A model for evaluating the efficacy of improving lactose intolerance and its application

Abstract

The present invention relates to the field of medicine, health food and food evaluation, and for the problem that the model modeling of the effect of lactose intolerance is difficult and the observation is not intuitive, a model for evaluating and improving the effect of lactose intolerance and its application are provided. The model is constructed as follows: zebrafish is treated with 1000-6000μg / mL lactose aqueous solution for 4-24 h, the treated zebrafish is taken under a dissecting microscope to take pictures, and the zebrafish intestinal area is analyzed; zebrafish egg yolk powder and 4000-8000μg / mL lactose aqueous solution are given for a total intervention of 3-5 h, the egg yolk powder and lactose are washed off, and the intestinal opacity of the zebrafish is observed to decrease after a period of time, and the treated zebrafish is taken under a dissecting microscope to take pictures, and the sum of the intestinal opacity of the zebrafish is analyzed. The present invention uses zebrafish as a model animal, and preferably selects suitable model parameters to obtain a model for evaluating the effect of lactose intolerance with fast modeling, high throughput, low cost and convenient detection.

Description

Technical Field

[0001] The invention relates to the fields of medicine, health food and food evaluation, and in particular to a model for evaluating the efficacy of improving lactose intolerance and its application. Background Art

[0002] Lactose is one of the short-chain fermentable carbohydrates (FODMAPs), which can cause intestinal symptoms by stimulating the activity of intestinal gas-producing bacteria. Lactose intolerance accounts for 69% of the total food intolerance population, ranking first, and 56.6% of them have multiple food intolerances at the same time, which seriously affects the quality of life of patients. The pathological mechanism of lactose intolerance mainly includes small intestinal digestive disorders and abnormal colon metabolism.

[0003] There are endless lactose-related products, foods, supplements and probiotics. Although there are many products that improve lactose-induced lactose intolerance symptoms, there is a lack of animal experiments when they claim efficacy. Most animal experiments use physical stimulation (water avoidance stress method) combined with oral administration of large amounts of lactose to induce a lactose intolerance liver depression and spleen deficiency model (for example, Wang Yitian, Chen Ting, Xue Hong, et al. Establishment and evaluation of a lactose intolerance liver depression and spleen deficiency model [J]. Chinese Journal of Integrated Traditional and Western Medicine for Digestion, 2022(006):030). The modeling time is as long as 13-15 days, and the phenomenon observation is not intuitive, and the definition of lactose intolerance is not accurate. In addition, most products predict efficacy based on possible ingredients, and no in vivo studies have been conducted on related ingredients. Commonly used model animals are rats, mice and rabbits, but the results are not intuitive and intestinal experiments often have high requirements for animals. If necessary, endpoint studies will be conducted on animals, causing their death, resulting in unnecessary waste. Therefore, it is very necessary to choose a suitable animal model to explore the mechanism of lactose intolerance and develop drugs, health foods and foods to improve lactose intolerance. Summary of the invention

[0004] In order to overcome the problems of difficult modeling and non-intuitive observation of models for evaluating the efficacy of lactose intolerance, the present invention provides a model for evaluating the efficacy of improving lactose intolerance and its application. Zebrafish is used as a model animal, and appropriate model parameters are optimized to obtain a model for evaluating the efficacy of lactose intolerance with fast modeling, high throughput, low cost and convenient detection.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A model for evaluating the efficacy of improving lactose intolerance, comprising the following construction steps:

[0007] (1) Construction of abdominal distension model: randomly selected zebrafish were treated with 1000-6000 μg / mL lactose aqueous solution for 4-24 h. The treated zebrafish were photographed under a dissecting microscope and the intestinal area of ​​the zebrafish was analyzed using image processing software.

[0008] (2) Construction of diarrhea model: Zebrafish were randomly selected and treated with 2-5 mg / mL egg yolk powder and 4000-8000 μg / mL lactose aqueous solution for 3-5 h. The egg yolk powder and lactose were washed off and the zebrafish were left for a period of time to observe that the intestinal opacity of the zebrafish decreased. The treated zebrafish were photographed under a dissecting microscope and the total intestinal opacity of the zebrafish was analyzed using image processing software.

[0009] There is no restriction on the order of the above steps (1) and (2).

[0010] Preferably, the zebrafish is a zebrafish of 3-9 dpf after fertilization, and more preferably a zebrafish of 3-5 dpf. Because the intestinal morphology of zebrafish is basically formed at 3 dpf after fertilization, the zebrafish of 3-5 dpf are in good condition and the experimental phenomenon is obvious, while the condition of zebrafish seriously deteriorates after 9 dpf if they are not fed.

[0011] Preferably, the concentration of the lactose aqueous solution in step (1) is 3000-5000 μg / mL, and more preferably 4000 μg / mL. The ratio of intestinal area expansion after 4 hours of stimulation with 4000 μg / mL lactose is significantly different from that after 2 hours, indicating that the zebrafish intestine is stable after stimulation with this concentration of lactose. Therefore, it can be determined that 4000 μg / mL lactose stimulation for 4 hours can induce acute abdominal distension in zebrafish in lactose intolerance, which is the optimal modeling condition.

