%-25% sugar, 0.5%-5% ammonium sulfate and 0.5%-5% diammonium phosphate; further preferably, the culture medium of the primary seed tank comprises 23%-25% sugar, 0.5%-1% ammonium sulfate and 0.5%-1% diammonium phosphate.

[0029] And / or, more preferably, further comprising: inoculating the Kluyveromyces marxianus seed solution obtained by culturing in the primary seed tank into the primary seed tank for further culturing to obtain the seed solution; more preferably, all the Kluyveromyces marxianus seed solution obtained by culturing in the primary seed tank is inoculated into the primary seed tank; and / or, more preferably, the culturing conditions in the primary seed tank are: temperature 30-37° C., time 25-30 h, and / or ventilation volume 1500-3600 m 3 / h; most preferably, the culture conditions in the first-level seed tank are: temperature 30-32°C, and / or time 25-26h; and / or, further preferably, based on the total mass of the culture medium, the culture medium of the first-level seed tank comprises 20-30% molasses, 1-5% ammonia water and 1-5% diammonium phosphate; most preferably, the culture medium of the first-level seed tank comprises 26%-28% molasses, 1.5%-2.5% ammonia water and 1%-2% diammonium phosphate.

[0030] And / or, preferably, in step 2, the temperature of the heat shock treatment is 80-95°C; more preferably, the temperature of the heat shock treatment is 85-95°C; and / or, the time of the heat shock treatment is 40-120s; more preferably, the time of the heat shock treatment is 40-100s.

[0031] And / or, preferably, the pH is adjusted after the heat shock treatment and then autolysis is carried out; more preferably, the pH is adjusted to 4-6 after the heat shock treatment; and / or, more preferably, the temperature of autolysis is 50-60°C; further preferably, the temperature of autolysis is 55-58°C; and / or, more preferably, the time of autolysis is 6-12h; further preferably, the time of autolysis is 8-12h.

[0032] And / or, preferably, in step 3, the complex enzyme includes one or more selected from the group consisting of papain, neutral protease and alkaline protease; more preferably, the complex enzyme is a combination of papain, neutral protease and alkaline protease; further preferably, the conditions for enzymolysis are: temperature 55-65°C, pH 5-7, and / or time 8-14h.

[0033] And / or, preferably, based on the mass of dry matter of the yeast milk obtained after fermentation and culturing in step 1, the added amount of the complex enzyme is 0.1% to 1.5%; preferably, the added amount of the complex enzyme is 0.6% to 1.1%.

[0034] And / or, preferably, based on the mass of dry matter of the yeast milk obtained after fermentation and culturing in step 1, the amount of papain added is 0.1% to 0.5%; more preferably, the amount of papain added is 0.1% to 0.3%; further preferably, the amount of papain added is 0.15% to 0.25%.

[0035] And / or, preferably, based on the mass of dry matter of yeast milk obtained after fermentation and culturing in step 1, the added amount of the neutral protease is 0.1% to 0.5%; more preferably, the added amount of the neutral protease is 0.15% to 0.5%; further preferably, the added amount of the neutral protease is 0.15% to 0.25%.

[0036] And / or, preferably, based on the mass of dry matter of yeast milk obtained after fermentation and culturing in step 1, the amount of alkaline protease added is 0.1% to 0.5%; more preferably, the amount of alkaline protease added is 0.15% to 0.5%; further preferably, the amount of alkaline protease added is 0.15% to 0.25%.

[0037] And / or, preferably, in step 4, the conditions for inactivating the enzyme and / or concentrating the solution are: a temperature of 80 to 95° C., and / or a time of 2 to 4 hours.

[0038] And / or, preferably, the drying is selected from spray drying or drum drying; preferably, the conditions of the spray drying are: feed flow rate 1.6-1.8m 3 , pressure 100~120bar, inlet air temperature 165~180℃, and / or, outlet air temperature 80~85℃.

[0039] Preferably, a Kluyveromyces marxianus hydrolyzate for feeding is prepared by the method for preparing the Kluyveromyces marxianus hydrolyzate for feeding.

[0040] In a third aspect, the present invention provides a Kluyveromyces marxianus protein for feeding, wherein the Kluyveromyces marxianus protein for feeding is prepared by drying the yeast milk obtained in step 1 of the method for preparing a Kluyveromyces marxianus hydrolyzate for feeding.

[0041] In a fourth aspect, the present invention provides a use of a hydrolyzate of Kluyveromyces marxianus or a protein of Kluyveromyces marxianus for feeding in the field of preparing feed.

[0042] Preferably, the feed Kluyveromyces marxianus hydrolyzate or the feed Kluyveromyces marxianus protein is used in the preparation of piglet feed, aquatic feed or ruminant feed.

[0043] And / or, preferably, use of the feed Kluyveromyces marxianus hydrolyzate or the feed Kluyveromyces marxianus protein in the field of preparing feed for improving diarrhea.

[0044] In a fifth aspect, the present invention provides a feed comprising the feed Kluyveromyces marxianus hydrolyzate or the feed Kluyveromyces marxianus protein.

[0045] Preferably, the addition amount of the Kluyveromyces marxianus hydrolyzate or the Kluyveromyces marxianus protein for feeding is 0.5% to 10% of the dry weight of the feed.

[0046] More preferably, the addition amount of the Kluyveromyces marxianus hydrolyzate or the Kluyveromyces marxianus protein for feeding is 0.5% to 5% of the dry weight of the feed.

[0047] More preferably, the feed is piglet feed, aquatic feed or ruminant feed; and / or, more preferably, the feed is feed for improving diarrhea.

[0048] Beneficial effects of the present invention

[0049] (1) The crude protein content of the Kluyveromyces marxianus hydrolyzate of the present invention is ≥50%, and the acid-soluble protein content is ≥32.5%; wherein the mass of the acid-soluble protein accounts for ≥65% of the crude protein mass, and the acid-soluble protein accounts for a higher content of the crude protein. The increase in the acid-soluble protein content can increase the digestibility and utilization rate of the raw materials and improve the growth performance of animals.

[0050] (2) The Kluyveromyces marxianus hydrolyzate of the present invention also contains rich nutrients and bioactive substances, such as manno-oligosaccharides, β-glucans, amino acids, small peptides and nucleic acids, which can improve the intestinal health of animals, enhance animal immunity and increase the utilization rate of feed.

[0051] (3) The enzymatic hydrolysis energy value, dry matter digestibility, protein digestibility and energy digestibility of the Kluyveromyces marxianus hydrolysate and Kluyveromyces marxianus protein of the present invention are all high. Among them, the enzymatic hydrolysis energy value, dry matter digestibility, protein digestibility and energy digestibility of the Kluyveromyces marxianus hydrolysate are all better than those of the Kluyveromyces marxianus protein.

[0052] (4) The Kluyveromyces marxianus protein of the present invention contains rich nutrients, wherein the crude protein content is ≥48%, the RNA content is ≥7%, the manno-oligosaccharide content is ≥12% and the β-glucan content is ≥15%.

[0053] (5) The Kluyveromyces marxianus hydrolyzate of the present invention can significantly improve the growth performance of piglets and significantly improve diarrhea.

[0054] Strain deposit information

[0055] Kluyveromyces marxianus AMCC 30634 was deposited in China Center for Type Culture Collection on February 27, 2023, with a deposit number of CCTCC NO: M 2023217, and a deposit address of: Wuhan University, Wuhan, China, zip code: 430072; telephone: (027) 68754052. Kluyveromyces marxianus AMCC 30634 has been disclosed in patent application number CN202310540115.9. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a product picture of the Kluyveromyces marxianus protein prepared in Example 1-2.

[0057] Figure 2 This is a product picture of the Kluyveromyces marxianus hydrolyzate prepared in Example 2-4. DETAILED DESCRIPTION

[0058] As described above, the object of the present invention is to provide a feed Kluyveromyces marxianus hydrolyzate and a preparation method and application thereof.

[0059] Kluyveromyces marxianus is a microorganism that can utilize a wide range of substrates (carbon sources), is resistant to high temperatures, grows fast, and has a strong protein secretion ability. It can make full use of the by-products of agriculture and fermentation industries as substances in fermentation nutrient sources and produce a large amount of microbial protein. It can not only reduce environmental pollution and turn waste into treasure, but also obtain a large amount of nutrients such as protein and vitamins, which can be used in animal feed and food, alleviate the food crisis, and has great market potential and economic value.

[0060] In the present invention, manno-oligosaccharides, β-glucans and nucleic acids (or nucleotides) are the main functional components in the Kluyveromyces marxianus hydrolyzate product: 1) Manno-oligosaccharides are mainly used as functional oligosaccharides. First, they help to adsorb intestinal pathogens and mycotoxins in animals (pigs and chickens, etc.), reducing the harm of intestinal risk factors to animals; second, as a prebiotic, they promote the reproduction of beneficial bacteria in the hind intestine and regulate intestinal flora; 2) β-glucan is also a functional polysaccharide, especially yeast-derived β-glucan, which is a highly effective immunopotentiator that can activate immune pathways, promote animal specific and nonspecific immune levels, and improve animal health levels; 3) Nucleic acids (or nucleotides) are mainly beneficial to supplement the insufficient intake of animals in feed, especially for young animals, as an additional supplement to promote the development of organs such as the early intestine of animals. The manno-oligosaccharides, β-glucans or nucleic acids (or nucleotides) components have related functions such as regulating intestinal immunity, and can be fed back on indicators such as feed intake and feed efficiency for the growth performance of livestock and poultry.

