CN117603865B

CN117603865B - Lactobacillus acidophilus and related preparation and application thereof

Info

Publication number: CN117603865B
Application number: CN202311575094.0A
Authority: CN
Inventors: 李光玉; 赵梦迪; 张媛媛; 李悦垚; 刘可园
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2023-11-23
Filing date: 2023-11-23
Publication date: 2024-09-17
Anticipated expiration: 2043-11-23
Also published as: CN117603865A

Abstract

The invention discloses lactobacillus acidophilus and a related preparation and application thereof, and relates to the field of microorganisms. The invention provides lactobacillus acidophilus GLA09 with a preservation number of CCTCC NO: M2023982. Compared with other probiotics, the strain GLA09 has better high-temperature tolerance and higher survival rate in the process of preparing pet food by spraying; secondly, the strain GLA09 also has higher artificial gastrointestinal fluid tolerance and self-coagulation property, and has better colonization capability in the gastrointestinal tract; moreover, the strain GLA09 has good inhibition effect on various pathogens, and can be applied to preventing and treating related diseases caused by the pathogens; in addition, the strain GLA09 also has high free radical scavenging capacity and can be used for resisting oxidation and aging.

Description

Lactobacillus acidophilus and related preparation and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to lactobacillus acidophilus and a related preparation and application thereof.

Background

In recent years, the number of pet dogs cultivated is gradually increased, and the social status of the pet dogs is gradually personified. The pet owners of the current generation are called 'pet parents', and the pets are ties and bridges for emotion communication among family members. As the number of pets increases, pet owners begin to focus on the scientificity of raising pets, and functional pet products are receiving a great deal of attention. A series of organic, natural, functional pet foods containing beneficial health ingredients have been developed. The use of probiotics as a non-toxic, residue-free and beneficial additive in pet foods is becoming increasingly widespread. Such as lactobacillus, bifidobacterium, enterococcus faecium, etc.

However, most of the pet probiotics sold in the market at present are not host sources, and the use effect is good. Considering that the host species has specificity, the probiotics from the intestinal tract of the host species can be better adapted to the gastrointestinal tract environment, and the probiotics function can be exerted. However, the canine lactobacillus acidophilus has fewer reports at present, and corresponding products on the market are fewer.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide lactobacillus acidophilus and a related preparation and application thereof.

The invention is realized in the following way:

In a first aspect, the embodiment of the invention provides lactobacillus acidophilus, which is named Lactobacillus acidophilus GLA and has a preservation number of CCTCC NO: M2023982.

In a second aspect, an embodiment of the present invention provides a microbial preparation comprising lactobacillus acidophilus as described in the previous embodiment.

In a third aspect, embodiments of the present invention provide a product comprising lactobacillus acidophilus as described in the previous embodiments and/or the microbial preparation as described in the previous embodiments.

In a fourth aspect, the present invention provides the use of lactobacillus acidophilus as described in the previous examples or a microbial preparation as described in the previous examples for the preparation of a product having anti-ageing and/or anti-oxidant properties.

In a fifth aspect, embodiments of the present invention provide the use of lactobacillus acidophilus as described in the previous embodiments or a microbial preparation as described in the previous embodiments for the inhibition of a pathogen or for the manufacture of a product for the inhibition of said pathogen or for the prevention and treatment of a related disease caused by said pathogen for non-diagnostic therapeutic purposes.

In a sixth aspect, the present invention provides the use of lactobacillus acidophilus as described in the previous examples or a microbial preparation as described in the previous examples for the manufacture of a product for the prevention and treatment of gastrointestinal disorders.

In a seventh aspect, embodiments of the present invention provide use of lactobacillus acidophilus as described in the previous embodiments or a microbial preparation as described in the previous embodiments in the preparation of a pet food.

The invention has the following beneficial effects:

the invention provides lactobacillus acidophilus GLA09, which has the following advantages:

the strain GLA09 has good high-temperature tolerance capability, the high-temperature resistant survival rate of 70 ℃ is more than 50%, and the strain GLA09 has higher survival rate in the process of preparing pet food by spraying, so that the processing cost is reduced;

the strain GLA09 has higher artificial gastrointestinal fluid tolerance and self-coagulation property, and compared with other probiotics, the strain GLA09 has better colonization capability in the gastrointestinal tract;

The strain GLA09 has excellent inhibition effect on various pathogens and can be applied to preventing and treating related diseases caused by the pathogens;

The strain GLA09 has high free radical scavenging capability, and can be used for preparing antioxidant and anti-aging products, such as anti-aging for pets and prolonging the service life of pets.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the colony morphology (A) and the cell morphology (B) of Lactobacillus acidophilus GLA 09;

FIG. 2 is a Lactobacillus acidophilus GLA09 evolutionary tree;

FIG. 3 is a graph showing the growth and acid production rate of Lactobacillus acidophilus GLA 09;

FIG. 4 shows the tolerance of Lactobacillus acidophilus GLA09 to acidity;

Fig. 5 is a bacteriostatic image of lactobacillus acidophilus GLA 09; wherein A: coli, B: staphylococcus aureus, C: salmonella, D: pseudomonas aeruginosa, E: listeria monocytogenes; a: bacterial suspension, b: cell-free supernatant, c: bacterial precipitation, d: MRS liquid culture medium;

FIG. 6 shows the results of a hemolysis test of Lactobacillus acidophilus GLA09; wherein A is the front surface of a blood plate, B is the back surface of the blood plate, a is staphylococcus aureus (positive control), and B is lactobacillus acidophilus GLA09;

