CN113854450A - Hydrogen-rich probiotic fermented grain composition and application thereof in preparation of product for preventing non-alcoholic fatty liver disease - Google Patents
Hydrogen-rich probiotic fermented grain composition and application thereof in preparation of product for preventing non-alcoholic fatty liver disease Download PDFInfo
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- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/169—Plantarum
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a hydrogen-rich probiotic fermented grain composition and application thereof in preparing a product for preventing non-alcoholic fatty liver, wherein the composition comprises the following components: probiotic fermented grain products and hydrogen rich water or magnesium hydride. The preparation method comprises the following steps: s1, crushing grains, and sieving the grains with a 60-80-mesh sieve according to a mass ratio of 1: adding pure water at a material-to-liquid ratio of 5-1: 7, stirring, heating and raising the temperature; s2, adding alpha-high temperature amylase with the addition amount of 9.5-10.5U/g, and performing enzymolysis to obtain a grain culture medium; s3, sterilizing the grain culture medium under high pressure, cooling, adding activated probiotic fermentation strain bacterial liquid, and fermenting for 24-40 hours on a shaking table with the rotating speed of 90-110 r/min to obtain probiotic fermented grain product liquid; s4, adding the additive into pure water to be dissolved and then sterilizing; s5, mixing and homogenizing the supernatant of the probiotic fermented grain product fermentation liquor and the sterilized additive; and S6, mixing the homogeneous liquid with hydrogen-rich water and filling. The composition can be used for preparing product for preventing non-alcoholic fatty liver disease.
Description
Technical Field
The invention relates to the technical field of health products and functional foods, in particular to a hydrogen-rich probiotic fermented grain beverage and a preparation method and application thereof, and especially relates to preparation of the probiotic fermented grain health beverage and application thereof in prevention of high-fat diet-induced non-alcoholic fatty liver disease.
Background
In recent years, as the onset of metabolic syndrome such as obesity, high Body Mass Index (BMI), abdominal obesity, hyperlipidemia, type 2 diabetes, and hypertension has increased due to changes in dietary structure and lifestyle, NAFLD has been considered as a liver manifestation of metabolic syndrome, as studies have shown that the above indications are clear risk factors for NAFLD. NAFLD prevalence has been as high as 25% worldwide; the results of system review and Meta data analysis show that the mean prevalence rate of NAFLD in asia is 29%, the prevalence rates from 1999 to 2017 are 25.28% for 1999-; the incidence of NAFLD in economically developed areas of China is as high as 15%, and the NAFLD has a trend of being younger and continuously increasing. NAFLD has now become one of the most common liver diseases worldwide.
The liver fat metabolism of NAFLD patients is disordered, so that a large amount of fat substances are accumulated in liver cells (simple fatty liver), and further the liver cells are subjected to steatosis, liver cell injury, inflammatory reaction, liver fibrosis and the like. Although early simple fatty liver is a benign stage of NAFLD and is easily reversed, 10-20% of simple fatty liver can progress to nonalcoholic steatohepatitis (NASH) without effective intervention, the incidence of NASH liver fibrosis is as high as 25%, and about 1.5-8% of patients can develop cirrhosis and possibly cause hepatocellular carcinoma. In addition, fatty liver not only affects the liver and gall system of a patient to cause liver-related diseases and death, but also is closely related to insulin resistance, hyperlipidemia, atherosclerotic diseases and the like to increase the risk of cardiovascular and cerebrovascular diseases. It poses great threat to human life health.
Long-term high calorie, high fat, high sugar dietary structure and sedentary immobility are important causes of fatty liver. Although there are clinically available drugs for NAFLD, no specific drugs for NAFLD, such as hypolipidemic drugs (e.g., statins and fibrates), drugs with insulin-sensitizing effect (e.g., metformin and thiazolidinediones), and the like are available, and although these drugs can reduce the levels of Triglyceride (TG), cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) to some extent and improve the liver function of patients with NAFLD, the safety of long-term administration (e.g., liver and kidney toxicity) is yet to be confirmed, and the therapeutic effects of some drugs are still controversial. Therefore, there is a need to search for effective target regulation measures to prevent or mitigate the development of NAFLD, depending on the pathogenesis of NAFLD.
The pathogenesis of NAFLD is not completely understood at present, and it was generally accepted earlier that the theory of "secondary attacks" was: namely, Insulin Resistance (IR) is taken as a common pathophysiological basis of metabolic syndrome, which is the initiation and central link (first hit) of the development of NAFLD, and the second hit comprises a lipid peroxidation process caused by liver lipid deposition, endotoxin-mediated toxic cytokines and the like, so that the adipose-derived liver cells are subjected to inflammation, necrosis and even fibrosis. In recent years, more and more testifies show that the pathogenic factors of NAFLD are relatively complex, the theory of 'secondary attack' gradually changes to the theory of 'multiple attack', and the multiple attack theory indicates that the secondary attack factors of overnutrition, insulin resistance, inflammation, oxidative stress, intestinal barrier damage, endoplasmic reticulum stress, heredity, epigenetic regulation and the like all participate in the generation and development process of NAFLD, so that if the oxidative stress damage and inflammatory response can be relieved through dietary supplement, and the body metabolism can be regulated, the generation and development of fatty liver can be effectively improved and delayed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a hydrogen-rich probiotic fermented grain composition, a preparation method and application thereof, and solves the technical problem of how to effectively prevent the occurrence and development of non-alcoholic fatty liver disease through daily dietary supplement.
