Branched chain fatty acid composition and use thereof in regulating brain nerve immunity

Abstract

A branched chain fatty acid composition, and a use thereof in regulating brain nerve immunity. The branched chain fatty acid composition comprises C15:0 and C17:0. By using the branched chain fatty acid composition to promote LPS-induced microglia, the tendency to promote the differentiation of microglia into M2-phenotype microglia is higher than the tendency to promote the differentiation of microglia into M1-phenotype microglia. Also, the secretion of anti-inflammatory factors such as IL-4 and IL-10 is promoted, and the secretion of pro-inflammatory factors such as NO, TNF-a and IL-1β is inhibited. C15:0 and C17:0 in the branched chain fatty acid composition have a good synergistic effect, and have the function of regulating brain nerve immunity.

Description

Branched-chain fatty acid compositions and their use in regulating brain neuroimmunity Technical Field

[0001] This invention relates to branched-chain fatty acid compositions and their use in regulating brain neuroimmunity, and more specifically to branched-chain fatty acid compositions containing C15:0 and C17:0 and their use in regulating brain neuroimmunity, belonging to the field of nutrient research. Background Technology

[0002] The human gut performs complex physiological functions, including absorption, digestion, motility, secretion, regulation of the gut microbiota, and maintenance of intact barrier function. The enteric nervous system plays a crucial role in coordinating these processes, exhibiting a high degree of autonomy. It forms an external connection with the central nervous system through the autonomic nervous system, creating the gut-brain axis. The gut is also the largest immune organ in the human body, and neuroimmunity is a complex and important physiological process involving the bidirectional regulation and interaction between the nervous and immune systems. Glial cells are immune cells of the central nervous system, and their participation in neuroinflammatory processes and the induction of nerve damage repair are key mechanisms for their immune function. For a long time, controlling neuroinflammatory diseases has been a focus of treatment for degenerative diseases and traumatic injuries of the central nervous system. Currently, there are no effective medical treatments for many neurodegenerative diseases, such as Alzheimer's disease and stroke. The gut-brain axis has become a breakthrough point in medical treatment. By regulating the gut microbiota through drugs or fecal microbiota transplantation, and altering the gut microenvironment, the gut microbiota can be continuously restored from multiple dimensions of the microenvironment, such as microbiota structure and short-chain fatty acids, thereby improving the occurrence and development of neurodegenerative diseases. For example, the study cited in Reference 1 found that gut microbiota imbalance can produce bacterial metabolites that affect peripheral immune cells. Once peripheral immune cells are affected, they promote inflammation in the central nervous system, leading to Tau accumulation and neurodegeneration in the brain. Short-term antibiotic treatment or aseptic feeding can remodel or eliminate gut microbiota and reduce its metabolites, thereby exerting a neuroprotective effect and reducing Tau accumulation and neurodegeneration. However, many mechanisms remain unclear.

[0003] Branched-chain fatty acids (BCFAs) are fatty acids with one or more branched alkyl groups or other functional groups on their alkyl chains. When the branched alkyl group is located on the second carbon atom from the alkyl end, it is called an isomer or iso-fatty acid; when it is located on the third carbon atom from the alkyl end, it is called an anteiso-fatty acid. Due to their unique branched structure, BCFAs possess excellent physicochemical properties: low freezing and pour points; good thermal and oxidative stability; good solubility; low foaming properties; and good air and water permeability in the membranes they form. BCFAs are widely distributed in nature, but in low amounts. It has been reported that certain amounts of BCFAs with different structures are present in the milk and internal tissues of ruminant animals (such as cow and sheep milk and meat products), and in human milk. BCFAs are abundant in vernix caseosa and the neonatal intestine. The BCFA profile of meconium shows that BCFAs are the main components of the gastrointestinal tract of normal, healthy, full-term newborns. Premature infants have significantly fewer BCFAs in their intestines than normally healthy infants and have a higher probability of developing necrotizing enterocolitis. Numerous studies have shown that branched-chain fatty acids (BCFAs) possess unique physiological regulatory functions, including anti-inflammatory, anti-cancer, and gut health-improving effects, thereby reducing the incidence of necrotizing enterocolitis in premature infants. Previous studies and patents have already confirmed the efficacy of branched-chain fatty acids in inhibiting intestinal inflammation. Results have shown that both iso-BCFAs and anteiso-BCFAs have significant anti-inflammatory effects on the intestines.