[0012] Preferably, the concentration of the egg yolk powder in step (2) is 2-4 mg / mL. When the concentration of the egg yolk powder is lower than 2 mg / mL and the zebrafish is given the feed for 4 h, the time is too short and the food intake of the zebrafish is uneven. Alternatively, due to the low concentration of the egg yolk powder, the total amount of egg yolk powder ingested by the zebrafish is uneven, and the model stability is poor.

[0013] Preferably, the concentration of egg yolk powder in step (2) is 2-3 mg / mL, and the concentration of lactose aqueous solution is 7000-8000 μg / mL

[0014] Preferably, in step (2), the concentration of egg yolk powder is 2.5 mg / mL, and the concentration of lactose aqueous solution is 8000 μg / mL.

[0015] Preferably, step (2) is left to stand for 1-4 h.

[0016] The present invention also provides an application of the model in evaluating the efficacy of a drug, a health food or a food in improving lactose intolerance, comprising the following steps: using the drug, the health food or the food as a sample,

[0017] 1) Randomly select zebrafish, treat with samples for 4-24 hours, remove the samples, treat with 1000-6000 μg / mL lactose aqueous solution for 4-24 hours, take pictures of the treated zebrafish under a dissecting microscope, and use image processing software to analyze the intestinal area of ​​zebrafish;

[0018] 2) Randomly select zebrafish, treat with samples for 4-24 h, remove the samples, and intervene with egg yolk powder and 4000-8000 μg / mL lactose aqueous solution for 3-5 h. Wash off the egg yolk powder and lactose, and leave for a period of time to observe that the intestinal opacity of the zebrafish decreases. Take pictures of the treated zebrafish under a dissecting microscope, and use image processing software to analyze the total intestinal opacity of the zebrafish.

[0019] Preferably, the sample is administered at a stage of 3 to 9 dpf.

[0020] As a preferred example, the samples are Ledish acid lactase, bifidobacterium lactobacillus triple live bacteria tablets, β-galactosidase-lactase and galacto-oligosaccharide. The above substances can be used to alleviate the symptoms of lactose-induced intestinal tract.

[0021] ① Ledis acidic lactase can supplement the insufficient amount of enzyme in the body, so that lactose can be broken down into more easily absorbed glucose and galactose in the small intestine. These two monosaccharides can be directly absorbed and utilized by the small intestine and will not reach the large intestine to be fermented by bacteria, thereby reducing discomfort symptoms and reducing the amount of undigested lactose reaching the large intestine, thereby reducing the production of gas and acid, and reducing or avoiding the indigestion symptoms related to lactose intolerance to achieve the therapeutic effect. However, it should be noted that when using lactase products such as Ledis, they are usually taken before or at the same time as eating foods containing lactose to ensure that lactase can start to work immediately and improve the digestion efficiency of lactose.

[0022] ② Bifidobacterium and Lactobacillus triple live bacteria tablets can achieve therapeutic effects by producing lactase, regulating intestinal flora, reducing intestinal inflammatory responses, enhancing intestinal barrier function, promoting nutrient absorption, and lowering intestinal pH. Similar to Ledis, when using Bifidobacterium and Lactobacillus triple live bacteria tablets to improve lactose intolerance, it usually takes a period of time to observe the effect, because they need to establish and maintain a sufficient number in the intestine to play the above role.

[0023] ③β-galactosidase can catalyze the hydrolysis of lactose, breaking it down into glucose and galactose, so that the decomposed monosaccharides can be absorbed by the epithelial cells of the small intestine. It can improve the absorption rate of lactose, reduce the production of intestinal gas, maintain intestinal health, maintain the balance of intestinal microecology, and avoid intestinal disorders caused by excessive fermentation.

[0024] ④Galactoligosaccharides can serve as a food source for beneficial bacteria such as bifidobacteria, promoting their growth and reproduction in the intestines. These beneficial bacteria help improve the structure of intestinal flora and enhance intestinal health. With the increase in the number of beneficial bacteria, especially those that can produce lactase, the lactase activity in the intestine can be indirectly increased, thereby contributing to the decomposition and absorption of lactose. In addition, galactoligosaccharides help regulate the pH value of the intestine by promoting the growth of beneficial bacteria, creating an environment that is more conducive to lactose digestion and absorption, while inhibiting the growth of harmful bacteria. The increase in beneficial bacteria helps improve intestinal peristalsis, speeds up the passage of food residues through the intestines, reduces the time that undigested lactose stays in the intestines, and reduces the discomfort caused by fermentation. Galacto-oligosaccharides can also promote the optimization of intestinal flora, which can reduce the amount of gas produced by fermenting undigested lactose in the intestines, thereby alleviating symptoms such as bloating and abdominal pain. A study showed that short-chain galactoligosaccharides can significantly improve lactose absorption and tolerance, and directly regulate colon bacteria to enable them to metabolize lactose more effectively, which is a potential effective way to improve lactose absorption and tolerance.

