A branched-chain fatty acid composition and its use in assisting in maintaining the health of the nervous system

Abstract

The present invention relates to a branched fatty acid composition and its use in assisting the maintenance of nervous system health, and belongs to the field of nutrient research. The branched fatty acid composition provided by the present invention comprises C14:0, C15:0, C16:0 and C17:0. After LPS-induced microglia are treated with the branched fatty acid composition, the trend that the microglia can be promoted to differentiate into M2 phenotype microglia is higher than the trend that the microglia are promoted to differentiate into M1 phenotype microglia, while promoting the secretion of anti-inflammatory factors such as IL-4 and IL-10, and inhibiting the secretion of pro-inflammatory factors such as NO, TNF-a, and IL-1β. C14:0, C15:0, C16:0 and C17:0 in the branched fatty acid composition have a good synergistic effect, which plays a role in regulating brain immunity and maintaining the health of the central nervous system.

Description

Technical Field

[0001] The present invention relates to a branched fatty acid composition and use thereof in assisting in maintaining the health of the nervous system, and more specifically to a branched fatty acid composition containing C14:0, C15:0, C16:0 and C17:0 and use thereof in regulating brain immunity, and belongs to the field of nutrient research. Background Art

[0002] The human intestine performs complex physiological functions, including absorption, digestion, motility, secretion, regulation of intestinal microbiota, and maintenance of intact barrier function. The enteric nervous system is crucial to the coordination of these processes. It has a high degree of autonomy and forms an external connection with the central nervous system through the autonomic nervous system, forming the gut-brain axis. The communication mechanism of the gut-brain axis includes three main pathways: first, intestinal microorganisms stimulate the enteric nervous system and then transmit information to the central nervous system through the vagus nerve of the autonomic nervous system; second, intestinal microorganisms produce or induce intestinal epithelial secretory cells to produce metabolites or neurotransmitters, such as 5-HT, GABA, dopamine, short-chain fatty acids (SCFAs), etc. These substances enter the circulatory system and eventually cross the blood-brain barrier to achieve the purpose of regulating the central nervous system; third, intestinal microorganisms regulate the immune system by producing microbial-associated molecular patterns (MAMPs), promoting immune cells or cytokines, such as IL-6, IL-1a, IL1b, TNFa, etc., which in turn affect the central nervous system. At present, the role of the gut-brain axis in neurodegenerative diseases has received increasing attention. Reference 1 discloses the interaction between the gut microbiome and oligodendrocytes, as well as the role of gut microbiome-derived metabolites in neurodegenerative diseases, pointing out that the gut microbiome can regulate oligodendrocyte maturation and myelin production, and that changes in the gut microbiome are related to the pathogenesis of neurodegenerative diseases. However, many mechanisms remain unclear.

[0003] Branched chain fatty acids (BCFA) refer to fatty acids with one or more branched alkyl groups (mainly methyl groups) on the carbon skeleton of fatty acids, usually saturated fatty acids. Branched alkyl groups located on the second carbon atom at the end of the alkyl group are called iso- or iso- fatty acids, and those located on the third carbon atom at the end of the alkyl group are called anteiso- or anteiso- fatty acids. Due to the specific branched chain structure, BCFA has low freezing point and pour point, good thermal stability and oxidative stability, good solubility, low foaming, and good air and water permeability of the formed membrane. BCFA is widely present in nature, but the content is low. It is reported that a certain amount of BCFA with different structures exists in the milk and internal tissues of ruminants (such as cow and sheep milk and meat products), and in human milk. BCFA is more abundant in vernix and the intestines of newborns. The BCFA spectrum of meconium shows that BCFAs are the main components of the gastrointestinal tract of normal and healthy full-term newborns. The BCFA in the intestines of premature infants is much less than that of normal and healthy infants, and the probability of suffering from necrotizing enterocolitis is higher. Many studies have shown that BCFA has unique physiological regulatory functions, including inhibiting inflammation, anti-cancer effects and improving the intestine, thereby reducing the incidence of necrotizing enterocolitis in premature infants.

[0004] Currently, many studies or patents have confirmed the effectiveness of branched-chain fatty acids in inhibiting intestinal inflammation. The results showed that both iso-BCFAs and anteiso-BCFAs have a significant effect in inhibiting intestinal inflammation.

[0005] For example, in Reference 2, a composition containing branched-chain fatty acids such as 14-methylpentadecanoic acid, 12-methyltridecanoic acid, and 13-methyltetradecanoic acid is disclosed. The composition can inhibit the generation of ROS in the LPS-induced calf intestinal epithelial cell inflammation model; and can regulate the level of inflammatory factors in the intestine of DSS-induced colitis mice and improve the gastrointestinal health of mice.

[0006] Reference 3 discloses that branched-chain fatty acids were added to the CaCo2 cell culture system and administered orally to mice with DSS-induced enteritis. The results showed that BCFAs can induce hyper-SUMOylation of intestinal proteins in CaCo2 cells, thereby reducing the expression of pro-inflammatory cytokines. In addition, BCFAs also inhibit intestinal inflammation caused by DSS, reduce the activity of intestinal cell deaminase, and promote intestinal epithelial integrity.