[0004] For example, cited reference 2 discloses a composition containing branched-chain fatty acids such as 14-methylpentadecanoic acid, 12-methyltridecanoic acid, and 13-methyltetradecanoic acid. Adding this composition to an LPS-induced cellular inflammation model inhibits the production of ROS in calf small intestinal epithelial cells; feeding this composition to mice with DSS-induced colitis regulates the levels of inflammatory factors in the mouse intestine and improves gastrointestinal health.

[0005] Reference 3 discloses the addition of branched-chain fatty acids (BCFAs) to a CaCo2 cell culture system and the administration of BCFAs via gavage to mice with DSS-induced enteritis. The results showed that BCFAs could induce hyperSUMOylation of intestinal proteins in CaCo2 cells, thereby reducing the expression of pro-inflammatory cytokines. In addition, BCFAs also inhibited DSS-induced intestinal inflammation, reduced the activity of intestinal cell deaminases, and promoted intestinal epithelial integrity.

[0006] Similarly, reference 4 also discloses the function of branched-chain fatty acids in inhibiting intestinal inflammation and enhancing intestinal resistance in mice with DSS-induced enteritis. Reference 5 also discloses that branched-chain fatty acids can reduce the risk of necrotizing enterocolitis (NEC) and increase the expression of IL-10 in the intestine.

[0007] References

[0008] Cited literature 1: Seo D, O'Donnell D, Jain N, et al. ApoE isoform–and microbiota-dependent progression of neurodegeneration in a mouse model of tauopathy[J].Science, 2023, 379(6628):eadd1236.

[0009] Reference 2: CN118416112B;

[0010] Reference 3: WO2023031226A1;

[0011] Reference 4: CN115300530A;

[0012] Reference 5: Ran-Ressler RR, et al. Branched chain fatty acids reduce the incidence of necrotizing enterocolitis and alter gastrointestinal microbial ecology in a neonatal rat model. PLoS One. 2011; 6(12): e29032. Summary of the Invention

[0013] The problem the invention aims to solve

[0014] The gut-brain axis is currently considered an important mechanism for the occurrence of neurodegenerative phenomena, and more research is focused on using the gut microbiota as a target to regulate the gut microecological environment and thus improve the occurrence and development of neurological diseases.

[0015] In the process of researching branched-chain fatty acids, this invention unexpectedly discovered that branched-chain fatty acids also have certain effects in regulating neuroimmunity in the brain and alleviating and improving neuroinflammation. Furthermore, branched-chain fatty acid compositions that can regulate inflammation caused by nerve damage by modulating microglial cell phenotypic polarization were screened.

[0016] It should be noted that the neuroinflammation mentioned in this statement refers to a non-pathological state in which the nerves are not fully healthy due to the presence of inflammatory factors.

[0017] Solution for solving the problem

[0018] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0019] [1]. A branched-chain fatty acid composition, wherein the branched-chain fatty acid comprises C15:0 and C17:0,

[0020] C15:0 and C17:0 are anti-isomers;

[0021] In terms of mass,

[0022] The ratio of the content of C15:0 to the content of C17:0 is 3:7 to 6:4.

[0023] [2]. The composition according to [1], wherein,

[0024] The ratio of the content of C15:0 to the content of C17:0 is 3.5:6.5 to 5.5:4.5.

[0025] [3]. The composition according to [1] or [2], wherein, based on the total mass of the branched-chain fatty acid composition,

[0026] The content of C15:0 is 30% to 60%, and the content of C17:0 is 40% to 70%.

[0027] [4]. Use of the composition described in any one of [1] to [3] in the preparation of a food that helps regulate brain neuroimmunity.

[0028] [5]. According to the use described in [4], the aid in regulating brain neuroimmunity includes regulating microglia polarization.

[0029] [6]. According to the use described in [5], wherein the regulation of microglia polarization includes at least one of inhibiting microglia polarization to the M1 phenotype, promoting microglia polarization to the M2 phenotype, increasing the amount of IL-4 and / or IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β, and / or NO released by microglia.

[0030] [7]. The use according to any one of [4] to [6], wherein the food is a confectionery, beverage, dairy product or baked food.

[0031] [8]. According to the use described in [7], the candy includes at least one of hard candy, gel candy, shortbread candy, compressed candy and aerated candy; the beverage includes at least one of carbonated beverage, tea beverage, coffee beverage, fruit and vegetable juice beverage and lactic acid bacteria beverage; the dairy product includes at least one of fermented milk, cheese and milk powder; the baked goods include at least one of bread, cake and biscuit.

