Application of lychee leavening in preparation of marker gene expression product for relieving intestinal aging and / or reducing intestinal cell aging

Abstract

The invention provides an application of a lychee leavening in preparation of a product for relieving intestinal aging and / or reducing expression of an intestinal cell aging marker gene, which proves that a metagen product, such as the lychee leavening, taking lychee as a raw material has the effect of improving occurrence and development of intestinal aging for the first time and can provide a new strategy for prevention of clinical diseases. Good application prospects are realized in the prevention of intestinal aging.

Description

Technical Field

[0001] The invention belongs to the field of food science and technology, and specifically relates to the application of litchi fermentation products in preparing products for alleviating intestinal cell aging and / or reducing the expression of intestinal aging marker genes. Background Art

[0002] The intestine is the main digestive organ and the largest immune organ of the human body. It absorbs nutrients and acts as a protective barrier against the external environment. Intestinal aging can cause major changes in the body, including decreased immune function, damaged intestinal barriers, and harmful molecules such as pathogens escaping the immune barrier and sneaking into the blood, causing serious damage to other organs. Therefore, developing targeted interventions to target biomarkers of aging-related intestinal failure will be beneficial for prevention or treatment, reducing the occurrence of aging diseases and improving the quality of life of the elderly.

[0003] There are many factors that affect intestinal aging, such as aging, irregular diet, lack of exercise, and mental stress. These factors will accelerate intestinal aging, reduce intestinal health, and cause a series of other health problems.

[0004] There are many intervention measures to combat intestinal aging. The first is dietary intervention, such as calorie restriction diet, which can significantly change the overall intestinal intake-absorption-conversion balance, reduce the intestinal load, and maintain a certain digestive vitality of the intestine to promote intestinal health. The second is drug intervention, such as classic anti-aging drugs, rapamycin, resveratrol, etc.

[0005] The existing products or drugs for improving the intestinal tract are generally drugs or non-food components. Drugs have certain side effects on the body, and non-food components also mean that they cannot be used frequently as common foods, and have certain limitations in terms of scope of use and safety. Summary of the invention

[0006] In order to solve the problems and shortcomings in the prior art, the present invention provides an application of litchi fermentation product in the preparation of a product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes. It is the first time to prove that postbiotic products with litchi as raw material, such as litchi fermentation product, have the effect of improving the occurrence and development of intestinal aging, which can provide a new strategy for the prevention of clinical diseases and has good application prospects in the prevention of intestinal aging.

[0007] According to a first aspect of the present invention, there is provided a use of a litchi fermentation product in the preparation of a product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes.

[0008] As a typical subtropical fruit, litchi has important economic significance. It is rich in flavonoids and phenolic acid substances. For example, ferulic acid has a positive effect on maintaining the balance of intestinal flora, and rutin has an improvement effect on intestinal inflammation. New postbiotics such as litchi fermented products obtained by fermentation process with litchi as raw material have been confirmed by some studies on their efficacy in the intestine. However, there are currently no reports on the role of litchi fermented products or other litchi-derived products in alleviating intestinal aging.

[0009] The present invention verifies for the first time that litchi fermented products can effectively improve the problem of intestinal aging. Specifically, through relevant model experiments, it is verified that litchi fermented products can effectively reduce the proportion of intestinal aging cells and the expression of intestinal aging marker genes. Therefore, it can alleviate intestinal aging from a fundamental microscopic level, improve intestinal health, and promote the health of the human intestine and other aspects.

[0010] Regarding the use of litchi fermentation products in the preparation of products that alleviate intestinal aging and / or reduce the expression of intestinal aging marker genes, since their main ingredients are common foods, they can effectively avoid the side effects and adverse effects of drugs or non-food ingredients, have a wider range of use and safety, have lower costs, and are more beneficial to the overall health level of humans or animals.

[0011] Preferably, the product prepared by using litchi fermentation product to alleviate intestinal cell aging, intestinal aging and / or reduce intestinal aging marker gene expression can be litchi fermentation product liquid, or a composition prepared by using litchi fermentation product. The composition can be a food or a health food. The main dosage forms are oral liquid, beverage, solid beverage, and soft candy, which are mainly taken by direct drinking, mixing or chewing.

