Functional nutritional composition and use thereof

Abstract

A functional nutritional composition. The composition comprises the following two necessary components: i) casein phosphopeptides, and ii) Bifidobacterium probiotics. In the composition, the ratio of i) the casein phosphopeptides to ii) the Bifidobacterium probiotics is configured as follows: an effective quantity of the casein phosphopeptides : an effective quantity of the Bifidobacterium probiotics is (50-380 mg):(1.00*109-1.20*1011 CFU). Compared with casein phosphopeptides or Bifidobacterium probiotics alone, the functional nutritional composition can enhance immunity more effectively and achieve a better anti-oxidation effect.

Description

Functional nutritional composition and use thereof Technical Field

[0001] The present invention belongs to the technical field of functional nutrient research, and specifically relates to a functional nutrient composition and uses of the composition, and more specifically relates to a functional nutrient composition and uses thereof in helping to enhance immunity and anti-oxidation. Background Art

[0002] Casein phosphopeptides (CPPs) are natural physiologically active peptides containing phosphoserine clusters (SerP) obtained by hydrolyzing bovine milk casein with single or combined proteases and then separating and purifying. Their structural characteristics are that they contain a common core site SerP-SerP-SerP-Glu-Glu. The main source of CPP is α s1 -, α s2 - and β-caseins contain 9, 12, and 5 phosphoserine residues, respectively, in bovine milk.

[0003] CPPs have three negatively charged amino acid sequences and two glutamic acid residues, which enable them to chelate divalent metal cations. This prevents the precipitation of calcium, iron, zinc, and magnesium in the alkaline environment of the digestive tract, making the mineral ions more readily absorbed and utilized (Reference 1). Numerous in vitro cell culture and animal studies have demonstrated that CPPs can enhance the absorption and utilization of calcium and zinc (Reference 2). Human studies have also confirmed that the addition of CPPs to infant rice-based cereals can enhance calcium and zinc absorption (Reference 3). Smialowska et al. demonstrated that CPPs bind more ferrous ions than cow's milk proteins, and that the iron-CPP complex is more resistant to enzymatic degradation (Reference 4). Studies have shown that CPPs increase magnesium transport across the Caco-2 cell monolayer, and this effect is amplified when magnesium and calcium are co-administered (Reference 5). Furthermore, a diet supplemented with magnesium and CPPs has positive effects on femoral length, serum parathyroid hormone levels, and urinary deoxypyridinoline in rats (Reference 6).

[0004] The ability of CPPs to bind divalent metal ions gives them a certain ability to stabilize free radicals. Chiu et al. found that CPPs produced by trypsin digestion reduced the formation of secondary oxidation products and stabilized free radicals in aqueous solution (reference 7). Recent studies have found that CPPs have antioxidant activity in intestinal and osteoblastic cells in the presence of a reagent that induces peroxyl radicals or ferrous chloride (reference 8).

[0005] In addition, CPPs have been shown to promote splenocyte proliferation and immunoglobulin production. Co-culturing β-casein (1-28) with various cultured cell lines of T cells, B cells, and monocytes induces cell proliferation and IgA production (Reference 9). Furthermore, CPPs induce the expression and production of anti-inflammatory cytokines, including IL-6 and IL-10, in splenocytes (Reference 10).

[0006] Therefore, CPPs are a series of bioactive peptides embedded in food-derived animal protein sequences that, upon enzymatic release, exhibit a variety of unique functions. They have been approved by China's National Health Commission as food fortifiers, and the national food safety standard GB14880-2012 permits their use in infant formula, grains and grain products, and beverages. Countries like Japan and Germany have incorporated CPPs into foods as functional ingredients, and China has also used them as a food fortifier in infant formula and health foods. Prior art also supports the use of CPPs in calcium-supplementing functional foods or health supplements.

[0007] Furthermore, probiotics are known to be important symbiotic bacteria in the human body. Depending on the strain and species, they possess various physiological functions, such as regulating digestive tract flora, digestive tract function, and enhancing immunity. In recent years, formula milk powders containing probiotics have become a hot topic among consumers. The main added strains are bifidobacteria and lactobacilli, aiming to improve the intestinal environment and immune system.

[0008] Bifidobacterium longum subsp. longum BB536 (BB536) is a member of the Bifidobacterium family and a component of the human intestinal flora. This strain has been approved for use in infant formula in the United States and Japan, and was approved by the National Health Commission for use in infant formula in May 2022. During pregnancy and in newborns, the immune system tends to favor a thymus-derived lymphocyte helper cell (Th2) response. While postnatal immune system maturation gradually develops with age, the development of a Th1 immune response can reset the Th1 / Th2 balance. Establishing a balanced Th1 / Th2 immune response is crucial for early immune development (Reference 11). Reference 12: To demonstrate the effect of BB536 on the development of immune responses in healthy infants, a double-blind, randomized, placebo-controlled intervention trial was conducted. The study found that fortifying infant formula with BB536 significantly altered the composition of the intestinal flora. Infants supplemented with BB536 had significantly higher levels of Bifidobacteria in the gut compared to the control group. BB536 also enhanced the immune response to vaccination in healthy full-term infants, beneficially impacting the Th1 / Th2 balance. The study used interferon-γ (IFN-γ) and interleukin-4 (IL-4) as representative cytokines for Th1 and Th2 cells, respectively, and used the IFNγ / IL-4 ratio to investigate the balance of Th1 and Th2 responses. The results showed that the proportions of IFN-γ-secreting cells and IFN-γ / IL-4-secreting cells increased in the BB536-supplemented group compared with the control group, and that a higher ratio of Bifidobacteriaceae to Enterobacteriaceae in infancy was associated with an increase in IFN-γ-secreting cells and IFN-γ / IL-4 in later life. These results suggest that BB536 has a positive effect on enhancing Th1 immune responses.

[0009] In addition, cited document 13 discloses an edible composition containing probiotics and casein phosphopeptide with digestive effects, but its effects or uses only relate to the effects on digestibility.

[0010] References:

[0011] Cited literature 1: Zidane, F., Mat′eos, A., Cakir-Kiefer, C., et al. Binding of divalent metal ions to 1–25β-caseinophosphopeptide: An isothermal titration calorimetry study. Food Chemistry, 2012, 132(1): 391-398.

[0012] Citation 2: Tsuchita, H., Suzuki, T., Kuwata, T. The effect of casein phosphopeptides on calcium absorption from calcium-fortified milk in growing rats. The British Journal of Nutrition, 2001, 85(1): 5-10.

[0013] Citation 3: Hansen, M., Sandstrom, B., Jensen, M., et al. Casein phosphopeptides improve zinc and calcium absorption from rice-based but not from whole-grain infant cereal. Journal of Pediatric Gastroenterology and Nutrition, 1997, 24(1): 56-62.

[0014] Citation 4: Smialowska, A., Matia-Merino, L., Carr, A. J. Assessing the iron chelation capacity of goat casein digest isolates. Journal of Dairy Science, 2017, 100(4), 2553-2563.

[0015] Citation 5: Cao, Y., Miao, J., Liu, G., et al. Bioactive peptides isolated from casein phosphopeptides enhance calcium and magnesium uptake in Caco-2 cell monolayers. Journal of Agricultural and Food Chemistry, 2017, 65(11): 2307-2314.

[0016] Citation 6: Liu, G., Miao, J., Sun, S., et al. The effect of dietary magnesium and casein phosphopeptides on bone metabolism in rats. Food & Function, 2017, 8(12): 4487-4495.