FIG. 7 shows growth and feeding status of GLA09 fed mice; wherein A is the condition of body weight, B is the condition of feed intake, C is the condition of daily gain, and D is the condition of average daily feed intake;

FIG. 8 is a graph of ileal and jejunal villus height and crypt depth HE staining of GLA09 fed mice; wherein A, B, C, D is the ileum HE staining picture of the mice in the CK group, the LA group, the MA group and the HA group respectively; E. f, G, H are the jejunum HE staining pictures of mice in CK group, LA group, MA and HA group, respectively;

FIG. 9 is a diagram of a genome circos;

FIG. 10 is a chart of classification of COG results;

FIG. 11 is a GO annotation result classification diagram;

FIG. 12 is a KEGG PATHWAY result classification chart.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In one aspect, the embodiment of the invention provides lactobacillus acidophilus, which is named Lactobacillus acidophilus GLA and has a preservation number of CCTCC NO: M2023982.

Specifically, lactobacillus acidophilus GLA09 is preserved in China center for type culture Collection (China center for type culture Collection) at 6 months and 19 days in 2023, wherein the preservation address is post code 430072 of university of Wuhan, and the preservation number is CCTCC NO: M2023982.

The 16S rRNA gene sequence of lactobacillus acidophilus GLA09 is shown in SEQ ID NO. 1.

In another aspect, the embodiment of the present invention also provides a microbial preparation, which contains the lactobacillus acidophilus described in the previous embodiment.

In some embodiments, the method of preparing the microbial formulation comprises: culturing the lactobacillus acidophilus.

The medium and the culture conditions for culturing lactobacillus acidophilus GLA09 are not particularly limited, and those currently used for lactobacillus acidophilus culture can be employed. The medium may specifically be an MRS medium. The culture temperature may be 20 to 40 ℃, specifically may be any one or more of 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40 ℃. The cultivation time may be 1 to 50 hours, and specifically may be in a range between any one or any two of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 hours.

In another aspect, the present embodiment also provides a product comprising the lactobacillus acidophilus of the previous embodiment and/or the microbial preparation of any of the previous embodiments;

In some embodiments, the product is a pharmaceutical or feed.

In some embodiments, the feed comprises pet feed.

In another aspect, the present embodiment also provides the use of lactobacillus acidophilus as described in the previous embodiment or the microbial preparation as described in any of the previous embodiments for the preparation of a product having anti-aging and/or antioxidant properties.

In another aspect, the present invention also provides the use of lactobacillus acidophilus as described in the previous examples or the microbial preparation as described in any of the previous examples for the inhibition of a pathogen or for the preparation of a product for the inhibition of said pathogen or for the preparation of a product for the control of a related disease caused by said pathogen for non-diagnostic therapeutic purposes.

In some embodiments, the pathogen comprises: any one or more of escherichia coli, staphylococcus aureus, salmonella, pseudomonas aeruginosa, and listeria monocytogenes.

In some embodiments, the controlling comprises: any one or more of prophylaxis, treatment or adjuvant therapy.

"Treating" herein includes alleviating a condition, reducing the rate at which a condition is raised or developed, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof.

For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination of the foregoing. For tumors, "treatment" includes clearing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or slowing tumor progression, or some combination thereof.

In another aspect, the embodiment of the invention also provides the application of the lactobacillus acidophilus or the microbial preparation in any of the previous embodiments in preparing products for preventing and treating gastrointestinal diseases.

In some embodiments, the gastrointestinal disorder comprises: diarrhea, gastritis, enteritis, colitis, appendicitis, crohn's disease, peptic ulcer, gastric cancer and intestinal cancer.

In some embodiments, the peptic ulcer comprises: esophageal ulcers, gastric ulcers, duodenal ulcers.

In some embodiments, the article of any of the preceding embodiments comprises: any one of medicine and feed.

In some embodiments, the feed comprises pet feed. In addition, on the other hand, the embodiment of the invention also provides application of the lactobacillus acidophilus described in the previous embodiment or the microbial preparation described in any of the previous embodiments in preparing pet feed.

Specifically, the types of pet feeds include animal feeds, plant feeds, and feed additives. The animal feed is derived from animal and slaughtered byproducts, including meat, viscera, blood powder, meat and bone powder, fish powder, and milk of livestock and fowl. The animal feed has good palatability, is easy to digest, and has high protein and vitamin contents. Common canine animal feeds are fish, meat, eggs, milk, and the like. The plant feed has various types and low price, and is a supplementary feed for meat dogs or large dogs. Wherein the plant feed comprises mature seeds of crops such as corn, rice, soybean, etc.; rhizomes of rhizome plants such as sweet potato, cassava, carrot, etc.; melon and fruit, such as pumpkin, etc.; agricultural and sideline industry processed products such as rice bran, sugar residue, bean curd residue, etc.; vegetables, and the like. These feeds are rich in starch and sugars. The feed additive is also called as an auxiliary material, and is used for feeding the pet with the auxiliary material so as to supplement the pet with a certain trace component. The feed additive has various functions, and is most commonly used for promoting the growth and development of dogs, preventing and treating diseases and the like. In some embodiments, the step of preparing the pet food comprises: spraying Lactobacillus acidophilus or microorganism preparation on the basic feed for pets. The probiotics are added into the pet feed in a spraying mode, so that the probiotics with high activity can be uniformly distributed in and/or on the surface of the feed, the survival rate of the probiotics is improved, and the loss rate of the probiotics in the transportation process is reduced.

