Yeast beta-glucan composition for preventing and relieving upper respiratory tract infection

Abstract

The invention relates to the field of food, in particular to a yeast beta-glucan composition for preventing and relieving upper respiratory tract infection. The composition provided by the invention mainly takes yeast beta-glucan and lactoferrin as main raw materials, and comprises the following components in percentage by weight: 0.1 to 80 percent of yeast beta-glucan and 0.1 to 80 percent of lactoferrin. The composition provided by the invention can also be prepared from other selective synergistic components and auxiliary materials through a series of processing technologies. The composition provided by the invention can ensure the complete structure and activity of the effective components, and has the effects of boosting immunity and preventing and relieving upper respiratory tract infection.

Description

Technical Field

[0001] This invention relates to the food industry, and in particular to a yeast β-glucan composition for preventing and alleviating upper respiratory tract infections. Background Technology

[0002] Upper respiratory tract infection (UPTI) ranks first in the number of patients seeking treatment at respiratory clinics. It is an infection caused by pathogens invading the body, primarily bacteria and viruses, but can also be caused by mycoplasma, chlamydia, etc. The disease is common in children and can occur year-round, but is more prevalent in winter and spring. Symptoms of UPTI vary in severity. Mild cases present with runny nose, nasal congestion, cough, low-grade fever, and sneezing, with relatively good mental status. Severe cases can lead to high fever (>39℃), poor mental status, headache, sore throat, loss of appetite, and even lower respiratory tract infections and complications. The disease is recurrent and difficult to treat, seriously impacting the healthy growth of children and placing a heavy burden on society and families. Current treatment for UPTI mainly involves antibacterial and antiviral therapy combined with symptomatic treatment such as antipyretics, analgesics, and antitussives. Antibacterial and antiviral treatments often employ broad-spectrum antibiotics and broad-spectrum antiviral drugs. These drugs have a strong inhibitory effect on pathogens such as bacteria and viruses, but long-term use of these drugs can easily lead to drug resistance and is accompanied by certain side effects, thus limiting their application.

[0003] Therefore, providing compositions that can prevent and alleviate upper respiratory tract infections is of significant practical importance. Summary of the Invention

[0004] In view of this, the present invention provides a yeast β-glucan composition for the prevention and relief of upper respiratory tract infections. The composition provided by the present invention mainly uses yeast β-glucan and lactoferrin as the main raw materials, with yeast β-glucan accounting for 0.1-80% and lactoferrin accounting for 0.1-80% by weight. The composition provided by the present invention can also be prepared by a series of processing techniques with other selective synergistic ingredients and excipients. The composition provided by the present invention ensures the complete structure and activity of the active ingredients, and has the effect of helping to enhance immunity and prevent and relieve upper respiratory tract infections.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a composition comprising yeast β-glucan and lactoferrin.

[0007] In some specific embodiments of the present invention, the components are included, by weight:

[0008] Yeast β-glucan 0.1–80 parts;

[0009] Lactoferrin 0.1–80 parts.

[0010] In some specific embodiments of the present invention, the components are included, by weight:

[0011]

[0012] In some specific embodiments of the present invention, the yeast β-glucan includes non-water-soluble or water-soluble yeast β-glucan;

[0013] The lactoferrin includes lactoferrin and / or whey protein powder containing lactoferrin.

[0014] In some specific embodiments of the present invention, the composition further includes inactive edible yeast, bovine colostrum powder, milk fat globule membrane whey protein, whey protein concentrate, yeast protein, soy protein isolate, elderberry powder, N-acetylneuraminic acid, lactic acid bacteria powder, 2'-fucosylated lactose, lactose-N-neotetrasaccharide, stachyose, taurine, vitamin C, or acceptable excipients.

[0015] In some specific embodiments of the present invention, the excipients include one or more of dairy raw materials, dietary fiber, sweeteners, flavorings, fruit and vegetable powders, tricalcium phosphate, or magnesium stearate.

[0016] In some specific embodiments of the present invention, the dairy raw materials include one or more of whole milk powder, skim milk powder, or demineralized whey powder;

[0017] The dietary fiber includes one or more of resistant dextrin, polydextrose, galactooligosaccharides, fructooligosaccharides, or isomaltooligosaccharides.

[0018] The sweetener includes one or more of glucose, sorbitol, erythritol, maltitol, D-mannitol, isomaltitol, xylitol, monk fruit powder, steviol glycosides, or sucralose.

[0019] In a second aspect, the present invention also provides for the use of the said composition in any of the following:

[0020] (I) Prepare products that improve or enhance immunity;

[0021] (II) Prepare products for the prevention, relief and / or treatment of upper respiratory tract infections.

[0022] In some specific embodiments of the present invention, the improvement or enhancement of immunity includes any of the following:

[0023] (I) Increase the thymus / body weight ratio;

[0024] (II) Increase the phagocytic index;

[0025] (III) Inhibit the degree of ear swelling;

[0026] (IV) Increase the number of hemolytic plaques;

[0027] (V) Increases the half-life of hemolysis;

[0028] (VI) Improve the phagocytic rate of macrophages;

[0029] (VII) Increase the phagocytic index of macrophages;

[0030] (VIII) Promotes ConA-induced transformation of mouse spleen lymphocytes;

[0031] (IX) Enhance NK cell activity.

[0032] Thirdly, the present invention also provides products for improving or enhancing immunity or preventing, alleviating and / or treating upper respiratory tract infections, characterized in that they comprise the composition as described in any one of claims 1 to 7.

[0033] In summary, this invention provides a composition and its application. The composition mainly uses yeast β-glucan and lactoferrin as the main raw materials, with yeast β-glucan accounting for 0.1-80% and lactoferrin accounting for 0.1-80% by weight. The composition provided by this invention can also be prepared by a series of processing techniques with other selective synergistic ingredients and excipients.