[0017] Citation 7: Chiu, S.C., Kitts, D.D. Antioxidant characterization of casein phosphopeptides from bovine milk. In Nutraceutical Beverages, 2003, 871: 279-289.

[0018] Citation 8: Bottani, M., Cattaneo, S., Pica, V., et al. In vitro antioxidant properties of digests of hydrolyzed casein and casein phosphopeptide preparations in cell models of human intestine and osteoblasts. Journal of Functional Foods, 2020, 64: 103673.

[0019] Citation 9: Kawahara, T., Katayama, D., Otani, H. Effect of beta-casein(1–28)on proliferative responses and secretory functions of human immunocompetent cell lines. Bioscience Biotechnology and Biochemistry, 2004, 68(10): 2091-2095.

[0020] Cited literature 10: Mullaiselvan, I., Kanagaraj, V., Sekar, S., et al. Studies on immunomodulatory effect of casein phospho peptide isolated from cultured dairy product. International Research Journal of Pure and Applied Chemistry, 2020, 21(23): 28-40.

[0021] Cited literature 11: Levy, O. Innate immunity of the newborn: basic mechanisms and clinical correlates. Nature Reviews Immunology, 2007, 7(5): 379-390.

[0022] Cited literature 12: Wu, BB, Yang, Y., Xu,

[0023] Reference 13: CN 110897166 A

[0024] Summary of the Invention

[0025] Problems to be solved by the invention

[0026] Based on the above existing research on casein phosphopeptides and probiotics, however, most of these studies are still limited to cell experiments on individual components. Therefore, even when examining animal experiments based on some mechanisms, further favorable conclusions may not be drawn. In addition, research on the synergistic effects of the two and possible (other) synergistic mechanisms has not yet been attempted.

[0027] After extensive further research, the present invention unexpectedly discovered that casein phosphopeptides and Bifidobacterium probiotics, when combined in a specific ratio, have a synergistic effect in enhancing immunity and / or anti-oxidation. Furthermore, the improvement of human immunity described in the present invention is achieved by promoting an increase in serum complement C3; and the improvement of human antioxidant capacity is achieved by improving the antioxidant capacity of the spleen. Such a synergistic effect has not been reported to date.

[0028] Therefore, the present invention aims to provide a safe and effective composition and its use, combining CPP, a component of breast milk, with long-term edible Bifidobacterium probiotics in an appropriate ratio to achieve a synergistic immune-enhancing and / or antioxidant effect. Furthermore, the aforementioned effects in the present invention are non-therapeutic.

[0029] Solutions for solving problems

[0030] [1] A functional nutritional composition for use in preparing a composition or food having the effect of improving human immunity and / or enhancing human antioxidant capacity, wherein the functional nutritional composition comprises the following two essential components:

[0031] i) casein phosphopeptides,

[0032] ii) Bifidobacterium probiotics;

[0033] Furthermore, in the functional nutritional composition, the ratio of i) casein phosphopeptide to ii) Bifidobacterium probiotics is configured as follows:

[0034] Effective amount of casein phosphopeptide: The effective amount of Bifidobacterium probiotics is (50mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU).

[0035] [2] The use according to [1], wherein the effective amount of casein phosphopeptide is: the effective amount of Bifidobacterium probiotics is (120mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU); the functional nutritional composition has the effect of improving human immunity and enhancing human antioxidant capacity.

[0036] [3] The use according to [1], wherein the improvement of human immunity is achieved by promoting the increase of serum complement C3; the improvement of human antioxidant capacity is achieved by providing spleen antioxidant capacity.

[0037] [4] The use according to any one of [1] to [3], wherein the Bifidobacterium probiotic comprises any one or more of Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum and Bifidobacterium longum subsp. infantis.

[0038] [5] The use according to [4], wherein the Bifidobacterium longum subsp. longum comprises Bifidobacterium longum subsp. longum BB536.

[0039] [6] The use according to any one of [1] to [5], wherein the functional nutritional composition may further comprise other nutritional ingredients, wherein the other nutritional ingredients comprise one or more of proteins, vitamins, monosaccharides, polysaccharides or plant extracts.

[0040] [7]. The use according to any one of [1] to [6], wherein the functional nutritional composition is liquid, semi-solid or solid.

[0041] [9]. According to the use described above, wherein the improvement of human immunity includes an increase in the content of immunoglobulins and / or complement.

[0042] Effects of the Invention

[0043] Through the implementation of the above technical solution, the present invention has the following advantages and can achieve the following technical effects:

[0044] 1) Through extensive screening and research, the present invention has developed a functional nutritional composition comprising casein phosphopeptides and Bifidobacterium probiotics in a specific ratio. Compared to casein phosphopeptides or Bifidobacterium probiotics alone, the composition provided by the present invention can more effectively enhance human immunity and improve the body's antioxidant capacity.

[0045] 2) The results of the present invention are verified based on animal experiments rather than cell experiments, and the synergistic mechanism of the two substances in the above-mentioned specific ratio is studied, that is, the effect of the composition in improving human immunity is achieved by promoting the increase of serum complement C3; the effect of the composition in improving human antioxidant capacity is achieved by providing spleen antioxidant capacity, while the above-mentioned mechanism and efficacy cannot be achieved when the two substances are used alone, regardless of the dosage.

[0046] In addition, the present invention unexpectedly discovered that, in some preferred embodiments, when the casein phosphopeptide and the Bifidobacterium probiotics in the composition are contained in specific amounts, the composition has particularly significant effects in enhancing immunity and providing antioxidant effects. DETAILED DESCRIPTION

[0047] The following describes the technical features of the present invention in detail. The technical features described below are described based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples.

[0048] It should be noted that:

[0049] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0050] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0051] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0052] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used means weight or mass percentage.

[0053] In this specification, the term "infant" refers to a human population under the age of 3.

[0054] In this specification, the term "children" refers to a group of human beings who are older than 3 years old and younger than 12 years old and are in the growth and development stage.

[0055] In this specification, the term "youth" refers to a group of people who are older than 12 years old and younger than 18 years old and are in the growth and development stage.

[0056] As used herein, the term "middle-aged and elderly" refers to a human population aged 45 years and above.

[0057] In this specification, "animal milk" is used to refer to the liquid obtained from the mammary glands of mammals during lactation. The term "animal milk" should be interpreted broadly and covers both raw milk (i.e., liquid obtained directly from the mammary gland) and standardized milk products (such as, for example, skim milk or whole milk).

[0058] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0059] In this specification, the "room temperature" used here means an indoor ambient temperature of "23±2°C".

[0060] In addition, unless otherwise defined, other technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0061] The technical solution of the present invention is mainly based on the following insights:

[0062] The composition of the present invention comprises two essential components. The combination of casein phosphopeptide and bifidobacterium probiotics in the composition exhibits a significant synergistic effect, having one or more of the following functions or effects: enhancing immunity by increasing the level of complement C3, and promoting immunity and providing antioxidant effects by increasing the levels of glutathione peroxidase, superoxide dismutase, catalase, glutathione sulfhydryltransferase, and glutathione reductase in the spleen.

[0063] The present invention studies the compounding of casein phosphopeptide and Bifidobacterium longum subspecies longum BB536 to form a composition through animal experiments. Based on the effects of the use of the composition on relevant indicators in mouse serum and spleen, it is unexpectedly found that the use of the nutritional composition formed by the compounding of the two can produce obvious synergistic effects in promoting immunity and anti-oxidation.

