Lactobacillus johnsonii and related products and applications thereof

Abstract

The invention discloses a lactobacillus johnsonii and related products and applications thereof, and relates to the field of microorganisms. The Lactobacillus johnsonii GJ231 provided by the invention has a preservation number of CCTCC NO: M 2023981. Compared with other lactobacillus johnsonii, GJ231 is acid-resistant and bile-resistant, has a higher survival rate in the intestine, has a better antibacterial effect on a variety of pathogens, and can be used to prevent and treat related diseases caused by pathogens; secondly, GJ231 has a better high temperature tolerance and has a higher survival rate in the process of spraying and preparing pet food; furthermore, GJ231 also has a strong antioxidant capacity, can reduce the peroxidation reaction caused by oxidative stress, and can be used for anti-aging and anti-oxidation; in addition, GJ231 also has a bacteriocin gene cluster, which may be used to produce bacteriocin, and has the potential to replace antibiotics and food preservation.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Lactobacillus johnsonii and related products and applications thereof. Background Art

[0002] Lactobacillus johnsonii (L. johnsonii) is classified in the genus Lactobacillus. Studies have shown that Lactobacillus johnsonii can activate intestinal CD206+ macrophages through the TLR1 / 2-STAT3 signaling pathway, promote their secretion of IL-10, and thus relieve ulcerative colitis in mice; Lactobacillus johnsonii can also protect heart function, inhibit myocardial injury cytokines and improve the integrity of the host intestinal barrier; oral Lactobacillus johnsonii can also increase the level of reduced glutathione in the blood of mice, thereby improving the mitochondrial morphology and function of the liver, reducing liver lipids, and improving systemic sugar metabolism. Therefore, Lactobacillus johnsonii has been widely used as a probiotic in functional products for humans and animals.

[0003] With the upgrading of pet consumption, pet owners have begun to pay attention to scientific pet care, and the demand for functional products has continued to increase. Probiotics, as a non-toxic, residue-free and probiotic functional supplement, are increasingly used in human and pet food, such as lactic acid bacteria, bifidobacteria, yeast, etc. in pet food. However, most of the pet probiotic products currently sold on the market are not host-derived, and the effects of their use vary. Considering that the host species is specific, it may exclude microorganisms in the intestines of other species.

[0004] At present, there are few reports on canine-derived Lactobacillus johnsonii, and no related products have been found on the market.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a Lactobacillus johnsonii and related products and applications thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a Lactobacillus johnsonii, whose name is Lactobacillus johnsonii GJ231 and whose preservation number is CCTCC NO: M 2023981.

[0009] In a second aspect, an embodiment of the present invention provides a microbial preparation containing the Lactobacillus johnsonii described in the above embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a fermentation product of Lactobacillus johnsonii, which is obtained by fermentation and culture of the Lactobacillus johnsonii described in the above embodiment.

[0011] In a fourth aspect, an embodiment of the present invention provides a product, which includes the Lactobacillus johnsonii described in the preceding embodiment or the microbial preparation described in the preceding embodiment or the fermentation product described in the preceding embodiment.

[0012] In a fifth aspect, embodiments of the present invention provide use of the Lactobacillus johnsonii described in the preceding embodiments, or the microbial preparation described in the preceding embodiments, or the fermentation product described in the preceding embodiments in the preparation of a product having anti-aging and / or antioxidant functions.

[0013] In a sixth aspect, an embodiment of the present invention provides the use of Lactobacillus johnsonii as described in the preceding embodiments, or the microbial preparation as described in the preceding embodiments, or the fermentation product as described in the preceding embodiments in inhibiting pathogens for non-diagnostic treatment purposes, or in preparing products that inhibit the pathogens, or in preparing products that prevent and treat related diseases caused by the pathogens.

[0014] In a seventh aspect, the embodiments of the present invention provide the use of Lactobacillus johnsonii as described in the preceding embodiments, or the microbial preparation as described in the preceding embodiments, or the fermentation product as described in the preceding embodiments in the preparation of bacteriocins or the preparation of products having any one or more of the following functions: (1) improving liver function; (2) promoting the development of immune organs; (3) promoting the growth of intestinal villi; (4) preventing and treating gastrointestinal diseases.

[0015] In an eighth aspect, embodiments of the present invention provide use of the Lactobacillus johnsonii described in the preceding embodiments, or the microbial preparation described in the preceding embodiments, or the fermentation product described in the preceding embodiments in the preparation of pet feed.

[0016] The present invention has the following beneficial effects:

[0017] 1. Lactobacillus johnsonii GJ231 is resistant to acid, bile salts, and artificial intestinal fluid, has good probiotic potential, and has a high survival rate in the intestine, which helps it to continue to exert its efficacy.

[0018] 2. Lactobacillus johnsonii GJ231 has good antibacterial ability and has good antibacterial effects on Staphylococcus aureus, Escherichia coli, Salmonella, Pseudomonas aeruginosa and Listeria monocytogenes, and can be used to prevent and treat diarrhea.

[0019] 3. Lactobacillus johnsonii GJ231 has strong antioxidant capacity and has high free radical scavenging ability of DPPH, ABTS and superoxide anion. It can reduce the peroxidation reaction caused by oxidative stress and can be used for anti-aging of pets and prolong their lifespan.

[0020] 4. Oral administration of Lactobacillus johnsonii GJ231 has no adverse effects on mice, and can improve the serum antioxidant capacity of mice (T-AOC and SOD content), reduce MDA content; improve liver function (BUN content is significantly reduced); promote the development of immune organs (spleen organ index is significantly increased) and villus length.

