Deodorizing disinfectant for pets as well as preparation method and application of deodorizing disinfectant

Abstract

The invention discloses a pet deodorization disinfectant as well as a preparation method and application thereof. The deodorization disinfectant is prepared from a metabolite of a specific strain lactobacillus salivarius NJAUTYRGJ-Y (the preservation number is CCTCC NO: M 20242787), a plant extract, a disinfectant and salt components. The deodorant disinfectant can be directly sprayed on pet excrement, urine and living environment, and efficient deodorization and disinfection are realized. The deodorization disinfectant has a remarkable deodorization effect on excrement, urine and ammonia gas, and the deodorization effect lasts for a long time; the antibacterial agent has a good antibacterial effect on common pathogenic bacteria (staphylococcus aureus, escherichia coli and klebsiella pneumoniae), and also has a good disinfection effect on excrement, urine and air. In addition, the deodorant disinfectant has good safety, has no toxic or side effect, and is suitable for safe disinfection of pet living environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of deodorizing materials, in particular to a pet deodorizing disinfectant solution and a preparation method and application thereof. BACKGROUND

[0002] Pet enriches human spiritual life, but also affects human health due to the odor and pathogenic bacteria it may carry. The odor of pets mainly comes from their own secretions and excretions, which is often formed by nitrogen-containing compounds, sulfur-containing inorganic compounds and volatile organic compounds. Such odor gases have the characteristics of easy volatilization, low perception threshold, and non-linear relationship between perception intensity and concentration. Long-term exposure to odor gases not only affects the living environment, but also may stimulate the mucous membranes of humans and animals, affect the respiratory, digestive, cardiovascular and endocrine systems, and cause harm to human and animal health. In addition, pets and their secretions and excretions often carry various pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae, which can increase the spread of these pathogenic bacteria through frequent contact between pets and between pets and humans, thereby seriously threatening human and animal health.

[0003] Currently, there are several methods for removing odors, including covering method, adsorption method, chemical method, solvent dissolution method, catalytic decomposition method and biological method. The covering method can only mask the odor and cannot eliminate it fundamentally. The chemical reaction method is to make the odor molecules and specific substances react chemically to generate odorless substances, which is the most effective deodorizing method for pets and the environment. However, clinical reports have shown that some deodorizing disinfectants can cause various adverse effects on animals and humans when used, including allergies, skin damage, respiratory tract irritation and other effects caused by the components of the deodorizing liquid.

[0004] Therefore, it is of great significance and has a wide market to develop a safe and effective pet deodorizing disinfectant product. SUMMARY

[0005] The present application aims to overcome the deficiencies in the prior art and provide a pet deodorizing disinfectant solution that has good deodorizing and disinfecting effects and safety, fast deodorizing speed, long deodorizing maintenance time, and is friendly to pets when used.

[0006] In a first aspect of the present application, a pet deodorizing disinfectant solution is provided, which comprises metabolic products of Lactobacillus salivarius, plant extracts, disinfectants and salt substances; wherein the Lactobacillus salivarius is Lactobacillus salivarius NJAUTYRGJ-Y, and the preservation number is CCTCC NO: M20242787.

[0007] In the present application, the Lactobacillus salivarius metabolite has both deodorizing and disinfecting functions. The organic acid in the Lactobacillus salivarius metabolite can reduce the environmental pH value, inhibit the growth and metabolism of odor-producing bacteria, and reduce the generation of odor substances such as ammonia and hydrogen sulfide; at the same time, the organic acid can also chemically react with odor molecules to change their chemical structure and reduce the odor intensity. The bacteriocin can inhibit the growth of harmful bacteria and reduce the possibility of odor generation from the source.

[0008] According to some embodiments of the present application, the nucleotide sequence of the 16S RNA of the Lactobacillus salivarius NJAUTYRGJ-Y is shown in SEQ ID NO. 1.

[0009] According to some embodiments of the present application, the optimal growth pH of the Lactobacillus salivarius NJAUTYRGJ-Y is 2-7; the Lactobacillus salivarius NJAUTYRGJ-Y has acid resistance, heat resistance and bile salt resistance.

[0010] According to some embodiments of the present application, the pet deodorizing and disinfecting solution comprises the following ingredients in mass percentage: 5-20% Lactobacillus salivarius metabolite, 10-25% plant extract, 4-12% salt substance and 0.1-0.3% disinfectant.

[0011] According to some embodiments of the present application, the mass percentage content of the Lactobacillus salivarius metabolite is 5-20%; preferably, the mass percentage content of the Lactobacillus salivarius metabolite is 8-15%; more preferably, the mass percentage content of the Lactobacillus salivarius metabolite is 10%.

[0012] According to some embodiments of the present application, the mass percentage content of the plant extract is 10-25%; preferably, the mass percentage content of the plant extract is 15-20%; more preferably, the mass percentage content of the plant extract is 18%.

[0013] According to some embodiments of the present application, the plant extract comprises Artemisia argyi extract and thyme extract.

[0014] In the present application, the plant extract (Artemisia argyi extract and thyme extract) has both deodorizing and disinfecting functions. The natural fragrance emitted by the Artemisia argyi extract and the thyme extract can effectively mask the odor on the pet and in the living environment, making the environmental air more fresh and pleasant. The volatile oil and flavonoid components in the Artemisia argyi extract and the thyme phenol in the thyme extract have inhibitory and killing effects on bacteria, fungi and viruses. They can destroy the cell membrane, protein and nucleic acid structure of microorganisms, thereby inhibiting the growth and reproduction of microorganisms, and synergistically enhance the disinfection effect with benzalkonium bromide and Lactobacillus salivarius metabolite.

[0015] According to some embodiments of the present application, the mass percentage content of the extract of Artemisia argyi is 5% to 15%, and the mass percentage content of the extract of Thymus vulgaris is 5% to 10%; preferably, the mass percentage content of the extract of Artemisia argyi is 10%, and the mass percentage content of the extract of Thymus vulgaris is 8%.

