Yeast mannose oligosaccharides, methods of making and using same

Abstract

The application provides a yeast mannose oligosaccharide and a preparation method and application thereof. The preparation method comprises the following steps: S1, mixing yeast cell walls with water to obtain a mixed solution; S2, adjusting the pH of the mixed solution to 4-9, performing first enzymolysis on the mixed solution by using a protease to form a first enzymolysis product; secondly, performing second enzymolysis on the first enzymolysis product by using a polysaccharide enzyme to obtain a second enzymolysis product; the amount of the protease is greater than or equal to 0.2% of the weight of the yeast cell walls, and the amount of the polysaccharide enzyme is greater than or equal to 0.1% of the weight of the yeast cell walls; S3, centrifuging the second enzymolysis product to obtain a supernatant and a centrifugal heavy phase; filtering the supernatant by using an ultrafiltration membrane, and the retentate is the yeast mannose oligosaccharide, the permeate is a yeast extract, and the centrifugal heavy phase is a yeast glucan. The yeast mannose oligosaccharide obtained by the application has high purity and high yield, the mannose oligosaccharide is clean, can be fermented by beneficial bacteria, and can be applied as a prebiotic in food.

Description

Technical Field

[0001] This invention relates to the field of yeast extraction technology, and more specifically, to a yeast mannan oligosaccharide, its preparation method, and its application. Background Technology

[0002] Prebiotics, proposed by Glenn Gibson et al. (1955), refer to "indigestible food components that can exert beneficial effects on the host by selectively stimulating the growth or activity of one or a few types of bacteria in the colon, thereby improving the host's health." These activated bacteria should be naturally beneficial, such as Bifidobacteria and Lactobacillus.

[0003] Currently, commonly used prebiotics include oligosaccharides, such as fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, and inulin. Some microalgae can also be used as prebiotics, such as spirulina and arthrophyllum. In addition, polysaccharides (such as trachoma azedarach polysaccharides and carrot nitrogenous polysaccharides), protein hydrolysates (such as casein hydrolysates, α-lactalbumin, lactoferrin, etc.), as well as vegetables, traditional Chinese medicine, and wild plants from natural plants can also be used as prebiotics.

[0004] Yeast cell walls are rich in polysaccharides, such as yeast β-glucan and mannan oligosaccharides. Mannan oligosaccharides are located in the outermost layer of the cell wall and have a molecular weight of approximately 20-200 kDa. Studies have found that they possess various biological activities, including enhancing animal immunity, regulating glucose and lipid metabolism, maintaining intestinal health, and promoting growth and providing antioxidant effects, making them a potential prebiotic. However, they are currently mostly used as feed additives, resulting in relatively low added value.

[0005] Currently, the main methods for preparing yeast mannan oligosaccharides include degradation methods, such as enzymatic degradation, oxidative acidification degradation, ultrasonic degradation, and irradiation modification degradation, as well as synthetic methods, such as microwave solid-state synthesis. Due to the high cost and technical difficulty of synthetic methods, industrial production currently mostly uses degradation methods (Liu Shiqi et al., Physiological Functions of Mannan Oligosaccharides and Research Progress in Livestock Production). However, even with degradation methods, the purity of the obtained mannan oligosaccharides is relatively low, generally around 40%.

[0006] CN 102051400 B provides a method for preparing a mannan protein product, the steps of which include: adjusting the pH of yeast cell walls to 7-10 and treating them at a temperature of 80-135℃; adjusting the temperature of the yeast cell walls to 30-70℃, adding alkaline protease for treatment, centrifuging to collect the supernatant, incubating the supernatant at low temperature for 1-20 hours, centrifuging to remove the precipitate; pulverizing the supernatant using a membrane with a shear molecular weight of 200-400 KD, collecting the permeate, and spray drying to obtain the yeast mannan protein product. However, the purity of the mannan obtained by this method is not high enough, less than 50%, and the mannan protein obtained by this method is mainly used for stabilizing tartrate and proteins in wine, limiting its application areas.

