Canine Weissella ZJUIDS-D034 with anti-aging effect on pets and its application

Abstract

The present invention discloses canine Weissella sp. ZJUIDS-D034 and its application with anti-aging efficacy for pets. The Weissella sp. ZJUIDS-D034 is classified and named as Weissella sp., and its deposit number is CGMCC 27039. The probiotics isolated from the intestinal tract of healthy dogs and derived from the intestinal tract of the host can better colonize in the intestinal tract of the host. Through DPPH clearance rate, hydroxyl radical scavenging ability, reducing ability and intervention experiments on D-galactose-induced aging mice, it is finally found that the present invention has good in vitro antioxidant capacity and has good anti-aging effects on D-galactose-induced aging mice. In addition, the present invention also has good acid tolerance and is sensitive to most antibiotics, which proves that the present invention has good probiotic properties. Therefore, the canine Weissella sp. ZJUIDS-D034 of the present invention can be used in the preparation of canine anti-aging probiotic preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Weissella separated from the intestinal tract of a dog and having the anti-aging effect on pets. Technical Background

[0002] In recent years, the number of pets has been increasing, and pets are playing an increasingly important role in people's lives. According to Packaged Facts' analysis of the US market, which accounts for the largest share of global pet consumption, from 2012 to 2022, the proportion of households with an elderly dog ​​(aged 7 and above) increased from 41.6% to 53.5%, and the proportion of elderly dogs is increasing. With the development of pet medical care, the life span of pet dogs will be longer, and the pet aging-related industry will definitely be one of the development trends of the pet industry.

[0003] Aging is an inevitable part of life. It is manifested as the gradual decline of the functions of cells, tissues and organs of the body over time, and cognitive and memory functions also decline. With the advent of aging, the incidence of some age-related chronic diseases, such as obstetric diseases, liver and kidney diseases, cataracts, heart disease, etc., will continue to increase.

[0004] At present, oxidative aging and inflammatory aging are hot topics in anti-aging research. Oxidative aging is mainly cell aging caused by oxidative stress: the biological oxidative metabolism process will produce reactive oxygen species ROS, which can be removed by antioxidants in the body. In the body, the production and removal of ROS are in dynamic balance. However, when the ROS in the body increases, this balance is broken, and the accumulation of ROS leads to oxidative stress. If oxidative stress persists, it will cause DNA damage, protein misfolding, mitochondrial mutations and cell damage, and eventually lead to cell aging; inflammatory aging is also called immune aging, which refers to the chronic and progressive increase in the body's inflammatory state during the natural aging process. The main reason is the imbalance between pro-inflammatory and anti-inflammatory cytokines in the body, which eventually leads to an increase in pro-inflammatory responses.

[0005] Studies have shown that there is an important relationship between intestinal microorganisms, oxidative aging and inflammatory aging of the body. Regulating and maintaining the balance of intestinal microbiota in elderly dogs may be a means of preventing and treating aging-related diseases and delaying aging. It is well known that probiotics play an important role in maintaining the balance of intestinal flora. Summary of the invention

[0006] The purpose of the present invention is to enrich the existing bacterial resources, make up for the vacancy of anti-aging probiotics and the vacancy of canine probiotic strains, and provide a canine Weissella ZJUIDS-D034 with anti-aging effects on pets. The strain d is classified and named: Weissella (Weissella sp.), which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 6, 2023, with a deposit number of: CGMCC 27039, and the deposit address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The ZJUIDS-D034 was screened from the canine intestine, and after separation, purification, and molecular biological identification, it was determined to be Weissella (Weissella sp.).

[0007] The Weissella ZJUIDS-D034 has the following characteristics:

[0008] (1) Colony and bacterial characteristics: After culturing on MRS medium for 48 h, the colonies had smooth edges, regular round shapes, and milky white color.

[0009] (2) In vitro antioxidant capacity: It has good in vitro antioxidant capacity, with the DPPH clearance rates of the fermentation supernatant being 89.47%, the DPPH clearance rates of the bacterial suspension being 25.92%, and the DPPH clearance rates of the sterile body fragment being 18.10%; the hydroxyl radical clearance rate of the fermentation supernatant being 32.32%; the reducing capacity of the fermentation supernatant being 5.54%, the reducing capacity of the bacterial suspension being 36.54%, and the reducing capacity of the sterile body fragment being 20.48%.

