CN119817812A - Nutritional composition and use of a nutritional composition - Google Patents
Nutritional composition and use of a nutritional composition Download PDFInfo
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Abstract
The present invention provides nutritional compositions and uses of nutritional compositions. The nutritional composition comprises medium-long chain fatty acid triglyceride (MLCT) and Milk Fat Globule Membrane (MFGM) in a mass ratio of 2:1 to 80:1, which in combination have synergistic effects of improving diarrhea, inhibiting staphylococcus aureus in vivo or in vitro, and/or modulating intestinal flora.
Description
Technical Field
The present disclosure relates to the field of food technology, in particular to a nutritional composition for inhibiting staphylococcus aureus and/or improving diarrhea, a food comprising the nutritional composition and the use of the nutritional composition.
Background
Infectious diarrhea is highly epidemic and has high incidence, and medical burden and economic burden caused by diarrhea in developing countries are serious, so that the infectious diarrhea is still a global public health problem which is worth focusing. The world health organization (World Health Organization, WHO) estimated that tens of millions of people worldwide had diarrhea every day, occurring for up to 17 hundred million cases per year, of which 220 tens of thousands died from severe diarrhea (Miao Xiaohui et al, journal of chinese digestion, 2013,33 (12): 793-802.). In addition, infectious diarrhea is a major cause of children's malnutrition due to high incidence and wide spread among children and serious harm to children's health (Ni Xin et al, chinese medicine science, 2020,10 (21): 249-256). Infectious diarrhea is an intestinal infectious disease caused by bacteria, fungi, viruses or parasites and mainly manifested by diarrhea.
Staphylococcus aureus is a gram-positive bacterium which is detected in 30% -50% of healthy adult human bodies, and about 20% of infected people can continuously carry the bacterium, and can cause chronic infection of other animals such as cattle, sheep, pigs, chickens and the like (Wu Yunpu and the like, experimental animal science, 2024,41 (03): 85-89). The diseases caused by staphylococcus aureus vary in severity, from moderate infections such as skin infections to fatal diseases such as severe pneumonia and septicemia, the course of disease treatment can be complicated by antibiotic resistance, and so far no effective vaccine is available, one of the most common causes of morbidity and mortality caused by infectious pathogens worldwide. Staphylococcus aureus induced chronic infections and antibiotic resistance, which pose serious challenges in the public health field. In addition, staphylococcus aureus is also an important pathogen that induces infectious diarrhea.
Medium-long chain fatty acid triglycerides (MLCT) have received considerable attention as a star product in novel structural lipids. The main structural feature of MLCT is that a glycerin skeleton is combined with medium chain fatty acid and long chain fatty acid. Early studies showed that MLCT has a number of health functions such as reducing blood lipid, inhibiting obesity, reducing diabetes and cardiovascular disease, and reducing risk of developing cancer (Lai Yundong, et al, chinese oil crop school report, 2024,46 (04): 719-727.). In addition, the fatty acid composition of MLCT is more similar to breast milk, is favorable for fat digestion and absorption, improves the absorption of lipid nutrients, inhibits the accumulation of body fat, and can quickly and stably supply (Cheng X, Jiang C, Jin J, Jin Q, Akoh CC, Wei W, Wang X. Medium- and Long-Chain Triacylglycerol: Preparation, Health Benefits, and Food Utilization. Annu Rev Food Sci Technol. 2024 Jun;15(1):381-408.). the known Milk Fat Globule Membrane (MFGM) which has important roles in improving the cognitive ability, improving the metabolism, reducing the incidence of infectious diseases and the like (Zhang Bo and the like, chinese journal of child health care, 2016,24 (01): 43-47).
No related report on the effect of MLCT and MFGM on staphylococcus aureus infection and diarrhea is found at home and abroad, and research on the effect of MLCT and MFGM and the combination thereof has important significance for developing foods and medicines with the effect of resisting infectious diarrhea.
