Application of neoagaro-oligosaccharide in preparation of dog food with effects of improving immunity of working dogs and improving intestinal flora

Abstract

The invention discloses application of neoagaro-oligosaccharide in preparation of dog food with effects of improving immunity of working dogs and improving intestinal flora, and belongs to the technical field of specific therapeutic activity of compounds. The invention also discloses a dog food with the efficacy of improving the immunity and the intestinal flora of the working dogs, the dog food comprises the neoagaro-oligosaccharide and a basic dog food, and the addition amount of the neoagaro-oligosaccharide is 0.05%-0.5%. The new agar oligosaccharide is added into the dog food for the working dogs, the functional activity of the new agar oligosaccharide is verified through detection and analysis of blood routine, immune indexes and intestinal flora, and the research finds that the new agar oligosaccharide can improve the bone marrow hematopoietic capacity of the working dogs, remarkably increase the content of IgA, IgM, IL-2 and IL-6 of organisms and enhance the immunity of the organisms of the working dogs; the intestinal microbial flora structure of the working dog can be improved, specific microbial abundance is formed, the growth of harmful flora is inhibited, and the abundance of beneficial flora is increased.

Description

Technical Field

[0001] This invention relates to the application of neo-agar oligosaccharides in the preparation of dog food that enhances the immunity and improves the intestinal flora of working dogs, belonging to the field of specific therapeutic activity technology of compounds. Background Technology

[0002] Neoagarooligo saccharides are oligosaccharides with a degree of polymerization of 2 to 10, generated by hydrolysis of agar polysaccharide by β-agarase. Their structure consists of repeating disaccharide units of β-D-galactose and 3,6-endo-α-L-galactose linked by β-1,3 glycosidic bonds. They are characterized by good water solubility and easy absorption, and can be used as fillers and dispersants for high-sweetness sweeteners in beverages, bread and other low-calorie foods.

[0003] Working dogs are professionally trained dogs within the public security system. The quality of their feed directly affects their growth, development, reproduction, and health, and also their safety. Therefore, feeding levels and dog food quality are decisive factors influencing the health of working dogs. Springer Spaniels are one of the commonly used working dogs. They are medium-sized, cheerful, outgoing, and extremely loyal to people. With training, they can participate in a variety of tasks. Researching dog food formulations to improve the physical performance of Springer Spaniels is a key focus of joint research by police officers and researchers.

[0004] Reticulocyte count (RET), percentage of high fluorescence intensity reticulocytes (HFR), and mean platelet volume (MPV) are important indicators for assessing bone marrow erythrocyte hematopoietic function and the state of cells in the blood. Among them, RET reflects the number of immature red blood cells in the bone marrow and is a direct indicator for assessing bone marrow erythrocyte hematopoietic function; its level reflects the bone marrow's erythrocyte hematopoietic capacity. HFR is one of the reticulocyte subclasses, representing the proportion of the youngest and most immature reticulocytes, and it can reflect the speed of bone marrow hematopoiesis. MPV reflects the average volume of a single platelet in peripheral blood and is related to platelet production, maturity, and function; its level reflects the rate of platelet turnover and the status of platelet production or maturation.

[0005] The gut microbiota plays an important role in the health and disease of dogs. It can participate in important physiological metabolic processes of the host, and the abundance of the gut microbiota can reflect the health of the gastrointestinal tract or the overall health of the body. It is also one of the important reference indicators for the pathogenesis of certain diseases and has important clinical significance. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides the application of neo-agar oligosaccharides in the preparation of dog food with the effects of improving the immunity and intestinal flora of working dogs, which belongs to the field of specific therapeutic activity technology of compounds.

[0007] This invention is achieved through the following technical solution: Application of Neo-Qiong oligosaccharides in the preparation of dog food that enhances the immunity and gut microbiota of working dogs.

[0008] Furthermore, the breed of the working dog is the Springer Spaniel.

[0009] Furthermore, the specific manifestations of the enhanced immunity are: increased reticulocyte count, increased percentage of high-fluorescence-intensity reticulocytes, and increased mean platelet volume.

