Composite mannan oligosaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal feed, in particular to compound mannan oligosaccharide as well as a preparation method and application thereof. The preparation method of the composite mannan oligosaccharide comprises the following steps: (a) carrying out water bath reaction on a mixed system containing a plant raw material solution, mannase and cellulase to obtain plant mannan oligosaccharide; (b) inoculating saccharomycetes into the plant mannan oligosaccharide for fermentation; and (c) adding cellulase and lysozyme into the mixed system after fermentation is finished, and carrying out autolysis reaction. The preparation method of the composite mannan oligosaccharide is simple and easy to operate, and the prepared composite mannan oligosaccharide has good intestinal pathogen adsorption and immunoregulation functions.

Description

Technical Field

[0001] The present invention relates to the technical field of animal feed, and in particular to a composite manno-oligosaccharide and a preparation method and application thereof. Background Art

[0002] In recent years, studies have found that long-term use of antibiotics is prone to drug resistance and residues in livestock products, and affects human health and damages the ecological environment through the food chain. Therefore, through the promulgation of laws, some antibiotics are prohibited or restricted from being used in feed, and then a green feed additive with low (or no) pollution and low (or no) residue is sought. As early as the 1960s, it was reported that oligosaccharides can be used as immunopotentiators. Manno-oligosaccharides have certain immunogenicity and can stimulate the body's immune response. Moreover, they can bind to the surface of certain toxins, viruses and eukaryotic cells as adjuvants for these exogenous antigens, slow down the absorption of antigens, increase the titer of antigens, and thus enhance the cellular and humoral immune responses of animals. It can also affect the immune system by stimulating the liver to secrete mannose-binding protein, which can adhere to the bacterial capsule and trigger a series of complements, thereby starting the immune system to respond. Manno-oligosaccharides can accelerate the recovery of the suppressed immune system of piglets, and can effectively reduce mortality and diarrhea rates.

[0003] Plant oligosaccharides are polymers formed by dehydration and condensation of 2 to 10 monosaccharides. They are divided into two categories: ordinary oligosaccharides and functional oligosaccharides. Ordinary oligosaccharides include sucrose, maltose, lactose, maltotriose and trehalose, which are familiar to people. Animals can digest and absorb these sugars, but they have no promoting effect on beneficial intestinal bacteria. Functional oligosaccharides mainly include manno-oligosaccharides, oligofructose, isomaltose oligosaccharides, lactulose and xylooligosaccharides, which cannot be digested and absorbed by animals. They are effective substances for promoting the proliferation of probiotics such as bifidobacteria. At the same time, functional oligosaccharides have the characteristics of low sweetness, low calories and high stability, and have good performance in moisture retention, acid resistance, heat resistance and anti-crystallization. A large number of studies have shown that as a new type of microecological effect additive, manno-oligosaccharides, it overcomes the shortcomings of all previous antibiotics, probiotics and enzyme preparations, with low dosage, pure natural, no residue and strong stability. It also has the dual functions of adsorbing intestinal pathogens and exerting immune regulation, thereby improving the production performance of animals. It is one of the most likely substitutes for antibiotics.

[0004] Yeast mannan is located in the outermost layer of the yeast cell wall, and together with the glucans in the middle and inner layers, it constitutes the main polysaccharide component of the yeast cell wall. Due to its functions of immune regulation, improving intestinal health, selectively adsorbing pathogenic microorganisms, and adsorbing mycotoxins, it is a green feed additive and food additive with great market potential.

[0005] Plant-based mannan oligosaccharides (plant-based raw materials containing mannan include palm kernel meal, coconut meal, konjac, guar gum, etc.) are easy to prepare, have high effective ingredients, and have a wide source of raw materials. However, their disadvantages are that they are easy to produce monosaccharides and lack the functions of selectively adsorbing pathogenic microorganisms and mycotoxins compared to yeast mannan. Yeast mannan has comprehensive effects in animal applications, but its disadvantages are that it is difficult to produce, has low product purity, and is costly.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a composite manno-oligosaccharide and its preparation method and application. The preparation method of the composite manno-oligosaccharide has the advantages of being simple and easy to operate, and the prepared composite manno-oligosaccharide has the advantages of good adsorption of intestinal pathogens and immune regulation.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] One aspect of the present invention relates to a method for preparing a complex manno-oligosaccharide, comprising the following steps:

[0010] (a) subjecting a mixed system containing a plant raw material liquid, mannanase and cellulase to a water bath reaction to obtain plant manno-oligosaccharides;

[0011] (b) inoculating yeast into the plant manno-oligosaccharide for fermentation;

[0012] (c) adding cellulase and lysozyme to the mixed system after the fermentation to carry out autolysis reaction.

