Application of protease to degradation of animal hair balls

Abstract

The present invention relates to a method of preventing or reducing hair ball formation or promoting hair ball elimination in an animal, the method comprising administering to the animal a composition comprising a polypeptide having protease activity.

Description

References to sequence lists This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Technical Field

[0001] This invention relates to a method for preventing or reducing hairball formation in animals or promoting hairball removal, the method comprising administering to the animal a composition comprising a polypeptide having protease activity; the use of a polypeptide having protease activity for preventing or reducing hairball formation in animals or promoting hairball removal; and a composition for use in the method. Background Technology

[0002] Cats are known for their meticulous grooming habits, in which they clean and maintain their fur with their tongues. This process inevitably results in the ingestion of shed hair, which usually passes harmlessly through the digestive tract. However, in many cases, the ingested hair accumulates in the stomach, forming a dense mass commonly known as a hairball or hairstone. While occasional expulsion of hairballs via vomiting is relatively common and often harmless, frequent or chronic occurrences can lead to significant health problems in cats, including gastrointestinal upset, loss of appetite, constipation, or even life-threatening obstructions that may require veterinary intervention.

[0003] Traditional methods of managing hairballs focus on minimizing hair intake or facilitating hair passage through the digestive system. These methods include regular brushing to reduce shed fur, dietary interventions (such as high-fiber foods) to promote gastrointestinal motility, and the application of lubricants or gels designed to coat the hair and facilitate its movement through the intestines. While these strategies may provide some relief, they do not address the structural composition of hairballs, which consist primarily of densely packed hair strands bound together by proteinaceous material secreted during digestion. This binding matrix makes hairballs resistant to natural decomposition, prompting their persistence in the gastrointestinal tract.

[0004] WO 2004 / 045300 discloses an animal feed composition that inhibits hairball formation and reduces the odor of animal excrement, wherein the composition may contain fungal acidic protease.

[0005] US 2003 / 0147940 discloses an agent for promoting the emptying of hair and hairballs from animals, the agent comprising at least two sources selected from seaweed, protease, and cellulose, wherein the protease source preferably includes papaya.

[0006] WO 99 / 65514 discloses animal food preparations for animals prone to hairballs, which contain a source of nutrients including bromelain, an enzyme derived from pineapple.

[0007] Although some patent disclosures involve protease-containing compositions that supposedly solve the fuzz problem, such products that are truly effective against fuzz are not yet commercially available.

[0008] This invention addresses this challenge by introducing a novel method that utilizes the enzymatic action of proteases to specifically target and degrade the protein matrix that holds hairballs together. By incorporating proteases into the diet of cats or other animals or administering them as supplements, the protein structures binding ingested hair can be broken down. This enzymatic degradation allows hairballs to dissolve or disintegrate more effectively, enabling them to pass naturally through the digestive system without causing harm. This solution not only reduces hairball formation and impact but also improves the overall gastrointestinal health and quality of life of cats and other hairball-prone animals.

[0009] This innovative approach offers a safe, targeted, and non-invasive alternative to existing hairball management strategies. By addressing the root cause of persistent hair—the protein matrix—this method provides a more effective and sustainable solution to common feline / animal health issues. Summary of the Invention

[0010] In one aspect, the present invention provides a method for preventing or reducing hairball formation in an animal or promoting hairball elimination, the method comprising administering to the animal a composition comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1.

[0011] Other aspects of the invention relate to the use of a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1 for preventing or reducing hairball formation or promoting hairball elimination in animals; and to compositions comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1, the compositions being used for preventing or reducing hairball formation or promoting hairball elimination in animals.

[0012] Sequence Overview SEQ ID NO:1 is Nocardia sphaeroides ( Nocardiopsis prasina ) of proteases.

[0013] SEQ ID NO:2 is a variant of SEQ ID NO:1. Attached Figure Description

[0014] Figure 1 The image shows a cat hairball treated with the protease according to the invention (right) and a control cat hairball not treated with the protease (left).

[0015] Figure 2 This shows a comparison between cat hairballs treated with the protease according to the invention and cat hairballs treated with several commercially available proteases and a control group without protease.

[0016] definition Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0017] Unless otherwise defined or clearly indicated by the context, 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 invention pertains.

[0018] Additives: The term "additive" refers to ingredients added to, for example, pet food to enhance nutritional value, shelf life, taste, or appearance. Pet food additives can be categorized into nutritional additives, technical additives, and sensory additives. Nutritional additives (such as vitamins and minerals) ensure pets receive essential nutrients, while technical additives (such as preservatives and antioxidants) extend shelf life and maintain quality, and sensory additives (such as flavorings and colorings) make food more palatable and appealing to pets and their owners.