[0034] The beneficial effects of the present invention include, but are not limited to:

[0035] 1) The composition provided by this invention can enhance immunity, and its effect on enhancing immunity is better than that of a single component;

[0036] 2) The composition provided by this invention can effectively prevent and alleviate recurrent upper respiratory tract infections, and its effect is superior to that of a single component;

[0037] 3) The composition provided by the present invention is obtained through a simple processing technology without heat treatment, which can ensure the integrity and activity of the active ingredients;

[0038] 4) The composition provided by this invention has a simple formula and clear components, all of which are food raw materials and food additives. It is easy to analyze, the active ingredients are easy to detect and quantify, it is highly safe, has no side effects, and can be taken by healthy people for a long time. Detailed Implementation

[0039] This invention discloses a yeast β-glucan composition for preventing and alleviating upper respiratory tract infections. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0040] The yeast β-glucan used in this invention is a polysaccharide derived from the yeast cell wall, a high-molecular-weight polysaccharide with β-1,3-D-glucan as the main chain and β-1,6-D-glucan as the branch chain. After oral administration, most of the yeast β-glucan is absorbed in the intestine to the Peyer's cluster lymph nodes, then migrates to the subbasal layer. There, it is specifically bound to receptors on the surface of immune cells, phagocytosed by macrophages, and then the large β-glucan molecules are internalized and fragmented into smaller β-glucan fragments within the macrophages. These smaller β-glucan fragments are ultimately taken up by circulating granulocytes, monocytes, or macrophages via complement receptors, initiating a series of immune responses and enhancing the body's resistance.

[0041] The lactoferrin used in this invention is an iron-binding glycoprotein belonging to the transferrin family. It is found in human breast milk and various secretions, with the highest concentration in breast milk. It possesses antimicrobial, immunomodulatory, iron-regulating, and intestinal cell proliferation and differentiation functions. The main mechanism of lactoferrin's antimicrobial effect is its high affinity for iron ions, creating an iron-deficient environment around pathogens, reducing microbial absorption of this essential element, and thus inhibiting microbial growth. Furthermore, lactoferrin is currently recognized as a protein with pleiotropic immunomodulatory activity. It can affect various immune cells, including lymphocytes and macrophages. Lactoferrin receptors exist on the surface of these immune cells, and lactoferrin exerts its biological effects by binding to these receptors.

[0042] The composition in the embodiments of the present invention may also include other functional raw materials as synergistic ingredients, such as inactive edible yeast, bovine colostrum powder, milk fat globule membrane whey protein, whey protein concentrate, yeast protein, soy protein isolate, elderberry powder, N-acetylneuraminic acid, lactic acid bacteria powder, 2'-fucosylated lactose, lactose-N-neotetrasaccharide, stachyose, taurine, vitamin C, etc. It may also include optional excipients, such as dairy raw materials (whole milk powder, skim milk powder, demineralized whey powder, etc.), dietary fiber (resistant dextrin, polydextrose, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, etc.), sweeteners (glucose, sorbitol, erythritol, maltitol, D-mannitol, isomaltitol, xylitol, monk fruit powder, steviol glycosides, sucralose, etc.), flavorings, fruit and vegetable powders, tricalcium phosphate, magnesium stearate, etc.

[0043] The compositions in the embodiments of this invention can also be formulated into drugs with pharmaceutically acceptable excipients. "Pharmaceutically acceptable excipients" should be compatible with the components in the compositions of this invention, meaning they can be mixed with the excipients without significantly reducing the composition's effectiveness in enhancing immunity and preventing and alleviating upper respiratory tract infections under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars such as lactose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; malt; gelatin; vegetable oils, peanut oil, corn oil, cocoa butter, and sesame oil, etc.; polyols such as glycerol, sorbitol, and mannitol; flavoring agents; tablets; stabilizers; isotonic salt solutions; phosphate buffers, etc., wherein preferred carriers are selected from physiological saline, glycerol, and phosphate buffers.

[0044] The compositions in the embodiments of the present invention can be formulated into any dosage form acceptable in the food or pharmaceutical field, such as tablets, capsules, granules, powders, or other similar forms.

[0045] The preparation process provided by this invention includes:

[0046] 1. Weighing: Weigh the raw and auxiliary materials according to the formula ratio;

[0047] 2. Sieving: Select a suitable sieve (20-60 mesh) and sieve the weighed raw and auxiliary materials for later use;

[0048] 3. Premixing: Select materials with a formulation ratio of less than 20% (except magnesium stearate) and put them into the hopper mixer for premixing treatment. The premixing time is 5 to 15 minutes.

[0049] 4. Mixing: Put the premixed portion and the remaining material into a hopper mixer and mix for 20-35 minutes, or into an airflow mixer and mix for 3-8 minutes;

[0050] 5. Tableting (tablets): Tableting is performed using a rotary tableting machine under a main pressure of 10-30KN; Packaging (powder): Packaging is performed using a strip packaging machine or a canning packaging machine.

[0051] The process provided by this invention does not involve homogenization, heat sterilization, or other steps, thus ensuring the activity of lactoferrin.

[0052] The raw materials and reagents used in the yeast β-glucan composition for preventing and alleviating upper respiratory tract infections provided by this invention are all commercially available.