[0032] Use of the branched-chain fatty acid composition described in any one of [9]. [1] to [3] in the preparation of food for the purpose of assisting in the maintenance of the health of the central nervous system.

[0033]

[0010] . According to the use described in [9], the assistance in maintaining the health of the central nervous system includes relieving neuroinflammation for non-therapeutic purposes.

[0034] The effects of the invention

[0035] The branched-chain fatty acid composition provided by this invention, when applied to LPS-induced microglia in the brain, promotes the differentiation of microglia into M2 phenotype microglia more than into M1 phenotype microglia, thereby promoting the secretion of anti-inflammatory cytokines such as IL-4 and IL-10 and inhibiting the secretion of pro-inflammatory cytokines such as NO, TNF-α, and IL-1β. The C15:0 and C17:0 components in the branched-chain fatty acid composition exhibit a good synergistic effect, playing a role in regulating brain neuroimmunity. Attached Figure Description

[0036] Figure 1 shows the expression levels of iNOS and CD206 mRNA in BV2 cells of each group as detected by RT-PCR; where **** represents p<0.0001. Detailed Implementation

[0037] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0038] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0039] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0040] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0041] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0042] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0043] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0044] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0045] In this invention, the terms “a”, “an”, or “the” can mean “one”, “one or more”, “at least one”, or “one or more”.

[0046] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0047] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, while specific related values ​​have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range.

[0048] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] This invention is mainly based on the following insights:

[0050] Branched-chain fatty acids (BCFAs) have been widely reported for improving gut health, but their other functions remain to be explored and studied. This invention uses LPS-induced microglia, the brain's neuroimmune cells, as an in vitro neuroinflammation model. First, BCFAs with low cytotoxicity and appropriate intervention concentrations are selected. Then, different concentrations of BCFAs are blended to obtain BCFA compositions, exploring how these compositions can improve neuroinflammation and thus regulate the brain's neuroimmune state.

[0051] I. Branched-chain fatty acid composition

[0052] The branched-chain fatty acid composition of the present invention comprises C15:0 and C17:0. In some preferred embodiments, both C15:0 and C17:0 are anteiso.

[0053] In this invention, "iso-branched fatty acid" or "iso-branched fatty acid (iso-BCFA)" refers to a fatty acid with a methyl branch on the penultimate carbon atom of the fatty acid molecule's carbon chain backbone. "Anti-iso-branched fatty acid" or "antiso-BCFA" refers to a fatty acid with a methyl branch on the penultimate carbon atom of the fatty acid molecule's carbon chain backbone.

[0054] Furthermore, regarding the content ratio of C15:0 to C17:0 in the fatty acid composition, it has been found that the technical effects of the present invention can be achieved by setting the following mass ratio: the content ratio of C15:0 to C17:0 is 3:7 to 6:4. In some preferred embodiments, the content ratio of C15:0 to C17:0 is 3.5:6.5 to 5.5:4.5, more preferably 6:13 to 7:6, and even more preferably 7:13 to 13:12. For example, the content ratio of C15:0 to C17:0 is 3:7, 3.5:7, 3.5:6.5, 4:6.5, 4:7, 4.5:7, 4.5:6.5, 5:7, 5.5:7, 5.5:6.5, 5.5:6, 6:7, 6.5:7, 7:7, 7.5:7, 8:7, 8.5:7, 9:7, 9.5:7, 10:7, 10.5:7, 3:6, 3.5:6, 4:6, 4.5:6, 5:6, 5.5:6, 6:6, 6.5:6, 7:6, 7.5:6, 8:6, 8.5:6, 9:6. 2.1:5, 2.2:5, 2.3:5, 2.4:5, 2.5:5, 3:5, 3.5:5, 4:5, 4.5:5, 5:5, 5.5:5, 5.5:4.5, 5.5:4, 6:5, 6.5:5, 7:5, 7.5:5, 1.68:4, 2:4, 3:4, 4:4, 5:4, 6:4, 6:13, 7:13, 8:13, 9:13, 10:13, 11:13, 12:13, 13:13, 14:13, 13:12, 12:12, 11:12, 10:12, 9:12, 8:12, 7:12.