There remains a need in the art for methods capable of ameliorating diarrhea, inhibiting staphylococcus aureus in vivo or in vitro, and/or modulating intestinal flora.
Disclosure of Invention
The present invention has been made in view of the above-mentioned problems in the art.
The first aspect of the present invention provides a nutritional composition comprising medium-long chain fatty acid triglycerides and milk fat globule membranes, wherein the mass ratio of medium-long chain fatty acid triglycerides to milk fat globule membranes is from 2:1 to 80:1.
In a second aspect the invention provides a food product comprising the nutritional composition according to the first aspect of the invention.
In a third aspect, the invention provides the use of a nutritional composition according to the first aspect of the invention for the preparation of a product for ameliorating (e.g. alleviating or treating) diarrhea.
In a fourth aspect, the present invention provides the use of a nutritional composition according to the first aspect of the invention for the preparation of a product for inhibiting staphylococcus aureus infections.
In a fifth aspect, the invention provides the use of a nutritional composition according to the first aspect of the invention for the preparation of a product for modulating intestinal flora, for example intestinal flora in a subject suffering from diarrhea.
In a sixth aspect the invention provides the use of a nutritional composition according to the first aspect of the invention for improving the non-therapeutic purpose of diarrhea.
In a seventh aspect, the invention provides the use of a nutritional composition according to the first aspect of the invention for non-therapeutic purposes for inhibiting staphylococcus aureus infections.
The present inventors have found that when the medium-long chain fatty acid triglyceride and the milk fat globule membrane are used in combination in a defined mass ratio, there is a synergistic effect between the two, which is capable of synergistically ameliorating diarrhea, in particular ameliorating (e.g. alleviating or treating) diarrhea caused by staphylococcus aureus, inhibiting staphylococcus aureus (in vivo or in vitro).
Drawings
Fig. 1 shows intestinal fluorescence intensity of zebra fish after sample treatment. P-values are the difference significance analyses for each example group versus the model control group. * P < 0.01 and P < 0.001.
Fig. 2 shows intestinal fluorescence intensity of zebra fish after composition sample treatment. P values are a significance analysis of differences between each example group and model control group, where P < 0.01 and P < 0.001.
Fig. 3 shows a typical graph of the intestinal tract of a sample-treated zebra fish.
Fig. 4 shows fluorescence intensity of staphylococcus aureus of zebra fish after sample treatment. P values are a difference significance analysis of each example group versus model control group, with P < 0.05, P < 0.01, and P < 0.001.
FIG. 5 shows a typical plot of fluorescence intensity of S.aureus from zebra fish after sample treatment.
Detailed Description
The following definitions are provided to facilitate understanding of the present invention by those skilled in the art. Unless defined otherwise, all 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. Preferred materials and methods are described herein, but any methods and materials similar or equivalent to those described herein can be used in the practice of the present test. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Definition of terms
Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. Moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically detailed may be, and have been, performed in a manner known per se, which will be appreciated by those skilled in the art.
As used herein, the term "medium-long chain fatty acid triglyceride" or "MLCT" is a particular triglyceride in which both medium and long chain fatty acids are present on the triglyceride backbone. As used herein, the term "medium-long chain fatty acid triglyceride" or "MLCT" is a particular triglyceride in which both medium and long chain fatty acids are present on the triglyceride backbone. MLCT is well known in the art and is prepared from edible vegetable oil and medium chain triglyceride as raw materials through transesterification reaction by lipase, and distillation separation, decolorization, deodorization and other processes. The medium-long chain fatty acid triglyceride may be derived from a medium-long chain fatty acid edible oil.
