Enzyme composition, complex additive containing same, and method for preparing same, and deodorizing cat litter

Abstract

The application relates to the technical field of pet product additives, in particular to an enzyme composition, a composite additive containing the same, a preparation method of the composite additive and deodorizing cat litter. The enzyme composition comprises urease, uric acid oxidase and creatinine enzyme; the composite additive comprises the enzyme composition and probiotic metabolites; the deodorizing cat litter is prepared by using the composite additive, can efficiently decompose urea, uric acid, creatinine and other uremic toxins, has the advantages of safety, stability, easy use and the like, and has a wide application prospect in the technical field of pet product additives.

Description

Technical Field

[0001] This application relates to the field of pet product additives technology, and in particular to enzyme compositions, compound additives containing the same, methods for preparing the same, and deodorizing cat litter. Background Technology

[0002] With the increasing rate of pet cat ownership, the foul odor generated during the use of indoor cat litter has become a core pain point for pet-owning families. The foul odor of cat litter mainly comes from uremic toxins (including urea, creatinine, uric acid, etc.) in cat urine, sulfur-containing compounds (hydrogen sulfide, methanethiol) and nitrogen-containing compounds (ammonia, skatole, indole) in feces. Among them, ammonia, produced by the decomposition of uremic toxins by putrefactive bacteria in the environment, is highly volatile and has a persistent odor. In addition, urate is insoluble in water and easily adheres to the surface of cat litter, making it difficult to completely remove with conventional cleaning methods. Its half-life can be as long as 2 to 6 years, which is the main cause of recurring foul odor.

[0003] Currently, cat litter odor control additives are mainly divided into three categories:

[0004] (1) Chemical additives (such as sodium bicarbonate, activated carbon, fragrance, etc.) can quickly mask or absorb malodors, but they cannot fundamentally decompose malodorous substances, which can easily cause secondary pollution, and fragrances may irritate the cat's respiratory tract.

[0005] (2) Single biological additives (such as single probiotic powder or single deodorizing enzyme) have weak deodorizing specificity. Among them, probiotic powder needs to be revived and multiplied in the cat litter environment to play a role, and the response speed is slow; single deodorizing enzyme can only decompose a certain type of malodorous substances, and it is difficult to deal with the complex malodor system in cat litter.

[0006] (3) Compound biological additives often use a combination of "probiotic powder + multi-enzyme compound + plant extract". For example, patent document CN120077959B describes a deodorizing cat litter composition with "probiotics (Lactobacillus rhamnosus + Lactobacillus salivarius) + compound enzymes (cellulase, protease, chitinase) + plant polypeptides" working together. It achieves broad-spectrum and long-lasting deodorization by inhibiting putrefactive bacteria through the secretion of bacteriocins by probiotics, decomposing fecal organic matter by compound enzymes, and killing mites by polypeptides. However, the compound enzymes in this technology are mainly for fecal organic matter and lack specific enzyme systems for urea, creatinine, uric acid and other uremic toxins in cat urine, resulting in low decomposition efficiency. Summary of the Invention

[0007] Based on this, embodiments of this application provide an enzyme composition, a composite additive containing the same, a method for preparing the same, and deodorizing cat litter, to improve the decomposition efficiency of urea, creatinine, uric acid, and other urea toxins in cat litter.

[0008] The objective of this application embodiment is achieved through the following technical solutions:

[0009] One or more embodiments of this application provide an enzyme composition comprising urease, uricase, and creatinine enzyme; based on the urease activity ≥1000 U / g, the uricase activity ≥800 U / g, and the creatinine enzyme activity ≥600 U / g, the mass ratio of the urease, uricase, and creatinine enzyme is (2~3):1:1.

[0010] One or more embodiments of this application provide a composite additive comprising the enzyme composition and probiotic metabolites.

[0011] In some embodiments of this application, the probiotic metabolites are products prepared by centrifuging, filtering, concentrating and drying the fermentation products obtained from fermenting probiotics.

[0012] In some embodiments of this application, the probiotic metabolites include metabolites of Lactobacillus plantarum with accession number CICC 25125 and / or metabolites of Lactobacillus rhamnosus with accession number CICC 6137.

[0013] Optionally, the ratio of the metabolites of *Lactobacillus plantarum* with accession number CICC 25125 to the metabolites of *Lactobacillus rhamnosus* with accession number CICC 6137 is (0.8~1.2):1.