[0013] The preparation method of the composite manno-oligosaccharide is simple and easy to operate, and the prepared composite manno-oligosaccharide has good intestinal pathogen adsorption and immune regulation functions.

[0014] Another aspect of the present invention also relates to the complex manno-oligosaccharide prepared by the preparation method of the complex manno-oligosaccharide.

[0015] The complex manno-oligosaccharide has the ability to promote animal growth, improve the health of animals, increase animal weight, and enhance animal immunity.

[0016] Another aspect of the present invention also relates to a feed, including the complex manno-oligosaccharide prepared by the preparation method of the complex manno-oligosaccharide or the complex manno-oligosaccharide.

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

[0018] (1) The method for preparing the composite manno-oligosaccharide provided by the present invention comprises preparing plant manno-oligosaccharides and culturing yeast cells at the same time, utilizing the growth of yeast cells to digest monosaccharides, thereby reducing the monosaccharide content in the plant manno-oligosaccharides, and obtaining yeast cell walls by promoting the autolysis of yeast cells through enzymes, thereby obtaining a high-content, low-cost, and fully effective composite manno-oligosaccharide.

[0019] (2) The composite manno-oligosaccharide provided by the present invention has good adsorption of intestinal pathogens and immune regulation functions, and has the advantages of promoting animal growth, improving the health status of animals, increasing animal weight, and improving animal immunity. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] One aspect of the present invention relates to a method for preparing a complex manno-oligosaccharide, comprising the following steps:

[0022] (a) subjecting a mixed system containing a plant raw material liquid, mannanase and cellulase to a water bath reaction to obtain plant manno-oligosaccharides;

[0023] (b) inoculating yeast into the plant manno-oligosaccharide for fermentation;

[0024] (c) adding cellulase and lysozyme to the mixed system after the fermentation to carry out autolysis reaction.

[0025] The method for preparing the composite manno-oligosaccharide comprises preparing plant manno-oligosaccharide and culturing yeast cells at the same time, utilizing the growth of yeast cells to digest monosaccharides, thereby reducing the monosaccharide content in the plant manno-oligosaccharide, and obtaining yeast cell walls by promoting the autolysis of yeast cells through enzymes, thereby obtaining a high-content, low-cost, and fully effective composite manno-oligosaccharide.

[0026] Furthermore, the plant raw material in the plant raw material liquid includes at least one of palm kernel meal, coconut meal, konjac or guar gum.

[0027] Furthermore, the water content of the plant raw material in the plant raw material liquid is <10%, including but not limited to any point value of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9% or a range value between any two of them.

[0028] Furthermore, the particle size of the plant raw material in the plant raw material liquid is 40 to 80 mesh, including but not limited to any point value of 40 mesh, 45 mesh, 50 mesh, 55 mesh, 60 mesh, 65 mesh, 70 mesh, 75 mesh or 80 mesh or a range value between any two of them.

[0029] Furthermore, the material-water ratio of the plant raw material liquid is 1g:2.2-10mL, including but not limited to 1g:2.2mL, 1g:3mL, 1g:5mL, 1g:7mL, 1g:9mL or 1g:10mL.

[0030] Furthermore, the mass ratio of the plant raw material, the mannanase and the cellulase in the plant raw material liquid is 100:0.1-10:0.1-1, including but not limited to 100:0.1:1, 100:3:0.8, 100:5:0.5, 100:8:0.3 or 100:10:0.1. Mannanase and cellulase are added in a certain amount to fully enzymatically hydrolyze mannan and cellulose in the plant raw material, which is conducive to obtaining plant manno-oligosaccharides.

[0031] Further, the temperature of the water bath reaction is 30-80°C, including but not limited to any point value of 30°C, 35°C, 40°C, 45°C, 50°C, 55, 60°C, 65°C, 70°C, 75°C or 80°C or a range value between any two of them.

[0032] Furthermore, the water bath reaction time is 3 to 25 h, including but not limited to any one of 3 h, 5 h, 8 h, 10 h, 13 h, 15 h, 18 h, 20 h, 23 h or 25 h, or a range between any two of them.

[0033] Limiting the water bath reaction temperature and time within a certain range can ensure that the water bath reaction is fully carried out and exert the best enzymatic hydrolysis effect of mannan and cellulase.