[0061] In the present invention, RNA is a type of nucleic acid, and nucleic acid mainly exists in the form of RNA in yeast cells. Nucleotides are monomers for further hydrolysis of nucleic acids, including AMP, GMP, UMP and CMP.

[0062] The present invention provides the following technical solutions:

[0063] Technical solution 1: A feed Kluyveromyces marxianus hydrolysate, characterized in that, based on the total mass of the feed Kluyveromyces marxianus hydrolysate, it includes a crude protein content of ≥50%, an acid-soluble protein content of ≥32.5%; and the mass of the acid-soluble protein accounts for a percentage of ≥65% of the crude protein mass.

[0064] Technical solution 2: The Kluyveromyces marxianus hydrolyzate for feeding according to technical solution 1, wherein the crude protein content is 50% to 60%; preferably, the crude protein content is 51% to 60%; more preferably, the crude protein content is 54% to 60%;

[0065] And / or, the content of acid-soluble protein is 35% to 50%; preferably, the content of acid-soluble protein is 40% to 50%; more preferably, the content of acid-soluble protein is 45% to 50%;

[0066] And / or, the mass percentage of acid-soluble protein to crude protein is 65% to 90%; preferably, the mass percentage of acid-soluble protein to crude protein is 70% to 90%; more preferably, the mass percentage of acid-soluble protein to crude protein is 75% to 90%; further preferably, the mass percentage of acid-soluble protein to crude protein is 80% to 90%.

[0067] Technical solution 3: The feed Kluyveromyces marxianus hydrolysate according to technical solution 1 or 2, wherein, based on the total mass of the feed Kluyveromyces marxianus hydrolysate, the feed Kluyveromyces marxianus hydrolysate further comprises manno-oligosaccharides, and the content thereof is ≥10.0%; preferably, the content of manno-oligosaccharides is 10% to 15%;

[0068] And / or, based on the total mass of the Kluyveromyces marxianus hydrolysate for feeding, the Kluyveromyces marxianus hydrolysate for feeding further comprises β-glucan, and the content thereof is ≥10.0%; preferably, the content of β-glucan is 10% to 20%; more preferably, the content of β-glucan is 15% to 20%;

[0069] And / or, based on the dry weight of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises RNA, the content of which is ≥5.0%; preferably, the content of RNA is 5% to 10%; more preferably, the content of RNA is 7% to 9%;

[0070] and / or, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises free amino acids, the content of which is ≥10%; preferably, the content of the free amino acids is 10% to 15%;

[0071] And / or, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises hydrolyzed amino acids, the content of which is ≥45%; preferably, the content of the hydrolyzed amino acids is 45% to 55%; more preferably, the content of the hydrolyzed amino acids is 47% to 52%;

[0072] And / or, based on the total mass of the Kluyveromyces marxianus hydrolysate for feeding, the Kluyveromyces marxianus hydrolysate for feeding further comprises small peptides, and the content thereof is ≥15%; preferably, the content of the small peptides is 20% to 37%; further preferably, the content of the small peptides is 29% to 37%; further preferably, the content of the small peptides is 31% to 36%.

[0073] Technical solution 4: The feed Kluyveromyces marxianus hydrolysate according to any one of technical solutions 1 to 3, wherein the feed Kluyveromyces marxianus hydrolysate is prepared using any Kluyveromyces marxianus disclosed in the prior art; preferably, the Kluyveromyces marxianus is prepared from Kluyveromyces marxianus AMCC 30634 (Kluyveromyces marxianus AMCC 30634), and the preservation number of the Kluyveromyces marxianus AMCC 30634 (Kluyveromyces marxianus AMCC 30634) is CCTCC NO: M 2023217.

[0074] Technical solution 5: A method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding as described in any one of technical solutions 1 to 4, characterized in that it comprises the following steps:

[0075] Step 1: fermenting and culturing Kluyveromyces marxianus to obtain yeast milk;

[0076] Step 2: subjecting the fermented Kluyveromyces marxianus to a heat shock treatment to perform autolysis;

[0077] Step 3: adding a composite enzyme to the solution obtained in step 3 for enzymolysis; and

[0078] Step 4: The enzymatic hydrolysate obtained in step 3 is inactivated, concentrated and dried to obtain Kluyveromyces marxianus hydrolysate.

[0079] Technical Solution 6: According to the method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding as described in Technical Solution 5, wherein in step 1, the fermentation culture comprises the following steps:

[0080] The Kluyveromyces marxianus is inoculated into a seed tank for culturing to obtain a seed solution; and the Kluyveromyces marxianus seed solution is then inoculated into a fermentation tank for culturing.

[0081] Technical solution 7: The method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding according to technical solution 6, wherein, in step 1, the volume percentage of the inoculation amount of the Kluyveromyces marxianus seed liquid inoculated into the fermentation tank is 20% to 40%;

[0082] and / or, the fermentation conditions in the fermenter are: temperature 30-37° C., time 25-30 h, ventilation 1-3 vvm, and / or, pH 4.5-6.5;

[0083] And / or, the culture medium of the fermentation tank includes corn syrup, wherein the mass percentage of the corn syrup is 50% to 100%; preferably, the culture medium of the fermentation tank also includes molasses, wherein the mass percentage of the molasses is 0% to 50%, and the sum of the mass percentages of molasses and corn syrup is 100%.

[0084] Technical Solution 8: The method for preparing a feed Kluyveromyces marxianus hydrolyzate according to Technical Solution 6 or 7, wherein in step 1, the culturing process in the seed tank comprises: inoculating Kluyveromyces marxianus into a primary seed tank for culturing to obtain a Kluyveromyces marxianus seed liquid.

[0085] Technical solution 9: The method for preparing a hydrolyzate of Kluyveromyces marxianus for feed according to any one of technical solutions 6 to 8, wherein in step 1, the culture conditions in the primary seed tank are: temperature 30-37°C, time 24-30h, and / or ventilation volume 15-30m 3 / h; More preferably, the fermentation conditions in the primary seed tank are: temperature 30-32°C, and / or time 24-25h;

[0086] And / or, based on the total mass of the culture medium, the culture medium of the primary seed tank includes 10% to 25% sugar, 0.5% to 5% ammonium sulfate and 0.5% to 5% diammonium phosphate; preferably, the culture medium of the primary seed tank includes 23% to 25% sugar, 0.5% to 1% ammonium sulfate and 0.5% to 1% diammonium phosphate.

[0087] Technical solution 10: The method for preparing a feed Kluyveromyces marxianus hydrolyzate according to technical solution 8 or 9, wherein the Kluyveromyces marxianus seed liquid obtained by culturing in a primary seed tank is inoculated into a primary seed tank for further culturing to obtain the Kluyveromyces marxianus seed liquid.

[0088] Technical solution 11: The method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding according to technical solution 10, wherein all the Kluyveromyces marxianus seed liquid obtained by culturing in the primary seed tank is inoculated into the primary seed tank;

[0089] And / or, the culture conditions in the primary seed tank are: temperature 30-37°C, time 25-30h, and / or ventilation volume 1500-3600m 3 / h; preferably, the fermentation conditions in the primary seed tank are: temperature 30-32°C, and / or time 25-26h;

[0090] And / or, based on the total mass of the culture medium, the culture medium of the first-level seed tank includes 20% to 30% molasses, 1% to 5% ammonia water and 1% to 5% diammonium phosphate; preferably, the culture medium of the first-level seed tank includes 26% to 28% molasses, 1.5% to 2.5% ammonia water and 1% to 2% diammonium phosphate.

[0091] Technical solution 12: The method for preparing a hydrolyzate of Kluyveromyces marxianus for feed according to any one of technical solutions 5 to 11, wherein in step 2, the temperature of the heat shock treatment is 80-95°C; preferably, the temperature of the heat shock treatment is 85-95°C; more preferably, in some specific embodiments, the temperature of the heat shock treatment is 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94 or 95°C, or a heat shock treatment temperature within a numerical range consisting of any two of the above specific numerical values ​​as endpoints;

[0092] and / or, the time of heat shock treatment is 40 to 120 s; preferably, the time of heat shock treatment is 40 to 100 s; more preferably, in some specific embodiments, the time of heat shock treatment is 40, 41 s, 42 s, 43 s, 44 s, 45 s, 46 s, 47 s, 48 ​​s, 49 s, 50 s, 51 s, 52 s, 53 s, 54 s, 55 s, 56 s, 57 s, 58 s, 59 s, 60 s, 61 s, 62 s, 63 s, 64 s, 65 s, 66 s, 67 , 68 s, 69 s, 70 s, 71 s, 72 s, 73 s, 74 s, 75 s, 76 s, 77 s, 78 s, 79 s, 80s, 81s, 82s, 83s, 84s, 85s, 86s, 87s, 88s, 89s, 90s, 91s, 92s, 93s, 94s, 95s, 96s, 97s, 98s, 99s, 100s, 101s, 102s, 103s, 104s, 105s, 106s, 107s, 108s, 109s, 110s, 111s, 112s, 113s, 114s, 115s, 116s, 117s, 118s, 119s or 120s, or a heat shock treatment time within a numerical range consisting of any two of the above specific numerical values ​​as endpoints;

[0093] and / or, heat shock treatment followed by pH adjustment and subsequent autolysis;

[0094] Preferably, the pH is adjusted to 4-6 after the heat shock treatment; more preferably, the pH is adjusted to 4.8-5.2 after the heat shock treatment; and / or, preferably, the temperature of the autolysis is 50-60°C; more preferably, the temperature of the autolysis is 55-58°C; further preferably, in some specific embodiments, the temperature of the autolysis is 55°C, 55.5°C, 56°C, 56.5°C, 57°C, 57.5°C, 58°C, 58.5°C, 59°C, 59.5°C or 60°C, or a temperature of the autolysis within a numerical range consisting of any two of the above specific values ​​as endpoints;

[0095] And / or, preferably, the autolysis time is 6 to 12 h; more preferably, the autolysis time is 8 to 12 h; further preferably, in some specific embodiments, the autolysis time is 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, 10, 10.5 h, 11 h, 11.5 h or 12 h, or an autolysis time within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0096] Technical solution 13: The method for preparing a hydrolyzate of Kluyveromyces marxianus for feed according to any one of technical solutions 5 to 12, wherein in step 3, the complex enzyme comprises one or more selected from the group consisting of papain, neutral protease and alkaline protease;

[0097] Preferably, the complex enzyme is a combination of papain, neutral protease and alkaline protease;

[0098] More preferably, the conditions for enzymatic hydrolysis are: temperature 55-65° C., pH 5-7, and / or time 8-14 h.

