CN116574650A - Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof - Google Patents

Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof Download PDF

Info

Publication number
CN116574650A
CN116574650A CN202310545734.7A CN202310545734A CN116574650A CN 116574650 A CN116574650 A CN 116574650A CN 202310545734 A CN202310545734 A CN 202310545734A CN 116574650 A CN116574650 A CN 116574650A
Authority
CN
China
Prior art keywords
aging
enterococcus faecalis
zjuids
capacity
mice
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310545734.7A
Other languages
Chinese (zh)
Inventor
任大喜
陈楠
师福山
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN202310545734.7A priority Critical patent/CN116574650A/en
Publication of CN116574650A publication Critical patent/CN116574650A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/22—Animal feeding-stuffs from material of animal origin from fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/35—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from potatoes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Abstract

The invention discloses a canine enterococcus faecalis ZJUIDS-D016 with anti-aging effect for pets and application thereof, wherein the enterococcus faecalis ZJUIDS-D016 is classified and named (Enterococcus faecalis) and the preservation number is CGMCC 27038. The invention is separated from the intestinal tracts of healthy dogs, probiotics derived from the intestinal tracts of the host can be better planted in the intestinal tracts of the host, and the canine enterococcus faecalis ZJUIDS-D016 provided by the invention has good in-vitro antioxidant capacity and good anti-aging effect on D-galactose aging mice through DPPH clearance rate, hydroxyl free radical clearance capacity, reducing capacity and intervention experiments on D-galactose aging mice. In addition, the invention has good acid tolerance and bile salt tolerance and is sensitive to most antibiotics, and the invention has good probiotic characteristics. Therefore, the invention can be used for preparing the anti-aging probiotic product for the pet dogs.