[0007] CN 104862355 B provides a method for extracting dextran and mannoprotein from yeast cell walls in a single step. The method involves homogenizing the yeast cell wall under high pressure, hydrolyzing it with an alkaline protease, centrifuging to obtain the heavy phase as dextran, and filtering the light phase using a nanofiltration membrane to obtain mannoprotein. However, high-pressure homogenization is very costly and noisy.

[0008] CN 109912700 A also provides a method for extracting dextran and mannoprotein from yeast cell walls in a single step. This method uses yeast cells as raw material. First, the yeast cells are subjected to high-temperature inactivation, centrifuged, and the supernatant is dried to obtain yeast extract, with the cell wall as the heavy phase. The yeast cell wall is then enzymatically hydrolyzed using β-1,6-glucanase, and centrifuged to obtain the enzymatic supernatant and precipitate. The enzymatic supernatant is then subjected to water extraction, alcohol precipitation, centrifugation, and drying to obtain mannoprotein. After ethanol recovery, the remaining liquid is concentrated and spray-dried to obtain the yeast extract. The enzymatic precipitate is defatted, treated with protease, and spray-dried to obtain yeast dextran. While the mannoprotein produced by this method has high purity, the yield is low, and the production process requires a large amount of ethanol, resulting in high production risks and costs. Furthermore, the dextran production method requires defatting with an organic solvent (petroleum ether), which may pose safety hazards if applied to food, and petroleum ether is highly volatile, making it neither environmentally friendly nor safe.

[0009] For the reasons mentioned above, it is necessary to provide a new method for preparing yeast mannan oligosaccharides to improve their purity and make them more suitable for use as yeast prebiotics in food. Summary of the Invention

[0010] The main objective of this invention is to provide a yeast mannan oligosaccharide, its preparation method, and its application, in order to solve the problems of low purity or low yield in the preparation of yeast mannan oligosaccharides in the prior art.

[0011] To achieve the above objectives, according to one aspect of the present invention, a method for preparing yeast mannan oligosaccharides is provided, comprising the following steps: Step S1, mixing yeast cell walls with water to obtain a mixture; Step S2, adjusting the pH of the mixture to 4-9, and then performing a first enzymatic hydrolysis with a protease to form a first hydrolysis product; then, performing a second enzymatic hydrolysis on the first hydrolysis product with a polysaccharide enzyme to obtain a second hydrolysis product; the amount of protease used is ≥0.2% of the weight of the yeast cell wall, and the amount of polysaccharide enzyme used is ≥0.1% of the weight of the yeast cell wall; Step S3, centrifuging the second hydrolysis product to obtain a supernatant and a centrifuged repetition phase; filtering the supernatant using an ultrafiltration membrane, the retained product is yeast mannan oligosaccharides, the permeate is yeast extract, and the centrifuged repetition phase is yeast glucan.

[0012] Furthermore, the pore size of the ultrafiltration membrane is 10–100 kD.

[0013] Furthermore, in step S2, the amount of protease used is 0.2-2% of the weight of the yeast cell wall, and the amount of polysaccharide enzyme used is 0.1-1% of the weight of the yeast cell wall.

[0014] Furthermore, the temperature for the first enzymatic hydrolysis is 45–60℃, and the temperature for the second enzymatic hydrolysis is 45–60℃.

[0015] Furthermore, the duration of the first enzymatic hydrolysis is 2-8 hours, preferably 3-7 hours, and more preferably 4-6 hours; the duration of the second enzymatic hydrolysis is 1-16 hours, preferably 2-14 hours, and more preferably 4-12 hours.

[0016] Furthermore, the protease is selected from one or more of alkaline proteases, plant peptidases, and complex proteases.

[0017] Furthermore, the polysaccharide enzyme is selected from one or more of cellulase, mannanase, complex polysaccharide enzyme, and cell wall-breaking enzyme.