[0021] 5. Lactobacillus johnsonii GJ231 has a bacteriocin gene cluster that can be used to produce bacteriocins, which has the potential to replace antibiotics and preserve food. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The colony morphology (A) and cell morphology (B) of Lactobacillus johnsonii GJ231;

[0024] Figure 2 This is the evolutionary tree of Lactobacillus johnsonii GJ231;

[0025] Figure 3 This is the growth and acid production rate curve of Lactobacillus johnsonii GJ231;

[0026] Figure 4 is the tolerance of Lactobacillus johnsonii GJ231 to acidity;

[0027] Figure 5 The results of the antibacterial test of Lactobacillus johnsonii GJ231; A is Escherichia coli, B is Staphylococcus aureus, C is Listeria monocytogenes, D is Salmonella, and E is Pseudomonas aeruginosa; well a is added with GJ231 bacterial suspension, well b is added with GJ231 supernatant, well c is added with GJ231 bacterial protein, and well d is added with MRS culture medium (blank control);

[0028] Figure 6 This is the result of the hemolysis test of Lactobacillus johnsonii GJ231;

[0029] Figure 7 The effect of feeding GJ231 mice on organ indexes; A to E are heart index, liver index, spleen index, kidney index and thymus index respectively;

[0030] Figure 8are the serum biochemical indexes of GJ231 mice fed with diet; A to H are aspartate aminotransferase, alanine aminotransferase, urea nitrogen, malondialdehyde, total antioxidant capacity, superoxide dismutase, catalase and glutathione peroxidase, respectively;

[0031] Fig. 9 The HE staining of the jejunum of mice fed with GJ231; A to B are the control group and the GJ231 gavage group, respectively;

[0032] Fig.10 This is the circlize map of the GJ231 genome;

[0033] Fig.11 This is the classification diagram of COG results;

[0034] Fig.12 This is a classification diagram of GO annotation results;

[0035] Fig.13 This is the classification diagram of KEGG pathway results;

[0036] Fig.14 The potential gene cluster of GJ231 bacteriocin; A to C are the predicted structure diagram of GJ231 bacteriocin, the predicted tertiary structure of bacteriocin, and the amino acid sequence of GJ231 bacteriocin is compared with the reported bacteriocin sequence. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0038] In one aspect, an embodiment of the present invention provides a Lactobacillus johnsonii, whose name is Lactobacillus johnsonii GJ231 and whose preservation number is CCTCC NO: M 2023981.

[0039] Specifically, Lactobacillus johnsonii GJ231 was deposited in the China Center for Type Culture Collection on June 12, 2023, and the deposit address is Wuhan University, Wuhan, China, with a postal code of 430072.

[0040] The 16S rRNA sequence of Lactobacillus johnsonii GJ231 is shown in SEQ ID NO:1.

[0041] On the other hand, an embodiment of the present invention provides a microbial preparation containing the Lactobacillus johnsonii described in the above embodiment.

[0042] In some embodiments, the microbial preparation may also include other probiotics.

[0043] On the other hand, an embodiment of the present invention provides a fermentation product of Lactobacillus johnsonii, which is obtained by fermentation and culture of the Lactobacillus johnsonii described in the above embodiment.

[0044] In some embodiments, there is no particular limitation on the culture medium and culture conditions for fermenting Lactobacillus johnsonii GJ231, and the culture medium and culture conditions currently used for culturing Lactobacillus johnsonii can be used.

[0045] In some embodiments, the culture medium may be MRS medium.

[0046] In some embodiments, the fermentation culture temperature may be 20-40° C., specifically any one or more of 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40° C. The culture time may be 1-50 h, specifically any one or a range between any two of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 h.

[0047] On the other hand, an embodiment of the present invention provides a product, which includes the Lactobacillus johnsonii described in the above embodiment or the microbial preparation described in any of the above embodiments or the fermentation product described in the above embodiments.

[0048] In some embodiments, the product is food, medicine, feed or health product.

[0049] In some embodiments, the feed comprises pet food.

[0050] On the other hand, an embodiment of the present invention provides the use of the Lactobacillus johnsonii described in the preceding embodiments or the microbial preparation described in any preceding embodiment or the fermentation product described in the embodiment in the preparation of a product having anti-aging and / or antioxidant functions.

[0051] In some embodiments, the antioxidant function may specifically include: reducing or avoiding peroxidation reactions caused by oxidative stress.

[0052] On the other hand, an embodiment of the present invention provides the use of Lactobacillus johnsonii as described in the preceding embodiments, or the microbial preparation as described in any preceding embodiment, or the fermentation product as described in the embodiment for inhibiting pathogens for non-diagnostic treatment purposes, or for preparing products for inhibiting the pathogens, or for preparing products for preventing and treating related diseases caused by the pathogens.

[0053] In some embodiments, the pathogens include any one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, and Listeria monocytogenes.

[0054] In some embodiments, the prevention and treatment includes any one or more of prevention, treatment or adjuvant therapy.

[0055] As used herein, "treating" includes alleviating a condition, reducing the rate at which a condition develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0056] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0057] On the other hand, the embodiments of the present invention provide the use of the Lactobacillus johnsonii described in the above embodiments or the microbial preparation described in any of the above embodiments or the fermentation product described in the embodiments in preparing bacteriocins or preparing products having any one or more of the following functions:

[0058] (1) Improve liver function;

[0059] (2) Promote the development of immune organs;

[0060] (3) Promote the growth of intestinal villi;

[0061] (4) Prevent and treat gastrointestinal diseases;

[0062] (5) Food preservation.

[0063] In some embodiments, the immune organ includes: spleen. The spleen is an important lymphatic organ located in the upper left part of the abdominal cavity (between the stomach fundus and the diaphragm in the left hypochondrium), flat and oval, dark red, soft and brittle, and easily ruptured and bleeds when hit violently.

[0064] In some embodiments, the gastrointestinal disease includes any one or more of diarrhea, gastritis, enteritis, colitis, appendicitis, Crohn's disease, peptic ulcer, gastric cancer and intestinal cancer;

[0065] In some embodiments, the peptic ulcer includes any one of esophageal ulcer, gastric ulcer, and duodenal ulcer;

[0066] In some embodiments, the prevention and treatment includes: any one or more of prevention, treatment or adjuvant therapy;

[0067] In some embodiments, the product includes: any one of medicine, food, feed and health care products;

[0068] In some embodiments, the feed comprises pet food.

