Composition for immune enhancement, immune improvement, or immune modulation comprising porcine brain enzymatic hydrolysate

Abstract

The present invention relates to a composition for immune enhancement, immune improvement, or immune modulation comprising a porcine brain enzymatic hydrolysate. The porcine brain enzymatic hydrolysate according to the present invention has an excellent immune enhancement, immune improvement, or immune modulation effect and exhibits no toxicity, and thus can be helpfully used as a health functional food composition, a pharmaceutical composition, or the like.

Description

Composition for enhancing immunity, improving immunity, or regulating immunity comprising enzymatic hydrolyzate of pig brain

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0012383, filed January 26, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for enhancing immunity, improving immunity, or regulating immunity, comprising an enzyme hydrolyzate of pig brain.

[0003] The immune system is a defense mechanism that protects the human body from various infectious agents entering from the outside, and is maintained through interactions between various immune cells such as white blood cells, macrophages, and natural killer cells.

[0004] In particular, macrophages are distributed throughout all tissues in the body and have the ability to remove unnecessary elements in the human body by performing phagocytosis on infectious pathogens such as bacteria and viruses as well as aging normal cells and cancer cells. In addition, macrophages play an important role as mediators that maximize secondary immune responses by presenting antigens and secreting various cytokines and physiologically active substances. Cytokines and physiologically active substances secreted by activated macrophages include IL-1β, IL-6, nitric oxide (NO), and TNF-α, which are known to exhibit direct toxicity against cancer cells.

[0005] Immunity is a resistance response against foreign substances such as pathogens, recognizing the presence of specific substances and eliminating them to maintain homeostasis in the body. Cell-mediated immunity involves T lymphocytes and natural killer cells (NK cells) secreting interferons, ILs, and TNF-alpha to remove foreign substances. Humoral immunity involves antibodies produced by B cells binding to antigens and eliminating foreign substances. The immune system is maintained through the interaction of these two immune mechanisms. Among the immunoglobulins (Ig) that play a crucial role in the immune response, IgG, in particular, plays a role in fighting bacteria in humoral immunity. Recent studies have reported that growth hormones and insulin-like growth factor (IGF)-1 affect immunoglobulin production levels. Since disease and infection primarily occur in conditions of compromised immune function, boosting immunity is crucial for maintaining health.

[0006] The present invention provides a food composition or health functional food composition for enhancing immunity, improving immunity, or regulating immunity, which contains a pig brain enzyme hydrolyzate.

[0007] The present invention also provides a pharmaceutical composition for enhancing immunity, improving immunity, or regulating immunity, which comprises a pig brain enzyme hydrolyzate.

[0008] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0009] The present invention provides a food composition or health functional food composition for enhancing immunity, improving immunity, or regulating immunity, which contains a pig brain enzyme hydrolyzate as an active ingredient.

[0010] In addition, the present invention provides a pharmaceutical composition for enhancing immunity, improving immunity, or regulating immunity, which contains pig brain enzyme hydrolyzate as an active ingredient.

[0011] The above pig brain enzymatic hydrolysate may be hydrolyzed by one or more protein-decomposing enzymes.

[0012] The above pig brain enzyme hydrolyzate may include at least one peptide consisting of sequence number 1 or sequence number 2.

[0013] The above peptide may be derived from pig brain enzyme hydrolyzate.

[0014] The above pig brain enzyme hydrolyzate can exhibit macrophage proliferation ability or TNF-α production ability.

[0015] The above pig brain enzyme hydrolyzate can increase natural killer cell activity.

[0016] The above composition can be formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquids and pills.

[0017] In addition, the present invention provides a method for improving memory, improving cognitive function, or preventing or treating a brain disease, which comprises a step of administering or ingesting to a subject a pharmaceutical composition containing a pig brain enzyme hydrolyzate as an active ingredient.

[0018] In addition, the present invention provides a pharmaceutical composition containing a pig brain enzyme hydrolyzate as an active ingredient for use in enhancing immunity, improving immunity, or regulating immunity.

