A probiotic fermented immunomodulatory nutritional supplement and a method for its preparation

Abstract

The present application relates to the technical field of nutritional supplement, in particular to a kind of probiotic fermentation type immunomodulatory nutritional supplement and preparation method thereof.The method uses oat beta-glucan and yeast beta-glucan as substrate, after buffer aqueous phase hydration, limited enzyme cutting and enzyme deactivation, access plant lactobacillus, rhamnose lactobacillus and animal bifidobacterium lactis subspecies, fermentation under low-oxygen closed condition, and after heat inactivation, concentration, add resistant dextrin and inulin, spray drying to obtain powder product.The method can make beta-glucan form partially exposed and structure-retained fermentation substrate state, give consideration to beta-glucan retention, oligosaccharide generation, powder dispersibility and storage stability, and the obtained supplement has good in vitro immunomodulatory potential.

Description

Technical Field

[0001] This invention relates to the field of nutritional supplement technology, and in particular to a probiotic fermented immunomodulatory nutritional supplement and its preparation method. Background Technology

[0002] β-glucan, derived from grains, yeast, and other raw materials, is frequently used in nutritional supplements, health foods, and fermented food formulations due to its dietary fiber properties and immune-regulating activities. In existing technologies, one approach focuses on extracting or enzymatically hydrolyzing β-glucan from grains such as oats, barley, and wheat to obtain soluble dietary fiber or prebiotic raw materials; another approach focuses on utilizing the particulate structure and immune-related activities of yeast β-glucan, using it as a functional component in combination with proteins, probiotics, or plant extracts.

[0003] Patent CN1966531A discloses the preparation of oat β-glucan products from oat bran through enzyme treatment and spray drying; patent CN114376231A discloses the application of yeast β-glucan in the preparation of immune-enhancing drugs or health foods; patent CN115518121A discloses a composition containing probiotics, yeast β-glucan, and plant extracts; and patent CN116004742A discloses a method for increasing the content of β-glucan and other components in barley bran through solid-state fermentation with lactic acid bacteria. These technologies demonstrate that β-glucan, along with probiotics and fermentation processes, already has a basis for application in nutritional foods.

[0004] However, in the preparation of powder-based immunomodulatory nutritional supplements, simple compounding is insufficient to achieve the fermented β-glucan composition; direct fermentation of high molecular weight or granular β-glucan results in insufficient substrate exposure, limiting cell utilization and oligosaccharide formation; and excessive enzymatic hydrolysis or fermentation can easily lead to decreased β-glucan structure retention, higher acidity, and poorer storage stability. Therefore, a nutritional supplement and its preparation method that can achieve a balance between fermentation transformation, structure retention, and powder dispersion stability are still needed. Summary of the Invention

[0005] In view of this, the purpose of this invention is to propose a probiotic fermented immunomodulatory nutritional supplement and its preparation method, so as to solve the problems that existing β-glucan nutritional supplements are prone to insufficient substrate utilization or excessive structural degradation during probiotic fermentation and transformation, and it is difficult to balance the degree of fermentation and transformation, β-glucan structure retention and powder dispersion stability.

[0006] To achieve the above objectives, the present invention provides a method for preparing a probiotic fermented immune-modulating nutritional supplement, comprising the following steps:

[0007] (1) Based on a total of 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, 72-83 parts by weight of oat β-glucan powder and 17-28 parts by weight of yeast β-glucan powder are added to food production water containing buffer salts and nutrients, and the sum of the amounts of oat β-glucan powder and yeast β-glucan powder is 100 parts by weight. After shear dispersion and hydration, a biphasic β-glucan hydration substrate is obtained.

[0008] (2) Add food-grade endo-β-glucanase to the biphasic β-glucan hydrate substrate and enzymatically digest it at pH 5.35-5.55 and 49-51℃ for 20-28 min. After the enzymatic digestion is completed, immediately raise the temperature to 91-93℃ and keep it at that temperature for 6-7 min to inactivate the β-glucanase.

[0009] (3) Cool the enzyme-inactivated material to 37°C, adjust the pH to 5.78-5.82 before inoculation, rehydrate the compound bacterial powder composed of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis, and inoculate it, and do not add glucose before inoculation.

[0010] (4) Place the inoculated material under low oxygen closed conditions with dissolved oxygen of 0.7-0.9 mg / L and ferment at 37°C for 15-17 h. Then raise the temperature to 80°C and keep it at 12 min to inactivate the fermentation cells.

[0011] (5) The inactivated fermentation broth was vacuum concentrated at 50°C and -0.085 MPa to a solid content of 27.2-27.8 wt%. Resistant dextrin and inulin were added, mixed, and spray-dried to obtain the probiotic fermented immunomodulatory nutritional supplement.

[0012] Preferably, the oat β-glucan powder has a β-glucan content of 70.5-73.0 wt%, a weight-average molecular weight of 360-450 kDa, and a D90 particle size of 126-145 μm; the yeast β-glucan powder has a β-glucan content of 84.0-85.0 wt%, an insoluble component mass fraction of 77.0-79.0 wt%, a D50 particle size of 17-20 μm, and a D90 particle size of 54-61 μm.