[0025] Therefore, the beneficial effects of the present invention are:

[0026] (1) Typical symptoms of lactose intolerance include abdominal pain, bloating, flatulence, diarrhea, and less frequently nausea and vomiting. Symptoms of lactose intolerance are closely related to hydrogen excretion and the amount of lactose ingested. Abdominal pain and bloating are caused by unabsorbed lactose fermented in the colon. The intestinal flora provides a salvage pathway for lactose digestion by breaking down lactose into short-chain fatty acids and gas. Unabsorbed lactose in the ileum and colon leads to acidification of the colonic contents and an increase in the osmotic load. This leads to increased secretion of electrolytes and fluids, as well as rapid transit time, leading to the occurrence of loose stools and diarrhea. In addition, there is a mutual influence between diarrhea and abdominal pain: during diarrhea, intestinal peristalsis accelerates, and the intestinal wall is frequently stimulated, which may cause abdominal pain. Abdominal pain may prompt patients to defecate frequently, resulting in symptoms of diarrhea. Therefore, the present invention evaluates abdominal distension by analyzing the intestinal lumen area of ​​zebrafish, evaluates diarrhea by analyzing the total intestinal opacity of zebrafish, and evaluates diarrhea by analyzing the total intestinal opacity of zebrafish. TRPA1a and COX-2 The expression of genes to evaluate abdominal pain, combined with the comprehensive evaluation of the three, can comprehensively and accurately evaluate the degree of lactose intolerance.

[0027] (2) The development of the gastrointestinal system is highly conserved between zebrafish and humans. The metabolic and regulatory pathways of intestinal and hepatocyte differentiation, digestion, and nutrient absorption and transport are highly conserved. The zebrafish intestine is divided into three histologically defined parts, including the foregut, midgut, and caudal gut. In zebrafish, the most anterior part of the digestive tract develops independently of the intestinal tube and may retain the "genetic footprint" of gastric ontogeny. Zebrafish express several digestive enzymes that are functionally identical to mammalian gastric markers, including renin, triphosphatase, several cathepsins, and lipase.

[0028] It is worth noting that because zebrafish are not mammals and do not drink milk, it is usually difficult for those skilled in the art to associate them with the study of lactose intolerance. The present invention takes into account that, on the one hand, the anatomical structure and tissue cell morphology of the zebrafish digestive tract are similar to those of the human digestive tract; on the other hand, several digestive enzymes are expressed in zebrafish, which are functionally the same as mammalian gastric markers, including renin, triphosphatase, several cathepsins and lipases, and the physiological changes of zebrafish and human digestive enzymes are consistent. Therefore, the present invention creatively uses zebrafish to construct a lactose intolerance research model, which has the advantages of short cycle, low cost, convenient detection and high throughput compared to existing mammalian models. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The following are typical images of zebrafish intestinal expansion induced by 1000 μg / mL lactose for 2, 4, 6, and 24 h.

[0030] Figure 2 The following are typical images of zebrafish intestinal expansion induced by 2000 μg / mL lactose for 2, 4, 6, and 24 h.

[0031] Figure 3 The following are typical images of zebrafish intestinal expansion induced by 4000 μg / mL lactose for 2, 4, 6, and 24 h.

[0032] Figure 4 The following are typical images of zebrafish intestinal expansion induced by 8000 μg / mL lactose for 2, 4, 6, and 24 h.

[0033] Figure 5 These are typical images of zebrafish intestinal fluorescence induced by lactose at 2, 4, 6, and 24 h.

[0034] Figure 6 This is a typical picture of zebrafish diarrhea induced by co-intervention of 4000 μg / mL lactose and egg yolk powder for 4 h and observed for 1 h.

[0035] Figure 7 The typical graphs are shown in which 4000 μg / mL lactose and egg yolk powder were co-interventionally induced to induce diarrhea in zebrafish for 4 h and then observed at 1 h and 2 h.

[0036] Figure 8 This is a typical picture of zebrafish diarrhea induced by co-intervention of 8000 μg / mL lactose and egg yolk powder for 4 h and observed for 2 h.

[0037] Fig. 9 This is a typical picture of the intestinal area of ​​zebrafish after treatment with Bifidobacterium-Lactobacillus triple live bacteria tablets and β-galactosidase-lactase.

[0038] Fig.10 This is a typical picture of the intestinal lumen area of ​​zebrafish after treatment with Ledis acid lactase.

[0039] Fig.11 A typical image of zebrafish intestinal opacity after treatment with Ledis lactase.

[0040] Fig.12 This is a typical picture of the intestinal lumen area of ​​zebrafish after galacto-oligosaccharide treatment.

[0041] Fig.13 A typical image of zebrafish intestinal opacity after galacto-oligosaccharide treatment. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below through specific embodiments.

[0043] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are all conventional methods in the art. Unless otherwise specified, parts are by weight, temperatures are expressed in ° C or at ambient temperature, and pressures are atmospheric pressure or near atmospheric pressure. There are many variations and combinations of reaction conditions (such as component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges) and conditions that can be used to optimize the purity and yield of the product obtained by the method, and only reasonable routine experiments will be required to optimize such method conditions.

[0044] Example

[0045] Experimental animals: Wild-type AB strain zebrafish.

[0046] Main instruments and reagents: dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); electric focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China).