[0064] The technical solution of the present invention is further described in detail below:

[0065] <Composition>

[0066] The present invention provides a functional nutritional composition comprising the following two essential components: i) casein phosphopeptide and ii) bifidobacterium probiotics, and in the composition, the ratio of component i) casein phosphopeptide and component ii) bifidobacterium probiotics can be prepared according to the following ratio:

[0067] Effective amount of casein phosphopeptide: The effective amount of Bifidobacterium probiotics is (50mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU).

[0068] It has been found that compared with casein phosphopeptide or Bifidobacterium probiotics alone, the composition provided by the present invention can more effectively enhance human immunity and also better improve the body's antioxidant capacity.

[0069] (Casein Phosphopeptide)

[0070] Casein phosphopeptide (CPP) is made from casein hydrolyzed with trypsin, then refined and purified. Its molecular structure consists of twenty to thirty amino acid residues, including 4 to 7 clustered phosphopeptides.

[0071] In principle, there are no specific limitations on the source of casein phosphopeptides used in the present invention. For example, in some specific embodiments, various commercially available products can be used. Currently, the purity of CPP in commercially available CPP products ranges from 12% to 90%. It should be noted that the weight percentage of CPP in the functional nutritional composition of the present invention is based on the amount of CPP in the CPP product.

[0072] In addition, it should be emphasized that the addition form of the above-mentioned casein phosphopeptide in the composition must comply with the permission of local laws and regulations. Within the permitted range, it can be added as a separate component. When it is not allowed to be added alone, other components that can be added containing this component can also be used to give the composition the required casein phosphopeptide (CPP).

[0073] (Bifidobacterium probiotics)

[0074] The present invention does not impose any particular limitation on the source of Bifidobacterium. Typically, it can be obtained through microbial fermentation or directly purchased from the market.

[0075] The present invention does not particularly limit the specific species of Bifidobacterium. In some embodiments, the Bifidobacterium probiotics described herein include any one or more of Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, and Bifidobacterium longum subsp. infantis. In some preferred embodiments, the Bifidobacterium probiotics described herein are Bifidobacterium longum subsp. longum.

[0076] The present invention does not particularly limit the specific strains of the above-mentioned strains. For example, the animal Bifidobacterium lactis subspecies includes BB12 strain, HN109 strain and Bi-07 strain, etc., Bifidobacterium bifidum includes R0071 strain, etc., the Bifidobacterium longum subspecies infantis includes R0033 strain, etc., Bifidobacterium longum subspecies long includes BB536 strain, etc., and the Bifidobacterium breve includes M-16V strain, etc. From the perspective of better imparting the above-mentioned synergistic effect to the composition, in some preferred embodiments, the Bifidobacterium longum subspecies long of the present invention is selected from the BB536 strain.

[0077] In addition, it should be emphasized that the addition form of the above-mentioned Bifidobacterium probiotics in the composition must comply with the specifications or permission of local laws and regulations. Within the permitted range, they can be added as a separate ingredient. When the separate addition form is not allowed, other components that can be added containing this ingredient can also be used to provide the composition with the required Bifidobacterium probiotics.

[0078] (Effective content ratio)

[0079] Furthermore, in order to obtain a better synergistic effect of enhancing immunity and anti-oxidation, in some specific embodiments, in the composition of the present invention, the ratio of i) casein phosphopeptide and ii) Bifidobacterium probiotics can be configured as follows:

[0080] Effective amount of casein phosphopeptide: The effective amount of Bifidobacterium probiotics is (50mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU).

[0081] It should be noted that in the products described below, the two are also expected to have such a proportional relationship.

[0082] It should be noted that although the above data are obtained based on the verification results of the animal experiments of the present invention, the specific content of casein phosphopeptide and Bifidobacterium probiotics in the composition should be within the above relationship and set in a manner that complies with local laws and regulations.

[0083] Furthermore, in some preferred embodiments of the present invention, the effective amount of casein phosphopeptide: the effective amount of Bifidobacterium probiotics is (120mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU); More preferably, the effective amount of casein phosphopeptide: the effective amount of Bifidobacterium probiotics is (180mg~360mg): (1.00×10 9 CFU~1.20×10 11 CFU); further preferred effective amount of casein phosphopeptide: effective amount of Bifidobacterium probiotics (200mg~350mg): (1.00×10 9 CFU~1.20×10 11 CFU). In addition, the ratios that can be cited include (180mg to 360mg): (2.00×10 9 CFU~1.20×10 11 CFU), (200mg~360mg): (5.00×10 9CFU~1.20×10 11 CFU), (180mg~360mg): (1.00×10 10 CFU~1.20×10 11 CFU), (180mg~360mg): (5.00×10 10 CFU~1.20×10 11 CFU), etc.

[0084] It should be noted that the above-mentioned ratio of effective contents represents the ratio of the effective contents of the two in the composition within the range of their respective measurement units, which does not mean the absolute concentration or content of each component in the composition or the product described below. For example, in some specific embodiments, in the composition or product, the content of casein phosphopeptide can be 0.1mg / 100g to 300mg / 100g, preferably 10mg / 100g to 300mg / 100g, more preferably 30mg / 100g to 300mg / 100g, and most preferably 40mg / 100g to 200mg / 100g. The content of the Bifidobacterium probiotic can be 10CFU / g to 10 13 CFU / g, preferably 10 5 CFU / g~10 12 CFU / g, more preferably 10 6 CFU / g~10 11 CFU / g, most preferably 10 7 CFU / g~10 9 CFU / g.

[0085] Furthermore, the above-mentioned composition has been found to synergistically enhance human immunity and antioxidant capacity. Experimental verification has shown that the improved / enhanced immunity of the composition is achieved by promoting increases in serum complement C3, while the enhanced antioxidant capacity of the composition is achieved by enhancing spleen antioxidant capacity. In particular, the synergistic effect is even greater when the two ingredients are used together at the higher end of the range.

[0086] Furthermore, it was found that if either substance is used alone, the above-mentioned mechanism and efficacy cannot occur regardless of the dosage.

[0087] Therefore, the functional composition of the present invention comprises at least the casein phosphopeptide and the probiotic bacteria of the genus Bifidobacterium described above.

[0088] In addition, there are no particular limitations on other components or other nutrients that can be used in the composition of the present invention. Without impairing the effects of the present invention, other edible ingredients, food additives or solvent components commonly used in the art can be used. Other nutrients include one or more of proteins, vitamins, monosaccharides, polysaccharides or plant extracts.

[0089] The functional composition of the present invention may be in a liquid state, or in a semi-solid state or a solid state.

[0090] <Product>

[0091] The present invention provides a product composition, which comprises the above composition.

[0092] In addition to the above-mentioned essential components, the product of the present invention may also include other ingredients according to the needs of the final product, and examples thereof include:

[0093] Plant product ingredients, including fruits or their extracts such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, rubber, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica chinensis and bilberries; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano, or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans, or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts, or their extracts; coffee or its extracts; and some botanical Chinese medicinal materials or their extracts that are both medicinal and edible.

[0094] Animal meat product ingredients, including meat product ingredients from pigs, cattle, sheep, aquatic products or poultry.

[0095] Animal dairy ingredients include fresh milk from cows, sheep, etc., as well as processed dairy products such as milk powder, whey protein or cheese.