[0069] In addition, the embodiments of the present invention also provide the use of the Lactobacillus johnsonii described in the above embodiments or the microbial preparation described in any of the above embodiments or the fermentation product described in the embodiments in the preparation of pet feed.

[0070] Specifically, the types of pet feed include animal feed, plant feed and feed additives. Animal feed comes from animals and their by-products after slaughter, including meat, viscera, blood meal, meat and bone meal, fish meal, milk, etc. of livestock and poultry. Animal feed has good palatability, is easy to digest, and has high protein and vitamin content. Common canine animal feeds include fish, meat, eggs, milk, etc. Plant feeds are of various types and low price, and are supplementary feeds for meat dogs or large dogs. Among them, plant feeds include mature seeds of crops, such as corn, rice, soybeans, etc.; roots and stems of root plants, such as sweet potatoes, potatoes, cassava, carrots, etc.; fruits and vegetables, such as pumpkins, zucchini, etc.; agricultural and sideline products, such as rice bran, sugar residue, tofu residue, etc.; green vegetables, etc. These feeds are rich in starch and sugar. Feed additives are also called "supplementary feeds". Feeding pets with "supplementary feeds" is to supplement pets with certain trace ingredients. There are many types of feed additives with different functions. The most common ones are used to promote dog growth and development, prevent and treat diseases, etc. In some embodiments, the step of preparing pet feed includes: spraying Lactobacillus johnsonii or microbial preparations on the pet base feed. Adding probiotics to pet feed by spraying can make the inside and / or surface of the feed evenly distributed with highly active probiotics, improve the survival rate of probiotics, and reduce the loss rate of probiotics during transportation.

[0071] In some embodiments, the step of preparing the pet food comprises: spraying Lactobacillus johnsonii or a microbial preparation on a basic pet food.

[0072] There is no special restriction on the specific process of spraying, and it can be implemented based on existing processes. In some embodiments, the spraying includes: high-temperature spraying or vacuum spraying. The high temperature can be 60-70°C, specifically any one of 60, 62, 64, 66, 68, 70°C or a range between any two. Lactobacillus johnsonii GJ231 has excellent high temperature tolerance, with a survival rate of more than 80% at 50°C, a survival rate of more than 70% at 60°C, and a high temperature survival rate of more than 60% at 70°C. Compared with other strains, it is more suitable for adding to pet food by spraying (higher survival rate), which improves the efficacy of pet feed while reducing the processing cost of pet feed.

[0073] In some embodiments, the pet includes: a canine.

[0074] In some embodiments, the canine includes: dogs. Compared with Lactobacillus johnsonii from other sources, Lactobacillus johnsonii GJ231 is better adapted to the gastrointestinal environment of canines, can better colonize and adhere to intestinal cells, and exert its probiotic function. At the same time, oral administration of native animal microorganisms will not cause gastrointestinal disorders caused by foreign microorganisms.

[0075] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0076] Example 1

[0077] 1. Isolation and identification of canine-derived Lactobacillus johnsonii

[0078] 1.1 Isolation culture medium

[0079] MRS medium, the specific components are shown in Table 1.

[0080] Table 1 MRS culture medium composition g / L

[0081]

[0082] pH 6.5±0.2, autoclave at 121℃ for 15min.

[0083] 1.2 Separation method

[0084] (1) Sample collection and microbial isolation: Two healthy adult female beagles were selected from the Jimo Animal Husbandry Experimental Farm in Qingdao, China. Rectal feces were collected by sterile swabs and quickly placed in a 50 mL sterile screw-capped test tube containing physiological saline. The samples were transported to the Special Economic Animal Nutrition Laboratory of Qingdao Agricultural University under low temperature refrigeration conditions, and gradient dilutions were immediately performed, with three plates per gradient, and the samples were separated and cultured by pouring method and inverted at 37°C.

[0085] (2) Isolation and purification: Take the above plates with various bacteria, select single lactic acid bacteria colonies with different colony morphologies, and separate and purify them by continuous streaking method. Repeat 3-4 times and examine under a microscope until pure strains are obtained.

[0086] (3) Observe the characteristics of the colonies and use Gram staining and microscopy to observe the bacterial morphology.

[0087] (4) Bacterial DNA was extracted according to the instructions of the Bacterial Genome Extraction Kit (D1600) of Beijing Solebaugh Technology Co., Ltd. The conserved sequences of lactic acid bacteria were amplified and sequenced using the universal bacterial primers: 27F, 1492R. The primer synthesis and sequencing were sent to Beijing Qingke Biotechnology Co., Ltd. for completion, and the 16S rRNA sequence was compared for homology. The amplification system of 16S rDNA was as follows, and the total volume of the amplification system was 50 μl.

[0088] <![CDATA[ddH2O]]> 19μl DNA plate 2μl Primer F 2μl Primer R 2μl 2xEs Taq MasterMix (with dye) 25μl

[0089] PCR reaction program: 94°C pre-denaturation for 5 min, 94°C denaturation for 45 s, 56°C annealing for 30 s, 72°C extension for 45 s, a total of 30 cycles, and a final extension at 72°C for 10 min and storage at 4°C.

[0090] 1.3 Isolation, identification and screening of Lactobacillus johnsonii

[0091] Through morphological observation, microscopic examination, enzymatic analysis and carbohydrate utilization experiments, combined with 16S rRNA sequencing, it was identified as Lactobacillus johnsonii and named GJ231. The 16S rRNA gene sequence is shown below. After 20 hours of culture, the colony has a convex middle, spherical or oval shape, and a smooth surface; it is facultative anaerobic, Gram-positive, and the bacteria are short rods with round ends. The colony morphology of GJ231 is as follows Figure 1 As shown in A, the morphology under microscope is as follows Figure 1 As shown in B.

[0092] Phylogenetic analysis of the 16S rRNA gene of GJ231 confirmed that they were closely related to Lactobacillus johnsonii. Figure 2 shown.

[0093] (1) The 16S rRNA gene sequence is shown in SEQ ID NO: 1, and is as follows (5'-3'):

[0094]

[0095] (2) Phylogenetic tree of GJ231

[0096] See Figure 2 .