[0013] Preferably, based on 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, the water used in food production is 1440-1500 parts by weight, and the buffer salts and nutrients include 1.9-2.2 parts by weight of sodium citrate dihydrate, 0.49-0.58 parts by weight of citrate monohydrate, 0.57-0.60 parts by weight of dipotassium hydrogen phosphate, 0.28-0.30 parts by weight of magnesium sulfate heptahydrate, 11.5-12.0 parts by weight of food-grade pea peptides, and 4.8-5.0 parts by weight of food-grade yeast extract.

[0014] Preferably, in step (1), oat β-glucan powder and yeast β-glucan powder are added at 57-59℃, sheared and dispersed at 1200rpm for 10min, and then stirred at 260rpm and heated to 61-63℃ for hydration for 25min.

[0015] Preferably, the food-grade endo-β-glucanase has an enzyme activity of 10,000 U / g and is added in an amount of 0.36-0.55 parts by weight; the reducing sugar release rate of the material obtained in step (2) before inoculation is 8.7-12.7 wt%, the β-glucan retention rate with a molecular weight of not less than 100 kDa is 52.3-65.1 wt%, and the viscosity retention rate is 43.6-57.4 wt%.

[0016] Preferably, based on 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, 0.64-0.73 parts by weight of sodium bicarbonate are added in step (3); the compound bacterial powder contains 0.18-0.20 parts by weight of freeze-dried Lactobacillus plantarum, 0.096-0.12 parts by weight of freeze-dried Lactobacillus rhamnosus, and 0.096-0.10 parts by weight of freeze-dried Bifidobacterium animalis subsp. lactis.

[0017] Preferably, in step (4), the low-oxygen sealed conditions are formed by filling the headspace of the fermenter with nitrogen, the stirring speed is 120 rpm during fermentation, the pH at the end of fermentation is 4.31-4.53, the residual β-glucan retention rate is 48.6-55.4 wt%, and the DP2 to 8 oligosaccharide ratio is 12.2-16.8 wt%.

[0018] Preferably, in step (5), 39.5-42.5 parts by weight of resistant dextrin and 15-20 parts by weight of inulin are added, based on a total of 100 parts by weight of oat β-glucan powder and yeast β-glucan powder; the dietary fiber content of the resistant dextrin is 88.0 wt%, the DE value is 8, and the average degree of polymerization of the inulin is 10.

[0019] Preferably, the inlet air temperature of the spray drying in step (5) is 152-158℃, the outlet air temperature is 77-80℃, the feed flow rate is 28-31mL / min, and the dried powder obtained by spray drying passes through a 250μm sieve.

[0020] Furthermore, the present invention also provides a probiotic fermented immune-modulating nutritional supplement, which is prepared by the aforementioned preparation method.

[0021] Preferably, the nutritional supplement is a powder with a β-glucan content of 20.1-22.4 wt%, a total dietary fiber content of 56.4-58.1 wt%, a 30-second dispersion residue rate of 5.9-9.2 wt%, a β-glucan retention rate of 92.6-94.8 wt% after being placed at 40℃ and 75%RH for 30 days, and a viable bacterial count of less than 10 CFU / g after inactivation.

[0022] The beneficial effects of this invention are:

[0023] This invention utilizes oat β-glucan and yeast β-glucan together as a fermentation substrate, distributing hydrated segments and particulate β-glucan components within the same system. This substrate provides segments that can be enzymatically cleaved and utilized by the cells, while preserving the particulate structure of yeast β-glucan. This helps avoid the problems of excessive degradation of single-grain β-glucan or insufficient utilization during fermentation of single-yeast β-glucan, thereby improving the integrity of the β-glucan composition in nutritional supplements.

[0024] By performing limited enzymatic digestion and timely inactivation before inoculation, the fermentation substrate is formed in a partially exposed but not fully destroyed state. This treatment helps release appropriate amounts of reducing sugars and oligosaccharide precursors derived from β-glucan, reducing the limitations imposed by the high-viscosity system on cell fermentation initiation, while inhibiting the loss of high-molecular-weight β-glucan due to continued enzymatic hydrolysis during fermentation. Therefore, the resulting fermentation product maintains a good balance between acid production, oligosaccharide formation, and β-glucan structure retention.

[0025] A closed, low-oxygen fermentation process using *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Bifidobacterium animalis* subsp. *lactobacter*, followed by heat inactivation at the fermentation endpoint, yields a complex composition containing inactivated bacterial cells, fermentation metabolites, β-glucan-derived oligosaccharides, and residual β-glucan. This composition exhibits a high relative value of macrophage phagocytic activity within the tested range, while avoiding storage and application fluctuations caused by retaining live bacteria in the finished product. Vacuum concentration followed by the addition of resistant dextrin and inulin, and then spray drying, further increases the total dietary fiber content of the powder, improves water dispersibility and storage stability of the dried powder, facilitating packaging, transportation, and reconstitution as a powder-based immunomodulatory nutritional supplement. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0027] The food production water, sodium citrate dihydrate, citrate monohydrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, food-grade pea peptides, food-grade yeast extract, food-grade endo-β-glucanase, freeze-dried Lactobacillus plantarum powder, freeze-dried Lactobacillus rhamnosus powder, freeze-dried Bifidobacterium animalis subsp. lactis powder, food-grade resistant dextrin, and inulin used in the following examples and comparative examples are all food-grade raw materials.

