Yeast dietary fiber capable of being used as freeze-drying protective agent and use thereof

Abstract

Provided are a yeast dietary fiber capable of being used as a freeze-drying protective agent and use thereof. Based on the dry weight of the yeast dietary fiber, the mass percentage of a crude protein in the yeast dietary fiber is 15%-75%, the mass percentage of glucan having a molecular weight of 300-550000 Da to the total glucan is 20%-65%, and the mass percentage of the total glucan is 15%-70%. Also provided is a freeze-drying protective agent. The freeze-drying protective agent comprises, in parts by weight, 10-90 parts of the yeast dietary fiber, 5-50 parts of sucrose, 5-20 parts of trehalose, 0.2-1 parts of manganese sulfate, and 0.8-3 parts of Vc. The yeast dietary fiber can provide polysaccharides, proteins, amino acids, etc., contained in general protective agents, and is an ideal substitute for skim milk as a freeze-drying protective agent, which is combined with a certain proportion of inorganic salts, sugars, etc., to form a freeze-drying protective agent having good freeze-drying protective efficiency, thereby having wide application prospects.

Description

A yeast dietary fiber that can be used as a freeze-drying protectant and its application

[0001] Related applications

[0002] This application claims priority to the earlier application No. 202411411695.2 filed on October 10, 2024 with the China National Intellectual Property Administration, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of microbial technology, specifically relating to a yeast dietary fiber that can be used as a freeze-drying protectant and its application. Background Technology

[0004] Freeze-drying protectants are essential raw materials in the production of live bacteria preparations such as lactic acid bacteria. They are generally composed of multiple components formulated in a specific way, with mainstream components including skim milk, amino acids, inorganic salts, glucose, trehalose, peptides, dextrin, polysaccharides, and glutathione. Currently, most freeze-drying protectant formulations use a large amount of skim milk, which is derived from animals and poses an allergen risk. Its application in relevant food certifications and special food fields carries certain regulatory and certification risks.

[0005] Yeast dietary fiber, also known as yeast polysaccharide, yeast cell wall hydrolysate, yeast derivative, yeast cell wall polysaccharide, etc., contains polysaccharides, proteins, amino acids, etc. Summary of the Invention

[0006] To address the issue of current freeze-drying preservatives using large amounts of skim milk, which poses an allergen risk, this invention proposes a yeast dietary fiber that can be used as a freeze-drying preservative, along with its application method. Yeast dietary fiber provides polysaccharides, proteins, and amino acids found in conventional preservatives, without the risk of allergens, making it an ideal alternative to skim milk as a freeze-drying preservative.

[0007] Specifically, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides the application of yeast dietary fiber in a freeze-drying protectant.

[0009] Preferably, the application of the yeast dietary fiber in the freeze-drying protectant is characterized in that, based on the dry weight of the yeast dietary fiber, the mass percentage of crude protein in the yeast dietary fiber is 15% to 75%, the mass percentage of glucan with a molecular weight of 300 to 550,000 Da is 20% to 65% of the total glucan, and the mass percentage of the total glucan is 15% to 70%.

[0010] Preferably, the crude protein content in the yeast dietary fiber is 27.8% to 65.3% by mass, the glucan with a molecular weight of 300 to 550,000 Da accounts for 28.2% to 42.5% of the total glucan by mass, and the total glucan by mass is 15.5% to 60.7%.

[0011] In a second aspect, the present invention provides a freeze-drying protectant, characterized in that, by weight, the freeze-drying protectant comprises 10-90 parts yeast dietary fiber, 5-50 parts sucrose, 5-20 parts trehalose, 0.2-1 parts manganese sulfate and 0.8-3 parts vitamin C.

[0012] Preferably, the freeze-drying protectant is characterized in that, by weight, the freeze-drying protectant comprises 30-70 parts yeast dietary fiber, 15-40 parts sucrose, 10-15 parts trehalose, 0.4-0.8 parts manganese sulfate and 1-2 parts vitamin C;

[0013] Preferably, the freeze-drying protectant comprises 30-40 parts yeast dietary fiber, and / or 15-50 parts sucrose, and / or 10-20 parts trehalose, and / or 0.2-0.4 parts manganese sulfate, and / or 0.8-1 parts vitamin C;

[0014] Preferably, based on the dry weight of yeast dietary fiber, the crude protein content in the yeast dietary fiber is 15% to 75%, the glucan with a molecular weight of 300 to 550,000 Da accounts for 20% to 65% of the total glucan content, and the total glucan content is 15% to 70%.

[0015] More preferably, the crude protein content in the yeast dietary fiber is 27.8% to 65.3% by mass, the glucan with a molecular weight of 300 to 550,000 Da accounts for 28.2% to 42.5% of the total glucan by mass, and the total glucan by mass is 15.5% to 60.7%.

[0016] Thirdly, the present invention provides a method for preparing the freeze-drying protectant, characterized in that the method includes mixing yeast dietary fiber, sucrose, trehalose, manganese sulfate and vitamin C.

[0017] Preferably, the method for preparing the freeze-drying protectant is characterized in that the preparation of the yeast dietary fiber includes the following steps:

[0018] (1) Enzymatically hydrolyze yeast cells or yeast cell walls to obtain an enzymatic hydrolysate;

[0019] (2) The enzyme hydrolysate was subjected to solid-liquid separation to remove the precipitate and obtain yeast dietary fiber.