[0010] (3) Acid resistance: After culturing at 37°C for 4 h in PBS with a pH of 2, the survival rate was 86%.

[0011] (4) Antibiotic sensitivity: The virus was sensitive to 6 antibiotics, namely, chloramphenicol, erythromycin, tetracycline, ceftriaxone, and ampicillin-penicillin; it was intermediate to lincomycin; and it was resistant to ciprofloxacin and gentamicin.

[0012] (5) In vivo anti-aging properties: After the present invention was used to intervene in D-galactose-induced aging mice, the oxidation level and inflammation level of the mice were significantly improved, indicating that the present invention has an alleviating effect on the oxidative aging and inflammatory aging of aging mice. The present invention has a certain anti-aging effect on pets.

[0013] Another object of the present invention is to provide the use of the Weissella ZJUIDS-D034 in the preparation of canine anti-aging probiotic preparations. The Weissella ZJUIDS-D034 provided by the present invention has resistance to both oxidative aging and inflammatory aging of D-galactose-induced aging mice, has good in vivo anti-aging ability, has good in vitro antioxidant ability - DPPH scavenging ability, hydroxyl free radical scavenging ability and reducing ability, has good acid resistance, can adapt to the gastrointestinal environment and has proliferation ability, and the strain is sensitive to multiple antibiotics. The Weissella ZJUIDS-D034 of the present invention has anti-aging effects on pets and has excellent probiotic properties.

[0014] The anti-aging probiotic preparation includes bacterial powder, milk powder, canned food (pet wet food), fermented yogurt, dog food and the like.

[0015] The advantages and positive effects achieved by the present invention are as follows: (1) The Weissella ZJUIDS-D034 provided by the present invention is derived from the canine intestine, has strong host adaptability, and fills the gap in canine-derived probiotics; (2) The present invention has anti-aging effects on pets, filling the gap in the efficacy of canine-derived probiotic products; (3) It provides a basis for the development of anti-aging probiotic products. The present invention is based on probiotics isolated from the canine intestine, and a strain of Weissella with anti-aging effects on pets is screened out. The strain also has good performance in acid resistance, and is sensitive to multiple antibiotics and has antibacterial activity. The Weissella of the present invention can provide a basis for the development of anti-aging probiotic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Attached Figure 1 This is the colony morphology of Weissella ZJUIDS-D034.

[0017] Attached Figure 2 This is the electrophoresis diagram of Weissella ZJUIDS-D034. Well 2 is ZJUIDS-D03, and well M is a marker. In the figure, M is 5000, 3000, 2000, 1000, 750, 500, 250, and 100 bp from top to bottom.

[0018] Attached Figure 3 Figure 2 is the protein carbonyl content in the serum of each group of mice.

[0019] Attached Figure 4 The graph shows the GSH content in the spleen of each group of mice.

[0020] Attached Figure 5 The graph shows the SOD content in the spleen of each group of mice.

[0021] Attached Figure 6 The graph shows the MDA content in the spleen of each group of mice.

[0022] Attached Figure 7The graph shows the IgG content in the liver of each group of mice.

[0023] Attached Figure 8 The graph shows the IL-1β content in the liver of each group of mice.

[0024] Attached Fig. 9 The graph shows the IL-6 content in the liver of each group of mice.

[0025] Attached Fig.10 The graph shows the TNF-α content in the liver of each group of mice. DETAILED DESCRIPTION

[0026] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in combination with the accompanying drawings and embodiments. Obviously, what is described is only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary persons in the art without creative work are within the scope of protection of this patent.

[0027] The materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0028] Example 1 Isolation, purification and identification of Weissella ZJUIDS-D034

[0029] 1.1 Sample Source

[0030] The fecal sample was from a healthy 7-year-old female Yorkshire Terrier. The sample was collected by anal swab, that is, 1 mL of normal saline was pre-filled into a 1.5 mL EP tube, and the anal swab of the dog was collected with a moistened cotton swab. Finally, the cotton swab head was inserted into the EP tube and soaked, and the wood part was cut off.