[0096] Functional added ingredients, including vitamins (vitamin A, β-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B 12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, biotin, L-carnitine, lutein, one or more); starch; modified starch; amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, L-leucine, etc.); dietary fiber (inulin, konjac flour, galacto-oligosaccharides, fructo-oligosaccharides, raffinose, polydextrose, isomaltooligosaccharide, soybean polysaccharide, cyclodextrin, resistant dextrin or soybean fiber, etc.); trace element supplements (which may include metal ion salts of organic acids, such as calcium citrate, calcium L-lactate, potassium gluconate, sodium citrate, ferrous gluconate, zinc gluconate, sodium selenite, copper gluconate, manganese gluconate and magnesium gluconate, etc.), fat supplements (such as saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structural lipids, OPL structural lipids, LPL structural lipids, DHA, EPA, ARA, phospholipids, etc.); nucleotide supplements; human milk oligosaccharides (such as 2'-FL, 3-FL, DFL, LNFPI, LNFP II, LNT, LNnT, 3'-SL, 6'-SL, DSLNT, etc.), etc.

[0097] Any acceptable excipients include, but are not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0098] The present invention does not specifically limit the specific type of the above-mentioned product. In some embodiments, the product is a food. In some embodiments, the product is a health food.

[0099] For different product categories, the present invention does not specifically limit the specific form of the product. For example, it can be in solid form, semi-solid form or liquid form.

[0100] The present invention does not specifically limit the target population of the product. For example, the product can be used for infants, children, teenagers or adults.

[0101] In some specific embodiments, the product of the present invention can be infant formula milk powder, baby food, children's formula milk powder, children's snacks, milk powder for pregnant women, milk powder for middle-aged and elderly people, or nutritional or dietary supplements.

[0102] In other specific embodiments, the products described in the present invention can be beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages, alcoholic beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), candies (jelly candies, hard candies, compressed candies, etc.), milk and dairy products (fresh milk from fresh cow (sheep) milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc.), pasta products (noodles, instant noodles, steamed buns, dumplings or wontons, as well as baked goods such as bread, cakes or biscuits), etc.

[0103] In some other specific embodiments, the product of the present invention is an oral preparation, which includes but is not limited to tablets, pills, granules, powders, sprays, teas, capsules or oral liquids.

[0104] The product provided by the present invention is suitable for all people in principle, and is particularly suitable for people with low immunity, or who need to enhance immunity, or who need antioxidants. The components in the product can also be adjusted accordingly for people with different characteristics.

[0105] Uses that help boost immunity

[0106] The present invention proposes to utilize casein phosphopeptides and Bifidobacterium probiotics to compound in a specific ratio, which can enhance immunity, and there is a synergistic effect between the two substances. Therefore, the composition provided by the present invention can be used to prepare products that help enhance immunity, and the products described in the present invention (such as compositions, foods or health foods) can also enhance immunity. Moreover, such efficacy exists for non-therapeutic purposes.

[0107] The immunity enhancement described in the present invention mainly includes the increase of complement content, especially, the complement includes complement C3.

[0108] Uses with antioxidant effects

[0109] The present invention proposes that casein phosphopeptides and Bifidobacterium probiotics, when combined in a specific ratio, can be used to increase antioxidant enzyme levels, with the two substances exhibiting a synergistic effect. Therefore, the composition provided by the present invention can be used to prepare products with antioxidant properties, and the products described in the present invention (e.g., compositions, foods, or health foods) also have antioxidant properties.

[0110] In some embodiments, the antioxidant effect of the present invention is not intended to prevent and / or treat a disease, and the oxidation effect has not reached the level of becoming a disease. In this case, the composition or product with antioxidant capacity can be a food or a health food.

[0111] The antioxidant effect of the present invention is mainly achieved by improving the antioxidant capacity of the spleen. More specifically, the antioxidant effect of the present invention includes an increase in the content of antioxidant enzymes. In some embodiments, the antioxidant enzymes include any one or more of glutathione peroxidase, superoxide dismutase, catalase, glutathione sulfhydryl transferase and glutathione reductase.

[0112] Example

[0113] The present invention will be further described in detail below through specific examples. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0114] <Raw Materials>

[0115] 1. Main reagents and instruments

[0116] 1.1 Main Reagents

[0117] 1% sodium pentobarbital (Merck, Germany), casein phosphopeptide (Green Extract), Bifidobacterium longum subsp. longum BB536 (Baishi Biotechnology), Mouse IgM and Mouse IgG kits (Biyuntian Biotechnology), Mouse C3 kit (Wuhan Yilai Ruite Biotechnology), glutathione peroxidase (GSH-P X ) kit (Nanjing Jiancheng Bioengineering), superoxide dismutase (SOD) and catalase (CAT) activity detection kit (Biyuntian Biotechnology), glutathione sulfhydryltransferase (GST) and glutathione reductase (GR) activity detection kit (Beijing Solebold Technology).

[0118] 1.2 Main instruments and equipment

[0119] 1 / 10,000 electronic balance (Beijing Sartorius Instrument System Co., Ltd.), electric constant temperature water bath (Tianjin Leicester Instrument Co., Ltd.), refrigerator (Qingdao Haier Co., Ltd.), vortex oscillator (Weiwosoke Technology (Beijing) Co., Ltd.), centrifuge (Shanghai Chemical Machinery Company), 3100 fully automatic biochemical analyzer (Hitachi, Japan), SPX-150B-Z biochemical incubator (Shanghai Boxun Industrial Co., Ltd.), Bio-rad 1575 plate washer (Bio-Rad, USA), ultra-low temperature refrigerator and Varioskan Flash microplate reader (Thermo Fisher Scientific, USA).

[0120] 2. Experimental Animals

[0121] We will purchase 132 SPF-grade C57BL / 6 mice in stable health. Housing conditions: 20-25°C, 50% relative humidity, a standard 12-hour light / 12-hour dark cycle, consistent feed and water, good ventilation, daily cleaning, and cage hygiene. Animal Welfare: During all experimental procedures, mice were treated appropriately, in compliance with the relevant regulations of the Laboratory Animal Ethics Committee, and in accordance with IAC and other relevant standard operating procedures (SOPs). Sacrifice: Mice were anesthetized and sacrificed by exsanguination of the inferior vena cava at the end of the experiment. Samples were collected, and the bodies were stored in a dedicated freezer container for safe and uniform disposal.

[0122] <Animal Experiments>

[0123] 1. Experimental Methods

[0124] Take 132 C57BL / 6 mice of SPF grade, raise at room temperature, and eat and drink freely. Adaptive feeding starts formal experiment after 3 days. Mice are randomly divided into 11 groups, 12 in each group. Be respectively blank control group (comparative example 1), give test article group (experimental example 1-10), experimental example 1-10 gavage sample, comparative example 1 gavage equal amount of normal saline, each group of mice continues gavage for 3 weeks, gavage 1 time a day, each group is fed maintenance feed. Specific experimental dosage is shown in the table below.

[0125] Comparative Examples and Experimental Examples were prepared by adding CPP and BB536 in the composition according to the ratios shown in Table 1 below.

[0126] Table 1. Animal experimental dosage

[0127] Note: The dosages of CPP and BB536 in this section are daily dosages, the same below.

[0128] 2. General observation

[0129] During the gavage, all mice were observed for their eating, drinking, and activity.

[0130] 3. Serum-related index detection

[0131] Three weeks after oral gavage, 11 groups of mice were anesthetized and bled out from the inferior vena cava. Blood samples were collected and placed at room temperature for 30 minutes. After the whole blood naturally coagulated and the serum was precipitated, it was centrifuged at 3000 rpm and 4°C for 10 minutes. The serum was collected and stored in a -80°C refrigerator for unified measurement.