[0055] In addition to the C15:0 and C17:0 fatty acids described above, the branched-chain fatty acid composition of the present invention may optionally contain or not contain other types of fatty acids. These other types of fatty acids may be branched or linear. Furthermore, the amount of these other types of fatty acids is not particularly limited in principle. In some preferred embodiments, the total content of the C15:0 and C17:0 fatty acids, based on the total mass of the branched-chain fatty acid composition, may be 10% to 100%, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0056] In some embodiments, the C15:O content, based on the total mass of the branched-chain fatty acid composition, is 30% to 60%, preferably 35% to 55%, more preferably 35% to 53%, and even more preferably 38% to 52%, for example 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%. %, 57%, 58%, 59%, 60%; the C17:0 is 40% to 70%, preferably 45% to 65%, more preferably 47% to 65%, even more preferably 48% to 62%, for example 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 58.5%, 58.7%, 58.8%, 59%, 60%, 61%, 61.5%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%.

[0057] This invention does not particularly limit the source of branched-chain fatty acids. For example, they can be isolated and extracted from natural substances containing branched-chain fatty acids, such as bacterial biofilms, animal sebum, mammary tissue, and plants. They can also be prepared through biological or chemical synthesis, such as using microorganisms or *C. elegans* for synthesis. Exemplarily, branched-chain fatty acids are synthesized in microorganisms or *C. elegans* using branched-chain amino acids as substrates through enzymatic reactions. Currently, the commonly used method in this field is to isolate and extract branched-chain fatty acids from lanolin. Lanolin undergoes a saponification reaction to prepare free lanolin alcohol and free lanolin acid soap. Branched-chain fatty acids from lanolin are then obtained through multiple steps, including alcohol-soap separation and fatty acid preparation and extraction.

[0058] II. Food

[0059] The food products of this invention contain or use the aforementioned branched-chain fatty acid compounds, thereby including C15:0 and C17:0 in the food products of this invention. In some preferred embodiments, both C15:0 and C17:0 are trans isomers.

[0060] This invention does not impose any particular limitation on the absolute content of C15:0 and C17:0 in food, as long as it complies with relevant laws and regulations, such as the quantitative limits for fatty acids in food as specified in GB 5009.168-2016 "National Food Safety Standard - Determination of Fatty Acids in Food". In some embodiments, the content of C15:0 is not higher than 0.0033 g / 100g in solid foods, for example, 0.0001 to 0.0033 g / 100g; and not higher than 0.0013 g / 100g in liquid foods, for example, 0.0001 to 0.0013 g / 100g. In some implementations, the content of C17:0 is no higher than 0.0066g / 100g in solid foods, for example 0.0001 to 0.0066g / 100g, and no higher than 0.0026g / 100g in liquid foods, for example 0.0001 to 0.0026g / 100g.

[0061] This invention does not specifically limit the types of food products, but may include candies, beverages, dairy products, or baked goods. Examples of candies include hard candies, shortbread candies, gel candies, compressed candies, and aerated candies. Examples of beverages include carbonated drinks, tea drinks, coffee drinks, fruit and vegetable juices, and lactic acid bacteria drinks. Examples of dairy products include fermented milk, cheese, and milk powder. Examples of baked goods include bread, cakes, and biscuits. Furthermore, the food products described in this invention can also be health foods, such as various types of oral preparations, including tablets, pills, granules, powders, capsules, and oral liquids.

[0062] In addition to C15:0 and C17:0 in the branched-chain fatty acid composition, in some embodiments, depending on the type of food and the final needs of the target audience, the food of the present invention may also contain any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0063] Examples of plant-based ingredients include fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, banana, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; and rice (indica rice). Grains or their extracts, including japonica rice, glutinous rice, cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans, etc.; nuts or their extracts, including walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, ginkgo nuts, etc.; coffee or its extracts; and some medicinal and edible plant-based Chinese medicinal materials or their extracts.

[0064] Animal dairy product ingredients can include fresh milk from mammals such as cows, sheep, and camels, as well as reprocessed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0065] Examples of animal meat product ingredients include those from pork, beef, mutton, seafood, or poultry.

[0066] Examples of functional additives include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, and functional polyunsaturated fatty acid supplements.

[0067] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0068] III. Uses that help regulate the brain's neuroimmune system

[0069] This invention unexpectedly discovered that branched-chain fatty acid compositions containing C15:0 and C17:0, particularly those containing trans-isomer C15:0 and trans-isomer C17:0, have a modulating effect on neuroimmunity in the brain. Furthermore, when C15:0 and C17:0 are combined, they exhibit a synergistic effect, further enhancing the modulating effect on neuroimmunity. Therefore, the branched-chain fatty acid compositions containing C15:0 and C17:0 described in this invention, as well as foods containing or using said branched-chain fatty acid compositions, all possess a modulating effect on neuroimmunity in the brain. However, the modulating effect on neuroimmunity described in this invention is not intended for the prevention or treatment of diseases.