[0031] 1.2 Isolation and purification of strains

[0032] Take 100 μL of the above turbid solution and spread it on MRS solid medium, and culture it at 37℃ for 48h. After the culture is completed, pick typical colonies and separate them by streaking on MRS solid medium several times until the colonies on the whole plate have the same morphology. Then pick a single colony and expand it in MRS liquid medium. The obtained bacterial solution is stored at -80℃ in 40% (w / v) glycerol.

[0033] 1.3 Identification of Weissella ZJUIDS-D034

[0034] 1.3.1 Colony characteristics

[0035] After culturing Weissella ZJUIDS-D034 in MRS medium for 48 hours, the surface edges were smooth, the shape was round, and the color was milky white. Figure 1.

[0036] 1.3.2 16S rDNA identification

[0037] The target strain genomic DNA was extracted using the Ezup column bacterial genomic DNA extraction kit. The extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. The bacterial universal primers 27F and 1492R were used to perform a 16S rDNA PCR experiment. After the PCR reaction was completed, the PCR product was taken for agarose gel detection and photography. The length of the amplified fragment was about 1500 bp, see attached Figure 2 The PCR product was sent to Shanghai Biotechnology Co., Ltd. for sequencing. The results are shown at the end of the patent. A BLAST sequence comparison was performed on the NCBI website. The results showed that the sequence had a homology of more than 99% with the 16S rDNA sequence identified in Weissella. The amplification primers and sequencing primers are shown in Table 1 below.

[0038] Table 1 Amplification primers and sequencing primers for Weissella ZJUIDS-D034

[0039]

[0040] The nucleotide sequence of Weissella ZJUIDS-D034 is shown in SEQ ID NO.1.

[0041] Example 2 Anti-aging effect of Weissella ZJUIDS-D034 on D-galactose-induced aging mice

[0042] 2.1 Establishment of D-galactose-induced aging mouse model

[0043] 24 mice were randomly divided into 3 groups: normal control group, D-galactose aging model group, Weissella ZJUIDS-D034 strain action group, with 8 mice in each group. The treatments are shown in Table 2.

[0044] Table 2 Animal experimental groups and treatments

[0045]

[0046] 2.2 Samples and measurement indicators

[0047] The present invention aims to illustrate the anti-aging effect of the present invention by alleviating the oxidative aging and inflammatory aging of mice. Therefore, after the animal experiment, the oxidation level and the inflammatory level of the mouse body are mainly detected.

[0048] After the animal experiment, the blood, liver and spleen of each group of mice were collected, and the levels of IgG, IL-1β, IL-6 and TNF-α in the liver of each group of mice were measured to evaluate the inflammation level of the mice; the protein carbonyl groups in the serum and the GSH, SOD and MDA contents in the spleen were used to evaluate the oxidation level of the mice.

[0049] 2.2.1 ZJUIDS-D034 alleviates oxidative aging in D-galactose-induced aged mice

[0050] (1) Protein carbonyl content in serum

[0051] Protein carbonyl is an early marker of the oxidation modification of various amino acids in proteins. Its content indicates the extent of protein oxidation damage and is the main indicator for measuring protein oxidation damage. Figure 3 As shown, it can be seen that after Weissella ZJUIDS-D034 intervened in D-galactose-induced aging mice, the protein carbonyl content in the mouse serum was significantly reduced (p<0.01), even lower than that of the normal control group.

[0052] (2) GSH, SOD, and MDA content in spleen

[0053] GSH (glutathione) is an important antioxidant in the body that can remove free radicals in the body. Since GSH itself is susceptible to oxidation by certain substances, it can protect the sulfhydryl groups in many proteins and enzymes from being oxidized by harmful substances, thereby ensuring the normal physiological functions of proteins and enzymes. The results of the GSH experiment are shown in the attached figure. Figure 4 As shown, it can be seen that after Weissella ZJUIDS-D034 intervened in D-galactose-induced aging mice, the GSH content in the spleen of mice was significantly (p<0.05), which was constantly approaching that of normal mice.