The specific process of spraying is not particularly limited, and can be performed based on the existing process. In some embodiments, the spraying comprises: high temperature spraying or vacuum spraying. The high temperature may be 60 to 80 ℃, and specifically may be any one or any two of 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 ℃.

Strain GLA09 has excellent high temperature tolerance, its survival rate at 50 ℃ exceeds 70%, survival rate at 60 ℃ exceeds 60%, high temperature resistant survival rate at 70 ℃ exceeds 50%, and it is more suitable for being added in pet food in a spray-coating manner (higher survival rate) than other strains, and the efficacy of pet feed is improved under the condition of reducing the processing cost of pet feed.

In some embodiments, the pet comprises: canines, including but not limited to dogs (pet dogs).

Compared with lactobacillus acidophilus from other sources, the strain GLA09 can be better suitable for the gastrointestinal tract environment of dogs and can play the probiotic function.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

1. Separation and identification of canine lactobacillus acidophilus

1.1 Medium for isolation

MRS medium, the specific composition is shown in Table 1:

TABLE 1 MRS Medium composition Table

Composition of the components	Weight of (E)	Composition of the components	Weight of (E)
				Peptone	10.0·g/L	Dipotassium hydrogen phosphate	2.0·g/L
Beef soaked powder	8.0·g/L	Diammonium hydrogen citrate	2.0·g/L
				Yeast extract powder	4.0·g/L	NaAc	5.0·g/L
Glucose	20.0·g/L	MgSO₄	0.2·g/L
				Agar-agar	14.0·g/L	MnSO₄	0.04·g/L
Tween 80	1.0·mL	Distilled water	1000·mL

The pH was 6.5.+ -. 0.2 and autoclaved at 121℃for 15min.

1.2 Separation method

(1) Sample collection and microorganism separation: 3 adult healthy female beagle dogs were selected in the chinese peninsula, i.e. the ink farm laboratory. Rectal feces were collected by sterile swabs and rapidly placed into 50mL sterile screw cap test tubes containing saline. The low-temperature refrigeration condition is transported to a special economic animal nutrition laboratory of Qingdao agricultural university, gradient dilution is immediately carried out, three plates are used for each gradient, separated culture is carried out by a pouring method, and the culture is carried out in an inverted way at 37 ℃.

(2) And (3) separating and purifying: and (3) taking the flat plate with various bacteria, selecting single bacterial colonies of lactic acid bacteria with different bacterial colony morphologies, and separating and purifying by adopting a continuous streaking method. Repeating for 3-4 times, and performing microscopic examination until pure seeds are obtained.

(3) Bacterial colony characteristics were observed, and bacterial morphology was observed using gram staining and microscopy.

(4) Bacterial DNA was extracted according to the instructions of the beijing solebao technology limited bacterial genome extraction kit (D1600), using bacterial universal primers: 27F, 1499R carries out amplification and sequencing on the obtained lactobacillus conserved sequence, and primer synthesis and sequencing are finished by Beijing qingke biotechnology Co, and the 16S rRNA sequence is subjected to homology comparison. The amplification system of 16S rDNA is shown in Table 2, and the total volume of the amplification system is 50. Mu.l.

TABLE 2 amplification System

ddH₂O	19·μl
		DNA·plate	2·μl
Primer·F	2·μl
		Primer·R	2·μl
2 XEs Taq Mastermix (containing dye)	25·μl

PCR reaction procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 45s, annealing at 56℃for 30s, extension at 72℃for 45s for 30 cycles, extension at 72℃for 10min and storage at 4 ℃.

1.3 Isolation, identification and Screen of Lactobacillus acidophilus

Morphological observation, microscopic examination, enzymatic analysis and carbohydrate utilization experiments, and sequencing and comparison in combination with 16S rRNA are carried out, and the lactobacillus acidophilus is identified, and is named GLA09, and the 16S rRNA gene sequence is shown as SEQ ID NO: 1.

After the bacterial strain is cultured for 24 hours, the middle of the bacterial colony is convex, the edge is neat, and the surface is smooth; facultative anaerobic, gram positive, rod-like. The colony morphology of GLA09 is shown in fig. 1a, and the microscopic morphology is shown in fig. 1B.

Phylogenetic analysis was performed on the 16S rRNA genes of GLA09, and it was confirmed that they were closely related to Lactobacillus acidophilus. Phylogenetic tree is shown in fig. 2.

2. Growth characteristics

2.1 Physiological and Biochemical Properties

And taking the separated bacteria liquid after the activation culture for 18 hours, and carrying out the physiological and biochemical characteristics identification of the lactobacillus by referring to the Bojie's bacteria identification manual, the common bacteria system identification manual and the lactobacillus classification identification and experimental method, and carrying out the identification of the biochemical characteristics such as a sugar fermentation test, a V-P test, a Methyl Red (MR) test, a gelatin test and the like.

2.2 Determination of growth Property and acid production Property

And inoculating the separated bacterial liquid after the activation culture for 18 hours into an MRS culture medium with an inoculum size of 3.0%, and culturing at a constant temperature of 37 ℃ for 24 hours, wherein the control group is a liquid culture medium without inoculating bacterial liquid. The absorbance values of the culture solutions OD600nm were sampled and measured at0, 1,2, 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours, and the pH was sampled and measured at0, 1,2, 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours. And drawing a growth curve and an acid production curve of the culture solution by taking time as an abscissa and taking pH value and OD600nm absorbance value of the culture solution as an ordinate.