[0099] Technical solution 14: The method for preparing a hydrolyzate of Kluyveromyces marxianus for feed according to any one of technical solutions 5 to 13, wherein, based on the mass of the dry matter of the yeast milk obtained after fermentation and culturing in step 1, the added amount of the complex enzyme is 0.1% to 1.5%; preferably, the added amount of the complex enzyme is 0.6% to 1.1%; more preferably, in some specific embodiments, the added amount of the complex enzyme is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%, or the added amount of the complex enzyme is within a numerical range consisting of any two of the above specific numerical values ​​as endpoints;

[0100] And / or, based on the mass of dry matter of yeast milk obtained after fermentation and culturing in step 1, the amount of papain added is 0.1% to 0.5%; preferably, the amount of papain added is 0.1% to 0.3%; more preferably, the amount of papain added is 0.15% to 0.25%; more preferably, for example, in some specific embodiments, the amount of papain added is 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.35%, 0.40%, 0.45% or 0.50%, or an amount of papain added within a numerical range consisting of any two of the above specific values ​​as endpoints;

[0101] And / or, based on the mass of dry matter of yeast milk obtained after fermentation and culturing in step 1, the addition amount of the neutral protease is 0.1% to 0.5%; preferably, the addition amount of the neutral protease is 0.15% to 0.5%; more preferably, the addition amount of the neutral protease is 0.15% to 0.25%; more preferably, in some specific embodiments, the addition amount of the neutral protease is 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45% or 0.50%, or an addition amount of the neutral protease within a numerical range consisting of any two of the above specific numerical values ​​as endpoints;

[0102] And / or, based on the mass of dry matter of yeast milk obtained after fermentation and culturing in step 1, the amount of alkaline protease added is 0.1% to 0.5%; preferably, the amount of alkaline protease added is 0.15% to 0.5%; further preferably, the amount of alkaline protease added is 0.15% to 0.25%; more preferably, in some specific embodiments, the amount of alkaline protease added is 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45% or 0.50%, or an amount of alkaline protease added within a numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0103] Technical solution 15: The method for preparing a hydrolyzate of Kluyveromyces marxianus for feed according to any one of technical solutions 5 to 14, wherein in step 4, the conditions for inactivating the enzyme and / or concentrating the enzyme are: a temperature of 80 to 95° C., and / or a time of 2 to 4 hours; preferably, the concentration is performed to a solid content of 30% to 40%;

[0104] And / or, the drying is selected from spray drying or drum drying; preferably, the conditions of the spray drying are: feed flow rate 1.6-1.8m 3 , pressure 100~120bar, inlet air temperature 165~180℃, and / or, outlet air temperature 80~85℃.

[0105] Technical solution 16: A hydrolyzate of Kluyveromyces marxianus for feeding, characterized in that it is prepared by the preparation method of the hydrolyzate of Kluyveromyces marxianus for feeding described in any one of technical solutions 5-15.

[0106] Technical solution 17: A feeding Kluyveromyces marxianus protein, characterized in that the feeding Kluyveromyces marxianus protein is prepared by drying the yeast milk obtained in step 1 of the method for preparing a feeding Kluyveromyces marxianus hydrolyzate described in any one of technical solutions 5-11.

[0107] Technical Solution 18: Use of the Kluyveromyces marxianus hydrolyzate described in Technical Solution 16 or the Kluyveromyces marxianus protein described in Technical Solution 17 in the field of feed preparation;

[0108] Preferably, the feed Kluyveromyces marxianus hydrolyzate or the feed Kluyveromyces marxianus protein is used in the preparation of piglet feed, aquatic feed or ruminant feed;

[0109] And / or, preferably, use of the feeding Kluyveromyces marxianus hydrolyzate or the feeding Kluyveromyces marxianus protein in the field of preparing feed for improving diarrhea.

[0110] Technical solution 19: A feed, characterized in that it comprises the feeding Kluyveromyces marxianus hydrolyzate described in any one of technical solutions 1-4, the feeding Kluyveromyces marxianus hydrolyzate described in technical solution 16, or the feeding Kluyveromyces marxianus protein described in technical solution 17;

[0111] Preferably, the addition amount of the Kluyveromyces marxianus hydrolyzate or the Kluyveromyces marxianus protein for feeding is 0.5% to 10% of the dry weight of the feed;

[0112] More preferably, the addition amount of the Kluyveromyces marxianus hydrolysate or the Kluyveromyces marxianus protein for feeding is 0.5% to 5% of the dry weight of the feed;

[0113] More preferably, the feed is piglet feed, aquatic feed or ruminant feed;

[0114] And / or, more preferably, the feed is a feed that improves diarrhea.

[0115] Technical solution 20: The Kluyveromyces marxianus protein for feeding according to technical solution 17, wherein the crude protein content is 47%-60%; preferably, the crude protein content is 48%-59%;

[0116] and / or, the crude fiber content is 0.3%-0.7%; preferably, the crude fiber content is 0.4%-0.6%;

[0117] and / or, the crude ash content is 6%-9%; preferably, the crude ash content is 6.3%-8.4%;

[0118] and / or, the crude fat content is 5.5%-6.5%; preferably, the crude fat content is 6%-6.3%;

[0119] and / or, the RNA content is 7%-8.5%; preferably, the RNA content is 7.2%-8.2%;

[0120] And / or, the content of manno-oligosaccharides is 12%-15%; preferably, the content of manno-oligosaccharides is 12.4%-14.7%;

[0121] And / or, the β-glucan content is 15%-17%; preferably, the β-glucan content is 15.4%-16.3%.

[0122] Technical Solution 21: A method for preparing the Kluyveromyces marxianus protein for feed as described in Technical Solution 17 or 20, characterized in that Kluyveromyces marxianus is fermented and cultured to obtain yeast milk.

[0123] Technical Solution 22: According to the method for preparing Kluyveromyces marxianus protein for feeding according to Technical Solution 21, the fermentation culture comprises the following steps:

[0124] The Kluyveromyces marxianus is inoculated into a seed tank for culturing to obtain a seed solution; and the Kluyveromyces marxianus seed solution is then inoculated into a fermentation tank for culturing.

[0125] Technical solution 23: The method for preparing Kluyveromyces marxianus protein for feeding according to technical solution 21 or 22, wherein the volume percentage of the inoculation amount of Kluyveromyces marxianus seed liquid inoculated into the fermentation tank is 20% to 40%;

[0126] and / or, the fermentation conditions in the fermenter are: temperature 30-37° C., time 25-30 h, ventilation 1-3 vvm, and / or, pH 4.5-6.5;

[0127] And / or, the culture medium of the fermentation tank includes corn syrup, wherein the mass percentage of the corn syrup is 50% to 100%; preferably, the culture medium of the fermentation tank also includes molasses, wherein the mass percentage of the molasses is 0% to 50%, and the sum of the mass percentages of molasses and corn syrup is 100%.

[0128] Technical Solution 24: A method for preparing Kluyveromyces marxianus protein for feed according to any one of Technical Solutions 21-23, wherein the culturing process in the seed tank comprises: inoculating Kluyveromyces marxianus into a primary seed tank for culturing to obtain Kluyveromyces marxianus seed liquid.

[0129] Technical solution 25: The method for preparing Kluyveromyces marxianus protein for feed according to any one of technical solutions 21-24, wherein the culture conditions in the primary seed tank are: temperature 30-37°C, time 24-30h, and / or ventilation volume 15-30m 3 / h; More preferably, the fermentation conditions in the primary seed tank are: temperature 30-32°C, and / or time 24-25h;

[0130] And / or, based on the total mass of the culture medium, the culture medium of the primary seed tank includes 10% to 25% sugar, 0.5% to 5% ammonium sulfate and 0.5% to 5% diammonium phosphate; preferably, the culture medium of the primary seed tank includes 23% to 25% sugar, 0.5% to 1% ammonium sulfate and 0.5% to 1% diammonium phosphate.

[0131] Technical Solution 26: A method for preparing Kluyveromyces marxianus protein for feed according to any one of Technical Solutions 21-25, wherein the Kluyveromyces marxianus seed liquid obtained by culturing in a primary seed tank is inoculated into a primary seed tank for further culturing to obtain the Kluyveromyces marxianus seed liquid.