[0054] The main function of SOD (superoxide dismutase) is to catalyze the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen. The generated superoxide anion free radicals are normal metabolic products in the body. However, the accumulation of free radicals will cause lipid peroxidation in the cell membrane and cause membrane fission, leading to cell damage or even death. SOD is the most important and best free radical scavenger in the body, maintaining the body's metabolic balance. SOD can remove and reduce excessive free radicals in the human body and delay aging, and is being studied as an anti-aging drug. The results of the SOD experiment are shown in the attached figure. Figure 5 As shown, it can be seen that after Weissella ZJUIDS-D034 intervenes in D-galactose-induced aging mice, the SOD content in the spleen of mice can be increased.

[0055] In the body, free radicals act on lipids to produce peroxidation reactions. The final oxidation product is MDA (malondialdehyde), which will cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, and has cytotoxicity. MDA content is an important parameter reflecting the body's antioxidant potential. It can reflect the body's lipid peroxidation rate and intensity, and can also indirectly reflect the degree of tissue peroxidation damage. The MDA test results are shown in the attached Figure 6 As shown, it can be seen that after Weissella ZJUIDS-D034 intervenes in D-galactose-induced aging mice, the MDA content in the spleen of mice can be reduced.

[0056] In summary, it can be seen that Weissella ZJUIDS-D034 can increase the content of antioxidant enzymes in the body of aging mice, reduce oxidative damage in the body, and have a certain resistance to oxidative aging in aging mice.

[0057] 2.2.2 ZJUIDS-D034 alleviates oxidative aging in D-galactose-induced aging mice

[0058] (1) IgG content in the liver

[0059] IgG is the main immunoglobulin in the body. Its content can reflect the body's immune ability. The higher the content, the stronger the immunity. The IgG test results are shown in the attached Figure 7 As shown, it can be seen that after Weissella ZJUIDS-D034 intervened in D-galactose-induced aging mice, the IgG content in the mouse liver was significantly (p<0.001).

[0060] (2) Contents of IL-1β, IL-6, and TNF-α in the liver

[0061] TNF-α, IL-1β and IL-6 are all pro-inflammatory cytokines, and their content is related to the level of inflammation in the body. Figure 8 , 9 As shown in Figures 10, it can be seen that after Weissella ZJUIDS-D034 intervened in D-galactose-induced aging mice, the levels of these three pro-inflammatory factors in the liver of mice were significantly (p<0.001).

[0062] In summary, it can be seen that Weissella ZJUIDS-D034 can increase the IgG content in the body of aging mice and enhance the body's immunity; reduce the content of proinflammatory cytokines TNF-α, IL-1β and IL-6 in the body, and have a certain resistance to inflammatory aging in aging mice.

[0063] Example 3 Determination of in vitro antioxidant capacity of Weissella ZJUIDS-D034

[0064] The DPPH scavenging rate, hydroxyl radical scavenging rate and reducing ability of Weissella ZJUIDS-D034 isolated from the canine intestine were determined.

[0065] 3.1 Sample preparation

[0066] The strains stored in the glycerol tube were streaked on MRS solid medium and cultured at 37°C for 48 hours. A single colony was picked with an inoculation loop and inoculated into sterile MRS liquid medium. The culture was cultured at 37°C for 18-24 hours to obtain a culture solution. The culture solution was adjusted with distilled water to a lactic acid bacteria concentration of 10 9 CFU / mL, centrifuge at 4°C, 8000×g for 20 min, collect the supernatant as the fermentation supernatant. Resuspend and wash the bacterial pellet after centrifugation with 0.02M PBS buffer (pH=7.4), centrifuge at 4°C, 8000×g for 20 min, repeat 3 times. Resuspend the washed bacterial cells in PBS buffer and adjust the bacterial concentration to 10 9 The bacterial suspension is subjected to ultrasonic disruption, and then centrifuged to obtain the supernatant to obtain a sterile disrupted product.

[0067] 3.2DPPH clearance rate

[0068] 1 mL of sample solution and 1 mL of 0.2 mM DPPH anhydrous ethanol solution were mixed by oscillation for 1 min, and then reacted at room temperature in the dark for 30 min. The supernatant was centrifuged and the absorbance was measured at 517 nm. 95% ethanol was used instead of DPPH as a control, and PBS was used instead of the sample solution as a blank.