[0097] 2. Growth characteristics

[0098] 2.1 Positive control strain

[0099] Canine (blue fox source) CCTCC NO.M ZJBF004 Lactobacillus johnsonii ZJBF004 was selected as a positive control, invention application (patent) number: CN201911265623.0.

[0100] 2.2 Physiological and biochemical characteristics

[0101] Take the isolated bacterial liquid after activation culture for 18 hours, and identify the physiological and biochemical characteristics of lactic acid bacteria with reference to the "Bergey's Bacterial Identification Manual", "Common Bacterial System Identification Manual" and "Lactic Acid Bacteria Classification and Identification and Experimental Methods", and identify the biochemical characteristics such as sugar fermentation test, VP test, methyl red (MR) test, gelatin test, etc.

[0102] 2.3 Growth performance and acid production performance determination

[0103] The isolated bacterial liquid after activation culture for 18 hours was inoculated into MRS medium at a 3.0% inoculum and cultured at 37°C for 24 hours. The control group was a liquid culture medium without bacterial liquid. The culture liquid OD was measured at 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 36, and 48 hours. 600 The absorbance value was measured at 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 36, and 48 hours. The pH and OD of the culture medium were measured with time as the horizontal axis. 600 The nm absorbance value is used as the ordinate, and the growth curve and acid production curve are drawn.

[0104] 2.4 Acid resistance test

[0105] The isolated bacterial suspension after activation culture for 18 hours was inoculated with an inoculum of 3.0% in MRS liquid medium with initial pH of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively, and cultured at 37°C. The OD was measured at 0, 3, 6, 9, 12, 18, 24, 30, 36, and 42 hours. 600 nm absorbance value, 3 parallels per group. After the determination is completed, the average value of the 3 parallel test results is calculated, and a curve is drawn based on the calculated results.

[0106] 2.5 High temperature resistance test

[0107] Take the isolated bacterial solution after 18 hours of activation culture, treat it in a water bath at 50℃, 60℃, 70℃ and 80℃ for 5 minutes, and put it in an ice box immediately after treatment. Take 100μL of culture solution for gradient dilution and spread it, calculate the number of viable bacteria by plate counting method, and calculate the survival rate with the number of viable bacteria at 0h as the control.

[0108] Survival rate

[0109] Among them, T initial 1 and T treatmeft 1 is the number of surviving bacteria at 0 min and 5 min, respectively (log CFU / mL).

[0110] 2.6 Gastrointestinal colonization ability

[0111] 2.6.1 Acid resistance test

[0112] Take the isolated bacterial culture after 18 hours of activation culture, centrifuge at 4°C, 10000rpm for 10 minutes, discard the supernatant, wash twice with sterile neutral PBS buffer (pH 7.0), and then resuspend in sterile PBS buffer (pH 2.5). After culturing at 37°C for 0 and 3 hours, spread 10× serial dilutions of the culture on MRS agar plates, and then incubate at 37°C for 24 hours. The acid tolerance of the isolated strains was evaluated by live colonies, and the test was repeated 3 times.

[0113] Survival rate

[0114] Among them, T initial 2 and T treatment 2 are the numbers of surviving bacteria (log CFU / mL) before incubation (0 h) and after incubation (3 h), respectively.

[0115] 2.6.2 Bile salt tolerance test

[0116] The isolated bacterial suspension after activation culture for 18 hours was centrifuged at 4°C, 10000rpm for 10 minutes, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0), and resuspended in PBS buffer containing 0.1% and 0.3% (w / v) porcine bile salts (Solarbio, China). After culturing at 37°C for 0, 1h, 4h and 6h, 10× serial dilutions of the culture were spread on MRS agar plates and then incubated at 37°C for 24h. The tolerance of the isolated strain to bile salts was evaluated by the viable bacterial colonies, and the test was repeated 3 times.

[0117] Survival rate

[0118] Among them, T initial3 and T treatment 3 are the numbers of surviving bacteria (log CFU / mL) before (0 h) and after (4 h) incubation, respectively.

[0119] 2.6.3 Artificial gastrointestinal fluid tolerance test

[0120] The configuration of artificial gastric juice and artificial intestinal juice refers to the 2010 edition of the "Chinese Pharmacopoeia".

[0121] Take the isolated bacterial suspension after activation culture for 18 hours, centrifuge at 4°C, 10000rpm for 10 minutes, discard the supernatant, wash twice with sterile neutral PBS buffer (pH 7.0), resuspend in artificial gastric juice, and incubate at 37°C, 200rpm for 1.5 hours. After incubation, wash the bacterial precipitate twice with sterile neutral PBS buffer (pH 7.0), centrifuge at 4°C, 10000rpm for 10 minutes, then resuspend in artificial intestinal juice, and incubate at 37°C, 200rpm for 3 hours. Before and after incubation in artificial gastric juice and intestinal juice, calculate the activity of bacterial precipitate by plate count dilution method. Repeat the experiment 3 times.

[0122] Survival rate

[0123] Among them, T initial 4 and T treatment 4 is the number of surviving bacteria (log CFU / mL) before and after incubation in artificial gastric or intestinal fluid, respectively.

[0124] 2.6.4 Surface hydrophobicity test

[0125] The bacterial adhesion to hydrocarbons (BATH) method was used to reflect the hydrophobicity of the strain surface through the affinity of lactic acid bacteria to hydrocarbons. The isolated bacterial solution after activation culture for 18 hours was centrifuged at 4°C, 10000rpm for 10 minutes, washed three times with sterile neutral PBS buffer (pH 7.0), and the OD of the bacterial solution was adjusted. 600 The absorbance value is 0.25±0.05 (A1) and it is set aside. Then, xylene and chloroform are added in a ratio of 1:1, vortexed for 3 minutes, and incubated at 37°C for 2 hours. Finally, carefully aspirate the aqueous phase and measure the OD 600 Absorbance value (A2). Repeat the test 3 times. The hydrophobicity calculation formula is as follows:

[0126] Hydrophobicity CSH (%) = (1-A2 / A1) × 100% (5);

[0127] A1 and A2 are the OD values ​​before and after incubation, respectively. 600 Absorbance value.