[0034] Furthermore, the viable count of the yeast is 10 10 ~10 100 CFU / g, including but not limited to 10 10 CFU / g, 10 20 CFU / g, 10 30 CFU / g, 10 40 CFU / g, 10 50 CFU / g, 1060 CFU / g, 10 70 CFU / g, 10 80 CFU / g, 10 90 CFU / g or 10 100 CFU / g or a range of values ​​between them.

[0035] Furthermore, the mass ratio of the yeast to the plant raw material in the plant raw material liquid is 0.05 to 0.1:100, including but not limited to 0.05:100, 0.06:100, 0.07:100, 0.08:100, 0.09:100 or 0.1:100.

[0036] After obtaining plant mannooligosaccharides, yeast is inoculated into the plant mannooligosaccharides for fermentation. During the growth process, yeast utilizes monosaccharides in plant mannooligosaccharides, reduces monosaccharides in plant mannooligosaccharides, and improves the selective adsorption of pathogenic microorganisms and mycotoxins by plant mannooligosaccharides. Limiting the inoculation amount of yeast within a certain range can ensure that yeast fully utilizes monosaccharides in plant mannooligosaccharides.

[0037] Furthermore, the fermentation temperature is 25-40°C, including but not limited to any one of 25°C, 30°C, 35°C or 40°C or a range between any two of them.

[0038] Furthermore, the fermentation time is 24 to 72 hours, including but not limited to any one of 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours or 72 hours, or a range between any two of the points.

[0039] Controlling the fermentation temperature and time within a certain range can not only ensure that the yeast has a good growth ability, but also ensure that the yeast can fully utilize the monosaccharides in the plant mannan oligosaccharides, which is beneficial to improving the ability of the plant mannan oligosaccharides to selectively adsorb pathogenic microorganisms and mycotoxins.

[0040] Further, the mannose content in the fermented mixed system is lower than 1% of the mannose content in the plant raw material liquid, and the fermentation is terminated. Reducing mannose in the raw material - not manno-oligosaccharides. Digesting monosaccharides to increase oligosaccharide content The manno-oligosaccharide content is based on the palm kernel meal raw material as the denominator, and it is determined that most of the mannose monosaccharide is fully utilized.

[0041] Furthermore, the temperature of the autolysis reaction is 40-90°C, including but not limited to any point value of 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C or a range value between any two of them.

[0042] Furthermore, the autolysis reaction time is 16 to 48 hours, including but not limited to any point value of 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours or 48 hours, or a range value between any two of them.

[0043] Carrying out the autolysis reaction of yeast at a certain temperature and time can fully autolyze the yeast to obtain the yeast cell wall and obtain yeast mannan.

[0044] Furthermore, the mass ratio of the plant raw material, the cellulase and the lysozyme in the plant raw material liquid is 100:0.1~1:0.1~1, including but not limited to 100:0.1:1, 100:0.3:0.8, 100:0.5:0.5, 100:0.8:0.3 or 100:1:0.1.

[0045] Another aspect of the present invention also relates to the complex manno-oligosaccharide prepared by the preparation method of the complex manno-oligosaccharide.

[0046] The complex manno-oligosaccharide has the ability to promote animal growth, improve the health of animals, increase animal weight and enhance animal immunity.

[0047] Another aspect of the present invention also relates to a feed, including the complex manno-oligosaccharide prepared by the preparation method of the complex manno-oligosaccharide or the complex manno-oligosaccharide.

[0048] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0049] Example 1

[0050] The preparation method of the composite manno-oligosaccharide provided in this embodiment comprises the following steps:

[0051] (1) The palm kernel meal raw material is crushed to 60 mesh and the moisture content of the raw material is controlled to be less than 10%;

[0052] (2) adding the palm kernel meal obtained in step (1) to water at a material-water ratio of 1:3; after mixing evenly, adding mannanase in an amount of 5% of the raw material and cellulase in an amount of 0.5% of the raw material, and heating and stirring in a water bath at 50° C. for 24 hours;

[0053] (3) cooling the mixed solution obtained in step (2) to 30° C.;

[0054] (4) The number of viable bacteria is 10 10 CFU / g yeast is inoculated onto the plant manno-oligosaccharide obtained in step (3) for liquid fermentation, wherein the inoculation amount is 0.1% of the mass of the palm kernel meal raw material; the fermentation temperature is 30° C., the fermentation time is 48 hours, and the mannose content in the solution is detected. If the content is lower than 1% of the raw material, the fermentation is terminated;

[0055] (5) heating the fermentation liquid obtained in step (4) to 70° C., adding cellulase in an amount equal to 1% of the palm kernel meal raw material and lysozyme in an amount equal to 0.5%, and stirring the fermentation liquid for 24 hours to promote the autolysis of the yeast cell wall;

[0056] (6) Filter the yeast solution obtained in step (5) through a plate and frame to obtain a filtrate, add a small amount of maltodextrin and spray-dry.