[0069] DPPH clearance rate = 1-(A i -A j ) / A c

[0070] A i : Sample solution + DPPH absorbance

[0071] A j : Sample solution + 95% ethanol absorbance

[0072] A c : PBS+DPPH absorbance.

[0073] 3.3 Hydroxyl radical scavenging rate

[0074] Take 1 mL of o-phenanthroline (2.5 mmol / L), add 1 mL of phosphate buffer (PBS concentration is 0.02 mol / L, pH = 7.4), 1 mL of distilled water, mix well, add 1 mL of ferrous sulfate (FeSO4 concentration is 2.5 mmol / L), mix well, add 1 mL of hydrogen peroxide (H2O2 mass fraction is 20 mmol / L), and measure its absorbance at 536 nm after 1.5 hours in a 37°C water bath as A. p ; Use 1mL of distilled water to replace 1mL of hydrogen peroxide for A b ; Use 1mL sample to replace 1mL distilled water as A s .

[0075] Hydroxyl free radical scavenging rate = (A s -A p ) / (A b -A p ).

[0076] 3.4 Reduction capability

[0077] In the reaction solution, 0.5 mL of sample solution, 0.5 mL of potassium ferricyanide (mass fraction 1%) and 0.5 mL of phosphate buffer (PBS concentration of 0.2 mol / L, pH = 6.6) were mixed in a 50°C water bath for 20 min. After the reaction, the solution was cooled and 0.5 mL of trichloroacetic acid (TAC mass fraction of 10%) was added to precipitate the protein. After centrifugation, 1 mL of the supernatant was reacted with 1 mL of ferric chloride (FeCl3 mass fraction of 0.1%), and the absorbance was measured at 700 nm. PBS or MRS liquid culture medium was used instead of the sample solution as a blank control.

[0078] Reduction ability = (A s -A b ) / A b

[0079] Among them A s : Absorbance of experimental group, A b : Absorbance of blank group.

[0080] Table 3 In vitro antioxidant activity of Weissella ZJUIDS-D034

[0081] index Fermentation supernatant Bacteria suspension Sterile body fragments DPPH removal rate / % 89.47±2.49 25.92±1.85 18.1±4.01 Hydroxyl free radical scavenging rate / % 32.32±12.99 - - Reduction ability / % 5.54±1.08 36.54±2.84 20.48±1.25

[0082] Example 4 Determination of acid and bile resistance of Weissella ZJUIDS-D034

[0083] 4.1 Acid resistance test

[0084] A single colony of Weissella ZJUIDS-D034 was selected and expanded in MRS liquid medium for 18 h. The expanded bacterial suspension was inoculated into MRS broth medium at a volume of 1% (v / v). After culturing at 37°C for 18 h, the concentration of the bacterial suspension reached 10 9 CFU / mL. 10% of the sample was inoculated into PBS at pH 2.0 and cultured at 37°C for 4 hours. Using PBS without pH adjustment as the control, the plate count method was used to count the live bacteria in the 0h and 4h samples and calculate their survival rate. The survival rate calculation formula is as follows:

[0085] In the above formula, N0 is the viable count of the test strain at 0h (CFU / mL); N t It is the number of viable bacteria (CFU / mL) of the test strain at 4 hours.

[0086] Table 4 Acid resistance characteristics of Weissella ZJUIDS-D034

[0087] index 4h survival rate Acid resistance pH = 2 0.86±0.1

[0088] Example 5 Antibiotic Sensitivity Test

[0089] Take a single colony of Weissella ZJUIDS-D034 that grows well on the MRS plate and inoculate it into 20 mL of sterile MRS liquid medium. After culturing at 37°C in an incubator for 24 hours, adjust the bacterial concentration to 10 9 CFU / mL, take 100μL of the culture solution of the bacteria to be tested and spread it evenly on the MRS plate, use tweezers to take the drug sensitivity paper and place it on the culture medium, press it lightly until it is fixed. Place the culture dish face up in a 37℃ incubator and culture for 24 hours, use a vernier caliper to measure the diameter of the drug sensitivity circle of each bacteria to each antibiotic and record the data.