[0007] Reference 4 discloses that branched-chain fatty acids can reduce the risk of necrotizing enterocolitis (NEC) and increase the expression of IL-10 in the intestine. In addition, reference 5 discloses that branched-chain fatty acids have the function of inhibiting intestinal inflammation in DSS-induced enteritis mice and enhancing the resistance of the mouse intestine.

[0008] Citations

[0009] Cited literature 1: Loh, JS, Mak, WQ, Tan, LKS et al. Microbiota-gut-brain axis and its therapeutic applications in neurodegenerative diseases. Sig TransductTarget Ther 9, 37 (2024).

[0010] Reference 2: CN118416112B;

[0011] Reference 3: WO2023031226A1;

[0012] Reference 4: Ran-Ressler RR, et al. Branched chain fatty acids reduce theincidence of necrotizing enterocolitis and alter gastrointestinal microbialecology in a neonatal rat model.PLoS One.2011;6(12):e29032.

[0013] Reference 5: CN115300530A. Summary of the invention

[0014] Problem that the invention aims to solve

[0015] Regarding the occurrence of neurodegenerative diseases, the gut-brain axis is an important research direction, and more research focuses on using the intestinal flora as a target to improve the occurrence and development of neurological diseases by regulating the intestinal microecological environment.

[0016] The present invention is based on the gut-brain axis principle. In the process of studying branched-chain fatty acids (BCFAs), it was found that BCFAs have certain effects in regulating brain immunity and alleviating and improving neuroinflammatory indicators.

[0017] It should be noted that the neuroinflammation mentioned in this specification refers to the non-disease state in which the nerves are not healthy and full due to the presence of inflammatory factors.

[0018] Solutions for solving problems

[0019] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0020] [1]. A branched fatty acid composition, wherein the branched fatty acid comprises C14:0, C15:0, C16:0 and C17:0;

[0021] The C14:0 includes isomeric forms (iso-C14:0);

[0022] The C16:0 includes isomeric forms (iso-C16:0);

[0023] The C15:0 includes an isomeric form (iso-C15:0) and an anti-isomer form (anteiso-C15:0);

[0024] The C17:0 includes an isomeric form (iso-C17:0) and an anteisomeric form (anteiso-C17:0);

[0025] Based on the total mass of each of the branched-chain fatty acids C14:0, C15:0, C16:0 and C17:0,

[0026] The content of iso-C14:0 is not less than 5%, the content of iso-C15:0 is not less than 10%, the content of anteiso-C15:0 is 15% to 23.5%, the content of iso-C16:0 is 10% to 20%, the content of iso-C17:0 is 15% to 30%, and the content of anteiso-C17:0 is 15% to 25%.

[0027] [2] The composition according to [1], wherein, based on the total weight of each of the branched-chain fatty acids C14:0, C15:0, C16:0 and C17:0,

[0028] The content of iso-C14:0 is 5% to 10%, the content of iso-C15:0 is 10% to 20%, the content of anteiso-C15:0 is 16% to 23.5%, the content of iso-C16:0 is 14% to 20%, the content of iso-C17:0 is 17% to 25%, and the content of anteiso-C17:0 is 20% to 25%.

[0029] [3] The composition according to [1] or [2], wherein, based on the total weight of each of the branched-chain fatty acids C14:0, C15:0, C16:0 and C17:0,

[0030] The content of iso-C14:0 is 5% to 7%, the content of iso-C15:0 is 10% to 15%, the content of anteiso-C15:0 is 17% to 23.5%, the content of iso-C16:0 is 14.5% to 19%, the content of iso-C17:0 is 18% to 22.5%, and the content of anteiso-C17:0 is 21% to 24.5%.

[0031] [4] Use of the branched fatty acid composition described in any one of [1] to [3] in the preparation of food for assisting in maintaining the health of the central nervous system.

[0032] [5]. The use according to [4], wherein the assistance in maintaining the health of the central nervous system includes alleviating neuroinflammation for non-therapeutic purposes.

[0033] [6] Use of the composition described in any one of [1] to [3] in the preparation of a food that helps regulate brain immunity.

[0034] [7]. The use according to [6], wherein the helping to regulate brain immunity includes regulating microglial polarization.

[0035] [8]. The use according to [7], wherein the regulation of microglia polarization comprises at least one of inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to 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.

[0036] [9]. The use according to any one of [4] to [8], wherein the food is candy, a beverage, a dairy product or a baked product.

[0037]

[10] . The use according to [9], wherein the candy comprises at least one of hard candy, jelly candy, crisp candy, compressed candy and aerated candy; the beverage comprises at least one of carbonated beverage, tea beverage, coffee beverage, fruit and vegetable juice beverage and lactic acid bacteria beverage; the dairy product comprises at least one of fermented milk, cheese and milk powder; and the baked food comprises at least one of bread, cake and biscuits.