Description

Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a canine enterococcus faecalis with an anti-aging effect on pets and application thereof. Is a canine enterococcus faecalis (Enterococcus faecalis) ZJUIDS-D016 separated from canine intestinal tract and application thereof.
Technical Field
In recent years, the number of pets has been increasing, and the pets have played an increasingly important role in the lives of people. According to the analysis of the united states market with the largest global pet consumption by packaged products, the proportion of dogs keeping a senior dog (aged 7 years and older) in their own dogs keeping families was increased from 41.6% to 53.5% and the proportion of senior dogs was increased from 2012 to 2022. With the development of pet medicine, the life of the pet dogs will be longer, and the pet aging-related industry will be one of the development trends of the pet industry.
Aging is a process that must be experienced during life and is manifested by a gradual decline in the functions of cells, tissues and organs of the body over time, as well as a decline in cognitive and memory functions. With aging, the incidence of chronic age-related diseases such as obstetric, hepatorenal, cataract, heart disease, etc. is increasing.
Oxidative aging and inflammatory aging are hot spots in anti-aging research. Oxidative aging is mainly cellular aging caused by oxidative stress: the biological oxidative metabolic process generates reactive oxygen species ROS and can be scavenged by antioxidants in the body. In organisms, ROS production and clearance are dynamically balanced. However, when ROS increase in the body, this balance is broken and ROS accumulate, resulting in oxidative stress. If oxidative stress persists, it can cause DNA damage, protein misfolding, mitochondrial mutations, and cellular damage, ultimately leading to cellular senescence; inflammatory aging is also known as immune aging, and refers to the chronic and progressive increase in inflammatory states of the body during natural aging. The main reason is the imbalance between pro-and anti-inflammatory cytokines in vivo, ultimately leading to an increase in the pro-inflammatory response.
There are studies showing 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 senior 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.
Disclosure of Invention
The invention aims to enrich the existing strain resources, make up for the vacancies of anti-aging probiotics and the vacations of canine probiotics strains, provide a canine enterococcus faecalis ZJUIDS-D016 with anti-aging effect for pets, and is a canine enterococcus faecalis ZJUIDS-D016 with anti-aging effect and excellent probiotics characteristics, screened from canine intestinal tracts, classified and named enterococcus faecalis (Enterococcus faecalis) ZJUIDS-D016, and deposited in China general microbiological culture Collection center for 4 months and 6 days in 2023, with the deposit number of: the preservation address of the CGMCC 27038 is 1 # 3 of North Chen West Lu in the Chaoyang area of Beijing city.
The canine enterococcus faecalis ZJUIDS-D016 sieve is selected from canine intestinal tract, and is identified as enterococcus faecalis (Enterococcus faecalis) after separation, purification and molecular biological identification.
The enterococcus faecalis ZJUIDS-D016 has the following characteristics:
(1) The bacterial colony and the bacterial body are characterized in that: culturing on MRS culture medium for 48 hr, wherein the colony surface has smooth edge, regular round shape and milky white color;
(2) Antioxidant capacity in vitro: has good in vitro antioxidation capability, the clearance rate of the fermentation supernatant DPPH is 86.21%, the clearance rate of the bacterial suspension DPPH is 24.96%, and the clearance rate of the sterile crushed DPPH is 14.79%; the clearance of hydroxyl radicals in fermentation supernatant is 1.94%, the clearance of hydroxyl radicals in bacterial suspension is 11.61%, and the clearance of hydroxyl radicals in aseptic crushed products is 0.7%; the reduction capacity of the bacterial suspension is 109.3 percent, and the reduction capacity of the aseptic crushed material is 24.21 percent;
(3) Acid resistance: culturing in PBS with pH of 2 at 37deg.C for 4 hr, with survival rate of 97%;
(4) Bile salt resistance: culturing in PBS with bile salt concentration of 0.2% at 37deg.C for 4 hr, with survival rate of 86%;
(5) Antibiotic sensitivity: are sensitive to 3 antibiotics, respectively: chloramphenicol, erythromycin, penicillin; the three antibiotics are mediated, respectively: tetracyclines, ceftriaxone and ampicillin; resistance to lincomycin;
(6) Anti-aging properties in vivo: after the invention is used for intervening the D-galactose-induced aging mice, the oxidation level and the inflammation level of the organism of the mice are obviously improved, which proves that the invention has an effect of relieving the oxidation aging and the inflammation aging of the aging mice, and has a certain anti-aging effect.
The invention also aims to provide the application of the enterococcus faecalis ZJUIDS-D016 in preparing a canine anti-aging probiotic product. The product comprises fungus powder, microbial inoculum, milk powder, fermented yoghurt, dog food and the like. The strain has good in-vivo anti-aging capability of pets, has an anti-oxidation effect on oxidation aging and inflammation aging of D-galactose aging mice, and has good in-vitro anti-oxidation capability, namely DPPH scavenging capability, hydroxyl free radical scavenging capability and reducing capability. The strain has good acid resistance and bile salt resistance, which indicates that the strain can adapt to gastrointestinal environment and has proliferation capability; the strain is sensitive to a variety of antibiotics.