[0020] Preferably, the preparation method is characterized in that, in step (1), before enzymatic hydrolysis, the yeast cells are prepared into yeast milk or the yeast cell walls are prepared into yeast cell wall solution;

[0021] Preferably, the concentration of yeast milk is 100–160 g / L;

[0022] And / or, the concentration of the yeast cell wall solution is 100–160 g / L;

[0023] In a further preferred embodiment, enzyme inactivation is performed after enzymatic hydrolysis at a temperature of 70-80℃.

[0024] Preferably, the preparation method is characterized in that, in step (1), the enzyme required for enzymatic hydrolysis is selected from glucanase, or a combination of glucanase and mannanase, or a combination of glucanase, mannanase and protease;

[0025] Preferably, the enzyme is added to the yeast milk or yeast cell wall solution at a mass percentage of 0.1-0.5% of the dry matter of the yeast milk or yeast cell wall.

[0026] And / or, the enzymatic hydrolysis time is 8-12 hours;

[0027] And / or, the enzymatic hydrolysis temperature is 45-55℃;

[0028] And / or, the enzymatic hydrolysis pH is 5.5-5.8.

[0029] Preferably, the preparation method is characterized in that, in step (2), the solid-liquid separation is performed using a centrifuge;

[0030] Preferably, the centrifuge speed is 4000-5000 rpm;

[0031] And / or, centrifuge for 10-15 minutes.

[0032] Preferably, the preparation method is characterized in that, after step (2), it further includes a concentration and drying step;

[0033] Preferably, the concentration is achieved using vacuum concentration;

[0034] And / or, the vacuum degree of the reduced pressure concentration is 0.05-0.08 MPa;

[0035] And / or, concentrate under reduced pressure to a dry matter content of 10-18 wt%;

[0036] And / or, the temperature for vacuum concentration is 75–85°C;

[0037] More preferably, the drying is carried out by spray drying or microwave drying;

[0038] And / or, the drying temperature is 140℃~150℃.

[0039] Fourthly, the present invention provides the application of the lyophilization protectant or the lyophilization protectant prepared by the method described above in microbial preparations.

[0040] Preferably, the application of the freeze-drying protectant in microbial preparations is characterized in that the microorganisms include Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophilus, and Bifidobacterium animalis.

[0041] Fifthly, the present invention provides a freeze-dried microbial preparation, characterized in that it contains freeze-dried microorganisms and the freeze-drying protectant or the freeze-drying protectant prepared by the method of preparing the freeze-drying protectant.

[0042] Preferably, in the lyophilized microbial preparation, the content of the lyophilization protectant in the lyophilized microbial preparation is 1% or more, preferably 5% or more, preferably 10% or more, preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, and more preferably 90% or more.

[0043] The beneficial effects of this invention are:

[0044] The yeast dietary fiber freeze-drying protectant of this invention provides polysaccharides, proteins, amino acids, etc., found in general freeze-drying protectants, making it an ideal alternative to skim milk as a freeze-drying protectant. When combined with a certain proportion of inorganic salts, sugars, and other substances without biosafety issues or allergen risks, it forms a freeze-drying protectant with excellent freeze-drying efficiency and broad application prospects. Yeast dietary fiber, as a freeze-drying protectant, provides a complex component, rather than a single functional component, making it a novel freeze-drying protectant raw material with more efficient freeze-drying protection. Detailed Implementation

[0045] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and comparative examples, but the present invention is not limited to the following technical solutions.

[0046] The purpose of this invention is to screen yeast dietary fiber as a freeze-drying protectant, provide a method for preparing yeast dietary fiber, and optimize a freeze-drying protectant formulation with yeast derivatives as the main raw material that is free of allergens and animal sources based on the combination of yeast dietary fiber with inorganic salts, sugars, etc.

[0047] Yeast dietary fiber, also known as yeast polysaccharide, yeast derivative, yeast cell wall polysaccharide, etc., is a hydrolysis product of yeast cells. It is mainly produced by hydrolyzing yeast cell wall components using glucanase, mannanase, protease, etc., to form hydrolysates containing nutrients such as polysaccharides, proteins, and amino acids, which are then spray-dried and have good dispersibility.

[0048] In one embodiment of the present invention, a freeze-drying protectant is provided, the freeze-drying protectant comprising 10-90 parts yeast dietary fiber, 5-50 parts sucrose, 5-20 parts trehalose, 0.2-1 parts manganese sulfate and 0.8-3 parts vitamin C;

[0049] Based on the dry weight of yeast dietary fiber, the crude protein content in the yeast dietary fiber is 27%–56%, and the glucan with a molecular weight of 300–550,000 Da accounts for 28%–42% of the total glucan content.

[0050] In another embodiment of the present invention, the weight parts of yeast dietary fiber in the freeze-drying protectant can be any value between 10 and 90, specifically 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80, or the weight parts of yeast dietary fiber within the numerical range formed by any two of the above specific values ​​as endpoints.

[0051] In another embodiment of the present invention, the weight parts of sucrose in the freeze-drying protectant can be any value between 5 and 50, specifically 10, 15, 20, 25, 30, 35, 40 or 45, or the weight parts of sucrose within the numerical range formed by any two of the above specific values ​​as endpoints.

[0052] In another embodiment of the present invention, the weight parts of trehalose in the freeze-drying protectant can be any value between 5 and 20, specifically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, or the weight parts of trehalose within the numerical range formed by any two of the above specific values ​​as endpoints.