[0038] Effects of the Invention

[0039] The branched fatty acid composition provided by the present invention comprises C14:0, C15:0, C16:0 and C17:0, and more specifically comprises iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0 and anteiso-C17:0. After the branched fatty acid composition is applied to microglia induced by LPS, the trend of promoting the differentiation of microglia into M2 phenotype microglia is higher than the trend of promoting the differentiation 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-a, and IL-1β. C14:0, C15:0, C16:0 and C17:0 in the branched fatty acid composition have a good synergistic effect, which plays a role in regulating brain immunity and maintaining the health of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The expression levels of iNOS and CD206 mRNA in BV2 cells of each group detected by RT-PCR are shown; where * represents p<0.05, ** represents p<0.01, and **** represents p<0.0001. DETAILED DESCRIPTION

[0041] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0042] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0043] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0044] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0045] In this specification, the 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 exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0046] Throughout the specification, “optional” and “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0047] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0048] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0049] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0050] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0051] In the present invention, the term "about" is used to define the numerical range and parameters of the present invention are approximate values, and the specific relevant values ​​are presented as accurately as possible. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" usually means that the actual value is within ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0052] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0053] The present invention is mainly completed based on the following insights:

[0054] Branched-chain fatty acids have been widely reported to be used to improve intestinal health, and their other functions have yet to be explored and studied. The present invention uses LPS-induced microglia as an in vitro neuroinflammation model, first selects branched-chain fatty acids with less cytotoxicity and intervention concentrations, and then mixes branched-chain fatty acids of different concentrations to obtain branched-chain fatty acid compositions, in order to explore branched-chain fatty acid compositions that can improve neuroinflammation and thus regulate brain immunity.

[0055] I. Branched-chain fatty acid composition

[0056] The branched fatty acid composition of the present invention comprises C14:0, C15:0, C16:0 and C17:0. In some preferred embodiments, the C14:0 and the C16:0 include isomeric forms (iso), i.e., iso-C14:0 and iso-C16:0; the C15:0 and C17:0 include isomeric forms (iso) and anteisomeric forms (anteiso), i.e., iso-C15:0, anteiso-C15:0, iso-C17:0, anteiso-C17:0.

[0057] In the present invention, the "isomeric" or "isomeric branched-chain fatty acid (iso-BCFA)" refers to a fatty acid with a methyl branch on the second to last carbon atom of the carbon chain backbone of the fatty acid molecule. The "anteisomeric" or "anteisomeric branched-chain fatty acid (anteiso-BCFA)" refers to a fatty acid with a methyl branch on the third to last carbon atom of the carbon chain backbone of the fatty acid molecule.

[0058] For branched fatty acid composition of the present invention, except necessary containing above-mentioned various branched fatty acids, optional, can also contain other branched or straight-chain fatty acids.The content of these other fatty acids can be below 50 mass %, below 30 mass % or below 10 mass % of composition.

[0059] In some embodiments, based on the total mass of the branched-chain fatty acids of C14:0, C15:0, C16:0 and C17:0, the content of iso-C14:0 is not less than 5%, preferably 5% to 10%, more preferably 5% to 7%, and more preferably 5.4% to 6.4%, for example, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.41%, 5.42%, 5.43%, 5.44%, 5.45%, 5.46%, 5.47%, 5.48%, 5.49%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.31%, 6.32%, 6.33%, 6.34%, %, 6.35%, 6.36%, 6.37%, 6.38%, 6.39%, 6.4%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%; the content of iso-C15:0 is not less than 10%, preferably 10% to 20%, more preferably 10% to 15%, more preferably 11% to 13.5%, for example 10%, 10.5%, 11%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14. .52%, 12.53%, 12.54%, 12.55%, 12.56%, 12.57%, 12.58%, 12.59%, 12.6%, 12.61%, 12.62%, 12.63%, 12.64%, 12.65%, 12.66%, 12.67%, 12.68%, 12.69%, 12.7%, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 15%, 16%, 17%, 18%, 19%, 20%; the content of anteiso-C15:0 is 15% % to 23.5%, preferably 16% to 23.5%, more preferably 17% to 23.5%, more preferably 17.8% to 23.3%, for example 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 17.6%, 17.7%, 17.71%, 17.72%, 17.73%, 17.74%, 17.75%, 17.76%, 17.78%, 17.79%, 17.8%, 17.9%, 18%, 18.5%, 19%, 19.5%, 20%, 20.1%, 20.2%, 20.3%, 20.4%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.1%, 23.2%, 23.21%, 23.22%, 23.23%, 23.24%, 23.25%, 23.26%, 23.27%, 23.28%, 23.29%, 23.3%, 23.4%, 23.5%; the content of iso-C16:0 is 10% to 20%, preferably 14% to 20%, more preferably 14.5% to 19%, more preferably 14.9% to 18.8%, for example 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 14.91%, 14.92%, 14.93%, 14.94%, 14.95%, 14.96%, 14.97%, 14.98%, 14.99%, 15.10%, 15.11%, 15.12%, 15.13%, 15.14%, 15.15 93%, 14.94%, 14.95%, 14.96%, 14.97%, 14.98%, 14.99%, 15%, 15.01%, 15.02%, 15.03%, 15.04%, 15.05%, 15.06%, 15.07%, 15.08%, 15.09%, 15.1%, 15.15%, 15.2%, 15.3%, 15.31%, 15.32%, 15.33%, 15.34%, 15.35%, 15.36%, 15.37%, 15.38%, 15.39%, 15.4%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.1 %, 18.2%, 18.3%, 18.4%, 18.5%, 18.51%, 18.52%, 18.53%, 18.54%, 18.55%, 18.56%, 18.57%, 18.58%, 18.59%, 18.6%, 18.7%, 18.8%, 18.9%, 19%, 20%; the content of iso-C17:0 is 15% to 30%, preferably 17% to 25%, more preferably 18% to 22.5%, more preferably 18.4% to 22.4%, for example 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.1%, 18.2%, 18.3%, 18 .4%, 18.41%, 18.42%, 18.43%, 18.44%, 18.45%, 18.46%, 18.47%, 18.48%, 18.49%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19%, 19.5%, 19.55%, 19.6%, 19.7%, 19.71%, 19.72%, 19.73%, 19.74%, 19.8%, 19.9%, 20%, 20.1%, 20.2%, 20.3%, 20.4%, 20.5%, 20.6%, 20.7%, 20.8%, 20.9%, 21%, 21.5%, 22%, 22.5%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%; the content of anteiso-C17:0 is 15% to 25%, preferably 20% to 25%, more preferably 21% to 24.5%, more preferably 22% to 24.3%, for example 15%, 16%, 17%, 18%, 19%, 20%, 20.5%, 21%, 21.5%, 22%, 22.1%, 22.2%, 22.3%, 22.4%, 22.5%, 22.6%, 22.7%, 22.8%, 22.81%, 22.82% , 22.83%, 22.84%, 22.85%, 22.86%, 22.87%, 22.89%, 22.9%, 23%, 23.1%, 23.2%, 23.3%, 23.4%, 23.5%, 24%, 24.1%, 24.2%, 24.21%, 24.22%, 24.23%, 24.24%, 24.25%, 24.26%, 24.27%, 24.28%, 24.29%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25%. .