[0019] The pig brain enzyme hydrolyzate according to the present invention has excellent immune enhancement, immune improvement, and immune regulation effects and does not exhibit toxicity, so it can be usefully used as a health functional food composition, a pharmaceutical composition, etc.

[0020] Figure 1 is a process diagram schematically showing a manufacturing process of a pig brain enzyme hydrolyzate according to one embodiment of the present invention.

[0021] Figure 2 is a graph showing the results of an analysis of the macrophage proliferation ability of a pig brain enzyme hydrolyzate according to one embodiment of the present invention (*, p<0.05; **, p<0.01; ***, p<0.001).

[0022] Figure 3 is a graph showing the results of analysis on the TNF-α production ability of pig brain enzyme hydrolyzate according to one embodiment of the present invention (*, p<0.05; **, p<0.01; ***, p<0.001).

[0023] FIG. 4 is a graph showing the results of confirming the relationship between the enzymatic hydrolyzate of pig brain and body weight change according to one embodiment of the present invention (control: control group, PBEH-L: low-concentration enzymatic hydrolyzate of pig brain administration group, PBEH-M: medium-concentration enzymatic hydrolyzate of pig brain administration group, PBEH-H: medium-concentration enzymatic hydrolyzate of pig brain administration group, Positive: positive control group, Normal: normal control group).

[0024] FIG. 5 is a graph showing the effect of enzymatic hydrolysate of pig brain according to one embodiment of the present invention on changes in cytokine concentrations in spleen cells induced by LPS (control: control group, PBEH-L: group administered low concentration enzymatic hydrolysate of pig brain, PBEH-M: group administered medium concentration enzymatic hydrolysate of pig brain, PBEH-H: group administered medium concentration enzymatic hydrolysate of pig brain, Positive-control: positive control group, Normal-congtriol: normal control group; subscripts (a, b, c, d) indicate significant differences between groups using Tukey's test at p < 0.05). The right vertical axis of FIG. 5 represents the Ratio IFN-γ / IL-4.

[0025] Figure 6 is an image analyzing the effect of administration of pig brain enzyme hydrolyzate according to one embodiment of the present invention on the spleen.

[0026] Figure 7 is a graph showing the results of confirming the effect of administration of pig brain enzyme hydrolyzate according to one embodiment of the present invention on the spleen through changes in spleen damage scores.

[0027] Hereinafter, the present invention will be described in detail.

[0028] The present invention provides a composition for enhancing immunity, improving immunity, or regulating immunity, which contains a pig brain enzyme hydrolyzate as an active ingredient.

[0029] The above composition may be a food composition, a health functional food composition, or a pharmaceutical composition.

[0030] The above pig brain enzymatic hydrolysate may be hydrolyzed by one or more protein-decomposing enzymes.

[0031] More preferably, the hydrolyzate can be produced by primary hydrolysis and secondary hydrolysis.

[0032] The above primary hydrolysis can be performed by adding a protein-decomposing enzyme to a solid solution (pig brain sludge solution), and can be performed at 40±20°C for 1 to 20 hours, but is not limited thereto.

[0033] The above secondary hydrolysis can be performed by adding a protein-decomposing enzyme to the solid solution that has undergone the primary hydrolysis, and can be performed at 40±20°C for 1 to 20 hours. However, it is not limited thereto.

[0034] The above pig brain enzyme hydrolyzate may include at least one peptide consisting of sequence number 1 or sequence number 2.

[0035] The above peptide may be included in the pig brain enzymatic hydrolysate at a concentration of 0.1 ppm to 300 ppm. More preferably, it may be included at a concentration of 1 to 30 ppm.

[0036] The above peptide may be derived from pig brain enzyme hydrolyzate.

[0037] The above pig brain enzyme hydrolyzate can exhibit macrophage proliferation ability or TNF-α production ability.

[0038] The above pig brain enzyme hydrolyzate can increase natural killer cell activity.

[0039] The above composition can be formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquids and pills.

[0040]

[0041] As used herein, “enhancement,” “improvement,” and “modulation” refer to any act of improving or beneficially altering the immune status by administration of the composition of the present invention.