[0028] The food-grade endo-β-glucanase has an enzyme activity of 10,000 U / g; the labeled viable counts of the *Lactobacillus plantarum* freeze-dried bacterial powder, *Lactobacillus rhamnosus* freeze-dried bacterial powder, and *Bifidobacterium animalis* subsp. *lactobacter* freeze-dried bacterial powder are all 1.0 × 10⁻⁶. 11 CFU / g.

[0029] The resistant dextrin has a dietary fiber content of 88.0 wt% and a DE value of 8; the inulin has an average degree of polymerization of 10.

[0030] Example 1:

[0031] (1) Take 80.0g of food-grade oat β-glucan powder, wherein the oat β-glucan powder has a β-glucan content of 72.0wt%, a weight-average molecular weight of 420kDa, a moisture content of 6.2wt%, and a D90 particle size of 132μm; take 22.0g of food-grade yeast β-glucan powder, wherein the yeast β-glucan powder has a β-glucan content of 85.0wt%, a mass fraction of insoluble components of 78.0wt%, a D50 particle size of 19μm, a D90 particle size of 58μm, and a moisture content of 5.8wt%; the total amount of β-glucan in the above oat β-glucan powder and yeast β-glucan powder is 76.3g;

[0032] (2) Add 1500.0g of food production water to the jacketed mixing tank, heat to 58℃, add 2.10g of sodium citrate dihydrate, 0.55g of citrate monohydrate, 0.60g of dipotassium hydrogen phosphate, 0.30g of magnesium sulfate heptahydrate, 12.0g of food-grade pea peptides and 5.0g of food-grade yeast extract, stir until dissolved, and then adjust the pH of the system to 5.45; the pea peptides have a protein mass fraction of 82.0wt%, and the proportion of peptides with a molecular weight less than 1000Da is 75.0wt%; the yeast extract has a total nitrogen mass fraction of 8.2wt%.

[0033] (3) Oat β-glucan powder and yeast β-glucan powder were added to the above aqueous phase at 58℃, and dispersed by shearing at 1200 rpm for 10 min. Then the mixture was stirred at 260 rpm and heated to 62℃ for hydration for 25 min to obtain biphasic β-glucan hydration substrate.

[0034] (4) Cool the biphasic β-glucan hydrate substrate to 50°C, add 0.46g of food-grade endo-β-glucanase with an enzyme activity of 10000U / g, maintain pH 5.45, 50°C and 180rpm stirring, and perform enzymatic digestion for 24min. After the enzymatic digestion is completed, immediately raise the temperature to 92°C and keep it at that temperature for 6min to inactivate the β-glucanase.

[0035] (5) Cool the material after enzyme inactivation to 37°C, add 0.70g of sodium bicarbonate, adjust the pH to 5.80 before inoculation, take 0.20g of freeze-dried Lactobacillus plantarum powder, 0.10g of freeze-dried Lactobacillus rhamnosus powder and 0.10g of freeze-dried Bifidobacterium animalis subsp. lactis powder, rehydrate the above powders with 30.0g of sterile water for 20min and then inoculate them into the material;

[0036] (6) Introduce nitrogen into the headspace of the fermenter for 3 minutes to make the dissolved oxygen in the fermentation system 0.8 mg / L. Then ferment for 16 hours at 37°C, 120 rpm and in a closed environment. Raise the temperature of the fermentation liquid to 80°C and keep it at that temperature for 12 minutes to inactivate the fermentation cells and stop the fermentation.

[0037] (7) The deactivated fermentation broth was vacuum concentrated at 50℃ and -0.085MPa to a solid content of 27.5wt%. 42.0g of food-grade resistant dextrin and 18.0g of inulin were added and stirred at 180rpm for 20min at 40℃.

[0038] (8) The concentrated fermented product with added carrier was spray-dried at an inlet air temperature of 155°C, an outlet air temperature of 78°C, and a feed flow rate of 30 mL / min. The dried powder was passed through a 250 μm sieve to obtain a probiotic fermented immune-regulating nutritional supplement.

[0039] Example 2:

[0040] (1) Take 86.0g of food-grade oat β-glucan powder, wherein the oat β-glucan powder has a β-glucan content of 70.5wt%, a weight-average molecular weight of 360kDa, a moisture content of 6.5wt%, a D90 particle size of 145μm, and take 18.0g of food-grade yeast β-glucan powder, wherein the yeast β-glucan powder has a β-glucan content of 84.0wt%, a mass fraction of insoluble components of 77.0wt%, a D50 particle size of 17μm, a D90 particle size of 54μm, and a moisture content of 5.6wt%. The total amount of β-glucan in the above oat β-glucan powder and yeast β-glucan powder is 75.75g.