[0047] 1. Exploration of abdominal distension modeling conditions

[0048] Example 1

[0049] Wild-type AB zebrafish at 5 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate, with 30 zebrafish in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well. Except for the blank control group, all other experimental groups were given 1000 μg / mL lactose in water, and 2, 4, 6, and 24 hours after lactose administration, 10 zebrafish were randomly selected from each group and photographed under a dissecting microscope and saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the intestinal area of ​​zebrafish. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0050] Example 2

[0051] The difference from Example 1 is that each experimental group was given 2000 μg / mL lactose in water.

[0052] Example 3

[0053] The difference from Example 1 is that each experimental group was given 4000 μg / mL lactose in water.

[0054] Example 4

[0055] The difference from Example 1 is that each experimental group was given 8000 μg / mL lactose in water.

[0056] Results Analysis

[0057] The results showed that in Example 1, no obvious intestinal expansion occurred after 2 h and 4 h of administration of 1000 μg / mL lactose to 5 dpf zebrafish. The phenomenon of intestinal area expansion did not occur until 6 h after stimulation with 1000 μg / mL lactose. The phenomenon of intestinal expansion can still be maintained by continuing to administer lactose for 24 h. Clinically, lactose intolerance is a condition in which abdominal distension occurs rapidly after eating. Therefore, it is necessary to change the experimental conditions to achieve the effect of short-term rapid abdominal distension. See Table 1 and Figure 1 .

[0058] Table 1. Experimental results of 1000 μg / mL lactose-induced zebrafish intestinal expansion for 2, 4, 6, and 24 h (pixels, mean ± SE, n = 10)

[0059]

[0060] Compared with the blank control group, *p<0.05, ***p<0.001.

[0061] In Example 2, after 2000 μg / mL lactose was administered to 5 dpf zebrafish for 2 hours, the intestinal cavity expansion was not obvious. After continuous administration of lactose for 4 hours, obvious intestinal cavity expansion occurred, but the difference was not obvious compared with 2 hours. After continuous administration of lactose for 6 hours, obvious intestinal cavity expansion occurred. After continuous administration of lactose for 24 hours, the phenomenon of intestinal cavity expansion can still be maintained. This shows that increasing the concentration of lactose can shorten the time of abdominal distension. Therefore, it is necessary to continue to increase the concentration of lactose to achieve the effect of short-term rapid abdominal distension. See Table 2 and Figure 2 .

[0062] Table 2. 2000 μg / mL lactose induced zebrafish intestinal expansion at 2, 4, 6 and 24 h (pixels, mean ± SE, n = 10)

[0063]

[0064] Compared with the blank control group, ***p<0.001.

[0065] In Example 3, after 2 hours of administration of 4000 μg / mL lactose to 5 dpf zebrafish, no obvious intestinal dilation occurred. However, after 4 hours of stimulation with 4000 μg / mL lactose, the intestinal area expanded, which was consistent with the expected time. The phenomenon of intestinal dilation can still be maintained by continuing to administer lactose for 6 and 24 hours. The data also showed that the intestinal area expansion ratio after 4 hours of stimulation with 4000 μg / mL lactose was significantly different from that after 2 hours, indicating that the zebrafish intestine was stable after stimulation with this concentration of lactose. Therefore, it can be determined that 4 hours of stimulation with 4000 μg / mL lactose can induce acute abdominal distension in zebrafish with lactose intolerance. However, it is still unknown whether continuing to increase the lactose concentration will lead to abdominal distension more quickly, so it is necessary to increase the lactose concentration for verification. See Table 3 and Figure 3 .

[0066] Table 3. 4000 μg / mL lactose induced zebrafish intestinal expansion at 2, 4, 6 and 24 h (pixels, mean ± SE, n = 10)

[0067]

[0068] Compared with the blank control group, **p<0.01, ***p<0.001.

[0069] In Example 4, 8000 μg / mL lactose was administered to 5 dpf zebrafish for 2 hours, and the intestinal area expanded, which was consistent with the expected time. Continuous administration of lactose for 4, 6 and 24 hours can still maintain the phenomenon of intestinal expansion. However, zebrafish with large doses of lactose will be in poor condition. Therefore, it can be determined that 4000 μg / mL lactose stimulation for 4 hours can induce acute abdominal distension in zebrafish with lactose intolerance, which is the best modeling condition. See Table 4 and Figure 4 .

[0070] Table 4. 8000 μg / mL lactose induced zebrafish intestinal expansion at 2, 4, 6 and 24 h (pixels, mean ± SE, n = 10)

[0071]

[0072] Compared with the blank control group, ***p<0.001.

[0073] 2. Exploration of diarrhea modeling conditions

[0074] Example 5

[0075] Wild-type AB zebrafish at 5 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well. Nile red 50 ng / mL was given at 8:30 in the morning for 3 hours to fill the intestine, and Nile red was removed at 11:30. Except for the blank control group, the other experimental groups were given lactose 1000, 2000, 4000 and 8000 μg / mL in water. After 2, 4, 6, and 24 hours of lactose administration, 10 zebrafish were randomly selected from each group and photographed under a dissecting microscope and saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the fluorescence intensity (pixel) of the zebrafish intestine. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0076] Example 6

[0077] Wild-type AB zebrafish at 5 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL per well. Egg yolk powder 1.5 mg / mL was given to fill the intestine. At the same time, except for the blank control group, all other experimental groups were given 4000 μg / mL lactose in water for a total of 4 h. After washing away the egg yolk powder and lactose and leaving for 1 h, 10 zebrafish were randomly selected from each group and photographed under a dissecting microscope and saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the total opacity (pixels) of the zebrafish intestine. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0078] Example 7

[0079] The difference from Example 6 is that ① 2.5 mg / mL of egg yolk powder was given to fill the intestine; ② the egg yolk powder and lactose were washed away and left for 1 h or 2 h.