[0042]

[0043] In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. The health functional food composition of the present invention has the advantage of not having side effects that may occur with long-term administration of liquid or powder formulation drugs, and is highly portable, so it can be taken as a supplement for enhancing immunity, improving immunity, or regulating immunity.

[0044] In the food composition or health functional food composition according to the present invention, the pig brain enzyme hydrolyzate may be included in an amount of 1% to 20% (weight %) based on the total weight of the composition, but is not necessarily limited thereto, and the mixing amount of the effective ingredient may be appropriately determined depending on each purpose of use, such as prevention, health, or treatment.

[0045] The formulation of health functional foods can be in the form of powders, granules, pills, tablets, capsules, or any other form of general food or beverage.

[0046] There is no particular limitation on the types of the above foods, and examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense can be included.

[0047] In general, when manufacturing food or beverages, in the case of liquid form, the effective ingredient may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and the present invention may also be used in an amount greater than the above range, as there is no problem in terms of safety.

[0048] Among the functional foods according to the present invention, the beverage may contain various flavoring agents or natural carbohydrates as additional ingredients, just like conventional beverages. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame may be used. The proportion of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0049] In addition to the above, the health functional food composition according to the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the health functional food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the functional food composition of the present invention.

[0050]

[0051] The pharmaceutical composition according to the present invention does not particularly limit the content thereof as long as it contains the above-mentioned effective ingredient, but preferably, the pig brain enzyme hydrolyzate may be contained in an amount of 1 to 50 wt%, more preferably 5 to 30 wt%, based on the total weight of the composition. However, the present invention is not limited thereto. In this case, if the effective ingredient is contained below the above-mentioned concentration range, there is a problem that it is difficult to exhibit a desirable preventive or therapeutic effect, and if the concentration range is exceeded, the change in the expected effect may be minimal.

[0052]

[0053] The pharmaceutical composition according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods, and may include appropriate carriers, excipients, or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0054] The carrier or excipient or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0055] When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0056] Solid preparations for oral administration can be prepared by mixing the above jasmone with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0057] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0058] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerol gelatin.

[0059] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, body weight, severity of the disease, drug form, route of administration, and duration of administration, but can be appropriately selected by those skilled in the art. For example, the dosage may be 0.0001 to 2,000 mg / kg per day, more preferably 0.001 to 2,000 mg / kg. The dosage may be administered once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0060] The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0061] Hereinafter, the present invention will be described in more detail through examples. The objectives, features, and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the examples described herein and may be embodied in other forms. The examples introduced herein are provided to ensure that those skilled in the art can sufficiently convey the spirit of the present invention. Therefore, the present invention should not be limited by the following examples.

[0062]

[0063] Example 1. Preparation of enzymatic hydrolyzate of pig brain

[0064] After thawing the pig brain in a defrosting device, the pig brain is bled and foreign substances are removed with tap water. The blood-free pig brain is crushed in a blender to facilitate defatting. The crushed pig brain is defatted with alcohol (ethanol, etc.) to remove fat and foreign substances. The defatted pig brain is subjected to primary enzymatic hydrolysis with a proteolytic enzyme (pepsin) for 1 to 20 hours. After the proteolytic enzyme (pepsin) is inactivated, and after the primary enzymatic hydrolysis is completed, filtration is performed for solid-liquid separation. After filtration, a proteolytic enzyme (pancreatin) is added to the primary hydrolyzed pig brain and secondary enzymatic hydrolysis is performed for 1 to 20 hours. After the secondary enzymatic hydrolysis is completed, the proteolytic enzyme is inactivated by heating, and the pig brain enzymatic hydrolyzed pig brain is subjected to secondary solid-liquid separation. Afterwards, 0.01 to 5 times (w / w) alcohol (ethanol) was added to the filtrate of the pig brain hydrolyzate, left for 1 to 30 hours, and filtered. The filtrate was concentrated, and the sterilized filtered liquid was divided into small portions and sterilized. The filtrate, which had undergone high-pressure steam sterilization, was placed in a freeze dryer and freeze-dried to ultimately obtain a high-purity pig brain enzymatic hydrolyzate (see Fig. 1).