[0070] Furthermore, the present invention provides the use of branched-chain fatty acid compositions containing C15:0 and C17:0 in the preparation of foods that help regulate brain neuroimmunity.

[0071] In some embodiments, the effect of assisting in regulating brain neuroimmunity includes regulating the polarization of microglia, the neuroimmune cells of the brain. In some specific embodiments, regulating microglia polarization includes at least one of inhibiting microglia polarization towards the M1 phenotype, promoting microglia polarization towards the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglia, and decreasing the amount of TNF-α, IL-1β, and / or NO released by microglia. In some specific embodiments, regulating microglia polarization includes inhibiting the tendency of microglia to polarize towards the M1 phenotype, promoting the tendency of microglia to polarize towards the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglia, and decreasing the amount of TNF-α, IL-1β, and / or NO released by microglia. In some specific implementations, the regulation of microglia polarization includes inhibiting the tendency of microglia to polarize towards the M1 phenotype, promoting the tendency of microglia to polarize towards the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β, and NO released by microglia.

[0072] Furthermore, the aforementioned role in regulating brain neuroimmunity includes promoting the development of brain nerves and the nervous system.

[0073] In some implementations, the nervous system is the central nervous system.

[0074] IV. Uses to assist in improving or maintaining the health of the central nervous system

[0075] This invention unexpectedly discovered that combining C15:0 and C17:0, for example, combining trans-isomer C15:0 and trans-isomer C17:0, can help improve or maintain the health of the central nervous system, especially improving discomfort or lack of well-being caused by neuroinflammation in a non-pathological state. Furthermore, when C15:0 and C17:0 are combined, they have a synergistic effect, further enhancing the effect of improving neuroinflammation. Therefore, the branched-chain fatty acid composition containing C15:0 and C17:0 described in this invention also has the effect of helping to improve or maintain the health of the central nervous system and improving discomfort or lack of well-being caused by neuroinflammation in a non-pathological state.

[0076] Furthermore, the present invention provides the use of a branched-chain fatty acid composition containing C15:0 and C17:0 in the preparation of a food for the purpose of assisting in the maintenance of the health of the central nervous system.

[0077] In some implementations, the assistance in maintaining central nervous system health includes non-therapeutic relief of neuroinflammation that does not reach a medically pathological level.

[0078] Example

[0079] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0080] 1. Instruments and reagents

[0081] Mouse microglia BV-2 cell line (Shanghai Enzyme Research Institute, catalog number CC-Y2022), fetal bovine serum (Vicente, catalog number 085-150), high glucose DMEM medium (Ciscotech, catalog number CA0004-500ML), antibiotics, culture flasks, well plates, centrifuge tubes, trypsin, dimethyl sulfoxide, lipopolysaccharide, kits, etc.

[0082] 2. Experimental Methods

[0083] 2.1 Cell Culture

[0084] Mouse microglia (BV2) cell line was cultured in high-glucose DMEM medium (containing 10% fetal bovine serum) and placed in a 5% CO2 incubator (37°C, saturated humidity).

[0085] 2.2 Cytotoxicity assay

[0086] BV2 microglia viability was detected using the CCK8 assay (CCK8 kit purchased from White Shark, catalog number BS350A).

[0087] Methods for evaluating the cytotoxicity of nutrients: BV2 cells in the logarithmic growth phase were selected and cultured at a density of 5 × 10⁶ cells per well. 4 Cells were seeded at a rate of [number] cells / mL in 96-well plates and allowed to adhere overnight. Then, different concentrations of branched-chain fatty acids were added for 12 hours. After treatment, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for 4 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0088] According to the formula: Cell viability = [(Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells)] × 100% (where experimental wells contain cells, culture medium, nutrients of different concentrations and CCK-8 solution; blank wells contain culture medium and CCK-8 solution, but not cells and nutrients; control wells contain cells, culture medium and CCK-8 solution, but not nutrients), the cell viability is finally calculated.

[0089] 2.3 Evaluation of anti-inflammatory efficacy

[0090] 1) Establishment of a microglial cell inflammation model and nutritional intervention methods

[0091] Select BV2 cells in the logarithmic growth phase, using 5 × 10⁶ cells per well. 4 Cells were seeded at a rate of 10 cells / mL in 96-well plates and allowed to adhere overnight before being divided into groups for intervention. (1) Control group: cells were cultured using conventional methods; (2) LPS group (model group): BV2 cells were treated with LPS (1 μg / mL) for 24 h to establish a cell neuroinflammation model; (3) Experimental group (example and comparative group): BV2 cells were stimulated with LPS for 24 h, and then 10 different combinations were added for 12 h of intervention.

