Nutritional mixtures of amino acids and enzymic preparations for promoting proliferation of canine gastrointestinal cells

Abstract

The invention discloses an amino acid and enzyme preparation nutrient mixture for promoting proliferation of gastrointestinal cells of dogs, and belongs to the technical field of pet food. According to the invention, the proliferation of intestinal epithelial cells and the secretion of Gastrin can be effectively promoted by supplementing arginine. In order to enhance the functional effect of the invention, lactase is especially added in the invention. On the basis of normal diet, 0.125%-0.5% of arginine and 0.00375%-0.015% of lactase are complementarily fed every day. The digestion and absorption of the dogs on nutrient substances of staple food grains can be improved, the parasitic quantity of intestinal escherichia coli of the dogs can be reduced, and excrement in a healthy form can be formed. In addition, the nutritional mixture can significantly promote the expression of epidermal growth factors (EGF). The invention discloses a mechanism for promoting proliferation of canine intestinal epithelial cells by arginine and lactase, and provides a new thought for prevention and treatment of canine digestive diseases.

Description

Technical Field

[0001] This invention belongs to the field of pet food technology, specifically involving a nutrient mixture of arginine and lactase that increases cell proliferation by promoting the production of canine epidermal growth factor (EGF), promotes the secretion of canine gastrin, improves the digestion and absorption of staple food nutrients in dogs, and alleviates inflammatory irritation caused by damage to the gastrointestinal tract. Background Technology

[0002] Common causes of gastrointestinal problems in dogs include overeating, acute gastritis, and heatstroke. Dogs are relatively resistant to hunger, but are prone to gastrointestinal upset due to overeating. They often don't chew their food properly, which can trigger physiological vomiting. A dog's stomach is irregularly pear-shaped, and its gastric juices contain a high amount of hydrochloric acid. This makes them highly capable of digesting high-protein foods, but due to insufficient chewing and a short intestine, their intestinal fermentation capacity is weak, resulting in poor digestion of roughage. The intestines are a vital digestive and absorptive organ for dogs, and their health directly impacts the overall health of the dog.

[0003] Arginine is one of the essential raw materials for protein synthesis in the body. By participating in the protein synthesis process, arginine provides the necessary material basis for cell growth and proliferation. As an amino acid, arginine, under the action of intestinal mucosal epithelial endocrine cells, can stimulate these cells to synthesize and secrete a number of small-molecule, highly efficient bioactive peptides, which are called gastrointestinal hormones or gut-brain peptides. Among them, gastrin is an important gastrointestinal hormone. Gastrin is mainly secreted by G cells in the gastric antrum and small intestinal mucosa. Its main function is to stimulate gastric acid secretion, improve the digestive capacity of proteins in the stomach, and nourish the gastrointestinal mucosa. This helps improve digestive function and ensures that the body can effectively absorb nutrients from food. Lactase, also known as β-galactosidase, is an enzyme that breaks down lactose into galactose and glucose. Lactose is a disaccharide unique to mammalian milk; therefore, lactase is mainly found in the mammalian intestine, especially in the brush border of the small intestinal mucosa. The main function of lactase is to help the body digest lactose, making it absorbable and usable by the intestines. Lactase breaks down lactose into galactose and glucose, two monosaccharides that can be directly absorbed by the small intestinal mucosal cells, enter the bloodstream, and provide energy for the body.

[0004] For decades, antibiotics added to animal feed have played a vital role in maintaining animal health. However, continued use of these drugs can lead to the development of highly resistant bacteria, the so-called "superbugs." Furthermore, antibiotic residues in animals pose a threat not only to animal health but also to human health. Exploring raw materials with minimal side effects and digestive benefits from natural amino acids and enzymes, effectively combining them, and developing products using modern pharmaceutical technologies is attracting increasing attention and possesses promising market prospects and significant social value. Summary of the Invention

[0005] This invention provides a dietary composition that can effectively improve canine intestinal function, thereby solving the problems mentioned in the background art.

[0006] This invention relates to a nutritional mixture of amino acids and enzymes for promoting the proliferation of canine gastrointestinal cells. The formulation of the above-mentioned supplemental nutritional mixture as a staple food includes, by weight percentage, 0.125-0.5% arginine and 0.00375-0.015% lactase daily on the basis of a normal diet.

[0007] Preferably, on the basis of a normal diet, the diet is supplemented with 0.25% arginine and 0.0075% lactase daily as a percentage by weight.