[0016] According to some embodiments of the present application, the mass percentage content of the salt is 4% to 12%; preferably, the mass percentage content of the salt is 6% to 10%; more preferably, the mass percentage content of the salt is 8%.

[0017] According to some embodiments of the present application, the salt comprises sodium chloride and magnesium sulfate.

[0018] In the present application, the salt (sodium chloride and magnesium sulfate) mainly plays a role in disinfection and stabilization. Sodium chloride can change the osmotic pressure of bacterial cells, cause the bacteria to lose water, and inhibit the growth and reproduction of the bacteria. Sodium chloride and magnesium sulfate jointly regulate the ionic strength of the solution, and increase the stability of the product.

[0019] According to some embodiments of the present application, the mass percentage content of the sodium chloride is 2% to 8%, and the mass percentage content of the magnesium sulfate is 2% to 4%; preferably, the mass percentage content of the sodium chloride is 5%, and the mass percentage content of the magnesium sulfate is 3%.

[0020] According to some embodiments of the present application, the mass percentage content of the disinfecting component is 0.1% to 0.3%; preferably, the mass percentage content of the disinfecting component is 0.2%.

[0021] According to some embodiments of the present application, the disinfecting component comprises benzalkonium bromide.

[0022] In the present application, benzalkonium bromide, as a cationic surfactant, can be adsorbed on the surface of bacteria with negative electricity, change the permeability of the cell membrane, cause the substances in the cells to leak out, and cause the bacteria to die. Benzalkonium bromide has a strong disinfecting effect on both Gram-positive bacteria and Gram-negative bacteria. 0.2% benzalkonium bromide has little adverse effect on the function of the metabolic products of Salivaricella saliva, and benzalkonium bromide and the metabolic products of Salivaricella saliva can have a synergistic disinfecting effect: the metabolic products of Salivaricella saliva can destroy the cell membrane and cell wall of harmful bacteria, or interfere with the physiological processes such as protein synthesis and nucleic acid replication of the bacteria, and the disinfecting mechanisms of the two are different. The synergistic effect of 0.2% benzalkonium bromide and the metabolic products of Salivaricella saliva can more comprehensively destroy the structure and function of bacteria, and enhance the disinfecting effect.

[0023] According to some embodiments of the present application, the pet deodorizing disinfectant solution comprises the following ingredients in mass percentage: 5-20% of Lactobacillus salivarius metabolites, 5-15% of Artemisia leaf extract, 5-10% of Thymus extract, 2-8% of sodium chloride, 2-4% of magnesium sulfate, and 0.1-0.3% of benzalkonium bromide.

[0024] According to some embodiments of the present application, the pet deodorizing disinfectant solution comprises the following ingredients in mass percentage: 10% of Lactobacillus salivarius metabolites, 10% of Artemisia leaf extract, 8% of Thymus extract, 5% of sodium chloride, 3% of magnesium sulfate, and 0.2% of benzalkonium bromide.

[0025] According to some embodiments of the present application, the pet deodorizing disinfectant solution further comprises ethanol and water; preferably, the water is deionized water.

[0026] In the present application, ethanol is used to dissolve the active ingredients in the plant extracts and benzalkonium bromide, so that they are better dispersed in the solution and the disinfection effect is enhanced. At the same time, when ethanol evaporates, it takes away part of the heat, so that the product can dry quickly after use, reducing the risk of skin diseases caused by a humid environment in pets. Deionized water is used as a solvent to uniformly dilute various ingredients, so that the product reaches a suitable concentration, facilitating use and storage; at the same time, deionized water does not contain impurity ions, avoiding the influence of impurities on the quality and stability of the product.

[0027] According to some embodiments of the present application, the pet deodorizing disinfectant solution has a pH value of 6.8-7.2.

[0028] In the second aspect of the present application, a preparation method of the above-mentioned pet deodorizing disinfectant solution is provided, comprising the following steps:

[0029] S1, slowly adding the disinfectant to the plant extract, so that the disinfectant is fully combined with the plant extract;

[0030] S2, under the condition of continuous stirring, adding Lactobacillus salivarius metabolites, and then adding the prepared solution of salt substances, continuing to stir for 20-60 min to obtain a mixed solution;

[0031] S3, adding ethanol and water to the mixed solution, adjusting the pH value to 6.8-7.2, and then using water to dilute to the required total volume.

[0032] According to some embodiments of the present application, the preparation method specifically comprises the following steps:

[0033] S1, slowly adding benzalkonium bromide to the plant extract, so that the benzalkonium bromide is fully combined with the plant extract;

[0034] S2, under the condition of continuous stirring, the Lactobacillus salivarius metabolic product is added, and then the prepared salt solution is added, and the stirring is continued for 20-60 min to obtain a mixed solution;

[0035] S3, ethanol and deionized water are added to the mixed solution, the pH value is adjusted to 6.8-7.2, and then the deionized water is used to dilute to the required total volume.

[0036] According to some embodiments of the present application, the deodorizing and disinfecting solution is prepared according to the proportions of 10% Lactobacillus salivarius metabolic product, 10% Artemisia leaf extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate, and 0.2% benzalkonium bromide.

[0037] According to some embodiments of the present application, the process specifically comprises the following steps: the benzalkonium bromide is slowly added to the mixture of Artemisia leaf extract and thyme extract, and stirred while adding, to ensure that the benzalkonium bromide is fully combined with the plant extracts; under the condition of continuous stirring, the Lactobacillus salivarius metabolic product is added; then, the prepared salt solution is added, and the stirring is continued for 30 min to ensure uniform distribution of the components; an appropriate amount of ethanol and deionized water is added to the mixed solution, and the pH value of the solution is carefully adjusted to the range of 6.8-7.2 using a pH adjuster. After adjusting the pH value, the deionized water is used to dilute to the required total volume, and then the stirring is continued until uniform.

[0038] In a third aspect of the present application, a use method of the above-mentioned pet deodorizing and disinfecting solution is provided, wherein the pet deodorizing and disinfecting solution is directly sprayed on pet feces, urine, and living space of the pet for deodorization and disinfection.