[0132] Technical solution 27: The method for preparing Kluyveromyces marxianus protein for feeding according to any one of technical solutions 21-26, wherein all the Kluyveromyces marxianus seed liquid obtained by culturing in the primary seed tank is inoculated into the primary seed tank;

[0133] And / or, the culture conditions in the primary seed tank are: temperature 30-37°C, time 25-30h, and / or ventilation volume 1500-3600m 3 / h; preferably, the fermentation conditions in the primary seed tank are: temperature 30-32°C, and / or time 25-26h;

[0134] And / or, based on the total mass of the culture medium, the culture medium of the first-level seed tank includes 20% to 30% molasses, 1% to 5% ammonia water and 1% to 5% diammonium phosphate; preferably, the culture medium of the first-level seed tank includes 26% to 28% molasses, 1.5% to 2.5% ammonia water and 1% to 2% diammonium phosphate.

[0135] Technical solution 28: The method for preparing Kluyveromyces marxianus protein for feeding according to any one of technical solutions 21-27, comprising the following steps:

[0136] Step 1: Inoculate Kluyveromyces marxianus into a primary seed tank for cultivation. The cultivation conditions in the primary seed tank are: temperature 30-37°C, time 24-30h, and / or ventilation volume 15-30m 3 / h; based on the total mass of the culture medium, the culture medium of the primary seed tank includes 10% to 25% sugar, 0.5% to 5% ammonium sulfate and 0.5% to 5% diammonium phosphate; then all the Kluyveromyces marxianus seed liquid obtained by culturing in the primary seed tank is inoculated into the primary seed tank to obtain the Kluyveromyces marxianus seed liquid, and the culture conditions in the primary seed tank are: temperature 30 to 37°C, time 25 to 30h, and / or ventilation volume 1500 to 3600m 3 / h, based on the total mass of the culture medium, the culture medium of the first-stage seed tank includes 20-30% molasses, 1-5% ammonia water and 1-5% diammonium phosphate;

[0137] Step 2: inoculating Kluyveromyces marxianus seed liquid into a fermenter for culturing, wherein the fermentation conditions in the fermenter are: temperature 30-37° C., time 25-30 h, ventilation volume 1-3 vvm, and / or pH 4.5-6.5, and the culture medium of the fermenter includes corn steep liquor, wherein the mass percentage of the corn steep liquor is 50%-100%, and the rest is molasses;

[0138] Step 3: separation and washing: the fermentation broth is separated by a centrifuge. The parameters vary according to different centrifuges, but the final separation of Kluyveromyces marxianus requires a yeast milk wet weight concentration of 20% to 60%; preferably, the yeast milk wet weight concentration is 25% to 30%;

[0139] Step 4: Inactivation and drying.

[0140] Unless otherwise specified, the various reagents / instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The experimental materials and instrument information used in the present invention are shown in the following table:

[0141] Table 1 Experimental materials and vendors

[0142]

[0143]

[0144] In the present invention, the source of papain is not limited. The inventors have found that any papain with an activity of 500,000-700,000 U / g, which is homemade or commercially available, can be used in the present invention. Preferably, the activity of papain is 550,000-650,000 U / g; more preferably, the activity of papain is 590,000-610,000 U / g.

[0145] In the present invention, the source of alkaline protease is not limited. The inventors have found that any alkaline protease with an activity of 400,000-600,000 U / g, which is homemade or commercially available, can be used in the present invention. Preferably, the alkaline protease has an activity of 450,000-550,000 U / g; more preferably, the alkaline protease has an activity of 490,000-510,000 U / g.

[0146] In the present invention, the source of neutral protease is not limited. The inventors have found that any self-made or commercially available neutral protease with an activity of 200,000-400,000 U / g can be used in the present invention. Preferably, the activity of neutral protease is 250,000-350,000 U / g; more preferably, the activity of neutral protease is 290,000-310,000 U / g.

[0147] In the present invention, the source of molasses is not limited, and homemade or commercially available molasses can be used in the present invention. Preferably, molasses with a total sugar content of ≥45% can be used in the present invention.

[0148] The steps of the detection method are as follows:

[0149] (1) The determination of water content in Kluyveromyces marxianus protein and Kluyveromyces marxianus hydrolysate in the present invention refers to the method for determining water content in feed in accordance with the national standard of the People's Republic of China GB / T 6435-2014.

[0150] (2) The crude ash content in the Kluyveromyces marxianus protein and the Kluyveromyces marxianus hydrolysate in the present invention is determined according to the method for determining the crude ash content in feeds in accordance with the National Standard of the People's Republic of China GB / T 6438-2007.

[0151] (3) The crude protein in the Kluyveromyces marxianus protein and the Kluyveromyces marxianus hydrolysate in the present invention is determined by referring to the National Standard of the People's Republic of China GB / T 6432-2018 Determination of Crude Protein in Feed by Kjeldahl Nitrogen Determination Method.

[0152] (4) The determination of inorganic nitrogen in the hydrolyzate of Kluyveromyces marxianus in the present invention refers to the national standard of the People's Republic of China GB / T 6432-2018 Determination of crude protein in feed by Kjeldahl nitrogen determination. The sample is not digested with sulfuric acid, but directly extracted with aqueous solution, and the total nitrogen content is detected by Kjeldahl nitrogen determination.

[0153] (5) The determination of acid-soluble protein in the hydrolyzate of Kluyveromyces marxianus in the present invention is carried out in accordance with the provisions of Appendix B of the National Standard of the People's Republic of China GB / T 22492-2008 on the determination of peptide content in soybean peptide powder B.4.1.

[0154] (6) Free amino acids in the Kluyveromyces marxianus hydrolyzate of the present invention: refer to the provisions of Appendix B B.4.2 of the National Standard of the People's Republic of China GB / T 22492-2008 on the determination of peptide content in soybean peptide powder.

[0155] (7) Hydrolyzed amino acids in the Kluyveromyces marxianus hydrolyzate of the present invention: refer to the national standard of the People's Republic of China GB / T 18246-2019 Determination of amino acids in feed. Hydrolyzed amino acids refer to the content of amino acids detected after all the proteins in the sample are hydrolyzed into monomeric amino acids.

[0156] (8) The determination of small peptides in the hydrolyzate of Kluyveromyces marxianus in the present invention refers to the method for determining the peptide content in soybean peptide powder in Appendix B of the National Standard of the People's Republic of China GB / T 22492-2008, wherein the content of small peptides is the acid-soluble protein content minus the free amino acid content.

[0157] (9) Determination of RNA in Kluyveromyces marxianus protein and Kluyveromyces marxianus hydrolysate of the present invention: Weigh 0.06-0.15 g of sample into a centrifuge tube, add 8 mL of cold (4°C) 0.25 mol / L HClO 4 Pour the mixture into a centrifuge tube and shake it evenly. Immediately place the centrifuge tube in a 4°C cold water bath and keep it warm for 15 minutes. Do not shake it during this period. Centrifuge at 4000 rpm for 10 minutes and gently pour off the clear liquid on the surface. Add 5 mL of 0.5 mol / L HClO to the precipitate. 4, oscillate to mix. Place the centrifuge tube in a 70℃ water bath, keep warm for 15 minutes, and oscillate every 3 to 4 minutes. Centrifuge at 4000 rpm for 10 minutes, aspirate 1mL of supernatant, dilute to 100mL with distilled water, mix well, and test. Rinse the cuvette with the sample to be tested, fill the cuvette and place it in the spectrophotometer. Wipe the surface. Measure the absorbance at 260nm, using distilled water as a blank. Record the absorbance, repeat the measurement once, and take the average of the two measurements.

[0158]

[0159] Where: A——absorbance of sample solution;

[0160] Dilution - When operating according to this method, the dilution is 100;

[0161] m——the mass of the sample weighed, mg;

[0162] DS——the percentage of dry matter in the sample, expressed in %;

[0163] 5——Add 0.5mol / l HClO 4 The volume of the solution after the reaction, in mL;

[0164] 0.03365——corresponds to the RNA content in the test solution when the absorbance is 1.00, mg / mL.

[0165] (10) The determination of β-glucan and manno-oligosaccharide in the Kluyveromyces marxianus protein and the Kluyveromyces marxianus hydrolysate in the present invention shall be carried out in accordance with the provisions of Appendix I of the National Standard of the People's Republic of China GB / T 20886.2-2021 Quality Requirements for Yeast Products Part 2: Yeast Processed Products.

[0166] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0167] Example 1: Preparation of Kluyveromyces marxianus protein

[0168] Example 1-1

[0169] Step 1: subjecting the raw materials used for fermentation of Kluyveromyces marxianus AMCC 30634 to high temperature sterilization; the Kluyveromyces marxianus AMCC 30634 has a deposit number of CCTCC NO: M 2023217 and was deposited in the China Center for Type Culture Collection on February 27, 2023.

[0170] Step 2: Seed pot cultivation

[0171] Kluyveromyces marxianus AMCC 30634 strains were taken out and rejuvenated by using the slant-F bottle-Ponzi tank method. All the rejuvenated strains were then inoculated into a primary seed tank. The culture medium of the primary seed tank included 25% mixed sugar (wherein the mixed sugar contained 30wt% molasses and 70wt% starch hydrolyzate) , 0.5% ammonium sulfate and 0.5% diammonium phosphate, and the rest was water, based on the mass of the culture medium. The culture conditions were: air volume 15-30m 3 / h, temperature 30℃, culture for 24h until the alcohol reaches 1.5% (based on the total mass of the cultured liquid), then transfer to the first-level seed tank for culture. Based on the mass of the culture medium, the culture medium of the first-level seed tank includes 27% molasses, 2% ammonia water and 1.5% diammonium phosphate, and the rest is water. The culture conditions are: ventilation volume 1500-3600m 3 / h, temperature 30℃, time 25 hours.