As used herein, the term "medium chain fatty acid" refers to fatty acids having 6 to 12 carbon atoms in the carbon chain, such as caproic acid, heptanoic acid, caprylic acid, nonanoic acid, capric acid, undecanoic acid, dodecanoic acid, and the like, and the term "long chain fatty acid" refers to fatty acids having 14 or more carbon atoms in the carbon chain, typically 14 to 30 carbon atoms, such as myristic acid, palmitic acid, oleic acid, linoleic acid, stearic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, and the like. The terms "medium-chain fatty acid triglyceride" and "long-chain fatty acid triglyceride" refer to the esterification reaction products of "medium-chain fatty acid" and "long-chain fatty acid" with glycerol, respectively, and the term "medium-chain fatty acid triglyceride" refers to triglycerides containing both medium-chain fatty acid residues and long-chain fatty acid residues in the molecular structure. The medium-long chain fatty acid triglyceride used in the present invention is not particularly limited, and medium-long chain fatty acid triglycerides generally used in the art can be used. The medium-long chain fatty acid triglycerides may be used in pure form, or in non-pure form enriched in medium-long chain fatty acid triglycerides. For example, the medium-long chain fatty acid triglycerides used in the present invention may contain C 6-C12 fatty acid residues and C 14-C30 fatty acid residues. The C 6-C12 fatty acid residue may be derived from one or more of caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, dodecanoic acid, and the like. The C 14-C30 fatty acid residue may be derived from one or more of myristic acid, palmitic acid, margaric acid, oleic acid, linoleic acid, stearic acid, nonadecanoic acid, eicosapentaenoic acid, heneicosanoic acid, docosatetraenoic acid, docosahexaenoic acid, tricosanoic acid, tetracosaenoic acid, pentacosapentaenoic acid, hexacosahexaenoic acid, arachidonic acid, and the like. In some embodiments, the C 6-C12 fatty acid residue is derived from one or more of caproic acid, caprylic acid, capric acid, and lauric acid, and/or the C 14-C30 fatty acid residue is derived from one or more of myristic acid, palmitic acid, oleic acid, linoleic acid, stearic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. In some embodiments, the weight ratio of C 6-C12 fatty acid residues to the C 14-C30 fatty acid residues is 0.124 to 2.000. In certain embodiments, the weight ratio of C 6-C12 fatty acid residues to the C 14-C30 fatty acid residues may be within a range defined by 0.124、0.125、0.130、0.140、0.150、0.160、0.170、0.180、0.190、0.200、0.300、0.400、0.500、0.600、0.700、0.800、0.900、1.000、1.100、1.200、1.300、1.400、1.500、1.600、1.700、1.800、1.900、2.000、 or any two of them. In some embodiments, the "medium-long chain fatty acid triglyceride" may be prepared by the method of patent CN115369132 a.
As used herein, the term "milk fat globule membrane" or "MFGM", english Milk Fat Globule Membrane, is a complex biological membrane structure present in breast milk. The milk fat globule membrane is a complex 3-layer phospholipid protein membrane composed of polar lipid, cholesterol, protein and the like, which is encapsulated on the surface of milk fat droplets. The source of the milk fat globule membrane of the present invention is not particularly limited in principle and may generally be obtained by extraction from animal milk or products thereof, which in some preferred embodiments may be cow milk, sheep milk, camel milk, horse milk or dairy products based thereon (e.g. cheese) or the like, more preferably may be extracted from cow milk, including cow colostrum or cow regular milk. The method for extracting MFGM from the animal milk or the product thereof is not particularly limited, and for example, the MFGM may be isolated by an acidification precipitation-centrifugation-isoelectric enrichment-drying method, or by a conventional membrane filtration method. The MFGM may be obtained by commercial products, including milk fat globule membrane whey protein powder, and the like. Milk fat globule membrane whey protein powder refers to a powdery product containing milk fat globule membrane, which is prepared from raw milk or whey by the steps of separation, concentration, drying and the like. Milk fat globule membrane milk protein powder refers to a powdery product containing milk fat globule membrane, which is prepared by taking raw milk or cream as raw materials and through the procedures of separation, concentration, drying and the like. Commercial sources of MFGM according to the present invention include Lacprodan cube MFGM-10, lacprodan cube PL-20, cor-Power cube SM2, lipid-rich MFGM fraction, or buttermilk concentrate BPC50, BPC60, G600, PC700, hilmarCor-Power, WPC7500MEGM ENRICHED WPC, etc.