[0039] According to one of the technical solutions in the method of the present application, at least the following beneficial effects are achieved: the present application prepares a pet deodorizing and disinfecting solution composed of Lactobacillus salivarius metabolic product, plant extract, disinfectant, and salt substance, which can be directly sprayed on pet feces, urine, and living space of the pet for deodorization and disinfection; compared with traditional disinfecting solution, the deodorizing and disinfecting solution has more significant disinfection effect and longer disinfection maintenance time, and also has persistent deodorizing effect; the pet deodorizing and disinfecting solution of the present application has good deodorizing and bacteriostatic effect, good safety, fast deodorizing speed, long deodorizing maintenance time, and is friendly to pets when used. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0041] Figure 1 is the acid resistance test result graph of Lactobacillus salivarius NJAUTYRGJ-Y in the embodiments of the present application; * indicates that the difference is significant (p<0.05) compared with the group with pH=5.7.

[0042] Figure 2 Figure 1 is a graph of the heat tolerance test results of Lactobacillus salivarius NJAUTYRGJ-Y in an embodiment of the present application.

[0043] Figure 3 Figure 2 is a graph of the bile salt tolerance test results of Lactobacillus salivarius NJAUTYRGJ-Y in an embodiment of the present application; * indicates significant difference (p<0.05) compared with the control group.

[0044] Figure 4 Figure 3 is a sequence alignment of Lactobacillus salivarius NJAUTYRGJ-Y in an embodiment of the present application; NR_112759.1 corresponds to the non-coding RNA sequence of Lactobacillus salivarius strain JCM 1231 in the NCBI database; NR_041498.1 corresponds to Lactobacillus hayakitensis strain KBL13; NR_104979.1 corresponds to Lactobacillus araffinosus strain LMG 23560; NR_112692.1 corresponds to Lactobacillus aviarius strain NBRC 102162; NR_113259.1 corresponds to Lactobacillus agilis strain JCM 1187; NR_112689.1 corresponds to Lactobacillus murinus strain NBRC 14221; g: NR_028623.1 corresponds to Lactobacillus equi strain YIT0455; h: NR_044703.2 corresponds to Lactobacillus aviarius strain DSM 20655; NR_179200.1 corresponds to Lactobacillus salitolerans strain YK43; j: NR_042677.1 corresponds to Liquorilactobacillus cacaonum strain LMG 24285.

[0045] Figure 5 Figure 4 is a graph of the paper disc diffusion method antibacterial test results in an embodiment of the present application; different letters in the figure indicate significant difference (p<0.05), the same letter indicates no significant difference between groups, and different letters indicate significant difference between groups.

[0046] Figure 6Figure is a result chart of feces odor score in the embodiment of the present application; different letters in the figure represent significant difference (p<0.05), the same letter represents no significant difference between groups, and different letters represent significant difference between groups.

[0047] Figure 7 Figure is a result chart of urine odor score in the embodiment of the present application; different letters in the figure represent significant difference (p<0.05), the same letter represents no significant difference between groups, and different letters represent significant difference between groups.

[0048] Figure 8 Figure is a result chart of odor score in the embodiment of the present application; A is the feces odor score; B is the urine odor score. * represents significant difference (p<0.05) compared with the con group, and # represents significant difference (p<0.05) compared with the group containing Lactobacillus salivarius metabolite deodorizing disinfectant.

[0049] Figure 9 Figure is the result of ammonia gas measured by pH method in the embodiment of the present application; * represents significant difference (p<0.05) compared with the con group, and # represents significant difference (p<0.05) compared with the group containing Lactobacillus salivarius metabolite deodorizing disinfectant.

[0050] Figure 10 Figure is the result of paper diffusion method antibacterial test in the embodiment of the present application; * represents significant difference (p<0.05) compared with the group containing Lactobacillus salivarius metabolite deodorizing disinfectant.

[0051] Figure 11 Figure is the result chart of colony after disinfection using deodorizing disinfectant in the embodiment of the present application; A: feces, B: urine, C: air.

[0052] Figure 12 Figure is the influence chart of deodorizing disinfectant on dog food intake and dog respiratory rate in the embodiment of the present application; A1, B1, C1 in the left chart represent the food intake of the three dogs before the test, and A2, B2, C2 represent the food intake of the three dogs after the test; A in the right chart represents before the test, and B, C, D, E, F represent the respiratory rate of the dog at 5min, 30min, 1h, 6h, 24h after spraying, respectively. ns represents no significant difference.

[0053] Figure 13 Figure is the influence chart of deodorizing disinfectant on mouse physiological activity in the embodiment of the present application; A: influence on mouse food intake, B: influence on mouse water intake, C: influence of deodorizing disinfectant on mouse weight; A1, B1, C1 in the chart are the food intake of the control group, the medium-dose group and the high-dose group before the test, respectively. A2, B2, C2 are the food intake of the control group, the medium-dose group and the high-dose group after the test, respectively. ns represents no significant difference.

[0054] Figure 14 Figure is the influence chart of deodorizing disinfectant on mouse intact skin in the embodiment of the present application.

[0055] Figure 15 Figure is a diagram of the influence of the deodorizing disinfectant solution on the broken skin of mice in the embodiments of the present application; A1, B1, C1, D1, E1 represent the conditions of the wounds of the control group at 1d, 2d, 4d, 7d, 14d respectively; A2, B2, C2, D2, E2 represent the conditions of the wounds of the test group at 1d, 2d, 4d, 7d, 14d respectively.

[0056] Figure 16 Figure is a diagram of the influence of the deodorizing disinfectant solution on the biochemical indexes of mice in the embodiments of the present application.