[0128] 2.6.5 Self-cohesion test

[0129] Take the isolated bacterial suspension cultured overnight for 18 hours, centrifuge at 10000rpm for 10 minutes at 4°C, wash twice with sterile neutral PBS buffer (pH 7.0), and then resuspend in an equal volume of sterile neutral PBS buffer (pH 7.0), measure the OD600 absorbance value (A3), then vortex for 10 seconds, place at 37°C, incubate for 2 hours, avoid stirring, carefully absorb the upper part, and measure the OD 600 Absorbance value (A4), repeat the test 3 times. The formula for calculating the self-aggregation rate is as follows:

[0130] Autoaggregationability (%) = (1-A4 / A3) × 100% (6);

[0131] Among them, A3 and A4 are the OD values ​​before and after incubation for 8 h, respectively. 600 Absorbance value.

[0132] 2.7 Antibacterial test

[0133] The antibacterial properties of the strains were determined by the Oxford cup agar diffusion method. The isolated bacterial suspension after activation culture for 18 hours was centrifuged at 4°C, 10000rpm for 10 minutes to obtain the cell-free supernatant (CFS). 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). 200uL of a 1×10 7 CFU / mL of pathogenic bacteria (Escherichia coli ATCC25922; Salmonella ATCC14028; Staphylococcus aureus ATCC25923; Pseudomonas aeruginosa ATCC27853; Listeria monocytogenes ATCC19115) were spread on TSA agar plates. After the bacterial solution was completely absorbed, 200 μL of the bacterial solution of the isolated strain, CFS, bacterial protein and MRS liquid culture medium (negative control) were inoculated into each well. The TSA plates were placed upright in a constant temperature incubator at 37°C for incubation. After 24 hours, the formation of inhibition zones was observed and the diameter of the inhibition zones was accurately measured.

[0134] 2.8 Antibiotic susceptibility testing

[0135] The antibiotic sensitivity of the strain was determined by the disc diffusion method. According to the recommendations for evaluating the safety of probiotics and combined with the common antibiotics on the market, the isolated bacterial solution after activation culture for 18 hours was centrifuged at 4°C, 10000rpm for 10 minutes, washed twice 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 the logarithmic growth phase (0.5McFarland suspension). Take 100uL of the McFarland suspension of the isolated strain and spread it on the MRS agar plate. Finally, the antibiotic paper was attached to the surface of the MRS agar plate within 15 minutes, and the plate was cultured at 37°C for 24 hours to observe whether an inhibition zone was formed, and the diameter of the inhibition zone was measured with a vernier caliper.

[0136] 2.9 Free radical scavenging test

[0137] 2.9.1DPPH free radical scavenging test

[0138] The DPPH scavenging ability of the isolated strains was determined using the Solebow DPPH detection kit. The operation was performed according to the instructions. In short, 100 μL of the isolated bacterial culture after 18 hours of culture was added to 900 μL of the extract, vortexed and mixed, centrifuged at 10000 rpm for 10 minutes at room temperature, 25 μL of the supernatant was added to 975 μL of the DPPH working solution, vortexed and mixed, and kept at room temperature in the dark for 30 minutes. The absorbance was recorded at 515 nm using an ELISA reader. The calculation formula for DPPH free radical scavenging rate is as follows:

[0139]

[0140] Among them: A assay 1 A is the absorbance of the sample to be tested; control 1 A is the absorbance of the supernatant and the mixture of anhydrous ethanol for the treatment of isolated strains; blank 1 is the absorbance of the mixture of the extract and the working solution.

[0141] 2.9.2 ABTS free radical scavenging test

[0142] The Solebow ABTS detection kit was used to determine the ABTS scavenging ability of the isolated strains. The operation was carried out according to the instructions. In short, 100 μL of the isolated bacterial solution after 18 hours of culture was added to 900 μL of the extract, vortexed and mixed, centrifuged at 10000 rpm for 10 minutes at room temperature, and the supernatant was taken and placed on ice for testing. 50 μL of the supernatant was added to 850 μL of ABTS working solution, and then 100 μL of reagent four working solution was added, mixed thoroughly, and allowed to stand at room temperature in the dark for 6 minutes. The absorbance at 405 nm was measured with an ELISA instrument, and blank and control were also performed. The calculation formula for the ABTS free radical scavenging rate is as follows:

[0143]

[0144] Among them: A assay 2 A is the absorbance of the sample to be tested; control 2 is the absorbance of the blank tube; A blank 2 The absorbance of the control tube.

[0145] 2.9.3 Superoxide anion free radical scavenging test

[0146] The superoxide anion free radical scavenging detection kit was used to determine the superoxide anion scavenging ability of the isolated strains according to the instructions. The absorbance values ​​of the blank tube and the test tube were measured at 530 nm and recorded as A 空白 and A 测定 The calculation formula is as follows:

[0147] Superoxide anion clearance rate (%) = (A 空白 -A 测定 ) / A 空白 ×100%(9).

[0148] 2.10 Safety Test

[0149] 2.10.1 Hemolysis test

[0150] Take the isolated bacterial suspension after activation culture for 18 hours and streak it on tryptic soy agar (TSA) containing 5.0% (w / v) sheep blood (Oxoid, Germany), incubate the plate at 37°C for 48 hours, and check the blood agar plate for signs of β-hemolysis (clear area around the colonies), α-hemolysis (green area around the colonies) or γ-hemolysis (no area around the colonies). Staphylococcus aureus (ATCC25923) strain was used as a positive control.

[0151] 2.10.2 Safety test in mice

[0152] For in vivo safety evaluation, 40 healthy Kunming mice with similar body weight were randomly divided into two groups, with 20 animals in each group. The mice in the control group (CON) were gavaged with 0.2 mL of sterile saline, while the mice in the probiotic group (GJ231) were gavaged with 0.2 mL of 1×10 isolated strains. 10 CFU / mL. Body weight and food intake were measured every 3 days, and the health status of the mice was recorded. After 28 days, the mice were fasted for 12 hours and then anesthetized with 1% sodium pentobarbital (50 mg / kg). Blood was collected by blood sampling from the abdominal aorta, and serum was collected by centrifugation (4000 rpm, 4 ° C, 10 min) for further analysis. After the mice were killed, the internal organs of the two groups of mice were dissected and observed, and organs such as the heart, liver, kidney, spleen and thymus were collected and weighed, and the organ coefficient was calculated as organ weight / body weight × 100.