[0053] The present invention will be further illustrated below with reference to the embodiments:

[0054] Formulations of Examples 1-7

[0055]

[0056]

[0057] Example 1

[0058] The components of the composition provided in this embodiment are as follows (by weight percentage): yeast β-glucan 10%, lactoferrin 15%, whole milk powder 50%, skim milk powder 24%, and tricalcium phosphate 1%. Weigh the above components according to the formula, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan, lactoferrin, and tricalcium phosphate to a hopper mixer and premix for 5 minutes. Then, add them together with the whole milk powder and skim milk powder to the hopper mixer and mix for 35 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0059] Example 2

[0060] The components of the composition provided in this embodiment are as follows: by mass percentage, yeast β-glucan 80%, lactoferrin 0.1%, whole milk powder 18.9%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved lactoferrin, tricalcium phosphate, and whole milk powder to a hopper mixer and premix for 15 minutes. Then, add the yeast β-glucan together to the hopper mixer and mix for 20 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0061] Example 3

[0062] The components of the composition provided in this embodiment are as follows: by mass percentage, yeast β-glucan 0.1%, lactoferrin 80%, whole milk powder 18.9%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan, tricalcium phosphate, and whole milk powder to a hopper mixer and premix for 10 minutes. Then, add the lactoferrin together to the hopper mixer and mix for 30 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0063] Example 4

[0064] The components of the composition provided in this embodiment are as follows: by mass percentage, yeast β-glucan 40%, lactoferrin 40%, whole milk powder 19%, and tricalcium phosphate 1%. Weigh the above components according to the formula, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved tricalcium phosphate and whole milk powder to a hopper mixer and premix for 15 minutes. Then, add it together with lactoferrin and yeast β-glucan to an airflow mixer and mix for 8 minutes. Finally, package the mixture using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0065] Example 5

[0066] The components of the composition provided in this embodiment are as follows (by weight percentage): yeast β-glucan 2%, lactoferrin 0.1%, whey protein powder (containing 40% lactoferrin) 1%, bovine colostrum powder 40%, elderberry powder 1%, inactive edible yeast 1%, lactic acid bacteria powder 0.4%, N-acetylneuraminic acid 0.2%, 2'-fucosylated lactose 2%, whole milk powder 43%, demineralized whey powder 8.8%, and tricalcium phosphate 0.5%. Weigh all the above ingredients according to the formula, and sieve the weighed materials through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan, lactoferrin, whey protein powder (containing 40% lactoferrin), elderberry powder, inactive edible yeast, lactic acid bacteria powder, N-acetylneuraminic acid, 2'-fucosylated lactose, tricalcium phosphate, and demineralized whey powder to a hopper mixer and premix for 10 minutes. Then, add the colostrum powder and whole milk powder to an airflow mixer and mix for 6 minutes. Finally, package the mixture using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0067] Example 6

[0068] The components of the composition provided in this embodiment are as follows (by weight percentage): 12% yeast β-glucan, 2% whey protein powder (containing 40% lactoferrin), 10% bovine colostrum powder, 1% inactive edible yeast, 25% whole milk powder, 49% sorbitol, and 1% magnesium stearate. Weigh the above components according to the formula, and sieve the weighed materials through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan, whey protein powder (containing 40% lactoferrin), bovine colostrum powder, and inactive edible yeast to a hopper mixer and premix for 5 minutes. Then, add the whole milk powder, sorbitol, and magnesium stearate to an airflow mixer and mix for 3 minutes. Finally, use a tableting machine to compress the tablets at a main pressure of approximately 15 kN, with a target tablet weight of 1.0 g / tablet, to obtain a tablet product that can be directly chewed. The recommended daily intake is 2 g per person.

[0069] Example 7

[0070] The components of the composition provided in this embodiment are as follows (by weight percentage): yeast β-glucan 1.2%, lactoferrin 1.5%, yeast protein 20%, whey protein concentrate 20%, soy protein isolate 50%, whole milk powder 6.8%, and monk fruit powder 0.5%. Weigh the above components according to the formula, and sieve the weighed materials through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan, lactoferrin, monk fruit powder, and whole milk powder to a hopper mixer and premix for 10 minutes. Then, add the yeast protein, whey protein concentrate, and soy protein isolate to the hopper mixer and mix for 30 minutes. Finally, package using a strip packaging machine, with packaging specifications of 10g / bag or 20g / bag. The recommended daily intake per person is 20g.

[0071] Comparative Examples 1-6:

[0072]

[0073] Comparative Example 1

[0074] The components of the composition provided in this comparative example are as follows: by mass percentage, lactoferrin 15%, whole milk powder 50%, skim milk powder 34%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved lactoferrin and tricalcium phosphate to a hopper mixer and premix for 10 minutes. Then, add the lactoferrin and tricalcium phosphate together to the hopper mixer and mix for 30 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0075] Comparative Example 2

[0076] The components of the composition provided in this comparative example are as follows: by mass percentage, yeast β-glucan 10%, whole milk powder 50%, skim milk powder 39%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan and tricalcium phosphate to a hopper mixer and premix for 10 minutes. Then, add them together with the whole milk powder and skim milk powder to the hopper mixer and mix for 30 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0077] Comparative Example 3

[0078] The components of the composition provided in this comparative example are as follows (by mass percentage): yeast β-glucan 0.09%, lactoferrin 0.09%, whole milk powder 50%, skim milk powder 48.82%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved yeast β-glucan, lactoferrin, tricalcium phosphate, and a portion of the whole milk powder (15%) to a hopper mixer and premix for 10 minutes. Then, add the remaining whole milk powder (35%) and skim milk powder to an airflow mixer and mix for 6 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0079] Comparative Example 4

[0080] The components of the composition provided in this comparative example are as follows: by mass percentage, yeast β-glucan 81%, lactoferrin 0.09%, whole milk powder 17.91%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and sieve the weighed material through a 20-60 mesh sieve for later use. Add the sieved lactoferrin, tricalcium phosphate, and whole milk powder to a hopper mixer and premix for 10 minutes. Then, add the yeast β-glucan together to an airflow mixer and mix for 6 minutes. Finally, package using a strip packaging machine, with packaging specifications of 1g / bag or 2g / bag. The recommended daily intake per person is 2g.

[0081] Comparative Example 5

[0082] The components in the composition provided in this comparative example are as follows: by mass percentage, yeast β-glucan 0.09%, lactoferrin 81%, whole milk powder 17.91%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and use a 20-60 mesh sieve to sieve the weighed materials and then reserve them; put the sieved yeast β-glucan, tricalcium phosphate and whole milk powder into a hopper mixer for premixing for 10 minutes; then put them into an air flow mixer together with lactoferrin and mix for 6 minutes; finally, use a stick packaging machine for packaging, and the packaging specifications are 1 g / bag or 2 g / bag. The recommended daily intake per person is 2 g.

