Bifidobacterium adolescentis from longevity dogs and cats and application thereof

Abstract

The invention discloses bifidobacterium adolescentis from longevity dogs and cats and application thereof, and belongs to the technical field of microorganisms. Bifidobacterium adolescentis with the effect of prolonging the life of mice is separated from fresh feces of longevity dogs and cats, and after mutagenesis, the bifidobacterium adolescentis for dogs and cats, which is more suitable for gastrointestinal environments of dogs and cats, is bred by taking growth speed, gastrointestinal digestion resistance and oxygen resistance as indexes. The mutant strain is preserved in the China Center for Type Culture Collection (CCTCC) on August 5, 2024, and the preservation number of the mutant strain is CCTCC NO: M20241747 and the preservation number of the mutant strain is CCTCC NO: M20241746. The bifidobacterium adolescentis disclosed by the invention can be applied to food (feed) of dogs and cats, and has the effects of reducing the food change stress rate of the dogs and the cats and improving the senescence index (NAD < + >).

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Bifidobacterium adolescentis which can withstand the gastrointestinal environment of pet dogs and cats and has good colonization ability and application thereof. Background Art

[0002] Bifidobacterium adolescentis is a Gram-positive, non-spore-forming, strictly anaerobic microorganism belonging to the genus Bifidobacterium. It is widely present in the intestines of mammals. In addition to preventing and treating diarrhea (patent: Method for the prophylaxis and treatment of diarrhoea in dogs, GB7614161; Bifidobacterium prepn. for cure and prevention of caninediarrhoea contg. Bifidobacterium seudolongum and or adolescentis isolated from canine digestive organ of faeces, JP85038372-B), anti-infection (paper: Search forpromising strains of probiotic microbiota isolated from different biotopes ofhealthy cats for use in the control of surgical infections, 2021), and maintaining intestinal health, it also has the function of prolonging animal lifespan (paper: Bifidobacterium adolescentis regulates catalase activity and host metabolism and improves healthspan and lifespan in multiple species, 2021; Heat-inactivated Bifidobacterium adolescentisameliorates colon senescence through Paneth-like-cell-mediated stem cellactivation, 2023).The Bifidobacterium adolescentis currently being studied and applied is not of dog or cat origin, but is concentrated on human and cattle sources. For example, strains such as CICC6178 and CICC6179 preserved by the China Industrial Microbiological Culture Collection are isolated from adult intestines and cow stomachs, and the Bifidobacterium adolescentis isolated by Pan et al. comes from human infant feces (paper: Effects of probiotics and nutrients addition on the microbial community and fermentation quality of peanut hull, 2019).

[0003] The prerequisite for Bifidobacterium adolescentis to play a probiotic role in the intestines of dogs and cats is that it can colonize normally, which requires it to be able to adapt to the gastrointestinal environment of dogs and cats. There are many differences between the gastrointestinal environment of dogs and cats and that of humans, cattle and other mammals, which are manifested in many aspects such as gastric juice pH, bile salt concentration, and large intestine oxygen content. The intestinal temperature of dogs is 37.5℃~39℃, the gastric juice pH is 1.5~2.5, the bile salt concentration can reach 1~4g / L, and the oxygen partial pressure of the large intestine microaerobic environment is 0.8%; the intestinal temperature of cats is 38℃~39.5℃, the gastric juice pH is 1~2, the intestinal bile salt concentration is 1~4g / L, and the oxygen partial pressure of the large intestine microaerobic environment is 0.8%. In addition, Bifidobacterium adolescentis mostly distributed in the large intestine of animals. The large intestine of herbivores is longer than that of omnivores, and that of omnivores is longer than that of carnivores. As carnivores, the large intestine of dogs and cats is much shorter than that of humans and cattle. The large intestine of an adult is 2 to 3 meters long, that of a cattle is 6.4 to 10 meters long, and that of dogs and cats is only 0.6 and 0.4 meters long, respectively. The longer the large intestine, the easier it is to form an anaerobic environment, coupled with the blocking effect of food residues after digestion and the consumption of oxygen by microbial fermentation. Therefore, unlike humans and cattle, it is difficult for dogs and cats to form a completely anaerobic environment (actually a micro-oxygen environment) inside the relatively short large intestine. Intestinal microorganisms have a certain degree of specificity in their choice of host. Therefore, Bifidobacterium adolescentis isolated from the intestines and excreta of non-pet dogs and cats (humans, cattle, etc.) is more adapted to the original host animal environment. If directly applied to dogs and cats, it has poor adaptability to the gastrointestinal environment of dogs and cats and is difficult to fully exert its beneficial functions. However, there is no report on the research on isolating Bifidobacterium adolescentis from the feces of long-lived dogs and cats, and obtaining through mutagenesis and selection that can better adapt to the gastrointestinal environment of dogs and cats, have good colonization ability in the intestines of dogs and cats, and have the effect of reducing the stress rate of food change and improving aging indicators in dogs and cats. Summary of the invention

[0004] The purpose of the present invention is to provide Bifidobacterium adolescentis for pet dogs and cats, which has the characteristics of adapting to the gastrointestinal environment of dogs and cats and having good colonization ability in the intestine, as well as the application of Bifidobacterium adolescentis for dogs and cats.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: Invention content 1:

[0007] The present invention provides a strain of Bifidobacterium adolescentis LifeAge20 derived from a long-lived cat, which is currently deposited in the China Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China. The preservation date is August 5, 2024, the preservation number is: CCTCCNO: M20241746, the preservation name is Bifidobacterium adolescentis LifeAge20, and the recommended classification name is Bifidobacterium adolescentis.