The invention has the advantages and positive effects that: (1) The enterococcus faecalis ZJUIDS-D016 provided by the invention is derived from the intestinal tract of dogs, has stronger host adaptability, and fills up the gap of the canine probiotics; (2) The invention has anti-aging effect, and makes up for the lack of the effect of the canine probiotics product; and (3) provides a foundation for developing anti-aging probiotic products. Based on probiotics separated from canine intestinal tracts, enterococcus faecalis with anti-aging effect is screened out. The strain also has good performance in acid resistance and bile salt resistance, is sensitive to various antibiotics, and has antibacterial activity. The enterococcus faecalis can provide a basis for developing anti-aging probiotic products.
Drawings
FIG. 1 is a colony morphology of enterococcus faecalis ZJUIDS-D016.
FIG. 2 is an electrophoresis chart of enterococcus faecalis ZJUIDS-D016, wherein the hole 1 is ZJUIDS-D016, the hole M is Marker, and the hole M is 5000 and 3000/2000/1000/750/500/250/100bp from top to bottom.
FIG. 3 is a graph showing the carbonyl content of protein in serum of each group of mice.
FIG. 4 is a graph showing GSH content in spleens of mice in each group.
FIG. 5 is a graph showing SOD levels in spleens of mice in each group.
FIG. 6 is a graph showing MDA content in spleens of mice in each group.
FIG. 7 is a graph showing the IgG content in the liver of each group of mice.
FIG. 8 is a graph showing IL-1β content in livers of mice in each group.
FIG. 9 is a graph showing IL-6 content in livers of mice in each group.
FIG. 10 is a graph showing TNF- α levels in the livers of mice in each group.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings and examples, in which it is evident that only some, but not all embodiments of the present invention are shown. All other embodiments, based on the examples of the invention, which are within the scope of the invention as claimed by a person of ordinary skill in the art without any inventive effort.
The materials used in the invention are conventional commercial products unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
EXAMPLE 1 isolation, purification and characterization of enterococcus faecalis ZJUIDS-D016
1.1 sample Source
A fecal sample from a healthy caucasian female of 4 years old. The sample is collected by adopting an anal swab mode, namely, 1mL of physiological saline is filled in a 1.5mL EP tube in advance, the anal swab of the dog is collected by using a wet cotton swab, and finally, the cotton swab head is inserted into the EP tube for soaking, and the wooden part is sheared off.
1.2 isolation and purification of Strain
100. Mu.L of the turbid liquid was spread on MRS solid medium and cultured at 37℃for 48 hours. After the culture is finished, picking a typical colony, carrying out streak separation on an MRS solid culture medium for a plurality of times until the colony morphology on the whole plate is consistent, and picking a single colony to the MRS liquid culture medium for expansion culture. The obtained bacterial liquid is preserved in 40% (w/v) glycerol at-80 ℃.
1.3 identification of enterococcus faecalis ZJUIDS-D016
1.3.1 colony characterization
After enterococcus faecalis ZJUIDS-D016 is cultured in MRS culture medium for 48 hr, the surface edges are smooth, and the shape is round and milky white, as shown in figure 1.
1.3.2 16S rDNA identification
Extracting target strain genome DNA by using Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out PCR experiment of 16S rDNA by using bacterial universal primers 27F and 1492R, taking PCR products after PCR amplification, carrying out agarose gel detection and photographing, wherein the amplified fragment length is about 1500bp, as shown in figure 2. The PCR products were sent to the biological engineering (Shanghai) Inc. for sequencing and BLAST sequence alignment on the NCBI website, which showed over 99% sequence homology to the identified 16S rDNA sequence of enterococcus faecalis. Amplification primers and sequencing primers are shown in Table 1 below.
TABLE 1 enterococcus faecalis ZJUIDS-D016 amplification primers and sequencing primers
The nucleotide sequence of enterococcus faecalis ZJUIDS-D016 is shown in SEQ ID NO. 1.
EXAMPLE 2 anti-aging effect of enterococcus faecalis ZJUIDS-D016 on D-galactose-senescent mice
2.1 establishment of D-galactose aging mouse model
24 mice were divided into 3 groups according to the random number table method: normal control group, D-galactose aging model group, enterococcus faecalis ZJUIDS-D016 strain acting group, 8 mice each. The treatment is shown in Table 2 below.
Table 2 animal experiment grouping and treatment
2.2 sample and measurement index
The invention aims to demonstrate that the invention has anti-aging effect by relieving oxidative aging and inflammatory aging of mice. Therefore, after the animal test is finished, the oxidation level and the inflammation level of the mouse body are mainly detected.
After the animal experiment is finished, collecting blood, liver and spleen of each group of mice, and respectively measuring the contents of IgG, IL-1 beta, IL-6 and TNF-alpha in the liver of each group of mice to evaluate the inflammation level of the organism of the mice; protein carbonyl in serum, GSH, SOD, MDA content in spleen, was used to evaluate the oxidation level of the mouse organism.
2.2.1ZJUIDS-D016 effect on alleviating oxidative aging of D-galactose-induced aging mice
(1) Protein carbonyl content in serum