[0053] In another embodiment of the present invention, the weight part of manganese sulfate in the freeze-drying protectant can be any value between 0.2 and 1, specifically 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95, or the weight part of manganese sulfate within the numerical range formed by any two of the above specific values ​​as endpoints.

[0054] In another embodiment of the present invention, the weight part of Vc in the freeze-drying protectant can be any value between 0.8 and 3, specifically 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6 or 2.8, or the weight part of Vc within the numerical range formed by any two of the above specific values ​​as endpoints.

[0055] In another embodiment of the present invention, a method for preparing yeast dietary fiber in the freeze-drying protectant is provided, comprising the following steps:

[0056] (1) Enzymatically hydrolyze yeast cells or yeast cell walls to obtain an enzymatic hydrolysate;

[0057] (2) The enzyme hydrolysate was subjected to solid-liquid separation to remove the precipitate and obtain yeast dietary fiber.

[0058] Step (2) is followed by concentration and drying steps. The concentration is carried out by vacuum concentration, and the vacuum degree of vacuum concentration is 0.05-0.08 MPa.

[0059] Vacuum concentration refers to lowering the boiling point of water using vacuum to evaporate and dry it. Vacuum degree refers to the rarefaction of a gas under vacuum conditions. If the pressure inside the measuring equipment is lower than atmospheric pressure, a vacuum gauge is required for pressure measurement. The value read from the vacuum gauge is called the vacuum degree. The vacuum degree value indicates that the actual system pressure is lower than atmospheric pressure, i.e.: Vacuum degree = Atmospheric pressure - Absolute pressure, Absolute pressure = Atmospheric pressure - Vacuum degree.

[0060] In another embodiment of the present invention, in step (1), the enzyme required for enzymatic hydrolysis is selected from dextranase, or a combination of dextranase and mannanase, or a combination of dextranase, mannanase and protease.

[0061] Preferably, the glucanase activity is greater than 1.5-1.8 × 10⁻⁶. 5 IU / g,

[0062] And / or mannanase activity is 1-1.2 × 10⁻⁶ 5 IU / g,

[0063] And / or protease activity greater than 6-6.5 × 10 5 IU / g,

[0064] The protease can be selected from one or more of papain, alkaline protease, neutral protease and acidic protease. In the embodiments of the present invention, protease ZF106 is a combination of alkaline protease, neutral protease and papain, wherein the weight ratio of alkaline protease, neutral protease and papain is 0-7:0-7:3-10, preferably, the weight ratio of alkaline protease, neutral protease and papain is 1-7:1-7:3-10.

[0065] The yeast used in this invention is *Saccharomyces cerevisiae* strain FX-2, which was isolated from fermented dough containing various wild-type microorganisms. Using the fermented dough as a sample, a dough extract was prepared, and a pure strain was isolated via dilution and plating. Identification was performed using 16S rRNA sequencing, and the results showed sequence homology greater than 99%, thus confirming that the isolated strain belongs to *Saccharomyces cerevisiae* and was named *Saccharomyces cerevisiae* strain FX-2. It was deposited at the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with accession number M2016418. The method for isolating and identifying this strain has been disclosed in patent application CN108220175A.

[0066] In some specific embodiments, the raw materials used in preparing the culture medium according to the present invention include peptone and yeast extract. Peptone and yeast extract primarily exist as organic nitrogen sources in the culture medium, providing the necessary nitrogen element for microbial growth during fermentation. Common organic nitrogen sources such as yeast extract and peptone decompose in the seed culture medium, releasing amino acids and small peptides, which then become nitrogen sources required for microbial growth. That is, when using peptone and yeast extract as common organic nitrogen sources to prepare the culture medium, the present invention does not particularly limit their source; they can be commercially available or prepared using conventional methods. Commercially available or conventionally prepared peptone with a total nitrogen content greater than or equal to 12.0 wt% and an amino nitrogen content greater than or equal to 3.0 wt% can be used in the present invention. Similarly, commercially available yeast extract with a total nitrogen content greater than or equal to 10.0 wt% and an amino nitrogen content greater than or equal to 5.0 wt% can be used in the present invention.

[0067] Preferably, in some specific embodiments, the yeast extract, by weight, further comprises: 2-2.5 ppm of vitamin B1, 37-40 ppm of vitamin B2, 113-116 ppm of vitamin B5, 15-20 ppm of vitamin B6, 6-10 ppm of vitamin B7, 25-28 ppm of vitamin B9, 3205-3210 ppm of choline, 1575-1580 ppm of inositol, 325-330 ppm of niacin, and 2-4 μg of vitamin B12 per 100 g of yeast extract.

[0068] And / or, based on the weight of the yeast extract, the potassium is 31910-31912 mg / kg, the sodium is 5735-5740 mg / kg, the calcium is 354-357 mg / kg, the magnesium is 2670-2675 mg / kg, the zinc is 77-83 mg / kg, and the iron is 77-83 mg / kg.

[0069] And / or the yeast extract comprises: free amino acids and hydrolyzed amino acids, wherein the free amino acid content comprises, by weight of the yeast extract: 1-2% free aspartic acid, 2-3% free threonine, 1.5-2% free serine, 6.5-7% free glutamic acid, 1-1.5% free glycine, 4-5% free alanine, 0.1-0.15% free cysteine, 2-3% free valine, 0.5-1% free methionine, 2-2.5% free isoleucine, 3.3-3.7% free leucine, 0.5-1% free tyrosine, 1.5-2% free phenylalanine, 2-2.5% free lysine, 0.1-1% free histidine, 1.5-2.5% free arginine, and 0.5-1% free proline.