[0008] The present invention provides a method for breeding the above-mentioned Bifidobacterium adolescentis LifeAge20, comprising the following steps:

[0009] (1) Fresh feces of long-lived cats were collected, and the suspension was diluted, plated and anaerobically cultured to obtain single colonies, which were identified by 16S rDNA sequencing. Bifidobacterium adolescentis was selected for anti-aging function identification, and wild cat-derived Bifidobacterium adolescentis was obtained.

[0010] The above-mentioned anti-aging function identification was carried out using rapid aging model mice as the experimental subjects and the lifespan of mice as the indicator.

[0011] (2) Wild feline Bifidobacterium adolescentis was induced to mutate, and growth rate, gastric digestion resistance, intestinal digestion resistance and oxygen resistance were used as indicators to select and obtain a feline Bifidobacterium adolescentis LifeAge20 that is more adaptable to the gastrointestinal environment of cats.

[0012] The above mutagenesis is accomplished by radiation mutagenesis.

[0013] The above growth rate is based on the bacterial concentration (OD 600 value) as an indicator to select strains with faster growth rate.

[0014] The above-mentioned resistance to gastric digestion is based on the survival rate of the strain after being treated in artificial gastric juice for a fixed period of time, and strains with stronger resistance to gastric digestion are selected.

[0015] The above-mentioned resistance to intestinal digestion is based on the survival rate of the strain after being treated in artificial intestinal fluid for a fixed period of time, and strains with stronger resistance to intestinal digestion are selected.

[0016] The above-mentioned oxygen tolerance is based on the survival rate of the strain after being cultured in a microaerobic environment for a fixed period of time, and strains with stronger oxygen tolerance are selected.

[0017] The present invention also provides application of the cat-derived Bifidobacterium adolescentis LifeAge20 in cat food (feed).

[0018] Furthermore, the cat-derived Bifidobacterium adolescentis LifeAge20 can also be applied to carnivorous animals with a gastrointestinal structure similar to that of cats, such as foxes.

[0019] Furthermore, cat food (feed) includes but is not limited to cat food, cat snacks, cat health food, etc.

[0020] Furthermore, the cat-derived Bifidobacterium adolescentis LifeAge20 has the effects of reducing the rate of food change stress and improving aging indicators (increasing serum NAD + content). Summary of the invention 2:

[0022] The present invention provides a strain of Bifidobacterium adolescentis LifeAge18 derived from a long-lived dog, which is currently deposited in the China Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China. The preservation date is August 5, 2024, the preservation number is: CCTCC NO: M20241747, the preservation name is Bifidobacterium adolescentis LifeAge18, and the recommended classification name is Bifidobacterium adolescentis (Bifidobacterium adolescentis).

[0023] The present invention provides a method for breeding the above-mentioned Bifidobacterium adolescentis LifeAge18, comprising the following steps:

[0024] (1) Fresh feces of long-lived dogs were collected, and the suspension was diluted, plated and anaerobically cultured to obtain single colonies, which were identified by 16S rDNA sequencing. Bifidobacterium adolescentis was selected for anti-aging function identification, and wild canine-derived Bifidobacterium adolescentis was obtained.

[0025] The above anti-aging identification was carried out using rapid aging model mice as test subjects and the lifespan of mice as an indicator.

[0026] (2) Wild canine Bifidobacterium adolescentis was induced to mutate, and the canine Bifidobacterium adolescentis LifeAge18 was selected based on growth rate, gastric digestion resistance, intestinal digestion resistance and oxygen resistance to obtain a canine Bifidobacterium adolescentis LifeAge18 that is more adaptable to the gastrointestinal environment of dogs.

[0027] The above mutagenesis is accomplished by radiation mutagenesis.

[0028] The above growth rate is based on the bacterial concentration (OD 600 value) as an indicator to select strains with faster growth rate.

[0029] The above-mentioned resistance to gastric digestion is based on the survival rate of the strain after being treated in artificial gastric juice for a fixed period of time, and strains with stronger resistance to gastric digestion are selected.

[0030] The above-mentioned resistance to intestinal digestion is based on the survival rate of the strain after being treated in artificial intestinal fluid for a fixed period of time, and strains with stronger resistance to intestinal digestion are selected.

[0031] The above-mentioned oxygen tolerance is based on the survival rate of the strain after being cultured in a microaerobic environment for a fixed period of time, and strains with stronger oxygen tolerance are selected.

[0032] The present invention also provides application of the canine-derived Bifidobacterium adolescentis LifeAge18 in dog food (feed).

[0033] Furthermore, the canine-derived Bifidobacterium adolescentis LifeAge18 can also be applied to omnivorous animals with a gastrointestinal structure similar to that of dogs, such as pigs.

[0034] Furthermore, dog food (feed) includes but is not limited to dog grain, dog snacks, dog health food, etc.

[0035] Furthermore, the canine-derived Bifidobacterium adolescentis LifeAge18 has the effects of reducing the stress rate of food change and improving aging indicators (increasing serum NAD + content).