[0012] Preferably, alleviating intestinal aging includes reducing the activity of lysosomal enzyme β-galactosidase; reducing the expression of intestinal cell aging marker genes includes reducing the expression of cyclin-dependent kinase inhibitor 2a and / or cyclin-dependent kinase inhibitor 1. Further experimental verification shows that litchi fermented products can effectively reduce the lysosomal contents in the intestine, reduce the activity of lysosomal enzyme β-galactosidase, and thus effectively slow down the aging of intestinal cells, so that the intestine shows a younger and healthier state macroscopically. At the same time, litchi fermented products can also reduce the expression of intestinal aging marker genes such as cyclin-dependent kinase inhibitor 2a and / or cyclin-dependent kinase inhibitor 1, which further slows down the aging of intestinal cells and improves the health of the intestine.

[0013] Preferably, the subject of the product for preparing the product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes using litchi fermentation product is human or animal. The animal can be any mammal, such as model animals, companion animals, farm animals, etc., such as mice, cats, dogs, cows, pigs, horses, etc.

[0014] Preferably, the dosage of litchi fermented product is 1-40000 mg / kg / day.

[0015] Preferably, the dosage of litchi fermented product is 26.1-130.5 mg / kg / day. Within this dosage range, the use of litchi fermented product can effectively alleviate intestinal aging and reduce the expression of intestinal cell aging marker genes. Too low a dose or too high a dose will have a poor effect on relieving intestinal aging or reducing the expression of intestinal cell aging marker genes. This is because too low a dose will result in poor effect of litchi fermented product, while too high a dose may cause some adverse reactions with the intestines, which is not conducive to relieving intestinal aging.

[0016] Preferably, the dosage of litchi fermented product is 78.3 mg / kg / day. At this dosage, the effect of using litchi fermented product to alleviate intestinal aging is the best, that is, there is a more obvious effect in reducing the proportion of intestinal senescent cells and the expression of intestinal cell senescence marker genes.

[0017] According to the second aspect of the present invention, there is provided a use of a lychee fermented product in the preparation of a product for improving intestinal motility, wherein improving intestinal motility includes improving the propulsion rate of the small intestine, and / or shortening the transit time of the colon and the whole intestine, and / or increasing the depth of the colon crypts. Relevant experiments have shown that the lychee fermented product can also effectively improve intestinal motility, mainly in terms of improving the propulsion rate of the small intestine, and / or shortening the transit time of the colon and the whole intestine, and / or increasing the depth of the colon crypts, and the improvement in these aspects also effectively alleviates intestinal aging and improves intestinal health.

[0018] The use of litchi fermentation products in the preparation of products for improving intestinal motility can also be used by humans or animals, with a dosage of 26.1 to 130.5 mg / kg / day, and an optimal dosage of 78.3 mg / kg / day.

[0019] Preferably, the litchi fermented product is obtained by fermenting a mixture of litchi juice and litchi pomace twice; the first fermentation process uses yeast or lactic acid bacteria to ferment at 4 to 60° C. in a sealed state for 10 to 168 hours; the second fermentation process uses acetic acid bacteria to ferment at 4 to 60° C. in a static state or with ventilation for 10 to 168 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of litchi fermentation on β-galactosidase staining in colon tissue of D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0021] Figure 2 The effect of litchi fermentation on aging marker genes in D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0022] Figure 3 The effect of litchi fermentation on intestinal transport capacity of D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0023] Figure 4 The effect of litchi fermentation liquid on the colon tissue morphology of D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation liquid group; middle: medium-dose litchi fermentation liquid group; high: high-dose litchi fermentation liquid group.

[0024] Figure 5 The effect of litchi fermentation on the expression of intestinal tight junction proteins in D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0025] Figure 6 The effect of litchi fermentation on serum D-lactic acid content in D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0026] Figure 7 The effect of litchi fermentation on the expression of intestinal proinflammatory factors in D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0027] Figure 8 The effect of litchi fermentation on blood antioxidant in D-gal model intestinal aging mice is shown. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group.

[0028] Fig. 9 , 10 Figure 11 shows the effect of litchi fermentation on the intestinal flora of D-gal model intestinal aging mice. Control: normal control group; D-gal: model group; low: low-dose litchi fermentation group; middle: medium-dose litchi fermentation group; high: high-dose litchi fermentation group; LF: litchi fermentation group. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0030] 1. Preparation of litchi fermentation:

[0031] (1) Pretreatment: 5 tons of litchi fruits were washed and squeezed;

[0032] (2) First fermentation: add 1 sugar to the mixture of fruit juice and pomace, inoculate 1 bacterial agent, seal and ferment at 30°C for 72 hours;

[0033] (3) Filtration: using a filter press to obtain a primary fermentation filtrate, wherein the mass of the primary fermentation filtrate is greater than 60% of the total mass of the litchi fruit;

[0034] (4) Second fermentation: add sugar 2 to the first fermentation filtrate, inoculate bacterial agent 2, and culture at 15°C for 5 days with aeration;

[0035] (5) Filtration and sterilization.