Protein carbonyl is an early sign of various amino acids in the oxidative modification process of protein, and the content of the protein carbonyl indicates the degree of oxidative damage of the protein, and is a main index for measuring the oxidative damage of the protein. The experimental results are shown in figure 3, and can be seen that the protein carbonyl content in the serum of the mice can be remarkably reduced (p < 0.01) even lower than that of the normal control group after the enterococcus faecalis ZJUIDS-D016 intervenes in the D-galactose-induced aging mice.
(2) GSH, SOD, MDA content in spleen
GSH (glutathione) is an important antioxidant in the body, and can scavenge free radicals in the body; because GSH itself is easily oxidized by some substances, it can protect sulfhydryl groups in molecules such as many proteins and enzymes from being oxidized by harmful substances in vivo, thereby ensuring normal exertion of physiological functions of the proteins and enzymes. The GSH experimental result is shown in figure 4, and can be seen that after the enterococcus faecalis ZJUIDS-D016 intervenes in the D-galactose aging mice, the GSH content in the spleens of the mice can be improved and the mice are continuously close to normal mice.
SOD (superoxide dismutase) has the main function of catalyzing the disproportionation of superoxide anion radicals into hydrogen peroxide and oxygen, and the generated superoxide anion radicals are normal metabolites in organisms. However, accumulation of free radicals will cause peroxidation of the lipids of the cell membrane to cause membrane fission, resulting in cell damage and even death. SOD is the most important and optimal free radical scavenger in organisms, maintaining the metabolic balance of the body. SOD has the effect of eliminating and reducing excessive free radicals in human bodies to delay aging, and is being studied as an anti-aging drug. The SOD experimental result is shown in figure 5, and can be seen that after the enterococcus faecalis ZJUIDS-D016 intervenes in the D-galactose aging mice, the SOD content in the spleens of the mice can be improved, even higher than that of normal group mice.
In vivo, the free radical acts on lipid to generate peroxidation, and the oxidation end product is MDA (malondialdehyde) which can cause cross-linking polymerization of living macromolecules such as protein, nucleic acid and the like, and has cytotoxicity. The MDA experimental results are shown in figure 6, and can be seen that after enterococcus faecalis ZJUIDS-D016 intervenes in a D-galactose aging mouse, the MDA content in the spleen of the mouse can be remarkably reduced (p < 0.05).
In conclusion, the enterococcus faecalis ZJUIDS-D016 can improve the content of antioxidant enzyme in the body of the aging mouse, reduce oxidative damage of the body, and has a certain resistance to oxidative aging of the aging mouse.
2.2.2ZJUIDS-D016 effect on alleviating oxidative aging of D-galactose-induced aging mice
(1) IgG content in liver
IgG is the main immunoglobulin of the body, and its content can reflect the immunity of the body. The higher the content, the stronger the immunity. The IgG experimental results are shown in figure 7, and can be seen that the IgG content in the liver of the mice can be remarkably improved (p < 0.05) after the enterococcus faecalis ZJUIDS-D016 intervenes in the D-galactose aging mice.
(2) IL-1 beta, IL-6, TNF-alpha content in liver
TNF-alpha, IL-1 beta and IL-6 are all pro-inflammatory cytokines, the levels of which are related to the inflammatory levels of the body. The experimental results are shown in figures 8, 9 and 10, and can be seen that the content of the three pro-inflammatory factors in the liver of the mice can be remarkably reduced (p < 0.001) after the enterococcus faecalis ZJUIDS-D016 intervenes in the D-galactose aging mice.
In conclusion, enterococcus faecalis ZJUIDS-D016 can be seen to improve the IgG content in the body of the aging mouse and improve the immunity of the body; reducing the content of pro-inflammatory cytokines TNF-alpha, IL-1 beta and IL-6 in the organism, and has certain resistance to inflammatory aging of aging mice.
Example 3 in vitro antioxidant Capacity assay of enterococcus faecalis ZJUIDS-D016
DPPH clearance, hydroxyl radical clearance and reducing ability of enterococcus faecalis ZJUIDS-D016 isolated from canine intestinal tract were measured.
3.1 sample preparation
Strains stored in glycerol tubes were streaked on MRS solid medium and incubated at 37℃for 48h. And (3) picking single colonies by using an inoculating loop, inoculating the single colonies into a sterilized MRS liquid culture medium, and standing and culturing for 18-24 hours at 37 ℃ to obtain a culture solution. The culture solution was adjusted to a lactic acid bacteria concentration of 10 with distilled water 9 CFU/mL,4 ℃, 8000 Xg centrifugal 20min, collecting the supernatant, namely fermentation supernatant. The centrifuged cell pellet was resuspended and washed with 0.02M PBS buffer (ph=7.4), and centrifuged at 8000×g for 20min at 4 ℃ for 3 replicates. Resuspending the washed cells in PBS buffer and adjusting the cell concentration to 10 9 CFU/mL to obtain the bacterial suspension. And (3) performing ultrasonic crushing on the thallus suspension, and centrifuging to obtain a supernatant after crushing to obtain a sterile thallus crushed material.
3.2DPPH clearance
1mL of the sample solution and 1mL of a 0.2mM DPPH absolute ethanol solution are mixed in an oscillating way for 1min, then the mixture is reacted for 30min at room temperature in a dark place, the supernatant is centrifugally taken to measure absorbance at 517nm, 95% ethanol is used for replacing DPPH as a control, and PBS is used for replacing the sample solution as a blank.
DPPH clearance = 1- (a) i -A j )/A c
Wherein A is i : sample solution+DPPH absorbance, A j : sample solution +95% ethanol absorbance, A c : PBS+DPPH absorbance.