[0080] Example 8

[0081] The difference from Example 7 is that 8000 μg / mL lactose was administered for co-intervention.

[0082] Results Analysis

[0083] In Example 5, 4000 μg / mL lactose was administered to 5 dpf zebrafish for 4 hours, and the intestinal fluorescence was enhanced, which was not in line with expectations. Continuous administration of lactose at 4000 and 8000 μg / mL for 6 and 24 hours still showed the phenomenon of enhanced fluorescence in the zebrafish intestine. The above experimental results are contrary to expectations, which may be because Nile red is a lipophilic stain, and Nile red has strong fluorescence in a fat-rich environment. After lactose is administered to zebrafish, lactose is absorbed in the body and converted into fat, resulting in enhanced fluorescence. Therefore, conventional Nile red staining indicators cannot be used to calibrate intestinal motility. Therefore, it is necessary to select other intestinal filling agents to study the diarrhea indicators of zebrafish lactose intolerance. See Table 5 and Table 5 for details. Figure 5 .

[0084] Table 5. Lactose-induced zebrafish intestinal fluorescence at 2, 4, 6, and 24 h (pixels, mean ± SE, n = 10)

[0085]

[0086] Compared with the blank control group, *p<0.05, ***p<0.001.

[0087] In Example 6, 4000 μg / mL lactose and 1.5 mg / mL egg yolk powder were given to 5 dpf zebrafish for a total of 4 hours. After the lactose and egg yolk powder were washed off and left for another 1 hour, there was still no decrease in intestinal opacity. During the experiment, it was found that 1.5 mg / mL egg yolk powder was given to zebrafish for 4 hours, which was a short time and the zebrafish's food intake was uneven. Or because of the low concentration of egg yolk powder, the total amount of egg yolk powder ingested by the zebrafish was uneven, and the overall group CV value was large. Therefore, the egg yolk powder concentration should be increased in the future. See Table 6 and Figure 6 .

[0088] Table 6. Results of 4000 μg / mL lactose and egg yolk powder co-intervention to induce zebrafish diarrhea for 4 h and observation for 1 h

[0089] (pixels, mean ± SE, n = 10)

[0090]

[0091] In Example 7, 4000 μg / mL lactose and 2.5 mg / mL egg yolk powder were given to 5 dpf zebrafish for a total of 4 h. After washing the lactose and egg yolk powder, there was still no significant decrease in intestinal opacity after 1 h and 2 h. However, the phenomenon of decreased intestinal opacity was observed after 2 h. Therefore, the lactose concentration can be increased in the future. See Table 7 and Figure 7 .

[0092] Table 7. 4000 μg / mL lactose and egg yolk powder co-intervention induced zebrafish diarrhea for 4 h and observed the results at 1 h and 2 h

[0093] (pixels, mean ± SE, n = 10)

[0094]

[0095] In Example 8, 8000 μg / mL lactose and 2.5 mg / mL egg yolk powder were administered to 5 dpf zebrafish for a total of 4 h, and then the lactose and egg yolk powder were washed off and left for another 2 h, resulting in a significant decrease in intestinal opacity. Therefore, the above conditions can be determined as the modeling conditions for the diarrhea model. See Table 8 and Figure 8 .

[0096] Table 8. Results of 8000 μg / mL lactose and egg yolk powder co-intervention to induce zebrafish diarrhea for 4 h and observation for 2 h

[0097] (pixels, mean ± SE, n = 10)

[0098]

[0099] Compared with the blank control group, **p<0.01.

[0100] Application Examples

[0101] Experimental animals: Wild-type AB strain zebrafish.

[0102] Main instruments and reagents:

[0103] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); electric focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); fully automatic sample rapid grinding instrument (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); fluorescence quantitative PCR instrument (CFX Connect, BIO-RAD, Singapore);

[0104] Lactose (lot number D2328302, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Bifidobacterium-Lactobacillus Triple Live Bacteria Tablets (lot number: 202312478, Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., China); β-galactosidase-lactase (lot number: C15483379202312478, Shanghai MacLean Biochemical Technology Co., Ltd., China); ChamQ Universal SYBR qPCR Master Mix (lot number 027E2201CD, Vazyme, China); Universal Genomic DNA Extraction Kit (lot number 2ZAP13, Hangzhou Kefit Biotechnology Co., Ltd., China); Universal RNAExtraction TL Kit C (Product No. TL2204001643C, Foshan Aowei Biotechnology Co., Ltd., China); Ledis acid lactase (Batch No. 615232_9039, Lactrase, Germany); Galacto-oligosaccharide (Batch No. C15916553, Shanghai MacLean Biochemical Technology Co., Ltd., China).