[0083] Comparative Example 6

[0084] The components in the composition provided in this comparative example are as follows: by mass percentage, yeast β-glucan 10%, lactoferrin 15%, whole milk powder 50%, skim milk powder 24%, and tricalcium phosphate 1%. Weigh the above components according to the formula amount, and use a 20-60 mesh sieve to sieve the weighed materials and then reserve them; dissolve the sieved above components at 50 °C, then homogenize at a temperature of 45 °C and a pressure of 15 Mpa, heat and sterilize at 85 °C for 30 s, concentrate, and finally obtain by spray drying.

[0085] Functional evaluation experiment on the composition in Effect Example 1 for enhancing immunity

[0086] 1. Materials and methods

[0087] 1.1. Experimental animals

[0088] BALB / C SPF level, male adult mice, weighing 14-16 g, provided by Hunan Slack Jingda Experimental Animal Co., Ltd. (Approval No.: SCXK(Xiang)2019-0004).

[0089] 1.2. Animal grouping and dosage

[0090] Before the experiment, use the random number table method to divide the mice into 11 groups, with 50 mice in each group, and then mark each group. Use distilled water by gavage as the negative control group, and set Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Example 1, Example 2, Example 3, Example 4, and Example 5 as 10 experimental groups. The samples are all set at 10 times the dose. The mice in each group are allowed to eat and drink freely, and the gavage volume of the test substance and the control group is 0.1 mL / 10 g.bw.

[0091] 1.3. Main instruments and reagents

[0092] Clean bench, 2-mercaptoethanol (2-ME), penicillin, streptomycin, 24-well culture plates, 96-well culture plates, surgical instruments, 8mm diameter punch, microscope, micro-coagulation test plate, centrifuge, MD SpectraMax M2 multi-functional enzyme-linked immunosorbent assay (ELISA) system, 96-well culture plates, 5% CO2 incubator, 200-mesh sieve; ink for injection, concanavalin A (ConA), MTT, dinitrofluorobenzene (DNFB), acetone, sesame oil, sheep red blood cells (SRBC), complement (guinea pig serum), SA buffer, agarose, Na2CO3, physiological saline, chicken red blood cells, methanol, Giemsa staining solution, YAC-1 cells, Hank's solution (pH 7.2–7.4), RPMI 1640 complete culture medium, NP 40 (1%), trypan blue, hydrochloric acid (1 mol / L), etc.

[0093] 1.4 Experimental Methods

[0094] 1.4.1 Measurement of mouse weight at three stages: Prepare an electronic balance with a suitable range, place a weighing box of appropriate size on it, and zero the balance; take the mice from the feeding box and place them in the weighing box. After the value stabilizes, read the value; if the value fluctuates, record the intermediate value. Weigh the mice before starting gavage, after 15 days of continuous gavage, and after 30 days of continuous gavage to record the initial, intermediate, and final weights.

[0095] 1.4.2 Carbon clearance assay (monocyte-macrophage function assay) and organ / body weight ratio determination: Mice were weighed after 30 days of continuous gavage. Indian ink diluted 3-4 times (10 mL / kg bw) was injected into the tail vein of each mouse according to body weight. 2 and 10 minutes after ink injection, 20 μL of blood was collected from the internal canthal venous plexus and immediately added to 2 mL of 0.1% Na₂CO₃ solution. Using Na₂CO₃ solution as a control, the optical density (OD) was measured at 600 nm, and the phagocytic index was calculated to represent the mouse's carbon clearance capacity. Immediately after blood collection, the mice were euthanized by dislocation. The liver, spleen, and thymus were dissected, blotted dry with filter paper, weighed, and the corresponding organ / body weight ratio was calculated.

[0096] 1.4.3 Delayed-type hypersensitivity (DTH) (Cellular immune function assay): After 30 days of continuous gavage, the skin on the abdomen of each mouse was shaved with a depilatory agent or razor (approximately 3cm × 3cm area), and then sensitized with 50μL of DNFB solution evenly. Five days after sensitization, 10μL of DNFB solution was applied evenly to both sides of the right ear of the mouse for challenge. 24 hours later, the mouse was euthanized by cervical dislocation, and both ears were cut off. Ear pieces with a diameter of 8mm were removed using a punch and weighed. The difference in weight between the left and right ears indicated the degree of DTH.

[0097] 1.4.4 Determination of the half-maximal hemolytic value (HC50) and detection of antibody-producing cells (Jerne modified slide method) (assay for humoral immune function): Mice were immunized by intraperitoneal injection of 0.2 mL of 2% (V / V) SRBC cell suspension after 30 consecutive days of gavage. Five days later, blood and spleen of mice were collected to determine the half-maximal hemolytic value (HC50) and the number of antibody-producing cells, respectively.

[0098] 1.4.4.1 Determination of the half-maximal hemolytic value (HC50): Blood was collected from mice after enucleation into centrifuge tubes. After standing for 1 hour, the tubes were centrifuged (2000 rpm) for 10 minutes to collect serum. The serum was diluted 300 times with physiological saline, and 1 mL was placed in a test tube. Then, 0.5 mL of 10% (V / V) SRBC and 1 mL of complement diluted (1:10) with physiological saline were added sequentially. A control tube without serum was prepared (using physiological saline instead). The tubes were incubated in a 37°C water bath for 15–30 minutes, and then the reaction was terminated by ice bath. After centrifuging the reaction solution (2000 rpm) for 10 minutes, 1 mL of the supernatant was collected in a new test tube, and 3 mL of DuPont's reagent was added. At the same time, 0.25 mL of 10% (V / V) SRBC was collected in another test tube, and DuPont's reagent was added to a final volume of 4 mL. After adding Duchenne's reagent to each tube, mix thoroughly and let stand for 10 minutes. Measure the optical density (OD) at 540 nm using the control tube as a blank, and calculate the half-hemolysis value (HC50).