[0014] In some embodiments of this application, the composite additive comprises, by weight percentage, 20% to 35% of the enzyme composition, 40% to 60% of the probiotic metabolites, and excipients.

[0015] In some embodiments of this application, the composite additive comprises, by mass percentage, 20% to 35% of the enzyme composition, 40% to 60% of the probiotic metabolites, 5% to 15% of the activity protectant, 3% to 8% of the dispersant, and 2% to 7% of the pH adjuster.

[0016] In some embodiments of this application, the composite additive satisfies one or more of the following conditions:

[0017] (1) The active protective agent comprises one or more of trehalose and xanthan gum; optionally, the mass ratio of trehalose to xanthan gum is 1:(1~2).

[0018] (2) The dispersant comprises one or more of magnesium stearate and silicon dioxide; optionally, the mass ratio of magnesium stearate to silicon dioxide is (2~3):1;

[0019] (3) The pH adjuster comprises one or more of citric acid and potassium dihydrogen phosphate; optionally, the mass ratio of citric acid to potassium dihydrogen phosphate is 1:(1~3).

[0020] One or more embodiments of this application provide a method for preparing the composite additive, the method comprising the step of mixing the enzyme composition, probiotic metabolites, activity protectant, dispersant and pH adjuster to prepare the composite additive.

[0021] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0022] (1) Before mixing, the enzyme composition, probiotic metabolites, active protectant, dispersant and pH adjuster are dried at 55℃~65℃ for 2 h~3 h, and then passed through an 80 mesh~100 mesh sieve respectively.

[0023] (2) Mix at 25℃~30℃ and 800 r / min~1000 r / min for 15 min~20 min;

[0024] (3) After mixing, the composite additive is cooled to 23°C~27°C and then passed through a 90-mesh sieve~110-mesh sieve.

[0025] One or more embodiments of this application provide a deodorizing cat litter, the deodorizing cat litter comprising the aforementioned composite additive and cat litter;

[0026] Optionally, the cat litter includes one or more of bentonite cat litter, tofu cat litter, mixed cat litter, and crystal cat litter;

[0027] Optionally, the composite additive is 0.5% to 2.0% of the cat litter mass.

[0028] Compared with traditional technologies, this application has the following advantages:

[0029] The enzyme composition provided in this application contains urease, uricase, and creatinine enzyme, which can efficiently decompose malodorous precursors such as urea, creatinine, and uric acid. When using this enzyme composition for cat litter deodorization, it can be used in conjunction with probiotic metabolites, resulting in a faster deodorization response and superior long-lasting deodorization performance. Detailed Implementation

[0030] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0033] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0034] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0035] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0036] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0037] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0038] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0039] In a first aspect, this application provides an enzyme composition comprising urease, uricase, and creatinine enzyme; based on the urease activity ≥1000 U / g, the uricase activity ≥800 U / g, and the creatinine enzyme activity ≥600 U / g, the mass ratio of the urease, uricase, and creatinine enzyme is (2~3):1:1, for example, 2:1:1, 2.5:1:1, or 3:1:1.

[0040] A second aspect of this application provides a composite additive comprising the enzyme composition and probiotic metabolites.

[0041] In some embodiments of this application, the probiotic metabolites are products prepared by centrifuging, filtering, concentrating and drying the fermentation products obtained from fermenting probiotics.

[0042] In some embodiments of this application, the probiotic metabolites include metabolites of Lactobacillus plantarum with accession number CICC 25125 and / or metabolites of Lactobacillus rhamnosus with accession number CICC 6137.

[0043] Optionally, the ratio of the metabolites of *Lactobacillus plantarum* with accession number CICC 25125 to the metabolites of *Lactobacillus rhamnosus* with accession number CICC 6137 is (0.8~1.2):1, for example, 0.8:1, 0.9:1, 1:1, 1.1:1, or 1.2:1.

[0044] In some embodiments of this application, the composite additive comprises, by weight percentage, 20% to 35% of the enzyme composition, for example, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%; and 40% to 60% of the probiotic metabolites, for example, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%; and excipients.