[0065]

[0066] Experimental Example 1. Identification of Peptides in Enzymatic Hydrolyzate of Pig Brain

[0067] The pig brain enzymatic hydrolyzate obtained in Example 1 was diluted to 1 / 2 by adding 100 ㎕ of DW to 100 ㎕ of the sample. The diluted solution was diluted to 1 / 20 by adding 950 ㎕ of DW to 50 ㎕, and the solution filtered through a syringe filter was subjected to sequence analysis using the HPLC system of ACQUITY UPLC I-Class PLUS (Waters) and the ms / ms ionization analysis of the Xevo TQ-XS (Waters) MS system.

[0068] <Analysis Conditions>

[0069] - Column: ACQUITY UPLC® BEH C18 (2.1 x 50 mm, 1.7 μm, Waters)

[0070] - Flow rate: 200 ㎕ / min

[0071] - Mobile phase A: H2O / FA=100 / 0.1(v / v)

[0072] Mobile phase B: Acetonitrile / FA = 100 / 0.1 (v / v)

[0073] - Gradient

[0074]

[0075] Minutes (min)A (%)B (%)0982598215841616703018595275952998235982

[0076] The results obtained by analyzing under the above device conditions are as follows. As a result of the analysis, a total of four peptides were analyzed, and among them, the following two peptides were identified as pig brain-derived peptides that showed the most stable reproducibility.

[0077] Peptide sequence derived from enzymatic hydrolysate of pig brain No. m / z RT (min) Charge Sequence Content in hydrolysate (ppm) brPEP-1403.227.61PSIS (SEQ ID NO. 1) 5-30 ppm brPEP-2418.728.42GPAGPQGPR (SEQ ID NO. 2) 5-30 ppm

[0078] A peptide having the same mass and ms / ms ionization pattern as the peptide identified from the above pig brain hydrolysate was synthesized from Anygen (www.anygen.com) and confirmed to be identical to the peptide present in the pig brain enzymatic hydrolysate, and subsequent experiments were conducted.

[0079] Experimental Example 2. Analysis of the immune-enhancing efficacy of hydrolysates containing pig brain-derived peptides (measurement of immune-enhancing factors in an in vitro model)

[0080] The immunostimulating effect was analyzed using the pig brain enzymatic hydrolysate (containing peptides of SEQ ID NO: 1 and SEQ ID NO: 2) prepared in Example 1. The content of the peptides in the pig brain enzymatic hydrolysate is 5 to 30 ppm, respectively.

[0081]

[0082] 2-1. Macrophage proliferation capacity

[0083] To investigate the effect on macrophage proliferation rate, MTT assay was used. Mouse macrophage cells, Raw264.7, were cultured at 2.5×10 4 After stabilizing by seeding at a concentration of cells / 1 ml / well, the sample was treated and then cultured for 24 hours at 37°C, 5% CO2 to evaluate the cell proliferation rate. 40 μl of MTT solution was added to 360 μl of cell culture medium and cultured for 4 hours. After dissolving the purple Crystal in DMSO solution, the absorbance value was measured at a wavelength of 450 nm using an ELISA reader (TECAN, Infinite M200 pro). The control group was treated with distilled water, a solvent in which the pig brain enzymatic hydrolysate was dissolved, without treating the sample (pig brain enzymatic hydrolysate), and the sample was treated to a final concentration of pig brain enzymatic hydrolysate (3.9–500 μg / mL), and the sample was treated at a ratio of 1% to the medium. At this time, the calculation was performed based on the control group as 100%.

[0084] As a result of the experiment, it was confirmed that the cell proliferation rate increased compared to the untreated group at all treatment concentrations when treated with the pig brain enzymatic hydrolyzate at various concentrations (3.9–500 μg / mL). This confirmed that the pig brain enzymatic hydrolyzate has the effect of promoting the proliferation of macrophages in a concentration-dependent manner without exhibiting cytotoxicity, and in particular, it was confirmed that the result showed an increase rate of approximately 38% compared to the control group at 250 μg / mL (Fig. 2).