The purpose of the invention is realized by the following scheme:
in a first aspect of the invention there is provided a hydrogen-rich probiotic fermented cereal composition comprising the following components:
probiotic fermented cereal product: 60-120 parts;
hydrogen-rich water: 180-440 parts. .
The second aspect of the invention provides the application of the hydrogen-rich probiotic fermented grain composition in preparing a product for preventing non-alcoholic fatty liver disease. The health product can reduce abdominal fat, and relieve fatty degeneration, oxidative stress and inflammatory reaction of liver caused by non-alcoholic fatty liver. Preferably, the concentration of the hydrogen-rich water is 0.8-2 mg/L.
Preferably, the beverage further comprises an additive, wherein the additive is at least one of a sweetening agent, an essence, a stabilizing agent and a thickening agent, and the specific dosage is added according to the standard allowable amount of the selected additive (see GB 2760-2014).
The third aspect of the present invention provides a method for preparing the hydrogen-rich probiotic fermented cereal composition, comprising the following steps:
s1, crushing grains, and sieving the grains with a 60-80-mesh sieve according to a mass ratio of 1: adding pure water at a material-to-liquid ratio of 5-1: 7, stirring, heating to 63-67 ℃;
s2, adding alpha-high temperature amylase with the addition amount of 9.5-10.5U/g, performing enzymolysis for 35-45 minutes to obtain a grain culture medium after the enzymolysis is finished;
s3, sterilizing the grain culture medium under high pressure, cooling, adding activated probiotic fermentation strain bacterial liquid according to the inoculation amount of 2-5%, fermenting for 24-40 hours on a shaking table with the rotating speed of 90-110 r/min to obtain probiotic fermentation grain product fermentation liquid, and centrifuging to obtain supernatant;
s4, adding the additive into pure water to be dissolved and then sterilizing;
s5, mixing and homogenizing the supernatant of the probiotic fermented grain product fermentation liquor and the sterilized additive;
s6, mixing the homogeneous liquid obtained in the step S5 with hydrogen-rich water, and filling; wherein the volume ratio of the supernatant of the probiotic fermented grain product fermentation liquid to the hydrogen-rich water is 60-120 parts: 180-440 parts.
Preferably, 60mL of the probiotic fermented grain stock solution is added, then sterilization solutions such as sucralose, sodium citrate and stabilizers are added, the mixture is homogenized, and the homogenized mixture is mixed with hydrogen-rich water (0.8-2 mg/L) and canned. Or processing probiotic fermented grain fermentation liquid into solid powder by freeze drying or spray drying, wherein each 60ml of solid powder is compatible with hydrogen-rich water, and can be mixed for drinking before use. The solid powder such as probiotics, grain powder and grain polyphenol can be used to be mixed with hydrogen-rich water (0.8-2 mg/L) and drunk before use.
Preferably, in step S1, the cereal is at least one of black barley, brown rice and quinoa.
Preferably, in step S3, the activated probiotic fermentation strain liquid contains viable bacteria with a count of 109CFU/ml; the number of viable bacteria at the end of fermentation was 108CFU/ml。
Preferably, the probiotic bacteria are lactic acid bacteria, including Lactobacillus, bifidobacterium, streptococcus, preferably Lactobacillus plantarum (Lactobacillus kisonensis), or are fermented in combination with other lactic acid bacteria.
In a third aspect of the invention, the hydrogen-rich probiotic fermented grain composition is applied to the preparation of health products or foods for preventing the non-alcoholic fatty liver. The health product can reduce abdominal fat, and relieve fatty degeneration, oxidative stress and inflammatory reaction of liver caused by non-alcoholic fatty liver.
Preferably, the probiotic fermented cereal in the food product is in the form of a fermentation broth, a solid powder, a capsule, a tablet or a pill. That is, the product is a hydrogen-rich fermented grain beverage, and solid powdered fermented grains, fermented grain capsules, fermented grain tablets or fermented grain pills can also be used in combination with hydrogen-rich water. Wherein the solid powdered fermented grain is prepared by freeze drying or spray drying; in the product, the probiotic fermented grains are used as main active ingredients and are matched with fruit and vegetable powder and/or probiotics to prepare hydrogen-rich probiotic beverage or hydrogen-rich water-compatible edible probiotic solid food.