[0045] In some embodiments of this application, the composite additive comprises, by mass percentage, 20% to 35% of the enzyme composition, for example, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%; 40% to 60% of the probiotic metabolites, for example, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%; 5% to 15% of the activity protectant, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%; 3% to 8% of the dispersant, for example, 3%, 4%, 5%, 6%, 7%, 8%; and 2% to 7% of the pH adjuster, for example, 2%, 3%, 4%, 5%, 6%, 7%.

[0046] In some embodiments of this application, the composite additive satisfies one or more of the following conditions:

[0047] (1) The active protective agent comprises one or more of trehalose and xanthan gum; optionally, the mass ratio of trehalose to xanthan gum is 1:(1~2), for example 1:1, 1:1.5, 1:2;

[0048] (2) The dispersant comprises one or more of magnesium stearate and silicon dioxide; optionally, the mass ratio of magnesium stearate to silicon dioxide is (2~3):1, for example 2:1, 2.5:1, 3:1;

[0049] (3) The pH adjuster comprises one or more of citric acid and potassium dihydrogen phosphate; optionally, the mass ratio of citric acid to potassium dihydrogen phosphate is 1:(1~3), for example 1:1, 1:2, 1:3.

[0050] A third aspect of this application provides a method for preparing the composite additive, the method comprising the step of mixing the enzyme composition, probiotic metabolites, activity protectant, dispersant and pH adjuster to prepare the composite additive.

[0051] In some embodiments of this application, the method satisfies one or more of the following conditions:

[0052] (1) Before mixing, the enzyme composition, probiotic metabolites, active protectant, dispersant and pH adjuster are dried at 55℃~65℃, for example 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65℃ for 2 h~3 h, for example 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 h; and then passed through 80 mesh to 100 mesh sieves, for example 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100 mesh sieves.

[0053] (2) At 25℃~30℃, for example 25, 26, 27, 28, 29, 30℃; at 800 r / min~1000 r / min, for example 800, 850, 900, 950, 1000 r / min; mix for 15 min~20 min, for example 15, 16, 17, 18, 19, 20 min;

[0054] (3) After mixing, the composite additive is cooled to 23°C to 27°C, for example, 23, 24, 25, 26, 27°C; and then passed through a 90-mesh sieve to a 110-mesh sieve, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 mesh sieves.

[0055] A fourth aspect of the embodiments of this application provides a deodorizing cat litter, the deodorizing cat litter comprising the aforementioned composite additive and cat litter;

[0056] Optionally, the cat litter includes one or more of bentonite cat litter, tofu cat litter, mixed cat litter, and crystal cat litter;

[0057] Optionally, the composite additive is 0.5% to 2.0% of the cat litter mass, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%.

[0058] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0059] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0060] All components used in the embodiments of this application are commercially available conventional raw materials. Specifically, *Lactobacillus plantarum* and *Lactobacillus rhamnosus* were purchased from the China Industrial Microbial Culture Collection Center, with accession numbers CICC 25125 and CICC 6137, respectively. Urease (activity 1200 U / g), uricase (activity 1000 U / g), and creatinine enzyme (activity 800 U / g) were purchased from Guangzhou Yuhong Technology Co., Ltd. Trehalose, xanthan gum, magnesium stearate, silicon dioxide, citric acid, and potassium dihydrogen phosphate were all food-grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0061] Example 1

[0062] (I) Preparation methods of probiotic metabolites

[0063] (1) Inoculate Lactobacillus plantarum into MRS medium (Man-Rogosa-Sharpe medium) and culture at 37℃ and 180 r / min for 24 h to prepare fermentation broth;

[0064] (2) Centrifuge the fermentation broth at 8000 r / min and 4℃ for 15 min to remove the bacterial precipitate;

[0065] (3) Take the supernatant and filter it through a 0.22 μm filter membrane to remove residual bacteria;

[0066] (4) The filtered supernatant was concentrated to 1 / 5 to 1 / 4 of its original volume at 60°C and a vacuum of -0.08 MPa to prepare a concentrated solution;

[0067] (5) The concentrate was freeze-dried at -80℃ for 24 h, pulverized and passed through an 80-mesh sieve to prepare probiotic metabolites;

[0068] Among them, the moisture content of probiotic metabolites is ≤3%, the organic acid content is ≥25%, and the antimicrobial peptide content is ≥5%.