[0041] (2) Add 1500.0g of food production water to the jacketed mixing tank, heat to 57℃, add 2.00g of sodium citrate dihydrate, 0.60g of citrate monohydrate, 0.60g of dipotassium hydrogen phosphate, 0.30g of magnesium sulfate heptahydrate, 12.0g of food-grade pea peptide and 5.0g of food-grade yeast extract, stir until dissolved, and then adjust the pH of the system to 5.35;

[0042] (3) Oat β-glucan powder and yeast β-glucan powder were added to the above aqueous phase at 57℃, and dispersed by shearing at 1200 rpm for 10 min. Then, the mixture was stirred at 260 rpm and heated to 61℃ for hydration for 25 min to obtain biphasic β-glucan hydration substrate.

[0043] (4) Cool the biphasic β-glucan hydrate substrate to 49°C, add 0.38g of food-grade endo-β-glucanase, maintain pH 5.35, 49°C and 180rpm stirring, digest for 20min, and immediately raise the temperature to 91°C and keep warm for 6min after digestion to inactivate β-glucanase.

[0044] (5) Cool the material after enzyme inactivation to 37°C, add 0.75g of sodium bicarbonate, adjust the pH to 5.82 before inoculation, take 0.20g of freeze-dried Lactobacillus plantarum powder, 0.10g of freeze-dried Lactobacillus rhamnosus powder and 0.10g of freeze-dried Bifidobacterium animalis subsp. lactis powder, rehydrate the above powders with 30.0g of sterile water for 20min and then inoculate them into the material;

[0045] (6) Introduce nitrogen into the headspace of the fermenter for 3 minutes to make the dissolved oxygen in the fermentation system 0.9 mg / L. Then ferment for 15 hours at 37°C, 120 rpm and in a closed environment. Raise the temperature of the fermentation liquid to 80°C and keep it at that temperature for 12 minutes to inactivate the fermentation cells and stop the fermentation.

[0046] (7) The deactivated fermentation broth was vacuum concentrated at 50℃ and -0.085MPa to a solid content of 27.2wt%. 44.0g of food-grade resistant dextrin and 16.0g of inulin were added and stirred at 180rpm for 20min at 40℃.

[0047] (8) The concentrated fermented product with added carrier was spray-dried at an inlet air temperature of 152°C, an outlet air temperature of 77°C, and a feed flow rate of 28 mL / min. The dried powder was passed through a 250 μm sieve to obtain a probiotic fermented immune-regulating nutritional supplement.

[0048] Example 3:

[0049] (1) Take 75.0g of food-grade oat β-glucan powder, wherein the oat β-glucan powder has a β-glucan content of 72.5wt%, a weight-average molecular weight of 450kDa, a moisture content of 6.0wt%, a D90 particle size of 126μm, and take 26.0g of food-grade yeast β-glucan powder, wherein the yeast β-glucan powder has a β-glucan content of 84.5wt%, a mass fraction of insoluble components of 79.0wt%, a D50 particle size of 20μm, a D90 particle size of 61μm, and a moisture content of 5.7wt%. The total amount of β-glucan in the above oat β-glucan powder and yeast β-glucan powder is 76.35g.

[0050] (2) Add 1500.0g of food production water to the jacketed mixing tank, heat to 59℃, add 2.15g of sodium citrate dihydrate, 0.50g of citrate monohydrate, 0.60g of dipotassium hydrogen phosphate, 0.30g of magnesium sulfate heptahydrate, 12.0g of food-grade pea peptide and 5.0g of food-grade yeast extract, stir until dissolved, and then adjust the pH of the system to 5.55;

[0051] (3) Oat β-glucan powder and yeast β-glucan powder were added to the above aqueous phase at 59℃, and dispersed by shearing at 1200 rpm for 10 min. Then the mixture was stirred at 260 rpm and heated to 63℃ for hydration for 25 min to obtain biphasic β-glucan hydration substrate.

[0052] (4) Cool the biphasic β-glucan hydrate substrate to 51°C, add 0.55g of food-grade endo-β-glucanase, maintain pH 5.55, 51°C and 180rpm stirring, digest for 28min, and immediately raise the temperature to 93°C and keep it warm for 7min after digestion to inactivate β-glucanase.

[0053] (5) Cool the material after enzyme inactivation to 37°C, add 0.65g of sodium bicarbonate, adjust the pH to 5.78 before inoculation, take 0.20g of freeze-dried Lactobacillus plantarum powder, 0.10g of freeze-dried Lactobacillus rhamnosus powder and 0.10g of freeze-dried Bifidobacterium animalis subsp. lactis powder, rehydrate the above powders with 30.0g of sterile water for 20min and then inoculate them into the material;

[0054] (6) Introduce nitrogen into the headspace of the fermenter for 3 minutes to make the dissolved oxygen in the fermentation system 0.7 mg / L. Then ferment for 17 hours at 37°C, 120 rpm and in a closed environment. Raise the temperature of the fermentation liquid to 80°C and keep it warm for 12 minutes to inactivate the fermentation cells and stop the fermentation.