The hydrogen-rich water can effectively reduce the blood lipid index rise induced by bad diet; reducing serum inflammatory cytokine levels; and shows good combined curative effect with other treatment means. The fermented black barley can reduce the degree of liver steatosis induced by high-fat diet, and regulate the imbalance of intestinal flora and metabolic disorder of organisms.
Compared with the prior art, the invention has the following beneficial effects:
1. the preparation method of the invention is one-time fermentation, the process is simple, the used fermentation substrate is whole grain food, and the cost is low.
2. From the implementation effect, the hydrogen-rich fermented grain beverage obtained by the preparation method of the invention is subjected to multi-angle analysis on the influence of liver steatosis degree, inflammatory factor level, oxidative stress and body lipid metabolism, and the result shows that the hydrogen-rich fermented grain beverage has the effect of specifically improving the generation and development of fatty liver.
3. The hydrogen-rich lactobacillus fermented black barley can remarkably reduce the fatty degeneration and oxidative stress condition of the liver induced by high-fat diet, reduce the abdominal fat index, reduce the inflammatory reaction and regulate the lipid metabolism.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a flow diagram of a process for preparing a hydrogen enriched fermented cereal beverage;
FIG. 2 is a graph comparing the effect of hydrogen-rich Lactobacillus fermented black barley on the perirenal lipid index of rats on a high-fat diet;
FIG. 3 is a graph comparing the effect of hydrogen-rich Lactobacillus fermented black barley on hepatic steatosis;
FIG. 4 is a graph comparing the effect of hydrogen-rich Lactobacillus fermented barley on the antioxidant activity of liver tissue;
FIG. 5 is a graph comparing the effect of hydrogen-rich Lactobacillus fermented barley on the inflammatory factors in the serum fraction;
FIG. 6 is a graph comparing the effect of hydrogen-rich Lactobacillus fermented black barley on lipid metabolism.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
NAFLD has now become one of the most common liver diseases worldwide, with its prevalence reaching 25% worldwide. If the dietary supplement can relieve oxidative stress injury and inflammatory reaction and regulate the metabolism of the organism, the occurrence and development of the fatty liver can be effectively improved and delayed.
The invention relates to the technical field of functional foods, and discloses preparation of a hydrogen-rich probiotic fermented cereal food and application of the hydrogen-rich probiotic fermented cereal food in preparation of health-care products and foods for relieving fatty liver caused by high-fat diet. The invention firstly discovers that the hydrogen-rich probiotic fermented cereal food can obviously reduce liver steatosis caused by high-fat diet and regulate body metabolism, provides preparation and application embodiments of the hydrogen-rich probiotic fermented cereal food, and can provide new ideas and technologies for developing and preventing obesity caused by high-fat diet and fatty liver related functional food. Therefore, the method has the characteristics of simplicity, convenience, practicability, low cost and the like, and has better market development and application prospects.
Example 1:preparation method of hydrogen-rich probiotic fermented black barley beverage(as shown in FIG. 1 (a))
(1) Crushing: pulverizing black barley, and sieving with 80 mesh sieve;
(2) enzymolysis gelatinization and saccharification: according to the following steps of 1: 5(m/m) of material-liquid ratio, adding pure water, stirring and heating to 65 ℃; adding alpha-high temperature amylase (10U/g) for enzymolysis for 40 min; autoclaving after saccharification;
(3) cooling and inoculating fermentation: cooling the sterilized enzymolysis solution, adding activated Lactobacillus plantarum bacterial solution (containing viable count of 10) according to 3% of inoculation amount under aseptic condition9CFU/ml), fermenting for 24h at 30 ℃ in a shaker (100rpm), and rapidly cooling to below 20 ℃ for later use;
(5) blending: adding 60mL of the fermented stock solution of the grains, adding sterilization liquid such as sucralose (0.05g/kg, maximum not more than 0.25g/kg) and stabilizer (xanthan gum, which can be added according to needs), mixing, homogenizing, mixing with hydrogen-rich water (0.8mg/L, 440mL), and canning.
Example 2:preparation method of hydrogen-rich probiotic fermented black barley beverage
(1) Crushing: pulverizing black barley, and sieving with 80 mesh sieve;
(2) enzymolysis gelatinization and saccharification: according to the following steps of 1: 5(m/m) of material-liquid ratio, adding pure water, stirring and heating to 65 ℃; adding alpha-high temperature amylase (10U/g) for enzymolysis for 40 min; autoclaving after saccharification;
(3) cooling and inoculating fermentation: cooling the sterilized enzymolysis solution, adding activated Lactobacillus plantarum bacterial solution (containing viable count of 10) according to 3% of inoculation amount under aseptic condition9CFU/ml), fermenting for 24h at 30 ℃ in a shaker (100rpm), and rapidly cooling to below 20 ℃ for later use;
(4) blending: adding 60mL of grain fermentation stock solution, adding sterilization solution such as sucralose, sodium citrate, stabilizer and the like, mixing, homogenizing, mixing with hydrogen-rich water (1.5mg/L, 440mL), and canning.