[0060] The present invention does not specifically limit the source of branched fatty acids. For example, they can be separated and extracted from natural substances containing branched fatty acids, such as bacterial biofilms, animal sebum, mammary tissue, and plants; they can also be prepared by biological or chemical synthesis, such as synthesis using microorganisms or Caenorhabditis elegans. For example, in microorganisms or Caenorhabditis elegans, branched fatty acids are synthesized by enzymatic reactions using branched-chain amino acids as substrates. At present, the common method in the art is to separate and extract branched fatty acids from lanolin. Free lanolin alcohol and free lanolin acid soap can be prepared by saponification of lanolin, and then the branched fatty acids in lanolin are obtained through multiple steps such as alcohol soap separation process and preparation and extraction of fatty acids.

[0061] II. Food

[0062] The food of the present invention contains or uses the branched-chain fatty acid complex, thereby containing C14:0, C15:0, C16:0 and C17:0. In some preferred embodiments, the C14:0 and the C16:0 are both isomeric, and the C15:0 and C17:0 are isomeric and trans-isomerized.

[0063] The present invention does not particularly limit the absolute content of C14:0, C15:0, C16:0 and C17:0 in food, as long as it complies with the requirements of relevant laws and regulations, for example, it complies with the quantitative limit of fatty acids in food in GB 5009.168-2016 "National Food Safety Standard Determination of Fatty Acids in Food". In some embodiments, the content of C14:0 is not higher than 0.0033g / 100g in solid food, for example, 0.0001 to 0.0033g / 100g; and not higher than 0.0013g / 100g in liquid food, for example, 0.0001 to 0.0013g / 100g. In some embodiments, the content of C15:0 is not higher than 0.0033 g / 100 g in solid foods, such as 0.0001 to 0.0033 g / 100 g; in liquid foods, it is not higher than 0.0013 g / 100 g, such as 0.0001 to 0.0013 g / 100 g. In some embodiments, the content of C16:0 is not higher than 0.0066 g / 100 g in solid foods, such as 0.0001 to 0.0066 g / 100 g; in liquid foods, it is not higher than 0.0026 g / 100 g, such as 0.0001 to 0.0066 / 100 g. In some embodiments, the C17:0 content in solid foods is not higher than 0.0066 g / 100 g, for example, 0.0001 to 0.0066 g / 100 g; and in liquid foods, the content is not higher than 0.0026 g / 100 g, for example, 0.0001 to 0.0026 g / 100 g.

[0064] The present invention does not particularly limit the types of food, and examples thereof include candies, beverages, dairy products or baked goods. For candies, examples include hard candies, crisp candies, gel candies, compressed candies and aerated candies. For beverages, examples include carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages and lactic acid bacteria beverages. For dairy products, examples include fermented milk, cheese and milk powder. For baked goods, examples include bread, cakes and biscuits. In addition, the food of the present invention can also be a health food, such as various types of oral preparations, including tablets, pills, granules, powders, capsules and oral liquids.

[0065] In addition to C14:0, C15:0, C16:0 and C17:0 in the branched-chain fatty acid composition, depending on the type of food and the final needs of the applicable objects, in some embodiments, the food described in the present invention also contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable excipients.