[0105] Application Example 1 - Bifidobacterium-Lactobacillus Triple Live Bacteria Tablets and β-galactosidase-lactase

[0106] Wild-type AB zebrafish at 4 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, and the volume of each well was 3 mL. After treatment with a triple live bacteria tablet of Bifidobacterium and Lactobacillus (500, 1000, 2000 μg / mL) and β-galactosidase-lactase (500, 1000, 2000 μg / mL) for 24 h, the samples were removed, and 4000 μg / mL lactose was given for 4 h. Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope and saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the intestinal area (pixels) of zebrafish. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0107] Results Analysis

[0108] The intestinal area of ​​zebrafish in the model control group was significantly increased compared with the blank control group, indicating that the 4000 μg / mL lactose induction for 4 h can successfully induce the lactose intolerance bloating model. After administering the bifidobacterium and lactobacillus triple live bacteria tablets and β-galactosidase-lactase, the intestinal area of ​​zebrafish was significantly reduced, indicating that the bifidobacterium and lactobacillus triple live bacteria tablets and β-galactosidase-lactase have the effect of significantly improving the bloating of lactose intolerance. See Table 9 and Fig. 9 .

[0109] Table 9. Intestinal area of ​​zebrafish after treatment with Bifidobacterium-Lactobacillus triple live bacteria tablets and β-galactosidase-lactase

[0110] (pixels, mean ± SE, n = 10)

[0111]

[0112] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0113] Application Example 2-Ledis Acid Lactase

[0114] 2.1 Evaluation of the efficacy of Lactase Acid Lactase on abdominal distension in lactose-intolerant zebrafish

[0115] Wild-type AB zebrafish at 4 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate, with 30 zebrafish treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well. After 24 h of treatment with Ledix acid lactase (62.5, 125, 250 μg / mL), the samples were removed, and 4 h of water-soluble lactose intervention was given. Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope and the pictures were saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the intestinal area (pixels) of zebrafish. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0116] Results Analysis

[0117] The intestinal area of ​​zebrafish in the model control group was significantly higher than that in the blank control group, indicating that the 4000 μg / mL lactose induction for 4 h can successfully induce the lactose intolerance abdominal distension model. After administration of Leddy acid lactase, the intestinal area of ​​zebrafish was significantly reduced, indicating that Leddy acid lactase has the effect of significantly improving the abdominal distension of lactose intolerance. See Table 10 and Fig.10 .

[0118] Table 10. Zebrafish intestinal area after treatment with Lactase (pixels, mean ± SE, n = 10)

[0119]

[0120] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0121] 2.2 Evaluation of the efficacy of Lactase Acid Lactase on diarrhea in lactose-intolerant zebrafish

[0122] Wild-type AB zebrafish at 4 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL per well. After treatment with Ledix acid lactase (62.5, 125, and 250 μg / mL) for 24 h, the samples were removed and 2.5 mg / mL of egg yolk powder was given to fill the intestine. At the same time, except for the blank control group, 8000 μg / mL lactose was given to the other experimental groups for 4 h. The egg yolk powder and lactose were washed off and left for 2 h. Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope and the pictures were saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the total opacity (pixels) of the zebrafish intestine. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0123] Results Analysis

[0124] The total intestinal opacity of zebrafish in the model control group was significantly lower than that in the blank control group, indicating that the lactose intolerance diarrhea model induced by 8000 μg / mL lactose and 2.5 mg / mL egg yolk powder co-intervention for 4 h was successfully established. After administration of Ledis acid lactase, the total intestinal opacity of zebrafish increased significantly, indicating that Ledis acid lactase has the effect of significantly improving lactose intolerance diarrhea. See Table 11 and Fig.11 .

[0125] Table 11. Total opacity of zebrafish intestine after treatment with Lactase (pixels, mean ± SE, n = 10)

[0126]

[0127] Compared with the model control group, *p<0.05, ***p<0.001.

[0128] 2.3 Study on the mechanism of diarrhea in lactose-intolerant zebrafish by using Lactase

[0129] Wild-type AB strain zebrafish at 4 days post-fertilization (4 dpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well, and three biological replicates were set up in parallel. After 24 hours of treatment with Ledis acid lactase (62.5, 125, 250 μg / mL), the samples were removed, and 2.5 mg / mL of egg yolk powder was given to fill the intestine. At the same time, except for the blank control group, the remaining experimental groups were given 8000 μg / mL lactose in water for a total of 4 hours, and then the egg yolk powder and lactose were washed off. After 2 hours of placement, the total RNA of each group of zebrafish was extracted using an automatic nucleic acid extractor, and the total RNA concentration and purity were determined using a UV-visible spectrophotometer. 2.00 μg of total RNA from zebrafish samples was taken, and 20.0 μL cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit, and q-PCR was used to detect AQP4 and claudin 1 Gene expression. β-actin was used as the internal reference for gene expression. AQP4 and claudin 1 Relative RNA expression of genes. Statistical results were expressed as mean ± SE. T-test was used between the two groups, and p < 0.05 indicated that the difference was statistically significant.