[0070] The hydrolyzed amino acid content includes, by weight of the yeast extract: 6-6.5% hydrolyzed aspartic acid, 2-3% hydrolyzed threonine, 2-3% hydrolyzed serine, 10-15% hydrolyzed glutamic acid, 2-3% hydrolyzed glycine, 5-6% hydrolyzed alanine, 0.5-1% hydrolyzed cysteine, 3-4% hydrolyzed valine, 0.5-1% hydrolyzed methionine, 3-4% hydrolyzed isoleucine, 4-5% hydrolyzed leucine, 1-2% hydrolyzed tyrosine, 2-3% hydrolyzed phenylalanine, 4-5% hydrolyzed lysine, 1-2% hydrolyzed histidine, 3-4% hydrolyzed arginine, and 2-3% hydrolyzed proline.

[0071] Preferably, in some specific embodiments, the peptone comprises: free amino acids and hydrolyzed amino acids, wherein the content of free amino acids is as follows: based on the weight of the peptone, free aspartic acid 0.05-0.10%, free threonine 0.50-1.00%, free serine 0.50-1.00%, free glutamic acid 0.50-1.00%, free glycine 0.05-0.10%, free alanine 0.10-0.50%, and free cysteine ​​0. 0.01-0.05%, Free valine 0.50-1.00%, Free methionine 0.50-1.00%, Free isoleucine 0.50-1.00%, Leucine 3.00-4.00%, Free tyrosine 0.50-1.00%, Free phenylalanine 2.00-3.00%, Free lysine 4.00-5.00%, Free histidine 0.10-0.50%, Free arginine 2.00-3.00%, Free proline 0.01-0.05%.

[0072] The hydrolyzed amino acid content is as follows (based on the weight of the yeast extract): hydrolyzed aspartic acid 6.00-6.50%, hydrolyzed threonine 2.00-2.50%, hydrolyzed serine 1.00-1.50%, hydrolyzed glutamic acid 20.00-25.00%, hydrolyzed glycine 1.50-2.00%, hydrolyzed alanine 2.00-4.00%, hydrolyzed cysteine ​​0.10-0.20%, and hydrolyzed valine 6.00%. -7.00%, hydrolyzed methionine 1.00-3.00%, hydrolyzed isoleucine 4.00-5.00%, hydrolyzed leucine 8.00-8.50%, hydrolyzed tyrosine 1.00-1.50%, hydrolyzed phenylalanine 4.50-5.00%, hydrolyzed lysine 7.00-7.50%, hydrolyzed histidine 2.50-3.00%, hydrolyzed arginine 3.00-3.50%, hydrolyzed proline 10.00-10.50%.

[0073] Unless otherwise stated, all reagents and instruments used in the embodiments and comparative examples of this invention are conventional commercially available products. Information on the sources of the instruments and reagents used in the embodiments and comparative examples of this invention is shown in Table 1 below.

[0074] Table 1

[0075] The vitamins contained in the yeast extract (model: FM888) used in the examples are as follows: vitamin B1 2.3 ppm, vitamin B2 38.8 ppm, vitamin B5 115.0 ppm, vitamin B6 18.0 ppm, vitamin B7 7.9 ppm, vitamin B9 26.7 ppm, vitamin B12 2.3 (ug / 100g), choline 3206.0 ppm, inositol 1577.7 ppm, and niacin 328.0 ppm.

[0076] The trace elements contained in the yeast extract (model: FM888) used in the examples are as follows: potassium 31911.66 mg / kg, sodium 5738.91 mg / kg, calcium 355.25 mg / kg, magnesium 2673.59 mg / kg, zinc 80.94 mg / kg and iron 80.21 mg / kg.

[0077] The yeast extract (model: FM888) used in the examples contained 35.1% free amino acids and 61.21% hydrolyzed amino acids.

[0078] The free amino acid content is as follows, based on the weight of the yeast extract: free aspartic acid 1.6%, free threonine 2.1%, free serine 1.7%, free glutamic acid 6.7%, free glycine 1.2%, free alanine 4.2%, free cysteine ​​0.1%, free valine 2.7%, free methionine 0.8%, free isoleucine 2.2%, leucine 3.5%, free tyrosine 0.9%, free phenylalanine 1.8%, free lysine 2.3%, free histidine 0.5%, free arginine 2.0%, and free proline 0.8%.

[0079] The hydrolyzed amino acid content is as follows: based on the weight of the yeast extract, hydrolyzed aspartic acid 6.23%, hydrolyzed threonine 2.71%, hydrolyzed serine 2.73%, hydrolyzed glutamic acid 12.33%, hydrolyzed glycine 2.74%, hydrolyzed alanine 5.17%, hydrolyzed cysteine ​​0.61%, hydrolyzed valine 3.84%, hydrolyzed methionine 0.84%, hydrolyzed isoleucine 3.65%, hydrolyzed leucine 4.72%, hydrolyzed tyrosine 1.65%, hydrolyzed phenylalanine 2.68%, hydrolyzed lysine 4.63%, hydrolyzed histidine 1.19%, hydrolyzed arginine 3.30%, and hydrolyzed proline 2.19%.