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The adolescent Bifidobacterium LifeAge20 for cats bred according to the method of the present invention showed good tolerance to the gastrointestinal environment of cats. The bacterial survival rates reached 30.00%, 38.33% and 51.38% respectively when treated for 2 hours under the conditions of the gastric environment, intestinal environment and microaerobic environment of cats. The adolescent Bifidobacterium LifeAge20 had good passage stability. After 10 generations of passage, the bacterial survival rates could still reach 18.80%, 41.60% and 48.39% when treated for 2 hours under the conditions of the gastric environment, intestinal environment and microaerobic environment of cats. The adolescent Bifidobacterium LifeAge20 had good colonization ability in the intestines of cats. After stopping feeding the bacteria for 7 days, the number of viable bifidobacteria detected in cat feces could still reach 4.37lg CFU / g. Bifidobacterium adolescentis LifeAge20 can reduce the stress rate of cats changing food and improve aging indicators. After feeding the staple food containing Bifidobacterium adolescentis LifeAge20 for 7 days, the stress rate of cats changing food was only 8%, which was 85.71% lower than the control. After feeding the staple food containing Bifidobacterium adolescentis LifeAge20 for 30 days, the NAD in cat serum + The content increased to 30.83%, a significant increase of 13.22% compared with the control group.

[0038] (2) The adolescent Bifidobacterium LifeAge18 for dogs bred according to the method of the present invention showed good tolerance to the gastrointestinal environment of dogs. The bacterial survival rates reached 44.47%, 35.39% and 53.52% respectively when treated for 2 hours under the conditions of the gastric environment, intestinal environment and microaerobic environment of dogs. The adolescent Bifidobacterium LifeAge18 had good passage stability. After 10 generations of passage, the bacterial survival rates could still reach 33.87%, 34.19% and 46.75% when treated for 2 hours under the conditions of the gastric environment, intestinal environment and microaerobic environment of dogs. The adolescent Bifidobacterium LifeAge18 had good colonization ability in the intestinal tract of dogs. After stopping feeding the bacteria for 7 days, the number of viable bifidobacteria detected in the feces of dogs could still reach 4.43lg CFU / g. Bifidobacterium adolescentis LifeAge18 can reduce the stress rate of dog food change and improve aging indicators. After feeding the staple food containing Bifidobacterium adolescentis LifeAge18 for 7 days, the stress rate of dog food change was only 6%, which was 83.33% lower than the control. After feeding the staple food containing Bifidobacterium adolescentis LifeAge18 for 30 days, the NAD in the dog serum was + The content increased to 42.41%, a significant increase of 24.92% compared with the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a scanning electron micrograph of cat-derived Bifidobacterium adolescentis, where A is wild strain of Bifidobacterium adolescentis (QC-Y); B is a mutagenic strain (Bifidobacterium adolescentis LifeAge20);

[0040] Figure 2 The transmission electron micrograph of cat-derived Bifidobacterium adolescentis, A is wild strain of Bifidobacterium adolescentis (QC-Y); B is mutated strain (Bifidobacterium adolescentis LifeAge20);

[0041] Figure 3 The scanning electron micrographs of canine-derived Bifidobacterium adolescentis, A is wild strain of Bifidobacterium adolescentis (QC-x); B is a mutant strain (Bifidobacterium adolescentis LifeAge18);

[0042] Figure 4 The transmission electron micrographs of canine-derived Bifidobacterium adolescentis, A is wild strain of Bifidobacterium adolescentis (QC-x); B is a mutant strain (Bifidobacterium adolescentis LifeAge18);

[0043] Figure 5 The phylogenetic tree of Bifidobacterium adolescentis LifeAge20 based on 16S rDNA gene sequence;

[0044] Figure 6 This is a phylogenetic tree of Bifidobacterium adolescentis LifeAge18 based on the 16S rDNA gene sequence. DETAILED DESCRIPTION

[0045] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific embodiments and with reference to data. It should be understood that these embodiments are only further explanations of the present invention and cannot be construed as limiting the scope of protection of the present invention. Technical engineers in this field may make some non-essential improvements and adjustments to the present invention based on the content of the above invention.

[0046] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.

[0047] The present invention relates to the following culture medium and reagents:

[0048] The preparation of artificial gastric juice was based on the method of Li Hong et al. (Experimental study on the effects of Fangfeng Jiere mixture on gastric juice secretion, gastric emptying and intestinal peristalsis, 2020) with slight modifications. The pepsin content was adjusted to 25U / mL, and the pH was adjusted to 1.5 when treating cat-derived strains, and to 2.0 when treating dog-derived strains.

[0049] The preparation of artificial intestinal fluid was based on the method of Xue Junmin (Effects of Tea Polyphenols on Intestinal Flora, Intestinal Enzyme Activity and Short-Chain Fatty Acids in Mice, 2018) with slight modifications. Pig bile powder (4 g / L) was added and the pH was adjusted to 7.2 when treating cat-derived strains and to 8.3 when treating dog-derived strains.

[0050] The method for preparing the intestinal digestion resistance screening medium is to add pig bile powder (4g / L) to the LB basic medium, and the pH is adjusted to 7.2 when culturing cat-derived strains, and the pH is adjusted to 8.3 when culturing dog-derived strains.

[0051] LB basal medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; pig bile powder and pepsin were purchased from Sigma-Aldrich; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0052] The criteria for judging golden feces of dogs and cats are as follows: banana-shaped, without cuts, moderately hard, elastic and soft.

[0053] PCR-based bacterial species identification: Universal primers 27F and 1492R were used to amplify the sequence of the 16S rDNA portion of the strain by PCR, and the target gene sequence amplified by PCR was sequenced. The strain species was determined by comparing the sequence with the NCBI database Blast.

[0054] The sequences of universal primers 27F and 1492R are as follows:

[0055] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0056] 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0057] Human Bifidobacterium adolescentis, used as a control strain in the evaluation test of intestinal colonization effect of cats and dogs, was purchased from the China Industrial Microbiological Culture Collection Center with the strain number CICC6175.