[0085]

[0086] 2-2. TNF-α production ability

[0087] Raw264.7 cells, a mouse macrophage cell line, were seeded in a 48-well plate at 2.5 × 10 4 The cells were dispensed and cultured at a concentration of 500 μl / well. After processing the sample, it was cultured for 48 hours at 37°C and 5% CO2, and the culture supernatant was collected and the concentration of TNF-α (tumor necrosis factor-α) was measured using an ELISA kit.

[0088] When the enzymatic hydrolysate of pig brain was treated at concentrations ranging from 15.6 to 500 μg / mL, changes in TNF-α secretion were observed. A tendency toward an increase in TNF-α secretion was observed in all treatment groups. In particular, a significant increase of approximately 16% was observed at a concentration of 250 μg / mL. These results suggest that the enzymatic hydrolysate of pig brain has the potential to enhance immune activity (Fig. 3).

[0089]

[0090] Experimental Example 3. Confirmation of the immune-enhancing efficacy of pig brain enzyme hydrolyzate in an immunocompromised animal model.

[0091] The immune-enhancing efficacy was evaluated using a cyclophosphamide-induced immunosuppressed animal model.

[0092] laboratory animals

[0093] Five-week-old, specific-pathogen-free (SPF) Wistar rats were obtained from Samtako Biokorea (Osan, Korea) and used in the experiment. During the rearing period, the rats were fed a regular solid diet (Samtako, Gyunggi, Korea). During the acclimation period, the rats were allowed to drink filtered drinking water ad libitum, which was replaced daily. During the rearing period, the temperature was 23±1℃, the humidity 50±5%, the noise level was 60 pons or less, the lighting time was 08:00~20:00 (12 h per day), the illumination intensity was 150~300 Lux, and the ventilation rate was 10~12 times per hour. This experiment was performed in compliance with the Animal Experiment Ethics Regulations (Approval from the Animal Ethics Committee: HSUIACUC-23-023).

[0094]

[0095] Immunodeficiency-induced model

[0096] Cyclophosphamide (CTX), known as an immunosuppressant, was purchased from Sigma-Aldrich (St. Louis, USA). This experiment was conducted at a dose of 5 mg / kg, which was identified as the most appropriate CTX dose for the test based on previous research. In this experiment, CTX, an immunosuppressant, was administered simultaneously with the test substance, and the dose was administered via forced oral gavage at a dose of 5 mL / kg.

[0097]

[0098] Experimental group setting and sample administration

[0099] During the breeding period, the diet was a general solid feed (Samtako, Gyunggi, Korea). After the acclimatization period, the experimental animals were separated using the egg block method to ensure a uniform average value between groups based on the blood leukocyte count obtained through a general blood analysis, and then individually identified using an ear punch. The experimental groups were divided into the normal control group, control group, low-concentration pig brain enzyme hydrolysate administration group (PBEH-L; 6 mg / kg), medium-concentration administration group (PBEH-M; 13 mg / kg), high-concentration administration group (PBEH-H; 26 mg / kg), and positive control group, and 10 animals were used in the experiment. The doses were 26 mg / kg bw, 13 mg / kg bw, and 6 mg / kg bw for rats based on the results of a previous study. (This corresponds to 250 mg / 60 kg, 125 mg / 60 kg, and 62.5 mg / 60 kg bw in humans.)

[0100]

[0101] Test group feed Immunodepression-inducing substance Oral administration Substance Administration Dose and method Administration period Normal control group Standard feed (casein 26 mg / kg BW) Immunodepression-inducing substance not treated Vehicle Oral 4 weeks Control group Cyclophosphamide (5 mL / kg, oral administration once) Vehicle Oral PBEH-L Pig brain enzyme hydrolyzate 6 mg / kg Oral administration (PBEH and CTX administered sequentially PBEH-M Pig brain enzyme hydrolyzate 13 mg / kg PBEH-H Pig brain enzyme hydrolyzate 13 mg / kg Positive control Red ginseng extract 100 mg / kg BW Oral administration (Red ginseng extract and CTX administered sequentially)

[0102] 3-1. Biomarker Measurement

[0103] 1) Weight and dietary intake

[0104] Weekly body weight changes, food intake, and fasting blood glucose were measured for each individual in all experimental groups at a set time once a week. Autopsies were performed under inhalation anesthesia, and blood samples were collected from the vena cava for analysis. Thymus and spleen tissues were removed, weighed, and spleen tissue was used for subsequent analysis.