[0080] The vitamins contained in the yeast extract (model: FM888) used in the examples are as follows: vitamin B1 2.3 ppm, vitamin B2 38.8 ppm, vitamin B5 115.0 ppm, vitamin B6 18.0 ppm, vitamin B7 7.9 ppm, vitamin B9 26.7 ppm, vitamin B12 2.3 (ug / 100g), choline 3206.0 ppm, inositol 1577.7 ppm, and niacin 328.0 ppm.

[0081] The peptone (model: FP318) used in the examples contained 18.4% free amino acids and 85.7% hydrolyzed amino acids.

[0082] The free amino acid content is as follows, based on the weight of the peptone: free aspartic acid 0.08%, free threonine 0.60%, free serine 0.50%, free glutamic acid 0.55%, free glycine 0.07%, free alanine 0.45%, free cysteine ​​0.05%, free valine 0.95%, free methionine 0.70%, free isoleucine 0.55%, leucine 3.10%, free tyrosine 0.75%, free phenylalanine 2.20%, free lysine 4.70%, free histidine 0.30%, free arginine 2.80%, and free proline 0.05%.

[0083] The hydrolyzed amino acid content is as follows: based on the weight of the peptone, hydrolyzed aspartic acid 6.50%, hydrolyzed threonine 2.20%, hydrolyzed serine 1.30%, hydrolyzed glutamic acid 20.50%, hydrolyzed glycine 1.80%, hydrolyzed alanine 3.00%, hydrolyzed cysteine ​​0.10%, hydrolyzed valine 6.60%, hydrolyzed methionine 2.00%, hydrolyzed isoleucine 4.80%, hydrolyzed leucine 8.20%, hydrolyzed tyrosine 1.10%, hydrolyzed phenylalanine 4.60%, hydrolyzed lysine 7.20%, hydrolyzed histidine 2.60%, hydrolyzed arginine 3.30%, and hydrolyzed proline 10.20%.

[0084] The seed fermentation medium formula in Example 1 is as follows: yeast extract FM888 30g / L, glucose 80g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.05g / L, and zinc sulfate 0.5g / L.

[0085] The commercial fermentation medium formula in Example 1 is as follows: yeast extract FM888 10g / L, ammonium sulfate 20g / L, glucose 80g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.05g / L, and zinc sulfate 0.5g / L.

[0086] The fermentation medium formula in Experiment Example 1 is as follows: glucose 20 g / L, yeast extract 20 g / L, peptone 5 g / L, potassium dihydrogen phosphate 2 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, manganese sulfate 0.25 g / L, magnesium sulfate 0.5 g / L, and Tween-80 3 g / L.

[0087] Example 1: Preparation of yeast dietary fiber

[0088] Example 1-1

[0089] 1. Yeast fermentation and yeast cell collection

[0090] (1) Seed fermentation: The Saccharomyces FX-2 strain preserved in glycerol tubes was activated and inoculated into sterilized seed fermentation medium at a volume percentage of 0.5% to obtain seed fermentation broth.

[0091] (2) Commercial fermentation: The seed fermentation broth was inoculated into a 5L fermenter at a volume percentage of 10%. The culture medium in the fermenter was the commercial fermentation medium. The commercial fermentation temperature was 30℃, the stirring speed was 500rpm, the ventilation rate was controlled at 4L / min, and the fermentation pH was 5.5. The fermentation time was 24h.

[0092] (3) Yeast cell collection: The above fermentation broth was centrifuged at 4000 rpm for 10 minutes, the supernatant was removed, the cells were resuspended in deionized water, and centrifuged again at 4000 rpm for 10 minutes to obtain yeast cells, which were then washed twice with deionized water.

[0093] 2. The collected yeast cells are enzymatically digested.

[0094] The centrifuged yeast cells were mixed with deionized water to prepare yeast milk with a concentration of 130 g / L (i.e., 130 g of dry matter per L of yeast milk). Glucanase was added to the yeast milk at a concentration of 0.2% of the dry matter mass. The pH was controlled at 5.5, and the mixture was incubated at 45°C for 12 hours for enzymatic hydrolysis. The enzyme was then inactivated by heating to 70°C.

[0095] 3. Obtain yeast dietary fiber products

[0096] The above enzymatic hydrolysate was centrifuged at 4000 rpm for 10 minutes, the supernatant was collected, concentrated under reduced pressure to a dry matter content of 10 wt%, a vacuum degree of 0.05 MPa, a concentration temperature of 75℃, and then spray-dried at 140℃ to obtain yeast dietary fiber. The percentages of total glucan content, the percentage of glucan with a molecular weight of 300–550,000 Da, and the percentage of crude protein are shown in Table 2. The detection methods for total glucan content (%), the percentage of glucan with a molecular weight of 300–550,000 Da in total glucan, and crude protein content (%) are as follows: The glucan content (%) is detected using a near-infrared spectroscopy (NIRS). Yeast dietary fiber is stirred evenly and then placed into a sample cup, completely covering the bottom of the cup. The sample height should not be less than half the height of the cup. The sample is then compacted, and the NIRS is used for scanning analysis. The percentage of glucan with a molecular weight of 300–550,000 Da in total glucan (glucan molecular weight distribution detection) is determined using high-performance gel filtration chromatography (HPLC). This method uses porous packing material as the stationary phase, separating the sample components based on differences in relative molecular mass, and then measuring using a differential detector. The crude protein content (%) is determined using the Kjeldahl method.