[0008] Better yet, supplement the diet with 0.5% arginine and 0.015% lactase daily, by weight percentage, on top of a normal diet.

[0009] In one or more embodiments, the canine intestinal epithelial cells are PETCC61 cells.

[0010] The arginine and lactase nutrient mixture of this invention promotes the production of canine epidermal growth factor (EGF), increases cell proliferation, promotes the secretion of canine gastrin, improves the digestion and absorption of staple food nutrients in dogs, and alleviates inflammatory irritation caused by gastrointestinal damage. The mixture includes the following steps:

[0011] 1) Investigate the effect of a nutrient mixture on the proliferation of canine intestinal epithelial cells (PETCC61). Compared with the control group, both arginine and lactase significantly promoted cell viability.

[0012] 2) Using epidermal growth factor (EGF) as an indicator of canine epithelial cell proliferation, increased EGF expression was detected at both the mRNA and protein levels after treatment with the nutrient mixture.

[0013] 3) Using gastrin secretion as an indicator of gastric motility, the nutrient mixture was found to upregulate gastrin secretion.

[0014] 4) To investigate gastrointestinal digestion and absorption, dogs were fed staple food with added nutrients, and the content of crude protein, crude fat, and carbohydrates in feces was measured. It was calculated that supplementing dogs with arginine and lactase daily on the basis of a normal diet helps to improve the digestion and absorption of nutrients from the staple food.

[0015] 5) Evaluate the health of the dog's gastrointestinal digestive system. Feed the dogs a staple food supplemented with a nutrient mixture, and record their fecal scores every 7 days. Count the E. coli in the dog's feces during the experiment. Supplementing with arginine and lactase helps dogs form healthy-looking feces and reduces the number of intestinal E. coli parasites.

[0016] 6) The gastrointestinal damage was evaluated, and a decrease in the serum inflammatory factor levels was detected in dogs fed a staple diet supplemented with nutrient mixture.

[0017] In one or more embodiments, the nutrient mixture that induces proliferation of canine intestinal epithelial cells, EGF expression, and gastrin secretion includes: 500 μM arginine and 20 mg / ml lactase.

[0018] The present invention has the following beneficial effects:

[0019] 1. Arginine and lactase promote the production of epidermal growth factor in dogs, increase cell proliferation rate, and promote the secretion of gastrin in dogs.

[0020] 2. Arginine and lactase help improve the digestion and absorption of nutrients in the dog's staple food, help the dog form healthy stools, reduce the number of E. coli in the intestines, and alleviate the inflammatory irritation caused by damage to the gastrointestinal tract. Attached Figure Description

[0021] Figure 1 The proliferation of canine intestinal epithelial cells after treatment with arginine and lactase.

[0022] Figure 2 This study aims to detect the secretion of gastrin in canine intestinal epithelial cells after treatment with arginine and lactase.

[0023] Figure 3 The expression of canine EGF mRNA and protein after treatment with arginine and lactase.

[0024] Figure 4 This study aimed to detect the cell proliferation rate and gastrin secretion in canine intestinal epithelial cells after EGF treatment and combined treatment with arginine and lactase. Detailed Implementation

[0025] The invention will be described in more detail below through examples and experimental cases. The inventors discovered that arginine is an important cellular metabolite, and lactase is an important cellular metabolic enzyme. Supplementation with arginine and lactase can promote the proliferation of canine gastrointestinal cells and simultaneously promote the secretion of gastrin. Therefore, supplementation with arginine and lactase can serve as an adjunct therapy to promote canine gastrointestinal function.

[0026] Example 1: Arginine and lactase promote the proliferation of canine intestinal epithelial cells

[0027] The effect of nutrients on the proliferation of canine intestinal epithelial cells (PETCC61) was determined using the CCK-8 assay. PETCC61 cells were seeded in 96-well plates at a density of 2 × 10⁶ cells per well. 3 / mL, added to cell culture medium and CCK-8 reagent, and incubated at 37℃ for 2h. Absorbance at 450nm was measured using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = (Experimental group A / Control group A) × 100%. The experiment was independently repeated 3 times. Compared with the control group, arginine and lactase significantly promoted cell viability ( Figure 1 These findings suggest that arginine and lactase may be important for gastrointestinal function.