[0055] (7) The deactivated fermentation broth was vacuum concentrated at 50℃ and -0.085MPa to a solid content of 27.8wt%. 40.0g of food-grade resistant dextrin and 20.0g of inulin were added and stirred at 180rpm for 20min at 40℃.

[0056] (8) The concentrated fermented product with added carrier was spray-dried at an inlet air temperature of 158°C, an outlet air temperature of 80°C, and a feed flow rate of 31 mL / min. The dried powder was passed through a 250 μm sieve to obtain a probiotic fermented immune-regulating nutritional supplement.

[0057] Example 4:

[0058] (1) Take 72.0g of food-grade oat β-glucan powder, wherein the oat β-glucan powder has a β-glucan content of 73.0wt%, a weight-average molecular weight of 410kDa, a moisture content of 6.1wt%, a D90 particle size of 130μm, and take 28.0g of food-grade yeast β-glucan powder, wherein the yeast β-glucan powder has a β-glucan content of 85.0wt%, a mass fraction of insoluble components of 78.5wt%, a D50 particle size of 18μm, a D90 particle size of 57μm, and a moisture content of 5.9wt%. The total amount of β-glucan in the above oat β-glucan powder and yeast β-glucan powder is 76.36g.

[0059] (2) Prepare biphasic β-glucan hydration substrates according to steps (2) and (3) of Example 1;

[0060] (3) Cool the biphasic β-glucan hydrate substrate to 50°C, add 0.48g of food-grade endo-β-glucanase, maintain pH 5.45, 50°C and 180rpm stirring, digest for 24min, and immediately raise the temperature to 92°C and keep warm for 6min after digestion to inactivate β-glucanase.

[0061] (4) Cool the material after enzyme inactivation to 37°C, add 0.70g of sodium bicarbonate, adjust the pH to 5.80 before inoculation, take 0.18g of freeze-dried Lactobacillus plantarum powder, 0.12g of freeze-dried Lactobacillus rhamnosus powder and 0.10g of freeze-dried Bifidobacterium animalis subsp. lactis powder, rehydrate the above powders with 30.0g of sterile water for 20min and then inoculate them into the material;

[0062] (5) Ferment, inactivation, concentration, addition of carrier and spray drying are carried out according to steps (6) to (8) of Example 1 to obtain probiotic fermented immune-modulating nutritional supplement.

[0063] Example 5:

[0064] The preparation was carried out at a 10x scale-up according to the method of Example 1, with the difference being: 800.0g of food-grade oat β-glucan powder and 220.0g of food-grade yeast β-glucan powder were taken, and 15000.0g of food production water, 21.0g of sodium citrate dihydrate, 5.5g of citrate monohydrate, 6.0g of dipotassium hydrogen phosphate, 3.0g of magnesium sulfate heptahydrate, 120.0g of food-grade pea peptide and 50.0g of food-grade yeast extract, 4.6g of food-grade endo-β-glucanase, 7.0g of sodium bicarbonate, 2.0g of freeze-dried Lactobacillus plantarum powder, 1.0g of freeze-dried Lactobacillus rhamnosus powder and 1.0g of freeze-dried Bifidobacterium animalis subsp. lactis powder, 420.0g of resistant dextrin and 180.0g of inulin were added;

[0065] Hydration, enzyme digestion, enzyme inactivation, inoculation, nitrogen replacement, fermentation, cell inactivation, vacuum concentration, and spray drying were all performed according to Example 1. The dried powder was passed through a 250 μm sieve to obtain a probiotic fermented immunomodulatory nutritional supplement.

[0066] Comparative Example 1:

[0067] Take the raw materials according to the amounts of oat β-glucan powder 80.0g, yeast β-glucan powder 22.0g, resistant dextrin 42.0g and inulin 18.0g as in Example 1; separately take 0.20g of freeze-dried Lactobacillus plantarum powder, 0.10g of freeze-dried Lactobacillus rhamnosus powder and 0.10g of freeze-dried Bifidobacterium animalis subsp. lactis powder, incubate at 80℃ for 12min to inactivate the cells, then mix with the above powders for 30min and pass through a 250μm sieve to obtain a dry-mixed nutritional supplement.

[0068] Comparative Example 2:

[0069] The difference between Comparative Example 2 and Example 1 is that yeast β-glucan powder was not added, and the amount of oat β-glucan powder was adjusted to 105.9g, so that the amount of β-glucan in the oat β-glucan powder was 76.25g. The other conditions were the same as in Example 1.

[0070] Comparative Example 3:

[0071] The difference between Comparative Example 3 and Example 1 is that oat β-glucan powder was not added, and the amount of yeast β-glucan powder was adjusted to 89.8g, so that the amount of β-glucan in the yeast β-glucan powder was 76.33g. The other conditions were the same as in Example 1.

[0072] Comparative Example 4:

[0073] The difference between Comparative Example 4 and Example 1 is that the addition of food-grade endo-β-glucanase and enzymatic digestion in step (4) are omitted, and the biphasic β-glucan hydrated substrate is directly cooled to 37°C before inoculation. The other conditions are the same as in Example 1.