[0097] Examples 1-2

[0098] 1. Enzymatic hydrolysis of yeast cell walls

[0099] Yeast cell walls (sold by Angel Yeast Co., Ltd.) were dissolved in water until the concentration of the yeast cell wall solution was 160 g / L (i.e., the dry matter content in each L of yeast cell wall solution was 160 g). First, glucanase and mannanase were added to the yeast cell wall solution at 0.15% of the dry matter mass of the yeast cell wall, respectively, and the pH was controlled at 5.8. The solution was incubated at 50℃ for 12 h. Then, the temperature was raised to 60℃, and protease was added to the yeast cell wall solution at 0.1% of the dry matter mass of the yeast cell wall. The pH was controlled at 5.8, and the solution was incubated for 10 h. Finally, the solution was heated to 75℃ to inactivate the enzymes.

[0100] 2. Obtain yeast dietary fiber products

[0101] The above enzymatic hydrolysate was centrifuged at 4000 rpm for 15 minutes, the supernatant was collected, and concentrated under reduced pressure to a dry matter content of 18 wt% at a vacuum degree of 0.08 MPa and a concentration temperature of 85℃. Yeast dietary fiber was then obtained by microwave drying at 150℃. The percentages of total glucan content, the percentage of glucan with a molecular weight of 300–550,000 Da, and the percentage of crude protein are shown in Table 2.

[0102] Examples 1-3

[0103] 1. Yeast fermentation and yeast cell collection

[0104] Same as Example 1-1.

[0105] 2. The collected bacterial cells were subjected to enzymatic hydrolysis.

[0106] The centrifuged yeast cells were mixed with deionized water to prepare yeast milk at a concentration of 100 g / L (i.e., 100 g of dry matter per liter of yeast milk). Glucanase was added to the yeast milk at a concentration of 0.5% of the yeast milk dry matter mass, and the pH was controlled at 5.8. Enzymatic hydrolysis was carried out at 55°C for 8 hours. Then, the temperature was raised to 60°C, and protease was added to the yeast milk at a concentration of 0.1% of the yeast milk dry matter mass, maintaining the pH at 5.8 for 10 hours. Finally, the enzyme was inactivated by heating to 75°C.

[0107] 2. Obtain yeast dietary fiber products

[0108] The above enzymatic hydrolysate was centrifuged at 5000 rpm for 10 minutes, the supernatant was collected, and concentrated under reduced pressure to a dry matter content of 18 wt% at a vacuum degree of 0.05 MPa and a concentration temperature of 75°C. Yeast dietary fiber was then obtained by microwave drying at 140°C. The percentages of total glucan content, the percentage of glucan with a molecular weight of 300–550,000 Da, and the percentage of crude protein are shown in Table 2.

[0109] Table 2

[0110] Experiment Example 1: Verification of the effect of yeast dietary fiber on freeze-dried product survival rate

[0111] 1. Prepare Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophilus, and Bifidobacterium animalis cells by fermentation.

[0112] Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophilus, and Bifidobacterium animalis were activated and then transferred to sterilized fermentation medium at an inoculum rate of 5% (V / V). The culture temperature was 37℃, and the rotation speed was 100 rpm. During the culture process, the pH was controlled by a constant concentration of 30% NaOH. The pH of the culture medium for Lactobacillus plantarum was 6.0, for Lactobacillus acidophilus it was 5.0, for Lactococcus lactis it was 6.2, for Bifidobacterium animalis it was 6.0, and for Streptococcus thermophilus it was 5.8. Samples were taken and diluted every 1 hour during fermentation, and the OD value was measured using a spectrophotometer. Fermentation was terminated when the biomass (OD600 value) no longer increased.

[0113] After fermentation, the fermentation broth was centrifuged at 5000 rpm for 10 minutes, the supernatant was discarded and the precipitate (bacterial cells) was collected. The precipitate was washed twice with an equal volume of physiological saline. After washing and centrifugation, the bacterial cells were collected to obtain Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis and Streptococcus thermophilus, respectively.

[0114] 2. The cells of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactococcus lactis*, *Streptococcus thermophilus*, and *Bifidobacterium animalis* were freeze-dried separately.

[0115] Collected *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactococcus lactis*, *Streptococcus thermophilus*, and *Bifidobacterium animalis* cells were mixed with the test formulation at a 1:1 ratio (cell wet weight: test formulation weight). The test formulation consisted of a freeze-drying protectant made from skim milk, with 2% (T1), 5% (T2), 10% (T3), 20% (T4), 30% (T5), and 50% (T6) of yeast dietary fiber from Example 1-1 used to replace a portion of the skim milk. The mixture was then combined with the cells. CK represented a mixture entirely of skim milk and yeast dietary fiber, while CT represented a mixture entirely of yeast dietary fiber from Example 1-1. This experiment was designed to verify whether yeast dietary fiber as a freeze-drying protectant could achieve more than 80% of the effect of using only skim milk as a freeze-drying protectant.

[0116] After diluting the mixed sample, perform plate counting to calculate the total viable count (V) before lyophilization. 冻干前 The mixed emulsified samples were placed in freeze-drying bottles, pre-frozen at -70℃ for 12 hours, and then freeze-dried in a vacuum freeze dryer.