[0010] Furthermore, the specific manifestation of the improvement in gut microbiota is: inhibition of Clostridium difficile (… Peptoclostridium ), Prevotella spp. Prevotellaceae_Ga6A1 ) and Megamonas spp. Megamonas The growth of harmful bacteria such as *Vibrio spp.* was significantly increased; *Vibrio spp.* Oscillospira ), genus Rocherton Roseburia Erwinia ( ) Erwinia ), Sphingosomalmonella ( candidatus stoquefichus Bifidobacterium spp. Bifidobacterium ) and Prevotella spp. Prevotellaceae_UCG001 The abundance of beneficial bacteria such as bacteria.

[0011] Furthermore, in specific applications, Neo-Qiong oligosaccharides are added to the basic dog food at a rate of 0.05% to 0.5% by weight.

[0012] A dog food designed to enhance the immunity and gut microbiota of working dogs includes a combination of neo-Qiong oligosaccharides and a basic dog food. The neo-Qiong oligosaccharides are added at a concentration of 0.05%–0.5% by weight. The basic dog food, which is readily available, comprises: chicken, beef bone meal, sweet potato meal, pea meal, fermented soybean meal, chicken fat, soybean oil, fish meal, yucca, astragalus, roasted licorice root, yam, jujube, atractylodes macrocephala, oyster shell powder, fish bone meal, and red algae powder. The additives include: dicalcium phosphate, calcium citrate, L-lysine hydrochloride, choline chloride, B vitamins (A, B, C, D, E), yeast selenium, taurine, chitosan oligosaccharides, isomaltooligosaccharides, and fructooligosaccharides.

[0013] This invention adds low-molecular-weight, low-polymerization-degree-of-polymerization neo-agar oligosaccharides to the diet of working dogs. Its functional activity was verified through blood routine tests, immune indicators, and intestinal flora analysis. The study found that neo-agar oligosaccharides can increase the number of reticulocytes, the percentage of high-fluorescence-intensity reticulocytes, and the mean platelet volume in working dogs. Reticulocyte count, percentage of high-fluorescence-intensity reticulocytes, and mean platelet volume reflect bone marrow hematopoietic function, indicating that neo-agar oligosaccharides can improve the bone marrow hematopoietic capacity of working dogs. This functional effect has not been found in other oligosaccharide studies. The study also showed that neo-agar oligosaccharides can significantly increase the levels of IgA, IgM, IL-2, and IL-6 in the body, enhancing the immunity of working dogs. The study also showed that neo-Qiong oligosaccharides can improve the gut microbiota structure of working dogs, form a unique microbial abundance, inhibit the growth of harmful bacteria, and increase the abundance of beneficial bacteria. For example, it significantly increases the abundance of Oscillatoria and Sphingomonas species. Oscillatoria has important scientific and commercial value in the gut microbiota and is regarded as a new generation of probiotics, while Sphingomonas has significant research and application value in food and medicine.

[0014] This invention adds neo-agar oligosaccharides as a functional ingredient to working dog food, which can effectively improve the immunity of working dogs and improve the intestinal flora, and has broad market and scientific research prospects.

[0015] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0016] Figure 1 : Plot showing the difference in Prevotella abundance between the high-dose group and the control group, where * indicates a significant difference ( P <0.05), ** indicates a significant difference ( P <0.01), *** indicates a highly significant difference ( P <0.001). The same applies below.

[0017] Figure 2 : Difference in rumen cocci abundance between the high-dose group and the control group.

[0018] Figure 3 : Difference in fecal bacteria abundance between the high-dose group and the control group.

[0019] Figure 4 : Difference in anaerobic bacteria abundance between the high-dose group and the control group.

[0020] Figure 5 : Difference in Holdmannella abundance between the high-dose group and the control group.

[0021] Figure 6 Figure showing the difference in abundance of *Rheumatoid arthritis* between the high-dose group and the control group.

[0022] Figure 7 : Abundance difference of Stylospira NK4A136 between high-dose group and control group.

[0023] Figure 8 : Abundance difference of rumen bacteria 005 between high-dose group and control group.