[0058] Example 1

[0059] (1) Isolation of bacterial strains

[0060] Fresh golden feces of long-lived cats (average age 20 years) were collected, resuspended in PBS buffer, and diluted to 10 by 10-fold gradient dilution method. -1 ~10 -6 Concentration, take 100 μL of each gradient and spread it on LB agar plate, and culture it in anaerobic environment at 39°C for 48 hours. Select the plate with uniform colony distribution and good growth (the number of colonies on a single plate is less than 200, and the bacteria are moist), and pick a single colony.

[0061] (2) Molecular identification

[0062] The selected single colonies were identified by PCR and the Bifidobacterium adolescentis strain was obtained. The two fastest growing strains were selected and named QC-Y1 and QC-Y2 respectively.

[0063] (3) Functional identification

[0064] The SAMP8 strain of accelerated aging model mice were fed with Bifidobacterium adolescentis QC-Y1 and QC-Y2 to evaluate their effects on the lifespan of the accelerated aging model mice. Thirty 8-week-old SAMP8 strain accelerated aging model mice were randomly divided into QC-Y1 group, QC-Y2 group and control group with 10 mice in each group. The two types of Bifidobacterium adolescentis were mixed with the mouse diet and re-prepared into a composite mouse diet. The bacterial concentration of Bifidobacterium adolescentis in the composite mouse diet was 1×10 8 CFU / g. The QC-Y1 group was fed with a compound mouse diet containing Bifidobacterium adolescentis QC-Y1, the QC-Y2 group was fed with a compound mouse diet containing Bifidobacterium adolescentis QC-Y2, and the control group was fed with a common mouse diet. Each group was fed 40 g per day and had free access to water. The time of death of the mice was recorded and the lifespan was counted.

[0065] The results are shown in Table 1. Both QC-Y1 and QC-Y2 of Bifidobacterium adolescentis significantly prolonged the lifespan of mice in the aging-accelerated model, and the effect of QC-Y2 was better than that of QC-Y1. The average lifespan of mice in the QC-Y2 group was increased by 131 days compared with the control group, that is, the lifespan was extended by 33.85%, and the average lifespan of mice in the QC-Y1 group was increased by 92 days, that is, the lifespan was extended by 21.60%. QC-Y2 was renamed QC-Y and used as the original bacteria for mutagenesis and screening.

[0066] Table 1 Effects of long-lived cat-derived Bifidobacterium adolescentis on the lifespan of aging-accelerated model mice

[0067] Group Name Average life span (days) QC-Y1 426 QC-Y2 518 Control group 387

[0068] (4) Mutation and selection of strains

[0069] The original strain of Bifidobacterium adolescentis was irradiated with 150Gy of cobalt-60 radiation for 10 minutes.

[0070] The mutated strain was inoculated into LB liquid medium at 1%, cultured at 39°C for 15 h under anaerobic conditions, and the bacterial concentration (OD) before and after culture was measured. 600 value), calculate the growth rate of the strain. The growth rate calculation formula is as follows:

[0071] V=OD 15 -OD0

[0072] Where V is the growth rate; OD 15 and OD0 is the OD of the bacterial solution at 15h and 0h of culture 600 value.

[0073] In the first round of screening, more than 300 strains were screened, from which 24 strains with larger single colony diameters were selected for the second round of screening. The three strains with the fastest growth rate were selected for re-screening of resistance to cat stomach, intestinal digestion and oxygen tolerance.

[0074] The QC-09, QC-23 and QC-24 strains grew fastest after 15 hours of culture, and their growth rates increased by 5466.67%, 5425.00% and 5908.33% respectively compared with the original strain QC-Y. These three strains were selected for rescreening.

[0075] During the rescreening, the strains obtained from the initial screening were cultured in an intestinal digestion resistance screening medium for 7 consecutive days, with simulated intestinal digestion screening twice a day, and simulated gastric digestion screening was added separately on the first and fourth days. During this process, the strains were cultured in a microaerobic environment (0.8% oxygen partial pressure) to culture aerobic strains.

[0076] After enrichment and cultivation of the three strains, the bacterial liquid was taken at a 2% inoculum volume and placed in artificial gastric juice and artificial intestinal juice for 2 hours, and another bacterial liquid was placed in a microaerobic environment with an oxygen partial pressure of 0.8% for 2 hours. The number of viable bacteria was determined by the gradient dilution plate method after 0 hours and 2 hours of treatment, and the survival rate was calculated. The survival rate calculation formula is as follows:

[0077]

[0078] Wherein, P is the survival rate, unit is %; n2 is the number of viable bacteria in the bacterial solution after 2 hours of treatment, unit is CFU; n0 is the number of viable bacteria in the bacterial solution at 0 hours of treatment, unit is CFU.

[0079] The results are shown in Table 2. Compared with the original strains, the three mutant strains have significantly improved gastric digestion resistance, intestinal digestion resistance and oxygen resistance. Among them, QC-09 has the highest survival rate in gastric digestion resistance and oxygen resistance, which are 30.00% and 51.38% respectively. The survival rate in intestinal digestion resistance is 38.33%, which is only 3.06% lower than that of QC-24 (41.39%). In terms of comprehensive gastric, intestinal digestion and microaerobic environment resistance, QC-09 was selected for subsequent tests.