[0018] Furthermore, the weight concentration of yeast cell walls in the mixture is 8–10%.

[0019] According to another aspect of the present invention, a yeast manno oligosaccharide is also provided, which is prepared by the above-described method for preparing yeast manno oligosaccharides.

[0020] According to another aspect of the present invention, the application of the above-mentioned yeast mannan oligosaccharide as a yeast prebiotic is also provided.

[0021] This invention provides a method for preparing yeast mannan oligosaccharides. The method involves preparing a mixture of yeast cell walls and water, followed by enzymatic hydrolysis using protease and polysaccharide enzymes. The hydrolysate is then centrifuged and filtered through an ultrafiltration membrane to simultaneously obtain yeast mannan oligosaccharides, yeast extract, and yeast glucan. Using this method, the yield of yeast mannan oligosaccharides is above 25%, and the purity can reach above 70%. The yield of yeast glucan is above 30%, and the purity can reach above 70%.

[0022] On the one hand, this invention can simultaneously yield three products: yeast extract, mannan oligosaccharides, and yeast glucan. The entire process is clean, without the use of organic solvents, strong acids, or strong alkalis, and requires no complex production equipment, resulting in low production costs. On the other hand, the yeast mannan oligosaccharides obtained by this invention have high purity and high yield. Most importantly, the mannan oligosaccharides prepared by this invention are relatively clean and can be fermented by beneficial bacteria to produce short-chain fatty acids, which can promote the proliferation of beneficial intestinal bacteria and inhibit the growth of harmful bacteria, making them suitable for use as prebiotics in food. Detailed Implementation

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0024] As described in the background section, existing methods for preparing yeast mannan oligosaccharides suffer from low purity, environmentally unfriendly production processes, and high costs. To address these issues, this invention provides a method for preparing yeast mannan oligosaccharides, comprising the following steps: Step S1, mixing yeast cell walls with water to obtain a mixture; Step S2, adjusting the pH of the mixture to 4-9, then performing a first enzymatic hydrolysis with a protease to form a first hydrolysis product; subsequently, performing a second enzymatic hydrolysis on the first hydrolysis product with a polysaccharide enzyme to obtain a second hydrolysis product; the amount of protease used is ≥0.2% of the yeast cell wall weight, and the amount of polysaccharide enzyme used is ≥0.1% of the yeast cell wall weight; Step S3, centrifuging the second hydrolysis product to obtain a supernatant and a centrifuged repetition phase; filtering the supernatant using an ultrafiltration membrane, the retained product is yeast mannan oligosaccharides, the permeate is yeast extract, and the centrifuged repetition phase is yeast glucan.

[0025] The above preparation method involves preparing a mixture of yeast cell walls and water, followed by enzymatic hydrolysis using proteases and polysaccharides. The hydrolysates are then separated by centrifugation and ultrafiltration, simultaneously yielding yeast mannan oligosaccharides, yeast extract, and yeast glucan. Using this method, the yield of yeast mannan oligosaccharides is above 25%, with a purity exceeding 70%. The yield of yeast glucan is above 30%, with a purity exceeding 70%.

[0026] On the one hand, this invention can simultaneously yield three products: yeast extract, mannan oligosaccharides, and yeast glucan. The entire process is clean, without the use of organic solvents, strong acids, or strong alkalis, and requires no complex production equipment, resulting in low production costs. On the other hand, the yeast mannan oligosaccharides obtained by this invention have high purity and high yield. Most importantly, the mannan oligosaccharides prepared by this invention are relatively clean and can be fermented by beneficial bacteria to produce short-chain fatty acids, including formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid. These are rapidly absorbed by the hindgut, storing energy and lowering osmotic pressure. Furthermore, short-chain fatty acids play an important role in maintaining the normal function of the large intestine and the morphology and function of colonic epithelial cells. Short-chain fatty acids also promote sodium absorption; butyric acid has a stronger effect in this regard than acetic acid and propionic acid, and it can increase the yield of lactobacilli while reducing the number of Escherichia coli. Therefore, the mannan oligosaccharides produced by this invention can promote the proliferation of beneficial intestinal bacteria (such as Bifidobacteria) and inhibit the growth of harmful bacteria (such as pathogens), making them suitable for use as prebiotics in food.