[0074] Comparative Example 5:

[0075] The difference between Comparative Example 5 and Example 1 is that the amount of food-grade endo-β-glucanase was adjusted to 0.90g, and the pH was maintained at 5.45, 50℃ and 180rpm for stirring. The enzyme digestion was carried out for 60min, and the other conditions were the same as in Example 1.

[0076] Comparative Example 6:

[0077] The difference between Comparative Example 6 and Example 1 is that: after enzymatic digestion for 24 min in step (4), the temperature is not raised to 92℃ and kept warm for 6 min, but the material is directly cooled to 37℃ and inoculated for fermentation. The other conditions are the same as in Example 1.

[0078] Comparative Example 7:

[0079] The difference between Comparative Example 7 and Example 1 is that 30.0g of food-grade glucose was added to the enzyme-inactivated material before inoculation in step (5), and the other conditions were the same as in Example 1.

[0080] Comparative Example 8:

[0081] The difference between Comparative Example 8 and Example 1 is that the fermentation time in step (6) was extended from 16h to 24h, and after the fermentation was completed, the fermentation cells were kept at 80℃ for 12min to inactivate the fermentation cells. The other conditions were the same as in Example 1.

[0082] Performance testing:

[0083] (1) pH measurement: The pH of the fermentation broth was measured according to the pH measurement method of GB5009.237-2016. The pH of the fermentation endpoint sample was measured directly at 25℃. For the powder, 10.0g of powder was added to 90.0g of water, stirred evenly, and then measured at 25℃.

[0084] (2) Detection of viable bacteria and colony count after inactivation: The viable bacteria count of lactic acid bacteria before heat inactivation was determined according to GB4789.35-2023; the total number of colonies of powder after heat inactivation was determined according to GB4789.2-2022, and the detection of viable bacteria under the lactic acid bacteria culture conditions was verified according to GB4789.35-2023.

[0085] (3) Determination of total dietary fiber: The total dietary fiber of powder was determined in accordance with GB5009.88-2023, and the results were expressed as a percentage of sample mass.

[0086] (4) Determination of β-glucan content and retention rate: Oat β-glucan raw materials were subjected to enzyme-colorimetric determination according to NY / T2006-2011; total β-glucan was determined by acid-enzyme hydrolysis combined with glucose quantification method for mixed fermentation products and powder samples, and β-glucan components with a molecular weight of not less than 100kDa were determined by SEC-MALS. Residual β-glucan retention rate = final residual β-glucan content / initial total β-glucan content × 100%.

[0087] (5) Determination of reducing sugar release rate: Take the material before inoculation and use the DNS method to determine the reducing sugar. The reducing sugar release rate is calculated as glucose = amount of reducing sugar before inoculation / total amount of initial β-glucan × 100%.

[0088] (6) Viscosity retention rate determination: In accordance with the viscosity measurement method of GB / T10247-2008, the apparent viscosity of the hydrated undigested substrate and the material before inoculation was measured at 25℃. Viscosity retention rate = apparent viscosity of material before inoculation / apparent viscosity of hydrated undigested substrate × 100%.

[0089] (7) Determination of DP2-8 oligosaccharide ratio: After the sample was deactivated at the end of fermentation, the high molecular weight components were removed by ethanol precipitation. The oligosaccharides of β-glucan with a degree of polymerization of 2-8 were determined by HPAEC-PAD or HPLC-ELSD. The DP2-8 oligosaccharide ratio = DP2-8 oligosaccharide amount / initial β-glucan total amount × 100%.

[0090] (8) Determination of powder dispersion residue rate: Take 10.0g of powder and add it to 200mL of water at 25℃. Stir at 300rpm for 30s, filter through a 250μm sieve, collect the material on the sieve, dry and weigh it. Dispersion residue rate in 30s = dry residue mass on the sieve / powder sample mass × 100%.

[0091] (9) Stability test: The powder was sealed in an aluminum-plastic composite bag and placed at 40℃ and 75%RH for 30 days. The β-glucan content before and after placement was measured. The retention rate = β-glucan content after placement / β-glucan content before placement × 100%.

[0092] (10) Evaluation of in vitro immunomodulatory potential: Each powder was prepared into an aqueous dispersion, and the supernatant and dispersible fine particles were collected by centrifugation. RAW264.7 cells were treated with 200 μg / mL for 24 h. The phagocytic activity was measured by the neutral red phagocytosis method, and the blank group was recorded as 100%. At the same time, the cell viability was measured by the CCK-8 method.