3.3 hydroxyl radical scavenger
Taking 1mL of o-phenanthroline (2.5 mmol/L), sequentially adding 1mL of phosphate buffer solution (PBS concentration is 0.02mol/L, pH=7.4), distilled water 1mL, fully mixing, adding 1mL of ferrous sulfate (FeSO 4 concentration is 2.5 mmol/L), mixing, and adding hydrogen peroxide (H 2 O 2 20 mmol/L) 1mL, absorbance at 536nm after 1.5h in a 37℃water bath was determined to be A p The method comprises the steps of carrying out a first treatment on the surface of the 1mL distilled water was used as A instead of 1mL hydrogen peroxide b The method comprises the steps of carrying out a first treatment on the surface of the 1mL of distilled water was replaced with 1mL of sample A s 。
Hydroxyl radical scavenging = (a s -A p )/(A b -A p )。
3.4 reducing ability
Sample solution 0.5mL, potassium ferricyanide (mass fraction 1%) 0.5mL and phosphate buffer (PBS concentration 0.2mol/L, pH=6.6) 0.5mL were mixed in a water bath at 50℃for 20min, cooled after the reaction and 0.5mL trichloroacetic acid (TAC mass fraction 10%) was added to precipitate protein, and after centrifugation 1mL supernatant was taken with 1mL ferric trichloride (FeCl) 3 Mass fraction 0.1%) and absorbance at 700 nm. A blank was prepared by replacing the sample solution with PBS or MRS broth.
Reduction ability= (a s -A b )/A b
Wherein A is s : absorbance of experimental group, A b : blank absorbance.
TABLE 3 in vitro Oxidation resistance of enterococcus faecalis ZJUIDS-D016
Index (I) Fermentation supernatant Thallus suspension Sterile crushed material
DPPH clearance/% 86.21±2.51 24.96±7.66 14.79±4
Hydroxyl radical scavenging/% 1.94±3.52 11.61±1.03 0.7±5
Reducing power/% - 109.3±30.23 24.21±1.22
Example 4 determination of acid and bile salt resistance of enterococcus faecalis ZJUIDS-D016
4.1 acid resistance test
Selecting enterococcus faecalis ZJUIDS-D016 single colony, performing amplification culture in MRS liquid culture medium for 18 hr, inoculating the amplified bacterial suspension into MRS broth culture medium at 1% (v/v), and culturing at 37deg.C for 18 hr until the bacterial concentration reaches 10 9 CFU/mL. Inoculated in 10% of PBS at pH 2.0 and incubated at 37℃for 4h. The living bacteria in the 0h and 4h samples are counted by adopting a plate counting method by taking PBS without pH adjustment as a control, and the survival rate is calculated according to the following calculation formula:
in the above, N 0 Viable count (CFU/mL) for 0h of test strain; n (N) t The strain was tested for viable count (CFU/mL) for 4h.
4.2 bile salt resistance test
Selecting enterococcus faecalis ZJUIDS-D016 single colony, performing amplification culture in MRS liquid culture medium for 18 hr, inoculating the amplified bacterial suspension into MRS broth culture medium at 1% (v/v), and culturing at 37deg.C for 18 hr until the bacterial concentration reaches 10 9 CFU/mL. Inoculated in 10% amount into PBS containing 0.2% (m/v) ox gall salt, and cultured at 37℃for 4 hours. The living bacteria in the 0h and 4h samples are counted by adopting a plate counting method by taking PBS without ox gall salt as a control, and the survival rate is calculated according to the following calculation formula:
in the above, N 0 Viable count (CFU/mL) for 0h of test strain; n (N) t The strain was tested for viable count (CFU/mL) for 4h.
TABLE 4 acid and bile salt resistance of enterococcus faecalis ZJUIDS-D016
Index (I) Survival rate at 4h
Acid tolerance ph=2 0.97±0.01
Bile salt tolerance 0.2% 0.86±0.1
EXAMPLE 5 antibiotic susceptibility test
Inoculating single colony of enterococcus faecalis ZJUIDS-D016 with good growth on MRS plate into 20mL aseptic MRS liquid culture medium, culturing at 37deg.C for 24 hr, and regulating bacterial concentration to 10 9 About CFU/mL, 100 mu L of the culture solution of the bacteria to be detected is evenly coated on an MRS plate, and a drug sensitive paper sheet is taken by forceps and placed on the culture medium, and is slightly pressed until the bacteria is fixed. The dishes were incubated in a 37℃incubator for 24 hours with the front side facing upwards, and the diameter of the drug sensitive loop of each strain for each antibiotic was measured with a vernier caliper and the data recorded.
TABLE 5 enterococcus faecalis ZJUIDS-D016 antibiotic susceptibility results
Medicine name Medicine content/tablet Diameter of drug sensitive ring Sensitive type
Chloramphenicol 30μg 23.43±1.09 S
Lincomycin 2μg 9.75±0.25 R
Erythromycin 15μg 23.9±0.24 S
Tetracycline 30μg 15.37±0.14 I
Ceftriaxone 30μg 15.34±1.19 I
Ampicillin (Amoxicillin) 10μg 19.14±0.96 I
Penicillin 10U 27.19±0.72 S
Note that: s-sensitivity; i-intermediation; r-resistance
EXAMPLE 6 preparation of enterococcus faecalis ZJUIDS-D016 anti-aging probiotic powder
6.1 preparation of enterococcus faecalis ZJUIDS-D016 bacterial mud
The enterococcus faecalis ZJUIDS-D016 single colony is picked and inoculated into 50mL MRS liquid culture medium, and placed in a 37 ℃ incubator for culture for 18 hours. Activated again in 250mL MRS liquid culture medium according to the inoculum size of 5%, and placed in a 37 ℃ incubator for 24 hours. Finally, the activated enterococcus faecalis ZJUIDS-D016 is subjected to high-density anaerobic culture in a 10L fermentation tank at an inoculum size of 5%, and is cultured for 18 hours at 37 ℃ and pH of 6.8. Then centrifuging at 8000r/min and 4deg.C for 15min, discarding supernatant, collecting bacterial precipitate, and rinsing bacterial cells with sterile PBS (pH 7.0) for 2 times. Thus obtaining the enterococcus faecalis ZJUIDS-D016 bacterial mud.
6.2 preparation of protectant
The lyoprotectant comprises 15% of skim milk powder, 5% of trehalose, 3% of sodium glutamate, 1% of glycerol and 0.5% of cysteine hydrochloride. Water was used as solvent. Sterilizing at 110deg.C.