[0172] Step 3: Commercial tank fermentation

[0173] The Kluyveromyces marxianus seed solution obtained in step 2 is inoculated into a fermenter at an inoculation rate of 30% by volume for fermentation culture. The fermentation medium in the fermenter is 100wt% corn steep liquor and a small amount of alkali, and the alkali is used to adjust the pH to 4.5. Culture conditions: fermentation temperature 30°C, pH 4.5, ventilation volume 1.3-2.8vvm, and culture time 25 hours.

[0174] Step 4: Separation

[0175] The fermentation liquid obtained in step 3 was separated by centrifuge at a speed of 7000 rpm, and the moisture content of the precipitate was determined by a rapid moisture detector. The wet weight of the obtained Kluyveromyces marxianus milk was 30 wt % (the wet weight refers to the mass percentage of the heavy phase bacteria obtained after centrifugation in the fermentation liquid).

[0176] Step 5: Inactivation and drying

[0177] The yeast milk obtained in step 4 is inactivated by keeping it at 80° C. for 4 hours, and then the inactivated material is spray-dried.

[0178] Example 1-2

[0179] Step 1: subjecting the raw materials used for fermentation of Kluyveromyces marxianus AMCC 30634 to high temperature sterilization; the Kluyveromyces marxianus AMCC 30634 has a deposit number of CCTCC NO: M 2023217 and was deposited in the China Center for Type Culture Collection on February 27, 2023.

[0180] Step 2: The steps of seed tank culture are the same as those of step 2 of Example 1-1.

[0181] Step 3: Commercial tank fermentation

[0182] The Kluyveromyces marxianus seed solution obtained in step 2 is inoculated into a fermenter at an inoculation rate of 30% by volume for fermentation culture, and the fermentation culture medium in the fermenter is 70wt% corn steep liquor, 30wt% molasses and a small amount of alkali, and the alkali is used to adjust the pH of the corn steep liquor to 5.5. Culture conditions: fermentation temperature 35°C, pH 5.5, ventilation volume 1.3-2.8vvm, and culture time 28 hours.

[0183] Step 4: The separation step is the same as the separation step in Example 1-1, and the wet weight of the obtained Kluyveromyces marxianus milk is 28 wt%.

[0184] Step 5: The steps of inactivation and drying are the same as those of step 5 in Example 1-1. The obtained Kluyveromyces marxianus protein product is shown in FIG. Figure 1 As shown, the product is yellow-brown powder.

[0185] Examples 1-3

[0186] Step 1: subjecting the raw materials used for fermentation of Kluyveromyces marxianus AMCC 30634 to high temperature sterilization; the Kluyveromyces marxianus AMCC 30634 has a deposit number of CCTCC NO: M 2023217 and was deposited in the China Center for Type Culture Collection on February 27, 2023.

[0187] Step 2: The steps of seed tank culture are the same as those of step 2 of Example 1-1.

[0188] Step 3: Commercial tank fermentation

[0189] The Kluyveromyces marxianus seed solution obtained in step 2 is inoculated into a fermenter at an inoculation rate of 30% by volume for fermentation culture, and the fermentation medium in the fermenter is 50wt% corn steep liquor, 50wt% molasses and a small amount of alkali, wherein the alkali is used to adjust the pH of the corn steep liquor to 6.5. Culture conditions: fermentation temperature is 37°C, pH is 6.5, ventilation is 1.3-2.8vvm, and culture time is 30 hours.

[0190] Step 4: The separation step is the same as the separation step in Example 1-1, and the wet weight of the obtained Kluyveromyces marxianus milk is 25 wt%.

[0191] Step 5: The steps of inactivation and drying are the same as those of step 5 in Example 1-1.

[0192] The products obtained in Examples 1-1 to 1-3 were tested, and the results are shown in Table 2 below:

[0193] Table 2 Kluyveromyces marxianus protein detection results

[0194] project Example 1-1 Example 1-2 Examples 1-3 Moisture,% 3.8 3.8 3.8 Crude protein, % 48.01 55.08 58.36 Crude fiber, % 0.4 0.5 0.6 Crude ash, % 8.4 6.7 6.3 Crude fat, % 6.2 6.0 6.3 RNA, % 7.24 7.41 8.11 Manno-oligosaccharide, % 12.44 13.08 14.66 β-Glucan, % 15.70 15.49 16.27

[0195] Example 2: Preparation of Kluyveromyces marxianus hydrolysate

[0196] Example 2-1

[0197] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0198] Step 1: Fermentation

[0199] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0200] Step 2: Autolysis

[0201] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 80° C. for 120 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the mixture was subjected to autolysis treatment at 55° C. for 6 hours.

[0202] Step 3: Enzymatic hydrolysis

[0203] The material obtained in step 2 is added with a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.1wt% papain, 0.1wt% alkaline protease and 0.1wt% neutral protease; the composite enzymolysis temperature is 60°C, the enzymolysis pH is 6.0, and the enzymolysis time is 10h.

[0204] Step 4: Enzyme inactivation and concentration

[0205] After mixing, the temperature was raised to 85° C. and kept for 3.5 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 35 wt %.

[0206] Step 5: Drying

[0207] The concentrated product was spray dried with a feed rate of 1.7 m 3 , the feed pressure is 110 bar, the inlet air temperature of the spray tower is 170°C, the outlet air temperature is 82°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0208] Example 2-2

[0209] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0210] Step 1: Fermentation

[0211] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0212] Step 2: Autolysis

[0213] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 90° C. for 80 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the mixture was kept at 60° C. for autolysis treatment for 8 hours.

[0214] Step 3: Enzymatic hydrolysis

[0215] The material obtained in step 2 is added with a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.5wt% papain, 0.1wt% alkaline protease and 0.1wt% neutral protease; the composite enzymolysis temperature is 65°C, the enzymolysis pH is 7.0, and the enzymolysis time is 8h.

[0216] Step 4: Enzyme inactivation and concentration

[0217] After mixing, the temperature was raised to 80° C. and kept for 4.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 30%.

[0218] Step 5: Drying

[0219] The concentrated product was spray dried with a feed rate of 1.8 m 3 , the feed pressure is 100 bar, the inlet air temperature of the spray tower is 165°C, the outlet air temperature is 80°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0220] Example 2-3

[0221] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0222] Step 1: Fermentation

[0223] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0224] Step 2: Autolysis

[0225] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 95° C. for 45 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the mixture was kept at 50° C. for autolysis treatment for 9 hours.

[0226] Step 3: Enzymatic hydrolysis

[0227] The material obtained in step 2 is added to a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.3wt% papain, 0.1wt% alkaline protease and 0.1wt% neutral protease; the composite enzymolysis temperature is 55°C, the enzymolysis pH is 5.0, and the enzymolysis time is 14h.

[0228] Step 4: Enzyme inactivation and concentration

[0229] After mixing, the temperature was raised to 95° C. and kept for 2.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 40%.

[0230] Step 5: Drying

[0231] The concentrated product was spray dried with a feed rate of 1.6 m 3 , the feed pressure is 120 bar, the inlet air temperature of the spray tower is 180°C, the outlet air temperature is 85°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0232] Embodiment 2-4

[0233] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0234] Step 1: Fermentation

[0235] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0236] Step 2: Autolysis

[0237] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 85° C. for 100 s. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the mixture was kept at 58° C. for autolysis treatment for 12 h.

[0238] Step 3: Enzymatic hydrolysis

[0239] The material obtained in step 2 is added to a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.3wt% papain, 0.2wt% alkaline protease and 0.2wt% neutral protease; the composite enzymolysis temperature is 58°C, the enzymolysis pH is 6.5, and the enzymolysis time is 12h.

[0240] Step 4: Enzyme inactivation and concentration

[0241] After mixing, the temperature was raised to 87° C. and kept for 3.5 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 32%.

[0242] Step 5: Drying

[0243] The concentrated product was spray dried with a feed rate of 1.8 m 3, the feed pressure is 100 bar, the inlet air temperature of the spray tower is 172°C, the outlet air temperature is 83°C, and after drying, the Kluyveromyces marxianus hydrolysate is obtained. The obtained Kluyveromyces marxianus hydrolysate product is shown in the figure Figure 2 As shown, the product is yellow-brown powder.

[0244] Embodiment 2-5

[0245] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0246] Step 1: Fermentation

[0247] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0248] Step 2: Autolysis

[0249] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 90° C. for 90 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the yeast milk was subjected to autolysis treatment at 53° C. for 10 hours.

[0250] Step 3: Enzymatic hydrolysis

[0251] The yeast milk obtained in step 2 is added with a composite enzyme for enzymolysis; based on the dry matter weight of the yeast milk, the composite enzyme includes 0.1wt% papain, 0.5wt% alkaline protease and 0.5wt% neutral protease; the composite enzymolysis temperature is 62°C, the enzymolysis pH is 5.5, and the enzymolysis time is 9h.

[0252] Step 4: Enzyme inactivation and concentration

[0253] After mixing, the temperature was raised to 90° C. and kept for 3.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 36%.