[0092] 2) NO level measurement

[0093] The nitrate reductase method was used to detect the NO (nitric oxide) level in BV2 microglia culture medium. Experimental grouping and nutrient intervention methods were as shown in 1) above. Cell culture medium was collected from each group. The NO detection kit was used according to the instructions, and the absorbance of each tube was measured at a wavelength of 550 nm and a light path of 0.5 cm. The NO content was then calculated.

[0094] 3) Measurement of inflammatory factor levels

[0095] The levels of IL-1β, TNF-α, IL-10, and IL-4 in the supernatant of BV2 cells were detected using ELISA. The experimental grouping and nutrient intervention methods were the same as described above. Cell supernatant was collected from each group, and the ELISA kit was operated according to the instructions. The absorbance values ​​of each group were measured sequentially at a wavelength of 405 nm using a microplate reader to detect the levels of IL-1β, TNF-α, IL-4, and IL-10.

[0096] 2.4 Detection of iNOS and CD206, phenotypic markers of microglia M1 and M2 phenotypes

[0097] The mRNA expression levels of iNOS and CD206-related genes were detected using qRT-PCR to determine the changes in the content of M1 and M2 cell populations. Third-generation BV2 microglia suspension was seeded in 24-well plates at a density of 4 × 10⁵ cells / well, and cells were grouped according to the experimental design. After treatment, cells were washed twice with PBS (200 μL per well). Trypsin digestion was performed for 2.5 min, and the digestion was terminated with 400 μL of culture medium. Cells were collected by pipetting into enzyme-free centrifuge tubes. Total RNA was extracted from each group of cells. cDNA was obtained by reverse transcription using a reverse transcription kit according to the prescribed procedure. The amplified cDNA was mixed with primers, fluorescent probes, and qPCR Master Mix. The appropriate PCR cycle program and parameters were set according to qPCR requirements, and the corresponding mRNA expression levels were analyzed using a real-time PCR detection system. Specific primer information is as follows:

[0098] iNOS:

[0099] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO.1),

[0100] Downstream: 5'-TGCAAGTGAAATCCGATGTGG-3' (SEQ ID NO.2);

[0101] CD206:

[0102] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO.3),

[0103] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO.4);

[0104] GAPDH:

[0105] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO.5),

[0106] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO.6).

[0107] 3. Detection of cytotoxicity of various branched-chain fatty acids

[0108] In this invention, four branched-chain fatty acids—iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0—were selected to investigate the regulatory effects of different combinations on neuroinflammation. First, the cytotoxic effects of the four branched-chain fatty acids on microglia were investigated, with treatments using concentrations of 5, 10, 25, 50, 75, and 100 μmol / L of branched-chain fatty acids, respectively. The results are shown in Table 1. The table shows that the cytotoxic effect of iso-C17:0 decreased to 90.79% at a concentration of 25 μmol / L, while the cell viability remained above 100% at a concentration of the other three branched-chain fatty acids. Therefore, in this invention, the concentration of branched-chain fatty acids was uniformly determined to be 25 μmol / L.

[0109] Table 1. Effects of different concentrations of branched-chain fatty acids on microglial cell viability (%)

[0110] 4. The inhibitory effect of branched-chain fatty acids on neuroinflammation

[0111] Microglia, as resident immune cells of the central nervous system, are key participants in neuroinflammation. LPS-induced treatment of microglia accelerates the transcription of inflammatory genes by activating the Toll-like receptor (TLR4) NF-κB signaling pathway, further leading to the differentiation of microglia into M1 and M2 phenotypes.

[0112] M1 phenotype microglia secrete pro-inflammatory cytokines such as NO, IL-1β, and TNF-α, exacerbating the inflammatory response. NO free radicals are synthesized by enzymes such as NOS (eNOS) and iNOS via the L-arginine pathway. Under normal physiological conditions, iNOS remains dormant in dormant cells. However, under pathological conditions, it produces large amounts of NO and plays a dual role in chronic infection and inflammation. Reducing NO production may be an effective strategy to improve inflammation. IL-1β has strong pro-inflammatory activity and can induce various pro-inflammatory mediators, such as cytokines and chemokines. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine produced by various cells in response to inflammatory responses and immune regulation, and can induce apoptosis.