[0105] The analysis results showed that the normal control group showed significantly greater weight gain than the control group, and the pig brain enzymatic hydrolyzate showed higher weight gain than the control group (Fig. 4). In addition, food intake was reduced in immunocompromised animals compared to the normal control group, and it was confirmed that the PBEH-L and PBEH-M groups increased as much as the normal control group (Table 4).

[0106]

[0107] Weight gain, food intake, tissue weight, blood sugar measurementWeight gain (g)Food intake (g)Speen (g)Thymus (g)Blood sugar (mg / dL)Control 175.4±20.3 c 24±1.7 b 672.7±199.7 b 540.5±198.6 b 122.1±14.4 b PBEH-L203.3±19.5 b 26±0.9 a 789.3±133.1 b 576.9±200.6 b 135.1±20.3 ab PBEH-M185.4±20.4 bc 25.1±0.6 ab 738.3±135.1 b 501.3±62.1 b 126.3±19 b PBEH-H193.9±21.4 b 25±0.8 ab 803.6±153 b 526±115.7 b 127.3±21.8 b Positive178.7±24 c 24.1±1.2 b 755.9±201.1 b503.7±124.7 b 135.1±18.3 ab Normal221.5±19.7 a 26.5±1.1 a 1098.3±206 a 734.8±123.6 a 138.3±22.7 a

[0108] Values ​​are expressed as mean ± standard deviation (n = 10). Superscripts (a, b, c) indicate significant differences between groups using Tukey's test at p < 0.05.

[0109] 2) Weight of immune function-related organs

[0110] Spleen and thymus weights, which are related to immune function, were significantly reduced in the control group compared to the normal control group, and spleen showed a tendency to increase in the pig brain enzyme hydrolyzate intake group (Table 4).

[0111]

[0112] 3) Blood sugar

[0113] Fasting blood glucose levels were measured using a blood glucose meter using tail blood draws. The control group had lower levels than the normal control group, while both the enzymatic hydrolyzed pig brain and the positive control group had higher levels than the control group (Table 4).

[0114]

[0115] 3-2. Hematological changes

[0116] Blood was collected from the abdominal vena cava after inhalation anesthesia and divided into EDTA and conical tubes for analysis. For routine hematological tests, blood was placed in EDTA tubes (DB Caribe, Ltd., USA) and spun on a roll mixer for approximately 30 minutes. White blood cells, lymphocytes, and granulocytes were measured using a hematological analyzer (Hemavet 950Fs, Drew Scientific Inc., TX, USA). The blood collected in the conical tube was subsequently used for cytokine analysis.

[0117] WBCGranulocyteslymphocyteMIDControl2.82±0.55 b 1.17±0.22 b 1.51±0.54 c 0.13±0.04 b PBEH-L3.11±1.01 b 1.1±0.46 b 1.88±0.61 bc 0.13±0.03 b PBEH-M3±0.51 b 1.09±0.25 b 1.78±0.45 bc 0.13±0.03 b PBEH-H3.13±0.73 b 1.08±0.25 b 1.91±0.5 b 0.13±0.04 b Positive3.18±0.95 b 1.12±0.31 b 1.93±0.73 b 0.12±0.03 b Normal7.05±1.86 a 1.65±0.53 a 5.18±1.55 a 0.22±0.08 a

[0118] Values ​​are expressed as mean ± standard deviation (n = 10). Superscripts (a, b, c) indicate significant differences between groups using Tukey's test at p < 0.05.