[0090] Table 5 Antibiotic sensitivity results of Weissella ZJUIDS-D034

[0091]

[0092]

[0093] Note: S-sensitive; I-intermediate; R-resistant

[0094] Example 6 Preparation of Weissella ZJUIDS-D034 anti-aging probiotic powder

[0095] 6.1 Preparation of Weissella ZJUIDS-D034 bacterial slurry

[0096] Pick a single colony of Weissella ZJUIDS-D034 and inoculate it in 50mL MRS liquid culture medium, and place it in a 37°C incubator for 18h. Activate it again in 250mL MRS liquid culture medium at a 5% inoculation amount, and place it in a 37°C incubator for 24h. Finally, the activated Weissella ZJUIDS-D034 is cultured in a 10L fermenter at a high-density anaerobic culture with a 5% inoculation amount, and cultured for 18h at 37°C and pH 6.8. After that, centrifuge it at 8000r / min and 4°C for 15min, discard the supernatant, collect the bacterial precipitate, and rinse the bacterial body twice with sterile PBS (pH 7.0). You can get Weissella ZJUIDS-D034 bacterial mud.

[0097] 6.2 Preparation of protective agent

[0098] The freeze-dried protective agent contains 15% skim milk powder, 5% trehalose, 3% sodium glutamate, 1% glycerol, and 0.5% cysteine ​​hydrochloride. Water is used as the solvent. Sterilize at 110°C for use.

[0099] 6.3 Preparation of Weissella ZJUIDS-D034 anti-aging bacterial powder

[0100] The prepared Weissella ZJUIDS-D034 bacterial sludge was mixed with the protective agent solution at a ratio of 1:5. It was pre-frozen at -40℃ for 5 hours to make it evenly frozen on the inner wall of the container, and then vacuum-freeze-dried for 18 to 20 hours to obtain Weissella ZJUIDS-D034 bacterial powder. After rehydration with physiological saline, it was washed twice, and the number of live bacteria in the Weissella ZJUIDS-D034 bacterial powder was measured to be 1.0×10 11 ~1×10 12 CFU / g.

[0101] Example 7 Preparation of anti-aging probiotic milk powder for pet dogs using Weissella ZJUIDS-D034

[0102] 7.1 Preparation of Weissella ZJUIDS-D034 bacterial powder

[0103] Referring to the above case, freeze-dried powder of Weissella ZJUIDS-D034 was prepared, and the number of viable bacteria in the powder was

[0104] 1.0×10 11 ~1×10 12 CFU / g

[0105] 7.2 Preparation of pet formula powder

[0106] Primary selection of raw materials: milk powder, fish meal, bone meal, grains, vegetable oil, additives: vitamins, trace elements, functional factors, others;

[0107] Automatic batching: put the obtained raw materials into the material bin according to the formula;

[0108] Crushing: crush the weighed materials through a crusher;

[0109] Mixing: Add vegetable oil and trace elements to the crushed materials and put them into the mixer to mix evenly;

[0110] Puffing: The mixed materials are made into granular materials through the puffing machine;

[0111] Drying: The mixed materials are dried in a dryer with the temperature controlled at 65-70 degrees;

[0112] Grading screening: The logistics is passed through the grading screen and the particles are controlled at 2.5-5 mm.

[0113] 7.3 Preparation of probiotic milk powder for pets

[0114] The bacterial powder prepared in 7.1 and the pet formula powder prepared in 7.2 were mixed evenly at a ratio of 1:100. The live bacteria in the final product were 8 CFU / g or above. After filling, the product is stored in the warehouse for sale.

[0115] Example 8 Preparation of canned anti-aging probiotics for pet dogs using Weissella ZJUIDS-D034

[0116] The main formula is as follows: beef 60-80%, carrageenan 0.5-1.2%, sodium D-isoascorbate 0.05-0.3%, water 15-30%, ZJUIDS-D034 bacterial powder (1.0×10 11 ~1×10 12 CFU / g).