[0027] It should be noted that in the above enzymatic hydrolysis process, the pH of the mixture is adjusted to 4-9, and then a first enzymatic hydrolysis is performed with protease to form the first hydrolysis product. Next, a second enzymatic hydrolysis is performed on the first hydrolysis product with polysaccharide enzyme to obtain the second hydrolysis product. The amount of protease is ≥0.2% of the yeast cell wall weight, and the amount of polysaccharide enzyme is ≥0.1% of the yeast cell wall weight. This allows the yeast cell wall to undergo relatively thorough proteolytic and polysaccharide hydrolysis sequentially, forming more yeast extract, yeast mannan oligosaccharides, and yeast glucans. Furthermore, the pH conditions and enzyme dosage mentioned above also create a molecular weight difference between the yeast extract and yeast mannan oligosaccharides, facilitating filtration separation. Moreover, performing polysaccharide enzyme hydrolysis after proteolytic hydrolysis optimizes the yield and purity of the three products.

[0028] To more fully separate yeast extract and mannan oligosaccharides, in a preferred embodiment, the ultrafiltration membrane has a pore size of 10–100 kDa (kilodaltons).

[0029] To further enzymatically hydrolyze proteins and polysaccharides in the cell wall, while minimizing raw material waste and further improving the prebiotic efficacy of mannan oligosaccharides, in a preferred embodiment, in step S2, the amount of protease used is 0.2–2% of the yeast cell wall weight, and the amount of polysaccharide enzyme used is 0.1–1% of the yeast cell wall weight. More preferably, the temperature for the first enzymatic hydrolysis is 45–60°C, and the temperature for the second enzymatic hydrolysis is 45–60°C. Within these temperature ranges, the protease and polysaccharide enzyme exhibit better activity.

[0030] In a preferred embodiment, the duration of the first enzymatic hydrolysis is 2–8 hours, preferably 3–7 hours, and more preferably 4–6 hours; the duration of the second enzymatic hydrolysis is 1–16 hours, preferably 2–14 hours, and more preferably 4–12 hours. Within these time ranges, the enzymatic hydrolysis is more complete.

[0031] Preferably, the protease includes, but is not limited to, one or more of alkaline protease, plant peptidase, and complex protease. Preferably, the polysaccharide enzyme includes, but is not limited to, one or more of cellulase, mannanase, complex polysaccharide enzyme, and cell wall-breaking enzyme.

[0032] Specifically, proteases and polysaccharides listed in the table below can be used.

[0033]

[0034]

[0035] More preferably, the weight concentration of yeast cell walls in the mixture is 8-10%.

[0036] In practice, after ultrafiltration, the permeate is concentrated and spray-dried to obtain small-molecule yeast extract. The retentate is spray-dried to obtain yeast mannan oligosaccharide product. The centrifuged heavy phase is then spray-dried to obtain yeast glucan product.

[0037] According to another aspect of the present invention, a yeast manno oligosaccharide prepared by the above-described preparation method is provided. As mentioned above, this yeast manno oligosaccharide has high purity, clean composition, and can promote the proliferation of beneficial intestinal bacteria (such as Bifidobacteria) and inhibit the growth of harmful bacteria (such as pathogens), and can be used as a prebiotic in food.

[0038] The present invention further provides the application of the above-mentioned yeast mannan oligosaccharide as a yeast prebiotic.

[0039] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0040] Example 1

[0041] Take 10 kg of yeast cell wall and add water to make up to 100 kg.