[0132] 3.1 Detection of immunoglobulin IgM, IgG and complement C3 levels

[0133] Serum samples from 12 mice were collected from each group, and immunoglobulin IgM, IgG, and complement C3 were detected using enzyme-linked immunosorbent assay (ELISA). The specific detection methods are as follows:

[0134] Standard configuration:

[0135] 1) After taking the ELISA kit out of the refrigerator, equilibrate it at room temperature (25-28°C) for 20 minutes.

[0136] 2) Prepare an appropriate amount of washing solution: dilute the washing solution (20X) to 1X with double-distilled water or deionized water. For example, 10 mL of washing solution (20X) is added to 190 mL of water and mixed to obtain a 1X washing solution.

[0137] 3) Add the standard diluent to one vial of standard according to the volume indicated on the standard label and incubate at room temperature for 15 minutes. Gently mix and pipette several times to completely dissolve the standard, bringing the standard concentration to 2000 pg / mL. Typically, two wells are assayed for each standard concentration, using 100 μL of standard per well, for a total of 200 μL. Additionally, 250 μL is required for dilution. Therefore, if the prepared volume of one vial of standard is less than 0.45 mL, use additional vials of standard and combine and mix thoroughly before use.

[0138] 4) Prepare five clean 1.5 mL centrifuge tubes and pre-add 250 μL of standard diluent to each tube. Perform serial dilutions of the standard to obtain six standard concentrations: 2000, 1000, 500, 250, 125, and 62.5 pg / mL. Finally, add the diluted standards to the wells of the pre-coated plate in sequence. Add the standard diluent directly to the wells to achieve a concentration of 0 pg / mL, for a total of seven standard concentrations.

[0139] Sample testing:

[0140] 1) Calculate and determine the number of pre-coated strips required for one experiment. Remove the required strips and place them in a 96-well rack. Place any strips that are not in use back into the aluminum foil bag, seal it, and store at 4°C.

[0141] 2) Standards must be prepared and a standard curve drawn for each experiment. It is also recommended to set up a background correction well, i.e., a blank well, by adding only TMB solution and stop solution to the well.

[0142] 3) Add 100 μL / well of sample or standard of varying concentrations to the corresponding wells, seal the wells with transparent film, and incubate at room temperature for 120 minutes. (Dilute serum samples 1:1 with sample analysis buffer. If the dilution ratio is large, add equal amounts of sample and sample analysis buffer, and make up the remaining volume to 100 μL with standard diluent. Record the sample dilution factor.)

[0143] 4) Wash the plate five times, patting dry on thick absorbent paper for the final wash. (Use 300 μL of wash solution per well, with a 15-30 second interval between injection and aspiration. Wash the plate five times. After the final wash, invert the plate onto thick absorbent paper and pat dry.)

[0144] 5) Add 100 μL / well of biotinylated antibody, seal the reaction wells with a transparent sealing film, and incubate at room temperature for 60 min.

[0145] 6) Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0146] 7) Add 100 μL / well of horseradish peroxidase-labeled streptavidin. Seal the wells with white sealing film and incubate at room temperature (25°C) in the dark for 20 minutes.

[0147] 8) Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0148] 9) Add 100 μL / well of TMB solution, seal the wells with white sealing film, and incubate at room temperature in the dark for 20 minutes.

[0149] 10) Add 50 μL / well of stop solution, mix well and immediately measure the A450 value.

[0150] 4. Detection of spleen-related indicators

[0151] Spleen tissue samples of 12 mice were collected from each group, and the samples were processed, homogenized, and centrifuged. The supernatant was used as the test sample. The antioxidant enzymes (GSH-P X , SOD, CAT, GST, GR) content, the specific detection method is as follows:

[0152] (1) GSH-P X Determination:

[0153] Sample pre-treatment:

[0154] a. Weigh 10 mg of tissue and rinse with ice-cold saline to remove blood, then wipe dry with filter paper.

[0155] b. Add 90 μL of physiological saline and grind with a tissue homogenizer to make a 10% tissue homogenate.

[0156] c. Centrifuge at 3000 rpm, 4°C for 15 min and collect the supernatant for testing.

[0157] GSH-P X Viability assay procedure:

[0158] Enzymatic Reaction: (Pre-warm Reagent 1 Application Solution at 37°C) Add 20 μL of 1 mmol / L GSH to the non-enzyme tube. Add 20 μL of 1 mmol / L GSH and 20 μL of the homogenate to be tested to the enzyme tube. Pre-warm at 37°C for 5 minutes. Add 10 μL of Reagent 1 Application Solution. Pre-warm at 37°C for 5 minutes. Add 200 μL of Reagent 2 Application Solution. Finally, add 20 μL of the homogenate to be tested to the non-enzyme tube. Mix thoroughly, centrifuge at 3500-4000 rpm for 10 minutes, and collect the supernatant for color development.

[0159] Color development reaction: Set up blank tubes, standard tubes, non-enzyme tubes, and enzyme tubes. Add 100 μL of GSH standard solvent solution, 20 μmol / L GSH standard solution, supernatant from the non-enzyme tube, and supernatant from the enzyme tube, respectively. Add 100 μL of Reagent 3 solution, 25 μL of Reagent 4 solution, and 5 μL of Reagent 5 solution. Mix thoroughly, let stand at room temperature for 15 minutes, and monitor at 412 nm.

[0160] GSH-P X Calculation of vitality:

[0161] Definition: One unit of enzyme activity is defined as the amount of GSH in the reaction system that decreases by 1 μmol / L per minute after deducting the effects of non-enzymatic reactions per milligram of protein.

[0162] Calculation formula: GSH-P X Activity = (OD value of non-enzyme tube - OD value of enzyme tube) ÷ (OD value of standard tube - OD value of blank tube) × concentration of standard tube (20 μmol / L) × dilution factor (5×) ÷ reaction time × (sample volume × sample protein content)

[0163] [Note] From the enzymatic reaction table, we can see that adding 200 μL of reagent 2 to 50 μL of reaction solution will result in a 5-fold dilution in the reaction solution, so multiply by 5.

[0164] (2) SOD determination:

[0165] Take an appropriate amount of tissue sample and homogenize it at 4°C or on ice by adding 100 μL of SOD sample preparation solution per 10 mg of tissue. Centrifuge at approximately 12,000 g for 5 minutes at 4°C and collect the supernatant as the sample to be tested.

[0166] a. Preparation of WST-8 / enzyme working solution: Prepare an appropriate amount of WST-8 / enzyme working solution for a 160 μL reaction volume. Evenly mix 151 μL SOD Assay Buffer, 8 μL WST-8, and 1 μL enzyme solution to make 160 μL of WST-8 / enzyme working solution. Prepare an appropriate amount of WST-8 / enzyme working solution based on the number of samples (including standards) to be tested.

[0167] b. Preparation of the Reaction Start Working Solution: Dissolve the Reaction Start Working Solution (40X) in the kit and mix thoroughly. Dilute the solution by adding 39μL of SOD Assay Buffer per 1μL of Reaction Start Working Solution (40X). Mix thoroughly to prepare the Reaction Start Working Solution. Prepare an appropriate amount of Reaction Start Working Solution based on the number of samples (including standards) to be tested. Store the prepared Reaction Start Working Solution at 4°C or on ice and use immediately.