[0024] Figure 9 : Difference in abundance of *Ruminococcus gauriensis* between the high-dose group and the control group.

[0025] Figure 10 : The difference in Clostridium difficile abundance between the low-dose group and the control group.

[0026] Figure 11 : The difference in abundance of Prevotella Ga6A1 between the low-dose group and the control group.

[0027] Figure 12 : Difference in abundance of Rochetomyces between the low-dose group and the control group.

[0028] Figure 13 : Difference in Erwinia abundance between the low-dose group and the control group.

[0029] Figure 14 Figure showing the difference in abundance of *Oscillatoria* between the low-dose group and the control group.

[0030] Figure 15 : Abundance difference of Prevotella unclassified-001 between low-dose group and control group.

[0031] Figure 16 : Difference in the abundance of sphingosine monoclonal bacteria between the low-dose group and the control group.

[0032] Figure 17 : Difference in the abundance of Megamonas between the low-dose group and the control group.

[0033] Figure 18 : Abundance difference of Prevotella Ga6A1 between high-dose and low-dose groups.

[0034] Figure 19 High-dose group vs. low-dose group Lachnoclostridium Abundance difference diagram.

[0035] Figure 20 : Abundance difference of Stylospirillum NK4A136 between high-dose and low-dose groups.

[0036] Figure 21 : Difference in Erwinia abundance between high-dose and low-dose groups.

[0037] Figure 22 : Abundance difference of Prevotella NK3B31 between high-dose group and low-dose group.

[0038] Figure 23 : Difference in Bifidobacterium abundance between high-dose and low-dose groups.

[0039] Figure 24 : A graph showing the difference in Howard's abundance between the high-dose group and the low-dose group. Detailed Implementation

[0040] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0041] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0042] The novel agar oligosaccharide used in this invention was prepared by referring to the method reported in the following literature: at 50°C, agarase was used to degrade a 0.5% agar solution for 3 hours; impurities were removed by centrifugation, and the solution was dried to obtain the novel agar oligosaccharide.

[0043] References: Jin Jia, Jiang Chengcheng, Mao Xiangchao, Heterologous expression and enzymatic properties of α-agarase OUC-GaJJ96 published in the Journal of Food Science and Technology in November 2020. The enzyme activity of OUC-GaJJ96 was determined by DNS method in the article.

[0044] The basic dog food used in this invention is provided by Kalyou dog food, with the product name "Complete Price, Full Life Dog Food". The manufacturer of the product is Rizhao Bafang Muge Agricultural and Animal Husbandry Technology Co., Ltd., and the product distributor is Qingdao Hairun Shenlan Biotechnology Co., Ltd., with production license number Q / 1121LBF003-2020.

[0045] Experiment 1: Determination of the molecular weight of neo-agar oligosaccharides The molecular weight of neo-agar oligosaccharides was analyzed using the HPGPC method. The HPLC conditions were as follows: mobile phase was 0.2 mol / L sodium nitrate solution; column: TSKgel G3000PWXL (300 mm × 7.8 mm, 7 μm); column temperature: 40℃; flow rate: 0.5 mL / min; injection volume: 20 μL; differential detector: detector temperature: 35℃; detection time: 30 min.

[0046] Results: The liquid chromatography information of the neo-agar oligosaccharides is shown in Table 1. It can be seen that the peak elution time of the neo-agar oligosaccharides is 17.352, the peak area accounts for 86.43%, its number-average molecular weight (Mn) is 649, and its weight-average molecular weight (Mw) is 807. Based on the molecular weight data, it can be determined that the neo-agar oligosaccharides used in the experiment have a high content of neo-agar tetrasaccharides.

[0047] Table 1. Liquid phase chromatogram information of neo-agar oligosaccharides

[0048] Example 1: Preparation of dog food containing neo-agar oligosaccharides Neo-Qiong oligosaccharides were added to the basal dog food at ratios of 0.05% and 0.5% of the basal dog food mass, respectively, and mixed well to obtain the low-dose group dog food and the high-dose group dog food, which were used for the following experiments.