[0042] Heat to 50℃, adjust pH to 9.0, add 20g alkaline protease (Alcalase 2.4L, Novozymes) for 8h enzymatic hydrolysis, then add 10g mannanase (25,000 U / g, Nanning Pangbo) for 16h enzymatic hydrolysis.

[0043] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (10KD). The permeate was concentrated and then spray-dried to obtain 3 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.5 kg of mannan oligosaccharide with a purity of 71%. The centrifuged recapital was then spray-dried to obtain 3 kg of yeast glucan with a purity of 70.5%.

[0044] Example 2

[0045] Take 10 kg of yeast cell wall and add water to make up to 100 kg.

[0046] Heat to 50℃, adjust pH to 4.0, add 200g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) for 2 hours of enzymatic hydrolysis, and then add 100g of cellulase (10,000 U / g, Nanning Pangbo) for 1 hour of enzymatic hydrolysis.

[0047] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 2.8 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.6 kg of mannan oligosaccharide with a purity of 72%. The centrifuged recapital phase was spray-dried to obtain 3.4 kg of yeast glucan with a purity of 70.3%.

[0048] Example 3

[0049] Take 10 kg of yeast cell wall and add water to make up to 100 kg.

[0050] Heat to 50℃, adjust pH to 5.0, add 40g of complex protease (100,000 U / g, Angel Yeast Co., Ltd.) for 2 hours of enzymatic hydrolysis, and then add 20g of complex polysaccharide enzyme (Viscozyme, Novozymes) for 14 hours of enzymatic hydrolysis.

[0051] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (10KD). The permeate was concentrated and then spray-dried to obtain 2.6 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.8 kg of mannan oligosaccharide with a purity of 71%. The centrifuged recapital was then spray-dried to obtain 3.4 kg of yeast glucan with a purity of 71%.

[0052] Example 4

[0053] Take 10 kg of yeast cell wall and add water to make up to 100 kg.

[0054] Heat to 50℃, adjust pH to 8.0, add 180g alkaline protease (Alcalase 2.4L, Novozymes) for 7h of enzymatic hydrolysis, then add 90g cell wall disrupting enzyme (100,000 U / g, Nanning Pangbo) for 2h of enzymatic hydrolysis.

[0055] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (50KD). The permeate was concentrated and then spray-dried to obtain 2.9 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.9 kg of mannan oligosaccharide with a purity of 70.3%. The centrifuged recapital phase was spray-dried to obtain 3.0 kg of yeast glucan with a purity of 74%.

[0056] Example 5

[0057] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0058] Heat to 50℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0059] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 3.0 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.6 kg of mannan oligosaccharide with a purity of 72%. The centrifuged recapital phase was spray-dried to obtain 3.1 kg of yeast glucan with a purity of 71.5%.

[0060] Example 6

[0061] Take 10 kg of yeast cell wall and add water to make up to 125 kg.

[0062] Heat to 50℃, adjust pH to 7.0, add 170g of complex protease (100,000 U / g, Angel Yeast Co., Ltd.) for 6 hours of enzymatic hydrolysis, and then add 60g of cellulase (10,000 U / g, Nanning Pangbo) for 2 hours of enzymatic hydrolysis.

[0063] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (50KD). The permeate was concentrated and then spray-dried to obtain 2.9 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.6 kg of mannan oligosaccharide with a purity of 71.5%. The centrifuged recapital phase was spray-dried to obtain 3.2 kg of yeast glucan with a purity of 70.7%.

[0064] Example 7

[0065] 1) Take 10 kg of yeast cell wall and add water to make up to 125 kg.

[0066] 2) Heat to 50℃, adjust pH to 6.0, add 210g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) for 4 hours of enzymatic hydrolysis, and then add 50g of mannanase (25,000 U / g, Nanning Pangbo) for 4 hours of enzymatic hydrolysis.