[0168] Sample determination:

[0169] a. Refer to the table below to set up sample wells and various blank control wells in a 96-well plate. Add the sample to be tested and other solutions in the order shown in the table. Add the reaction starter solution and mix thoroughly. Note: The reaction begins immediately after adding the reaction starter solution. Operate at low temperatures or use a dispenser to minimize errors caused by the timing of adding the reaction starter solution between wells. For the sample well, add 20 μL of the sample to be tested, 160 μL of WST-8 / enzyme working solution, and 20 μL of reaction starter solution. For the blank control well (Blank 1), add 20 μL of SOD assay buffer, 160 μL of WST-8 / enzyme working solution, and 20 μL of reaction starter solution. For the blank control well (Blank 2), add 40 μL of SOD assay buffer and 160 μL of WST-8 / enzyme working solution. For the blank control well (Blank 3), add 20 μL of the sample to be tested, 20 μL of SOD assay buffer, and 160 μL of WST-8 / enzyme working solution.

[0170] b. Incubate at 37°C for 30 min.

[0171] c. Measure the absorbance at 450 nm.

[0172] Calculation of total SOD activity in samples:

[0173] a. Calculation of inhibition percentage:

[0174] The inhibition percentage was calculated according to the following formula: Inhibition percentage = [(A blank control 1 - A blank control 2) - (A sample - A blank control 3)] / (A blank control 1 - A blank control 2) × 100%

[0175] If the sample has no color and does not contain antioxidants, then A blank control 2 = A blank control 3. In this case, the calculation formula can be simplified to the following form (blank control 3 can be omitted during simplification):

[0176] Inhibition percentage = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%

[0177] If the calculated inhibition percentage is less than 30% or greater than 70%, the sample should generally be retested. Try to keep the inhibition percentage within the 30-70% range. If the calculated inhibition percentage is too high, the sample should be diluted appropriately. If the calculated inhibition percentage is too low, a new sample with a higher concentration should be prepared.

[0178] b. Definition of SOD enzyme activity unit: When the inhibition percentage in the above xanthine oxidase coupled reaction system is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity unit. Note: There are many ways to define the SOD activity unit, and different activity units need to be appropriately converted according to their definitions.

[0179] c. Calculation of SOD enzyme activity:

[0180] The calculation formula of SOD enzyme activity is as follows: SOD enzyme activity units in the test sample = SOD enzyme activity units in the detection system = inhibition percentage / (1-inhibition percentage) units

[0181] d. If the sample is a homogenate of cells or tissues, the SOD activity unit can be converted to U / g or U / mg protein based on the protein concentration and dilution factor of the sample.

[0182] (3) CAT assay:

[0183] Sample preparation:

[0184] The tissue was lysed with Western lysis buffer and the protein concentration of the samples was determined.

[0185] Standard curve determination:

[0186] a. Transfer 0, 12.5, 25, 50, or 75 μL of the prepared 5 mM hydrogen peroxide solution to a 1.5 mL or 0.5 mL plastic centrifuge tube. Add catalase assay buffer to a final volume of 100 μL and mix thoroughly. The hydrogen peroxide concentrations are 0, 0.625, 1.25, 2.5, and 3.75 mM, respectively.

[0187] b. Take 4 μL of each sample and add it to one well of a 96-well plate. Add 200 μL of colorimetric working solution. Incubate at 25°C for at least 30 minutes before measuring A520.

[0188] Sample measurement:

[0189] a. Add 10 μL of sample and 30 μL of catalase assay buffer or 40 μL of catalase assay buffer to the sample well and control well, respectively, and mix thoroughly. Then, add 10 μL of 250 mM hydrogen peroxide solution and mix quickly with a pipette. Incubate at 25°C for 5 minutes.

[0190] b. Add 450 μL of catalase reaction stop solution and mix by inversion or Vortex mixing to terminate the reaction. Complete steps c and d below within 15 minutes of terminating the reaction.

[0191] c. Add 40 μL of catalase assay buffer to a clean plastic centrifuge tube, then add 10 μL of the stopped and mixed reaction system above and mix thoroughly.

[0192] d. Take 10 μL of the 50 μL system from the previous step and add it to one well of a 96-well plate. Add 200 μL of color development solution.

[0193] e. Measure A520 after incubation at 25°C for at least 30 min.

[0194] Calculation of catalase activity in samples:

[0195] a. Calculate the standard curve. A520 = k[micromoles of hydrogen peroxide] + b. Calculate the values ​​of k and b from the standard curve. (For example, the standard curve formula in this manual is: y = 70.281x + 0.06, i.e., k = 70.281, b = 0.06, then A520 = 70.281 × [micromoles of hydrogen peroxide] + 0.06)

[0196] b. Calculate the residual micromoles of hydrogen peroxide in the sample. Residual micromoles of hydrogen peroxide = (A520-b) / k

[0197] c. Definition of catalase activity unit: 1 unit of enzyme activity (1 unit) can catalyze the decomposition of 1 micromole of hydrogen peroxide in 1 minute at 25°C and pH 7.0.

[0198] d. Calculation of catalase activity in tissue samples:

[0199] [Sample catalase activity] = [micromoles of hydrogen peroxide consumed] × [dilution factor] / ([reaction minutes] × [sample volume] × [protein concentration])

[0200] The unit of [sample catalase activity] is units / mg protein.

[0201] [Micromoles of hydrogen peroxide consumed] = [Micromoles of residual hydrogen peroxide in the blank control] - [Micromoles of residual hydrogen peroxide in the sample].

[0202] [Dilution factor] = 250.

[0203] [Reaction minutes] refers to the actual reaction time.

[0204] [Sample volume] is 10 μL, which is expressed in mL as 10 / 1000 mL.

[0205] [Protein concentration] Protein concentration in the sample, in mg / mL.

[0206] (4) GST assay:

[0207] Accurately weigh 10 mg of spleen and add 100 μL of Reagent 1 at a ratio of 1:10 tissue mass (mg) to Reagent 1 volume (μL). Homogenize on ice. Centrifuge at 8000g at 4°C for 10 min. Remove the supernatant and place on ice for analysis. Preheat Reagent 2 to 37°C for 15 min. Add 20 μL of sample or Reagent 1 to the assay well and blank well, followed by 180 μL of Reagent 2 and 20 μL of Reagent 3 to each well. Immediately measure the absorbance (A1) at 340 nm. Incubate at 37°C for 5 min. Afterward, remove and quickly dry the well and measure the absorbance (A2). Calculate ΔA = (A2 assay - A1 assay) - (A2 blank - A1 blank). Activity unit definition: One unit of enzyme activity is the catalytic binding of 1 μmol of CDNB to GSH per minute per gram of sample at 37°C.

[0208] GST activity (U / g mass) = ΔA ÷ (ε × d) × 10 6 ×V total ÷ (V sample ÷ V total × W) ÷ T = 0.38 × ΔA ÷ W

[0209] ε: product molar extinction coefficient, 9.6×10 3 L / mol / cm; d: 96-well plate optical diameter, 0.6 cm; 10 6 : Unit conversion factor, 1 mol = 1 × 10 6 μmol; Vtotal: total volume of reaction system, 220 μL = 2.2 × 10 -4 L; Cpr: supernatant protein concentration (mg / mL), determined using a BCA assay kit; W: sample mass, g; Vsample: supernatant volume added to the reaction system, 20 μL = 0.02 mL; Vsampletotal: reagent volume added, 1 mL; T: reaction time, 5 min;

[0210] (5) GR determination:

[0211] Accurately weigh 10 mg of spleen and add 100 μL of Reagent 1 to a 1:10 ratio of tissue mass (mg) to Reagent 1 volume (μL). Homogenize on ice. Centrifuge at 10,000 rpm at 4°C for 10 minutes. Remove the supernatant and place on ice for analysis. Preheat a portion of Reagent 1 at 37°C for 15 minutes, depending on the sample size. Add 20 μL of sample and 150 μL or 170 μL of Reagent 1 to the assay well and a blank well, respectively. Then, add 10 μL of Reagent 2 and 20 μL of Reagent 3 to each well and immediately measure the absorbance at 340 nm (A1). Incubate at 37°C for 3 minutes. After reaction, remove the sample, quickly dry it, and measure the absorbance (A2). Calculate ΔA = A2 - A1. Activity unit: One unit of enzyme activity is defined as the oxidation of 1 μmol of NADPH per gram of sample per minute at 37°C, pH 8.0.