[0254] Step 5: Drying

[0255] The concentrated product was spray dried with a feed rate of 1.7 m 3 , the feed pressure is 115 bar, the inlet air temperature of the spray tower is 170°C, the outlet air temperature is 81°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0256] Embodiment 2-6

[0257] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0258] Step 1: Fermentation

[0259] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0260] Step 2: Autolysis

[0261] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 95° C. for 60 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the yeast milk was subjected to autolysis treatment at 55° C. for 8 hours.

[0262] Step 3: Enzymatic hydrolysis

[0263] The yeast milk obtained in step 2 is added with a composite enzyme for enzymolysis; based on the dry matter weight of the yeast milk, the composite enzyme includes 0.2wt% papain, 0.2wt% alkaline protease and 0.2wt% neutral protease; the composite enzymolysis temperature is 64°C, the enzymolysis pH is 5.9, and the enzymolysis time is 8h.

[0264] Step 4: Enzyme inactivation and concentration

[0265] After mixing, the temperature was raised to 83° C. and kept for 3.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 38%.

[0266] Step 5: Drying

[0267] The concentrated product was spray dried with a feed rate of 1.6 m 3 , the feed pressure is 120 bar, the inlet air temperature of the spray tower is 175°C, the outlet air temperature is 83°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0268] The Kluyveromyces marxianus hydrolyzate products obtained in Examples 2-1 to 2-6 were tested, and the results are shown in the following table:

[0269] Table 3 Detection results of Kluyveromyces marxianus hydrolysate

[0270]

[0271] Comparative Example 1-1

[0272] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0273] Step 1: Fermentation

[0274] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0275] Step 2: Autolysis

[0276] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 90° C. for 80 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the mixture was kept at 45° C. for autolysis treatment for 12 hours.

[0277] Step 3: Enzymatic hydrolysis

[0278] The material obtained in step 2 is added to a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.3wt% papain, 0.2wt% alkaline protease and 0.2wt% neutral protease; the composite enzymolysis temperature is 50°C, the enzymolysis pH is 4.0, and the enzymolysis time is 10h.

[0279] Step 4: Enzyme inactivation and concentration

[0280] After mixing, the temperature was raised to 80° C. and kept for 4.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 30%.

[0281] Step 5: Drying

[0282] The concentrated product was spray dried with a feed rate of 1.8 m 3 , the feed pressure is 100 bar, the inlet air temperature of the spray tower is 165°C, the outlet air temperature is 80°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0283] Comparative Example 1-2

[0284] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0285] Step 1: Fermentation

[0286] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0287] Step 2: Autolysis

[0288] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 90° C. for 80 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the yeast milk was subjected to autolysis treatment at 65° C. for 4 hours.

[0289] Step 3: Enzymatic hydrolysis

[0290] The material obtained in step 2 is added to a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.3wt% papain, 0.2wt% alkaline protease and 0.2wt% neutral protease; the composite enzymolysis temperature is 60°C, the enzymolysis pH is 6.0, and the enzymolysis time is 7h.

[0291] Step 4: Enzyme inactivation and concentration

[0292] After mixing, the temperature was raised to 80° C. and kept for 4.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 30%.

[0293] Step 5: Drying

[0294] The concentrated product was spray dried with a feed rate of 1.8 m 3 , the feed pressure is 100 bar, the inlet air temperature of the spray tower is 165°C, the outlet air temperature is 80°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0295] Comparative Examples 1-3

[0296] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0297] Step 1: Fermentation

[0298] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0299] Step 2: Autolysis

[0300] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 90° C. for 80 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the yeast milk was subjected to autolysis treatment at 55° C. for 9 hours.

[0301] Step 3: Enzymatic hydrolysis

[0302] The material obtained in step 2 is added to a composite enzyme for enzymolysis; based on the dry matter weight of the yeast milk, the composite enzyme includes 0.05wt% papain, 0.05wt% alkaline protease and 0.05wt% neutral protease; the composite enzymolysis temperature is 70°C, the enzymolysis pH is 8.0, and the enzymolysis time is 8h.

[0303] Step 4: Enzyme inactivation and concentration

[0304] After mixing, the temperature was raised to 80° C. and kept for 4.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 30%.

[0305] Step 5: Drying

[0306] The concentrated product was spray dried with a feed rate of 1.8 m 3 , the feed pressure is 100 bar, the inlet air temperature of the spray tower is 165°C, the outlet air temperature is 80°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0307] Comparative Examples 1-4

[0308] A method for preparing Kluyveromyces marxianus hydrolysate comprises the following steps:

[0309] Step 1: Fermentation

[0310] The Kluyveromyces marxianus AMCC 30634 strain is fermented and cultured to obtain yeast milk; the yeast milk is the yeast milk obtained in step 4 of Example 1-2; the Kluyveromyces marxianus AMCC 30634 strain has a preservation number of CCTCC NO: M2023217, and was preserved in the China Center for Type Culture Collection on February 27, 2023.

[0311] Step 2: Autolysis

[0312] The yeast milk obtained in step 1 was subjected to heat shock treatment at a temperature of 90° C. for 80 seconds. After the heat shock was completed, citric acid was added to adjust the pH to 5.0, and the yeast milk was subjected to autolysis treatment at 55° C. for 9 hours.

[0313] Step 3: Enzymatic hydrolysis

[0314] The material obtained in step 2 is added to a composite enzyme for enzymolysis; based on the dry matter weight of yeast milk, the composite enzyme includes 0.6wt% papain, 0.6wt% alkaline protease and 0.6wt% neutral protease; the composite enzymolysis temperature is 50°C, the enzymolysis pH is 6.0, and the enzymolysis time is 15h.

[0315] Step 4: Enzyme inactivation and concentration

[0316] After mixing, the temperature was raised to 80° C. and kept for 4.0 hours to inactivate the enzyme and concentrate. The dry matter content of the concentrated product was 30%.

[0317] Step 5: Drying

[0318] The concentrated product was spray dried with a feed rate of 1.8 m 3 , the feed pressure is 100 bar, the inlet air temperature of the spray tower is 165°C, the outlet air temperature is 80°C, and the Kluyveromyces marxianus hydrolyzate is obtained after drying.

[0319] The Kluyveromyces marxianus hydrolyzate products prepared in Comparative Examples 1-1 to 1-4 were tested, and the results are shown in the following table:

[0320] Table 4 Detection results of different Kluyveromyces marxianus hydrolysates

[0321] Test items Comparative Example 1-1 Comparative Example 1-2 Comparative Examples 1-3 Comparative Examples 1-4 Crude ash, % 6.47 6.12 6.24 6.39 Crude protein, % 54.15 55.09 54.47 54.08 Inorganic nitrogen, % 1.01 0.96 1.12 0.95 Acid soluble protein, % 28.17 30.47 29.46 34.33 Acid soluble protein / crude protein, % 52.02 55.31 54.08 63.48 Free amino acids, % 9.82 10.17 8.79 20.15 Hydrolyzed amino acids, % 45.87 47.11 45.95 46.24 Small peptides, % 18.35 20.3 20.67 14.18 RNA, % 7.01 7.32 6.91 7.22 β-Glucan, % 17.19 18.15 16.54 17.39 Manno-oligosaccharide, % 12.44 12.76 13.01 13.29

[0322] Application Example 1

[0323] Application Example 1 is an in vitro biomimetic digestibility evaluation test of Kluyveromyces marxianus protein.

[0324] The Kluyveromyces marxianus protein samples prepared in Example 1-2 and the Kluyveromyces marxianus hydrolyzate samples prepared in Example 2-4 were taken for in vitro digestion test, and the SDS-III monogastric animal bionic digestion system (Hunan Zhongben Intelligent Technology Development Co., Ltd.) was used for pig bionic digestion.

[0325] Principle: Based on the systematic summary of the variation of the main digestive enzymes in the digestive tract of growing pigs, the State Key Laboratory of Animal Nutrition of Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, designed a simulated enzyme spectrum and hydrolysis environment of Durbin × Chang × Da hybrid growing pigs under typical dietary conditions, and developed a monogastric animal bionic digestion system (SDS-Ⅲ) that can fully automatically simulate the digestion process in pigs. At 39°C and in a phosphate buffer system, the feed completes the digestion process of a 30kg growing pig in the monogastric animal bionic digestion system. After the hydrolyzate is separated by a dialysis bag with a molecular weight cutoff of 14,000 Daltons, the material in the bag is dried and defatted, and the total energy content of the residue is determined. At the same time, the total energy content of the sample is determined. The energy value of the feed enzymatic hydrolyzate is determined by the difference method.

[0326] First, the nutritional components of the Kluyveromyces marxianus protein sample prepared in Example 1-2 and the Kluyveromyces marxianus hydrolyzate sample prepared in Example 2-4 were analyzed. Sample processing method: The sampled sample was divided into about 200 g by quartering, and the sample was crushed with a plant grinder or mortar until it passed through a test sieve (60 mesh) with a pore size of 0.30 mm, and sealed in a sample bag for storage as a test sample. Weigh 1 g of feed sample (accurate to 0.0002 g) and place it in a simulated digestive tube equipped with a dialysis bag. Simultaneously measure the dry matter content of the sample and enter the simulated pig gastrointestinal digestive system.