[0069] 1. Detection of live bacteria and bacterial remains

[0070] Weigh 10 g of probiotic metabolites and add them to 100 mL of sterile sodium chloride-peptone buffer (pH 7.0). Mix well to prepare a 1:10 test solution. Perform viable cell detection on the test solution: Incubate on tryptic soy agar at 32°C for 60 h. If no colonies grow, the result is considered as no viable cells detected. Prepare a suspension from the test solution and examine it under a microscope: Observe using a bacterial counting chamber under a phase-contrast microscope. If no intact bacterial morphology or fragments are found, the result is considered as no bacterial debris detected.

[0071] 2. Moisture content determination (refer to GB / T 6435-2014)

[0072] Weigh 2-5 g of probiotic metabolites and place them evenly in a weighing bottle that has been dried to constant weight at 80℃ and a vacuum degree ≤13 kPa. Place the weighing bottle in a vacuum drying oven preheated to 80℃, with the cap partially open. After closing the oven door, evacuate to a pressure ≤13 kPa and dry at 80℃±2℃ and ≤13 kPa for 4 hours. After drying, slowly introduce dry air to restore normal pressure, remove the weighing bottle, cap it, and place it in a desiccator to cool for 30 minutes. Weigh the bottle. Repeat the drying process until constant weight is achieved (the difference between two consecutive weighings should be less than 2 mg).

[0073] Moisture content (%) = (mass of sample before drying - mass of sample after drying) / mass of sample before drying × 100%.

[0074] 3. Determination of organic acid content (refer to NY / T 4685-2025)

[0075] Organic acids in the sample were extracted by ultrasonic extraction with sulfuric acid solution (30 mmol / L), and gradient elution was performed using a C18 reversed-phase column with phosphate buffer solution as the mobile phase. The sample was detected at 210 nm by a UV detector and quantified by external standard method.

[0076] 4. Determination of antimicrobial peptide content (refer to NY / T 4746-2025)

[0077] Samples were extracted ultrasonically and centrifuged. The supernatant was enriched and purified using a solid-phase extraction column (such as a WCX column) and eluted with methanol and ammoniated methanol. ZORBAX Eclipse Plus C18 was used as the stationary phase, and 0.1% formic acid-acetonitrile and 0.1% formic acid-water were used as the mobile phases. The column temperature was 35℃, the flow rate was 1.0 mL / min, and separation was performed using a gradient elution program. Quantitative detection was performed at a wavelength of 276 nm using the external standard method.

[0078] (II) Components of Composite Additives

[0079] A cat litter deodorizing compound additive based on a combination of probiotic metabolites and enzymes, comprising the following components by weight percentage (the sum of the weight percentages of each component is 100%):

[0080] (1) 50% of the metabolites of Lactobacillus plantarum

[0081] (2) Enzyme composition 30% (urease: uricase: creatinine enzyme = 2:1:1),

[0082] (3) 8% active protectant (trehalose: xanthan gum = 1:1),

[0083] (4) Dispersant 6% (magnesium stearate: silicon dioxide = 2:1), and,

[0084] (5) 6% pH adjuster (citric acid: potassium dihydrogen phosphate = 1:2);

[0085] Adding a pH adjuster adjusts the pH of the compound additive to 5.5.

[0086] (III) Preparation method of composite additives

[0087] (1) Pretreatment: Place the Lactobacillus plantarum metabolite powder, enzyme composition, activity protectant, dispersant and pH adjuster in a vacuum drying oven at 60℃ for 2.5 h to remove moisture (moisture content ≤3%), and then pass them through a 90-mesh sieve for later use;

[0088] (2) Mixing: The pretreated active protectant and enzyme composition were added to a high-speed mixer and mixed at 28°C and 1100 r / min for 12 min to prepare an enzyme-protectant complex;

[0089] (3) Compound preparation: Add the pretreated Lactobacillus plantarum metabolites, dispersant and pH adjuster to the enzyme-protectant complex in sequence, and mix at 28℃ and 900 r / min for 18 min to prepare crude compound additive;

[0090] (4) Post-processing: The crude compound additive is placed in a sterile environment and cooled to 25°C, passed through a 100-mesh sieve, and aseptically packaged to prepare the compound additive.

[0091] The mixing process in steps (2) and (3) is carried out in a sterile and dry environment (relative humidity ≤40%) to avoid contamination by bacteria and moisture affecting the activity of additives, and to ensure product quality stability.