[0153] The middle section of the mouse jejunum (about 1 cm) was collected, and the intestinal contents were gently rinsed with physiological saline, and then the sample was quickly immersed in 4% paraformaldehyde for tissue fixation for 24 hours. The tissue was then trimmed and washed, dehydrated, transparent, waxed, embedded, sliced, HE stained, and then the stained sections were observed under an optical microscope.

[0154] 2.11 Whole genome sequencing of strain

[0155] The Illumina high-throughput sequencing platform NovaSeq 6000 was used for joint sequencing and assembled using Unicycler (version: 0.5.0, https: / / github.com / rrwick / Unicycler). The assembled genome was predicted by Prokka (version: 1.14.6, https: / / github.com / tseemann / prokka) software for coding gene prediction. MinCED (version: 0.4.2) was used to predict clustered regularly interspaced short palindromic repeats (CRISPR) in the genome. The predicted gene sequences were analyzed using COG, KEGG, UniProt and RefSeq BLAST+ (version: 2.11.0+), and the predicted gene sequences were compared with these functional databases to obtain gene function annotation results. The software Hmmer (version: 3.3.2) was used for functional annotation based on the Pfam and TIGERFAMs databases, and the genome circle map was drawn using the R package.

[0156] 2.12 Bacteriocin Prediction

[0157] BAGEL4 ( http: / / bagel4.molgenrug.nl / index.php ) was used to obtain the amino acid sequence of GJ231 bacteriocin, Uniprot ( https: / / www.uniprot.org / blast / ) was used to mine the functional genes encoding bacteriocin, and Structure Assessment ( https: / / swissmodel.expasy.org / assess ) was used for visualization.

[0158] 3. Results and Analysis

[0159] 3.1 Growth characteristics

[0160] 3.1.1 Physiological and biochemical characteristics

[0161] Some physiological and biochemical characteristics of Lactobacillus johnsonii GJ231 are shown in Table 2. Lactobacillus johnsonii GJ231 can utilize glucose, maltose, sucrose, raffinose, inulin and galactose.

[0162] Table 2 Physiological and biochemical characteristics of Lactobacillus johnsonii GJ231

[0163] project result project result Escin - Arabinose - Cellobiose - fructose - maltose + glucose + Mannitol - Rhamnose - Salicin - L-Rhamnose - Sorbitol - Xylose - sucrose + MRI testing - Raffinose + VP test - Inulin + Nitrate gas production - Galactose + Hydrogen sulfide test -

[0164] Note: + indicates a positive reaction; - indicates a negative reaction.

[0165] 3.1.2 Growth performance and acid production performance determination

[0166] like Figure 3 As shown, Lactobacillus johnsonii GJ231 grew slowly from 0 to 3 h and was in the growth retardation phase. 600 The absorbance value of nm increased rapidly, and it was in the logarithmic growth phase. After 18 hours, the absorbance of the bacterial solution tended to be stable, and it entered the stable growth phase. The pH curve of the acid production of the bacterial solution was consistent with the growth curve. The pH dropped slowly within 3 hours; the pH drop rate accelerated after 3 hours, and the pH change of the bacterial solution tended to be gentle after 18 hours, stabilizing at about 3.7.

[0167] 3.1.3 Acid resistance of strains

[0168] Depend on Figure 4 It can be seen that the growth of strain GJ231 was completely inhibited when pH ≤ 3.0. As the culture time increased, OD 600 The absorbance value of nm did not change. At pH = 4.0, the strain could grow slowly, but the OD 600 The absorbance value of nm was significantly lower than that at pH = 5.0, 6.0, and 7.0. When pH ≥ 5.0, strain GJ231 could grow normally, but when it reached the stable phase, OD 60There are some differences in the absorbance values ​​of nm, among which, OD value pH = 6.0> pH = 7.0> pH = 5.0, and the number of viable bacteria is related to OD 600 The change trend of the absorbance values ​​was consistent with that of the

[0169] 3.1.4 High temperature tolerance of strains

[0170] As shown in Table 3, the survival rate of strain GJ231 at 50°C is over 80%, the survival rate at 60°C is over 70%, and the survival rate at 70°C is over 60%, indicating that Lactobacillus johnsonii GJ231 has good high temperature resistance and can be directly sprayed (70°C) on the surface of pet food to directly exert its efficacy and reduce processing costs.

[0171] Table 3 High temperature tolerance of Lactobacillus johnsonii GJ231

[0172]

[0173] Note: Note: Data in the same row do not contain the same lowercase letters, which indicates a significant difference (P<0.05); data with the same letters or no letters indicate no significant difference (P>0.05).

[0174] 3.2 Colonization ability of strains in the gastrointestinal tract

[0175] 3.2.1 Tolerance of strains to acid and bile salts

[0176] As shown in Table 4, strain GJ231 has strong tolerance to acid and bile salts. The survival rate of the strain was 73.04% after being inoculated in MRS liquid medium with pH = 2.5 for 3 hours. The survival rates of the strain after being inoculated in MRS liquid medium with a bile salt concentration of 0.1% for 1 hour, 4 hours and 6 hours were 98.60%, 85.23% and 73.52%; the survival rates of the strain after being inoculated in MRS liquid medium with a bile salt concentration of 0.3% for 1 hour, 4 hours and 6 hours were 86.65%, 72.82% and 68.78%. Compared with Lactobacillus johnsonii ZJBF004, GJ231 had a slightly higher survival rate for 0.3% bile salt for 6 hours.