[0327] Among them: 1) The parameters for simulating digestion in the stomach stage are: temperature 39°C, buffer flow rate 140mL / min, digestion time 3h, residual liquid discharge 4min. 2) The parameters for simulating digestion in the small intestine stage are: temperature 39°C, buffer flow rate 140mL / min, small intestine digestion buffer period 1h, small intestine digestion time 5h, residual liquid discharge 4min. 3) The parameters for simulating digestion in the large intestine stage are: temperature 39°C, buffer flow rate 140mL / min, large intestine digestion time 21h. The cleaning solution is 1500mL / time, each cleaning is 160min, and a total of 6 cleanings.

[0328] After being processed by the simulated porcine gastrointestinal digestive system, the undigested residue in the dialysis bag was transferred to a 90 mm culture dish of known absolute dry weight without loss, dried at 65°C until there was no water mark (usually 12 to 14 hours), and then dried at 105°C to constant weight. The nutritional components of the digested residue were analyzed and the digestibility was calculated. Among them, the total energy value was determined using a fully automatic oxygen bomb calorimeter.

[0329] The calculation formula of enzyme hydrolysis energy value is as follows:

[0330]

[0331] Among them, the unit of enzyme hydrolysis energy value is joule / kilogram (J / kg), the unit of total energy value of feed sample is joule (J), the unit of total energy value of undigested residue is joule (J), and the dry matter weight of feed sample is gram (g).

[0332] The formula for calculating the in vitro digestibility of nutrients is as follows:

[0333]

[0334] Among them, m a The absolute content of nutrients in the sample (absolute content refers to the content after drying), in g, m b The absolute content of nutrients in the residue (absolute content refers to the content after dryness) is expressed in grams. The digestibility of dry matter, energy, protein and amino acids are all calculated using the formula for the in vitro digestibility of nutrients.

[0335] Repeatability: When testing the in vitro digestibility of nutrients, 5 parallel samples were weighed for each test sample; the enzymatic hydrolysis energy value was also measured by taking 5 parallel samples, and the average value was taken as the analysis result. The enzymatic hydrolysis energy value and in vitro digestibility of some nutrients in the two samples are shown in the following table.

[0336] Table 5 Enzymatic hydrolysis energy values ​​of samples and in vitro digestibility of some nutrients

[0337]

[0338] All samples in the table were tested repeatedly, and the values ​​in the table represent the mean ± standard deviation.

[0339] The results showed that the total energy value of Kluyveromyces marxianus protein sample was 20.09MJ / kg. Based on the mass of Kluyveromyces marxianus protein sample, the three amino acids with the highest content were glutamic acid 5.73%, aspartic acid 3.38% and alanine 2.89%. The enzymatic hydrolysis energy value of Kluyveromyces marxianus protein was 13.05MJ / kg, with energy digestibility of 66.69%, dry matter digestibility of 65.68%, protein digestibility of 91.33% and total amino acid digestibility of 91.00%. The three amino acids with the highest digestibility were arginine, alanine and serine.

[0340] The total energy value of the sample of Kluyveromyces maximus hydrolysate for feeding is 20.71MJ / kg. Based on the mass of the sample of Kluyveromyces maximus hydrolysate for feeding, the three amino acids with the highest content are glutamic acid 6.09%, aspartic acid 3.92% and alanine 3.07%. The enzymatic hydrolysis energy value of Kluyveromyces maximus hydrolysate is 13.71MJ / Kg, the energy digestibility is 72.85%, the dry matter digestibility is 70.70%, the protein digestibility is 93.01%, and the total amino acid digestibility is 94.00%. The three amino acids with the highest digestibility are arginine, alanine and serine. The in vitro digestibility of the main nutrients of Kluyveromyces maximus hydrolysate is better than that of the commercially available brewer's yeast hydrolysate (Fubang feed raw material yeast hydrolysate type III NA102, 25kg / bag, sold by Angel Yeast (Chongzuo) Co., Ltd.) and Kluyveromyces maximus protein.

[0341] The "enzymatic hydrolysis energy value" of Kluyveromyces marxianus hydrolysate is higher than that of Kluyveromyces marxianus protein and commercially available Saccharomyces cerevisiae hydrolysate, indicating that it has higher energy available for animal use after being used in feed.

[0342] Application Example 2

[0343] To evaluate the effects of different doses of Kluyveromyces marxianus hydrolysate on growth performance of piglets.

[0344] This experiment used 300 nursery piglets with similar body weight (10.80±0.17kg) and basically the same body condition. According to the principle of similar body weight, they were randomly divided into 5 treatment groups, namely a control group (CON group) and four experimental groups, namely 0.5% MKS addition group (MKS refers to Kluyveromyces marxianus hydrolysate, and MKS here is the Kluyveromyces marxianus hydrolysate prepared in Example 2-4), 1% MKS addition group, 3% MKS addition group and 5% MKS addition group, where 0.5% MKS, 1% MKS, 3% MKS and 5% MKS refer to the mass percentage of MKS in the basic diet. Among them, the control group used a corn-soybean meal-based basic diet, and the group with MKS added used MKS to replace part of the fish meal and soybean meal in the corn-soybean meal-based basic diet formula, and adjusted it to an isonitrogenous and isoenergetic diet. The design of the experimental diet formula for piglets is shown in Table 6 below.

[0345] Table 6 Design of experimental diet formula for piglets

[0346]

[0347]

[0348] The control group and the experimental group were guaranteed to have sufficient feed in the trough for the piglets to eat freely, and water was supplied by the automatic drinking fountain. The amount of feed added to each pen every day was recorded in detail, and the remaining feed was collected and weighed the next day. The pre-feeding period was 5 days, and the experimental period was 28 days. The following growth performance tests were carried out, and the results are shown in Table 7.

[0349] (1) Average initial weight (initial weight): At the beginning of the formal experiment, weigh each animal on an empty stomach in the morning and calculate the average.

[0350] (2) Average ending weight (final weight): Weigh each animal on an empty stomach on the morning of the day the experiment ends, and calculate the average.

[0351] (3) Average daily gain (ADG): (average final weight - average initial weight) / number of days in the experiment.

[0352] (4) Average daily feed intake (ADFI) = total feed intake / number of experimental days.

[0353] (5) Feed-to-gain ratio (F / G) = average daily weight gain / average daily feed intake.

[0354] (6) Diarrhea rate

[0355] During the experiment, the diarrhea of ​​piglets was observed and recorded every day. One day of diarrhea for each piglet was considered as one diarrhea episode. After the experiment, the diarrhea rate of each group was calculated. The calculation formula of diarrhea rate is as follows:

[0356]

[0357] Among them, the unit of total number of diarrhea episodes is head, the unit of number of test pigs is head, and the unit of test days is day.

[0358] Table 7 Growth performance of weaned piglets in the experimental group and the control group

[0359]

[0360]

[0361] The values ​​in the table represent: mean ± standard deviation; "*" indicates significant difference after statistical analysis.

[0362] The results showed that adding 0.5-5% Kluyveromyces marxianus hydrolyzate to the nursery piglet diet to replace part or all of the fish meal in the formula can maintain the growth performance of the nursery piglets and reduce the effect of diarrhea.

[0363] In summary, the present invention prepares a Kluyveromyces marxianus protein for feeding, wherein the crude protein content is ≥48%, the manno-oligosaccharide content is ≥12%, the β-glucan content is ≥12%, and the RNA content is ≥7%. The Kluyveromyces marxianus hydrolysate prepared by further hydrolysis is rich in acid-soluble protein with higher digestibility, the acid-soluble protein content is ≥35%, wherein the acid-soluble protein accounts for ≥65% of the crude protein ratio, the protein digestibility is ≥90%, and the total amino acid digestibility is ≥93%. The Kluyveromyces marxianus protein or Kluyveromyces marxianus hydrolysate of the present invention has a wider range of raw material utilization, has a higher digestibility as a single-cell protein raw material for feeding, can increase animal feed intake, promote animal growth, increase breeding benefits, and has broad application prospects.

[0364] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A Kluyveromyces marxianus hydrolyzate for feeding, characterized in that: Based on the total weight of the Kluyveromyces marxianus hydrolysate for feeding, the crude protein content is ≥50%, the acid-soluble protein content is ≥32.5%, and the weight of the acid-soluble protein accounts for ≥65% of the crude protein weight.

2. The Kluyveromyces marxianus hydrolyzate for feeding according to claim 1, wherein The crude protein content is 50% to 60%; preferably, the crude protein content is 51% to 60%; more preferably, the crude protein content is 54% to 60%; And / or, the content of acid-soluble protein is 35% to 50%; preferably, the content of acid-soluble protein is 40% to 50%; more preferably, the content of acid-soluble protein is 45% to 50%; And / or, the mass percentage of acid-soluble protein to crude protein is 65% to 90%; preferably, the mass percentage of acid-soluble protein to crude protein is 70% to 90%; more preferably, the mass percentage of acid-soluble protein to crude protein is 75% to 90%; further preferably, the mass percentage of acid-soluble protein to crude protein is 80% to 90%.