[0092] (iv) Preparation of deodorizing cat litter

[0093] Add the above-mentioned compound additive to the bentonite cat litter at 1.0% of the cat litter mass, mix evenly for 12 minutes, and prepare deodorizing bentonite cat litter.

[0094] Example 2

[0095] (I) Preparation methods of probiotic metabolites

[0096] (1) The metabolites of Lactobacillus plantarum and Lactobacillus rhamnosus were inoculated into MRS medium (Man-Rogosa-Sharpe medium) and cultured at 37℃ and 180 r / min for 36 h to prepare fermentation broth;

[0097] (2) Centrifuge the fermentation broth at 10000 r / min and 4℃ for 20 min to remove the bacterial precipitate;

[0098] (3) Take the supernatant separately and filter it through a 0.22 μm filter membrane to remove residual bacteria;

[0099] (4) The filtered supernatant was concentrated to 1 / 5 to 1 / 4 of its original volume at 60°C and a vacuum of -0.09 MPa to prepare a concentrated solution;

[0100] (5) The concentrate was freeze-dried at -80℃ for 36 h, then pulverized and passed through a 100-mesh sieve.

[0101] (6) Prepare probiotic metabolites by mixing Lactobacillus plantarum metabolites and Lactobacillus rhamnosus metabolites at a mass ratio of 1:1.

[0102] Among them, the moisture content of probiotic metabolites is ≤3%, the organic acid content is ≥25%, and the antimicrobial peptide content is ≥5%.

[0103] (II) Components of Composite Additives

[0104] A cat litter deodorizing compound additive based on a combination of probiotic metabolites and enzymes, comprising the following components by weight percentage (the sum of the weight percentages of each component is 100%):

[0105] (1) 45% probiotic metabolites (Lactobacillus plantarum metabolites: Lactobacillus rhamnosus mixed metabolites = 1:1),

[0106] (2) Enzyme composition 35% (urease: uricase: creatinine enzyme = 3:1:1),

[0107] (3) 10% active protectant (trehalose: xanthan gum = 1:2),

[0108] (4) 5% dispersant (magnesium stearate: silicon dioxide = 3:1), and,

[0109] (5) pH adjuster 5% (citric acid: potassium dihydrogen phosphate = 1:3);

[0110] Adding a pH adjuster adjusts the pH of the compound additive to 6.5.

[0111] (III) Preparation method of composite additives

[0112] (1) Pretreatment: The mixed metabolite powder, enzyme composition, activity protectant, dispersant and pH adjuster were placed in a vacuum drying oven at 60°C for 2 h to remove moisture (moisture content ≤3%). After drying, they were passed through an 80-mesh sieve for later use.

[0113] (2) Mixing: Add the pretreated active protectant and enzyme composition to a high-speed mixer and mix at 25°C and 1000 r / min for 15 min to prepare the enzyme-protectant complex;

[0114] (3) Compounding: Add the pretreated mixed metabolites, dispersant and pH adjuster to the enzyme-protectant complex in sequence, and mix at 25℃ and 800 r / min for 20 min to prepare crude compound additive;

[0115] (4) Post-processing: The crude compound additive is placed in a sterile environment and cooled to 23°C, passed through a 100-mesh sieve, and aseptically packaged to prepare the compound additive;

[0116] The mixing process in steps (2) and (3) is carried out in a sterile and dry environment (relative humidity ≤40%) to avoid contamination by bacteria and moisture affecting the activity of additives, and to ensure product quality stability.

[0117] (iv) Preparation of deodorizing cat litter

[0118] Add the above-mentioned compound additive to the tofu cat litter at 0.8% of the cat litter mass and mix evenly for 10 minutes to prepare deodorizing tofu cat litter.

[0119] Example 3

[0120] (I) Preparation methods of probiotic metabolites

[0121] (1) Inoculate Lactobacillus rhamnosus into MRS medium and culture at 37℃ and 180 r / min for 30 h to prepare fermentation broth;

[0122] (2) Centrifuge the fermentation broth at 9000 r / min and 4℃ for 18 min to remove the bacterial precipitate;

[0123] (3) Take the supernatant and filter it through a 0.22 μm filter membrane to remove residual bacteria;

[0124] (4) The filtered supernatant was concentrated to 1 / 5 to 1 / 4 of its original volume at 60°C and a vacuum of -0.085 MPa to prepare a concentrated solution;

[0125] (5) The concentrated liquid was freeze-dried at -80℃ for 30 h, pulverized and passed through a 90-mesh sieve to prepare probiotic metabolites;

[0126] Among them, the moisture content of probiotic metabolites is ≤3%, the organic acid content is ≥25%, and the antimicrobial peptide content is ≥5%.