[0036] In the above process of preparing litchi fermented product, sugar 1 is fructose: brown sugar = 1:1; the added amount of sugar 1 is 2% (w / w); the bacterial agent 1 is yeast: lactic acid bacteria at 400:1; the yeast is sake yeast; the lactic acid bacteria are Lactobacillus paracasei: Lactobacillus reuteri: Lactobacillus gasseri at 1:1:1; the added amount of bacterial agent 1 is 0.9‰ of the total mass of litchi.

[0037] Sugar 2 is lactose; the amount of sugar 2 added is 15% of the total mass of the primary fermentation filtrate; bacterial agent 2 is 1:1 Acetobacter aceticus: Acetobacter aceti; the amount of bacterial agent 2 added is 1‰ of the total mass of the primary fermentation filtrate; the culture method is aeration culture with an aeration volume of 1 mL / min / mL of fermentation liquid.

[0038] The litchi fermented material is prepared through the above steps.

[0039] 2. Animal experiment design

[0040] Six-week-old male SPF C57BL / 6 mice, weighing 23±1g / mouse, were purchased from Vitallife. The breeding conditions were room temperature 22±2℃, light and dark cycle 12h each, and they were adapted for one week after purchase.

[0041] Experimental drug preparation:

[0042] aD-galactose: D-galactose powder (Sigma, A3134) was accurately weighed and the model was established at a dose of 500 mg / kg / day according to the weight of the mouse, and dissolved in distilled water. The intraperitoneal injection volume for each mouse was 100 μL.

[0043] b. Litchi fermented product: Litchi fermented product was administered according to the weight of mice, with the dosage of 26.1 mg / kg / day for the low-dose group, 78.3 mg / kg / day for the medium-dose group, and 130.5 mg / kg / day for the high-dose group. The intragastric volume for each mouse was 100 μL.

[0044] Animal Grouping:

[0045] Six-week-old male SPF C57BL / 6 mice were randomly divided into three groups, with 6 mice in each group, namely normal control group, D-gal aging group, and litchi fermentation product group. The normal control group was intraperitoneally injected and gavaged with normal saline every day, the other groups were intraperitoneally injected with D-galactose every day, the D-gal aging group was gavaged with normal saline every day, and the litchi fermentation product group was gavaged with litchi fermentation solution every day for 8 weeks. Each group of mice was intraperitoneally and gavaged once a day, and the experimental period was 56 days. During the experiment, mice in each group had free food and water, and the mice were weighed regularly every week.

[0046] Test materials:

[0047] After the last oral administration to mice, relevant mouse behavioral experiments were immediately carried out the next day. After the test, the colon, ileum, duodenum, etc. of the mice were obtained and their lengths were measured. Part of the tissue was preserved in tissue fixative, and the other part was quickly frozen and placed in a -80°C refrigerator for use.

[0048] 3. Determination of whole intestinal transport capacity

[0049] Each group of mice was first gavaged with 100 μL of the corresponding intervention solution, and 30 minutes later, they were gavaged with 100 μL of ink, placed in metabolic cages for separate culture, and allowed to resume diet and drinking water. The first black stool excretion time was calculated at 4 h.

[0050] 4. Colonic transport capacity determination

[0051] Use a pusher to push about 3 cm from the mouse anus to ensure that the mouse colon is empty. Then use the pusher to slowly push a small steel ball (usually painted with an easily identifiable color) from the mouse anus into the mouse colon. Then put the mouse in a metabolic cage and monitor the discharge of the steel ball. Record the time when the mouse first discharges the steel ball.

[0052] 5. Intestinal propulsion rate

[0053] Each group of mice was gavaged with 100 μL of carmine solution. After 30 minutes, the mice were killed by cervical dislocation. The abdominal cavity was opened and all the intestines were taken out. The intestines were measured under tension-free conditions. S1 represents the total length of the mouse intestines, and S2 represents the propulsion length of the carmine solution in the mouse intestines. The mouse intestine propulsion rate was calculated according to the formula: D = S2 / S1 × 100%.