[0093] Table 1. Preparation process parameters and structural parameters of each sample

[0094] Example 1 10.4 58.8 49.2 4.38 51.0 14.5 Example 2 8.7 65.1 57.4 4.53 55.4 12.2 Example 3 12.7 52.3 43.6 4.31 48.6 16.8 Example 4 10.0 56.7 47.8 4.34 50.2 14.0 Example 5 10.6 59.1 50.0 4.41 51.7 14.1 Comparative Example 1 2.3 93.8 88.4 5.62 93.1 2.4 Comparative Example 2 14.8 45.7 38.5 4.35 43.0 17.6 Comparative Example 3 4.7 82.2 62.4 4.86 66.8 6.2 Comparative Example 4 2.6 87.5 78.2 4.83 73.0 4.9 Comparative Example 5 23.9 31.4 22.6 4.21 34.6 24.7 Comparative Example 6 12.9 50.1 40.5 4.19 36.2 25.8 Comparative Example 7 10.5 59.0 49.4 4.12 52.0 7.8 Comparative Example 8 10.3 58.7 49.1 3.92 37.5 23.2

[0095] Table 2. Powder endpoint properties and in vitro evaluation results for each sample.

[0096] Example 1 21.1 57.6 6.8 94.2 162 96.8 <10 Example 2 22.4 58.1 8.5 94.8 152 97.5 <10 Example 3 20.1 56.4 5.9 92.6 157 96.2 <10 Example 4 21.0 57.9 9.2 93.8 165 95.9 <10 Example 5 21.3 57.4 7.1 94.0 160 97.1 <10 Comparative Example 1 38.5 68.2 18.4 98.1 121 98.3 <10 Comparative Example 2 17.6 55.5 10.8 91.7 139 96.4 <10 Comparative Example 3 29.8 62.1 16.2 95.6 145 95.8 <10 Comparative Example 4 30.2 64.8 22.8 96.0 129 97.0 <10 Comparative Example 5 14.4 51.7 4.2 88.1 134 94.9 <10 Comparative Example 6 15.0 50.9 3.9 86.5 130 94.2 <10 Comparative Example 7 18.5 52.6 7.6 92.0 132 96.6 <10 Comparative Example 8 15.5 49.8 4.1 85.3 126 93.8 <10

[0097] Data Analysis:

[0098] As shown in Tables 1 and 2, Examples 1 to 5 all formed a partially exposed β-glucan substrate state before inoculation while still retaining its structure. The reducing sugar release rate before inoculation was 8.7wt%-12.7wt%, the ≥100kDa β-glucan retention rate was 52.3wt%-65.1wt%, and the viscosity retention rate was 43.6wt%-57.4wt%. This state maintained the fermentation endpoint pH at 4.31-4.53, the residual β-glucan retention rate was 48.6wt%-55.4wt%, and the DP2-8 oligosaccharide ratio was 12.2wt%-16.8wt%. The powder simultaneously exhibited low dispersion residue and high relative in vitro phagocytic activity.

[0099] Although Comparative Example 1 had a high content of powdered β-glucan and total dietary fiber, the proportion of DP2-8 oligosaccharides was only 2.4 wt%, and the relative value of RAW264.7 phagocytic activity was 121%, which was lower than that of Examples 1-5. This indicates that simply dry-mixing β-glucan, carrier and inactivated bacteria cannot form the same composition as fermentation treatment.

[0100] In Comparative Example 2, after removing yeast β-glucan, the reducing sugar release rate increased to 14.8 wt%, but the retention rates of ≥100 kDa β-glucan and residual β-glucan decreased to 45.7 wt%-43.0 wt%, respectively. In Comparative Example 3, after removing oat β-glucan, the reducing sugar release rate was only 4.7 wt%, the fermentation endpoint pH was 4.86, and the DP2-8 oligosaccharide ratio was 6.2 wt%. These results indicate that using oat β-glucan and yeast β-glucan together as substrates is beneficial for balancing hydrated segments, retaining particulate components, and utilizing fermentable sugar ends.

[0101] In Comparative Example 4, after omitting limited enzymatic digestion before inoculation, the reducing sugar release rate was only 2.6 wt%, the viscosity retention rate was 78.2 wt%, and the fermentation endpoint pH was 4.83, indicating that the lack of substrate pre-exposure is detrimental to fermentation initiation. In Comparative Example 5, after using excess enzyme and extending the digestion period, the ≥100 kDa β-glucan retention rate decreased to 31.4 wt%, and the residual β-glucan retention rate decreased to 34.6 wt%. In Comparative Example 6, inoculated under conditions where the enzyme was not inactivated, the residual β-glucan retention rate at the fermentation endpoint was 36.2 wt%, indicating that continuous enzymatic digestion during the fermentation stage leads to excessive structural degradation.

[0102] In Comparative Example 7, the pH at the fermentation endpoint decreased to 4.12 after the addition of glucose, but the DP2-8 oligosaccharide ratio was only 7.8 wt%, and the relative phagocytic activity of RAW264.7 was 132%, indicating that the rapid acid production from the added monosaccharide cannot replace the fermentation conversion around the β-glucan substrate. In Comparative Example 8, after extending fermentation to 24 h, the endpoint pH decreased to 3.92, the residual β-glucan retention rate decreased to 37.5 wt%, and the 30-day β-glucan retention rate in the powder decreased to 85.3 wt%, indicating that the fermentation endpoint needs to be controlled within a range that balances oligosaccharide generation and structural retention.