[0067] 3) After enzymatic hydrolysis, centrifuge the sample. Filter the supernatant using an ultrafiltration membrane (100KD). Concentrate the permeate and spray-dry to obtain 3.0 kg of small molecule yeast extract. Spray-dry the retentate to obtain 2.5 kg of mannan oligosaccharide with a purity of 70.3%. Spray-dry the centrifuged recurrent phase to obtain 3.1 kg of yeast glucan with a purity of 70.5%.

[0068] Example 8

[0069] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0070] Heat to 50℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) for 4 hours of enzymatic hydrolysis, and then add 110g of mannanase (25,000 U / g, Nanning Pangbo) for 4 hours of enzymatic hydrolysis.

[0071] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100 kDa). The permeate was concentrated and then spray-dried to obtain 3.0 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.7 kg of mannan oligosaccharide with a purity of 70.1%. The centrifuged recapital phase was spray-dried to obtain 2.9 kg of yeast glucan with a purity of 70.3%.

[0072] Example 9

[0073] Take 10 kg of yeast cell wall and add water to make up to 100 kg.

[0074] Heat to 45℃, adjust pH to 9.0, add 20g alkaline protease (Alcalase 2.4L, Novozymes) for 8h enzymatic hydrolysis, then add 10g mannanase (25,000 U / g, Nanning Pangbo) for 16h enzymatic hydrolysis.

[0075] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (10KD). The permeate was concentrated and then spray-dried to obtain 2.6 kg of small molecule yeast extract. The retentate was spray-dried to obtain 3.0 kg of mannan oligosaccharide with a purity of 70.5%. The centrifuged recapital phase was spray-dried to obtain 3.1 kg of yeast glucan with a purity of 70.5%.

[0076] Example 10

[0077] Take 10 kg of yeast cell wall and add water to make up to 100 kg.

[0078] Heat to 60℃, adjust pH to 9.0, add 20g alkaline protease (Alcalase 2.4L, Novozymes) for 8h enzymatic hydrolysis, then add 10g mannanase (25,000 U / g, Nanning Pangbo) for 16h enzymatic hydrolysis.

[0079] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (10KD). The permeate was concentrated and then spray-dried to obtain 2.8 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.8 kg of mannan oligosaccharide with a purity of 70.8%. The centrifuged recapital phase was spray-dried to obtain 3.1 kg of yeast glucan with a purity of 70.6%.

[0080] Example 11

[0081] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0082] Heat to 40℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0083] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 2.2 kg of small molecule yeast extract. The retentate was spray-dried to obtain 3.0 kg of mannan oligosaccharide with a purity of 68.1%. The centrifuged recapital phase was spray-dried to obtain 3.1 kg of yeast glucan with a purity of 68.5%.

[0084] Example 12

[0085] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0086] Heat to 65℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0087] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100 kDa). The permeate was concentrated and then spray-dried to obtain 2.3 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.8 kg of mannan oligosaccharide with a purity of 68.4%. The centrifuged recapital phase was spray-dried to obtain 3.2 kg of yeast glucan with a purity of 67.3%.

[0088] Example 13

[0089] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0090] Heat to 50℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0091] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (5KD). The permeate was concentrated and then spray-dried to obtain 2.1 kg of small molecule yeast extract. The retentate was spray-dried to obtain 3.5 kg of mannan oligosaccharide with a purity of 68.5%. The centrifuged recapital phase was spray-dried to obtain 3.1 kg of yeast glucan with a purity of 70.5%.

[0092] Example 14

[0093] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0094] Heat to 50℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0095] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (200 kDa). The permeate was concentrated and then spray-dried to obtain 3.5 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.2 kg of mannan oligosaccharide with a purity of 70%. The centrifuged recapital was spray-dried to obtain 2.9 kg of yeast glucan with a purity of 71.5%.

[0096] Comparative Example 1 (with reference to Example 5)

[0097] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0098] Heat to 50℃, adjust pH to 3.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0099] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 2.1 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.2 kg of mannan oligosaccharide with a purity of 65%. The centrifuged recapital was spray-dried to obtain 3.9 kg of yeast glucan with a purity of 66%.