[0130] Results Analysis

[0131] Aquaporins (AQPs) are a family of integral membrane proteins that are widely distributed on human and mammalian cell membranes and are closely related to transmembrane water transport. AQP4 plays an important role in gastrointestinal water transport and regulation of cell homeostasis. Reduced AQP4 gene expression leads to reduced colonic water absorption and diarrhea. Upregulating the expression level of AQP4 in intestinal mucosal tissue promotes water absorption, thereby achieving the purpose of antidiarrhea. The tight junction protein Claudin-1 is expressed in the intestine, esophagus, lung and other tissues, especially in the liver, kidney, skin and other tissues. Claudin-1 is mainly located in the cell membrane, nucleus, cytoplasm and tight junctions between two cells. Downregulation of Claudin-1 expression can be used as an important indicator of intestinal mucosal barrier dysfunction. When diarrhea occurs, the expression of Claudin-1 will be downregulated.

[0132] Under the experimental conditions, Ledix acid lactase has the effect of relieving diarrhea, which is manifested by upregulating AQP4 and claudin 1 Relative gene expression. See Table 12 for details.

[0133] Table 12. Effect of Ledix acid lactase on AQP4 and claudin 1 Effect of gene expression (mean ± SE, n = 3)

[0134]

[0135] Compared with the model control group, *p<0.05, **p<0.05, ***p<0.001.

[0136] 2.3 Study on the mechanism of abdominal pain in lactose-intolerant zebrafish by using Lactase

[0137] Wild-type AB strain zebrafish at 4 days post-fertilization (4 dpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well, and three biological replicates were set up in parallel. After 24 hours of treatment with Ledis acid lactase (62.5, 125, 250 μg / mL), the samples were removed, and 2.5 mg / mL of egg yolk powder was given to fill the intestine. At the same time, except for the blank control group, the remaining experimental groups were given 8000 μg / mL lactose in water for a total of 4 hours, and then the egg yolk powder and lactose were washed off. After 2 hours of placement, the total RNA of each group of zebrafish was extracted using an automatic nucleic acid extractor, and the total RNA concentration and purity were determined using a UV-visible spectrophotometer. 2.00 μg of total RNA from zebrafish samples was taken, and 20.0 μL cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit, and q-PCR was used to detect TRPA1a and COX-2 Gene expression. β-actin was used as the internal reference for gene expression. TRPA1a and COX-2 Relative RNA expression of genes. Statistical results were expressed as mean ± SE. T-test was used between the two groups, and p < 0.05 indicated that the difference was statistically significant.

[0138] Results Analysis

[0139] TRPA1 (transient receptor potential cation channel subfamily A member 1) and COX-2 (cyclooxygenase-2) are two molecules closely related to pain perception. TRPA1 is a non-selective cation channel that is mainly involved in pain and temperature perception. It is considered a "nociceptor" that is activated when it senses harmful stimuli (such as cold, heat, mechanical damage or chemical stimulation), thereby inducing pain sensation. COX-2 (cyclooxygenase-2) is an inducible enzyme that is involved in the synthesis of prostaglandins. Prostaglandins are a class of lipid mediators involved in inflammatory responses. They can cause pain, fever and inflammation, which mainly characterize chronic pain. TRPA1a Increased gene expression indicates that the body will experience abdominal pain symptoms. COX-2 Gene expression is upregulated.

[0140] Under the experimental conditions, Ledix acid lactase has the effect of relieving abdominal pain, which is manifested by downregulating TRPA1a and COX-2 Relative gene expression. See Table 13 for details.

[0141] Table 13. Effect of Ledix acid lactase on TRPA1a and COX-2 Effect of gene expression (mean ± SE, n = 3)

[0142]

[0143] Compared with the model control group, *p<0.05, **p<0.05, ***p<0.001.

[0144] Application Example 3-Galacto-oligosaccharide

[0145] 3.1 Evaluation of the efficacy of galacto-oligosaccharides on abdominal distension in lactose-intolerant zebrafish

[0146] Wild-type AB zebrafish at 4 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate, with 30 zebrafish treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well. After 24 h of treatment with oligosaccharides (500, 1000, and 2000 μg / mL), the samples were removed. After 4 h of intervention with 4000 μg / mL lactose in water, 10 zebrafish were randomly selected from each group and photographed under a dissecting microscope and saved. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the intestinal area (pixels) of zebrafish. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0147] Results Analysis

[0148] The intestinal area of ​​zebrafish in the model control group was significantly higher than that in the blank control group, indicating that the 4000 μg / mL lactose induction for 4 h can successfully induce the lactose intolerance abdominal distension model. After administration of oligosaccharides, the intestinal area of ​​zebrafish was significantly reduced, indicating that oligosaccharides have the effect of significantly improving the abdominal distension of lactose intolerance. See Table 14 and Fig.12 .

[0149] Table 14. Zebrafish intestinal area after galacto-oligosaccharide treatment (pixels, mean ± SE, n = 10)

[0150]

[0151] Compared with the model control group, **p<0.01, ***p<0.001.

[0152] 3.2 Evaluation of the efficacy of galacto-oligosaccharides on diarrhea in lactose-intolerant zebrafish

[0153] Wild-type AB zebrafish at 4 days post-fertilization (dpf) were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL per well. After 24 h of treatment with oligosaccharides (500, 1000, and 2000 μg / mL), the samples were removed and 2.5 mg / mL of egg yolk powder was given to fill the intestine. At the same time, except for the blank control group, 8000 μg / mL of lactose was given to the remaining experimental groups for 4 h. The egg yolk powder and lactose were washed off and left for 2 h. Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope and the images were saved. Data were collected using NIS-ElementsD 3.20 advanced image processing software to analyze the total opacity (pixels) of the zebrafish intestine. Statistical analysis results were expressed as mean ± SE. T-test was used between the two groups, and p<0.05 indicated that the difference was statistically significant.