[0177] Table 4 Survival rate of Lactobacillus johnsonii GJ231 in acid and different bile salt concentrations

[0178] project Cultivation time / h GJ231 survival rate / % ZJBF004 survival rate / % pH 2.5 3 73.04±1.61 - 0.1% Bile Salts 1 98.60±2.36 - 0.1% Bile Salts 4 85.23±0.65 - 0.1% Bile Salts 6 73.52±0.80 - 0.3% Bile Salts 1 86.65±0.39 - 0.3% Bile Salts 4 72.82±5.13 - 0.3% Bile Salts 6 68.78±2.06 65.39

[0179] 3.2.2 Tolerance of strains to artificial gastrointestinal fluid

[0180] As shown in Table 5, Lactobacillus johnsonii GJ231 has a strong survival ability in artificial gastrointestinal fluid. The survival rate of the strain was 60.45% after 1.5 hours of inoculation in artificial gastric fluid; the survival rate of the strain was 97.03% after 3 hours of inoculation in artificial intestinal fluid; and the survival rate was 59.07% after 4.5 hours of co-digestion with artificial gastrointestinal fluid. Among them, the survival rate in artificial gastric fluid was lower than that of Lactobacillus johnsonii ZJBF004, but the survival rate in artificial intestinal fluid was higher than that of Lactobacillus johnsonii ZJBF004.

[0181] Table 5 Survival rate of Lactobacillus johnsonii GJ231 in artificial gastrointestinal fluid

[0182]

[0183] 3.3 Surface hydrophobicity and self-aggregation properties of strains

[0184] Bacteria are defined according to the surface hydrophobicity standard, generally setting CSH%>50% as highly hydrophobic, CSH% between 20% and 50% as moderately hydrophobic, and CSH%<20% as non-hydrophobic. Self-aggregation ability is generally divided into three categories: low (16%-35%), medium (35%-50%) and high (above 50%). As shown in Table 6, the surface hydrophobicity and self-aggregation rate of Lactobacillus johnsonii GJ231 are higher than those of Lactobacillus johnsonii ZJBF004, indicating that GJ231 can adhere to and colonize the gastrointestinal tract better, which is crucial for probiotics to continue to function and improve host health.

[0185] Table 6 Surface hydrophobicity and self-aggregation rate of Lactobacillus johnsonii GJ231

[0186]

[0187] 3.4 Antibacterial properties of strains

[0188] Figure 5 The results in Table 7 show that the diameter of the inhibition zone of the strain against Salmonella is greater than 20 mm, the diameter against Staphylococcus aureus is greater than 18 mm, the diameter of the inhibition zone against Listeria monocytogenes is 16-18 mm, and the diameter of the inhibition zone against Escherichia coli and Pseudomonas aeruginosa is 14-16 mm, and the antibacterial effects of the bacterial suspension and the supernatant are similar, so it is speculated that the antibacterial effect may be produced by its metabolites (organic acids, bacteriocins, exopolysaccharides, etc.). Compared with Lactobacillus johnsonii ZJBF004, GJ231 has better antibacterial effects on Escherichia coli and Salmonella, but its antibacterial effect on Staphylococcus aureus is lower than that of Lactobacillus johnsonii ZJBF004.

[0189] Table 7 Lactobacillus johnsonii GJ231 antibacterial test results

[0190]

[0191] 3.5 Antibiotic susceptibility of strains

[0192] The diameter of the drug sensitive sheet is 6 mm, so the diameter of the inhibition zone greater than 7 mm is considered to be antibacterial, and less than or equal to 7 mm is considered to be non-antibacterial. As shown in Table 8, strain GJ231 has different sensitivities to various common antibiotics. It is highly sensitive to minocycline, cefoperazone and chloramphenicol, with an inhibition zone diameter greater than 23 mm, moderately sensitive to piperacillin, cefazolin and cefuroxime, with an inhibition zone diameter greater than 15 mm, and the inhibition zone diameters of gentamicin and tetracycline are between 7-14 mm, and it is insensitive to these antibiotics.

[0193] Table 8 Antibiotic sensitivity of Lactobacillus johnsonii GJ231

[0194]

[0195]

[0196] Note: +++: 23-30mm; ++: 15-22mm; +: 7-14mm; -: no inhibition zone.

[0197] 3.6 Free radical scavenging ability of strains

[0198] As shown in Table 9, the scavenging rates of Lactobacillus johnsonii GJ231 for DPPH, ABTS and superoxide anion free radicals were 68.23%, 90.80% and 86.07%, respectively. The DPPH free radical scavenging rate of GJ231 was significantly higher than that of Lactobacillus johnsonii ZJBF004, indicating that GJ231 had a higher free radical scavenging ability.

[0199] Table 9 Free radical scavenging rate of Lactobacillus johnsonii GJ231

[0200] project GJ231 free radical scavenging rate / % ZJBF004 free radical scavenging rate / % DPPH 68.23±0.64 31.1 ABTS 90.80±1.89 - Superoxide anion 86.07±0.79 -

[0201] 3.7 Safety test results

[0202] 3.7.1 Hemolysis test results

[0203] Staphylococcus aureus was used as a positive control ( Figure 6 ), obvious β-hemolysis was observed (clear areas around the colonies), but GJ231 was non-hemolytic (no areas around the colonies, Figure 6 johnsonii GJ231 is safe and non-pathogenic, and in vivo experiments can be conducted to further verify its probiotic properties.

[0204] 3.7.2 Mouse test results

[0205] Clinical observations during the mouse test phase showed that all mice were in good mental condition, could eat and drink normally, had normal feces, and no experimental mice died during the test. After the test, no abnormal pathological histological changes or bacterial translocation were found in their internal organs during the autopsy, indicating that the strain has no toxic side effects for clinical application in mice.

[0206] This example also verifies how GJ231 affects the organ coefficients of mice. Figure 7 As shown in the figure, there was no significant difference in the organ weights of heart, liver, spleen and kidney between the CK group and the probiotic group. This indicates that strain GJ231 has no adverse effects on animal growth and development, is safe, and has no toxic side effects; the thymus weight of mice in the probiotic group was significantly higher than that in the control group, indicating that oral administration of GJ231 promoted the development of immune organs in mice.