[0057] Figure 17 Figure is a diagram of the influence of the deodorizing disinfectant solution on the liver, lung and kidney of mice in the embodiments of the present application. DETAILED DESCRIPTION

[0058] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is described and explained below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application. Based on the embodiments provided in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0059] Obviously, the accompanying drawings in the following description are only some examples or embodiments of the present application, and for those of ordinary skill in the art, the present application can be applied to other similar scenarios without creative efforts based on these drawings. In addition, it can be understood that although the efforts made in this development process can be complex and lengthy, for those of ordinary skill in the art related to the content disclosed in the present application, some designs, manufacturing or production changes based on the technical content disclosed in the present application are only routine technical means and should not be understood as insufficient disclosure of the present application.

[0060] In the present application, the phrase "embodiments" means that the specific features, structures or properties described in conjunction with the embodiments can be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily mean the same embodiment, nor is it an independent or alternative embodiment to other embodiments. Those of ordinary skill in the art explicitly and implicitly understand that the embodiments described in the present application can be combined with other embodiments without conflict.

[0061] Unless otherwise defined, technical terms and scientific terms used in the present application shall have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms "a", "an", "one", "this", and similar referents in the context of describing the application are to be construed to be inclusive, both singular and plural, unless otherwise indicated. The terms "comprising", "comprises" and "comprised of" as well as conjugations thereof, are used herein to mean that the process, method, composition, or product includes, but is not limited to, those elements specifically recited. The term "consisting essentially of" as used herein means including, but not limited to, those elements specifically recited in the claim, and optionally including additional elements that do not materially affect the basic and novel characteristics of the composition or method. The term "consisting of" as used herein means including, but not limited to, those elements specifically recited in the claim, and no additional elements. The term "a plurality" or "a number of" as used herein means two or more. The term "and / or" as used herein means "and", "or" or both. The term "a" or "an" as used herein means "one or more". The term "at least one" as used herein means "one or more". The term "first", "second", "third", etc. as used herein merely denote different classes, and do not imply a specific order or sequence unless otherwise indicated.

[0062] The reagents and consumables described in the present application are all commercially available unless otherwise specified.

[0063] Example 1: Isolation and screening of Lactobacillus salivarius NJAUTYRGJ-Y

[0064] This example describes in detail the steps of isolation, screening and identification of Lactobacillus salivarius NJAUTYRGJ-Y.

[0065] ① Isolation and screening

[0066] Healthy dogs were selected, and sterile cotton swabs were used to gently wipe the oral mucosa, gums and tongue surface of the dogs. The cotton swabs were placed into a sampling tube containing sterile normal saline and shaken well to obtain a bacterial suspension. The bacterial suspension was inoculated onto MRS agar medium containing calcium carbonate by the spread plate method, and the plate was placed in a 37℃ incubator and cultured anaerobically for 48h. After the culture ended, single colonies with typical lactic acid bacteria colony characteristics such as round shape, smooth surface, neat edges and milky white color were selected from the plate. On this basis, colonies with strong acid-producing ability and large calcium dissolution ring diameter were selected, which were preliminarily judged to be Lactobacillus salivarius. The preliminarily selected colonies were subjected to Gram staining, and rod-shaped Gram-positive bacteria were selected. Further, the colonies were picked onto a glass slide, and 3% hydrogen peroxide solution was added dropwise. No gas bubbles were generated, which met the characteristics of Lactobacillus salivarius.

[0067] The bacterial genomic DNA extraction kit was used to extract DNA from the strains after rescreening. The extracted DNA was used as a template for PCR amplification of the 16S rRNA gene with universal primers. The PCR reaction conditions were set as 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, a total of 30 cycles, and finally 72℃ extension for 10 min. The amplified 16S rRNA gene fragments were sequenced. The measured sequence was subjected to BLAST comparison in the GenBank database of NCBI to determine whether it was Lactobacillus salivarius.

[0068]

Preservation Information

[0069] ②Acid tolerance experiment

[0070] OD 600 is the absorbance of the bacterial culture solution at 600 nm wavelength, which can be used to measure the growth density of bacteria. As shown in Figure 1 compared with the optimal pH 5.7 of Lactobacillus salivarius, the growth density of Lactobacillus salivarius NJAUTYRGJ-Y was significantly reduced when the pH was 2 and 7; when the pH was in the range of 3 to 5, the bacterial growth density increased significantly, and the growth density increased with the increase of culture time. This shows that Lactobacillus salivarius NJAUTYRGJ-Y has certain selectivity and adaptability to pH value, and its growth will be inhibited when pH≤2 or ≥7, and its growth density is positively correlated with culture time in a suitable pH environment.

[0071] ③Heat tolerance experiment

[0072] As shown in Figure 2 compared with the control group, the growth density of Lactobacillus salivarius NJAUTYRGJ-Y did not change significantly after being heated at 46℃ for 30 min. This shows that Lactobacillus salivarius NJAUTYRGJ-Y has good heat resistance and can maintain high activity during processing and storage.

[0073] ④Bile salt tolerance experiment

[0074] As shown in Figure 3 compared with the control group, the growth density of Lactobacillus salivarius NJAUTYRGJ-Y did not change significantly when the bile salt concentration was 0.1%; when the bile salt concentration was 0.2% and 0.3%, the growth density decreased significantly. This shows that Lactobacillus salivarius NJAUTYRGJ-Y has certain bile salt tolerance.

[0075] ⑤16S rDNA sequence information

[0076] Sequence alignment was performed using the Basic Local Alignment Search Tool (BLAST). The top ten sequences with the highest similarity to *Lactobacillus salivarius* NJAUTYRGJ-Y all originated from different strains of *Lactobacillus salivarius*. Figure 4

[0077] Deodorization mechanism of Lactobacillus salivarius metabolites:

[0078] (1) Lactobacillus salivarius metabolites include organic acids such as lactic acid and acetic acid, bacteriocins, proteases, amylases, vitamins, etc. The deodorization mechanism of Lactobacillus salivarius metabolites is as follows:

[0079] Organic acids: Lactobacillus salivarius metabolites include organic acids such as lactic acid and acetic acid, which can lower the pH of the environment. Most odor-producing bacteria thrive in neutral or weakly alkaline environments, and acidic environments can inhibit their growth and reproduction. At the same time, acidic environments can affect the chemical stability and volatility of odorants. For example, ammonia can react with acid under acidic conditions to form ammonium salt, which has much lower volatility than ammonia, thereby reducing the concentration of ammonia in the air and reducing odor.