[0080] Table 2 Resistance of Bifidobacterium adolescentis from long-lived cats to gastric digestion, intestinal digestion and oxygen

[0081] Survival rate of gastric digestion resistance (%) Survival rate of intestinal digestion resistance (%) Oxygen tolerance survival rate (%) QC-Y 1.48 12.45 7.21 QC-09 30.00 38.33 51.38 QC-23 5.58 29.29 33.65 QC-24 9.35 41.39 19.57

[0082] (5) Strain identification

[0083] Using the genomic DNA of the mutant strain QC-09 of Bifidobacterium adolescentis as a template, the strain was identified by PCR and confirmed to be Bifidobacterium adolescentis. The mutant strain QC-09 was renamed Bifidobacterium adolescentis LifeAge20 and a phylogenetic tree was drawn ( Figure 5 ), which is closer to Bifidobacterium adolescentis in evolutionary relationship.

[0084] The cat-derived Bifidobacterium adolescentis derived from the above-mentioned long-lived cat is now preserved in the China Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China. The preservation date is August 5, 2024, the preservation number is: CCTCCNO: M20241746, the preservation name is Bifidobacterium adolescentis LifeAge20, and the recommended classification name is Bifidobacterium adolescentis (Bifidobacterium adolescentis).

[0085] (6) Test of cell culture stability

[0086] The original strain and the identified mutant strain QC-09 were inoculated into LB liquid medium at 2%, and cultured anaerobically at 39°C for 24 hours. This operation was repeated to culture the strain and pass 10 generations. Then, centrifuged at 4°C and 4000r / min for 10 minutes, washed with sterile saline, and the concentration of the bacterial solution was adjusted to 10 9 CFU / mL.

[0087] Evaluation of gastric digestion resistance and passage stability: 1 mL of bacterial solution was added to 9 mL of artificial gastric fluid, cultured at 39°C and 90 r / min for 2 h, and the number of viable bacteria at 0 and 2 h was determined by the plate count method.

[0088] Evaluation of intestinal digestion resistance and passage stability: 1 mL of bacterial solution was added to 9 mL of artificial intestinal fluid, cultured at 39°C and 90 r / min for 2 h, and the number of viable bacteria at 0 and 2 h was determined by the plate count method.

[0089] Evaluation of oxygen-resistant culture stability: 5 mL of bacterial solution was placed in an anaerobic (0.2% oxygen partial pressure) and microaerobic (0.8% oxygen partial pressure) environment for 2 h, and the number of viable bacteria at 0 and 2 h was determined by the plate count method.

[0090] The survival rate was calculated according to the following formula:

[0091]

[0092] Wherein, P is the survival rate, unit is %; n2 is the number of viable bacteria in the bacterial solution after 2 hours of treatment, unit is CFU; n0 is the number of viable bacteria in the bacterial solution at 0 hours of treatment, unit is CFU.

[0093] The results are shown in Table 3. The 10th generation QC-09 showed good stability in passage, with survival rates of 18.80% and 41.60% in gastric digestion and intestinal digestion environments, respectively. It also had a certain oxygen resistance, with a survival rate of 48.39% in a microaerobic environment (0.8% oxygen partial pressure).

[0094] Table 3 Results of the stability test of the passage of Bifidobacterium adolescentis from longevity cats

[0095] QC-Y(10th generation) QC-09(10th generation) Survival rate of gastric digestion resistance (%) 0.33 18.80 Survival rate of intestinal digestion resistance (%) 2.94 41.60 Survival rate in anaerobic environment (%) 81.24 93.26 Microaerobic environment survival rate (%) 5.73 48.39

[0096] (7) Evaluation of the effect of intestinal colonization in cats

[0097] 24 healthy cats were selected, weighing 3-5kg and aged 1-2 years old. The breeds included Ragdoll, British Shorthair, American Shorthair, Garfield, Chinese Rural Cat and Maine Coon, 4 of each kind, and evenly divided into blank group, human group, QC-Y group and QC-09 group, 6 cats in each group (one of each breed). The experiment lasted for 40 days, divided into adaptation period (0-7 days), colonization period (8-21 days) and observation period (22-40 days).

[0098] The bacterial solution was concentrated and then put into capsules to make bacterial preparation capsules (1×10 7 CFU / capsule), and cats were fed bacterial preparation capsules once a day and 1 capsule each time during the adaptation period and colonization period. The capsules fed to cats in the human, QC-Y and QC-09 groups contained human adolescent Bifidobacterium, QC-Y original bacteria and selected QC-09 mutagenic bacteria, respectively, and cats in the blank group were fed empty capsules without bacterial agents. Feeding bacterial preparation capsules was stopped after entering the observation period. During the experiment, cat feces were collected every 7 days, and the plate counts of Bifidobacterium adolescentis were performed and the number of colonized live bacteria was calculated.

[0099] The results are shown in Table 4. The colonization ability of QC-09 mutant bacteria is better than that of QC-Y original bacteria and human adolescent Bifidobacterium. On the 21st day of the experiment, the number of colonized live bacteria in the QC-09 group reached 5.93lg CFU / g, which was 0.77lg CUF / g higher than that of the QC-Y group and 3.65lg CFU / g higher than that of the human group. Seven days after stopping feeding Bifidobacterium adolescentis, that is, on the 28th day of the experiment, the number of colonized live bacteria in the human group and QC-Y group decreased rapidly, while the number of colonized live bacteria in the QC-09 group could still reach 4.37lg CFU / g, which was 1.74lgCUF / g higher than that of the QC-Y group and 3.02lg CFU / g higher than that of the human group. It can be seen that the colonization ability of QC-09 mutant bacteria in cats is stronger than that of QC-Y original bacteria and human adolescent Bifidobacterium.