2.3 Acid resistance test

The separated bacteria liquid after the activation culture for 18 hours is inoculated into MRS liquid culture media with initial pH of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 respectively according to the inoculum size of 3.0 percent, and the culture is carried out at the constant temperature of 37 ℃. The OD600nm absorbance values were measured at 0, 3, 6, 9, 12, 18, 24, 30, 36, 42h, respectively, 3 replicates per group. And after the measurement is completed, taking 3 groups of parallel test results to calculate an average value, and drawing a curve according to the calculated results.

2.4 High temperature resistance test

And (3) taking the separated bacterial liquid after the activation culture for 18 hours, respectively treating the bacterial liquid in water baths at 50 ℃, 60 ℃ and 70 ℃ for 5 minutes, and immediately placing the treated bacterial liquid in an ice box. The viable count was calculated by plate counting by applying 100. Mu.L of the culture medium after gradient dilution, and the survival rate was calculated by taking the viable count of 0h as a control. Pediococcus canis GLP06 was used as a positive control.

Wherein T _initial and T _treatment 1 are the number of surviving bacteria at 0min and 5min, respectively (l

g CFU/mL)。

2.5 Gastrointestinal tract colonization ability

2.5.1 Acid resistance test

The isolated bacterial solution after 18h of activation culture was taken, centrifuged at 10000rpm at 4℃for 10min, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0) and resuspended in sterile PBS buffer (pH 2.5). After incubation at 37 ℃ for 0 and 3 hours, 10 x serial dilutions of the cultures were plated on MRS agar plates, followed by incubation at 37 ℃ for 24 hours, and the isolated strain was assessed for acid tolerance by viable colonies, and the test repeated 3 times. Pediococcus canis GLP06 was used as a positive control.

Wherein T _initial and T _treatment 2 are the number of surviving bacteria (lg CFU/mL) before (0 h) and after (3 h) incubation, respectively.

2.5.2 Bile salt resistance test

The isolated bacterial solution after 18h of activation culture was taken, centrifuged at 10000rpm at 4℃for 10min, the supernatant was discarded, and washed twice with sterile neutral PBS buffer (pH 7.0) and resuspended in PBS buffer containing 0.1% and 0.3% (w/v) pig bile salt (Solarbio, china). After incubation at 37 ℃ for 0 and 4 hours, 10 x serial dilutions of the cultures were plated on MRS agar plates, followed by incubation at 37 ℃ for 24 hours, and isolated strains were assessed for resistance to bile salts by viable bacterial colonies, and the test repeated 3 times. Pediococcus canis GLP06 was used as a positive control.

Wherein T _initial and T _treatment 3 are the number of surviving bacteria (lg CFU/mL) before (0 h) and after (4 h) incubation, respectively.

2.5.3 Test for resistance to intestinal juice of Artificial stomach

The preparation of the artificial gastric juice and the artificial intestinal juice is referenced in 2010 edition of Chinese pharmacopoeia.

Taking the separated bacterial liquid after the activation culture for 18 hours, centrifuging for 10 minutes at 4 ℃ and 10000rpm, discarding the supernatant, washing twice by using sterile neutral PBS buffer (pH 7.0), re-suspending in artificial gastric juice, and incubating for 1.5 hours at 37 ℃ and 200 rpm. After incubation, the bacterial pellet was washed twice with sterile neutral PBS buffer (pH 7.0), centrifuged at 10000rpm for 10min at 4℃and then resuspended in artificial intestinal fluid and incubated at 200rpm for 2h at 37 ℃. Bacterial precipitation activity was calculated by plate count dilution before and after incubation in artificial gastric juice and intestinal juice and repeated 3 times. Pediococcus canis GLP06 was used as a positive control.

Wherein T _initial and T _treatment are the number of viable bacteria (lg CFU/mL) before and after incubation in artificial gastric or intestinal fluid, respectively.

2.5.4 Surface hydrophobicity test

The hydrophobic properties of the strain surface are reacted by the affinity of lactic acid bacteria for hydrocarbon compounds using bacterial hydrocarbon compound adhesion (bacterium adhesion to hydrocarbons, bat). And (3) taking a bacterial solution of the isolated bacteria after the activation culture for 18 hours, centrifuging at 10000rpm for 10min, washing with a sterile neutral PBS buffer (pH 7.0) for 3 times, and adjusting the absorbance value of the bacterial solution OD ₆₃₀ to be 0.25+/-0.05 (A1) for standby. Then, xylene and chloroform were added in a ratio of 1:1, vortexed for 3min, and incubated at 37℃for 8h. Finally, the aqueous phase was carefully aspirated, the OD ₆₃₀ absorbance value (A2) was measured and the 3 experiments repeated. Pediococcus canis GLP06 was used as a positive control. The formula for calculating the hydrophobicity is as follows:

the hydrophobicity CSH (%) = (1-A2/A1) ×100% (5);

Wherein A1 and A2 are OD ₆₃₀ absorbance values before and after 8 hours of incubation, respectively.