[0117] 3. Determination of freeze-dried product survival rate

[0118] After freeze-drying, collect the freeze-dried samples and record their mass. Then perform plate counting to calculate the total viable count (V) of the freeze-dried samples. 冻干后 ), calculate the freeze-dried survival rate of the sample (i.e., V). 冻干后 / V 冻干前 (×100%), the results are shown in Table 3.

[0119] Table 3

[0120] The results of this experiment show that the freeze-drying protectant made by using yeast dietary fiber as the freeze-drying protectant or by using yeast dietary fiber to replace 2-50% of the skim milk in the control group CK freeze-drying protectant, is basically the same as the freeze-drying protectant efficiency (i.e. freeze-drying survival rate) when using 100% skim milk as the freeze-drying protectant. It can reach more than 80% of the effect of using skim milk as the freeze-drying protectant. This indicates that yeast dietary fiber has the function of being a freeze-drying protectant and can replace skim milk as a freeze-drying protectant.

[0121] Example 2: A freeze-drying protectant based on yeast dietary fiber

[0122] Example 2-1

[0123] According to the weight parts, 10 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 50 parts of sucrose, 20 parts of trehalose, 0.25 parts of manganese sulfate and 1 part of vitamin C to obtain 1000g of freeze-drying protectant.

[0124] Example 2-2

[0125] According to the weight parts, 30 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 30 parts of sucrose, 12 parts of trehalose, 0.25 parts of manganese sulfate and 1 part of vitamin C to obtain 1000g of freeze-drying protectant.

[0126] Example 2-3

[0127] According to the weight parts, 50 parts of yeast dietary fiber from Example 1-1 were mixed with 10 parts of sucrose, 6 parts of trehalose, 0.25 parts of manganese sulfate and 1 part of vitamin C to obtain 1000g of freeze-drying protectant.

[0128] Examples 2-4

[0129] According to the weight parts, 90 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 5 parts of sucrose, 5 parts of trehalose, 1 part of manganese sulfate and 3 parts of vitamin C to obtain 1000g of freeze-drying protectant.

[0130] Examples 2-5

[0131] According to the weight parts, 10 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 5 parts of sucrose, 5 parts of trehalose, 0.8 parts of manganese sulfate and 2 parts of vitamin C to obtain 1000g of freeze-drying protectant.

[0132] Examples 2-6

[0133] Based on the weight, 90 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 50 parts of sucrose, 20 parts of trehalose, 0.4 parts of manganese sulfate and 0.8 parts of vitamin C to obtain 1000g of freeze-drying protectant.

[0134] Examples 2-7

[0135] Based on the weight, 30 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 15 parts of sucrose, 10 parts of trehalose, 0.2 parts of manganese sulfate and 0.9 parts of vitamin C to obtain 1000g of freeze-drying protectant.

[0136] Examples 2-8

[0137] Based on the weight, 70 parts of yeast dietary fiber obtained in Example 1-1 were mixed with 40 parts of sucrose, 15 parts of trehalose, 0.24 parts of manganese sulfate and 1.1 parts of vitamin C to obtain 1000g of freeze-drying protectant.

[0138] Comparative Example 1

[0139] By weight, compared with Example 2-1, no yeast dietary fiber was added, and the remaining components and

[0140] Same as Example 2-1, mixed to obtain 1000g of freeze-drying protectant.

[0141] Comparative Example 2

[0142] Based on the weight parts, compared with Example 2-1, the yeast dietary fiber was 1 part, the vitamin C was 0.5 parts, and the remaining components were the same as in Example 2-1. The mixture yielded 1000g of freeze-drying protectant.

[0143] Experiment Example 2: Verification of the effect of yeast dietary fiber-based freeze-drying protectant on freeze-dried product survival rate

[0144] 1. Prepare Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophilus, and Bifidobacterium animalis cells by fermentation.

[0145] The preparation method is the same as in Experiment 1.

[0146] 2. The cells of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactococcus lactis*, *Streptococcus thermophilus*, and *Bifidobacterium animalis* were freeze-dried separately.

[0147] The collected *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactococcus lactis*, *Streptococcus thermophilus*, and *Bifidobacterium animalis* cells were respectively mixed with the freeze-drying protectants in Examples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, and 2-8 at a mixing ratio of 1:1 (cell wet weight: freeze-drying protectant). CK represents the mixture of skim milk as the freeze-drying protectant and the cells.

[0148] After diluting the mixed sample, perform plate counting to calculate the total viable count (V) before lyophilization. 冻干前 The mixed emulsified samples were placed in freeze-drying bottles, pre-frozen at -70℃ for 12 hours, and then freeze-dried in a vacuum freeze dryer.

[0149] 3. Determination of freeze-dried product survival rate

[0150] After freeze-drying, collect the freeze-dried samples and record their mass. Then perform plate counting to calculate the total viable count (V) of the freeze-dried samples. 冻干后 ), calculate the freeze-dried survival rate of the sample (i.e., V). 冻干后 / V 冻干前 (×100%), the results are shown in Table 4.

[0151] Table 4

[0152] The experimental results show that the freeze-drying protectant formed by combining yeast dietary fiber with sucrose, trehalose, manganese sulfate, vitamin C, etc. in a certain proportion can achieve the same freeze-drying survival rate as skim milk on the relevant strains listed in this experiment, without using animal-derived ingredients such as skim milk. The results indicate that the freeze-drying protectant formulation has a good freeze-drying protection effect, and also show that the freeze-drying protectant can replace skim milk as a freeze-drying protectant.