As used herein, "staphylococcus aureus", also known as "staphylococcus aureus", belongs to the genus staphylococcus, is representative of gram-positive bacteria, is a common food-borne pathogenic microorganism, and is widely found in the natural environment. Staphylococcus aureus can produce enterotoxins under appropriate conditions, causing food poisoning. Enterotoxin is a single-chain small molecular protein, has a molecular weight of about 26-29 k Da, relatively low molecular weight, has thermal stability, and can damage intestinal tracts of human bodies to cause symptoms such as vomiting and diarrhea.
As used herein, a "kit" refers to a packaged set of related components, such as one or more compounds or compositions, and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants. The kit comprises medium-long chain fatty acid triglyceride and/or milk fat globule membrane. The "kit" is used to inhibit staphylococcus aureus in vitro, including inhibiting the growth, proliferation, and/or production of metabolites of staphylococcus aureus.
Detailed Description
Nutritional composition
The first aspect of the present invention provides a nutritional composition comprising medium long chain fatty acid triglycerides (MLCT) and Milk Fat Globule Membranes (MFGM), wherein the mass ratio of medium long chain fatty acid triglycerides to milk fat globule membranes is from 2:1 to 80:1.
The present inventors have found that when the medium-long chain fatty acid triglyceride and the milk fat globule membrane are used in combination in a defined mass ratio, there is a synergistic effect between the two, which is capable of synergistically ameliorating diarrhea, in particular ameliorating (e.g. alleviating or treating) diarrhea caused by staphylococcus aureus, and/or inhibiting (in vivo or in vitro) staphylococcus aureus.
In some embodiments, the nutritional composition consists of medium long chain fatty acid triglycerides (MLCT) and Milk Fat Globule Membrane (MFGM).
In an embodiment, the mass ratio of medium-long chain fatty acid triglyceride to milk fat globule membrane in the nutritional composition is in the range of 2:1 to 80:1, e.g. 2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、15:1、20:1、25:1、30:1、35:1、40:1、45:1、50:1、55:1、60:1、65:1、70:1、75:1、80:1 or any two of them. Preferably, the mass ratio of medium-long chain fatty acid triglyceride to milk fat globule membrane is from 2:1 to 40:1, within said range the synergistic effect is more pronounced, in particular being able to synergistically improve diarrhea, in particular synergistically improve (e.g. alleviate or treat) diarrhea caused by staphylococcus aureus, and synergistically (in vivo or in vitro) inhibit staphylococcus aureus. More preferably, the mass ratio of medium-long chain fatty acid triglyceride to milk fat globule membrane is from 3:1 to 40:1, within which range the synergistic effect in improving diarrhea, in particular synergistically improving (e.g. alleviating or treating) diarrhea caused by staphylococcus aureus is more pronounced. Further preferably, the mass ratio of medium-long chain fatty acid triglyceride to milk fat globule membrane is from 3:1 to 20:1, preferably from 3:1 to 10:1, within which range the synergistic effect in inhibiting staphylococcus aureus (in vivo or in vitro) is more pronounced.
Product(s)
The second aspect of the invention relates to a food product comprising the nutritional composition according to the first aspect of the invention.
All the above description of the first aspect of the present invention applies here and will not be repeated here.
In some embodiments, the food product may be, for example, a functional food, a health food, or a health food.
In some embodiments, the food product is a regular food product or a special food product. As an example, the food product may be selected from dairy products, candies, beverages, bread and biscuits, for example the food product may be selected from milk powder or fermented food products. In some embodiments, the milk powder is an infant milk powder, such as an infant formula.
In some embodiments, the food product is an infant food product, such as an infant formula.