[0154] Results Analysis

[0155] The total intestinal opacity of zebrafish in the model control group was significantly lower than that in the blank control group, indicating that the lactose intolerance diarrhea model induced by co-intervention of 8000 μg / mL lactose and 2.5 mg / mL egg yolk powder for 4 h was successfully established. After administration of galacto-oligosaccharides, the total intestinal opacity of zebrafish increased significantly, indicating that galacto-oligosaccharides have the effect of significantly improving lactose intolerance diarrhea. See Table 15 and Fig.13 .

[0156] Table 15. Total opacity of zebrafish intestine after galacto-oligosaccharide treatment (pixels, mean ± SE, n = 10)

[0157]

[0158] Compared with the model control group, *p<0.05, ***p<0.001.

[0159] 3.3 Study on the mechanism of diarrhea in lactose-intolerant zebrafish by galacto-oligosaccharides

[0160] Wild-type AB strain zebrafish at 4 days post-fertilization (dpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). A blank control group and a model control group were set up, with a volume of 3 mL in each well, and three biological replicates were set up in parallel. After 24 hours of treatment with oligosaccharides (500, 1000, and 2000 μg / mL), the samples were removed and 2.5 mg / mL of egg yolk powder was given to fill the intestine. At the same time, except for the blank control group, the remaining experimental groups were given 8000 μg / mL lactose in water for a total of 4 hours, and then the egg yolk powder and lactose were washed off. After 2 hours of standing, the total RNA of each group of zebrafish was extracted using an automatic nucleic acid extractor, and the total RNA concentration and purity were determined using a UV-visible spectrophotometer. 2.00 μg of total RNA from zebrafish samples was taken, and 20.0 μL cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit, and q-PCR was used to detect AQP4 and claudin 1 Gene expression. β-actin was used as the internal reference for gene expression. AQP4 and claudin 1 Relative RNA expression of genes. Statistical results were expressed as mean ± SE. T-test was used between the two groups, and p < 0.05 indicated that the difference was statistically significant.

[0161] Results Analysis

[0162] Under the experimental conditions, galacto-oligosaccharides have the effect of relieving diarrhea, which is manifested by upregulating AQP4 and claudin 1 Relative gene expression. See Table 16 for details.

[0163] Table 16. Sample pairs AQP4 and claudin 1 Effect of gene expression (mean ± SE, n = 3)

[0164]

[0165] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0166] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a model for evaluating the efficacy of improving lactose intolerance, characterized in that: The construction steps are as follows: (1) Treat zebrafish with 1000-6000 μg / mL lactose aqueous solution for 4-24 h, take pictures of the treated zebrafish under a dissecting microscope, and analyze the intestinal area of ​​the zebrafish; (2) Zebrafish were treated with 2-5 mg / mL egg yolk powder and 4000-8000 μg / mL lactose aqueous solution for 3-5 h. The egg yolk powder and lactose were washed off and the zebrafish were left for a period of time. The intestinal opacity of the zebrafish decreased. The treated zebrafish were photographed under a dissecting microscope and the total intestinal opacity of the zebrafish was analyzed.

2. The method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 1, characterized in that: The zebrafish were 3-9 dpf.

3. The method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 2, characterized in that: The zebrafish were 3-5 dpf.

4. The method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 1, characterized in that: The concentration of the lactose aqueous solution in step (1) is 3000-5000 μg / mL.

5. The method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 4, characterized in that: The concentration of the lactose aqueous solution in step (1) is 4000 μg / mL.

6. The method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 1, characterized in that: The concentration of egg yolk powder in step (2) is 2-3 mg / mL.

7. The method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 1, characterized in that: The concentration of the lactose aqueous solution in step (2) is 7000-8000 μg / mL.

8. A method for constructing a model for evaluating the efficacy of improving lactose intolerance according to claim 1, 6 or 7, characterized in that: Step (2) Leave for 1-4 h.

9. Use of the model constructed by any one of the construction methods of claims 1 to 8 in evaluating the efficacy of improving lactose intolerance, comprising the following steps: Taking medicine, health food or food as samples, 1) After the zebrafish samples were treated for 4-24 hours, the samples were removed and treated with 1000-6000 μg / mL lactose aqueous solution for 4-24 hours. The treated zebrafish were photographed under a dissecting microscope to analyze the intestinal area of ​​the zebrafish; 2) After the zebrafish samples were treated for 4-24 hours, the samples were removed and treated with egg yolk powder and 4000-8000 μg / mL lactose aqueous solution for 3-5 hours. The egg yolk powder and lactose were washed off and the zebrafish intestine was left for a period of time to observe the decrease in opacity. The treated zebrafish were photographed under a dissecting microscope to analyze the total intestinal opacity of the zebrafish.

10. The use according to claim 9, characterized in that: The dosing stage of samples was 3-9 dpf.

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