[0049] Experiment 2: Effects of Neo-Qiong Oligosaccharides on Immunity and Gut Microbiota in Springer Spaniels (1) Experimental grouping Eighteen Springer Spaniels, aged 2-4 years and weighing 20-25 kg, were selected and randomly divided into three groups: control group, low-dose group, and high-dose group, with 6 dogs in each group, 3 males and 3 females.

[0050] The low-dose group was fed dog food containing 0.05% Neo-Qiong oligosaccharide (i.e., the low-dose group dog food prepared in Example 1), the high-dose group was fed dog food containing 0.5% Neo-Qiong oligosaccharide (i.e., the high-dose group dog food prepared in Example 1), and the control group was fed basal dog food.

[0051] The kennel is cleaned every morning and fed at 2 pm, with 500g of dog food each time. This feeding is done for 30 consecutive days. After 30 days, the following indicators are tested.

[0052] (2) Detection of routine blood indicators Blood samples were collected from the anterior brachial vein, approximately 5 mL from each police dog. The sample was placed in an EDTA anticoagulant tube, gently inverted to thoroughly mix the blood and anticoagulant, and centrifuged at 3000 rpm, 4°C for 15 min. 100 μL of anticoagulant whole blood was pipetted into a 1.5 mL EP tube and stored at -80°C for later use. 100 μL of the blood sample was then analyzed using an automated hematology analyzer for relevant parameters.

[0053] The measured indicators included: RET, HFR, MPV, white blood cell count (WBC), neutrophils (NEUT), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), eosinophil count (EO), and mean corpuscular hemoglobin concentration (MCHC). The results are shown in Table 2. In the table, * indicates a significant difference (…). P <0.05), ** indicates a significant difference ( P <0.01), *** indicates a highly significant difference ( P <0.001).

[0054] Table 2. Results of routine blood tests

[0055] As shown in Table 1, compared with the control group, RET in both the low-dose and high-dose groups showed an increasing trend, with significant differences. P <0.05); HFR and MPV also increased to varying degrees, with significant differences between the low-dose group and the control group (P<0.05). Furthermore, changes in WBC and NEUT revealed that different concentrations of neo-agar oligosaccharides promoted the growth of immune cells, with a significant difference in WBC between the high-dose group and the control group (P<0.05). P <0.05).

[0056] (3) Detection of immune indicators Blood samples were collected using the same method as above and stored at -80°C. Various immune indicators were analyzed and detected using reagent kits purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0057] The results of the immune marker tests are shown in Table 3. In the table, * indicates a significant difference (*). P <0.05), ** indicates a significant difference ( P <0.01), *** indicates a highly significant difference ( P <0.001).

[0058] Table 3. Results of immune marker detection

[0059] As shown in Table 2, IgA, as the main antibody for mucosal immunity, can enhance the body's local defense against pathogens. This study found that the high-dose group had a significant improvement compared to the control group and the low-dose group. P <0.05); IgM has a certain inhibitory effect on the body's immunity or chronic inflammation, and the content in the low-dose group was significantly different from that in the control group ( P<0.001), the content in the high-dose group was significantly different from that in the control group ( P <0.01); IL-2 is a T cell growth factor that can promote T cell proliferation and differentiation and enhance cellular immunity. This study found that the content of IL-2 in the high-dose group was significantly higher than that in the control group and the low-dose group. P <0.01); IL-6 can induce B cells to differentiate into plasma cells and secrete antibodies (such as IgG, IgA, IgM), while enhancing the activity of cytotoxic T cells (CTLs) and participating in antiviral and antitumor immunity. This study found that the content of IL-6 in the low-dose group was significantly different from that in the control group. P <0.01).

[0060] (4) Collection and testing of fecal samples At the end of the experiment, samples were taken. Hands were washed before collecting fecal samples, and approximately 2 g of the portion of the feces from the middle section, away from air and the ground, was scooped out using a sterile spoon and placed in cryovials. The samples were labeled with information, temporarily stored in liquid nitrogen, and then stored at -80°C. 16S RNA sequencing of the gut microbiota was performed to analyze changes in gut microbiota structure.