[0099] 1.4.4.2 Antibody-producing cell detection (Jerne modified slide method): Spleens of mice immunized with SRBCs for 5 days were taken and placed in a small petri dish containing Hank's solution. The spleen was gently ground and filtered through a 200-mesh sieve to prepare a single-cell suspension. After centrifugation (1000 rpm × 10 min) and washing twice with Hank's solution, the cells (suspension) were resuspended in 5 mL of RPMI 1640 culture medium, counted, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6 Prepare 10 μL / mL solution. Dissolve the surface culture medium by heating, mix with an equal volume of 2×Hank's solution (pH 7.2–7.4), and dispense into small test tubes (0.5 mL per tube). Then, add 50 μL of 10% SRBC and 20 μL of spleen cell suspension (5×10⁻⁶ cells / mL) to each tube sequentially. 6 Mix the agarose solution (number of cells / mL) quickly and pour it onto a slide that has been coated with a thin layer of agarose. After the agar has solidified, place the slide upside down on a slide holder and incubate in a CO2 incubator for 1–1.5 hours. Then, add complement diluted with physiological saline (1:8) into the groove of the slide holder and continue incubating for another 1–1.5 hours. After that, remove the slide and count the number of hemolytic plaques to reflect the number of antibody-producing cells.

[0100] 1.4.5. Mouse peritoneal macrophage phagocytosis of chicken erythrocytes experiment (semi-in vivo method) (mononuclear-macrophage function assay): After 30 days of continuous gavage, mice were intraperitoneally injected with 2% chicken erythrocyte suspension (1 mL) for 30 min, then euthanized by cervical dislocation, fixed in a supine position on a wax plate, the abdominal wall skin was cut open in the middle, and 2 mL of physiological saline was injected into the peritoneal cavity. The mouse plate was rotated for 1 min, and then 1 mL of peritoneal fluid was aspirated and evenly distributed onto two glass slides. The slides were placed in an enamel box lined with damp gauze and incubated at 37°C for 30 min. After incubation, the slides were removed, rinsed in physiological saline to remove unattached cells, and air-dried. They were fixed with 1:1 acetone-methanol solution for 20 min, stained with 4% (V / V) Giemsa-phosphate buffer for 3 min, rinsed with distilled water, and air-dried. Macrophages were counted under an oil immersion microscope (100 cells / slide), and the phagocytic percentage and phagocytic index were calculated.

[0101] 1.4.6 ConA-induced mouse spleen lymphocyte transformation experiment (cellular immune function assay) and mouse NK cell activity assay: After 30 days of continuous gavage, mice were euthanized by cervical dislocation, and the spleen was aseptically removed and placed in a small petri dish containing an appropriate amount of sterile Hank's solution. The spleen was gently torn apart with tweezers and filtered through a 200-mesh sieve to prepare a single-cell suspension. The cell suspension was divided into two parts, which were used for the ConA-induced mouse spleen lymphocyte transformation experiment and the mouse NK cell activity assay, respectively.

[0102] 1.4.6.1 ConA-induced mouse spleen lymphocyte transformation assay (MTT method): After washing with Hank's solution and centrifuging (1000 rpm × 10 min) twice, the cells (suspension) were resuspended in 1 mL of RPMI 1640 complete culture medium. The number of viable cells was counted (above 95%) by trypan blue staining, and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 Cells / mL. Each cell suspension was added in two portions to a 24-well plate (1 mL per well). 75 μL of ConA solution was added to one well, with the other well serving as a control. After 68 hours of incubation, 0.7 mL of supernatant was aspirated from each well, and 0.7 mL of serum-free RPMI 1640 medium and 50 μL MTT were added. Incubation continued for 4 hours. After incubation, 1 mL of acidic isopropanol was added to each well, and the mixture was pipetted to completely dissolve the purple crystals. The mixture was then aliquoted into 96-well plates, with three replicates per well. The optical density (OD) was measured at 570 nm. The difference between the OD values ​​of wells with and without ConA represented the lymphocyte proliferation capacity.

[0103] 1.4.6.2 Mouse NK cell activity assay (lactate dehydrogenase (LDH) assay): Target cells (YAC-1 cells) were passaged 24 hours before the experiment. Before application, the cells were washed three times with Hank's solution and the cell concentration was adjusted to 4 × 10⁻⁶ cells / mL using RPMI 1640 complete culture medium.5 Spleen cell suspension was washed with Hank's solution, centrifuged twice (1000 rpm × 10 min), and the supernatant was discarded. The cells were then sponged up, and 0.5 mL of sterile water was added for 20 seconds to lyse the red blood cells. Then, 0.5 mL of 2× Hank's solution and 8 mL of 1× Hank's solution were added sequentially, centrifuged (1000 rpm) for 10 min, resuspended in 1 mL of RPMI 1640 complete medium, diluted with 1% glacial acetic acid, and counted. Trypan blue staining was performed to count the number of viable cells (should be above 95%), and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 7 100 μL each of target cells and effector cells (treated spleen cell suspension) (effector-target ratio 50:1) were added to a 96-well culture plate; 100 μL each of target cells and culture medium were added to the target cell spontaneous release wells; 100 μL each of target cells and NP 40 (1%) were added to the target cell maximum release wells. Each well was prepared in triplicate. After incubation at 37°C for 4 hours in a 5% CO2 incubator, the culture plate was centrifuged (1500 rpm) for 5 min. 100 μL of supernatant was aspirated from each well and placed in a 96-well culture plate, along with 100 μL of LDH matrix solution. The reaction was allowed to proceed for 3 min, and 30 μL of 1 mol / L HCl was added to each well. The optical density (OD) was measured at 490 nm to calculate NK cell activity.