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The application of yeast dietary fiber in freeze-drying protectants.

2. The application of yeast dietary fiber in freeze-drying protectants according to claim 1, characterized in that, Based on the dry weight of yeast dietary fiber, the crude protein content of the yeast dietary fiber is 15%–75%, the glucan with a molecular weight of 300–550,000 Da accounts for 20%–65% of the total glucan content, and the total glucan content is 15%–70%. Preferably, the crude protein content in the yeast dietary fiber is 27.8% to 65.3% by mass, the glucan with a molecular weight of 300 to 550,000 Da accounts for 28.2% to 42.5% of the total glucan by mass, and the total glucan by mass is 15.5% to 60.7%.

3. A freeze-drying protectant, characterized in that, By weight, the freeze-drying protectant comprises 10-90 parts yeast dietary fiber, 5-50 parts sucrose, 5-20 parts trehalose, 0.2-1 parts manganese sulfate and 0.8-3 parts vitamin C.

4. The freeze-drying protectant according to claim 3, characterized in that, By weight, the freeze-drying protectant comprises 30-70 parts yeast dietary fiber, and / or 15-40 parts sucrose, and / or 10-15 parts trehalose, and / or 0.4-0.8 parts manganese sulfate, and / or 1-2 parts vitamin C; Preferably, the freeze-drying protectant comprises 30-40 parts yeast dietary fiber, and / or 15-50 parts sucrose, and / or 10-20 parts trehalose, and / or 0.2-0.4 parts manganese sulfate, and / or 0.8-1 parts vitamin C. Preferably, based on the dry weight of yeast dietary fiber, the crude protein content in the yeast dietary fiber is 15% to 75%, the glucan with a molecular weight of 300 to 550,000 Da accounts for 20% to 65% of the total glucan content, and the total glucan content is 15% to 70%. More preferably, the crude protein content in the yeast dietary fiber is 27.8% to 65.3% by mass, the glucan with a molecular weight of 300 to 550,000 Da accounts for 28.2% to 42.5% of the total glucan by mass, and the total glucan by mass is 15.5% to 60.7%.

5. A method for preparing the freeze-drying protectant according to claim 3 or 4, characterized in that, The method includes mixing yeast dietary fiber, sucrose, trehalose, manganese sulfate, and vitamin C.

6. The method for preparing the freeze-drying protectant according to claim 5, characterized in that, The preparation of the yeast dietary fiber includes the following steps: (1) Enzymatically hydrolyze yeast cells or yeast cell walls to obtain an enzymatic hydrolysate; (2) The enzyme hydrolysate was subjected to solid-liquid separation to remove the precipitate and obtain yeast dietary fiber.

7. The preparation method according to claim 6, characterized in that, In step (1), before enzymatic hydrolysis, the yeast cells are prepared into yeast milk or the yeast cell walls are prepared into yeast cell wall solution; Preferably, the concentration of yeast milk is 100–160 g / L; And / or, the concentration of the yeast cell wall solution is 100–160 g / L; In a further preferred embodiment, the enzyme is inactivated after enzymatic hydrolysis at a temperature of 70-80℃.

8. The preparation method according to claim 6 or 7, characterized in that, In step (1), the enzyme required for enzymatic hydrolysis is selected from glucanase, or a combination of glucanase and mannanase, or a combination of glucanase, mannanase and protease; Preferably, the enzyme is added to the yeast milk or yeast cell wall solution at a mass percentage of 0.1-0.5% of the dry matter of the yeast milk or yeast cell wall. And / or, the enzymatic hydrolysis time is 8-12 hours; And / or, the enzymatic hydrolysis temperature is 45-55℃; And / or, the enzymatic hydrolysis pH is 5.5-5.

8.

9. The preparation method according to any one of claims 6-8, characterized in that, In step (2), solid-liquid separation is performed using a centrifuge; Preferably, the centrifuge speed is 4000-5000 rpm; And / or, centrifuge for 10-15 minutes.

10. The preparation method according to any one of claims 6-9, characterized in that, Step (2) is followed by concentration and drying steps; Preferably, the concentration is achieved using vacuum concentration; And / or, the vacuum degree of the reduced pressure concentration is 0.05-0.08 MPa; And / or, concentrate under reduced pressure to a dry matter content of 10-18 wt%; And / or, the temperature for vacuum concentration is 75–85°C; More preferably, the drying is carried out by spray drying or microwave drying; And / or, the drying temperature is 140℃~150℃.

11. The application of the lyophilization protectant according to claim 3 or 4, or the lyophilization protectant prepared by the method according to any one of claims 5-10, in microbial preparations.

12. The application of the lyophilization protectant according to claim 11 in microbial preparations, characterized in that, The microorganisms include Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophilus, and Bifidobacterium animalis.

13. A freeze-dried microbial preparation, characterized in that, The freeze-dried microorganisms and the freeze-drying protectant as described in claim 3 or 4, or the freeze-drying protectant prepared by the method described in any one of claims 5-10.

14. The freeze-dried microbial preparation according to claim 13, wherein, The content of the freeze-drying protectant in the freeze-dried microbial preparation is 1% or more, preferably 5% or more, preferably 10% or more, preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, and more preferably 90% or more.

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