[0061] Wilcoxon analysis revealed significant differences in species at the genus level between the high-dose group and the control group in *Prevotella* genus. Prevotella ), genus *Trichophyton* ( Lachnospiraceae ), Rumenococcus ( Ruminococcus_ gauvreauii There are significant differences in ) P <0.01), in Clostridium praosporum ( Faecalibacterium ), Rumenococcus ( Ruminococcus _torques ), Holdmanella ( Holdemanella ), Russula ( Muribaculaceae There are significant differences in ) P <0.05). The abundance differences of some species between the high-dose group and the control group are shown in the figure below. Figures 1-9 As shown.

[0062] Wilcoxon analysis of genus-level differences between the LDG and CG groups showed that, compared with the control group, the low-dose group had significantly higher levels of *Clostridium difficile* species. Peptoclostridium ), Prevotella spp. Prevotellaceae_Ga6A1 Megamonas genus ( Megamonas There are significant differences in ) P <0.01), indicating that low doses of neo-agar oligosaccharide have a certain inhibitory effect on the growth of the above-mentioned bacterial groups; in *Vibrio spp.* ( Oscillospira ), genus Rocherton Roseburia Erwinia ( ) Erwinia ), Sphingosomalmonella ( Candidatus stoquefichus Bifidobacterium spp. Bifidobacterium ), Prevotella spp. Prevotellaceae_UCG001 There are significant differences in ) P <0.05). The abundance differences of some species between the low-dose group and the control group are shown in the figure below. Figures 10-17 As shown.

[0063] Wilcoxon analysis was used to analyze the significant differences in bacterial genus between the HDG and LDG groups. Compared with the low-dose group, the high-dose group showed a higher proportion of *Trichophyton* species. Lachnospiraceae_NK4A136 There are significant differences in ) P <0.01), in the genus Prevotella ( Prevotellaceae_Ga6A1 ), Lachnoclostridium genus ( Lachnoclostridium Erwinia ( ) Erwinia ), Howard spp. Howardella Bifidobacterium spp. Bifidobacterium ), Prevotella spp. Prevotellaceae_NK3B31 There are significant differences in ) P <0.05). The abundance differences of some species between the high-dose and low-dose groups are shown in the figure below. Figures 18-24 As shown.

[0064] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. Application of neo-agar oligosaccharides in the preparation of dog food that can improve the immunity and gut microbiota of working dogs.

2. The application according to claim 1, characterized in that: The working dog is a Springer Spaniel.

3. The application according to claim 1, characterized in that, The specific manifestations of improved immunity are: increased reticulocyte count, increased percentage of high-fluorescence-intensity reticulocytes, and increased mean platelet volume.

4. The application according to claim 1, characterized in that, The specific manifestations of improving the gut microbiota include: inhibiting Clostridium difficile (… Peptoclostridium ), Prevotella spp. Prevotellaceae_Ga6A1 ) and Megamonas spp. Megamonas The growth of the bacterial flora was significantly increased; a significant increase in the number of *Vibrio spp.* ( Oscillospira ), genus Rocherton Roseburia Erwinia ( ) Erwinia ), Sphingosomalmonella ( candidatus stoquefichus Bifidobacterium spp. Bifidobacterium ) and Prevotella spp. Prevotellaceae_UCG001 ) Abundance of bacterial communities.

5. The application according to claim 1, characterized in that: In practical applications, add Neo-Qiong oligosaccharides to basic dog food at a rate of 0.05% to 0.5% by weight.

6. The application according to claim 5, characterized in that: The amount of the new oligosaccharide added is 0.05% or 0.5%.

7. A dog food that enhances the immunity and gut microbiota of working dogs, characterized in that: Including Neo-Qiong oligosaccharides and basic dog food, the amount of Neo-Qiong oligosaccharides added is 0.05% to 0.5% by weight.

8. The dog food according to claim 7, which has the effect of improving the immunity and intestinal flora of working dogs, is characterized in that: It consists of Neo-Qiong oligosaccharides and basic dog food, with Neo-Qiong oligosaccharides added at a rate of 0.05% or 0.5%.

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