2.5.5 Self-cohesion test

Taking an isolated bacterial solution cultured overnight for 18 hours, centrifuging at 4 ℃ for 10 minutes at 10000rpm, washing 2 times by using sterile neutral PBS buffer (pH 7.0), then re-suspending in an equal volume of sterile neutral PBS buffer (pH 7.0), measuring an OD ₆₃₀ absorbance value (A3), then carrying out vortex oscillation for 10 seconds, placing at 37 ℃ for incubation for 8 hours, avoiding stirring, carefully absorbing the upper part, measuring an OD ₆₃₀ absorbance value (A4), taking the Pediococcus canis GLP06 as a positive control, and repeating 3 times of experiments. The self-condensation rate is calculated as follows:

Autoaggregation ability(％)＝(1-A4/A3)×100％ (6)；

Wherein A3 and A4 are OD ₆₃₀ absorbance values before and after 8h incubation, respectively.

2.6 Bacteriostasis test

The bacteriostasis of the strain is measured by an oxford cup agar diffusion method, an isolated bacterial solution after 18h of activation culture is taken, the bacterial solution is centrifuged for 10min at 4 ℃ and 10000rpm, a cell-free supernatant (CFS) is removed, and bacterial sediment (BP) is resuspended in an equal volume of sterile neutral PBS buffer (pH 7.0). A sterile oxford cup was placed on a TSA agar plate containing tryptone (15.0 g/L), soytone (5.0 g/L), sodium chloride (5.0 g/L) and agar (15.0 g/L), 200. Mu.L of a pathogen (E.coli ATCC25922; salmonella ATCC14028; staphylococcus aureus ATCC25923; pseudomonas aeruginosa ATCC27853; listeria monocytogenes ATCC 19115) at a concentration of 1X 107CFU/mL was plated on the TSA agar plate, after the bacteria had been absorbed completely, 200uL of the isolated strain of BS, CFS, BP and MRS liquid medium were inoculated per well, the TSA plate was incubated in a constant temperature incubator at 37℃for 24 hours, and the formation of a zone of inhibition was observed, and the diameter of the zone of inhibition was precisely measured. Pediococcus canis GLP06 was used as a positive control.

2.7 Antibiotic susceptibility test

The antibiotic susceptibility of the strains was determined by means of a paper sheet diffusion method. According to the recommendations for evaluating the safety of probiotics in combination with antibiotics common on the market, 27 antibiotics were selected for testing in the form of 6mm paper sheets (BIO-KONT, china): penicillin, oxacillin, ampicillin, piperacillin, cefalexin, cefuroxime, ceftazidime, cefoperazone, amikacin, gentamicin, kanamycin, streptomycin, tetracycline, minocycline, erythromycin, azithromycin, norfloxacin, lincomycin, vancomycin, polymyxin B, compound neonomine, chloramphenicol, doxycycline, levofloxacin, florfenicol, ciprofloxacin.

The isolated bacterial solution after 18h of activation culture was taken, centrifuged at 10000rpm for 10min at 4℃and washed 2 times with sterile neutral PBS buffer (pH 7.0) and then resuspended in sterile neutral PBS buffer (pH 7.0) to obtain a bacterial solution in logarithmic growth phase (0.5 McFarland suspension). 100uL of the bacterial suspension of the isolated strain was plated on MRS agar plates. And finally, sticking an antibiotic paper sheet on the surface of an MRS agar plate within 15min, culturing the plate at a constant temperature of 37 ℃ for 24h, observing whether a bacteriostasis ring is formed or not, and measuring the diameter of the bacteriostasis ring by using a vernier caliper.

2.8 Free radical scavenging test

2.8.1DPPH free radical scavenging test

The DPPH removing capacity of the isolated strain is determined by using a Soxhlet DPPH detection kit, and according to the operation of the specification, in short, 100 mu L of the isolated bacterial liquid after 18h culture is sucked, 900 mu L of the extracting liquid is added, vortex oscillation and mixing are carried out, centrifugation is carried out at 10000rpm at room temperature for 10min, 25 mu L of the supernatant is taken and added into 975 mu L of the DPPH working solution, vortex mixing is carried out, and the mixture is kept at rest for 30min at room temperature in a dark place. Absorbance was recorded at 515nm using a microplate reader. Pediococcus canis GLP06 was used as a positive control. The DPPH radical scavenging rate was calculated as follows:

Wherein: a _assay ¹ is absorbance of a sample to be detected; a _control ¹ is absorbance of the mixed solution of the supernatant fluid treated by the isolated strain and the absolute ethyl alcohol; a _blank ¹ is absorbance of the mixture of the extracting solution and the working solution.

2.8.2ABTS free radical scavenging test

The DPPH removing capacity of the isolated strain is determined by using a Soxhlet DPPH detection kit, and according to the operation of the specification, in short, 100 mu L of the isolated bacterial liquid after 18h culture is sucked, 900 mu L of the extracting liquid is added, vortex oscillation and uniform mixing are carried out, centrifugation is carried out for 10min at 10000rpm at room temperature, and the supernatant is taken and placed on ice for detection. 50 mu L of supernatant is added into 850 mu L of ABTS working solution, 100 mu L of reagent four working solution is added, and the mixture is fully and uniformly mixed and kept stand at room temperature in a dark place for 6min. The absorbance at 405nm was measured with a microplate reader, and a blank and a control were simultaneously made. Pediococcus canis GLP06 was used as a positive control. The ABTS radical scavenging rate was calculated as follows:

Wherein: a _assay ² is absorbance of a sample to be detected; a _control ² is blank tube absorbance; a _blank ² controls the absorbance of the tube.