[0119] White blood cell analysis using a blood analyzer revealed a significant decrease in WBCs in the control group compared to the normal control group. Granulocytes, a component of WBCs, were also significantly reduced in the control group compared to the normal control group.

[0120] Lymphocytes were significantly lower in the control group than in the normal control group, but showed a tendency to increase in the positive control group and PBEH-H group compared to the control group. MID (middle corpuscles) is correlated with monocytes, eosinophils, and basophils, and was included less in the control group than in the normal control group, and there was no difference between the positive control group and the pig brain enzymatic hydrolysate and the control group.

[0121]

[0122] 3-3. Analysis of natural killer cell activity

[0123] 5×10 spleen cells (Effector cells) in a 96-well plate 6 cells / ml and AR42J cells (target cells) were added at a ratio of effector cells and target cells (1:20) and cultured for 24 hours in a 37°C, 5% CO2 incubator. After culture, LDH was measured using a CytoTox detection kit (Takara), and formazan formed by the oxidation of NAD in the reaction solution was measured for absorbance at 490 nm and compared with the control group.

[0124]

[0125] NK Cell activityNK cell activity (%)Control51.4±9.1 c PBEH-L81.7±±11.5 b PBEH-M73±15.5bc PBEH-H93.6±15 b Positive76.6±7.6 b Normal100±14.4 a

[0126] Values ​​are expressed as mean ± standard deviation (n = 10). Superscripts (a, b, c) indicate significant differences between groups using Tukey's test at p < 0.05.

[0127] NK cell activity decreased by approximately 50% in the control group compared to the normal control group, but significantly increased in the pig brain enzymatic hydrolysate group compared to the control group. In particular, the PBEH-H group was confirmed to have increased compared to the positive control group and recovered to a level almost similar to the normal control group.

[0128]

[0129] 3-4. Analysis of splenocyte and B cell proliferation capacity

[0130] To investigate the effect on the proliferation rate of immune cells, the WST-1 assay kit (ITSBio, Seoul, Korea) was used. The isolated splenocytes were 3 × 10 6 The proliferation rate of splenocytes was evaluated by culturing them for 48 hours at a concentration of 3 × 10 cells / well. 6 B cell proliferation was evaluated by treating splenocytes at a concentration of cells / well with LPS and culturing them at 37°C and 5% CO2 for 48 hours. After adding 10% volume of tetrazolium (WST, Sigma-Aldrich) to each well and incubating for 2 hours, the cell activity was compared by measuring the optical density (OD) at 405 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

[0131]

[0132] Cell proliferation assay (%) = (absorbance of sample treatment group / absorbance of control group) × 100

[0133]

[0134] In a state where inflammation was not induced by LPS, the proliferation rate of splenocytes was reduced by approximately 20% in the control group compared to the normal control group, but the pig brain enzyme hydrolyzate-treated group showed a dose-dependent increase and a proliferation rate of splenocytes similar to that of the positive control group and the normal control group.

[0135] When spleen cells were stimulated with LPS, the proliferation rate of spleen cells in the control group decreased by approximately 28% compared to the normal control group, and it was confirmed that the pig brain enzyme hydrolyzate treatment group and the positive control group showed a higher proliferation rate than the control group (Table 7).

[0136] Measurement of proliferation capacity of splenocytes and B cells Splenocyte proliferation rate (%) B cell proliferation rate (%) Control 51.4±9.1 c 72.4±11.5 b PBEH-L81.7±11.5 b 79.1±7.5 b PBEH-M73±15.5 bc 75.2±11.5 b PBEH-H93.6±15 b 89.6±7.6 a Positive76.6±7.6 b 87.4±10 a Normal100±14.4 a 100±16.7 a

[0137] Values ​​are expressed as mean ± standard deviation (n = 10). Superscripts (a, b, c) indicate significant differences between groups using Tukey's test at p < 0.05.

[0138] 3-5. Cytokines analysis in the spleen

[0139] 4 × 10 of the spleen cells obtained in 3-1 above 6After culturing in a 48-well plate at a cell / well concentration, LPS was treated at a concentration of 1 μg / mL. Afterwards, the cells were cultured for 24 hours at 37°C and 5% CO₂, and the culture fluid was collected, and the concentrations of IFN-γ and IL-4 were measured using an ELISA kit.