[0212] GR enzyme activity (U / g mass) = [(ΔA test tube - ΔA blank tube) ÷ (ε × d) × V total × 10 6 ]÷(V sample ÷ V sample total × W)÷T=0.536×(ΔA test tube - ΔA blank tube)÷W

[0213] ε: NADPH molar extinction coefficient 6.22×10 3 L / mol / cm; d: cuvette optical path, 1 cm; V: total volume of the reaction system, 200 μL = 2 × 10 -4 L; 106: unit conversion factor, 1mol = 1×10 6 μmol; Cpr: supernatant protein concentration, mg / mL; W: sample mass, g; V: sample volume added to the reaction system, 20 μL = 2 × 10 -2 mL; V total sample volume: 1 mL; T: reaction time, 3 min.

[0214] 5. Statistical Analysis

[0215] Experimental data are presented as mean ± standard deviation (mean ± SD). Each experiment was repeated at least three times. GraphPad Prism 8.0 software was used for statistical analysis. One-way ANOVA was used to analyze the statistical differences between the groups. p > 0.05 indicated no statistical significance, and p < 0.05 indicated a significant difference.

[0216] 6. Experimental Results

[0217] 6.1 General Observations

[0218] The mice were agile, had shiny fur, ate and drank normally, and were in good condition.

[0219] 6.2 Results of serum-related index testing

[0220] Immunoglobulin is a protein with antibody activity that exists in blood or body fluids. It can kill pathogenic microorganisms under the synergistic action of complement and is an important part of the body's humoral immunity.

[0221] IgG is the immunoglobulin with the highest content in serum. It is synthesized in the spleen and lymph nodes. It is the main immunoglobulin mediating humoral immunity and is also the main immunoglobulin for serological diagnostic tests.

[0222] IgM is the earliest immunoglobulin produced by the body, and is mainly secreted and synthesized by plasma cells in the spleen and lymph nodes. The body's immune level can be reflected by the immunoglobulin content in the serum.

[0223] Complement C3 is the complement component with the highest content in serum. It is mainly synthesized by macrophages and liver and plays an important role in both the classical complement activation pathway and the alternative complement activation pathway.

[0224] 6.2.1 Immunoglobulin IgM and IgG content test results

[0225] To evaluate the effects of the test substances alone or in combination on the immune function of mice, the levels of immunoglobulins IgM and IgG in the serum of mice in each group were determined. The results are shown in Table 2.

[0226] Table 2. Serum immunoglobulin IgM and IgG levels in mice of each group (mean ± SD, n = 12)

[0227] Note: * Compared with the blank control group, p<0.05, *** Compared with the blank control group, p<0.001, ns indicates no statistical significance compared with the blank control group.

[0228] As shown in Table 2, when C57BL / 6 mice were treated with different doses of CPP, there was no significant change in the IgM and IgG levels of mice in the CPP low-dose, CPP medium-dose and CPP high-dose groups compared with the blank control group.

[0229] When C57BL / 6 mice were treated with different doses of BB536, the IgM and IgG levels in the high-dose BB536 group were significantly increased compared with the blank control group.

[0230] 6.2.2 Complement C3 content test results

[0231] To evaluate the effects of the test substances alone or in combination on the immune function of mice, the levels of complement C3 in the serum of mice in each group were measured. The results are shown in Table 3.

[0232] Table 3. Complement C3 levels in serum of mice in each group (mean ± SD, n = 12)

[0233] Note: ** Compared with the blank control group, p<0.01, *** Compared with the blank control group, p<0.001, ns indicates no statistical significance compared with the blank control group.

[0234] As shown in Table 3, when CPP and BB536 were used alone in C57BL / 6 mice, the complement C3 content of mice did not change significantly regardless of the dosage, that is, the use of CPP and BB536 alone had no effect on the complement C3 content of mice;

[0235] However, when CPP levels were increased above the mid-dose, when combined with various doses of BB536, significantly elevated complement C3 levels were achieved, thus demonstrating a synergistic effect between the two.

[0236] 6.3 Results of spleen-related index testing

[0237] Glutathione peroxidase (GSH-Px) is an important peroxide-degrading enzyme widely present in the body, which can remove lipid hydroperoxides and thus reduce damage to the body.

[0238] Superoxide dismutase (SOD) is a key member of the antioxidant enzyme system in biological systems. It catalyzes the dismutation of superoxide anion free radicals, thereby reducing or eliminating excessive superoxide anion free radicals produced during the body's metabolism. It plays a vital role in preventing disease and improving human immunity.

[0239] Catalase (CAT) is an enzyme scavenger that can effectively catalyze the decomposition of H2O2, causing it to lose the effect of active oxygen, thereby protecting the body.

[0240] Glutathione S-transferases (GSTs) are a family of proteins with diverse physiological functions, primarily present in the cytoplasm. GSTs are a crucial component of the body's detoxification enzyme system, catalyzing the covalent binding of various chemicals and their metabolites to the sulfhydryl groups of GSH, converting electrophilic compounds into hydrophilic substances that are readily excreted in bile or urine, thereby degrading and eliminating various potentially toxic substances from the body. Therefore, GSTs play a crucial biological role in protecting cells from damage by electrophilic compounds.

[0241] Glutathione reductase (GR) is a flavoprotein oxidoreductase found in both eukaryotic and prokaryotic organisms. GR is a key enzyme in the glutathione redox cycle. GR catalyzes the reduction of GSSG from NADPH to GSH, helping to maintain the GSH / GSSG ratio in the body. GR plays a key role in scavenging reactive oxygen species during oxidative stress reactions. GR also participates in the ascorbic acid-glutathione cycle. GR catalyzes the reduction of GSSG from NADPH to GSH, while simultaneously dehydrogenating NADPH to NADP. + .

[0242] 6.3.1 Glutathione peroxidase (GSH-Px) content test results

[0243] In order to evaluate the effects of the test substances alone or in combination on the antioxidant activity of mice, the GSH-Px content in the spleen of each group of mice was measured. The results are shown in Table 4.

[0244] Table 4. GSH-P in spleen of mice in each group X Content (mean±SD, n=12)

[0245] Note: ** Compared with the blank control group, p<0.01, *** Compared with the blank control group, p < 0.001, ns means there is no statistical significance compared with the blank control group; ## Compared with the CPP low-dose group (50 mg / kg.bw), p < 0.01; && Compared with the CPP medium dose group (150 mg / kg.bw), p < 0.01, &&& Compared with the CPP medium-dose group (150 mg / kg.bw), p < 0.001; ^^^ Compared with the high-dose CPP group (350 mg / kg.bw), p < 0.001.

[0246] As shown in Table 4, both Experiment 3 using CPP alone and Experiment 5 using BB536 alone had a negative effect on GSH-P X The content of PEG-1 and PEG-2 had no effect, while the combination of the two in Experiment 6 showed a synergistic effect.

[0247] In addition, Experimental Example 7 also significantly increased GSH-P compared to Experimental Examples 2 and 5. X The content of GSH-P in Experimental Example 8 was significantly increased compared with Experimental Example 1 and Experimental Example 5. X Therefore, further increasing the amount of BB536 will also produce the same effect.