[0104] 1.5 Statistical Analysis of Experimental Data: The experimental data were statistically analyzed using SPSS 27.0.1 software (analysis of variance and Q test, with data transformation performed if necessary).

[0105] 2. Results

[0106] 2.1 Carbon clearance capacity, organ / body weight ratio, and phagocytic index test in mice: After 30 days of continuous gavage, mice were injected with ink via the tail vein. Blood was collected from the inner canthus vein 2 and 10 minutes later to measure the carbon clearance capacity (expressed as phagocytic index a). The mice were then immediately euthanized by dislocation, and the spleen and thymus were harvested and weighed to calculate the organ-to-body weight ratio. The results and body weight of the mice at each stage (see 1.4.1) are shown in Table 1.

[0107] Table 1

[0108]

[0109]

[0110] Note: * and ** indicate that the differences compared with the negative control group were statistically significant (P<0.05) and highly significant (P<0.01), respectively.

[0111] As shown in Table 1, during the experiment, all groups of animals responded normally, and their body weight increased to a certain extent. There were no significant differences in body weight gain among the groups or between different stages (P>0.05). Organ coefficient tests showed that the spleen / body weight ratio and thymus / body weight ratio in Comparative Examples 1, 2, and 3 were not significantly different from the negative control group; however, the thymus / body weight ratio in Comparative Examples 4 and 5, and Examples 1, 2, 3, 4, and 5 was significantly higher than the negative control group (P<0.05, P<0.01). Phagocytic index results showed that the phagocytic index in Comparative Example 2 was significantly higher than that in the negative control group (P<0.05), and the phagocytic index in Comparative Examples 4 and 5, and Examples 1, 2, 3, 4, and 5 was significantly higher than that in the negative control group (P<0.01).

[0112] 2.2 Delayed-type hypersensitivity (DTH) test in mice: After 30 days of continuous gavage, the right ear was sensitized with DNFB after hair removal. The degree of ear swelling and body weight at each stage of the test are shown in Table 2.

[0113] Table 2

[0114]

[0115]

[0116] Note: ** indicates that the difference was highly significant compared with the negative control group (P<0.01).

[0117] During the experiment, all groups of animals responded normally. Table 2 shows that the weight of all groups of animals increased to some extent, but there was no significant difference in weight gain or weight at different stages (P>0.05). After sensitizing the right ear with DNFB, the degree of ear swelling in Comparative Examples 1, 2, 3, 4, Example 2, and 5 was not significantly different from that in the negative control group (P>0.05), while the degree of ear swelling in Comparative Example 5, Example 1, Example 3, and Example 4 was significantly improved compared with the negative control group (P<0.01).

[0118] 2.3 Mouse half-hemolytic value (HC50) and antibody-producing cell count assay: After 30 days of continuous gavage, mice were immunized with SRBC cell suspension via intraperitoneal injection for 5 days. Blood and spleen samples were then collected to determine the half-hemolytic value (HC50) and antibody-producing cell count, respectively. The body weight and corresponding measurement results at each stage of the experiment are shown in Table 3.

[0119] Table 3

[0120]

[0121]

[0122] Note: * and ** indicate that the differences compared with the negative control group were statistically significant (P<0.05) and highly significant (P<0.01), respectively.

[0123] As shown in Table 3, all groups experienced some degree of weight gain, but the differences in weight gain and weight at different stages were not significant (P>0.05). After SRBC immunization, the number of hemolytic plaques in mice in Comparative Example 2, Comparative Example 5, and Example 3 groups was significantly higher than that in the negative control group (P<0.05); the number of hemolytic plaques in mice in Comparative Example 4, Examples 1, 2, 4, and 5 groups was significantly higher than that in the negative control group (P<0.01). The half-maximal hemolytic index (H2O) of mice in Comparative Example 1, Comparative Example 5, and Examples 1, 3, 4, and 5 groups was significantly higher than that in the negative control group (P<0.01), while the remaining groups showed no significant difference from the negative control group.

[0124] 2.4. Phagocytosis of Chicken Red Blood Cells by Mouse Peritoneal Macrophages: After 30 days of continuous gavage, mice were injected intraperitoneally with a suspension of chicken red blood cells. The phagocytic percentage and phagocytic index of mouse macrophages were determined using a semi-in vivo method. The body weight and corresponding measurement results at each stage of the experiment are shown in Table 4.

[0125] Table 4

[0126]

[0127] Note: * and ** indicate that the differences compared with the negative control group were statistically significant (P<0.05) and highly significant (P<0.01), respectively.

[0128] As shown in Table 4, the body weight of mice in each group increased to some extent during the experiment, but there was no significant difference in body weight gain or body weight at different stages (P>0.05). The phagocytic rate of mice in Comparative Examples 1-5 and Examples 1-5 was higher than that of the negative control group. Among them, there was no significant difference in Comparative Examples 1 and 3, but significant differences were found in Comparative Examples 2 and 4, and Examples 1, 2, and 4 (P<0.05). The differences in Comparative Examples 5, Examples 3, and 5 were extremely significant (P<0.01). Regarding the phagocytic index, there was no difference between Comparative Examples 1, 2, and 4, and Example 2 and the control group (P>0.05). Comparative Examples 3 and 5, and Example 3 were significantly higher than the control group (P<0.05). Examples 1, 4, and 5 were higher than the control group, and the differences were extremely significant (P<0.01).

[0129] 2.5 ConA-induced mouse spleen lymphocyte transformation and NK cell activity assay: Mice were euthanized by cervical dislocation after 30 days of continuous gavage. Spleens were aseptically harvested and prepared into single-cell suspensions for ConA-induced mouse spleen lymphocyte transformation and NK cell activity assays. Body weight and corresponding measurement results at each stage of the experiment are shown in Table 5.

[0130] Table 5

[0131]

[0132] Note: * and ** indicate that the differences compared with the negative control group were statistically significant (P<0.05) and highly significant (P<0.01), respectively.