[0028] Example 2: Arginine and lactase promote the secretion of gastrin by canine intestinal epithelial cells

[0029] The concentration of gastrin in the supernatant of PETCC61 cell culture was quantitatively detected in vitro using a canine gastrin enzyme-linked immunosorbent assay (ELISA) kit. Reagents, samples, and standards were prepared. The prepared samples and standards were added and reacted at 37°C for 90 minutes. The plates were washed, antibodies were added, and the plates were incubated at 37°C for 60 minutes. The plates were washed three times, enzymes were added, and the plates were incubated at 37°C for 30 minutes. The plates were washed five times, and then the chromogenic reagent solution was added, and the plates were incubated at 37°C for 30 minutes. A stop solution was added. The OD value was measured within 10 minutes using a microplate reader. The gastrin content was calculated. Arginine and lactase significantly promoted gastrin secretion. Figure 2 It can help improve a dog's digestive function.

[0030] Example 3: Arginine / lactase-EGF axis promotes canine intestinal epithelial cell proliferation and gastrin secretion.

[0031] EGF signaling plays a crucial role in cell proliferation. We determined the effect of EGF on canine intestinal epithelial cell proliferation and found that EGF stimulation significantly enhanced the proliferation of PETCC61 cells. Similarly, combined treatment with arginine and lactase also promoted the proliferation of PETCC61 cells. Both arginine and lactase can induce EGF expression at both the mRNA and protein levels. Figure 3ELISA results showed that EGF levels were as effective as combined arginine and lactase in promoting gastrin secretion. The arginine / lactase-EGF-gastrin axis collectively promotes digestive function in dogs. Figure 4 ).

[0032] Example 4: Arginine and lactase help improve the digestion and absorption of nutrients in dog food.

[0033] Twelve healthy adult dogs of two breeds, including Poodles and Chinese Rural Dogs, were selected for a feeding experiment. They were evenly divided into three groups (control group, experimental group 1, experimental group 2, and experimental group 3), with three dogs in each group. The control group and experimental group 1 received a normal diet (30% crude protein, 10% crude fat, 10% crude ash, 5% crude fiber, 1% calcium) supplemented with 0.25g arginine and 0.01g lactase per 200g of food. Experimental group 2 received the same diet (30% crude protein, 10% crude fat, 10% crude ash, 5% crude fiber, 1% calcium) supplemented with 0.5g arginine and 0.02g lactase per 200g of food. Experimental group 3 received the same diet (30% crude protein, 10% crude fat, 10% crude ash, 5% crude fiber, 1% calcium) supplemented with 1g arginine and 0.03g lactase per 200g of food. The experiment lasted for 28 days. Each dog was provided with 200g of commercially available complete dog food per day, with free access to water. Five days after the start of the adaptive feeding test, feces from each group of dogs were collected using the full fecal collection method. The feces were weighed, dried, pulverized, and weighed again for testing. The crude protein, crude fat, and carbohydrate content in the feces were determined, and the apparent digestibility of the protein, fat, and carbohydrates in the test samples was calculated. The apparent digestibility was calculated using formula (1):

[0034]

[0035] In the formula, D is the apparent digestibility of a certain nutrient, in %; c1 is the content of the nutrient in feces, in %; m1 is the total mass of feces, in g; c0 is the content of the nutrient in the diet, in %; and m0 is the mass of the ingested diet, in g.

[0036] Compared with the control group, the apparent digestibility of protein, fat, carbohydrates and dry matter in the staple food of the three experimental dogs increased by 0.92%, 1.79%, 2.00% and 0.36% respectively (Table 1). This indicates that supplementing the diet with 1g of arginine and 0.03g of lactase daily on the basis of normal diet helps to improve the digestion and absorption of nutrients in the staple food of dogs.

[0037] Table 1 Apparent digestibility of nutrients

[0038]

[0039] #There was a significant difference compared to the control group, p<0.05

[0040] Example 5: Arginine and lactase help dogs form healthy-looking stools and reduce the number of E. coli in the intestines.

[0041] During the feeding period, the fecal scores of the dogs were recorded every 7 days, and the scores were based on the standards in Table 2.