[0127] (II) Components of Composite Additives

[0128] A cat litter deodorizing compound additive based on a combination of probiotic metabolites and enzymes, comprising the following components by weight percentage (the sum of the weight percentages of each component is 100%):

[0129] (1) 55% of the metabolites of Lactobacillus rhamnosus

[0130] (2) Enzyme composition 25% (urease: uricase: creatinine enzyme = 2.5:1:1),

[0131] (3) 7% active protectant (trehalose: xanthan gum = 1:1.5),

[0132] (4) Dispersant 7% (magnesium stearate: silicon dioxide = 2.5:1), and,

[0133] (5) 6% pH adjuster (citric acid: potassium dihydrogen phosphate = 1:1);

[0134] Adding a pH adjuster adjusts the pH of the compound additive to 6.0.

[0135] (III) Preparation method of composite additives

[0136] (1) Pretreatment: The Lactobacillus rhamnosus metabolite powder, enzyme composition, activity protectant, dispersant and pH adjuster were placed in a vacuum drying oven at 60°C for 3 hours to remove moisture (moisture content ≤3%). After drying, they were passed through a 100-mesh sieve for later use.

[0137] (2) Mixing: Add the pretreated active protectant and enzyme composition to a high-speed mixer and mix at 30°C and 1200 r / min for 10 min to prepare the enzyme-protectant complex;

[0138] (3) Compound preparation: Add the pretreated Lactobacillus rhamnosus metabolites, dispersant and pH adjuster to the enzyme-protectant complex in sequence, and mix at 30℃ and 1000 r / min for 15 min to prepare crude compound additive;

[0139] (4) Post-processing: The crude compound additive is placed in a sterile environment and cooled to 27°C, passed through a 100-mesh sieve, and aseptically packaged to prepare the compound additive;

[0140] The mixing process in steps (2) and (3) is carried out in a sterile and dry environment (relative humidity ≤40%) to avoid contamination by bacteria and moisture affecting the activity of additives, and to ensure product quality stability.

[0141] (iv) Preparation of deodorizing cat litter

[0142] Add the above-mentioned compound additive to the mixed cat litter (bentonite: tofu = 1:1) at 1.5% of the cat litter mass, mix evenly for 15 minutes, and prepare deodorizing mixed cat litter.

[0143] Performance testing

[0144] The compound additives from Examples 1-3 were added to cat litter at a mass ratio of 1:100 to prepare deodorizing cat litter. A control group was prepared using commercially available deodorizing cat litter (Master Plant Tofu Cat Litter - King82 Super Deodorizing). The experimental results are as follows:

[0145] (a) Deodorization effect test

[0146] Using the closed container method, the above four groups of cat litter and the blank control group (cat litter without compound additives) were placed in closed containers of the same size, and an equal amount of simulated cat urine (containing 0.996 g / L urea, 0.0498 g / L creatinine, and 0.015 g / L uric acid) was added. The odor removal rate (using ammonia, hydrogen sulfide, and skatole as detection indicators) was measured at 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h.

[0147] 1. Detection of ammonia

[0148] The Nessler's reagent spectrophotometric method specified in HJ 533-2009 was adopted: ammonia gas was absorbed into a dilute sulfuric acid solution, reacted with Nessler's reagent to form a yellow complex, and the absorbance was measured at a wavelength of 420 nm.

[0149] 2. Detection of hydrogen sulfide

[0150] The detection is performed according to GB / T 14678-93 Gas Chromatography-Flame Photometric Detection Method. After the gas sample is separated by the chromatographic column, the sulfur-containing compounds produce characteristic spectra in the flame photometric detector, and the quantification is based on the peak height or peak area.

[0151] 3. Detection of skatole (3-methylindole)

[0152] High-performance liquid chromatography-fluorescence detection (HPLC-FLD) was used. After extraction with methanol, the sample was separated using a C18 reversed-phase column. The fluorescence detector was used to measure the emission at an excitation wavelength of 270-285 nm and an emission wavelength of 340-360 nm. Quantification was performed using the external standard method.