Example 3:preparation method of hydrogen-rich probiotic fermented black barley beverage
(1) Crushing: crushing the black barley and then sieving the crushed black barley with a 80-mesh sieve;
(2) enzymolysis gelatinization and saccharification: according to the following steps of 1: 5(m/m) of material-liquid ratio, adding pure water, stirring and heating to 65 ℃; adding alpha-high temperature amylase (10U/g) for enzymolysis for 40 min; autoclaving after saccharification;
(3) cooling and inoculating fermentation: cooling the sterilized enzymolysis solution, adding activated Lactobacillus plantarum bacterial solution (containing viable count of 10) according to 3% of inoculation amount under aseptic condition9CFU/ml), fermenting for 24h at 30 ℃ in a shaker (100rpm), and rapidly cooling to below 20 ℃ for later use;
(4) blending: adding 60mL of grain fermentation stock solution, adding sterilization solution such as sucralose, sodium citrate, stabilizer and the like, mixing, homogenizing, mixing with hydrogen-rich water (2mg/L, 440mL), and canning.
Example 4:preparation method of hydrogen-rich probiotic fermented black barley beverage(as shown in (b) of FIG. 1)
(1) Crushing: pulverizing black barley, and sieving with 80 mesh sieve;
(2) enzymolysis gelatinization and saccharification: according to the following steps of 1: 6.3(m/m) of material-liquid ratio, adding pure water, stirring and heating to 65 ℃; adding alpha-high temperature amylase (10U/g) for enzymolysis for 40 min; autoclaving after saccharification;
(3) cooling and inoculating fermentation: cooling the sterilized enzymolysis solution, adding activated Lactobacillus plantarum bacterial solution (containing viable count of 10) according to the inoculation amount of 2% under aseptic condition9CFU/ml), fermenting for 39h at 30 ℃ in a shaking table (100rpm), and rapidly cooling to below 20 ℃ for later use;
(4) spray drying to prepare powder: the sugar-containing vacuum concentrated milk is used as a protective agent, is uniformly mixed with a zymocyte liquid according to the mass ratio of 1:3, is subjected to hot air spray drying, is subjected to spray drying for 1 hour when the temperature in a tower is controlled to be 72-75 ℃, is taken out of powder and is packaged, and is mixed with hydrogen-rich water (the concentration is 1.5mg/L) for drinking before drinking, wherein the drinking amount of the hydrogen-rich water is not limited.
Example 5:preparation method of quinoa drink fermented by hydrogen-rich probiotics
(1) Crushing: crushing quinoa and sieving the crushed quinoa with a 80-mesh sieve;
(2) enzymolysis gelatinization and saccharification: according to the following steps of 1: 5(m/m) of material-liquid ratio, adding pure water, stirring and heating to 65 ℃; adding alpha-high temperature amylase (10U/g) for enzymolysis for 40 min; autoclaving after saccharification;
(3) cooling and inoculating fermentation: cooling the sterilized enzymolysis solution, adding activated Lactobacillus plantarum bacterial solution (containing viable count of 10) according to 3% of inoculation amount under aseptic condition9CFU/ml), fermenting for 24h at 30 ℃ in a shaker (100rpm), and rapidly cooling to below 20 ℃ for later use;
(4) blending: adding 60mL of grain fermentation stock solution, adding sterilization solution such as sucralose, sodium citrate, stabilizer and the like, mixing, homogenizing, mixing with hydrogen-rich water (concentration: 0.8-2mg/L, 440mL), and canning.
(5) Or spray drying to obtain powder, and mixing with hydrogen-rich water for drinking.
Example 6: primary efficacy test of hydrogen-enriched probiotic fermented cereal drink
1. Animal experiment intervention method and main index analysis (the hydrogen-rich probiotic fermented black barley beverage prepared in example 4 is selected)
(1) Animal grouping and feeding and intervention
Male SD rats (4 weeks old, 90-110g), after one week of acclimation feeding, the experimental animals were divided into: normal control group (NC, 8), high fat diet group (HF, 8), high fat diet + hydrogen rich water group (FL, 8), high fat diet + hydrogen rich fermented rye drink group (FBL, 8), high fat diet + fermented rye group (FB, 8). A feeding period: about 12 weeks (depending on the molding). The room temperature is maintained at 25 +/-1 ℃, the relative humidity is 45-65%, and the lighting is performed alternately in light and dark for 12 hours (6:00-18: 00). During the experiment the rats in each group had free diet and water. The stomach-filling dose of the fermented rye liquid is 1mL/100g BW for times, and the fermented rye liquid is drunk together with hydrogen-rich water (1.5ppm) (note: considering the control of the intervention amount in the animal experiment operation, the pulp fermented rye and the hydrogen-rich water are separately given to the animals in the experiment, the hydrogen-rich water is freely drunk, when the hydrogen-rich probiotic fermented cereal product is actually prepared, fermented cereal powder or liquid can be quantitatively drunk in a way of being mixed with the hydrogen-rich water, the specific preparation process is shown in example 1, and the intervention lasts for 12 w. The food intake was accurately measured every morning for a fixed period of time and the weight of each group of rats was measured every week for a fixed period of time.