[0100] Table 4 Changes in the number of viable colonized bacteria of Bifidobacterium adolescentis

[0101]

[0102] (8) Food change stress rate and aging index NAD + Content test

[0103] Cats were provided with 60g of complete cat food every day. After 30 days of feeding, cats without food change stress (no vomiting, diarrhea, soft stool, listlessness, loss of appetite or indigestion) were selected for grouping. The experiment was divided into two groups, the control group and the experimental group, with 50 cats in each group. The breeds included Dongshou, British Shorthair, American Shorthair, Ragdoll, Chinese Rural Cat, Maine Coon and Garfield, and each group had half male and half female.

[0104] The cats in the control group were fed a complete cat food 2, and the cats in the experimental group were fed a complete cat food 2 supplemented with Bifidobacterium adolescentis LifeAge20 (added at 1×10 8 CFU / kg), and each cat was fed 60g of cat food per day.

[0105] Seven days after the cats changed their diet, the proportion of cats in each group that had diet change stress was recorded. Diet change stress rate (%) = number of cats that still had stress symptoms seven days after the diet change × 100 / total number of cats in the group. Cats were considered to have diet change stress if they had symptoms such as vomiting, diarrhea, loose stools, listlessness, loss of appetite or indigestion.

[0106] After 21 days of changing cat food, follow the NAD + Kit instructions for detecting cat serum NAD + content.

[0107] As shown in Table 5, Bifidobacterium adolescentis LifeAge20 helps reduce the stress rate of cats changing their diet. Seven days after changing their diet, the stress rate of cats in the test group was only 8%, which was significantly reduced by 85.71% compared with the control group.

[0108] Table 5 Stress rate of cats after changing food

[0109]

[0110] As shown in Table 6, Bifidobacterium adolescentis LifeAge20 helps improve NAD, a key indicator of aging in cats. + Serum NAD of cats in the experimental group before changing diet + The content was 26.31nmol / L, which was not significantly different from the control group. + The content increased to 30.83nmol / L, a significant increase of 17.18% compared to before the diet change and a significant increase of 13.22% compared to the control group.

[0111] Table 6 NAD in cat serum + content

[0112] Control group Experimental Group Before changing diet (nmol / L) 26.52 26.31 Change food for 21 days (nmol / L) 27.23 30.83

[0113] Example 2

[0114] (1) Isolation of bacterial strains

[0115] Fresh golden feces of long-lived dogs (average age 18 years) were collected, resuspended in PBS buffer, and diluted to 10 by 10-fold gradient dilution method. -1 ~10 -6 Concentration, take 100 μL of each gradient and spread it on LB agar plate, and culture it in anaerobic environment at 38°C for 48 hours. Select the plate with uniform colony distribution and good growth (the number of colonies on a single plate is less than 200, and the bacteria are moist), and pick a single colony.

[0116] (2) Molecular identification

[0117] The selected single colonies were identified by PCR and Bifidobacterium adolescentis was obtained. The two fastest growing strains were selected and named QC-x1 and QC-x2 respectively.

[0118] (3) Functional identification

[0119] The SAMP8 strain of accelerated aging model mice were fed with Bifidobacterium adolescentis QC-x1 and QC-x2 to evaluate their effects on the lifespan of the accelerated aging model mice. Thirty SAMP8 strain accelerated aging model mice were randomly divided into QC-x1 group, QC-x2 group and control group with 10 mice in each group. The two types of Bifidobacterium adolescentis were mixed with the mouse diet and re-prepared into a composite mouse diet. The bacterial concentration of Bifidobacterium adolescentis in the composite mouse diet was 1×10 8 CFU / g. The QC-x1 group was fed with a compound mouse diet containing Bifidobacterium adolescentis QC-x1, the QC-x2 group was fed with a compound mouse diet containing the mutant strain of Bifidobacterium adolescentis QC-x2, and the control group was fed with a common mouse diet. Each group was fed 40 g per day and had free access to water. The time of death of the mice was recorded and the lifespan was counted.

[0120] The results are shown in Table 7. Both QC-x1 and QC-x2 of Bifidobacterium adolescentis significantly prolonged the lifespan of mice in the aging-accelerated model, and the effect of QC-x2 was better than that of QC-x1. The average lifespan of mice in the QC-x2 group was increased by 172 days compared with the control group, that is, the lifespan was extended by 42.68%, and the average lifespan of mice in the QC-x group was increased by 92 days, that is, the lifespan was extended by 18.31%. QC-x2 was renamed QC-x and used as the original bacteria for mutagenesis and screening.

[0121] Table 7 Effects of Bifidobacterium adolescentis from long-lived dogs on the lifespan of mice with accelerated aging model

[0122] Group Name Average life span (days) QC-x1 486 QC-x2 575 Control group 403

[0123] (4) Mutation and selection of strains

[0124] The original strain of Bifidobacterium adolescentis was irradiated with 150Gy of cobalt-60 radiation for 10 minutes.

[0125] The strain was inoculated into LB liquid medium at 1%, cultured at 38°C for 15 h under anaerobic conditions, and the bacterial concentration (OD) before and after culture was measured. 600 value), calculate the growth rate of the strain. The growth rate is calculated according to the following formula:

[0126] V=OD 15 -OD0

[0127] Where V is the growth rate; OD15 and OD0 is the OD of the bacterial solution at 15h and 0h of culture 600 value.