[0207] Figure 8 The effects of GJ231 addition on serum biochemical indices of mice were shown. Compared with the control group, the serum BUN and MDA levels of the probiotic group mice were significantly reduced, and the T-AOC and SOD levels were significantly increased. There were no significant differences in the serum AST, ALT, CAT and GSH-PX levels between the CK group and the probiotic group mice.

[0208] This example also verifies the effect of feeding GJ231 on the intestinal morphology of mice. Fig. 9 As shown, the length of jejunal villi in the probiotic group mice was longer, indicating that feeding GJ231 not only had no adverse effects on the intestinal development and intestinal health of mice, but also promoted the length of the jejunal villi of mice and improved the digestion and absorption of nutrients.

[0209] 3.8 Strain whole genome sequencing results

[0210] To understand the properties of probiotics and explore the prebiotic potential of GJ231, whole genome sequencing was performed. Fig.10 The results of the complete circular genome map of GJ231 are shown in Figure 2. After filtering out adapters, short fragments, and low-quality data, the size of the assembled complete genome was 1.763M.

[0211] The GJ231 genome contains three rings, a circular chromosome (Bacteria|ctg_1), a circular unclassified (Bacteria|ctg_2), and a circular plasmid (Bacteria|ctg_3), with G+C contents of 34.70%, 34.32%, and 35.73%, respectively, belonging to a medium-sized genome. These bacteria usually have strong metabolism, tolerance, and adaptability. The COG, GO, and KEGG results of the strain GJ231 genome are shown in Figure 2. Figure 11-13As shown in the figure. COG annotation results showed that GJ231 was mainly enriched in carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, and general function prediction only. GO annotation results showed that the top three enriched biological processes were translation, phosphoenolpyruvate-dependent sucrose phosphotransferase system, and carbohydrate metabolism; the top three enriched cellular components were plasma membrane, integral component of membrane, and cytoplasm; the top three enriched molecular functions were ATP binding, DNA binding, and metal ion binding. The top four signal pathways annotated by KEGG were: Global and overview maps (284), Carbohydrate metabolism (120), Membrane transport (98), and Translation (80).

[0212] 3.9 Bacteriocin prediction and comparison

[0213] Many symbiotic bacteria can produce antimicrobial small molecules called bacteriocins, which can inhibit / eliminate specific target bacteria and affect the composition of the microbiome. Therefore, bacteriocins have attracted more and more attention as potential tools for microbiome editing. The whole genome analysis of GJ231 also predicted two bacteriocins (Gassericin T and Gassericin E). The bacteriocin gene cluster is shown in the figure below: Fig.14 A. The main amino acid sequence and protein three-dimensional structure are as follows Fig.14 B-C. Bacteriocins isolated from GJ231 have many potential uses in pet food, including replacement of antibiotics and food preservation, as the thermal stability and wide pH range of bacteriocins can provide the product with an appropriate shelf life. In addition, their broad spectrum of activity helps combat spoilage microorganisms and pathogens.

[0214] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Lactobacillus johnsonii, characterized in that Its name is Lactobacillus johnsonii GJ231, the accession number is CCTCC NO: M 2023981.

2. A microbial preparation, characterized in that: The method comprises the Lactobacillus johnsonii according to claim 1.

3. A fermentation product of Lactobacillus johnsonii, characterized in that The method is obtained by fermenting and culturing the Lactobacillus johnsonii according to claim 1.

4. A product, characterized in that It comprises the Lactobacillus johnsonii described in claim 1 or the microbial preparation described in claim 2 or the fermentation product described in claim 3; the product is a medicine or feed.

5. The product according to claim 4, characterized in that The feed includes pet food.

6. Use of the Lactobacillus johnsonii according to claim 1, the microbial preparation according to claim 2, or the fermentation product according to claim 3 in the preparation of medicines or feeds having anti-aging and / or anti-oxidation functions.

7. Use of the Lactobacillus johnsonii according to claim 1, the microbial preparation according to claim 2, or the fermentation product according to claim 3 in inhibiting pathogens for non-diagnostic treatment purposes, preparing drugs for inhibiting the pathogens, or preparing drugs for preventing and treating related diseases caused by the pathogens, characterized in that: The pathogens include any one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa and Listeria monocytogenes.

8. The use according to claim 7, characterized in that: The prevention and treatment includes: any one or more of prevention, treatment or auxiliary treatment.

9. Use of the Lactobacillus johnsonii according to claim 1, the microbial preparation according to claim 2, or the fermentation product according to claim 3 in the preparation of bacteriocins or in the preparation of medicines having any one or more of the following functions: (1) Improve liver function; (2) Promote the development of immune organs; (3) Promote the growth of intestinal villi; (4) Prevent and treat gastrointestinal diseases; (5) Food preservation.

10. The use according to claim 9, characterized in that: The immune organs include: thymus.

11. The use according to claim 9, characterized in that: The gastrointestinal diseases include any one or more of diarrhea, gastritis, enteritis, appendicitis, Crohn's disease and peptic ulcer.

12. The use according to claim 9, characterized in that: The peptic ulcer includes any one of esophageal ulcer, gastric ulcer and duodenal ulcer.

13. The use according to claim 9, characterized in that: The prevention and treatment includes: any one or more of prevention, treatment or auxiliary treatment.

14. Use of the Lactobacillus johnsonii according to claim 1, the microbial preparation according to claim 2, or the fermentation product according to claim 3 in preparing feed.

15. Use according to claim 14, characterised in that The feed has any one or more of the following functions: (1) Promote the development of immune organs; (2) Promote the growth of intestinal villi; (3) Food preservation.

16. The use according to claim 15, characterized in that The immune organs include: thymus.

17. The use according to claim 14, characterized in that: The feed includes pet food.

18. The use according to claim 17, characterized in that The step of preparing the pet feed comprises: spraying the Lactobacillus johnsonii or the microbial preparation on the pet basic feed.

19. The use according to claim 18, characterized in that The pets include canines.

20. The use according to claim 19, characterized in that The canines include dogs.

Images