[0248] 6.3.2 Superoxide dismutase (SOD) content test results

[0249] In order to evaluate the effects of the test substances alone or in combination on the antioxidant activity of mice, the SOD content in the spleen of each group of mice was measured. The results are shown in Table 5.

[0250] Table 5. SOD content in spleen of mice in each group (mean ± SD, n = 12)

[0251] Note: * Compared with the blank control group, p<0.05, ** Compared with the blank control group, p<0.01, *** Compared with the blank control group, p < 0.001, ns means there is no statistical significance compared with the blank control group; # Compared with the CPP low-dose group (50 mg / kg.bw), p < 0.05; && Compared with the CPP medium dose group (150 mg / kg.bw), p < 0.01, &&& Compared with the CPP medium-dose group (150 mg / kg.bw), p < 0.001; ^^^ Compared with the high-dose CPP group (350 mg / kg.bw), p < 0.001.

[0252] As shown in Table 5, Experimental Example 3 using CPP alone and Experimental Example 5 using BB536 alone had no effect on the SOD content, while Experimental Example 6 using the combination of the two showed a synergistic effect.

[0253] In addition, Experimental Example 7 also significantly increased the SOD content compared to Experimental Example 2 and Experimental Example 5, and Experimental Example 8 also significantly increased the SOD content compared to Experimental Example 1 and Experimental Example 5. Therefore, further increasing the dosage of BB536 also has the same effect.

[0254] 6.3.3 Catalase (CAT) content test results

[0255] In order to evaluate the effects of the test substances alone or in combination on the antioxidant activity of mice, the CAT content in the spleen of each group of mice was measured. The results are shown in Table 6.

[0256] Table 6. CAT content in spleen of mice in each group (mean ± SD, n = 12)

[0257] Note: ** Compared with the blank control group, p<0.01, *** Compared with the blank control group, p < 0.001, ns means there is no statistical significance compared with the blank control group; ## Compared with the CPP low-dose group (50 mg / kg.bw), p < 0.01; &&&Compared with the CPP medium-dose group (150 mg / kg.bw), p < 0.001; ^^^ Compared with the high-dose CPP group (350 mg / kg.bw), p < 0.001.

[0258] As shown in Table 6, Experimental Example 3 using CPP alone and Experimental Example 5 using BB536 alone had no effect on the CAT content, while Experimental Example 6 using the combination of the two showed a synergistic effect.

[0259] In addition, Experimental Example 7 also significantly increased the CAT content compared to Experimental Examples 2 and 5, and Experimental Example 8 also significantly increased the CAT content compared to Experimental Examples 1 and 5. Therefore, further increasing the amount of BB536 also has the same effect.

[0260] 6.3.4 Glutathione S-transferase (GST) content test results

[0261] In order to evaluate the effects of the test substances alone or in combination on the antioxidant activity of mice, the GST content in the spleen of each group of mice was measured. The results are shown in Table 7.

[0262] Table 7. GST content in spleen of mice in each group (mean ± SD, n = 12)

[0263] Note: * Compared with the blank control group, p<0.05, ** Compared with the blank control group, p<0.01, *** Compared with the blank control group, p < 0.001, ns means there is no statistical significance compared with the blank control group; # Compared with the CPP low-dose group (50 mg / kg.bw), p < 0.05; && Compared with the CPP medium dose group (150 mg / kg.bw), p < 0.01, &&& Compared with the CPP medium-dose group (150 mg / kg.bw), p < 0.001; ^^ Compared with the CPP high-dose group (350 mg / kg.bw), p < 0.01, ^^^ Compared with the high-dose CPP group (350 mg / kg.bw), p < 0.001.

[0264] As shown in Table 7, Experimental Example 3 using CPP alone and Experimental Example 5 using BB536 alone had no effect on the GST content, while Experimental Example 6 using a combination of the two showed a synergistic effect.

[0265] In addition, Experimental Example 7 also significantly increased the GST content compared to Experimental Examples 2 and 5, and Experimental Example 8 also significantly increased the GST content compared to Experimental Examples 1 and 5. Therefore, further increasing the amount of BB536 also has the same effect.

[0266] 6.3.5 Glutathione Reductase (GR) Content Test Results

[0267] In order to evaluate the effects of the test substances alone or in combination on the antioxidant effect of mice, the GR content in the spleen of each group of mice was measured. The results are shown in Table 8.

[0268] Table 8. GR content in spleen of mice in each group (mean ± SD, n = 12)

[0269] Note: * Compared with the blank control group, p<0.05, ** Compared with the blank control group, p<0.01, *** Compared with the blank control group, p < 0.001, ns means there is no statistical significance compared with the blank control group; # Compared with the CPP low-dose group (50 mg / kg.bw), p < 0.05; & Compared with the CPP medium dose group (150 mg / kg.bw), p < 0.05, && Compared with the CPP medium-dose group (150 mg / kg.bw), p < 0.01; ^^ Compared with the CPP high-dose group (350 mg / kg.bw), p < 0.01, ^^^ Compared with the high-dose CPP group (350 mg / kg.bw), p < 0.001.

[0270] As shown in Table 8, Experimental Example 3 using CPP alone and Experimental Example 5 using BB536 alone had no effect on the GR content, while Experimental Example 6 using a combination of the two showed a synergistic effect.

[0271] In addition, Experimental Example 7 also significantly increased the GR content compared to Experimental Examples 2 and 5, and Experimental Example 8 also significantly increased the GR content compared to Experimental Examples 1 and 5. Therefore, further increasing the amount of BB536 also has the same effect.

Claims

1. Use of a functional nutritional composition in preparing a composition or food having the effect of improving human immunity and / or enhancing human antioxidant capacity, characterized in that: The functional nutritional composition comprises the following two essential components: i) casein phosphopeptides, ii) Bifidobacterium probiotics; Furthermore, in the functional nutritional composition, the ratio of i) casein phosphopeptide to ii) bifidobacterium probiotics is configured as follows: Effective amount of casein phosphopeptide: Effective amount of Bifidobacterium probiotics is (50mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU).

2. The use according to claim 1, characterized in that The effective amount of casein phosphopeptide: the effective amount of Bifidobacterium probiotics is (120mg~380mg): (1.00×10 9 CFU~1.20×10 11 CFU); the functional nutritional composition has the effects of improving human immunity and enhancing human antioxidant capacity.

3. The use according to claim 1, characterized in that The improvement of human immunity is achieved by promoting the increase of serum complement C3; the improvement of human antioxidant capacity is achieved by providing spleen antioxidant capacity.

4. The use according to any one of claims 1 to 3, characterized in that: The Bifidobacterium probiotics include any one or more of Bifidobacterium adolescentis, Bifidobacterium animalis subspecies animalis, Bifidobacterium animalis subspecies lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subspecies longum and Bifidobacterium longum subspecies infantis.

5. The use according to claim 4, characterized in that The Bifidobacterium longum subspecies longum includes Bifidobacterium longum subspecies longum BB536.

6. The use according to any one of claims 1 to 3, characterized in that: The functional nutritional composition further comprises other nutritional ingredients, which include one or more of proteins, vitamins, monosaccharides, polysaccharides or plant extracts.

7. The use according to any one of claims 1 to 3, characterized in that: The functional nutritional composition is liquid, semi-solid or solid.

8. The use according to any one of claims 1 to 3, characterized in that: The improvement of human immunity includes increasing the content of immunoglobulin and / or complement.

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