[0066] As for the plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica chinensis and bilberry or their extracts; vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; rice (indica rice , japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow rice, buckwheat, soybean, broad bean, pea, mung bean, red bean, kidney bean and other cereals or their extracts; walnut, pistachio, cashew, hazelnut, almond, apricot, pine nut, peanut, melon seed, chestnut, macadamia, ginkgo and other nuts or their extracts; coffee or its extract; and some edible and medicinal plant-based Chinese medicinal materials or their extracts.

[0067] As for animal dairy ingredients, examples 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, concentrated whey protein powder, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0068] Examples of animal meat ingredients include meat ingredients from pigs, cattle, sheep, aquatic animals or poultry.

[0069] Functional added ingredients include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, and the like.

[0070] As for any acceptable excipients, examples include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0071] III. Uses that help regulate brain immunity

[0072] The present invention unexpectedly found that a branched fatty acid composition comprising C14:0, C15:0, C16:0 and C17:0, especially a branched fatty acid composition comprising iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0, has the effect of regulating brain immunity. Moreover, when iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0 are combined, the six have a synergistic effect, which can further enhance the effect of brain immune regulation. Therefore, the branched-chain fatty acid composition containing iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0 of ​​the present invention and the food added with or using the branched-chain fatty acid composition have the effect of regulating brain immunity. The purpose of regulating brain immunity of the present invention is not to prevent and treat diseases.

[0073] Furthermore, the present invention provides the use of a branched fatty acid composition containing iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0 in preparing foods that help regulate brain immunity.

[0074] In some embodiments, the effect of helping to regulate brain immunity includes regulating microglial polarization. In some specific embodiments, the regulation of microglial polarization includes inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, increasing the amount of IL-4, IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β and / or NO released by microglia. In some specific embodiments, the regulation of microglial polarization includes inhibiting the trend of microglia polarization to M1 phenotype, promoting the trend of microglia polarization to M2 phenotype, increasing the amount of IL-4, IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β and / or NO released by microglia. In some specific embodiments, the regulation of microglial polarization includes inhibiting the tendency of microglial cells to polarize toward the M1 phenotype, promoting the tendency of microglial cells to polarize toward the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglial cells, and reducing the amount of TNF-α, IL-1β and NO released by microglial cells.

[0075] Furthermore, the effect of helping to regulate brain immunity includes promoting the development of brain nerves and nervous system.

[0076] In some embodiments, the nervous system is the central nervous system.

[0077] IV. Uses to assist in improving or maintaining the health of the central nervous system

[0078] The present invention unexpectedly found that the combination of C14:0, C15:0, C16:0 and C17:0, such as iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, anteiso-C17:0, can play a role in assisting the improvement or maintenance of the health of the central nervous system, especially improving the discomfort or poor health caused by non-disease neuroinflammation. Moreover, when iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, anteiso-C17:0 are combined, the six have a synergistic effect, which can further enhance the effect of improving neuroinflammation. Therefore, the branched-chain fatty acid composition containing iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0 described in the present invention also has the effect of assisting in improving or maintaining the health of the central nervous system and improving the discomfort or poor health caused by non-pathological neuroinflammation.

[0079] Furthermore, the present invention provides use of a branched-chain fatty acid composition containing iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0 in preparing food for assisting in maintaining the health of the central nervous system.

[0080] In some embodiments, the assistance in maintaining the health of the central nervous system includes alleviating and improving neuroinflammation for non-therapeutic purposes, such neuroinflammation does not reach a medically pathological level.

[0081] Example

[0082] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0083] 1. Instruments and Reagents

[0084] Mouse microglial cell line BV-2 cells (Shanghai Enzyme Research Biotechnology, catalog number CC-Y2022), fetal bovine serum (Vicente, catalog number 085-150), high-glucose DMEM medium (Scott, catalog number CA0004-500ML), double antibody, culture bottles, well plates, centrifuge tubes, trypsin, dimethyl sulfoxide, lipopolysaccharide, kits, etc.

[0085] 2. Experimental Methods

[0086] 2.1 Cell culture

[0087] Mouse microglial cell line (BV2) was cultured in high-glucose DMEM medium (containing 10% fetal bovine serum) in a 5% CO 2 incubator (37° C., saturated humidity).

[0088] 2.2 Cytotoxicity assay

[0089] The CCK8 method (CCK8 kit was purchased from White Shark, catalog number BS350A) was used to detect the activity of BV2 microglia cells.

[0090] Evaluation method of cytotoxicity of nutrients: BV2 cells growing in logarithmic phase were selected and 5×10 4 Cells were seeded in 96-well plates at a density of 100 cells / mL. After overnight attachment, branched-chain fatty acids of different concentrations were added for treatment for 12 hours. After treatment, 10 μL of CCK8 solution was added to each well and incubated at 37°C for 4 hours. The absorbance at 450 nm was measured using an ELISA reader.

[0091] According to the formula: cell survival rate = [(absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100% (wherein the experimental wells contain cells, culture medium, nutrients of different concentrations and CCK-8 solution; the blank wells contain culture medium and CCK-8 solution, but no cells and nutrients; the control wells contain cells, culture medium and CCK-8 solution, but no nutrients), the cell survival rate is finally calculated.