[0019] Animals: The term "animal" refers to any kind of animal, including farm animals and pets. However, the present invention is particularly directed to animals kept as pets (also known as "companion animals"), including but not limited to cats, dogs, rabbits, guinea pigs, and hamsters. In some preferred embodiments, the animal is a cat.

[0020] Animal food: The term "animal food" refers to a food composition designed to feed animals. Animal food can specifically be "pet food," that is, a food composition designed to feed pet animals (e.g., cats or dogs). Animal food (including pet food) is generally suitable for feeding specific animal species; for example, cat food is suitable for feeding cats, and dog food is suitable for feeding dogs. Animal food (such as pet food) can be of different types, such as dry, wet, semi-moist, raw, or freeze-dried, and will generally contain essential nutrients, including protein, fat, carbohydrates, vitamins, and minerals.

[0021] Protease activity: The term "protease" refers to an enzyme that catalyzes the hydrolysis of peptide bonds and possesses peptidase activity (EC 3.4; also known as proteolytic activity or protease activity). The EC 3.4 group includes several subgroups, including EC 3.4.21 (serine endopeptidase), which further contains subgroups, including EC 3.4.21.62 (subtilisin). The terms "protease" and "peptide with protease activity" are used interchangeably herein. The protease used in this invention is commercially available from Novonesis A / S under the trade names Targut® Digest or Yuchongxi® Intestinal Comfort. Protease activity can be determined using assays known in the art, in which a substrate comprising a peptide bond relevant to the specificity of the protease in question is employed. Suitable protease substrates include pNA-substrates, such as Suc-AAPF-pNA (available, for example, from Sigma, S-7388). Another example is Protazyme AK (azurite-stained cross-linked casein prepared as tablets by Megazyme, T-PRAK). Example 2 of WO 01 / 58276 describes a suitable protease activity assay using Suc-AAPF-pNA.

[0022] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0023] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) is used. J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) determines the sequence identity between two amino acid sequences as the output of "longest identity," an algorithm such as the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000). Trends Genet. The method is implemented in the Niedel procedure of [Trends in Genetics] 16: 276-277 (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel procedure to report the longest identity, the non-brief (-nobrief) option must be specified on the command line. The Niedel-marked "Longest Identity" output is calculated as follows: (Identical residues × 100) / (Alignment length - Total number of vacancies in the alignment) Supplements: The term "supplement" refers to products designed to provide additional nutrients beyond those typically found in, for example, regular pet food. They can help address specific health issues, support overall health, or fill nutritional gaps in a pet's diet. Pet food supplements are available for a wide range of pets, including dogs, cats, and others, and come in various forms such as powders, liquids, tablets, chewables, or capsules.

[0024] Variant: The term "variant" refers to a polypeptide with protease activity that includes at least one substitution, insertion (including extension), and / or deletion (e.g., truncation) at one or more positions compared to the protease of SEQ ID NO: 1. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, particularly one amino acid) adjacent to and immediately following the amino acid occupying a given position. Detailed Implementation

[0025] As described above, the present invention provides a method for preventing or reducing hairball formation in animals or promoting hairball elimination, the method comprising administering to the animal a composition comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1. In one embodiment, the method of the present invention is used to prevent hairball formation in animals. In a related embodiment, the method of the present invention is used to reduce hairball formation in animals. In another embodiment, the present invention is used to promote hairball elimination in animals. However, it will be apparent that these embodiments are not independent of each other, and the ultimate objective of the present invention is to solve the problem of hairballs in animals by preventing and / or reducing hairball formation and / or promoting hairball elimination.

[0026] The protease-active polypeptide used in any of the uses, methods, or compositions disclosed herein preferably has at least 75% sequence identity with SEQ ID NO: 1. More preferably, the polypeptide has at least 80% sequence identity with SEQ ID NO: 1, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one particular embodiment, the protease-active polypeptide has 100% sequence identity with SEQ ID NO: 1. In another particular embodiment, the protease-active polypeptide has at least 99% sequence identity with SEQ ID NO: 1 and comprises or is composed of the amino acid sequence of SEQ ID NO: 2.