[0133] As shown in Table 5, there were no significant differences in body weight and weight gain among the mice in each group during the experiment (P>0.05). Comparative Example 1, Example 1, and Group 5 mice showed ConA-induced splenic lymphocyte transformation (OD). 570 The difference was higher in the control group than in the negative control group, and the difference was statistically significant (P<0.05). The OD values ​​of groups 5, 3, and 4 were significantly higher than those of the negative control group. 570 The difference was higher than that of the negative control group, and the difference was extremely significant (P<0.01); the NK activity of mice in the control group 4, Example 2 and 5 was higher than that of the negative control, and the difference was significant (P<0.05); the NK activity of mice in the example group 1 and 4 was higher than that of the negative control, and the difference was extremely significant (P<0.01).

[0134] 3. Discussion of Results

[0135] During the experiment, none of the test substances had a significant effect on mouse body weight and growth; compared with the negative control group:

[0136] Comparative Example 1 only increased the half-hemolysis value after SRBC immunization and promoted the transformation of splenic lymphocytes in mice after ConA stimulation; Comparative Example 2 only increased the carbon clearance phagocytic index, the number of hemolytic plaques after SRBC immunization, and the phagocytic rate of macrophages after intraperitoneal injection of chicken erythrocytes; Comparative Example 3 only increased the phagocytic index of macrophages after intraperitoneal injection of chicken erythrocytes, with no significant effect on other indicators; Comparative Example 4 increased the spleen / body weight ratio and thymus / body weight ratio, carbon clearance phagocytic index, the number of hemolytic plaques after SRBC immunization, the phagocytic rate of macrophages after intraperitoneal injection of chicken erythrocytes, and NK cell activity in mice; Comparative Example 5 increased the spleen / body weight ratio and thymus / body weight ratio, carbon clearance phagocytic index, the number of hemolytic plaques and half-hemolysis value after SRBC immunization, the phagocytic rate and phagocytic index of macrophages after intraperitoneal injection of chicken erythrocytes, promoted the transformation of splenic lymphocytes in mice after ConA stimulation, and inhibited ear swelling after DNFB sensitization.

[0137] Example 1 increased the spleen / body weight ratio and thymus / body weight ratio, carbon clearance phagocytic index, number of hemolytic plaques and half-hemolysis value after SRBC immunization, macrophage phagocytic rate and phagocytic index after intraperitoneal injection of chicken erythrocytes, and NK cell activity in mice. It also promoted the transformation of splenic lymphocytes in mice stimulated with ConA and inhibited ear swelling after DNFB sensitization. Example 2 increased the spleen / body weight ratio and thymus / body weight ratio, carbon clearance phagocytic index, number of hemolytic plaques after SRBC immunization, macrophage phagocytic rate after intraperitoneal injection of chicken erythrocytes, and NK cell activity in mice. Example 3 increased the thymus / body weight ratio, carbon clearance phagocytic index, number of hemolytic plaques and half-hemolysis value after SRBC immunization, macrophage phagocytic rate and phagocytic index after intraperitoneal injection of chicken erythrocytes, promoted the transformation of splenic lymphocytes in mice stimulated with ConA, and inhibited ear swelling after DNFB sensitization. Example 4 increased the spleen / body weight ratio and thymus / body weight ratio, carbon clearance phagocytic index, number of hemolytic plaques and half-hemolysis value after SRBC immunization, phagocytic rate and phagocytic index of macrophages after intraperitoneal injection of chicken erythrocytes, and NK cell activity in mice; promoted the transformation of splenic lymphocytes in mice stimulated with ConA; and inhibited ear swelling after DNFB sensitization. Example 5 increased the spleen / body weight ratio and thymus / body weight ratio, carbon clearance phagocytic index, number of hemolytic plaques and half-hemolysis value after SRBC immunization, phagocytic rate and phagocytic index of macrophages after intraperitoneal injection of chicken erythrocytes, and NK cell activity in mice; and promoted the transformation of splenic lymphocytes in mice stimulated with ConA.

[0138] The criteria for determining whether a health food product has an immune-enhancing effect are as follows, according to the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)": Positive results in any two of the following four aspects: cellular immune function (mouse spleen lymphocyte transformation experiment, delayed-type hypersensitivity reaction experiment), humoral immune function (antibody-producing cell detection, serum hemolysin determination), monocyte-macrophage function (mouse carbon clearance experiment, mouse peritoneal macrophage phagocytosis of chicken red blood cells experiment), and NK cell activity. Specifically, a positive result in either of the two cellular immune function assays, or a positive result in either of the two dose groups of any one experiment, indicates a positive cellular immune function assay. Similarly, a positive result in either of the two humoral immune function assays, or a positive result in either of the two dose groups of any one experiment, indicates a positive humoral immune function assay. A positive result in either of the two monocyte-macrophage function assays, or a positive result in either of the two dose groups of any one experiment, indicates a positive monocyte-macrophage function assay. Finally, a positive result in one or more dose groups of the NK cell activity assay indicates a positive NK cell activity assay. Based on the above criteria, it can be preliminarily determined that Comparative Examples 4, 5, and Examples 1-5 all have the effect of enhancing immunity, while Comparative Examples 1, 2, and 3 do not. That is, the combination of lactoferrin and yeast β-glucan is more effective in enhancing immunity than lactoferrin or yeast β-glucan alone. Comparing the experimental data of Examples 2 and 3 with Comparative Examples 4 and 5, it can be seen that when the formulation ratio exceeds the range protected by the patent, the effect does not increase further.

[0139] Example 2: Evaluation experiment on the prevention and relief of upper respiratory tract infection by the composition.

[0140] 1. Experimental Methods

[0141] 1.1 Inclusion criteria for subjects

[0142] Children with clinical manifestations of upper respiratory tract infection, such as cough, sputum production, wheezing, runny nose, rales, and wheezing, meet the diagnostic criteria for acute upper respiratory tract infection.