[0092] 2.3 Evaluation of anti-inflammatory efficacy

[0093] 1) Establishment of microglial inflammation model and nutrient intervention method

[0094] BV2 cells in logarithmic phase were selected and 5×10 4 The number of cells / mL was inoculated in a 96-well plate, and the cells were grouped and intervened after overnight adherence. (1) Control group: conventional cell culture was used; (2) LPS group (model group): LPS (1 μg / mL) was used to intervene BV2 cells for 24 hours to establish a cellular neuroinflammation model; (3) Experimental group (example and comparative example): After LPS stimulated BV2 cells for 24 hours, 10 compositions were added for intervention for 12 hours.

[0095] 2) NO level measurement

[0096] The NO (nitric oxide) level in the culture medium of BV2 microglia was detected by nitrate reductase method. The experimental grouping and nutrient intervention method were as shown in 1) above, and the cell culture medium of each group of cells was collected. According to the instructions of the NO detection kit, the absorbance value of each tube was measured at a wavelength of 550nm and a light path of 0.5cm, and the NO content was calculated.

[0097] 3) Determination of inflammatory factor levels

[0098] ELISA was used to detect the content of IL-1β, TNF-α, IL-10, and IL-4 in the supernatant of BV2 cells. The experimental grouping and nutrient intervention methods were the same as those in 1) above. The cell supernatant of each group of cells was collected, and the ELISA kit instructions were followed. The absorbance values ​​of each group were measured in turn at a wavelength of 405 nm using an ELISA reader to detect the amount of IL-1β, TNF-α, IL-4, and IL-10.

[0099] 2.4 Detection of iNOS and CD206, markers of microglial M1 and M2 phenotypes

[0100] qRT-PCR was used to detect the mRNA expression levels of iNOS and CD206-related genes and then determine the changes in the content of M1 cell population and M2 cell population. The third-generation BV2 microglial cell suspension was inoculated in a 24-well culture plate with a cell density of 4×10 5 Each well was divided into groups according to the experimental design. After treatment, the cells were washed twice with PBS, using 200 μL per well. Digested with trypsin for 2.5 min, and digested with 400 μL of culture medium. The cells were blown up with a pipette and collected in enzyme-free centrifuge tubes. Total RNA of each group of cells was extracted. Reverse transcription was performed using a reverse transcription kit according to the operating procedures to obtain cDNA, and the amplified cDNA was mixed with primers, fluorescent probes and qPCR Master Mix. The corresponding PCR cycle program and parameters were set according to the requirements of qPCR, and the corresponding mRNA expression level was analyzed in the real-time PCR detection system. The specific primer information is as follows:

[0101] iNOS:

[0102] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO. 1),

[0103] Downstream: 5′-TGCAAGTGAAATCCGATGTGG-3′ (SEQ ID NO. 2);

[0104] CD206:

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

[0106] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO. 4);

[0107] GAPDH:

[0108] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO. 5),

[0109] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0110] 3. Cytotoxicity test of branched-chain fatty acids

[0111] In this study, five branched-chain fatty acids, iso-C13:0, anteiso-C13:0, iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, iso-C17:0, anteiso-C17:0, and iso-C20:0, were selected to investigate the regulatory effects of different compositions on neuroinflammation. First, the toxic effects of nine branched-chain fatty acids on microglia were investigated. The results are shown in Table 1. It can be seen from the table that the cytotoxic effect of iso-C17:0 decreased to 90.79% when the concentration reached 25 micromoles / liter, and the cell viability of the other eight branched-chain fatty acids was still above 100% at a concentration of 25%, so the concentration of branched-chain fatty acids in this study was uniformly determined to be 25 micromoles / liter.

[0112] Table 1 Effects of different concentrations of branched-chain fatty acids on microglial cell viability (%)

[0113]

[0114] 4. Inhibitory effects of branched-chain fatty acids on neuroinflammation

[0115] Microglia, as resident immune cells in the central nervous system, are key players in neuroinflammation. LPS-induced microglia activate the Toll-like receptor (TLR4) NF-kB signaling pathway, accelerate the transcription of inflammatory genes, and further cause the differentiation of microglia M1 phenotype and M2 phenotype.

[0116] M1 phenotype microglia secrete proinflammatory cytokines such as NO, IL-1β, and TNF-a, exacerbating the inflammatory response. NO free radicals are synthesized by enzymes such as NOS (eNOS) and iNOS through the l-arginine pathway. Under normal physiological conditions, iNOS is dormant in dormant cells. However, under pathological conditions, it produces a large amount of NO and plays a dual role in chronic infection and inflammation. Reducing the production of NO may be an effective strategy to improve inflammation; IL-1β has strong proinflammatory activity and can induce a variety of proinflammatory mediators, such as cytokines and chemokines; tumor necrosis factor-a (TNF-α) is a pleiotropic cytokine produced by a variety of cells during inflammatory responses and immune regulatory stimulation, which can induce cell apoptosis.

[0117] The M2 type can secrete anti-inflammatory cytokines such as IL-4 and IL-10 to reduce neuroinflammatory reactions. Among them, IL-4 is a typical type II immune cytokine, which plays an important immunomodulatory role in type II immune diseases such as asthma, allergic dermatitis and urticaria. Studies have shown that IL-4 can promote the differentiation of macrophages into M2 macrophages, thereby exerting an anti-inflammatory effect; interleukin 10 is a multi-cell-derived, multifunctional cytokine that regulates cell growth and differentiation, participates in inflammatory and immune responses, and is a recognized inflammatory and immunosuppressive factor.