[0080] Enzymes: Lactobacillus salivarius metabolites contain various enzymes such as proteases and lipases. Proteases can break down protein odorants in the environment into small peptides and amino acids, reducing the odor produced by the decomposition of proteins by harmful bacteria. Lipases break down fatty substances to prevent their oxidation and rancidity.

[0081] Bacteriocins: Bacteriocins in Lactobacillus salivarius metabolites can reduce the number of odor-producing harmful bacteria in the environment by disrupting the integrity of harmful bacterial cell membranes, interfering with nucleic acid and protein synthesis, etc.

[0082] (2) Odor components that can be removed by Lactobacillus salivarius metabolites also include:

[0083] Sulfides: Hydrogen sulfide, methyl mercaptan and other sulfides are common odor components in animal urine and feces. Organic acids in Lactobacillus salivarius metabolites can reduce the volatility of sulfides by lowering the pH of the environment. In addition, bacteriocins and organic acids in the metabolites can inhibit the growth of harmful bacteria that produce sulfides, reducing the generation of hydrogen sulfide and methyl mercaptan.

[0084] Volatile fatty acids: such as butyric acid, propionic acid, etc., have a strong acid odor. Organic acids in Lactobacillus salivarius metabolites can inhibit the activity of microorganisms that produce volatile fatty acids by lowering the pH of the environment. In addition, enzymes in the metabolites can promote the catabolism of volatile fatty acids, converting them into odorless or low-odor substances.

[0085] Aromatic heterocyclic organic compounds: indole and skatole (3-methylindole) are important components of fecal odor. Lactobacillus salivarius metabolites can inhibit the growth and metabolism of bacteria that produce indole and skatole, and can also inhibit the conversion of tryptophan and other precursor substances to indole and skatole, thereby improving the odor of the environment.

[0086] (3) The saliva lactobacillus metabolite for pet deodorization and disinfection solution also has the following functional effects:

[0087] Synergistic bacteriostasis: The bacteriocin in the saliva lactobacillus metabolite can specifically inhibit specific harmful bacteria such as Staphylococcus aureus and Escherichia coli. When combined with benzalkonium bromide, it can synergistically inhibit bacteria and enhance disinfection effect.

[0088] Regulation of microecological balance: The organic acids in the saliva lactobacillus metabolite can reduce the pH value of the environment and inhibit the survival and reproduction of harmful bacteria. At the same time, it creates suitable living conditions for beneficial bacteria in the environment and promotes the colonization of beneficial bacteria.

[0089] Promote the decomposition of organic matter: The saliva lactobacillus metabolite contains various enzyme substances such as protease, amylase, and lipase, which can accelerate the decomposition of organic waste such as feces.

[0090] Example 2: Optimization of the ratio of pet deodorization and disinfection solution

[0091] This example describes in detail the optimization process of the ratio of a pet deodorization and disinfection solution.

[0092] The formula of the basic pet deodorization and disinfection solution is: 10% Artemisia leaf extract (purchased from Ningshan Guosheng Biotechnology Co., Ltd.), 8% thyme extract (purchased from Ningshan Guosheng Biotechnology Co., Ltd.), 5% sodium chloride, 3% magnesium sulfate, and 0.2% benzalkonium bromide. Therefore, only the optimal ratio of saliva lactobacillus metabolite needs to be explored on this basis. According to the ratio of 5%-50% saliva lactobacillus metabolite, 10% Artemisia leaf extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate, and 0.2% benzalkonium bromide, multiple groups of experiments were set up. At the same time, 5%-50% saliva lactobacillus metabolite (including 5%, 10%, 20%, 30%, 40%, and 50% saliva lactobacillus metabolite, respectively), deodorization and disinfection solution containing 5%-50% saliva lactobacillus metabolite, and only using the basic deodorization and disinfection solution as a control were used for deodorization and disinfection tests. The deodorization and disinfection effects of each group were analyzed, the differences between different ratios were compared, and the ratio scheme with lower cost while ensuring good disinfection and deodorization effect was selected.

[0093] Among them, the preparation method of saliva lactobacillus metabolite is as follows: saliva lactobacillus is inoculated into MRS broth medium at an inoculation amount of 10%, and cultured anaerobically at 37℃ for 48h. After the culture is completed, the culture solution is centrifuged at 10000rpm for 30min to separate the supernatant and the bacterial body. The bacterial body is resuspended with PBS, and then ultrasonically broken in an ice bath for 1h. Subsequently, it is centrifuged at 10000rpm for 30min, and the supernatant is mixed with the culture solution and filtered using a 0.22μm sterile filter membrane to obtain a sterile saliva lactobacillus metabolite solution.

[0094] Through the optimization screening of the present embodiment, it is determined that 10% Lactobacillus salivarius metabolites can ensure better disinfection and deodorization effect and lower cost.

[0095] The paper disc diffusion method inhibition test results show that the deodorizing disinfectant containing 10% Lactobacillus salivarius metabolites has a significantly better inhibitory effect on Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae than the deodorizing disinfectant without adding metabolites. In addition, compared with the 10% Lactobacillus salivarius metabolite group, the inhibitory effect of the group with a concentration higher than 10% does not change significantly. Figure 5 At the same time, the odor score results show that all concentrations of Lactobacillus salivarius metabolites and deodorizing disinfectants containing Lactobacillus salivarius metabolites have good deodorizing effect, but only the deodorizing disinfectant containing Lactobacillus salivarius metabolites has the advantages of fast-acting and long-lasting, and the 10% Lactobacillus salivarius metabolite concentration can achieve ideal deodorizing effect. Figure 6 Figure 7 The above results show that 10% Lactobacillus salivarius metabolites are the optimal concentration of deodorizing disinfectant.