[0143] 1.2 Grouping

[0144] The recruited participants were randomly divided into experimental group and control group 1–3 using a random number table, with 50 participants in each group. In the experimental group, there were 30 males and 20 females, aged 2–14 years, with a mean age of 9.4 years. In control group 1, there were 27 males and 23 females, aged 2–14 years, with a mean age of 9.8 years. In control group 2, there were 29 males and 21 females, aged 2–14 years, with a mean age of 9.5 years. In control group 3, there were 25 males and 25 females, aged 2–14 years, with a mean age of 9.8 years. There were no significant differences in gender or age among the four groups (P>0.05), making them comparable.

[0145] 1.3 Intervention Methods

[0146] The experimental group used the composition prepared in Example 1, dissolved in warm water, 1g each time, twice a day. The control groups 1-3 used the compositions prepared in Comparative Examples 1, 2, and 3, respectively, dissolved in warm water, 1g each time, twice a day. Both groups were treated for one week.

[0147] 1.4 Evaluation Criteria

[0148] Three criteria were set: markedly effective, effective, and ineffective. Markedly effective means that clinical symptoms have completely disappeared and normal life has been restored; effective means that all or most of the symptoms have been significantly eliminated, but mild symptoms may still appear, and only a partial return to normal life has been achieved; ineffective means that there is no significant change in symptoms and signs.

[0149] 2. Experimental Results

[0150] 2.1 Evaluation of intervention effectiveness

[0151] The effectiveness rates of intervention for different groups of subjects are shown in Table 6.

[0152] Table 6

[0153]

[0154] The above experimental results show that the total effective rate of the experimental group taking the composition for preventing and alleviating upper respiratory tract infections prepared in Example 1 of this invention reached 98% for subjects with upper respiratory tract infections. The total effective rates of control groups 1 and 2 were 78% and 70%, respectively, while control group 3 had almost no effect. Therefore, the composition provided by this invention has a better effect on preventing and alleviating upper respiratory tract infections compared to a single component.

[0155] Example 3: Detection of active lactoferrin content in the composition

[0156] Literature reports that lactoferrin is a heat-sensitive substance. Various conventional heat treatments in dairy processing will more or less cause changes in the structure or biological activity of lactoferrin. The higher the intensity of heat treatment that lactoferrin is subjected to, the greater the degree of denaturation and inactivation.

[0157] The method for detecting lactoferrin, as specified in GB 5009.299-2024 (National Food Safety Standard - Determination of Lactoferrin in Food), involves enrichment and purification using a heparin affinity column, followed by separation using reversed-phase high-performance liquid chromatography (RP-HPLC) and quantitative detection using a UV detector. This method can only detect lactoferrin with intact structure and activity.

[0158] 1. Experimental Methods

[0159] The lactoferrin content in the samples of Examples 1 to 6 and Comparative Example 6 was determined using the method specified in GB 5009.299-2024.

[0160] 2. Experimental Results

[0161] The results of lactoferrin content detection in different groups of samples are shown in Table 7.

[0162] Table 7

[0163]

[0164] The above experimental results show that the samples prepared in Examples 1-7 of this invention achieved 95%-121.6% of the theoretical value when tested using the GB 5009.299-2024 detection method, while the sample prepared in Comparative Example 6 only achieved 24% of the theoretical value. This indicates that the product prepared by the process of this invention can better guarantee the intact structure and activity of lactoferrin.

[0165] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, Including yeast beta-glucan and lactoferrin.

2. The composition according to claim 1, characterized in that, Based on parts by weight, it comprises the following components: Yeast β-glucan 0.1–80 parts; Lactoferrin 0.1–80 parts.

3. The composition according to claim 1, characterized in that, Based on parts by weight, it comprises the following components:

4. The composition according to any one of claims 1 to 3, characterized in that, The yeast β-glucan includes non-water-soluble or water-soluble yeast β-glucan; The lactoferrin includes lactoferrin and / or whey protein powder containing lactoferrin.

5. The composition according to any one of claims 1 to 4, characterized in that, The composition further includes inactive edible yeast, bovine colostrum powder, milk fat globule membrane whey protein, whey protein concentrate, yeast protein, soy protein isolate, elderberry powder, N-acetylneuraminic acid, lactic acid bacteria powder, 2'-fucosylated lactose, lactose-N-neotetrasaccharide, stachyose, taurine, vitamin C, or acceptable excipients.

6. The composition according to claim 5, characterized in that, The excipients include one or more of the following: dairy raw materials, dietary fiber, sweeteners, flavorings, fruit and vegetable powders, tricalcium phosphate, or magnesium stearate.

7. The composition according to claim 6, characterized in that, The dairy raw materials include one or more of whole milk powder, skim milk powder, or demineralized whey powder; The dietary fiber includes one or more of resistant dextrin, polydextrose, galactooligosaccharides, fructooligosaccharides, or isomaltooligosaccharides. The sweetener includes one or more of glucose, sorbitol, erythritol, maltitol, D-mannitol, isomaltitol, xylitol, monk fruit powder, steviol glycosides, or sucralose.

8. The use of the composition according to any one of claims 1 to 7 in any one of the following: (I) Prepare products that improve or enhance immunity; (II) Prepare products for the prevention, relief and / or treatment of upper respiratory tract infections.

9. The application as described in claim 8, characterized in that, The improvement or enhancement of immunity includes any of the following: (I) Increase the thymus / body weight ratio; (II) Increase the phagocytic index; (III) Inhibit the degree of ear swelling; (IV) Increase the number of hemolytic plaques; (V) Increases the half-life of hemolysis; (VI) Improve the phagocytic rate of macrophages; (VII) Increase the phagocytic index of macrophages; (VIII) Promotes ConA-induced transformation of mouse spleen lymphocytes; (IX) Enhance NK cell activity.

10. Products that improve or enhance immunity or prevent, alleviate, and / or treat upper respiratory tract infections, characterized in that, Includes the composition as described in any one of claims 1 to 7.

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