2.8.3 Superoxide anion radical scavenging assay

The superoxide anion scavenging capacity of the isolated strain was determined using a Soxhaust superoxide anion radical scavenging assay kit, operating according to the instructions. The absorbance of the blank tube and the measurement tube was measured at 530nm and was designated as A _{Blank space} and A _Measurement, respectively. The calculation formula is as follows:

superoxide anion clearance (%) = (a blank-a assay)/a blank×100% (9).

2.9 Safety test

2.9.1 Hemolysis test

The isolated bacterial liquid after 18h of activation culture was streaked on Trypsin Soybean Agar (TSA) containing 5.0% (w/v) sheep blood (Oxoid, germany), the plate was cultured at 37℃for 48h, and the blood agar plate was examined for signs of beta-hemolysis (clear area around colonies), alpha-hemolysis (green area around colonies) or gamma-hemolysis (no area around colonies). Staphylococcus aureus (ATCC 25923) strain was used as a positive control.

2.9.2 Safety test of mice

For in vivo safety evaluation, 80 growth-healthy, body-weight-approximated queen mice were randomly divided into 4 groups of 20 animals each. Control (CK) mice were perfused with 0.2mL of sterile saline, while the other three groups were perfused with 0.2mL of isolated strains 2X 10 ⁹(LA09),2×10¹⁰ (MA 09) and 2X 10 ¹¹ (HA 09) CFU/mL, respectively. Body weight and feed intake were measured every 3 days and the health status of mice was recorded. After a period of 28 days, the mice were fasted for 12 hours and then anesthetized with 1% sodium pentobarbital (50 mg/kg). The abdominal aorta was sampled to collect blood and serum was collected by centrifugation (4000 rpm,4 ℃,10 min) for further analysis. After the mice were sacrificed, visceral toxicity was observed in each group of mice by dissection, and organs such as liver, kidney, spleen and thymus were collected and weighed, and the organ coefficients were calculated as organ weight/body weight×100.

The mice were collected at about 1cm from the midjejunum and midileum, and after rinsing the intestinal contents gently with physiological saline, the samples were rapidly immersed in 4% paraformaldehyde for tissue fixation for 24 hours. Tissue finishing and washing, dehydration, transparency, wax dipping, embedding, sectioning, HE staining were then performed, and the stained sections were then observed under an optical microscope and the villus height and crypt depth were measured using Image J.

2.10 Strain Whole genome sequencing

Joint sequencing was performed using an Illumina high throughput sequencing platform NovaSeq 6000, and genome assembly was performed using Unicycler (version: 0.5.0, https:// gitsub.com/rrwick/Unicycler). The assembled genome was predicted for the coding gene by Prokka (version: 1.14.6, https:// gitsub.com/tseemann/prokka) software. The genome was predicted using MinCED (Version: 0.4.2) of regularly spaced short palindromic repeats (CRISPR) clustered. Predicted gene sequences were analyzed using COG, KEGG, uniProt and RefSeq blast+ (version: 2.11.0 +) and compared to these functional databases to obtain gene function annotation results. Functional annotation was performed using software Hmmer (version: 3.3.2) based on Pfam and TIGERFAMS databases, and genome loop maps were drawn using R-packages.

3. Results and analysis

3.1 Growth Properties

3.1.1 Physiological and Biochemical Properties

The physiological and biochemical characteristics of the Lactobacillus acidophilus GLA09 moiety are shown in Table 3. The Lactobacillus acidophilus GLA09 can be monosaccharide such as galactose, cellobiose, maltose, sucrose, raffinose, inulin, etc.

TABLE 3 physiological and biochemical characteristics of Lactobacillus acidophilus GLA09

Project	Results	Project	Results
				Esculin	-	Arabinose (Arabic sugar)	-
Cellobiose	+	Fructose	-
				Maltose	+	Glucose	-
Mannitol (mannitol)	-	Rhamnose (rhamnose)	-
				Salicin	-	L-rhamnose	-
Sorbitol	-	Xylose	-
				Sucrose	+	MR test	-
Raffinose	+	V-P test	-
				Inulin	+	Nitrate produced gas	-
Galactose	+	Hydrogen sulfide test	-

+: A positive reaction; -: negative reaction.

3.1.2 Determination of growth Property and acid production Property

As shown in FIG. 3, lactobacillus acidophilus GLA09 grows slowly in 0-2 h, the absorbance value of 3-18 hOD ₆₀₀ nm rises rapidly in the slow growth period, the absorbance value of the bacterial liquid tends to be stable after 18h in the logarithmic growth period, and the bacterial liquid enters the stable growth period. The pH curve change of the acid production of the bacterial liquid is consistent with the growth curve, and the pH is slowly reduced within 2 hours; the descending rate of 3hpH is accelerated, the pH change of the bacterial liquid after 18 hours is gradually gentle, and the bacterial liquid is stabilized at about 4.0.

3.1.3 Acid resistance of Strain

As shown in FIG. 4, the strain GLA09 was completely inhibited from growing at pH 3.0 or less, and the absorbance at OD ₆₀₀ nm was unchanged with the prolonged culture time. At ph=4.0, the strain grew slowly, but the OD ₆₀₀ nm absorbance values were significantly lower than at ph=5.0, 6.0, 7.0. When the pH value is more than or equal to 5.0, the strain GLA09 can normally grow, but when the strain reaches a stable period, the absorbance value of OD ₆₀₀ nm is different to a certain extent, wherein the OD value of pH=7.0 > pH=6.0 > pH=5.0, and the viable count is consistent with the change trend of the absorbance value of OD ₆₀₀ nm.