[0096] Example 3: Preparation of pet deodorizing disinfectant

[0097] The present embodiment describes in detail a pet deodorizing disinfectant and its preparation method, and the component ratio is (mass percentage): 10% Lactobacillus salivarius metabolites, 10% Artemisia leaf extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate, 0.2% benzalkonium bromide. In addition, it also includes an appropriate amount of ethanol, and deionized water is used as a solution to prepare the pet deodorizing disinfectant.

[0098] The preparation method of the pet deodorizing disinfectant includes: slowly adding benzalkonium bromide to the mixture of Artemisia leaf and thyme extract, stirring while adding, to ensure that benzalkonium bromide and plant extract are fully combined; under continuous stirring, add Lactobacillus salivarius metabolites; then, add the prepared salt solution, continue stirring for 30 min to ensure uniform distribution of all components; add an appropriate amount of ethanol and deionized water to the mixed solution, and carefully adjust the pH value of the solution to the range of 6.8-7.2 using a pH adjuster. After adjusting the pH value, make up to the required total volume with deionized water, and then continue to stir uniformly.

[0099] The present embodiment lists other pet deodorizing disinfectants with different component ratios, and the component ratios are (mass percentage):

[0100] (1) 10% Lactobacillus salivarius metabolites, 8% Artemisia leaf extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate, 0.2% benzalkonium bromide.

[0101] ​(2) 10% Lactobacillus salivarius metabolite, 12% Artemisia leaf extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate, 0.2% benzalkonium bromide.

[0102] (3) 10% Lactobacillus salivarius metabolite, 8% Artemisia leaf extract, 5% thyme extract, 5% sodium chloride, 3% magnesium sulfate, 0.2% benzalkonium bromide.

[0103] (4) 10% Lactobacillus salivarius metabolite, 8% Artemisia leaf extract, 10% thyme extract, 5% sodium chloride, 3% magnesium sulfate, 0.2% benzalkonium bromide.

[0104] (5) 10% Lactobacillus salivarius metabolite, 8% Artemisia leaf extract, 8% thyme extract, 4% sodium chloride, 2% magnesium sulfate, 0.2% benzalkonium bromide.

[0105] (6) 10% Lactobacillus salivarius metabolite, 8% Artemisia leaf extract, 8% thyme extract, 6% sodium chloride, 4% magnesium sulfate, 0.2% benzalkonium bromide.

[0106] In addition, an appropriate amount of ethanol is also included, and deionized water is used as a solution to prepare the pet deodorizing disinfectant. The preparation method is the same as above. The pet deodorizing disinfectants prepared above have similar effects, and the effect of 10% Lactobacillus salivarius metabolite, 10% Artemisia leaf extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate, and 0.2% benzalkonium bromide is the best (the pet deodorizing disinfectant prepared under the optimal conditions is used in the following examples). The preparation method of the Lactobacillus salivarius metabolite is the same as that in Example 2.

[0107] Example 4: Deodorizing effect experiment of pet deodorizing disinfectant

[0108] This example describes in detail the deodorizing effect experiment of the pet deodorizing disinfectant described above, and comparative tests are carried out in terms of deodorization and ammonia removal.

[0109] 4.1 Feces and urine deodorizing effect test

[0110] Take 2g of feces and 10mL of urine respectively and place them in a culture dish. Use a spray bottle to spray the deodorizing disinfectant on the surface of the feces and urine. Score according to the standard of the odor rating table (Table 1) at 1min before spraying and at a certain time after spraying. Take the average score of the scores of four people to evaluate the deodorizing effect of the deodorizing disinfectant on the odor of feces and urine.

[0111] Table 1. Odor rating standard table

[0112]

[0113] The deodorizing effect test of feces and urine was carried out using saliva lactobacillus metabolites, base deodorizing disinfectant without saliva lactobacillus metabolites, and deodorizing disinfectant containing saliva lactobacillus metabolites respectively. The results show that the deodorizing effect is slow when only saliva lactobacillus metabolites are used, and the deodorizing effective time is short when base deodorizing disinfectant without saliva lactobacillus metabolites is used. The deodorizing disinfectant containing saliva lactobacillus metabolites improves both of the above deficiencies, and the deodorizing effect of the deodorizing disinfectant containing saliva lactobacillus metabolites is better than that of the other two groups (whether for feces or urine). Figure 5

[0114] 4.2 pH value method for detecting the ammonia removal effect of self-developed deodorizing disinfectant

[0115] The deodorizing disinfectant was sprayed in the container, 10% ammonia solution and boric acid solution were each put in the container for 25 mL, and then the container was sealed. The pH detector was used to measure the pH value of boric acid before and after the deodorizing disinfectant was sprayed. The control group was sprayed with normal saline.

[0116] Saliva lactobacillus metabolites, deodorizing disinfectant containing saliva lactobacillus metabolites, and deodorizing disinfectant without saliva lactobacillus metabolites all have good ammonia removal effect. However, over time, the ammonia removal effect of the deodorizing disinfectant containing saliva lactobacillus metabolites is significantly better than that of the base deodorizing disinfectant without saliva lactobacillus metabolites after 2 hours. Figure 6

[0117] The above experimental results prove that the pet deodorizing disinfectant with 10% saliva lactobacillus metabolites has better deodorizing performance.

[0118] Example 5: Disinfection effect experiment of pet deodorizing disinfectant

[0119] The example describes in detail the disinfection effect experiment of the above pet deodorizing disinfectant.

[0120] 5.1 Paper disc diffusion test

[0121] The autoclaved blank drug sensitivity sheet was placed in the deodorizing disinfectant containing different concentrations of bacterial liquid diluents, and then it was pasted on the culture dish coated with Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae after standing for 5 min. The results were observed after incubation at 37℃ for 24 h, and the average value of the inhibition zone diameter was calculated.