[0140] As a result of the test, in the spleen, the IFN-γ concentration was higher in the normal control group than in the control group, and in the PBEH group, the concentration increased in a dose-dependent manner. In particular, the PBEH-H group showed an IFN-γ concentration similar to the normal control group. The IL-4 concentration was higher in the control group than in the normal control group, and it was confirmed that the concentration was significantly lower in the PBEH-H group than in the control group. In addition, the Th1 / Th2 ratio calculated using the IFN-γ and IL-4 concentrations was higher in the normal control group than in the control group, and the PBEH-H group showed a result that the concentration was significantly higher than in the control group, confirming that it regulates the Th1 / Th2 ratio (Fig. 5).

[0141]

[0142] 3-6. Histological analysis of the spleen

[0143] Spleen tissue was first washed with saline and then fixed by sequential perfusion with 4% paraformalide solution (pH 7.2). The fixed tissue was embedded in paraffin, sectioned at 5 μm thickness, and stained with H&E to assess cell damage, cell aggregation, viable cells, and red pulp congestion.

[0144] As a result, the cell damage in the white pulp part of the spleen showed a higher score in the normal control group than in the control group, and the positive control group decreased to a level similar to that of the normal control group. It was also confirmed that the white pulp part of the spleen in the group that consumed the enzymatic hydrolyzate of pig brain also decreased in a dose-dependent manner (Fig. 6, Fig. 7).

[0145] On the other hand, the congestion of the red pulp area showed a higher score in the control group than in the normal control group, indicating that the congestion of the red pulp area was high. Although the congestion of the red pulp area was not alleviated in the positive control group, it was confirmed that PBEH-M and PBEH-H alleviated the congestion of the red pulp area.

[0146]

[0147] Project Information Project Number G0203100-02 Ministry Name Ministry of Agriculture, Food and Rural Affairs Research Management Specialist Organization Korea Food Research Institute Research Project Name Food Functionality Evaluation Support Project Research Project Name Effects and Mechanism of Pig Brain Enzyme Hydrolysate on Immunity Enhancement Research Lead Organization Unimed Pharmaceutical Co., Ltd. Research Period 2023.04~23.12

Claims

1. A food composition for enhancing immunity, improving immunity, or regulating immunity, containing pig brain enzyme hydrolyzate as an active ingredient.

2. In claim 1, A food composition characterized in that the above pig brain enzymatic hydrolyzate is hydrolyzed by one or more protein-decomposing enzymes.

3. In claim 1, A food composition characterized in that the above pig brain enzyme hydrolyzate comprises at least one peptide consisting of sequence number 1 or sequence number 2.

4. In claim 1, A food composition characterized in that the above peptide is derived from a pig brain enzyme hydrolyzate.

5. In claim 1, A food composition characterized in that the above pig brain enzyme hydrolyzate exhibits macrophage proliferation ability or TNF-α production ability.

6. In claim 1, A food composition characterized in that the above pig brain enzyme hydrolyzate increases natural killer cell activity.

7. In claim 1, A food composition characterized in that the above pig brain enzyme hydrolyzate increases natural killer cell activity.

8. In claim 1, A food composition characterized in that the composition is formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquids, and pills.

9. A pharmaceutical composition for enhancing immunity, improving immunity, or regulating immunity, comprising pig brain enzyme hydrolyzate as an active ingredient.

10. In claim 9, A pharmaceutical composition characterized in that the pig brain enzyme hydrolyzate comprises at least one peptide consisting of sequence number 1 or sequence number 2.

11. A method for improving memory, improving cognitive function, or preventing or treating brain diseases, comprising a step of administering or ingesting to a subject a pharmaceutical composition containing pig brain enzyme hydrolyzate as an active ingredient.

12. Use of a pharmaceutical composition containing pig brain enzyme hydrolyzate as an active ingredient for enhancing immunity, improving immunity, or regulating immunity.

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