[0118] Therefore, regulating the transformation of microglia to M2 is a potential strategy to inhibit neuroinflammation. The present invention selects the effects of different branched-chain fatty acids mixed in different proportions on LPS-induced BV2 microglial inflammation.

[0119] Examples 1 to 3

[0120] According to the method in step 1) of 2.3 above, a microglial inflammation model was established and nutrient intervention was performed. The composition of branched-chain fatty acids is shown in Table 2. The concentration of branched-chain fatty acids in the microglial inflammation model was 25 μmol / L. After the intervention, the NO level, inflammatory factor level, and iNOS and CD206 content were detected.

[0121] Comparative Examples 1 to 8

[0122] According to the method in step 1) of 2.3 above, a microglial inflammation model was established and nutrient intervention was performed. The composition of branched-chain fatty acids is shown in Table 2. The concentration of branched-chain fatty acids in the microglial inflammation model was 25 μmol / L. After the intervention, the NO level, inflammatory factor level, and iNOS and CD206 content were detected.

[0123] Table 2 Combination design of different ratios of different branched-chain fatty acids (%)

[0124] iso-C14:0 iso-C15:0 anteiso-C15:0 iso-C16:0 iso-C17:0 anteiso-C17:0 Example 1 6.36 12.56 17.79 18.51 22.39 22.39 Example 2 5.44 13.5 23.28 16.47 18.47 22.84 Example 3 5.51 11 23.27 14.94 21 24.28 Comparative Example 1 5.52 12.84 23.79 15.08 19.71 23.06 Comparative Example 2 4.8 12.33 15.5 15.32 27.22 24.83 Comparative Example 3 5.68 12.64 23.72 15.34 20.7 21.92 Comparative Example 4 4.68 18 20.17 14.94 20 22.21 Comparative Example 5 4.8 11.5 27 15.15 19.55 22 Comparative Example 6 4.6 11.5 17.9 25 19 22 Comparative Example 7 4.8 13.2 23.3 18.2 16 24.5 Comparative Example 8 4.8 13.2 23.3 18.2 22.5 18

[0125] By applying different branched fatty acid compositions to a microglial inflammation model, the inventors unexpectedly discovered that six medium-chain branched fatty acids, iso-C14:0, iso-C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0, have regulatory effects on neuroinflammation. The specific results are as follows:

[0126] Table 3 Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia

[0127] NO(μM / L) TNF-a (ng / L) IL-1β (ng / L) Control group 16.62±1.27 191.69±12.51 12.36±0.53 Model Group 29.35±0.36 254.96±2.71 20.32±0.53 Example 1 17.38±2.33 191.42±4.23 8.03±1.08 Example 2 19.65±2.30 192.39±16.84 8.17±0.80 Example 3 15.44±0.55 201.83±2.21 8.67±0.48 Comparative Example 1 23.89±2.86 209.33±10.10 7.74±1.19 Comparative Example 2 21.59±2.00 192.94±5.36 7.60±0.66 Comparative Example 3 20.50±1.39 204.61±20.08 7.43±0.86 Comparative Example 4 18.19±1.38 203.36±3.96 8.56±0.79 Comparative Example 5 24.26±2.23 203.78±4.59 6.56±0.76 Comparative Example 6 20.43±0.96 201.83±19.22 8.42±1.08 Comparative Example 7 25.59±1.00 194.61±10.42 8.65±0.33 Comparative Example 8 24.35±1.86 199.19±17.92 7.52±0.71

[0128] Table 3 shows the effects of different combinations of branched-chain fatty acids on LPS-induced BV2 microglia pro-inflammatory factors. It can be seen from the table that after LPS induction, microglia produced significant increases in pro-inflammatory factors NO, TNF-a and IL-1β, indicating that LPS induction caused microglia to be severely damaged and produce a large number of pro-inflammatory factors. In the experimental group and the control group treated with different branched-chain fatty acid compositions and LPS, it can be seen that the pro-inflammatory factors have been reduced to varying degrees.

[0129] After one-way ANOVA significance analysis, it was found that the proinflammatory factor IL-1β in the embodiments and comparative examples was not only significantly lower than that in the model group (p < 0.0001), but also significantly lower than that in the control group (p < 0.0001); for the proinflammatory factor TNF-a, the 11 compositions of the present invention were significantly lower than those in the model group (p < 0.0001), and there was no difference with the control group (p > 0.05). Inter-group comparison found that the content of TNF-a in Examples 1 and 2 was the lowest; for the proinflammatory factor NO, the present invention found that Examples 1, 2, 3 and Comparative Examples 3 and 4 were significantly lower than those in the treatment group (p < 0.0001), and there was no difference with the control group (p > 0.05).

[0130] In general, in terms of regulating pro-inflammatory factors, Example 1, Example 2, Example 3 and Comparative Example 3, Comparative Example 4 showed quantitative effects.

[0131] Table 4 Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia

[0132]

[0133]

[0134] Table 4 shows the effects of different combinations of branched-chain fatty acids on LPS-induced anti-inflammatory factors in BV2 microglia. It can be seen from the table that the production of anti-inflammatory factor IL-4 after treatment with the combination of Example 1, Example 2, Example 3, Comparative Example 5, Comparative Example 7 and Comparative Example 8 is higher than that of the model group, but after one-way ANOVA difference significance analysis, it is found that Example 1, Example 2, Example 3, Comparative Example 5 and the model group are significantly different (p < 0.05), and there is no significant difference between Comparative Example 7 and Comparative Example 8 (p > 0.05); further analysis of the effects of each combination on the anti-inflammatory factor IL-10 after treatment, the results show that Example 1, Example 2 and Example 3 have the greatest degree of increasing the production of IL-10. After difference analysis, it is found that the IL-10 content of the three reaches the effect of the control group (p > 0.05), especially Example 1, whose IL-10 content is significantly higher than that of the model group (p < 0.05).

[0135] Through the above analysis, it is found that Example 1, Example 2 and Example 3 have significant effects on regulating microglial inflammation after LPS induction, and their content ranges are 5.44%-6.36%, 11%-13.5%, 17.79%-23.28%, 14.94%-18.51%, 18.47%-22.39%, and 22.39%-24.28%, respectively. When the content of any type of branched-chain fatty acid exceeds this range, the overall effect of the composition in regulating inflammation will be affected.

[0136] In order to determine whether the inhibition of BV2 cell inflammatory response by branched-chain fatty acid composition is related to phenotypic polarization, this study used RT-qPCR to detect the mRNA expression of iNOS, a M1 phenotype marker, and CD206, a M2 phenotype marker, in BV2 cells. Figure 1 As shown. As can be seen from the figure, the iNOS marker characterizing M1 cell polarization in the three embodiments was significantly lower than that in the model group (p < 0.0001), and there was no significant difference from the control group (p > 0.05). At the same time, the mRNA expression of CD206 in the three embodiments was significantly higher than that in the treatment group (p < 0.0001), and there was no significant difference from the control group (p > 0.01). The above results further prove that the branched fatty acid composition shown in the three embodiments has the effect of regulating LPS-induced BV2 cell neuroinflammation, can promote the differentiation of microglia to M2 type, promote the production of anti-inflammatory factors, and has the effect of regulating the brain's immune balance function.

Claims

1. A branched-chain fatty acid composition, characterized in that: The branched-chain fatty acids include C14:0, C15:0, C16:0 and C17:0; The C14:0 includes isomeric forms (iso-C14:0); The C16:0 includes isomeric forms (iso-C16:0); The C15:0 includes an isomeric form (iso-C15:0) and an anti-isomer form (anteiso-C15:0); The C17:0 includes an isomeric form (iso-C17:0) and an anteisomeric form (anteiso-C17:0); Based on the total mass of each of the branched-chain fatty acids C14:0, C15:0, C16:0 and C17:0, The content of iso-C14:0 is not less than 5%, the content of iso-C15:0 is not less than 10%, the content of anteiso-C15:0 is 15% to 23.5%, the content of iso-C16:0 is 10% to 20%, the content of iso-C17:0 is 15% to 30%, and the content of anteiso-C17:0 is 15% to 25%.

2. The composition according to claim 1, characterized in that Based on the total mass of each of the branched-chain fatty acids C14:0, C15:0, C16:0 and C17:0, The content of iso-C14:0 is 5% to 10%, the content of iso-C15:0 is 10% to 20%, the content of anteiso-C15:0 is 16% to 23.5%, the content of iso-C16:0 is 14% to 20%, the content of iso-C17:0 is 17% to 25%, and the content of anteiso-C17:0 is 20% to 25%.

3. The composition according to claim 1 or 2, characterized in that Based on the total mass of each of the branched-chain fatty acids C14:0, C15:0, C16:0 and C17:0, The content of iso-C14:0 is 5% to 7%, the content of iso-C15:0 is 10% to 15%, the content of anteiso-C15:0 is 17% to 23.5%, the content of iso-C16:0 is 14.5% to 19%, the content of iso-C17:0 is 18% to 22.5%, and the content of anteiso-C17:0 is 21% to 24.5%.

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

5. The use according to claim 4, characterized in that The assistance in maintaining the health of the central nervous system includes alleviating neuroinflammation for non-therapeutic purposes.

6. Use of the composition according to any one of claims 1 to 3 in the preparation of food that helps regulate brain immunity.

7. The use according to claim 6, characterized in that Such contributions to the regulation of brain immunity include modulation of microglial polarization.

8. The use according to claim 7, characterized in that The regulation of microglial polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, increasing the amount of IL-4 and / or IL-10 released by microglial cells, and reducing the amount of TNF-α, IL-1β and / or NO released by microglial cells.

9. The use according to any one of claims 4 to 8, characterized in that: The food is candy, beverage, dairy product or baked food.

10. The use according to claim 9, characterized in that The candies include at least one of hard candies, jelly candies, crisp candies, compressed candies and aerated candies; the beverages include at least one of carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages and lactic acid bacteria beverages; the dairy products include at least one of fermented milk, cheese and milk powder; the baked goods include at least one of bread, cakes and biscuits.

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