6.3 preparation of enterococcus faecalis ZJUIDS-D016 anti-aging powder
The enterococcus faecalis ZJUIDS-D016 bacterial mud prepared by the method is fully and uniformly mixed with the protective agent solution according to the proportion of 1:5. Pre-freezing for 5 hours at the temperature of minus 40 ℃ to uniformly freeze the enterococcus faecalis ZJUIDS-D016 bacterial powder on the inner wall of a container, and then performing vacuum freeze drying for 18-20 hours. After rehydration with physiological saline, the mixture is washed twice, and the viable count in the enterococcus faecalis ZJUIDS-D016 bacterial powder is measured to be 1.0x10 11 ～1×10 12 CFU/g。
Example 7 preparation of anti-aging probiotic milk powder for pet dogs Using enterococcus faecalis ZJUIDS-D016
7.1 preparation of enterococcus faecalis ZJUIDS-D016 bacterial powder
Referring to the above cases, the enterococcus faecalis ZJUIDS-D016 bacterial powder freeze-dried bacterial powder is prepared, wherein the viable count of the bacterial powder is 1.0X10 11 ～1×10 12 CFU/g
7.2 preparation of Pet formula powder
Primary selection of raw materials: milk powder, fish meal, bone meal, cereal and vegetable oil, additives: vitamins, trace elements, functional factors and others;
automatic batching: throwing the obtained material raw materials into a material bin according to a formula;
crushing: crushing the weighed materials by a crusher;
mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniform mixing;
puffing: making the mixed materials into granular materials through a bulking machine;
and (3) drying: drying the mixed materials by a dryer, wherein the temperature is controlled to be 65-70 ℃;
and (3) classifying and screening: the stream was passed through a classifying screen with the particles controlled at 2.5-5 mm.
7.3 preparation of probiotic milk powder for pets
Bacteria prepared in 7.1Powder and 7.2 pet formula powder prepared according to 1:100, and the viable bacteria leaving the factory in the final product is 10 8 CFU/g or more. After the product is filled, the product is put in storage for sale.
Example 8 preparation of anti-aging Probiotics can for pet dogs Using enterococcus faecalis ZJUIDS-D016
The main formula is as follows: 60-80% of beef, 0.5-1.2% of carrageenan, 0.05-0.3% of D-sodium erythorbate, 15-30% of water, and ZJUIDS-D016 bacterial powder (1.0X10) 11 ～1×10 12 CFU/g)。
A preparation method of canned food for dogs: the method comprises the specific steps of uniformly mixing and stirring minced beef, carrageenan, water and D-sodium erythorbate, then carrying out vacuum steam stripping, sterilizing at 70-90 ℃, carrying out mixed adsorption on the sterilized product and bacterial powder, and finally canning for use. ZJUIDS-D016 effective bacteria guarantee value 10 in product 9 CFU/g。
Example 9 preparation of anti-aging probiotic fermented yogurt for pet dogs Using enterococcus faecalis ZJUIDS-D016
9.1 processing technique flow of yoghourt:
raw material preheating, homogenizing, blending, sterilizing, cooling, preparing, inoculating, fermenting, after-ripening, refrigerating
9.2 operating points
(1) Raw materials: 2L of fresh goat milk;
(2) Preheating: placing the mixture into a container and heating to 63 ℃;
(3) Homogenizing: homogenizing in a homogenizer under 15-25 MPa), pouring the mixed solution into an iron tank, adding 100g white sugar, and sterilizing in water bath at 90deg.C for 10min;
(4) And (3) blending: adding ingredients into goat milk, and dissolving;
(5) Sterilizing: sterilizing the added sugar goat milk in water bath at 90 ℃ for 10min;
(6) And (3) cooling: cooling sterilized goat milk to 40-50deg.C for use;
(7) Preparing a starter: enterococcus faecalis ZJUIDS-D016 strain is inoculated in a test tube containing sterilized skimmed milk (12% w/v) under aseptic condition, and cultured at 37deg.C for 20 hr. The inoculation amount for each passage is 2-4% (v/v), the passage is carried out for 2-3 times to restore activity, and the culture medium is placed in a refrigerator at 4 ℃ for preservation;
(8) Inoculating and fermenting: under aseptic condition, the activated enterococcus faecalis ZJUIDS-D016 is inoculated with an inoculum size of 2-4% (v/v). Fermenting at 42 deg.C for 6-10 hr;
(9) Post-ripening: after fermentation, placing the mixture in a refrigerator at the temperature of 4 ℃ for after-ripening for 12-24 hours;
(10) Filling and refrigerating: after finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.
Example 10 preparation of anti-aging feed for pet dogs Using enterococcus faecalis ZJUIDS-D016
10.1 production flow of pet feed
Raw material preparation, material mixing, conveying, extrusion puffing, conveying, drying, oil spraying, seasoning and screening (packaging)
10.2 device configuration
Powder mixer/pulverizer-feeder-bulking machine-air feeder/lifter-multi-layer oven-cooler-oil sprayer-roller-lifter-vibrating screen-packaging machine
10.3 ingredients
26% of iced chicken meat, 20% of dehydrated chicken meat powder, 18% of dehydrated fish meal, 9% of sweet potato powder, 6% of chicken fat, 5% of potato powder, 4% of peas, 3% of iced salmon sides, 3% of egg yolk powder, 1.2% of freeze-dried egg yolk, 1.2% of freeze-dried beef liver, 1% of freeze-dried chicken meat, iced chicken liver and enterococcus faecalis ZJIUIDS-D016 bacterial powder in example 6.
ZJUIDS-D016 effective bacteria guarantee value 10 in product 9 CFU/g。
The present invention is not limited to the above-mentioned embodiments, and any person skilled in the art, based on the technical solution of the present invention and the inventive concept thereof, can be replaced or changed within the scope of the present invention.