[0057] Example 2

[0058] The preparation method of the composite manno-oligosaccharide provided in this embodiment comprises the following steps:

[0059] (1) The coconut meal raw material is crushed to 40 mesh, and the moisture content of the raw material is controlled to be less than 10%;

[0060] (2) adding the coconut meal raw material obtained in step (1) into water at a material-water ratio of 1:2.2; after mixing evenly, adding mannanase in an amount of 10% of the raw material and cellulase in an amount of 0.1% of the raw material, and heating and stirring in a water bath at 30° C. for 24 hours;

[0061] (3) The number of viable bacteria is 10 100 CFU / g yeast is inoculated onto the plant manno-oligosaccharide obtained in step (2) for liquid fermentation, wherein the inoculation amount is 0.05% of the mass of the coconut meal raw material; the fermentation temperature is 25° C., the fermentation time is 72 hours, and the mannose content in the solution is detected. If the content is lower than 1% of the raw material, the fermentation is terminated;

[0062] (4) heating the fermentation liquid obtained in step (3) to 90° C., adding 0.1% of cellulase and 0.1% of lysozyme to the coconut meal raw material, and stirring the fermentation liquid for 16 h to promote autolysis of yeast cell walls;

[0063] (5) Filter the yeast solution prepared in step (4) through a plate and frame to obtain a filtrate, add a small amount of carrier and spray-dry.

[0064] Example 3

[0065] The preparation method of the composite manno-oligosaccharide provided in this embodiment comprises the following steps:

[0066] (1) the konjac raw material is crushed to 50 meshes, and the raw material moisture content is controlled to be less than 10%;

[0067] (2) adding the konjac raw material obtained in step (1) to water, with a material-water ratio of 1:10; after mixing, adding mannanase in an amount of 0.1% of the raw material and cellulase in an amount of 1% of the raw material, and heating and stirring in a water bath at 80° C. for 3 hours;

[0068] (3) cooling the mixed solution obtained in step (2) to 30° C.;

[0069] (4) The number of viable bacteria is 10 50 CFU / g yeast is inoculated onto the plant manno-oligosaccharide obtained in step (3) for liquid fermentation, wherein the inoculation amount is 0.06% of the mass of the konjac raw material; the fermentation temperature is 40° C., the fermentation time is 24 hours, and the mannose content in the solution is detected. If the content is lower than 1% of the raw material, the fermentation is terminated;

[0070] (5) heating the fermentation liquid obtained in step (4) to 40° C., adding 0.3% of cellulase to the konjac raw material and 1% of lysozyme, stirring the fermentation liquid for 48 h to promote autolysis of yeast cell walls;

[0071] (6) Filter the yeast from step (5) through a plate and frame to obtain a filtrate, add a small amount of carrier and spray-dry.

[0072] Example 4

[0073] The preparation method of the composite manno-oligosaccharide provided in this embodiment comprises the following steps:

[0074] (1) The guar gum raw material is crushed to 80 mesh, and the moisture content of the raw material is controlled to be less than 10%;

[0075] (2) adding the guar gum raw material obtained in step (1) into water at a material-water ratio of 1:7; after mixing evenly, adding mannanase in an amount of 5% of the raw material and cellulase in an amount of 0.6% of the raw material, and heating and stirring in a water bath at 65° C. for 18 hours;

[0076] (3) cooling the mixed solution obtained in step (2) to 30° C.;

[0077] (4) The number of viable bacteria is 10 30 CFU / g yeast is inoculated onto the plant manno-oligosaccharide obtained in step (3) for liquid fermentation, wherein the inoculation amount is 0.08% of the mass of the guar gum raw material; the fermentation temperature is 35° C., the fermentation time is 50 hours, and the mannose content in the solution is detected. If the content is lower than 1% of the raw material, the fermentation is terminated;

[0078] (5) heating the fermentation liquid obtained in step (4) to 60° C., adding cellulase in an amount equal to 0.7% of the guar gum raw material and lysozyme in an amount equal to 0.7%, and stirring the fermentation liquid for 20 hours to promote the autolysis of the yeast cell wall;

[0079] (6) Filter the yeast from step (5) through a plate and frame to obtain a filtrate, add a small amount of carrier and spray-dry.