3.1.4 Strain tolerance to high temperatures

As shown in Table 4, the survival rate of the strain GLA09 at 50 ℃ is over 70%, the survival rate at 60 ℃ is over 60%, the survival rate at 70 ℃ is over 50%, and the survival rate of the strain GLA09 at 70 ℃ is significantly higher than that of the strain GLP06, which indicates that the Lactobacillus acidophilus GLA09 has better high temperature resistance.

TABLE 4 high temperature tolerance of Lactobacillus acidophilus GLA09

3.2 Colonization ability of strains in the gastrointestinal tract

3.2.1 Strain tolerance to acid and bile salts

As can be seen from Table 5, strain GLA09 is more tolerant to acids and bile salts. The survival rate of the strain after 3 hours of inoculation in MRS liquid culture medium with pH=2.5 is 75.86%, and the viable count is about 2.53×10 ⁶ CFU/mL. The survival rate of the strain after being inoculated into MRS liquid culture medium with the bile salt concentration of 0.1 percent for 4 hours is 82.51 percent, and the viable count is about 4.43 multiplied by 10 ⁶ CFU/mL; the survival rate of the strain after being inoculated into MRS liquid culture medium with the concentration of bile salt of 0.3 percent for 4 hours is 59.19 percent, and the viable count is about 6.30X10. 10 ⁴ CFU/mL.

TABLE 5 survival of Lactobacillus acidophilus GLA09 in acid and various bile salt concentrations

3.2.2 Resistance of the Strain to Artificial gastrointestinal fluids

As can be seen from table 6, lactobacillus acidophilus GLA09 survived significantly more bacteria in artificial gastrointestinal fluids than strain GLP06. The survival rate of the strain after being inoculated in the artificial gastric juice for 1.5 hours is 94.33%, and the viable count is about 1.12 multiplied by 10 ⁸ CFU/mL; the survival rate of the strain after being inoculated in the artificial intestinal juice for 2 hours is 98.83%, the viable count is about 5.7X10 ⁹ CFU/mL, the survival rate after being digested together with the artificial intestinal juice for 3.5 hours is 69.62%, and the viable count is about 4.03X10 ⁶ CFU/mL.

TABLE 6 survival of Lactobacillus acidophilus GLA09 in Artificial gastrointestinal fluids%

Strain	Artificial gastric juice	Artificial intestinal juice	Artificial stomach intestine liquid
				GLP06	61.94±0.66^a	90.44±0.31^a	54.83±0.26^a
GLA09	94.33±2.09^b	98.83±1.01^b	69.62±0.39^b

3.3 Strain surface hydrophobicity and self-aggregation Property

Bacteria are generally set to be highly hydrophobic by a surface hydrophobicity definition standard with CSH% >50%, moderately hydrophobic between 20% and 50% and non-hydrophobic with CSH% < 20%. Self-agglomerating ability is generally divided into: low (16% -35%), medium (35% -50%) and high (more than 50%). As can be seen from table 7, lactobacillus acidophilus GLA09 was less hydrophobic than strain GLP06 but significantly more self-assembling than strain GLP06.

TABLE 7 Lactobacillus acidophilus GLA09 surface hydrophobicity and self-aggregation

3.4 Antibacterial Properties of Strain

As shown in FIG. 5, the bacterial suspension and the cell-free supernatant of Lactobacillus acidophilus GLA09 can effectively inhibit the growth and proliferation of 5 pathogenic bacteria, and the bacterial precipitation and MRS culture medium have no antibacterial effect. The results in Table 8 show that the strain has a zone of inhibition of greater than 20mm for E.coli, salmonella and Pseudomonas aeruginosa, greater than 19mm for Listeria monocytogenes and greater than 18mm for Staphylococcus aureus. The antibacterial effect of the strain GLA09 bacterial suspension and the supernatant is obviously superior to that of the strain GLP06.

TABLE 8 antibacterial test results of Lactobacillus acidophilus GLA09

Note that: the same row of data shoulder marks have no letters or the same letters indicate that the difference is not significant (P > 0.05), and different lower case letters indicate that the difference is significant (P < 0.05).

3.5 Antibiotic sensitivity of Strain

The diameter of the drug sensitive tablet is 6mm, so that the antibacterial effect is realized when the diameter of the antibacterial ring is larger than 7mm, and the antibacterial effect is not realized when the diameter is smaller than or equal to 7 mm. As can be seen from Table 9, the strain GLA09 is susceptible differently to various common antibiotics. The drug has strong sensitivity to penicillin, piperacillin, cefalexin, cefazolin, cefuroxime, tetracycline, minocycline, chloramphenicol, doxycycline, cefoperazone and florfenicol, the diameter of a bacteriostasis ring is larger than 23mm, the drug is moderately sensitive to ampicillin and vancomycin, the diameter of the bacteriostasis ring is larger than 15mm, the diameter of the drug inhibition ring is 7-14mm, and the drug inhibition ring is insensitive to ceftazidime, kanamycin, streptomycin, azithromycin, norfloxacin, lincomycin, polymyxin B and compound neonomine and ciprofloxacin.

TABLE 9 Lactobacillus acidophilus GLA09 antibiotic sensitivity

Note that: diameter of zone of inhibition (mm): +++:23-30mm; ++:15-22mm; +:7-14mm; -: there is no inhibition zone.