3. The Kluyveromyces marxianus hydrolyzate for feeding according to claim 1 or 2, wherein The hydrolyzate of Kluyveromyces marxianus for feeding further comprises manno-oligosaccharides, the content of which is ≥10.0% based on the total mass of the hydrolyzate of Kluyveromyces marxianus for feeding; preferably, the content of manno-oligosaccharides is 10% to 15%; And / or, based on the total mass of the Kluyveromyces marxianus hydrolysate for feeding, the Kluyveromyces marxianus hydrolysate for feeding further comprises β-glucan, and the content thereof is ≥10.0%; preferably, the content of β-glucan is 10% to 20%; more preferably, the content of β-glucan is 15% to 20%; And / or, based on the dry weight of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises RNA, the content of which is ≥5.0%; preferably, the content of RNA is 5% to 10%; more preferably, the content of RNA is 7% to 9%; and / or, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises free amino acids, the content of which is ≥10%; preferably, the content of the free amino acids is 10% to 15%; And / or, based on the total mass of the Kluyveromyces marxianus hydrolysate, the Kluyveromyces marxianus hydrolysate further comprises hydrolyzed amino acids, the content of which is ≥45%; preferably, the content of the hydrolyzed amino acids is 45% to 55%; more preferably, the content of the hydrolyzed amino acids is 47% to 52%; And / or, based on the total mass of the Kluyveromyces marxianus hydrolysate for feeding, the Kluyveromyces marxianus hydrolysate for feeding further comprises small peptides, and the content thereof is ≥15%; preferably, the content of the small peptides is 20% to 37%; further preferably, the content of the small peptides is 29% to 37%; further preferably, the content of the small peptides is 31% to 36%.

4. The Kluyveromyces marxianus hydrolyzate for feeding according to any one of claims 1 to 3, wherein The feed Kluyveromyces marxianus hydrolyzate is prepared by using Kluyveromyces marxianus AMCC 30634 (Kluyveromyces marxianus AMCC30634), and the preservation number of the Kluyveromyces marxianus AMCC 30634 (Kluyveromyces marxianus AMCC30634) is CCTCC NO:M 2023217.

5. A method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: fermenting and culturing Kluyveromyces marxianus to obtain yeast milk; Step 2: subjecting the fermented Kluyveromyces marxianus to a heat shock treatment to perform autolysis; Step 3: adding a composite enzyme to the solution obtained in step 3 for enzymolysis; and Step 4: The enzymatic hydrolysate obtained in step 3 is inactivated, concentrated and dried to obtain Kluyveromyces marxianus hydrolysate.

6. The method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to claim 5, wherein: In step 1, the fermentation culture comprises the following steps: The Kluyveromyces marxianus is inoculated into a seed tank for culturing to obtain a seed solution; and the Kluyveromyces marxianus seed solution is then inoculated into a fermentation tank for culturing.

7. The method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to claim 6, wherein: In step 1, the volume percentage of the inoculation amount of Kluyveromyces marxianus seed solution inoculated into the fermentation tank is 20% to 40%; and / or, the fermentation conditions in the fermenter are: temperature 30-37° C., time 25-30 h, ventilation 1-3 vvm, and / or, pH 4.5-6.5; And / or, the culture medium of the fermentation tank includes corn syrup, wherein the mass percentage of the corn syrup is 50% to 100%; preferably, the culture medium of the fermentation tank also includes molasses, wherein the mass percentage of the molasses is 0% to 50%, and the sum of the mass percentages of molasses and corn syrup is 100%.

8. The method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to claim 6 or 7, wherein: In step 1, the culturing process in the seed tank includes: inoculating Kluyveromyces marxianus into the primary seed tank for culturing to obtain Kluyveromyces marxianus seed liquid.

9. The method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding according to any one of claims 6 to 8, wherein: In step 1, the culture conditions in the primary seed tank are: temperature 30-37°C, time 24-30 hours, and / or ventilation volume 15-30m 3 / h; More preferably, the fermentation conditions in the primary seed tank are: temperature 30-32°C, and / or time 24-25h; And / or, based on the total mass of the culture medium, the culture medium of the primary seed tank includes 10% to 25% sugar, 0.5% to 5% ammonium sulfate and 0.5% to 5% diammonium phosphate; preferably, the culture medium of the primary seed tank includes 23% to 25% sugar, 0.5% to 1% ammonium sulfate and 0.5% to 1% diammonium phosphate.

10. The method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to claim 8 or 9, wherein: The Kluyveromyces marxianus seed solution obtained by culturing in the primary seed tank is inoculated into the primary seed tank for further culturing to obtain the Kluyveromyces marxianus seed solution.

11. The method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to claim 10, wherein: All the Kluyveromyces marxianus seed liquid obtained by culturing in the primary seed tank is inoculated into the primary seed tank; And / or, the culture conditions in the primary seed tank are: temperature 30-37°C, time 25-30h, and / or ventilation volume 1500-3600m 3 / h; preferably, the fermentation conditions in the primary seed tank are: temperature 30-32°C, and / or time 25-26h; And / or, based on the total mass of the culture medium, the culture medium of the first-level seed tank includes 20% to 30% molasses, 1% to 5% ammonia water and 1% to 5% diammonium phosphate; preferably, the culture medium of the first-level seed tank includes 26% to 28% molasses, 1.5% to 2.5% ammonia water and 1% to 2% diammonium phosphate.

12. The method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding according to any one of claims 5 to 11, wherein: In step 2, the temperature of the heat shock treatment is 80-95° C.; preferably, the temperature of the heat shock treatment is 85-95° C.; and / or, the time of the heat shock treatment is 40-120 seconds; preferably, the time of the heat shock treatment is 40-100 seconds; and / or, heat shock treatment followed by pH adjustment and subsequent autolysis; Preferably, the pH is adjusted to 4-6 after the heat shock treatment; more preferably, the pH is adjusted to 4.8-5.2 after the heat shock treatment; and / or, preferably, the temperature of autolysis is 50-60°C; more preferably, the temperature of autolysis is 55-58°C; and / or, preferably, the time of autolysis is 6-12h; more preferably, the time of autolysis is 8-12h.

13. The method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to any one of claims 5 to 12, wherein: In step 3, the complex enzyme includes one or more selected from the group consisting of papain, neutral protease and alkaline protease; Preferably, the complex enzyme is a combination of papain, neutral protease and alkaline protease; More preferably, the conditions for enzymatic hydrolysis are: temperature 55-65° C., pH 5-7, and / or time 8-14 h.

14. The method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding according to any one of claims 5 to 13, wherein: Based on the mass of dry matter of the yeast milk obtained after fermentation and culturing in step 1, the added amount of the complex enzyme is 0.1% to 1.5%; preferably, the added amount of the complex enzyme is 0.6% to 1.1%; And / or, based on the mass of dry matter of the yeast milk obtained after fermentation and culturing in step 1, the amount of papain added is 0.1% to 0.5%; preferably, the amount of papain added is 0.1% to 0.3%; more preferably, the amount of papain added is 0.15% to 0.25%; And / or, based on the mass of dry matter of yeast milk obtained after fermentation and culturing in step 1, the amount of neutral protease added is 0.1% to 0.5%; preferably, the amount of neutral protease added is 0.15% to 0.5%; more preferably, the amount of neutral protease added is 0.15% to 0.25%; And / or, based on the mass of dry matter of the yeast milk obtained after fermentation and culturing in step 1, the amount of alkaline protease added is 0.1% to 0.5%; preferably, the amount of alkaline protease added is 0.15% to 0.5%; more preferably, the amount of alkaline protease added is 0.15% to 0.25%.

15. The method for preparing the hydrolyzate of Kluyveromyces marxianus for feeding according to any one of claims 5 to 14, wherein: In step 4, the conditions for inactivating the enzyme and / or concentrating are: a temperature of 80 to 95° C., and / or a time of 2 to 4 hours; preferably, the concentration is performed to a solid content of 30% to 40%; And / or, the drying is selected from spray drying or drum drying; preferably, the conditions of the spray drying are: feed flow rate 1.6-1.8m 3 , pressure 100~120bar, inlet air temperature 165~180℃, and / or, outlet air temperature 80~85℃.

16. A hydrolyzate of Kluyveromyces marxianus for feeding, characterized in that: The hydrolyzate is prepared by the method for preparing the Kluyveromyces marxianus hydrolyzate for feeding according to any one of claims 5 to 15.

17. A Kluyveromyces marxianus protein for feeding, characterized in that: The Kluyveromyces marxianus protein is prepared by drying the yeast milk obtained in step 1 of the method for preparing a Kluyveromyces marxianus hydrolyzate according to any one of claims 5 to 11.

18. Use of the Kluyveromyces marxianus hydrolyzate for feeding according to claim 16 or the Kluyveromyces marxianus protein for feeding according to claim 17 in the field of preparing feed; Preferably, the feed Kluyveromyces marxianus hydrolyzate or the feed Kluyveromyces marxianus protein is used in the preparation of piglet feed, aquatic feed or ruminant feed; And / or, preferably, use of the feeding Kluyveromyces marxianus hydrolyzate or the feeding Kluyveromyces marxianus protein in the field of preparing feed for improving diarrhea.

19. A feed, characterized in that: It comprises the feed Kluyveromyces marxianus hydrolyzate according to any one of claims 1 to 4, the feed Kluyveromyces marxianus hydrolyzate according to claim 16, or the feed Kluyveromyces marxianus protein according to claim 17; Preferably, the addition amount of the Kluyveromyces marxianus hydrolyzate or the Kluyveromyces marxianus protein for feeding is 0.5% to 10% of the dry weight of the feed; More preferably, the addition amount of the Kluyveromyces marxianus hydrolysate or the Kluyveromyces marxianus protein for feeding is 0.5% to 5% of the dry weight of the feed; More preferably, the feed is piglet feed, aquatic feed or ruminant feed; And / or, more preferably, the feed is a feed that improves diarrhea.

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