[0122] Saliva lactobacillus metabolites, deodorizing disinfectant containing saliva lactobacillus metabolites, and deodorizing disinfectant without saliva lactobacillus metabolites were used respectively for the paper disc diffusion method antibacterial test of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. As shown in Figure 7 , the deodorizing disinfectant containing saliva lactobacillus metabolites has significantly better disinfection effect on the three kinds of bacteria than the other two groups. ​​

[0123] 5.2 Test tube dilution method to determine MIC

[0124] Take 0.1 mL of deodorizing disinfectant dilution, add 1 x 10 6 CFU / mL of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae bacterial suspension, and set up sterile normal saline as negative control, only add bacteria as positive control. After 24h culture at 37℃, spread on agar medium, and observe bacterial growth after 24h culture at 37℃. Calculate MIC value.

[0125] From Table 5, the MIC of the self-developed deodorizing disinfectant for Staphylococcus aureus and Escherichia coli is 1:8, and for Klebsiella pneumoniae is 1:4. Therefore, the self-developed deodorizing disinfectant has significant bacteriostatic effect on Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, and the bacteriostatic effect of the deodorizing disinfectant on the three pathogenic bacteria as a whole shows concentration dependence.

[0126] Table 2. Test tube dilution method test results

[0127]

[0128] In Table 2, - and + represent whether there are colonies in the medium. The negative control is sterile normal saline, and there is no colony growth. The negative control is solid medium inoculated with bacteria, and colonies grow.

[0129] 5.3 Fecal, urine and environmental disinfection effect test of self-developed deodorizing disinfectant

[0130] Take 0.1 mL of diluted fecal and urine liquid culture medium and spread on the plate for 24h, then count the colonies. Select the dilution concentration with 30-300 colonies as the test fecal and urine dilution concentration, mix with the deodorizing disinfectant as the test group, and mix with normal saline as the control group.

[0131] The environmental disinfection test was carried out in a relatively closed space. Before and 10 min after spraying the deodorizing disinfectant, the culture dish with medium was placed on a table about 1m high in the room, and the plate was placed for 5 min and then covered. After 24h culture at 37℃, the number of bacteria in the plate was determined. According to the change of colony number, the deodorizing disinfectant for air disinfection effect was evaluated.

[0132] From Figure 8(A: feces; B: urine; C: air) can be seen, the number of colonies in the test group and the number of colonies in the control group are very significant, and after calculation, the bactericidal rate of the deodorizing disinfectant for bacteria in feces can reach 99.05%, the bactericidal rate for bacteria in urine can reach 99.09%, and the bactericidal rate for bacteria in air is 82.79%. The experiment proves that the self-developed deodorizing disinfectant has good bactericidal and disinfecting effect on feces, urine and air.

[0133] Example 6: Safety experiment of pet deodorizing disinfectant

[0134] The example details the safety experiment of the above-mentioned pet deodorizing disinfectant.

[0135] 6.1 Test on the influence of deodorizing disinfectant on the food intake and respiratory rate of dogs

[0136] 10 minutes before feeding every day, 10 mL of the self-developed deodorizing disinfectant was sprayed on the faces of three test dogs, and then the food intake of the dogs was continuously recorded for 5 days to evaluate the influence of the deodorizing disinfectant on the food intake of the dogs. The respiratory rates of the dogs before and after spraying the deodorizing disinfectant were recorded to preliminarily evaluate whether the deodorizing disinfectant has adverse effects on the respiratory system of the dogs. The results are shown in Table 1. Figure 9 The experimental results show that the deodorizing disinfectant has no obvious influence on the food intake of the three dogs with different body types, indicating that the deodorizing disinfectant has no obvious influence on the appetite of the dogs. The deodorizing disinfectant has no obvious influence on the respiratory rate of the dogs, and the respiratory rates of the three dogs are within the normal range before and after the use of the deodorizing disinfectant, which preliminarily indicates that the deodorizing disinfectant has no obvious adverse effects on the respiration of the dogs.

[0137] 6.2 Test on the irritability of the deodorizing disinfectant to dogs

[0138] Three healthy test dogs were selected, and the deodorizing disinfectant was sprayed on the whole body of the dogs at a distance of 30 cm above the dogs and on the cages where the dogs lived, once a day for 5 consecutive days. It was observed and recorded whether the dogs had adverse reactions after spraying. The results show that the dogs did not have related symptoms after the use of the deodorizing disinfectant on the three dogs, indicating that the deodorizing disinfectant has no obvious irritability to the dogs.

[0139] 6.3 Test on the influence of the deodorizing disinfectant on the food intake, water intake and body weight of mice

[0140] Fifteen healthy mice were selected and divided into a control group, a medium-dose group and a high-dose group, with 5 mice in each group. The control group was sprayed with normal saline, the medium-dose group was sprayed with the deodorizing disinfectant diluted by one time, and the high-dose group was sprayed with the deodorizing disinfectant, twice a day in the cages. The daily food consumption, the daily water intake and the body weight of the mice were weighed to detect the influence of the deodorizing disinfectant on the three indicators of the mice.

[0141] As shown in Table 2, the deodorizing disinfectant has no obvious influence on the food intake, water intake and body weight of the mice, indicating that the deodorizing disinfectant has no obvious influence on the three indicators of the mice. Figure 10The results show that the daily food intake, water intake and body weight of mice in each group have no significant change before and after using the deodorizing disinfectant, indicating that the deodorizing disinfectant independently developed by the test has no significant effect on the physiological indicators of mice.

[0142] 6.4 Skin irritation test on mice

[0143] The skin irritation test refers to the skin irritation related specifications in the cosmetic safety technology specification. The test mice are divided into intact skin test group, intact skin control group, damaged skin test group and damaged skin control group, with 5 mice in each group. The back area of the mice is depilated before application, and the skin of the damaged group of mice is cut with a "one" character. The deodorizing disinfectant and normal saline are applied to the depilated area of the test group and control group of mice respectively every day, and the depilated area of each group is observed and recorded for 14 days. According to the mouse skin redness (Table 3) and mouse skin irritation reaction score standard (Table 4), the average score of irritation is calculated, and the average score of irritation = (total score of erythema + total score of edema) / total number of animals.

[0144] Table 3. Skin irritation score standard

[0145]

[0146] Table 4. Skin irritation evaluation standard

[0147]

[0148] Table 5. Evaluation of mouse skin irritation test

[0149]

[0150] In Table 5, "-" means no irritation; "+" means mild irritation; "++" means moderate irritation; and "+++" means strong irritation.

[0151] As shown in Table 5, Figure 11 and Figure 12 It can be seen that the deodorizing disinfectant has no abnormal symptoms such as erythema and edema on the skin of mice within two weeks of use on mice with intact skin, indicating that the deodorizing disinfectant has no significant irritation to the intact skin of mice. Compared with the control group using normal saline, the test group using the deodorizing disinfectant in the damaged skin group has approximately the same wound condition and recovery speed, and the deodorizing disinfectant has no significant adverse effect on damaged skin.

[0152] 6.5 Effect of deodorizing disinfectant on serum biochemical indicators of mice

[0153] The experimental mice were divided into a control group, a medium-dose group, and a high-dose group, with 5 mice in each group. The control group was sprayed with 5 mL of physiological saline, the medium-dose group was sprayed with 5 mL of a 1:1 diluted deodorizing disinfectant, and the high-dose group was sprayed with 5 mL of deodorizing disinfectant in each group's cage twice a day for 14 days. The mice were then sacrificed, and their blood was collected to measure alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine levels.

[0154] Depend on Figure 13 The study investigated the effects of the deodorizing disinfectant on serum urea nitrogen, creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels in mice two weeks after spraying. The results showed no significant differences (P > 0.05) between the low-dose group, the high-dose group, and the control group; all levels fluctuated within the normal range. This experiment demonstrates that the deodorizing disinfectant has no significant adverse effects on serum biochemical indicators in mice and no significant toxicity to ICR mice, indicating that the deodorizing disinfectant has good safety.

[0155] 6.6 Effects on the liver, lungs, and kidneys of mice

[0156] The mice were divided into a control group and an experimental group, with 5 mice in each group. The control group was sprayed with physiological saline, while the experimental group was sprayed with deodorizing disinfectant in each cage twice a day for 14 days. The mice were then sacrificed, and their livers, lungs, and kidneys were collected to prepare tissue sections. After staining with hematoxylin-eosin solution, the sections were observed under a microscope.

[0157] Depend on Figure 14 The liver sections from mice showed a central vein at the center of the liver lobules, with a well-preserved radial structure radiating outwards from the central vein. Lung sections revealed intact alveoli and bronchioles without sclerosis or other lesions. Kidney sections showed intact glomerular structures, good kidney cell morphology, clear and intact glomerular and tubular structures, and intact renal vascular structures without obvious lesions. No significant abnormalities were found in the lung and kidney sections, indicating that this deodorizing disinfectant had no significant adverse effects on the liver, lungs, or kidneys of mice.

[0158] The pet deodorizing disinfectant of this invention has a very significant deodorizing effect on feces, urine, and ammonia, and can maintain the effect for a long time; it has a good antibacterial effect on three common pathogens (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae), and has a good disinfection effect on feces, urine, and air; and it has good safety and no toxic side effects.

[0159] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A pet deodorizing and disinfecting liquid, characterized in that, The pet deodorizing and disinfecting solution contains Lactobacillus salivarius metabolites, plant extracts, disinfectants, and salts; wherein the Lactobacillus salivarius is Lactobacillus salivarius NJAUTYRGJ-Y, with accession number CCTCC NO: M20242787.

2. The pet deodorizing and disinfecting liquid as described in claim 1, characterized in that, The nucleotide sequence of the 16S RNA of *Lactobacillus salivarius* NJAUTYRGJ-Y is shown in SEQ ID NO.

1.

3. The pet deodorizing and disinfecting solution as described in claim 1, characterized in that, The pet deodorizing and disinfecting solution contains the following ingredients by weight percentage: 5%–20% Lactobacillus salivarius metabolites, 10%–25% plant extracts, 4%–12% salts and 0.1%–0.3% disinfectant.

4. The pet deodorizing and disinfecting solution as described in claim 1, characterized in that, The plant extract includes Artemisia argyi extract and thyme extract; and / or The disinfectant contains benzalkonium bromide; and / or The salt components include sodium chloride and magnesium sulfate.

5. The pet deodorizing and disinfecting solution as described in claim 4, characterized in that, The pet deodorizing and disinfecting solution contains the following ingredients by weight percentage: 5%–20% Lactobacillus salivarius metabolites, 5%–15% Artemisia argyi extract, 5%–10% Thyme extract, 2%–8% sodium chloride, 2%–4% magnesium sulfate, and 0.1%–0.3% benzalkonium bromide.

6. The pet deodorizing and disinfecting liquid as described in claim 5, characterized in that, The pet deodorizing and disinfecting solution contains the following ingredients by weight percentage: 10% Lactobacillus salivarius metabolites, 10% Artemisia argyi extract, 8% thyme extract, 5% sodium chloride, 3% magnesium sulfate and 0.2% benzalkonium bromide.

7. The pet deodorizing and disinfecting liquid as described in claim 1, characterized in that, The pet deodorizing and disinfecting solution also contains ethanol and water.

8. The pet deodorizing and disinfecting liquid as described in claim 1, characterized in that, The pH value of the pet deodorizing and disinfecting solution is 6.8 to 7.

2.

9. The method for preparing the pet deodorizing and disinfecting liquid according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1. Slowly add the disinfectant to the plant extract to allow the disinfectant and plant extract to fully mix; S2. While stirring continuously, add the Lactobacillus salivarius metabolites, then add the prepared salt solution, and continue stirring for 20-60 minutes to obtain a mixed solution. S3. Add ethanol and water to the mixed solution, adjust the pH value to 6.8-7.2, and then dilute with water to the required total volume.

10. The method of using the pet deodorizing and disinfecting solution as described in any one of claims 1 to 8, characterized in that, The pet deodorizing and disinfecting solution is sprayed directly onto pet feces, urine, and the pet's living space for deodorization and disinfection.

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