[0113] M2 macrophages secrete anti-inflammatory cytokines such as IL-4 and IL-10, reducing neuroinflammatory responses. IL-4, a typical type II immune cytokine, plays a crucial immunomodulatory role in type II immune diseases such as asthma, allergic dermatitis, and urticaria. Studies have shown that IL-4 can promote macrophage differentiation into M2 macrophages, thereby exerting an anti-inflammatory effect. Interleukin-10, a multicellular, multifunctional cytokine, regulates cell growth and differentiation, participates in inflammatory and immune responses, and is a recognized inflammatory and immunosuppressive factor.

[0114] Therefore, regulating the conversion of microglia to the M2 type is a potential strategy for suppressing neuroinflammation. This invention investigated the effects of different proportions of branched-chain fatty acids on LPS-induced inflammation in BV2 microglia.

[0115] Examples 1-5

[0116] A microglial cell inflammation model was established following step 1) in section 2.3 above, and nutritional intervention was implemented. The branched-chain fatty acid composition is shown in Table 2, and the concentration of branched-chain fatty acids in the microglial cell inflammation model was 25 μmol / L. After the intervention, NO levels, inflammatory factor levels, and the contents of iNOS and CD206 were measured.

[0117] Comparative Examples 1-5

[0118] A microglial cell inflammation model was established following step 1) in section 2.3 above, and nutritional intervention was implemented. The branched-chain fatty acid composition is shown in Table 2, and the concentration of branched-chain fatty acids in the microglial cell inflammation model was 25 μmol / L. After the intervention, NO levels, inflammatory factor levels, and the contents of iNOS and CD206 were measured.

[0119] Table 2. Design of different combinations of branched-chain fatty acids (%)

[0120] By applying different branched-chain fatty acid compositions to a microglial inflammation model, the inventors unexpectedly discovered the regulatory effects of two medium-chain branched-chain fatty acids of type α (antisomeric form) on neuroinflammation. The specific results are as follows:

[0121] Table 3. Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia.

[0122] Table 3 shows the effects of different combinations of branched-chain fatty acid treatments on LPS-induced pro-inflammatory factors in BV2 microglia.

[0123] As shown in the table, the levels of pro-inflammatory factors NO, TNF-α, and IL-1β in microglia significantly increased after LPS induction, indicating that LPS induction caused severe damage to microglia and the production of a large number of pro-inflammatory factors. In the experimental and control groups treated with different branched-chain fatty acid compositions in combination with LPS, the levels of pro-inflammatory factors decreased to varying degrees, as shown in Table 3. As can be seen from the table, the NO production in the examples and comparative examples was lower than that in the model group. The NO production in Example 1 was 15.71 μM / L, reaching the level of the control group before LPS treatment. After one-way ANOVA, it was found that the NO production in Examples 1 to 5 was significantly lower than that in the model group (p < 0.0001). Comparative Examples 1 and 2 were also significantly lower than that in the model group (p < 0.0001). However, the effect of Comparative Example 3 was worse. Although the NO production was lower than that in the model group, it was found to be not significantly different from that in the model group after the differential significance analysis (p > 0.05). Although Comparative Examples 4 and 5 were significantly lower than that in the model group (p < 0.01 and p < 0.05), the NO production in Comparative Examples 4 and 5 was still higher than that in Examples 1 to 5. Furthermore, after differential significance analysis, it was found that the NO production in Examples 1 to 3 was significantly lower than that in Comparative Example 5 (p < 0.01), and the NO production in Example 1 was significantly lower than that in Comparative Example 4 (p < 0.05).

[0124] Table 3 shows that different branched-chain fatty acid compositions have a stronger inhibitory effect on TNF-a. Examples 1 to 5 all significantly reduced TNF-a to the level of the control group. In particular, the amount of TNF-a produced in Examples 1 to 4 was lower than that in the control group. Comparative Examples 1 to 5 also inhibited the production of TNF-a, but the effect was not as good as that of the examples. The amount of TNF-a produced was higher than that in Examples 1 to 5.

[0125] The branched-chain fatty acid compositions all showed significant inhibitory effects on the anti-inflammatory factor IL-1β. Compared with the model group, each group showed a significant reduction (p < 0.0001). However, the IL-1β production in Examples 1-5 was lower than that in Comparative Examples 1-5. After significance analysis, Example 1 was significantly lower than Comparative Examples 1-5 (p < 0.01 and p < 0.0001), and Examples 2-5 were extremely significantly lower than Comparative Examples 2-5 (p < 0.0001). This indicates that when the anteiso-C15:0 concentration is 35%-53%, it has a significant inhibitory effect on the production of anti-inflammatory factors.