In some embodiments, the food product may be a powder, tablet, or liquid.
In some embodiments, the content of medium-long chain fatty acid triglycerides may be 0.1% to 30.0% based on the dry weight of the food product. As an example, the content of medium-long chain fatty acid triglycerides may be 0.1、0.2、0.5、0.8、1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0、13.0、14.0、15.0、16.0、17.0、18.0、19.0、20.0、21.0、22.0、23.0、24.0、25.0、26.0、27.0、28.0、29.0、30.0% by weight on a dry basis, or within a range defined by any two of them, based on the weight of the product. The content of milk fat globule membrane can be determined according to the ratio of the two is calculated.
In some embodiments, the milk fat globule membrane may be present in an amount of 0.0005% to 30% based on the dry weight of the product. As an example, the content of milk fat globule membrane may be 0.0005、0.001、0.005、0.01、0.05、0.1、0.2、0.5、0.8、1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0、13.0、14.0、15.0、16.0、17.0、18.0、19.0、20.0、21.0、22.0、23.0、24.0、25.0、26.0、27.0、28.0、29.0、30.0% by weight on a dry basis, or any two of them, based on the weight of the product.
Furthermore, it will be readily appreciated by a person skilled in the art that the food product may comprise, in addition to the nutritional composition of the first aspect of the invention, one or more selected from raw milk, desalted whey powder, concentrated whey protein, lactose, edible vegetable blend oil, fructooligosaccharides, galactooligosaccharides, nucleotides, choline, vitamins, minerals, DHA and taurine.
For example, when the food is milk powder, the milk powder may comprise proteins such as alpha-lactalbumin, milk fat globular membrane protein, sugars such as lactose, lipids, minerals such as calcium, iron, phosphorus, etc., vitamins, other additives such as whey powder, choline bitartrate, docosahexaenoic acid, arachidonic acid, walnut oil, etc., in addition to the nutritional composition according to the first aspect of the invention.
The food product may be produced using preparation methods commonly used in the art and will not be described in detail herein.
Application of
The invention also relates to the use of the nutritional composition of the first aspect of the invention.
In a third aspect, the use is in the manufacture of a product for ameliorating (e.g. alleviating or treating) diarrhea.
In some embodiments, the diarrhea is bacterial diarrhea. The bacterial diarrhea may be caused, for example, by a staphylococcus aureus infection.
In a fourth aspect, the use is in the manufacture of a product for inhibiting staphylococcus aureus infection.
In some embodiments, the inhibition is in vivo inhibition or in vitro inhibition.
In some embodiments, the inhibiting comprises inhibiting growth, proliferation, or metabolite production of staphylococcus aureus.
In a fifth aspect, the use is in the manufacture of a product for modulating intestinal flora (e.g. intestinal flora in a diarrhea subject). In some embodiments, the intestinal flora comprises staphylococcus aureus.
In a sixth aspect, the use is for non-therapeutic purposes for ameliorating diarrhea.
In some embodiments, the diarrhea is bacterial diarrhea. The bacterial diarrhea may be caused, for example, by a staphylococcus aureus infection.
In a seventh aspect, the use is for non-therapeutic purposes for inhibiting staphylococcus aureus infection.
In some embodiments, the inhibition is in vivo inhibition or in vitro inhibition.
In some embodiments, the inhibiting comprises inhibiting growth, proliferation, or metabolite production of staphylococcus aureus.
All the above description of the first aspect of the present invention applies here and will not be repeated here.
The product of the third to fifth aspects of the invention may for example be a food product or a medicament.
The above description of the second aspect of the present invention applies to the food product, and is not repeated here.
For the medicament, in some embodiments, the dosage forms include, but are not limited to, tablets, granular powders, capsules, and liquids.