[0103] Example 5 is a 10x scaled-up sample, and its key process data are similar to those of Example 1, indicating that the preparation route can still obtain similar reducing sugar release rate, β-glucan structure retention rate, oligosaccharide ratio, powder dispersibility and in vitro evaluation results under scaled-up conditions.

[0104] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A method for preparing a probiotic fermented immune-modulating nutritional supplement, characterized in that, Includes the following steps: (1) Based on a total of 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, 72-83 parts by weight of oat β-glucan powder and 17-28 parts by weight of yeast β-glucan powder are added to food production water containing buffer salts and nutrients, and the sum of the amounts of oat β-glucan powder and yeast β-glucan powder is 100 parts by weight. After shear dispersion and hydration, a biphasic β-glucan hydration substrate is obtained. (2) Add food-grade endo-β-glucanase to the biphasic β-glucan hydrate substrate and enzymatically digest it at pH 5.35-5.55 and 49-51℃ for 20-28 min. After the enzymatic digestion is completed, immediately raise the temperature to 91-93℃ and keep it at that temperature for 6-7 min to inactivate the β-glucanase. (3) Cool the enzyme-inactivated material to 37°C, adjust the pH to 5.78-5.82 before inoculation, rehydrate the compound bacterial powder composed of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis, and inoculate it, and do not add glucose before inoculation. (4) Place the inoculated material under low oxygen closed conditions with dissolved oxygen of 0.7-0.9 mg / L and ferment at 37°C for 15-17 h. Then raise the temperature to 80°C and keep it at 12 min to inactivate the fermentation cells. (5) The inactivated fermentation broth was vacuum concentrated at 50°C and -0.085 MPa to a solid content of 27.2-27.8 wt%. Resistant dextrin and inulin were added, mixed, and spray-dried to obtain the probiotic fermented immunomodulatory nutritional supplement.

2. The method for preparing the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, The oat β-glucan powder has a β-glucan content of 70.5-73.0 wt%, a weight-average molecular weight of 360-450 kDa, and a D90 particle size of 126-145 μm; the yeast β-glucan powder has a β-glucan content of 84.0-85.0 wt%, an insoluble component mass fraction of 77.0-79.0 wt%, a D50 particle size of 17-20 μm, and a D90 particle size of 54-61 μm.

3. The preparation method of the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, Based on 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, the water used in food production is 1440-1500 parts by weight. The buffer salts and nutrients include 1.9-2.2 parts by weight of sodium citrate dihydrate, 0.49-0.58 parts by weight of citrate monohydrate, 0.57-0.60 parts by weight of dipotassium hydrogen phosphate, 0.28-0.30 parts by weight of magnesium sulfate heptahydrate, 11.5-12.0 parts by weight of food-grade pea peptides, and 4.8-5.0 parts by weight of food-grade yeast extract.

4. The preparation method of the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, In step (1), oat β-glucan powder and yeast β-glucan powder are added at 57-59℃, sheared and dispersed at 1200rpm for 10min, then stirred at 260rpm and heated to 61-63℃ for hydration for 25min.

5. The method for preparing the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, The food-grade endo-β-glucanase has an enzyme activity of 10,000 U / g and is added in amounts of 0.36-0.55 parts by weight. The reducing sugar release rate of the material obtained in step (2) before inoculation is 8.7-12.7 wt%, the β-glucan retention rate with a molecular weight of not less than 100 kDa is 52.3-65.1 wt%, and the viscosity retention rate is 43.6-57.4 wt%.

6. The method for preparing the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, Based on a total of 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, 0.64-0.73 parts by weight of sodium bicarbonate are added in step (3); the compound bacterial powder contains 0.18-0.20 parts by weight of freeze-dried Lactobacillus plantarum, 0.096-0.12 parts by weight of freeze-dried Lactobacillus rhamnosus, and 0.096-0.10 parts by weight of freeze-dried Bifidobacterium animalis subsp. lactis.

7. The method for preparing the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, In step (4), the low-oxygen sealed conditions are formed by filling the headspace of the fermenter with nitrogen. The stirring speed during fermentation is 120 rpm. The final pH of fermentation is 4.31-4.53, the residual β-glucan retention rate is 48.6-55.4 wt%, and the DP2 to 8 oligosaccharide ratio is 12.2-16.8 wt%.

8. The method for preparing the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, Based on a total of 100 parts by weight of oat β-glucan powder and yeast β-glucan powder, 39.5-42.5 parts by weight of resistant dextrin and 15-20 parts by weight of inulin are added in step (5); the dietary fiber content of the resistant dextrin is 88.0 wt%, the DE value is 8, and the average degree of polymerization of the inulin is 10.

9. The method for preparing the probiotic fermented immune-modulating nutritional supplement according to claim 1, characterized in that, In step (5), the inlet air temperature of the spray drying is 152-158℃, the outlet air temperature is 77-80℃, the feed flow rate is 28-31mL / min, and the dried powder obtained by spray drying passes through a 250μm sieve.

10. A probiotic fermented immune-modulating nutritional supplement, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.