[0128] In the first round of screening, more than 300 strains were screened, from which 14 strains with larger single colony diameters were selected for the second round of screening. The three strains with the fastest growth rate were selected for re-screening of resistance to canine gastric and intestinal digestion and oxygen tolerance.

[0129] The QC-a, QC-c and QC-e strains grew fastest after 15 hours of culture, and their growth rates increased by 2921.43%, 2628.57% and 2535.71% respectively compared with the original strain QC-x. These three strains were selected for rescreening.

[0130] During the rescreening, the strains obtained from the initial screening were cultured in an intestinal digestion resistance screening medium for 7 consecutive days, with simulated intestinal digestion screening twice a day, and simulated gastric digestion screening was added separately on the first and fourth days. During this process, the strains were cultured in a microaerobic environment (0.8% oxygen partial pressure) to culture aerobic strains.

[0131] After enrichment and cultivation of the three strains, the bacterial liquid was taken at a 2% inoculum volume and placed in artificial gastric juice and artificial intestinal juice for 2 hours, and another bacterial liquid was placed in a microaerobic environment with an oxygen partial pressure of 0.8% for 2 hours. The number of viable bacteria was determined by the gradient dilution plate method at 0h and 2h, and the survival rate was calculated. The survival rate calculation formula is as follows:

[0132]

[0133] Wherein, P is the survival rate, unit is %; n2 is the number of viable bacteria in the bacterial solution after 2 hours of treatment, unit is CFU; n0 is the number of viable bacteria in the bacterial solution after 0 hours of treatment, unit is CFU.

[0134] The results are shown in Table 8. The three mutant strains showed significant improvements in gastric digestion resistance, intestinal digestion resistance and oxygen resistance compared with the original strains. Among them, QC-a had the highest survival rate in these three environments, reaching 44.47%, 35.39% and 53.52%, respectively. Therefore, QC-a was molecularly identified and used for subsequent tests.

[0135] Table 8 Gastric digestion resistance, intestinal digestion resistance and oxygen resistance of Bifidobacterium adolescentis from long-lived dogs

[0136] Survival rate of gastric digestion resistance (%) Survival rate of intestinal digestion resistance (%) Oxygen tolerance survival rate (%) QC-x 12.93 9.35 6.86 QC-a 44.47 35.39 53.52 QC-c 35.22 25.73 35.47 QC-e 24.81 20.28 22.62

[0137] (5) Strain identification

[0138] Using the genomic DNA of the mutant strain QC-a of Bifidobacterium adolescentis as a template, the strain was identified by PCR and confirmed to be Bifidobacterium adolescentis. The mutant strain QC-a was renamed Bifidobacterium adolescentis LifeAge18 and a phylogenetic tree was drawn ( Figure 6 ), which is closer to Bifidobacterium adolescentis in evolutionary relationship.

[0139] The canine-derived Bifidobacterium adolescentis derived from the above-mentioned long-lived dog is currently deposited in the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The preservation date is August 5, 2024, the preservation number is: CCTCCNO: M20241747, the preservation name is Bifidobacterium adolescentis LifeAge18, and the recommended classification name is Bifidobacterium adolescentis (Bifidobacterium adolescentis).

[0140] (6) Test of cell culture stability

[0141] The original strain and the identified mutant strain QC-a were inoculated into LB liquid medium at 2%, and cultured anaerobically at 38°C for 24 hours. This operation was repeated to culture the strain and pass 10 generations. Then, centrifuged at 4°C and 4000r / min for 10 minutes, washed with sterile saline, and the concentration of the bacterial solution was adjusted to 10 9 CFU / mL.

[0142] Evaluation of gastric digestion resistance and passage stability: 1 mL of bacterial solution was added to 9 mL of artificial gastric fluid, cultured at 38°C and 90 r / min for 2 h, and the number of viable bacteria at 0 and 2 h was determined by the plate count method.

[0143] Evaluation of intestinal digestion resistance and passage stability: 1 mL of bacterial solution was added to 9 mL of artificial intestinal fluid, cultured at 38°C and 90 r / min for 2 h, and the number of viable bacteria at 0 and 2 h was determined by the plate count method.

[0144] Evaluation of oxygen-resistant culture stability: 5 mL of bacterial solution was placed in an anaerobic (0.2% oxygen partial pressure) and microaerobic (0.8% oxygen partial pressure) environment for 2 h, and the number of viable bacteria at 0 and 2 h was determined by the plate count method.

[0145] The survival rate was calculated according to the following formula:

[0146]

[0147] Wherein, P is the survival rate, unit is %; n2 is the number of viable bacteria in the bacterial solution after 2 hours of treatment, unit is CFU; n0 is the number of viable bacteria in the bacterial solution at 0 hours of treatment, unit is CFU.

[0148] The results are shown in Table 9. The 10th generation QC-a showed good stability in passage, with survival rates of 33.87% and 34.19% in gastric digestion and intestinal digestion environments, respectively. It also had a certain oxygen resistance, with a survival rate of 46.75% in a microaerobic environment (0.8% oxygen partial pressure).

[0149] Table 9 Results of the stability test of the passage of Bifidobacterium adolescentis from longevity dogs

[0150] QC-x(10th generation) QC-a(10th generation) Survival rate of gastric digestion resistance (%) 0.84 33.87 Survival rate of intestinal digestion resistance (%) 3.26 34.19 Survival rate in anaerobic environment (%) 82.38 91.56 Microaerobic environment survival rate (%) 6.36 46.75

[0151] (7) Evaluation of the effect of canine intestinal colonization

[0152] 24 healthy dogs weighing 9-14kg and aged 1-2 years old were selected, including Beagle, Schnauzer, Corgi, Pomeranian, Bichon and Chihuahua, 4 of each kind, and evenly divided into blank group, human group, QC-x group and QC-a group, 6 in each group (one of each kind). The experiment lasted for 40 days, divided into adaptation period (0-7 days), colonization period (8-21 days) and observation period (22-40 days).