[0153] Odor removal rate (%) = (blank concentration - sample concentration) / blank concentration × 100%

[0154] The results showed that the deodorizing cat litter in Examples 1, 2, and 3 of this application had an odor removal rate of ≥85.2% after 2 hours, ≥95.6% after 24 hours, and maintained above 90.3% after 72 hours. In contrast, the control group cat litter had an odor removal rate of ≤72.1% after 2 hours, ≤85.4% after 24 hours, and decreased to 75.3% after 72 hours. This indicates that the cat litter containing the compound additive with the enzyme composition provided in this application has a faster deodorizing response and better long-lasting effect.

[0155] (ii) Activity stability test

[0156] Four groups of compound additives from Examples 1-3 and the control group were stored in an environment of 40°C and 60% relative humidity for 7 days. The activity retention rate of the enzyme composition and the content retention rate of probiotic metabolites (antimicrobial peptides, organic acids) were measured before and after storage.

[0157] 1. Determination of activity retention rate

[0158] The activities of urease, uricase, and creatinine enzyme in the enzyme composition before and after treatment were determined by spectrophotometry.

[0159] Activity retention rate (%) = (enzyme activity after treatment / initial enzyme activity) × 100%.

[0160] 2. Determination of antimicrobial peptides and organic acids

[0161] The determination was performed according to the determination method in Example 1.

[0162] Content retention rate (%) = (Component content after 7 days of storage / Initial component content) × 100%.

[0163] The results showed that after 7 days of storage, the composite additives in Examples 1, 2, and 3 of this application retained ≥90.8% of the uremic toxin decomposing enzyme activity and ≥90.1% of the antimicrobial peptide and organic acid content, indicating that the composite additives containing the enzyme composition provided in this application have stable activity and can exert their activity for a long time.

[0164] (iii) Clumping strength of cat litter

[0165] The clumping strength of cat litter was determined according to standards such as GB / T 43839-2024 "Safety Technical Requirements for Companion Animal (Pet) Products". During the test, the cat litter clumping was placed under the pressure plate of the testing machine and pressed down at a speed of 5 mm / min. The maximum pressure (peak force) when the clumping broke was recorded, and the clumping strength was calculated according to the formula P=F / S (P is the compressive strength, F is the peak pressure, and S is the cross-sectional area under force).

[0166] The results showed that the active protective agent, dispersant, and pH adjuster provided in this application did not affect the clumping performance of the cat litter. After addition, the clumping strength of the cat litter was ≥150 g, with good clumping properties, not easy to break apart, and easy to clean. At the same time, the additive has good dispersibility and can be used with various mainstream cat litters such as bentonite, tofu, and mixed types, with a wide range of applications, solving the problem that traditional compound additives easily affect cat litter clumping.

[0167] In summary, this application addresses the core odor source in cat litter—urine, uric acid, and creatinine—by providing an enzyme composition (urease + uricase + creatinine enzyme). Urease decomposes urea into ammonia and carbon dioxide; uricase decomposes water-insoluble uric acid into allantoin (odorless, easily soluble in water, and flushed out with the litter); and creatinine enzyme decomposes creatinine into creatine and subsequent odorless byproducts. The three enzymes work synergistically to achieve comprehensive and targeted decomposition of urine in cat urine, eliminating the odor generated by urine decomposition at its source. Simultaneously, the organic acids and antimicrobial peptides in the probiotic metabolites directly inhibit the growth of putrefactive bacteria in the cat litter that decompose urine and produce odor, reducing the production of odorous substances. This achieves a synergistic deodorization effect of "targeted decomposition + source inhibition," solving the problems of incomplete deodorization and recurring odors associated with existing additives.

[0168] This application adds a specific proportion of activity protectant and pH adjuster to adjust the pH value of the additive to 5.5~6.5, which is suitable for the normal pH environment of cat litter. This can effectively protect the activity of the enzyme composition and probiotic metabolites, and prevent their activity from decreasing due to temperature, humidity and pH fluctuations during cat litter processing (drying, mixing) and storage. At the same time, the probiotic metabolites do not need to be regenerated and are not affected by the humidity and pH value of cat litter. The activity stability is significantly better than that of traditional probiotic powder additives, solving the technical pain point of unstable activity of traditional biological additives.