[0126] Table 4. Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia.

[0127] Table 4 shows the effects of different combinations of branched-chain fatty acid treatments on LPS-induced anti-inflammatory factors in BV2 microglia. The table shows that different branched-chain fatty acid combinations have different effects on the production of anti-inflammatory factors. Regarding the anti-inflammatory factor IL-10, this invention found that Examples 1-5 and Comparative Example 1 all increased the production of IL-10, while the production levels of IL-10 in Comparative Examples 2-5 were close to or lower than the model group, showing no effect on IL-10 production. After significant difference analysis, it was found that Examples 1-5 had no significant difference from the control group (p>0.05), further verifying that the examples could achieve the same effect on the production of anti-inflammatory factor IL-10 as the untreated control group. Table 4 also shows the effects of each group on the production of anti-inflammatory factor IL-4. The results show that the overall effect of branched-chain fatty acids in promoting IL-4 production is not very good, but the production levels of IL-4 in Examples 1-5 were higher than the model group, while the IL-4 levels in Comparative Examples 1-5 were lower than the model group, indicating that the branched-chain fatty acid combinations in the comparative examples did not promote IL-4 production. Combining the production of the two anti-inflammatory factors mentioned above, it is demonstrated that when the proportion of anteiso-C15:0 is 35%-53%, it has a significant effect on promoting the production of the anti-inflammatory factor IL-10.

[0128] To determine whether the inhibition of BV2 cell inflammatory response by the branched-chain fatty acid composition is related to phenotypic polarization, this invention used RT-qPCR to detect the mRNA expression of the M1 phenotypic marker iNOS and the M2 phenotypic marker CD206 in BV2 cells. The results are shown in Figure 1. As can be seen from the figure, the expression level of iNOS, a marker of M1 cell polarization, was significantly lower in all five examples than in the model group (p < 0.0001), especially in Examples 1 and 2, where the mRNA expression level of iNOS was significantly lower than that in the control group. Conversely, the mRNA expression level of CD206 was significantly higher in all five examples than in the model group (p < 0.0001), and the CD206 production in Examples 1 and 3 was close to that in the control group. These results further demonstrate that the branched-chain fatty acid compositions of the five examples have a regulatory effect on LPS-induced neuroinflammation in BV2 cells, can promote microglia differentiation into the M2 type, and promote the production of anti-inflammatory factors.

Claims

1. A branched-chain fatty acid composition, characterized in that, The branched-chain fatty acids comprise C15:0 and C17:

0. C15:0 and C17:0 are anti-isomers; In terms of mass, The ratio of the content of C15:0 to the content of C17:0 is 3:7 to 6:

4.

2. The composition according to claim 1, characterized in that, The ratio of the content of C15:0 to the content of C17:0 is 3.5:6.5 to 5.5:4.

5.

3. The composition according to claim 1 or 2, characterized in that, Based on the total mass of the branched-chain fatty acid composition, The content of C15:0 is 30% to 60%, and the content of C17:0 is 40% to 70%.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a food that helps regulate brain neuroimmunity.

5. The use according to claim 4, characterized in that, The benefits of this approach to regulating brain neuroimmunity include modulating microglia polarization.

6. The use according to claim 5, characterized in that, The regulation of microglia polarization includes at least one of the following: inhibiting microglia polarization towards the M1 phenotype, promoting microglia polarization towards the M2 phenotype, increasing the amount of IL-4 and / or IL-10 released by microglia, and decreasing the amount of TNF-α, IL-1β, and / or NO released by microglia.

7. The use according to any one of claims 4 to 6, characterized in that, The food products mentioned are candies, beverages, dairy products, or baked goods.

8. The use according to claim 7, characterized in that, The candy includes at least one of hard candy, gel candy, shortbread candy, compressed candy, and aerated candy; the beverage includes at least one of carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages; the dairy product includes at least one of fermented milk, cheese, and milk powder; and the baked goods include at least one of bread, cakes, and biscuits.

9. Use of the branched-chain fatty acid composition according to any one of claims 1 to 3 in the preparation of a food for the purpose of assisting in the maintenance of the health of the central nervous system.

10. The use according to claim 9, characterized in that, The support for maintaining the health of the central nervous system includes relieving neuroinflammation for non-therapeutic purposes.

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