Collecting samples: after 12 weeks of intervention, rats were weighed, anesthetized with 2% sodium pentobarbital (4mg/100g), and sacrificed after dislocation of the cervical vertebrae. Then taking out the abdominal aorta blood from each group, standing for 1h at room temperature, and centrifuging to obtain serum; the abdominal cavity was opened, liver and perirenal fat were carefully removed, weighed, liver tissue (0.6cm × 0.6cm × 0.6cm) was fixed in 4% paraformaldehyde at the same position in each group of rats, paraffin sections were prepared after the fixation was completed, remaining tissue was washed with physiological saline, excess water was removed with clean filter paper, cut into small pieces and dispensed into 2mL centrifuge tubes, rapidly frozen in liquid nitrogen, and transferred to a-80 ℃ low temperature refrigerator for storage.
(2) Perirenal fat index
Perirenal fat index (%) ═ perirenal fat mass/rat mass 100
(3) Liver lipid content detection
After homogenizing the liver, 50mg was accurately weighed, 1mL of an extractant (methanol: chloroform: 2:1, v: v) was added, vortexed, sonicated for 10min, and 200. mu.L of physiological saline was taken out, vortexed, 12000rpm, 10min, and centrifuged at 4 ℃. Discarding the supernatant, transferring the lower layer liquid to a sampling bottle, concentrating to dryness, and accurately weighing the lipid by a differential method.
The triglyceride of rat liver (TG, A110-2-1, single reagent GPO-PAP method), total cholesterol (TC, A111-2-1, single reagent GPO-PAP method) and free fatty acid (NEFA, A042-1-1, double reagent enzyme method) are all determined by Nanjing institute of bioengineering institute, Inc. The specific pretreatment steps are as follows: accurately weighing 50mg of each group of samples, adding 10 times of volume of absolute ethyl alcohol for homogenate, centrifuging and taking supernatant to prepare liver tissue extract to be tested. Taking 10 mu L of liver tissue extract in TG detection for determination; taking 10 mu L of liver tissue extract in TC detection for determination; mu.L of liver tissue extract was measured in the NEFA assay.
(4) Analysis of antioxidant index
Rat liver superoxide dismutase SOD (A001-3), glutathione peroxidase GSH-PX (A005), catalase CAT (A007-1-1) activity analysis and level detection of glutathione GSH (A061-2) and malondialdehyde MDA (A003-1) are carried out by adopting Nanjing to build a biological Limited company kit, and the operation is carried out according to the instruction. The pretreatment steps are as follows: accurately weighing 100mg of each group of samples, adding 10 times of physiological saline for homogenate, centrifuging and taking supernate to prepare liver tissue extracting solution for later use; measuring the liver tissue extract after 50 mul of physiological saline is diluted by 160 times in SOD detection; 200 mu L of physiological saline is taken to be diluted by 20 times in GSH-Px detection to carry out determination on the liver tissue extracting solution; in the MDA assay, 50. mu.L of liver tissue extract was taken and assayed. In GSH detection, 50mg of each group of samples are accurately weighed, 5 times volume of physiological saline is added to prepare liver tissue extract, and 100 mu L of supernatant is centrifuged to be measured.
(5) Serum cytokine detection
Serum cytokines of rats in each group were detected by RayBiotech IL-10(Rat IL-10ELISA, ELR-IL10-1), Leptin (Rat Leptin ELISA, ELR-LEPTIN-1), TGF-beta (Human TGF-beta ELISA, ELH-TGFb1-1), TNF-alpha (Rat TNF-alpha ELISA, ELR-TNFa-1) assay kits, and the procedures were performed exactly as indicated in the instructions.
(6) Lipid metabolism analysis
After homogenizing the liver, 50mg was accurately weighed, 1ml of an extractant (methanol: chloroform: 2:1, v: v) was added, vortexed, sonicated for 10min, taken out, 200. mu.L of physiological saline was added, vortexed, 12000rpm, 10min, and centrifuged at 4 ℃. The liver tissue (already in the form of a sheet) was removed with forceps, the supernatant was discarded, and the lower layer liquid was transferred to a sampling bottle (weighed) and concentrated to dryness.
The double solvent is DCM, isopropanol and methanol which are 1:1:2, 200-500 mu L (fixed volume) for re-dissolution, and a larger volume is needed for re-dissolution in a high-fat group, otherwise, the double solvent is suspended too much, and the re-dissolution volume is determined in the high-fat group.
Chromatographic conditions are as follows: a Vanqish UHPLC system (ultra high performance liquid chromatography-quadrupole orbitrap mass spectrometer, Thermofisiher, US), an ACQUITY UPLC BEH C18 column (100X 2.1mm, 1.7 μm, Waters Co., US) equipped with a Q active plus mass spectrometer was used. The column temperature was 55 ℃ and the amount of sample was 1. mu.L. Mobile phase a was an acetonitrile solution containing 10mM ammonium formate and 0.1% formic acid (acetonitrile: water 60:40, v: v) and mobile phase B was an isopropanol solution containing 10mM ammonium formate and 0.1% formic acid (isopropanol: water 90:10, v: v). A gradient elution method is adopted, the A/B gradient in 17 minutes is 95/5-0/100, and the flow rate is 0.4 mL/min.
To ensure consistent performance of the assay system, mixed Quality Control (QC) samples were prepared by combining all samples in equal amounts. Before analysis, 10 QC samples are injected to balance the system; during the analysis, each 10 samples were injected and then 1 QC sample was taken to evaluate the system stability.
Mass spectrum conditions: the ion source was operated in two electrospray ionization modes, positive (ESI +, 3.8kV) and negative (ESI-, 3.0 kV). Using DDA scanning mode, carrying out 6 MS/MS scans after 1 full scan; adopting collision energy of NEC, 15, 30 and 45eV respectively; inducing the dissociated gas: nitrogen (99.999%). Full scan range: 150-2000 amu, full scan resolution 70000, AGC: 1e6, IT: 100 ms; dd-MS/MS resolution 17500, AGC: 5e5, IT: 50 ms. The capillary temperature was 320 ℃ and the ion lens voltage frequency (s-lens RF level) was 50V.
Data processing: raw data information is collected by software Xcalibur (thermodissher, US), and is imported into software Lipidsearch4.2 (thermodissher, US) to perform peak extraction, peak alignment, and normalization processing, so as to obtain a data matrix containing ion mass-to-charge ratio (m/z), Retention Time (RT), and peak area. Lipidsearch4.2 identifies compounds based on the exact molecular weight, molecular formula, fragmentation in MS/MS of the substance.
2. Statistical treatment
The results are expressed as mean ± standard deviation (mean ± SD) and the statistical analysis software SPSS19.0(IBM, usa) and compared between groups using one-way ANOVA, P <0.05 being statistically significant.
EXAMPLES results
The mechanism of fatty liver is complex, and mainly comprises liver lipid metabolism disorder, insulin resistance, oxygen stress, lipid peroxidation, immune response, genetic factors and the like. The important links are lipid metabolism disorder, increase of Free Fatty Acid (FFA), increase of tumor necrosis factor (TNF-alpha), enhancement of transforming growth factor (TGF-beta) activity and the like. The results of the invention show that the hydrogen-rich lactobacillus fermented black barley can remarkably reduce the fatty degeneration and oxidative stress conditions of the liver induced by high-fat diet, reduce the abdominal fat index, reduce the inflammatory response and regulate the lipid metabolism. The experimental results are set forth below:
(1) influence of hydrogen-rich lactobacillus fermented black barley on perirenal lipid index of rat with high fat diet
Abdominal fat (abdominal fat) is mainly visceral fat, and in rat experiments abdominal fat is mainly composed of perirenal fat and peritesticular fat. After the traditional Chinese medicine composition is taken for a long time, the calorie is excessive, and the abdominal fat is not taken, so that the visceral fat is increased easily, and the abdominal obesity is caused, and is one of risk factors of heart disease, stroke, fatty liver, type II diabetes and the like. The invention observes the increase condition of the perirenal fat of rats in each group by detecting the perirenal fat index. It was found that rats (HF) on high-fat diet alone significantly increased the perirenal lipid index (P <0.01) compared to Normal Control (NC) rats, while lactobacillus hydrogensus fermented black barley (FBL) significantly decreased the perirenal lipid index (P <0.05) compared to HF and better than rats (FB) on hydrogen-rich water (FL) and fermented rye alone (as shown in fig. 2).
(2) Effect of hydrogen-rich Lactobacillus fermented Black barley on hepatic steatosis
The long-term high fat diet can cause fatty liver due to liver steatosis, and it can be seen from fig. 3 that the liver lipid content of the pure high fat diet group is very significantly increased (p <0.0001), the mean value reaches 46.62%, which is 7 times of the liver lipid content (6.56%) of the rats in the NC group, while the liver lipid content of the rats in the different dry pre-groups is very significantly decreased (p <0.0001), wherein the dry prognosis liver lipid content (22.61%) of lactobacillus hydrogenus fermented black barley is lower than that of the pure FL group (24.72%) and FB group (24.68%).
Further, the analysis results of liver tissue triglyceride TG, total cholesterol TC and free fatty acid NEFA show that the contents of TC, TG and NEFA in the liver tissues of the rats in the HF group are respectively 4.33 times, 1.63 times and 1.27 times of those in the NC group, and the difference is obvious. The TC, TG and NEFA content of the liver of each intervention group rat is reduced overall, wherein the TC and TG content of the rats in the FBL group is 25.68 and 45.67 mu mol/g respectively, and is lower than that of the rats in the FL group (27.41 and 51.94 mu mol/g) and the rats in the FB group (26.71 and 50.85 mu mol/g), the TC content is reduced by 18.45 percent compared with that of the rats in the HF group (31.49 mu mol/g), and the difference is very obvious (p < 0.0001). While all three interventions effectively reduced NAFLD rat liver free fatty acid levels, with significant differences from the HF group (p <0.05) and no significant differences from the NC group (as shown in figure 3). The result shows that the hydrogen-rich probiotic grain beverage can improve the liver biochemical indexes of the NAFLD rat and effectively reduce the liver steatosis of the NAFLD rat.
(3) Influence of hydrogen-rich lactobacillus fermented black barley on antioxidant activity of liver tissue
The oxidation and the antioxidation of a normal human body are in a relative balance state, but the insulin resistance and the excessive peripheral fat mobilization exist in the body of a patient with the non-alcoholic fatty liver disease, so that a large amount of free fatty acid can be generated, and the beta oxidation overload is further caused. As the progress progresses slowly, the dynamic balance between the antioxidant and the antioxidant is imbalanced, and finally lipid peroxidation is caused. Lipid peroxidation can generate a large amount of active and highly toxic intermediate products, which affect the normal function of liver cells, and inflammatory reaction causes inflammatory cells to infiltrate the liver parenchyma.
The oxidative stress injury and the oxidation resistance of the liver of a rat are evaluated by detecting indexes such as Malondialdehyde (MDA) content, Superoxide dismutase (SOD) activity, Glutathione peroxidase (GSH-Px) activity and Glutathione (GSH) content of lipid peroxidation products, and the results are shown in fig. 4: the MDA content of the liver tissue of the rat in the HF group is increased by 20.05 percent compared with that of the rat in the NC group, which shows that the NAFLD rat body has lipid peroxidation injury; the liver MDA level of the rats in the FBL group is obviously reduced by 21.42 percent compared with that in the HF group (p is less than 0.05); in addition, the liver tissue GSH-Px activity (238.58vs.346.80U/mg prot) and GSH content (1.093vs. 13.813. mu. mol/g prot) of the HF group rat were also significantly decreased compared to the NC group, indicating liver tissue oxidation-antioxidant imbalance. After the hydrogen-rich lactobacillus fermented rye is dried, the GSH-Px activity (297.51U/mg prot) and the GSH content (3.438 mu mol/g prot) of the rat are obviously improved compared with the HF group (p is less than 0.05), and the intervention effect is better than that of the FL group and the FB group, which shows that the hydrogen-rich lactobacillus fermented rye can effectively reduce the oxidative stress injury of the liver tissue.
(4) Influence of hydrogen-rich lactobacillus fermented black barley on serum part inflammatory factors
Leptin (Leptin) is an adipocyte factor synthesized and released by fat cells in the body, which not only regulates the total amount of food intake and energy consumption, but also plays an important role in reproduction, angiogenesis, immune system defense reaction, and the like. In addition, leptin can effectively promote the synthesis and release of insulin, so that the oxidation reaction process of liver tissues and fatty acid is accelerated, and the synthesis and release of proinflammatory mediator factors can be obviously stimulated. Under normal conditions, human liver tissues cannot detect the expression of leptin, and when hepatic stellate cells are activated and hepatic fibrosis occurs, the expression level of leptin is obviously increased, which indicates that the leptin plays an important role in the development of hepatic fibrosis; serum tumor necrosis factor-alpha (TNF-alpha) is an important cytokine for clinically regulating insulin resistance, and the generation amount of the serum TNF-alpha is controlled by stabilizing the immunologic function of adipose tissues and livers in bodies of patients, so that the aim of treatment is finally fulfilled; the research shows that the TGF-beta level in serum shows an obvious rising trend along with the aggravation of the fatty liver degree and is closely related to the inflammatory change of the liver tissue seen in liver biopsy.
The inflammatory factor test results of the present invention are shown in fig. 5: the levels of Leptin (Leptin) and transforming growth factor (TGF-beta) of the HF group rats are obviously increased (p is less than 0.05), which is respectively 2.77 times and 1.61 times of the level of NC group rats, and the level of tumor necrosis factor alpha (TNF-alpha) is 1.38 times of the level of NC group rats; the mean values of TGF-beta and TNF-alpha levels of rat serum in the FBL group are respectively reduced by 30.41% and 41.33% compared with HF (p is less than 0.05), and the Leptin level in the FBL group is reduced by 53.25% compared with HF group, which shows that the lactobacillus-rich fermented rye can effectively reduce the inflammatory reaction of fatty liver rat body and the oxidative stress injury thereof, and the effect is better than that of pure hydrogen-rich water or fermented rye dry pre-treatment group.
(5) Regulation effect of hydrogen-rich lactobacillus fermented black barley on organism metabolism
Metabolomics results are shown in figure 6: high fat diet induced fatty liver causes significant changes in various metabolites in rat body, including serum metabolite docosahexaenoic acid (DHA), fecal metabolite glycocholic acid, L-cystine, 7-ketocholesterol, and liver metabolite lysophosphatidylserine (22:6), lysophosphatidylcholine (20:4), etc. It shows that the liver steatosis obviously affects the biosynthesis of unsaturated fatty acid, the synthesis of primary bile acid, partial amino acid metabolism and the like. After the hydrogen-rich lactobacillus fermented rye is dried, the metabolic disorder caused by NAFLD can be obviously relieved, and the FBL group metabolic spectrum shows greater similarity with the NC group.
In conclusion, the preparation process of the hydrogen-rich lactobacillus fermented cereal beverage and the embodiment of the effect of the hydrogen-rich lactobacillus fermented cereal beverage on the non-alcoholic fatty liver disease can effectively relieve steatosis, improve the oxidative stress state and inflammatory reaction of an organism and regulate the metabolism of the organism, and the hydrogen-rich lactobacillus fermented cereal beverage has a good application prospect in combination with the condition of big data investigation of sub-health people at present.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (9)
1. A hydrogen-rich probiotic fermented cereal composition is characterized by comprising the following components in parts by volume:
probiotic fermented cereal product: 60-120 parts;
hydrogen-rich water: 180-440 parts.
2. The application of a hydrogen-rich probiotic fermented grain composition in preparing a product for preventing nonalcoholic fatty liver, which is characterized by comprising the following components in percentage by volume: probiotic fermented cereal product: 60-120 parts; hydrogen-rich water: 180-440 parts; the concentration of the hydrogen-rich water is 0.8-2 mg/L.
3. Use of the hydrogen-rich probiotic fermented cereal composition according to claim 2 for the preparation of a product for the prevention of non alcoholic fatty liver disease, wherein the hydrogen-rich probiotic fermented cereal composition further comprises an additive which is at least one of a sweetener, an essence, a stabilizer, a thickener.
4. Use of a hydrogen-rich probiotic fermented cereal composition according to claim 2 for the preparation of a product for the prevention of non alcoholic fatty liver disease, characterized in that the preparation method of said hydrogen-rich probiotic fermented cereal composition comprises the following steps:
s1, crushing grains, and sieving the grains with a 60-80-mesh sieve according to a mass ratio of 1: adding pure water at a material-to-liquid ratio of 5-1: 7, stirring, heating to 63-67 ℃;
s2, adding alpha-high temperature amylase with the addition amount of 9.5-10.5U/g, performing enzymolysis for 35-45 minutes to obtain a grain culture medium after the enzymolysis is finished;
s3, sterilizing the grain culture medium under high pressure, cooling, adding activated probiotic fermentation strain bacterial liquid according to the inoculation amount of 2-5%, fermenting for 24-40 hours on a shaking table with the rotating speed of 90-110 r/min to obtain probiotic fermentation grain product fermentation liquid, and centrifuging to obtain supernatant;
s4, adding the additive into pure water to be dissolved and then sterilizing;
s5, mixing and homogenizing the supernatant of the probiotic fermented grain product fermentation liquor and the sterilized additive;
s6, mixing the homogeneous liquid obtained in the step S5 with hydrogen-rich water, and filling; wherein the volume ratio of the supernatant of the probiotic fermented grain product fermentation liquid to the hydrogen-rich water is 60-120 parts: 180-440 parts.
5. The use of the hydrogen-enriched probiotic fermented cereal composition according to claim 4 for the preparation of a product for the prevention of non-alcoholic fatty liver disease, wherein after step S3, the probiotic fermented cereal product broth is processed into solid powder by freeze-drying or spray-drying and added before use.
6. The use of the hydrogen-rich probiotic fermented cereal composition according to claim 4 for the preparation of a product for the prevention of non-alcoholic fatty liver disease, wherein in step S1, the cereal is at least one of black barley, brown rice and quinoa.
7. The use of the hydrogen-enriched probiotic fermented grain composition according to claim 4 for preparing a product for preventing nonalcoholic fatty liver disease, wherein in step S3, the activated probiotic fermented strain bacterial liquid contains 10 viable bacteria9CFU/ml; the number of viable bacteria at the end of fermentation was 108CFU/ml。
8. Use of a hydrogen-rich probiotic fermented cereal composition according to claim 4, in the manufacture of a product for the prevention of non alcoholic fatty liver disease, wherein the probiotic is a lactic acid bacterium.
9. Use of the hydrogen-rich probiotic fermented cereal composition according to claim 2 for the preparation of a product for the prevention of non alcoholic fatty liver disease, characterized in that the health product or food product comprising the probiotic fermented cereal product is a fermentation broth, a solid powder, a capsule, a tablet or a pill.
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