[0100] Comparative Example 2 (with reference to Example 5)

[0101] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0102] Heat to 50℃, adjust pH to 10.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0103] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 2.5 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.0 kg of mannan oligosaccharide with a purity of 64%. The centrifuged recapital phase was spray-dried to obtain 3.9 kg of yeast glucan with a purity of 62%.

[0104] Comparative Example 3 (with reference to Example 5)

[0105] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0106] Heat to 50℃, adjust pH to 6.0, add 10g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours, then add 50g of mannanase (25,000 U / g, Nanning Pangbo) and hydrolyze for 4 hours.

[0107] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 1.8 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.1 kg of mannan oligosaccharide with a purity of 70%. The centrifuged recapital was spray-dried to obtain 3.8 kg of yeast glucan with a purity of 63%.

[0108] Comparative Example 4 (with reference to Example 5)

[0109] Take 10 kg of yeast cell wall and add water to make up to 110 kg.

[0110] Heat to 50℃, adjust pH to 6.0, add 50g of plant polypeptide enzyme (soybean polypeptide hydrolase, 600,000 U / g, Nanning Pangbo) for 4 hours of enzymatic hydrolysis, and then add 8g of mannanase (25,000 U / g, Nanning Pangbo) for 4 hours of enzymatic hydrolysis.

[0111] After enzymatic hydrolysis, the sample was centrifuged, and the supernatant was filtered through an ultrafiltration membrane (100KD). The permeate was concentrated and then spray-dried to obtain 2.5 kg of small molecule yeast extract. The retentate was spray-dried to obtain 2.1 kg of mannan oligosaccharide with a purity of 67.3%. The centrifuged recapital phase was spray-dried to obtain 3.5 kg of yeast glucan with a purity of 65.8%.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the preparation of yeast manno-oligosaccharides, characterized in that, The method comprises the following steps: S1, mixing the yeast cell wall with water to obtain a mixture, wherein the weight concentration of the yeast cell wall in the mixture is 8-10%; S2, adjusting the pH of the mixture to 4-9, and then performing first enzymolysis on the mixture with a protease to form a first enzymolysis product; secondly, performing second enzymolysis on the first enzymolysis product with a polysaccharase to obtain a second enzymolysis product; S3, centrifuging the second enzymolysis product to obtain a supernatant and a centrifugal heavy phase; filtering the supernatant with an ultrafiltration membrane, and the retentate is the yeast mannose oligosaccharide, the permeate is a yeast extract, and the centrifugal heavy phase is a yeast glucan; The membrane pore size of the ultrafiltration membrane is 10-100 kD; In the step S2, the amount of the protease is 0.2-2% of the weight of the yeast cell wall, and the amount of the polysaccharase is 0.1-1% of the weight of the yeast cell wall; The temperature of the first enzymolysis is 45-60°C, and the time of the first enzymolysis is 2-8 h; the temperature of the second enzymolysis is 45-60°C, and the time of the second enzymolysis is 1-16 h; The protease is selected from one or more of alkaline protease, plant polypeptide enzyme, and complex protease; and the polysaccharase is selected from one or more of cellulase, mannanase, complex polysaccharase, and cell wall breaking enzyme.

2. The method of producing mannosyl-oligosaccharides of yeast according to claim 1, characterized in that, The time of the first enzymolysis is 3-7 h.

3. The method of producing mannosyl-oligosaccharides of yeast according to claim 1, characterized by, The time of the first enzymolysis is 4-6 h.

4. The method of producing mannosyl-oligosaccharides of yeast according to claim 1, characterized by, The time of the second enzymolysis is 2-14 h.

5. The method of producing mannosyl-oligosaccharides of yeast according to claim 1, characterized by, The time of the second enzymolysis is 4-12 h.

6. A yeast manno-oligosaccharide, characterized in that, Prepared by the method of any one of claims 1-5.

7. The yeast mannose oligosaccharide of claim 6 as a yeast prebiotic.

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