[0080] Comparative Example 1

[0081] The preparation method of the composite manno-oligosaccharide provided in this comparative example comprises the following steps:

[0082] (1) to (2) are the same as in Example 1;

[0083] (3) The plant enzymatic hydrolysis solution in step (2) is filtered through a plate and frame to obtain a filtrate, and a small amount of maltodextrin is added and spray-dried.

[0084] Experimental Example 1

[0085] The components of the complex manno-oligosaccharides prepared in each example and comparative example were analyzed using T / ESL 32004-2024. The results are shown in Table 1.

[0086] Table 1 Nutrient composition analysis results of complex manno-oligosaccharides

[0087]

[0088] From the data in Table 1, it can be seen that most of the products obtained in Example 1 are functional manno-oligosaccharides, while the comparative example only contains plant manno-oligosaccharides and a large amount of mannose.

[0089] Experimental Example 2

[0090] The composite manno-oligosaccharide product prepared in Example 1 and the plant-based manno-oligosaccharide product prepared in Comparative Example 1 were respectively used as sow feed additives, and the mass of each additive accounted for 0.1% of the mass of the sow basic feed, to verify the use effect of each product. The specific operation method is as follows:

[0091] 200 large long binary pregnant sows with similar parity and expected delivery date, 30 days before the expected delivery date, were divided into 5 groups using a completely randomized block design, corresponding to 5 areas (delivery rooms), namely control group I (control group) 40 heads, experimental group II 40 heads, experimental group III 40 heads, experimental group IV 40 heads, and experimental group V 40 heads. After 7 days of the pre-trial period, the full trial period began. During the trial, the sows were fed at 07:30 every day. The control group I was fed with the basic diet of pregnant sows, and 25g of blank premix was added. The basic diet was prepared according to NRC and was prepared in two stages: late pregnancy and lactation. In the experimental groups II, III, IV and V, 25g of MOS premix was added to the basic diet every morning, and the MOS content was 1.5g. It was mixed with the basic diet and fed twice a day. The experiment adopted single-pen feeding, and disinfection, immunization and deworming were carried out according to the routine procedures and methods of the pig farm. Drinking water was free. The composition and nutritional level of the basic diet during the peripartum period (late pregnancy-lactation period) are shown in Table 1. A dedicated person was responsible for the pig numbering and recording of various indicators. The feeding and management measures of each experimental group and its suckling piglets were consistent, and the production performance of sows in each group and the health status of piglets were tracked and recorded. The results are shown in Table 2.

[0092] Table 2 Effect of feed additives

[0093]

[0094] It can be seen from Table 2 that the composite manno-oligosaccharide prepared by the present invention has the best effect and can promote the growth of pigs, improve the health of pigs, and increase the weight of pigs.

[0095] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for preparing a composite manno-oligosaccharide, It is characterized in that The following steps are involved: (a) subjecting a mixed system containing a plant raw material liquid, mannanase and cellulase to a water bath reaction to obtain plant manno-oligosaccharides; (b) inoculating yeast into the plant manno-oligosaccharide for fermentation; (c) adding cellulase and lysozyme to the mixed system after the fermentation to carry out autolysis reaction.

2. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The temperature of the water bath reaction is 30 to 80°C; And / or, the water bath reaction time is 3 to 25 hours.

3. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The fermentation temperature is 25-40°C; And / or, the fermentation time is 24 to 72 hours.

4. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The temperature of the autolysis reaction is 40 to 90° C. And / or, the autolysis reaction time is 16 to 48 hours.

5. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The mass ratio of the plant raw material, the mannanase and the cellulase in the plant raw material liquid is 100:0.1-10:0.1-1.

6. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The viable count of the yeast is 10 10 ~10 100 CFU / g; And / or, the mass ratio of the yeast to the plant raw material in the plant raw material liquid is 0.05-0.1:

100.

7. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The mass ratio of the plant raw material, the cellulase and the lysozyme in the plant raw material liquid is 100:0.1-1:0.1-1.

8. The method for preparing the complex manno-oligosaccharide according to claim 1, It is characterized in that The plant raw material in the plant raw material liquid includes at least one of palm kernel meal, coconut meal, konjac or guar gum.

9. The complex manno-oligosaccharide prepared by the method for preparing complex manno-oligosaccharide according to any one of claims 1 to 8.

10. A feed, It is characterized in that The invention relates to a complex manno-oligosaccharide prepared by the method for preparing the complex manno-oligosaccharide according to any one of claims 1 to 8 or a complex manno-oligosaccharide according to claim 9.

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