[0153] During the adaptation and colonization period, dogs were fed with bacterial preparation capsules (1×10 7 CFU / capsule), once a day, 1 capsule each time. The capsules fed to the dogs in the human, QC-x and QC-a groups contained human adolescent Bifidobacterium, QC-x original bacteria and selected QC-a mutagenic bacteria, respectively, and the dogs in the blank group were fed empty capsules without bacterial agents. After entering the observation period, the feeding of bacterial preparation capsules was stopped. During the experiment, the feces of the dogs were collected every 7 days, and the plate counts of Bifidobacterium adolescentis were performed and the number of colonized live bacteria was calculated.

[0154] The results are shown in Table 10. The colonization ability of QC-a mutant bacteria is better than that of QC-x original bacteria and human adolescent Bifidobacterium. On the 21st day of the experiment, the number of colonized live bacteria in the QC-a group reached 6.04lg CFU / g, which was 0.95lg CUF / g higher than that of the QC-x group and 3.42lg CFU / g higher than that of the human group. Seven days after stopping feeding Bifidobacterium adolescentis, that is, on the 28th day of the experiment, the number of colonized live bacteria in the human group and QC-x group decreased rapidly, while the number of colonized live bacteria in the QC-a group could still reach 4.43lg CFU / g, which was 1.62lgCUF / g higher than that of the QC-x group and 2.77lg / CFU / g higher than that of the human group. It can be seen that the colonization ability of QC-a mutant bacteria in dogs is stronger than that of QC-x original bacteria and human adolescent Bifidobacterium.

[0155] Table 10 Changes in the number of viable colonized bacteria of Bifidobacterium adolescentis

[0156]

[0157] (8) Food change stress rate and aging index NAD + Content test

[0158] The dogs were provided with 80g of complete dog food every day. After 30 days of feeding, the dogs without food change stress (no vomiting, diarrhea, soft stool, listlessness, loss of appetite or indigestion) were selected for grouping. The experiment was divided into two groups, the control group and the experimental group, with 50 dogs in each group, including beagles, schnauzers, corgis, Pomeranians, bichon frise and Chihuahuas, and half of each group was male and half was female.

[0159] The dogs in the control group were fed a complete dog food 2, and the dogs in the experimental group were fed a complete dog food 2 supplemented with Bifidobacterium adolescentis LifeAge18 (added at 1×10 8 CFU / kg), and each dog was fed 80g of dog food per day.

[0160] Seven days after the dogs changed their diet, the proportion of dogs in each group that had diet change stress was recorded. Diet change stress rate (%) = number of dogs that still had stress symptoms seven days after the diet change × 100 / total number of dogs in the group. Dogs were considered to have diet change stress if they had symptoms such as vomiting, diarrhea, loose stools, listlessness, loss of appetite or indigestion.

[0161] After the dog has changed its food for 21 days, follow the NAD + The steps in the kit instructions are to detect canine serum NAD + content.

[0162] As shown in Table 11, Bifidobacterium adolescentis LifeAge18 helps to reduce the stress rate of dogs changing their diet. Seven days after changing their diet, the stress rate of dogs in the test group was only 6%, which was significantly reduced by 83.33% compared with the control group.

[0163] Table 11 Stress rate of dogs after changing their diet

[0164] Blank Group Experimental Group Stress rate (%) 36 6

[0165] As shown in Table 12, Bifidobacterium adolescentis LifeAge18 helps improve NAD, a key indicator of canine aging. + Serum NAD of the experimental group dogs before changing diet + The content was 33.69nmol / L, which was not significantly different from the control group. + The content increased to 42.41nmol / L, a significant increase of 25.88% compared to before the food change and a significant increase of 24.92% compared to the control group.

[0166] Table 12 NAD in dog serum + content

[0167] Blank Group Experimental Group Before changing diet (nmol / L) 33.27 33.69 Change food for 21 days (nmol / L) 33.95 42.41

Claims

1. A strain of Bifidobacterium adolescentis LifeAge20 derived from a long-lived cat, characterized in that: It is currently deposited in the China Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China. The preservation date is August 5, 2024, the preservation number is: CCTCC NO: M20241746, the preservation name is Bifidobacterium adolescentis LifeAge20, and the recommended classification name is Bifidobacterium adolescentis.

2. A strain of Bifidobacterium adolescentis LifeAge18 from a long-lived dog, characterized in that It is currently deposited in the China Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China. The preservation date is August 5, 2024, the preservation number is: CCTCC NO: M20241747, the preservation name is Bifidobacterium adolescentis LifeAge18, and the recommended classification name is Bifidobacterium adolescentis.

3. The use of Bifidobacterium adolescentis LifeAge20 derived from long-lived cats according to claim 1 in cat food or feed, characterized in that: It can reduce the stress rate of food change and improve aging indicators (increase serum NAD + content) effect.

4. The use of Bifidobacterium adolescentis LifeAge18 derived from longevity dogs according to claim 2 in dog food or feed, characterized in that: It can reduce the stress rate of food change and improve aging indicators (increase serum NAD + content) effect.

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