[0169] All components of this application are biologically derived or food-grade excipients. The probiotic metabolites and enzyme compositions are all natural bioactive substances, and the pH adjusters, dispersants, and activity protectants are all food-grade materials. There are no chemical fragrances or harmful preservatives, and it will not cause secondary pollution. At the same time, it has no irritating odor, will not irritate the cat's respiratory tract or skin, and will not pollute the pet-owning family environment. It meets the environmental protection and safety requirements of modern pet ownership and is different from the defects of traditional chemical additives that are prone to secondary pollution and strong irritation.

[0170] The preparation method described in this application does not require complex granulation or encapsulation processes; it can be completed simply through drying, mixing, and sieving. The process is simple and highly efficient. The probiotic metabolites can be prepared through conventional microbial fermentation, and the enzyme composition is a commercially available conventional enzyme preparation. The raw materials are readily available, and the cost is lower than that of traditional compound additives consisting of "probiotic powder + multiple enzymes + plant extracts," facilitating large-scale industrial production and widespread application.

[0171] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0172] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above content of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An enzyme composition, characterized in that, The enzyme composition comprises urease, uricase, and creatinine enzyme; based on the activity of urease ≥1000 U / g, the activity of uricase ≥800 U / g, and the activity of creatinine enzyme ≥600 U / g, the mass ratio of urease, uricase, and creatinine enzyme is (2~3):1:

1.

2. A composite additive, characterized in that, The compound additive comprises the enzyme composition of claim 1 and probiotic metabolites.

3. The composite additive according to claim 2, characterized in that, The probiotic metabolites are products prepared by centrifuging, filtering, concentrating and drying the fermentation products obtained from fermenting probiotics.

4. The composite additive according to claim 3, characterized in that, The probiotic metabolites include metabolites of Lactobacillus plantarum with accession number CICC 25125 and / or metabolites of Lactobacillus rhamnosus with accession number CICC 6137. Optionally, the ratio of the metabolites of *Lactobacillus plantarum* with accession number CICC 25125 to the metabolites of *Lactobacillus rhamnosus* with accession number CICC6137 is (0.8~1.2):

1.

5. The composite additive according to any one of claims 2 to 4, characterized in that, The compound additive comprises, by weight percentage, 20% to 35% of the enzyme composition, 40% to 60% of the probiotic metabolites, and excipients.

6. The composite additive according to claim 5, characterized in that, By mass percentage, the compound additive comprises 20% to 35% of the enzyme composition, 40% to 60% of the probiotic metabolites, 5% to 15% of the activity protectant, 3% to 8% of the dispersant, and 2% to 7% of the pH adjuster.

7. The composite additive according to claim 6, characterized in that, The composite additive satisfies one or more of the following conditions: (1) The active protective agent comprises one or more of trehalose and xanthan gum; optionally, the mass ratio of trehalose to xanthan gum is 1:(1~2). (2) The dispersant comprises one or more of magnesium stearate and silicon dioxide; optionally, the mass ratio of magnesium stearate to silicon dioxide is (2~3):1; (3) The pH adjuster comprises one or more of citric acid and potassium dihydrogen phosphate; optionally, the mass ratio of citric acid to potassium dihydrogen phosphate is 1:(1~3).

8. A method for preparing the composite additive according to claim 6 or 7, characterized in that, The method includes the step of preparing the composite additive by mixing the enzyme composition, probiotic metabolites, activity protectant, dispersant and pH adjuster.

9. The method for preparing composite additives according to claim 8, characterized in that, The method satisfies one or more of the following conditions: (1) Before mixing, the enzyme composition, probiotic metabolites, active protectant, dispersant and pH adjuster are dried at 55℃~65℃ for 2 h~3 h, and then passed through an 80 mesh~100 mesh sieve respectively. (2) Mix at 25℃~30℃ and 800 r / min~1000 r / min for 15 min~20 min; (3) After mixing, the composite additive is cooled to 23°C to 27°C and then passed through a 90-mesh sieve to a 110-mesh sieve.

10. A deodorizing cat litter, characterized in that, The deodorizing cat litter comprises the composite additive as described in claim 6 or 7 and the cat litter. Optionally, the cat litter includes one or more of bentonite cat litter, tofu cat litter, mixed cat litter, and crystal cat litter; Optionally, the composite additive is 0.5% to 2.0% of the cat litter mass.