[0042] Table 2 Dog feces scoring criteria

[0043]

[0044] During the feeding test period, the fecal scores of the control group dogs were all ≥3.5 points, and the feces were relatively soft and formed. After feeding different proportions of arginine and lactase, the fecal scores of experimental group 1 were <3.5 points on day 28 of feeding; experimental group 2 were <3.5 points on day 21 of feeding; and experimental group 3 were <3.5 points on day 14 of feeding (Table 3). This indicates that supplementing a normal diet with 1g of arginine and 0.03g of lactase daily helps dogs form healthy-looking feces.

[0045] Table 3 Dog feces score

[0046]

[0047] After feeding each group of dogs, fecal samples were taken and 10 mL of physiological saline was added to each 1 g of sample. The samples were dispersed by pipetting and allowed to stand. The supernatant was collected for testing. Eosin methylene blue agar was used for the test. The samples were anaerobically cultured at 38°C for 48 hours. The number of E. coli was determined by the serial dilution plate count method, and the logarithm was recorded. The E. coli reduction rate was calculated according to formula (2):

[0048]

[0049] In the formula, P is the reduction rate of Escherichia coli in feces, in %; n0 is the number of Escherichia coli in feces before the feeding test, in CFU / g; and n1 is the number of Escherichia coli in feces after 28 days of feeding, in CFU / g.

[0050] The fecal E. coli counts of dogs during the experiment were performed, and the results are shown in Table 4. In the control group, the E. coli inhibition rate was 21.78% after 28 days of regular diet feeding. In experimental groups 1, 2, and 3, after daily supplementation with different proportions of arginine and lactase on top of a normal diet, the number of E. coli in feces decreased by 54.37%, 66.87%, and 83.27% respectively compared to before feeding (Table 4). The fecal E. coli inhibition rates in experimental groups 2 and 3 were significantly higher than those in the control group (p < 0.05). This indicates that daily supplementation with 1g of arginine and 0.03g of lactase on top of a normal diet helps reduce the number of intestinal E. coli parasites in dogs.

[0051] Table 4. Quantity and inhibition rate of Escherichia coli in canine feces

[0052]

[0053] #There was a significant difference compared to the control group, p<0.05

[0054] Example 6: Serum inflammatory factor levels decreased in dogs fed with arginine and lactase.

[0055] Blood samples were collected from dogs in each group on day 0 and day 28 of feeding, and serum was separated and tested according to the kit instructions. In the control group, the serum IL-1β level decreased by 2.08% after 28 days of feeding with the usual staple diet. In experimental groups 1, 2, and 3, the serum IL-1β level decreased by 0.43%, 10.80%, and 9.06% respectively after feeding with different proportions of arginine and lactase (Table 5). The serum IL-1β levels in experimental groups 2 and 3 were significantly lower than those in the control group (p < 0.05). In the control group, serum TNF-α levels increased by 5.59% after 28 days of feeding with the normal staple diet. In experimental group 1, serum TNF-α levels increased by 0.90% after daily supplementation with 0.25g arginine and 0.01g lactase on top of the normal diet. In experimental groups 2 and 3, serum TNF-α levels decreased by 10.01% and 27.61% respectively after feeding with different proportions of arginine and lactase (Table 6). Serum TNF-α levels in experimental groups 2 and 3 were significantly lower than those in the control group (p < 0.05).

[0056] Table 5. IL-1β levels in canine serum

[0057]

[0058] #There was a significant difference compared to the control group, p<0.05

[0059] Table 6. Serum TNF-α content in dogs

[0060]

[0061] #There was a significant difference compared to the control group, p<0.05

[0062] The compounds of this invention are available from commercial sources or can be synthesized according to or with reference to the disclosed methods. Other aspects of this invention will be apparent to those skilled in the art from the disclosure herein.

Claims

1. A nutrient mixture of amino acids and enzymes for promoting the proliferation of canine gastrointestinal cells, characterized in that... The formula composition of the supplemental nutrient mixture staple food includes: 0.125-0.5% arginine and 0.00375-0.015% lactase per day on the basis of a normal diet, by weight percentage.

2. A nutrient mixture of amino acids and enzymes for promoting the proliferation of canine gastrointestinal cells, characterized in that... The formula for the supplemental nutrient mixture staple food includes, by weight percentage, 0.25% arginine and 0.0075% lactase daily on the basis of a normal diet.

3. A nutrient mixture of amino acids and enzymes for promoting the proliferation of canine gastrointestinal cells, characterized in that... The formula for the supplemental nutrient mixture staple food includes, by weight percentage, 0.5% arginine and 0.015% lactase daily on the basis of a normal diet.

Images