In some embodiments, the composition, food or pharmaceutical product is administered orally to a human at a dose of 0.1 to 30 g/day, e.g., 0.1/day, 0.2/day, 0.3/day, 0.4/day, 0.5/day, 0.6/day, 0.7/day, 0.8/day, 0.9/day, 1/day, 2/day, 3/day, 4/day, 5/day, 6/day, 7/day, 8/day, 9/day, 10/day, 11/day, 12/day, 13/day, 14/day, 15/day, 16/day, 17/day, 18/day, 19/day, 20/day, 21/day, 22/day, 23/day, 24/day, 25/day, 26/day, 27/day, 28/day, 29/day, or 30/day or any range therebetween. The dosage of the milk fat globule membrane can be converted according to the proportion of the milk fat globule membrane to the medium-long chain fatty acid triglyceride.
In some embodiments, the milk fat globule membrane is administered at a dose of 0.0005 to 30 g/day, e.g., 0.0005 g/day, 0.0006/day, 0.0007/day, 0.0008/day, 0.0009/day, 0.001/day, 0.002/day, 0.003/day, 0.004/day, 0.005/day, 0.006/day, 0.007/day, 0.008/day, 0.009/day, 0.01/day, 0.02/day, 0.03/day, 0.04 g/day, 0.05/day, 0.06/day, 0.07/day, 0.08/day, 0.09/day, 1/day, 2/day, 3/day, 4/day, 5/day, 6/day, 7/day, 8/day, 9/day, 10/day, 11/day, 12/day, 13/day, 14/day, 15/day, 16/day, 17/day, 18/day, 19/day, 20/day, 21/day, 22/day, 23/day, 24/day, 25/day, 26/day, 27/day, 28/day, 29/day, or 30/day or any range therebetween. The medium-long chain fatty acid triglycerides and milk fat globules film may be formulated into compositions or formulations for administration to a subject. Or the medium-long chain fatty acid triglyceride and the milk fat globule membrane may be taken separately, e.g. simultaneously or separately, without being formulated into the same composition. In the case of separate administration, the medium-long chain fatty acid triglyceride and milk fat globule membrane may be taken at intervals during the day. Long chain fatty acid triglycerides and milk fat globule membranes can also be taken several times a day. The time of application of the two components at intervals or the number of separate applications is readily determinable by one of skill in the art.
Examples
The present invention will be more readily understood by reference to the following examples, which are merely illustrative of certain aspects and embodiments of the present invention and are not intended to limit the invention.
Unless stated to the contrary, the reagents used in this example were all commercially available materials or conventional materials. In all the following examples, medium-long chain fatty acid triglyceride (MLCT) was derived from medium-long chain fatty acid edible oil (purity 69.+ -.10%), and was produced by Qingdao sea-Zhi Yuan life technology Co., ltd, and the production lot was Y1505-22120101.
Milk Fat Globule Membrane (MFGM), available from Arla under the product name Lacprodan MFGM-10, batch P510216, phospholipid content of 8±2%, protein content of 71.5±5 wt%, sphingomyelin g/100g phospholipid of 25±10g.
Vancomycin was derived from Shanghai Michelin Biochemical technologies Co., ltd, white powder, lot number C12976208.
EXAMPLE 1 MLCT or MFGM diarrhea improving efficacy evaluation
Wild type AB strain zebra fish were randomly selected 4 days after fertilization (4 dpf) in 6-well plates, and 30 zebra fish were treated per well (experimental group). The samples were given water-soluble (concentrations are shown in Table 1), positive control vancomycin at a concentration of 1000. Mu.g/mL, and normal and model control groups were set at a capacity of 3 mL per well. After treatment at 28 ℃ of 8h, each experimental group was given nile red as a fluorescent indicator of intestinal content in water, fed overnight, the next day, the samples were washed off and nile red, and water-soluble administration of the samples was continued (concentrations see table 1, fig. 1). Except for the normal control group, the rest experimental groups are all water-soluble to staphylococcus aureus to establish a zebra fish diarrhea model. After 30 h ℃ treatment, 10 zebra fish are randomly selected from each experimental group, photographed under a fluorescence microscope, analyzed and data are collected by NIS-ELEMENTS D3.20.20 advanced image processing software, the intestinal fluorescence signal intensity of the zebra fish is analyzed, and the effect of improving diarrhea induced by staphylococcus aureus of the sample is evaluated according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS 26.0 software, p <0.05 indicated that the differences were statistically significant.