Claims (4)

1. The canine enterococcus faecalis ZJUIDS-D016 with the anti-aging effect for pets is characterized in that the classification of the strain is named enterococcus faecalis (Enterococcus faecalis) ZJUIDS-D016, and the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 months and 6 days in 2023, wherein the preservation number is: CGMCC 27038.
2. Use of enterococcus faecalis ZJUIDS-D016 according to claim 1 for the preparation of a canine anti-aging probiotic product.
3. The product according to claim 2, comprising a bacterial powder, a bacterial agent, a milk powder, a fermented yoghurt, a canine food.
4. The use according to claim 2, wherein the strain has resistance to oxidative aging and inflammatory aging in D-galactose-induced aging mice, and has excellent in vitro antioxidant capacity-DPPH scavenging capacity, hydroxyl radical scavenging capacity and reducing capacity, acid resistance and bile salt resistance, adaptability to gastrointestinal environment, proliferation capacity, sensitivity to various antibiotics, and thus excellent in vivo anti-aging capacity in pets.
CN202310545734.7A 2023-05-16 2023-05-16 Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof Pending CN116574650A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310545734.7A CN116574650A (en) 2023-05-16 2023-05-16 Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310545734.7A CN116574650A (en) 2023-05-16 2023-05-16 Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof

Publications (1)