[0054] 6. H&E staining of colon tissue

[0055] Colon tissue was fixed with 4% formaldehyde and then rinsed with PBS to prepare paraffin sections. Hematoxylin was used to stain the nucleus and eosin was used to stain the cytoplasm. After dehydration with gradient ethanol, sections were made and finally sealed with neutral resin for observation under a microscope.

[0056] 7. β-galactosidase activity staining of colon tissue

[0057] First, prepare intestinal frozen sections, then fix the samples with β-galactosidase staining fixative, add staining working solution and incubate at 37°C overnight, wash away excess staining solution with PBS, dehydrate with gradient ethanol, and seal with neutral resin for observation.

[0058] 8. Detection of aging markers and intestinal tight junction protein gene expression levels

[0059] Intestinal tissue was obtained and ground with Trizol solution to extract total RNA, which was then reverse transcribed to obtain cDNA. Then, β-actin was selected as the internal reference gene to analyze the expression levels of cyclin-dependent kinase inhibitor 2a (P16), cyclin-dependent kinase inhibitor 1 (P21), tight junction proteins (ZO-1, Occludin, Claudn-1) and pro-inflammatory factors (Thf-a, IL-1β, IL-6, Cxcl1, MCP1).

[0060] 9. Blood antioxidant and D-lactic acid index detection

[0061] After the mice were broken in the neck, their eyeballs were removed and fresh blood was collected in a coagulant tube. The tube was left to stand for 2 h and centrifuged at 3000 rpm for 15 min at room temperature. The supernatant was taken as mouse serum, and ELISA kits were used to detect antioxidant indicators (MDA, GSH-PX, SOD) and D-lactic acid content.

[0062] 10. Intestinal flora analysis

[0063] Collect mouse feces in sterile centrifuge tubes and send the samples to Meiji Bio for intestinal flora sequencing and analysis.

[0064] Example 1: Litchi fermented product restores the proportion of senescent cells and expression of senescence marker genes in intestinal senescent mice

[0065] The lysosomal contents in senescent cells usually increase, which increases the activity of the lysosomal enzyme β-galactosidase. X-Gal (5-bromo-4-chloro-3-indole-β-D-galactoside) is the substrate of β-galactosidase. X-Gal itself is colorless. After being hydrolyzed by β-galactosidase, it produces galactose and blue 5-bromo-4-chloro-indigo. In β-galactosidase staining, senescent cells show blue signals, such as Figure 1 It can be seen that the aging of the model group is more obvious than that of the normal control group.

[0066] Cyclin-dependent kinase inhibitor 2a (P16) and cyclin-dependent kinase inhibitor 1 (P21) are both negative regulators of the cell cycle. P16 inhibits cyclin-dependent kinases CDK4 and CDK6, while P21 inhibits the cylin-CDK complex. They both block the cell cycle at the G1 phase and are highly expressed in senescent cells. Figure 2 As shown, after litchi fermentation (liquid) intervened in D-gal modeled intestinal aging mice, the intestinal cell aging marker genes P16 and P21 decreased significantly, among which the medium dose had the best effect, restoring to the level of the normal control group.

[0067] Example 2: Litchi fermented product improves intestinal motility and crypt morphology in aged mice

[0068] like Figure 3 As shown in the results, after D-gal modeling of aging mice, the small intestinal propulsion rate of mice decreased significantly compared with the normal control group, and the colon transit time and whole intestinal transit time increased significantly compared with the control group. The results showed that the intestinal motility of aging mice after D-gal intervention decreased significantly. After intervention with low, medium and high doses of litchi fermented products (liquid), the intestinal motility can be restored to a certain extent, among which the medium dose of litchi fermented liquid has the best effect. Compared with the model group, it significantly increased the small intestinal propulsion rate, significantly shortened the colon and whole intestinal transit time, and was able to restore the intestinal motility to the same level as the normal control group.

[0069] like Figure 4The results of H&E staining of the intestines of each group of mice are shown. Compared with the mice in the normal control group, the crypt depth of the colon of the D-gal aging group mice was significantly reduced and the arrangement was sparse and loose, which is a clear feature of intestinal aging, which will lead to the decline of intestinal peristalsis. The crypt depth of the colon of the mice in the litchi fermented liquid (liquid) intervention group was significantly longer than that of the D-gal aging group mice, and the arrangement was also more compact and orderly.