[0117] Preparation method of canned dog food: The specific steps include mixing beef mince, carrageenan, water and sodium D-isoascorbate, then vacuum degassing, sterilizing at 70-90 degrees Celsius, mixing and adsorbing the sterilized product with bacterial powder, and finally canning for use. The effective bacterial guarantee value of Weissella ZJUIDS-D034 in the product is 10 9 CFU / g.

[0118] Example 9 Preparation of anti-aging probiotic fermented yogurt for pet dogs using Weissella ZJUIDS-D034

[0119] 9.1 Yogurt processing technology:

[0120] Raw materials - preheating - homogenization - preparation - sterilization - cooling - preparation

[0121] Inoculation - fermentation - ripening - refrigeration.

[0122] 9.2 Operation points

[0123] (1) Raw materials: 2L fresh goat milk;

[0124] (2) Preheating: Place in a container and heat to 63°C;

[0125] (3) Homogenization: Pour the mixture into a homogenizer and homogenize at a pressure of 15-25 MPa), pour the mixture into an iron can, add 100 g of sugar, and sterilize in a 90°C water bath for 10 min;

[0126] (4) Preparation: Add ingredients to goat milk and dissolve;

[0127] (5) Sterilization: Sterilize the sweetened goat milk in a 90°C water bath for 10 min;

[0128] (6) Cooling: After sterilization, the goat milk is cooled to 40-50°C for later use;

[0129] (7) Preparation of starter culture: Weissella ZJUIDS-D034 was inoculated into a test tube containing sterilized skim milk (12%, w / v) under a sterile environment and cultured at 37°C for 20 hours. The inoculation volume for each subculture was 2-4% (v / v), and the culture was subcultured 2-3 times to restore vitality and stored in a refrigerator at 4°C;

[0130] (8) Inoculation and fermentation: Under sterile conditions, inoculate the activated Weissella ZJUIDS-D034 at an inoculation rate of 2-4% (v / v). Ferment at a constant temperature of 42°C for 6-10 hours;

[0131] (9) Post-ripening: After fermentation, place in a refrigerator at 4°C for 12-24 hours;

[0132] (10) Filling and refrigeration: After ripening, fill into 250 mL sterilized glass bottles and send to cold storage for refrigeration.

[0133] Example 10 Preparation of anti-aging feed for pet dogs using Weissella ZJUIDS-D034

[0134] 10.1 Pet Food Production Process

[0135] Raw material preparation - mixing - conveying - extrusion - conveying - drying - oil spraying - seasoning - screening (packaging)

[0136] 10.2 Equipment Configuration

[0137] Mixer / crusher—loader—expander—air conveyor / elevator—multi-layer oven—cooler—oil sprayer—drum—elevator—vibrating screen—packaging machine

[0138] 10.3 Ingredients

[0139] 26% frozen chicken, 20% dehydrated chicken meal, 18% dehydrated fish meal, 9% sweet potato powder, 6% chicken fat, 5% potato powder, 4% peas, 3% frozen salmon belly, 3% egg yolk powder, 1.2% freeze-dried egg yolk, 1.2% freeze-dried beef liver, 1% freeze-dried chicken, frozen chicken liver, and Weissella ZJUIDS-D034 bacterial powder in Example 6.

[0140] The effective bacteria value of Weissella ZJUIDS-D034 in the product is guaranteed to be 10 9 CFU / g.

[0141] The above are only preferred specific implementation modes of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of a strain of Weissella canis ZJUIDS-D034 in the preparation of an anti-aging probiotic preparation for dogs, characterized in that: The strain was classified and named: Weissella Weissella sp. ), which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 6, 2023, with the deposit number: CGMCC 27039; the Weissella ZJUIDS-D034 has resistance to the oxidative aging and inflammatory aging of D-galactose-induced aged mice, has good in vivo anti-aging ability, and has good in vitro antioxidant ability - DPPH scavenging ability, hydroxyl free radical scavenging ability and reducing ability, acid resistance, good adaptation to the gastrointestinal environment and proliferation ability, and is sensitive to multiple antibiotics.

2. The use according to claim 1, characterized in that: The anti-aging probiotic preparation includes bacterial powder, milk powder, pet wet food, fermented yogurt, and dog food.

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