Publication Number Publication Date
CN116574650A true CN116574650A (en) 2023-08-11

Family

ID=87540801

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310545734.7A Pending CN116574650A (en) 2023-05-16 2023-05-16 Dog enterococcus faecalis ZJUIDS-D016 with anti-aging effect on pets and application thereof

Country Status (1)

Country Link
CN (1) CN116574650A (en)

Similar Documents

Publication Publication Date Title
CN111235070B (en) Breast milk infant source lactobacillus plantarum BF _15 and application thereof
Sun et al. Effects of Bacillus subtilis natto on performance and immune function of preweaning calves
CA2304977C (en) Equine feed product containing lactobacillus
CN110835616B (en) Active substance of lactobacillus paracasei GKS6, composition containing same and application of active substance in promoting longevity
CN110144304B (en) Lactobacillus casei strain and application thereof
US20110269165A1 (en) Lactic acid bacteria and their cellular components inducing immunoregulatory function, and method of obtaining the same
CN108102959B (en) Humanized lactobacillus plantarum ZY08 for reducing cholesterol and application thereof
EA006428B1 (en) Strains of lactic acid bacteria and use thereof
CN113088463B (en) Lactobacillus acidophilus with probiotic characteristics and application thereof
JP2009269906A (en) Lactobacillus isolated strain having anti-inflammatory activity and use thereof
BRPI0417793B1 (en) composition comprising canine probiotic bifidobacterium globosum
CN113462613B (en) Lactobacillus plantarum ZJUIDS04 capable of reducing blood sugar and application thereof
CN112877241B (en) Human lactobacillus fermentum ZJUIDS06 and application thereof
CN108384735A (en) Lactobacillus plantarum CCFM1019, its fermented food and its application in medicine preparation
CN110833565B (en) Active substance of lactobacillus plantarum GKM3, composition containing same and application of active substance in promoting longevity
JP6952879B2 (en) New Bacillus amyloliquefaciens strain and method for producing fermented soybean using it
CN106071103B (en) A kind of method of lactobacillus plantarum fermented bean dregs
US20230218688A1 (en) Lactobacillus helveticus zjuids12 for treating alcoholic liver disease and application thereof
CN112715660A (en) Bifidobacterium longum dietary composition for intervening chronic nephritis and uremia and application thereof
CN116064286A (en) Lactobacillus helveticus ZJUIDS11 for improving nonalcoholic liver disease and application thereof
CN110835615B (en) Active substance of bifidobacterium lactis GKK2, composition containing same and application of active substance and composition in promoting longevity
García et al. Effect of fermented whey with a probiotic bacterium on gut immune system
CN108018248B (en) Lactobacillus casei capable of regulating flora structural disorder caused by antibiotics
CN116064285B (en) Lactobacillus rhamnosus ZJUIDS07 capable of reducing blood sugar and application thereof
CN114908009B (en) Lactobacillus mucilaginosus PR63 and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination