Pediococcus pentosaceus King33 for relieving excrement odor of pet dogs and cats as well as fungicide and application of pediococcus pentosaceus King33

Abstract

The invention provides pediococcus pentosaceus King33 for relieving excrement odor of pet dogs and cats as well as a microbial agent and application of the pediococcus pentosaceus King33, and belongs to the technical field of microorganisms. The preservation number of the pediococcus pentosaceus King33 is CGMCC (China General Microbiological Culture Collection Center) No.31050, the pediococcus pentosaceus King33 is classified and named as pediococcus pentosaceus, and the preservation date is June 24, 2024. According to the pediococcus pentosaceus King33 disclosed by the invention, the odor in cat excrement can be reduced, and the excrement quality is improved; according to the pediococcus pentosaceus King33 disclosed by the invention, the content of volatile organic compounds and biogenic amines in cat feces can be reduced; the pediococcus pentosaceus King33 disclosed by the invention can be used for relieving body odor, halitosis and urine odor of dogs.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pediococcus pentosaceus King33 for alleviating the odor of feces of pet dogs and cats, and a bacterial agent and application thereof. Background Art

[0002] With the rapid development of modern society, more and more people are raising pets to relieve work stress and fatigue. At the same time, pet cleaning and deodorization have also become the mainstream needs of "poop shovelers". The components of the odor produced by pets are not only harmful to the respiratory system and skin of pets, but also have a certain impact on human eyes, respiratory system, blood transportation, etc. The odor produced by pets generally comes from bad breath in the mouth, odor in the soles of the feet, odor in the hair, odor in the skin and odor in the digestive system (i.e. odor in excrement). For dogs and cats, the biggest source of odor is the odor in the digestive system (i.e. odor in excrement), especially after consuming excessive high-protein diets and when the stomach and intestines are in a pathological state or indigestion, the foul smell in the feces will also increase. Considering that dogs and cats are more sensitive to odors than humans and have the habit of licking, higher safety requirements are placed on related products.

[0003] At present, the main methods to reduce the odor of cat feces are to use deodorizing cat litter and closed cat litter box. New cat litters with deodorizing factors such as dandelion extract, compound enzyme preparations or probiotics have stronger deodorizing and antibacterial functions than traditional ordinary tofu cat litter, but they are expensive, need to be replaced regularly, and are only for external use, which only treats the symptoms but not the root cause. The use of closed cat litter boxes can be physically isolated, but the odor still exists when feces accumulate or the excretion site is changed. In addition, studies have shown that adding different deodorizing raw materials (enzyme preparations, plant extracts, yucca extracts, montmorillonite, zeolite and bentonite, etc.) to the diet can help reduce the production of harmful gases such as ammonia, thereby reducing odor, but the odorous gas in dog and cat feces is a mixture of multiple components. In addition to ammonia, there are also a variety of biogenic amines and volatile organic compounds. Therefore, there is an urgent need for a healthy, safe, efficient, easy-to-operate, time-saving, labor-saving and cost-saving method.

[0004] There are many types of probiotics, which are widely distributed. They mainly come from the host intestines and have the functions of regulating gastrointestinal function, preventing or improving diarrhea, improving digestion and absorption, and improving immunity. Studies have shown that Bacillus subtilis and Bacillus licheniformis can reduce the odor of dog feces, reduce the content of phenols and quinoline, and the concentration of decay compounds while improving intestinal health (Silvino TB, Cristina DLD, Menezes MCS, et al. Bacillus subtilis and Bacillus licheniformis reduce faecal protein catabolites concentration and odour in dogs. [J]. BMC veterinary research, 2020, 16 (1): 116.). There are no reports on the use of lactic acid bacteria such as Pediococcus pentosaceus to improve the odor of dog and cat feces. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a Pediococcus pentosaceus King33 and its bacterial agent and application for alleviating the odor of pet dog and cat feces, which can improve the intestinal health of dogs and cats through a healthy, safe, efficient, easy-to-operate, time-saving, labor-saving and cost-saving method, while reducing the malodor in dog and cat feces, improving the quality of dog and cat feces, and reducing the content of biogenic amines and volatile organic compounds in dog and cat feces.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A Pediococcus pentosaceus King33 for alleviating the odor of pet dog and cat feces, wherein the Pediococcus pentosaceus King33 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on June 24, 2024, with a deposit number of CGMCC No.31050 and a classification name of Pediococcus pentosaceus Pediococcus pentosaceus The storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a zip code of 100101.

[0007] A bacterial agent prepared using the Pediococcus pentosaceus King33; In the bacterial agent, the number of bacteria is 0.8-1.2×10 10 cfu / mL; In the bacterial agent, the Pediococcus pentosaceus King33 is a live bacterium or an inactivated bacterium; When the Pediococcus pentosaceus King33 is a live bacterium, the preparation method of the bacterial agent is to activate and ferment the Pediococcus pentosaceus King33 to obtain a bacterial solution, collect the bacterial bodies after centrifugation, resuspend them in reconstituted skim milk to obtain a bacterial suspension, freeze-dry them to obtain bacterial powder, and then mix them with oligomaltose to obtain a bacterial agent; When the Pediococcus pentosaceus King33 is an inactivated bacterium, the preparation method of the bacterial agent is to activate and ferment the Pediococcus pentosaceus King33 to obtain a bacterial liquid, collect the bacterial bodies after centrifugation, resuspend them in reconstituted skim milk to obtain a bacterial suspension, sterilize and freeze-dry to obtain bacterial powder, and then mix it with oligomaltose to obtain a bacterial agent.

[0008] The application of Pediococcus pentosaceus King33 in preparing products for alleviating the odor of pet dog and cat feces.

[0009] Compared with the prior art, the present invention has the following beneficial effects: (1) The Pediococcus pentosaceus King33 of the present invention can reduce the odor in cat feces and improve the quality of feces; (2) The Pediococcus pentosaceus King33 of the present invention can reduce the content of volatile organic compounds and biogenic amines in cat feces; (3) The Pediococcus pentosaceus King33 of the present invention can relieve dog body odor, bad breath, and urine odor. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The feces odor evaluation results of fresh feces in Example 4; Figure 2 The results of the feces odor evaluation of feces 6 hours after defecation in Example 4; Figure 3 The body odor scores of the test dogs in Example 8 on the first and 30th days of the test; Figure 4 The halitosis scores of the test dogs in Example 8 on the first and 30th days of the test; Figure 5 The urine odor scores of the test dogs in Example 8 on the 1st and 30th days of the test. DETAILED DESCRIPTION

[0011] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0012] The preparation method of the MRS plate culture medium is as follows: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, and 15g of agar are dissolved in distilled water and the volume is fixed to 1L; after mixing the above raw materials, the pH value is naturally adjusted, after stirring evenly, sterilizing at 121°C and 0.1MPa for 20min, pouring the sterilized culture medium into a plate, cooling it down and setting it aside for use.

[0013] The preparation method of the MRS liquid culture medium is as follows: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, and 0.2g of magnesium sulfate heptahydrate are dissolved in distilled water and the volume is fixed to 1L; after mixing the above raw materials, the pH is adjusted to 6.8, the mixture is stirred evenly, and sterilized at 121°C and 0.1MPa for 20min.

[0014] Example 1 Isolation and Identification of Strain 1. Strain screening and purification (1) Source of the strain: healthy cat feces, collected in Nanyangguan Village, Fangcun Town, Tai'an City, Shandong Province in August 2023.

[0015] (2) Sample preparation: ① Place 10 mL of sterilized saline (0.85%) in a sterile conical flask, then add 1 g of the healthy cat feces from step (1), shake, and set aside; ② Dilute the solution in step ① to prepare samples with different concentration gradients, which are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, and 8#, for use.

[0016] (3) Cultivation of strains: Since the concentrations of solutions 1#, 2#, 3#, and 4# were too high to isolate single colonies, 100 μL of each of solutions 5#, 6#, 7#, and 8# was spread on MRS plate culture medium using a spreader and cultured at 37°C under anaerobic conditions for 48 h. (4) Isolation and purification: Pick a single colony with a diameter of 1-2 mm, milky white, round, smooth and rounded surface, neat edges, opaque, and a bulge in the middle, and streak it into three zones on an MRS plate culture medium. After streaking, culture it at 37°C under anaerobic conditions for 48 h. Repeat the above operation for a total of 3 times to purify the pure strain. Add the pure strain to a glycerol tube and store it at -80°C.

[0017] 2. Identification The single colony obtained after separation and purification was sent for identification, and the identification unit was Jinan Tianyi Biotechnology Co., Ltd.; during the identification process, the upstream primer used was 27F (sequence shown in SEQ ID NO.1), and the downstream primer was 1492R (sequence shown in SEQ ID NO.2). The nucleotide sequence of 16S rDNA of the strain obtained during the identification process is shown in SEQ ID NO.3.

[0018] According to the identification results, the strain was Pediococcus pentosaceus Pediococcus pentosaceus The identified strain was named Pediococcus pentosaceus King33 and sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation. The preservation information is as follows: Classification name: Pediococcus pentosaceus Pediococcus pentosaceus , Deposit date: June 24, 2024, Deposit number: CGMCC No.31050, Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0019] Example 2 Preparation of Pediococcus pentosaceus King33 bacterial agent 1. Take the preserved Pediococcus pentosaceus King33 and activate it on the MRS plate medium, pick a single colony and transfer it to 100mL MRS liquid medium, culture it at 37℃ for 12h to prepare the seed solution, then inoculate it into MRS liquid medium at a 1% (v / v) inoculation amount, culture it at 37℃ for 24h to obtain the bacterial solution; 2. After centrifugation of the bacterial solution, the cells were collected, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a concentration of 1.0×10 10 cfu / mL of bacterial suspension, and freeze-drying the bacterial suspension to obtain bacterial powder.

[0020] 3. According to the concentration of bacterial suspension, mix the bacterial powder with oligomalto-oligosaccharide (purchased from Baolingbao Biotechnology Co., Ltd.) to make a bacterial cell count of 1.0×10 10 cfu / g of Pediococcus pentosaceus King33 inoculum.

[0021] Example 3 Preparation of Pediococcus pentosaceus King33 bacterial agent 1. Take the preserved Pediococcus pentosaceus King33 and activate it on MRS plate culture medium, pick a single colony and transfer it to 100 mL MRS liquid culture medium, culture it at 37℃ for 12 hours to prepare seed solution, and then inoculate it into MRS liquid culture medium at an inoculum volume of 1% (v / v), culture it at 37℃ for 24 hours to obtain bacterial solution.

[0022] 2. After centrifugation of the bacterial solution, the cells were collected, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a concentration of 1.0×10 10 cfu / mL of bacterial suspension.

[0023] 3. The bacterial suspension was sterilized at 121°C for 30 min, and then freeze-dried to obtain bacterial powder. According to the number of live bacteria in the bacterial suspension, it was mixed with oligomaltose (purchased from Baolingbao Biotechnology Co., Ltd.) to prepare a solution containing 1.0×10 inactivated Pediococcus pentosaceus King33 cells. 10 cfu / g of Pediococcus pentosaceus King33 bacterial agent. After testing, no live bacteria were found in the Pediococcus pentosaceus King33 bacterial agent prepared in this example.

[0024] Example 4 Evaluation of feces odor In cooperation with a cat breeding center in Shandong Province, 20 healthy adult civet cats were collected as test cats, half male and half female, with an average weight of (3.84±0.97) kg and an average age of (1.27±0.61) years. They were randomly divided into a control group and a test group, with 10 cats in each group, half male and half female. All test cats were raised in a single cage in a 76×51×115 cm cat cage. The test period was 30 days in total, the first 14 days were the dietary adaptation period, and the 15th to 28th days were the test period. Before the start of the test, each test cat was physically examined to ensure that they were in good health. During the test period, all cats were fed the same basic cat food. The product component analysis guarantee value of the cat food is shown in Table 1. During the dietary adaptation period, both groups of cats were fed only basic cat food, and during the test period, the control group was fed only basic cat food. In addition to feeding the basic cat food, the test group was supplemented with 1.5 g / d / cat of the Pentosaceae King33 bacterial agent prepared in Example 2.

[0025] On the 28th day of the test period, the feces odor was evaluated. Five cats were randomly selected from each group, and fresh feces were collected. After mixing, 5.0 g was taken and placed in a 100 mL conical bottle and covered with the same breathable sealing film. No other information was marked on the conical bottle except for the A and B bottles. Twenty assessors evaluated fresh feces (30 minutes after defecation) and feces 6 hours after defecation (simulating home conditions) according to the scoring criteria in Table 2.

[0026] The results of the fecal odor evaluation of fresh feces are as follows Figure 1As shown in Figure 2, the feces odor evaluation results of feces 6 hours after defecation are as follows: Figure 2 shown by Figure 1 and Figure 2 It can be seen that 85% of the evaluators (17 people) believed that the stench of the fresh feces of the test group cats was less than that of the control group; 75% of the evaluators (15 people) believed that the stench of the feces of the test group cats was less than that of the control group 6 hours after defecation. Only 5% of the evaluators (1 person) believed that the stench of the fresh feces of the test group cats was higher than that of the control group; 5% of the evaluators (1 person) believed that the stench of the feces of the test group 6 hours after defecation was higher than that of the control group. In addition, 10% of the evaluators (2 people) were neutral about the comparison of the stench of the fresh feces of the test group and the control group cats (believing that the odor of the test group was the same as that of the control group); 20% of the evaluators (4 people) were neutral about the comparison of the stench of the feces of the test group and the control group cats 6 hours after defecation. Therefore, whether it is the fresh feces of the cats or the feces 6 hours after defecation, the vast majority of evaluators believe that the stench of the test group is much less than that of the control group.

[0027] Table 1 Guaranteed values ​​of product composition analysis

[0028] Note: Additives include the natural steroid saponin (derived from yucca), taurine, manno-oligosaccharides, choline chloride, calcium carbonate, egg yolk lecithin, potassium chloride, amino acid zinc complex, glycine copper chelate, glycine iron chelate, amino acid manganese complex, calcium iodate, vitamin A acetate, vitamin D3, thiamine nitrate, dl-α-tocopheryl acetate, riboflavin, pyridoxine hydrochloride, cyanocobalamin, niacin, folic acid, D-calcium pantothenate, and D-biotin.

[0029] Table 2 Odor scoring criteria

[0030] Example 5 Evaluation of stool quality consistency During the test period (days 15-28) of the experiment in Example 4, the feces consistency of all cats in the test group and the experimental group was evaluated. Since the feces consistency score is a subjective evaluation, it was evaluated by the same researcher using a 5-point scale (Table 3). After the feces consistency score of all cats was scored every day during the test period, all the scores were recorded, and the average of the feces consistency scores of the two groups of cats was calculated.

[0031] According to the recorded results of fecal consistency score (Table 4), the fecal consistency score of the control group during the test period was between 2.70 and 3.60 points, and soft stools appeared on the 21st and 22nd days, while the fecal consistency score of the test group was between 3.60 and 3.80 points, which was relatively stable. This shows that after feeding Pediococcus pentosaceus King33 inoculant, the fecal consistency score of the test group has improved and become more stable, which is conducive to maintaining intestinal homeostasis.

[0032] Table 3 Stool consistency score

[0033] Note: 3-4 points is the best condition, and <3 points is soft stool.

[0034] Table 4 Stool consistency scoring results

[0035] Example 6 Analysis of Volatile Organic Compounds in Feces On the 26th day of the experiment in Example 4, fresh feces of all cats were collected for determination of volatile organic compounds. Phenols, quinoline, indole, and skatole were analyzed by Shimadzu GC-MS 2010 Plus gas chromatograph coupled with TQ8040 mass spectrometer according to the method of Bastos et al. (Silvino TB, Cristina DLD, Menezes MCS, et al. Bacillus subtilis and Bacillus licheniformis reduce faecal protein catabolites concentration and odour in dogs. [J]. BMC veterinary research, 2020, 16(1): 116.), and the chromatographic column was Shimadzu SH-Rtx-5MS (30m×0.25mm×0.25μm).

[0036] The test results are shown in Table 5. The volatile organic compounds in the feces of the test group cats were significantly reduced, and the concentrations of phenols, quinoline, indole, and skatole were all significantly reduced.

[0037] Table 5 Average peak area percentage of volatile organic compounds (%)

[0038] Example 7 Analysis of biogenic amine content in feces Amino acid fermentation decomposition metabolites are considered to be the main odor components of feces, which are conducive to the survival of bacteria with pathogenic potential. Under certain circumstances, they are toxic and may have a negative impact on intestinal function. Amino acids are decarboxylated to form amines under the action of bacterial amino acid decarboxylase, such as arginine and ornithine decarboxylation to putrescine, lysine decarboxylation to cadaverine, histidine decarboxylation to histamine, etc., which are potentially toxic to the human body. Undigested amino acids can produce a variety of corruption compounds through corruption. In addition to phenols, quinoline, indole, and skatole in Example 5, amines are also one of the main substances.

[0039] On day 27 of the experiment in Example 4, the biogenic amines in fresh feces collected within 15 minutes after defecation were analyzed according to the method described by Urrego et al. (Gonzalez IMU, ODFLM, Karine SMD, et al. Effects of different protein sources on fermentationmetabolites and nutrient digestibility of brachycephalic dogs. [J]. Journal of nutritional science, 2017, 6e43.).

[0040] The analysis results are shown in Table 6. Compared with the control group, the content of putrescine, cadaverine and histamine in the feces of cats in the test group was significantly reduced. In addition, the content of other types of biogenic amines, such as spermidine, tyramine, phenylethylamine and tryptamine, also showed a decreasing trend. In this study, the addition of Pediococcus pentosaceus King33 reduced the concentration of potentially toxic decay compounds in the intestinal mucosa, including putrescine, cadaverine, histamine, phenols, quinoline, indole and skatole, which made the fresh feces and the feces 6 hours after defecation less smelly.

[0041] Table 6 Analysis results of biogenic amine content in feces (mg / kg)

[0042] Example 8 Test on the function of relieving dog body odor, breath odor and urine odor 20 Welsh Corgis were selected, half male and half female, with an average weight of (12.65±1.47) kg and an average age of (3.27±1.38) years old, and were randomly divided into a control group and a test group, with 10 dogs in each group, half male and half female, one dog in one house, and the management method was the same. All dogs were fed the same dog food, twice a day, once in the morning and once in the evening, for a total of 30 days. In addition to feeding the dog food, the test group was supplemented with the Pediococcus pentosaceus King33 bacterial agent prepared in Example 3 once a day, with an amount of 4.5 g / d / dog. The nutritional composition table of the basic dog food is shown in Table 7.

[0043] During the test, the dogs were not bathed and their teeth were not brushed. Three experienced experimenters were appointed as odor judges. They placed a semi-wet cotton swab infected with odor 1 cm below the nostrils of the odor judges and scored after sniffing for 3 seconds. The odor extraction method is shown in Table 8. After odor extraction, the odor size was divided into 6 levels in order, with scores of 1 to 6 points respectively. The odor level is shown in Table 9. The total score of each group was counted and data analysis was performed.

[0044] The body odor scores of the experimental dogs on the first and 30th days of the experiment are shown in Figure 3 The results of the bad breath scores of the test dogs on the first and 30th days of the test are shown in Figure 4 The urine odor scores of the test dogs on the first and 30th days of the test are shown in Figure 5 ;Depend on Figure 3-5 It can be seen that on the 30th day, the odor score of the test group decreased significantly, and the odor score of the control group showed a slight upward trend, and there was a significant difference between the two. This shows that in this experiment, the Pediococcus pentosaceus King33 bacterial agent prepared in Example 3 has obvious effects on removing body odor, bad breath, and urine odor of the test dogs, further indicating that the inactivated Pediococcus pentosaceus King33 can also play a role through its cell structure and metabolites.

[0045] Table 7 Guaranteed values ​​of dog food nutritional composition analysis (based on dry matter, %)

[0046] Table 8 Odor extraction method

[0047] Table 9 Description of odor intensity levels

Claims

1. A Pediococcus pentosaceus King33 for alleviating the odor of pet dog and cat feces, characterized in that: The Pediococcus pentosaceus King33 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on June 24, 2024, with a deposit number of CGMCC No.31050 and a classification name of Pediococcus pentosaceus. Pediococcus pentosaceus .

2. A bacterial agent prepared using the Pediococcus pentosaceus King33 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that In the bacterial agent, the number of bacteria is 0.8-1.2×10 10 cfu / mL.

4. The bacterial agent according to claim 2, characterized in that In the bacterial agent, the Pediococcus pentosaceus King33 is a live bacterium or an inactivated bacterium; When the Pediococcus pentosaceus King33 is a live bacterium, the preparation method of the bacterial agent is to activate and ferment the Pediococcus pentosaceus King33 to obtain a bacterial solution, collect the bacterial bodies after centrifugation, resuspend them in reconstituted skim milk to obtain a bacterial suspension, freeze-dry them to obtain bacterial powder, and then mix them with oligomaltose to obtain a bacterial agent; When the Pediococcus pentosaceus King33 is an inactivated bacterium, the preparation method of the bacterial agent is to activate and ferment the Pediococcus pentosaceus King33 to obtain a bacterial liquid, collect the bacterial bodies after centrifugation, resuspend them in reconstituted skim milk to obtain a bacterial suspension, sterilize and freeze-dry to obtain bacterial powder, and then mix it with oligomaltose to obtain a bacterial agent.

5. Use of the Pediococcus pentosaceus King33 according to claim 1 in preparing a product for alleviating the odor of pet dog and cat feces.

Images