3.6 Free radical scavenging ability of Strain

As can be seen from Table 10, the clearance of Lactobacillus acidophilus GLA09 to DPPH, ABTS and superoxide anion radical was 64.34%, 98.19% and 86.36%, respectively. The DPPH radical clearance of strain GLA09 was significantly higher than that of strain GLP06.

TABLE 10 Lactobacillus acidophilus GLA09 free radical clearance

3.7 Safety test results

3.7.1 Hemolysis test results

With staphylococcus aureus as a positive control (fig. 6 a), it was seen that there was significant β -hemolysis (clear area around the colony), but GLA09 was γ -hemolysis (no area around the colony), and it was possible to preliminarily judge whether the strain GLA09 was safe and nonpathogenic.

3.7.2 Mouse test results

All mice are clinically observed to have good mental condition, can eat and drink water normally, and have no abnormal feces, and the mice are not dead during the test. After the test is finished, the viscera of the strain are observed through sectioning, and no abnormal pathological histological change and bacterial translocation are observed, so that the strain has no toxic or side effect on the clinical application of mice.

Body weight, food intake and organ coefficients are common indicators for assessing animal health, and are used to assess the safety of isolated strains. First, the effect of GLA09 supplementation on mouse body weight and food intake was observed. There were no significant differences between mice fed GLA09 at different doses in body weight, feed intake, average Daily Gain (ADG) and Average Daily Food Intake (ADFI) and CK groups (fig. 7). The above results indicate that GLA09 addition had no adverse effect on the body weight and food intake of mice.

TABLE 11 influence of organ index in GLA09 fed mice

Remarks: CK, control group (0.2 mL of sterile physiological saline was filled); LA, MA and HA were respectively 2X 10 ⁹、2×10¹⁰ and 2X 10 ¹¹ CFU/mL Lactobacillus acidophilus GLA09 for drenching.

How GLA09 affects organ coefficients in mice was also studied. Table 11 shows organ weight data of GLA09 added mice. The CK group and the experimental group mice have no significant differences in heart, liver, spleen and kidney, thymus weights. This shows that strain GLA09 has no adverse effect on animal growth and development, and is safe and has no toxic side effect.

TABLE 12 ileum and jejunum villus length versus crypt depth ratio in GLA09 fed mice

The effect of GLA09 feeding on the intestinal morphology of mice was also studied. Fig. 8 (a-D) shows HE staining pictures of ileal villus height and crypt depth of mice, and fig. 8 (E-H) is jejunum HE staining results. As shown in fig. 8 and table 12, there was no significant difference in the ileum and jejunum villus length, crypt depth, and ratios thereof. From this, it can be demonstrated that feeding GLA09 had no adverse effect on the intestinal development and intestinal health of mice.

3.8 Results of Whole genome sequencing of Strain

To understand the characteristics of probiotics and explore the probiotic potential of GLA09, whole genome sequencing was performed. The results of the complete circular genomic profile of GLA09 are shown in fig. 9. The complete genome of GLA09 consists of a 2.103M circular chromosome and a circular plasmid, with (g+c) contents of 34.94% and 33.38%, respectively, and genome sizes of 2,042,740bp, belonging to a medium-sized genome, these bacteria are generally very metabolic, tolerant and adaptable. The COG, GO and KEGG results for strain GLA09 genome are shown in fig. 10, 11 and 12.

COG annotation results show that GLA09 is mainly enriched with ribosomal structures and biosynthesis, carbohydrate transport and metabolism, and primary functions predict transcription and amino acid transport metabolism. GO annotation results show, biological processes

(Biological process) enrichment of the first three are translation, mediated DNA translocation and DNA integration, respectively; the first three of the cell components (cellular component) are respectively membrane integral components, cytoplasm and cytoplasmic membranes; the first three of the molecular functions (molecular function) are ATP binding, DNA binding and transferase activity, respectively. KEGG notes the first five signal paths: global and overview (Global and overview maps, 329), carbohydrate metabolism (Carbohydrate metabolism, 128), transmembrane transport (Membrane transport, 124), translation (Translation, 79) and amino acid metabolism (Amino acid metabolism, 76).

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The lactobacillus acidophilus is characterized by being named Lactobacillus acidophilus GLA-09 and having a preservation number of CCTCC NO: M2023982.

2. A microbial preparation comprising the Lactobacillus acidophilus according to claim 1.

3. A product comprising the lactobacillus acidophilus of claim 1 and/or the microbial preparation of claim 2;

The product is a medicine or a feed.

4. A product according to claim 3, wherein the feed comprises pet feed.

5. Use of lactobacillus acidophilus as claimed in claim 1 or a microbial formulation as claimed in claim 2 for non-diagnostic therapeutic purposes for the inhibition of pathogens or for the manufacture of a product for the inhibition of said pathogens; the pathogen includes: any one or more of pseudomonas aeruginosa and listeria monocytogenes; the product is a medicine or a feed.

6. Use of the lactobacillus acidophilus as claimed in claim 1 or the microbial preparation as claimed in claim 2 in the preparation of pet food.

7. The use of claim 6, wherein the step of preparing pet food comprises: spraying the lactobacillus acidophilus or the microbial preparation on the basic pet feed.

8. The use according to claim 6, wherein the pet comprises: a canine.

9. The use of claim 8 wherein the canine comprises: and (3) a dog.