[0070] In addition, the present invention also conducted relevant experiments on the effects of litchi fermentation on the gene expression of tight junction proteins, D-lactic acid levels, expression of pro-inflammatory factors, antioxidant levels, and intestinal flora in mice.

[0071] (1) Tight junction protein gene expression: Figure 5 As shown, the expression levels of intestinal tight junction protein genes (ZO-1, Occludin, Claudn-1) in the D-gal aging group were significantly downregulated, while after intervention with medium-dose fermented litchi ferment (liquid), the expression levels significantly rebounded and returned to the level of the normal control group.

[0072] (2) D-lactic acid level: Figure 6 As shown, the use of litchi fermented products, especially medium doses of litchi fermented products, was similar to the D-lactic acid level in the normal control group.

[0073] (3) Expression of pro-inflammatory factors: Figure 7 As shown in the data, compared with the normal control group, the use of litchi fermented products can make the expression levels of pro-inflammatory factors (Tnf-a, IL-1β, IL-6, Cxcl1, MCP1) lower, closer to the level of the normal control group, especially the medium amount of litchi fermented products, which is similar to the expression level of pro-inflammatory factors in the normal control group.

[0074] (4) Antioxidant level: Fig. 9 As shown, Ace and Chao reflect community richness, Shannon and Simpson indexes reflect community diversity. The richness and diversity of intestinal flora were closer to those of the normal control group when litchi fermented products were used, especially the medium dose of litchi fermented products, the richness and diversity of intestinal flora were closer to those of the normal control group.

[0075] like Fig.10 , 11 As shown, from the characterization results at the phylum and genus levels, it can be seen that the types and distribution of intestinal flora using litchi fermented products, especially medium-dose litchi fermented products, are closer to those of the normal control group.

[0076] In summary, it can be seen that the use of litchi fermented products has little effect on intestinal tight junction protein gene expression, D-lactic acid levels, pro-inflammatory factor expression, antioxidant levels, intestinal flora, etc., that is, it will not produce obvious side effects on the intestine, which reflects the safe use of litchi fermented products as related products such as food or oral compositions, and is more conducive to promoting the overall health level of humans or animals.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. Application of litchi fermented products in the preparation of products for alleviating intestinal aging and / or reducing the expression of genes that are markers of intestinal cell aging.

2. The use of the litchi fermented product as claimed in claim 1 in the preparation of a product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes, characterized in that: Alleviation of intestinal aging includes reducing the activity of the lysosomal enzyme β-galactosidase; Reducing the expression of intestinal cell senescence marker genes includes reducing the expression of cyclin-dependent kinase inhibitor 2a and / or reducing the expression of cyclin-dependent kinase inhibitor 1.

3. Use of the litchi fermented product according to any one of claims 1 to 2 in the preparation of a product for intestinal aging and / or reducing the expression of intestinal cell aging marker genes, characterized in that: The target users are humans or animals.

4. The use of the litchi fermented product as claimed in claim 3 in the preparation of a product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes, characterized in that: The dosage of the litchi fermented product is 1 to 40000 mg / kg / day.

5. The use of the litchi fermented product as claimed in claim 4 in the preparation of a product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes, characterized in that: The dosage of the litchi fermented product is 26.1-130.5 mg / kg / day.

6. Application of litchi fermentation product in the preparation of a product for improving intestinal motility, characterized in that: Improving intestinal motility includes increasing the small intestinal propulsion rate, and / or shortening the colon and whole intestinal transit time, and / or increasing the depth of colon crypts.

7. The use of the litchi fermented product as claimed in claim 6 in preparing a product for improving intestinal motility, characterized in that: The target users are humans or animals.

8. The use of the litchi fermented product as claimed in claim 7 in the preparation of a product for improving intestinal motility, characterized in that: The dosage of the litchi fermented product is 26.1-130.5 mg / kg / day.

9. The use of the litchi fermented product as claimed in claim 8 in preparing a product for improving intestinal motility, characterized in that: The dosage of the litchi fermented product is 78.3 mg / kg / day.

10. The use of the litchi fermented product according to claim 1 in the preparation of a product for alleviating intestinal aging and / or reducing the expression of intestinal cell aging marker genes, characterized in that: The litchi fermented product is obtained by fermenting a mixture of litchi juice and litchi pomace twice; The first fermentation process uses yeast and / or lactic acid bacteria to ferment at 4-60°C in a sealed state for 10-168 hours; The second fermentation process uses acetic acid bacteria to ferment at 4-60°C for 10-168 hours with static and / or aeration.

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