Following the above procedure, zebra fish was dosed at different doses of MLCT, MFGM, respectively, to evaluate the efficacy of two single components at different concentrations (intestinal fluorescence intensity) to improve diarrhea. The doses given are shown in Table 1, and the results of the administration are shown in Table 1 and FIG. 1.
TABLE 1 Single-component diarrhea improving efficacy (intestinal fluorescence intensity) efficacy evaluation test results
Note that P-value is the result of analysis of the significance of the difference in intestinal fluorescence intensity between the medium-long chain fatty acid triglyceride group or MFGM group and the model control group.
As can be seen from the results in table 1 and fig. 1, after the 4dpf wild type AB strain zebra fish is induced by staphylococcus aureus, the intestinal fluorescence intensity is significantly reduced (P < 0.05) compared with the normal control group, which indicates that modeling of the staphylococcus aureus induced diarrhea model is successful. The positive control (vancomycin 1000 mug/mL) and the MFGM (15.6 mug/mL, 31.2 mug/mL, 41.6 mug/mL) both have the effect of significantly improving diarrhea, and are characterized in that the intestinal fluorescence intensity of the zebra fish is stronger (P < 0.05) compared with the model control group. The long-chain fatty acid triglyceride in the monomer has the strongest diarrhea improving effect at the concentration of 250 mug/mL, and when the concentration is further increased (500 mug/mL, 750 mug/mL), the diarrhea improving effect is weakened or similar, so that the concentration of 250 mug/mL is the optimal concentration of the medium-chain fatty acid triglyceride for improving the diarrhea effect. The monomer MFGM showed the strongest effect of improving diarrhea at a concentration of 31.2. Mu.g/mL, but the effect of improving diarrhea was weaker in zebra fish when its concentration was further increased (41.6. Mu.g/mL), and not in zebra fish when its concentration was increased to 62.5. Mu.g/mL. Thus, 31.2 μg/mL is the optimal concentration for MFGM to improve diarrhea efficacy.
Evaluation of diarrhea improving efficacy of the compositions of examples 2 MLCT and MFGM
Zebra fish of the wild type AB strain of 4 dpf were randomly selected in 6-well plates, and 30 zebra fish were treated per well (experimental group). The samples were given water-soluble (concentrations are shown in Table 2, FIG. 3), positive control vancomycin at a concentration of 1000. Mu.g/mL, and normal control and model control were set at the same time, with a capacity per well of 3 mL. After treatment at 28 ℃ of 8 h, each experimental group was given nile red as a fluorescent indicator of intestinal content in water, fed overnight, the next day, the samples were washed off and nile red, and water-soluble administration of the samples was continued (concentrations see table 2, fig. 3). Except for the normal control group, the rest experimental groups are all water-soluble to staphylococcus aureus to establish a zebra fish diarrhea model. After 30 h ℃ treatment, 10 zebra fish are randomly selected from each experimental group, photographed under a fluorescence microscope, analyzed and data are collected by NIS-ELEMENTS D3.20.20 advanced image processing software, the intestinal fluorescence signal intensity of the zebra fish is analyzed, and the effect of improving diarrhea induced by staphylococcus aureus of the sample is evaluated according to the statistical analysis result of the index.
The dose and results are shown in table 2. The administration results are shown in table 2, fig. 2 and fig. 3.
Table 2 results of evaluation of efficacy of compositions for improving diarrhea (intestinal fluorescence intensity)
Note that P-value is the result of analysis of the significance of the difference in intestinal fluorescence intensity between the medium-long chain fatty acid triglyceride group or MFGM group and the model control group.
As can be seen from the results in table 2, fig. 2 and fig. 3, the compositions of examples 3 to 10 all had the effect of improving diarrhea, which is shown by the enhanced intestinal fluorescence intensity (P < 0.05), compared to the model control group. Compared with the effect of the separate MLCT (with the concentration of 250 mug/mL) and the separate MFGM (with the concentration of 31.2 mug/mL) under the optimal concentration, the intestinal fluorescence intensity values of the zebra fish in the examples 3-9 of the MLCT and the MFGM are obviously enhanced, which indicates that the synergy between the MLCT and the MFGM in the aspect of improving diarrhea exists, and the intestinal fluorescence intensity values of the zebra fish in the examples 4-8 of the MLCT and the MFGM in the ratio of (3-40) of the MLCT and the MFGM are further enhanced, which indicates that the synergy between the MLCT and the MFGM in the aspect of improving diarrhea is more obvious.
Efficacy test of the compositions of examples 3 MLCT and MFGM against staphylococcus aureus
Zebra fish of the wild type AB strain of 4 dpf were randomly selected in 6-well plates, and 30 zebra fish were treated per well (experimental group). Samples were given water-soluble (concentrations are shown in Table 3), positive control vancomycin at a concentration of 1000. Mu.g/mL, and model control group was set at a capacity of 3 mL per well. After 24h treatment at 28 ℃, all experimental groups were water-soluble to staphylococcus aureus to establish a zebra fish staphylococcus aureus infection model. After the staphylococcus aureus is marked and treated by CM-DiI at 28 ℃ for 6 h, 10 zebra fishes are randomly selected from each experimental group, photographed under a fluorescence microscope, analyzed and data are collected by NIS-ELEMENTS D3.20 advanced image processing software, the fluorescence intensity of the intestinal staphylococcus aureus of the zebra fishes is analyzed, and the antibacterial efficacy of the sample is evaluated according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS 26.0 software, p <0.05 indicated that the differences were statistically significant.
The doses administered are shown in table 3. The administration results are shown in Table 3, FIGS. 4 and 5.
TABLE 3 evaluation of efficacy against Staphylococcus aureus
Note that the P-value is a result of analysis of the significance of the difference in fluorescence intensity between medium-long chain fatty acid triglyceride group or MFGM group and model control group staphylococcus aureus.
As can be seen from the results in table 3, fig. 4 and fig. 5, the positive control (vancomycin, 1000 μg/mL) and the long-chain fatty acid triglyceride (250 μg/mL), MFGM (31.2 μg/mL) and examples 2 to 10 all have significant anti-staphylococcus aureus effects, and are specifically shown to be weaker in fluorescence intensity (P < 0.05) of the zebra fish staphylococcus aureus compared to the model control. The ratio between MLCT and MFGM (2-40) was such that the fluorescence intensity of Staphylococcus aureus in examples 3-8 of 1, which showed a synergistic effect between MLCT and MFGM, was weaker than that of long-chain fatty acid triglyceride (250. Mu.g/mL) and MFGM (31.2. Mu.g/mL) in the monomer, the ratio between MLCT and MFGM (3-20) was such that the fluorescence intensity of Staphylococcus aureus in examples 4-7 of 1, which showed a stronger synergistic effect between MLCT and MFGM, was further decreased, and the ratio between MLCT and MFGM (3-10) was such that the synergistic effect between CT and MFGM in Staphylococcus aureus was further enhanced.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.
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| CN106578134A (en) * | 2016-12-31 | 2017-04-26 | 东北农业大学 | Infant formula milk powder for preventing infectious diarrhea |
| CN114431306A (en) * | 2020-11-02 | 2022-05-06 | 丰益(上海)生物技术研发中心有限公司 | Breast milk-like oil composition and preparation method and application thereof |
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