[0027] The polypeptide used in this invention, having protease activity and having at least 70% sequence identity with SEQ ID NO: 1, can be, for example, derived from the genus Nocardia (…). Nocardiopsis The peptide is a wild-type protease polypeptide of a species or different bacterial species. Alternatively, it can be a variant of the peptide of SEQ ID NO: 1 (e.g., a genetically engineered variant) having one or more substitutions, insertions, and / or deletions compared to SEQ ID NO: 1, wherein the variant may, for example, have improved stability or improved efficacy compared to the parent protease having SEQ ID NO: 1. Such variants will typically contain one or more substitutions, such as 1-20 substitutions, for example 1-15 or 1-10 substitutions. In a specific embodiment, the variant has a single amino acid substitution relative to SEQ ID NO: 1 and comprises or is composed of the amino acid sequence of SEQ ID NO: 2.

[0028] The amount of peptides with protease activity in the composition used in the methods of the present invention can be determined by those skilled in the art, for example, based on the type of animal to which the composition is to be used and the nature of the composition (e.g., animal food, or supplement or additive). In the case of pet food, the composition may contain peptides in amounts such as about 0.01 wt% to 2 wt%, typically about 0.05 wt% to 1 wt%, for example about 0.1 wt% to 0.5 wt%, such as about 0.1 wt% to 0.2 wt%. In the case of supplements or additives, the amount of peptides in the composition will be higher, for example up to about 12 wt%, typically up to about 10 wt%, such as about 1 wt% to 10 wt%, more typically about 2 wt% to 10 wt%, such as about 4 wt% to 8 wt%.

[0029] The method of the present invention is intended for use in any animal where hairballs may be a problem, but is particularly intended for use in pets (companion animals). Given the widespread popularity of cats as pets worldwide and the fact that hairballs are a common problem in cats, the invention is especially suitable for use in cats. Furthermore, the invention is intended for use in other pets where hairballs may be a problem, such as rabbits, dogs, guinea pigs, or hamsters.

[0030] The composition comprising a polypeptide having protease activity used in this invention can be any composition suitable for ingestion by the animals under discussion, and will typically be pet food, or a pet food supplement or additive. In a preferred embodiment, the composition is pet food.

[0031] It will be apparent that, in the case of pet food, pet food is generally designed for use in specific animals; for example, cat food will be particularly suitable for feeding cats, dog food will be particularly suitable for feeding dogs, rabbit food will be particularly suitable for feeding rabbits, and guinea pig food will be particularly suitable for feeding guinea pigs. Each of these types of animal food (including the form in which they are produced and sold (e.g., dry, wet, etc.)), the ingredients they contain, and the methods used to produce them are well known to those skilled in the art.

[0032] The following provides information about cat food as an exemplary type of animal food, in which peptides with protease activity can be used for hairball degradation. However, it should be remembered that the methods and compositions of the present invention are also suitable for feeding animals other than cats, such as rabbits, dogs, guinea pigs, or hamsters, and those skilled in the art will be familiar with pet food compositions and ingredients for these or other animals.

[0033] Cat food comes in several forms, such as dry, wet, semi-moist, raw, or freeze-dried, which can affect its composition. Dry cat food (kibble) has a low moisture content (about 6%-10%) and is typically made from a combination of meat, grains, and other ingredients. Dry cat food is economical, convenient, and has a long shelf life.

[0034] Wet cat food (canned food) has a high moisture content of about 70%-85% and is often more palatable and closer to a cat's natural diet. It is typically made with a higher amount of animal-based protein than dry cat food.

[0035] Semi-moist cat food contains approximately 25%-35% water and is typically made with artificial flavorings and preservatives. Due to health concerns regarding sugar content and additives, semi-moist cat food is less common than before.

[0036] Raw or freeze-dried cat food is made from raw ingredients and is often freeze-dried or frozen for preservation. This mimics a cat's natural diet, but must be handled carefully to avoid contamination.

[0037] In addition, there are prescription and special diets designed for cats with specific health problems (such as urinary tract problems, obesity, kidney disease).

[0038] Cat food is formulated to meet the dietary needs of obligate carnivores, and its main components are protein, fat, vitamins and minerals, moisture, and additives / preservatives.

[0039] Essential proteins for cats and other animals, including pets, are typically obtained from chicken, beef, fish, turkey, lamb, eggs, and / or organ meats, and are typically found in high-quality cat food at approximately 30%–50% of the dry matter content. The function of protein is to support muscle development, provide energy, and maintain overall health.

[0040] Fats are typically in the form of animal fats, fish oils, and / or vegetable oils (e.g., sunflower oil or flaxseed oil), and are usually present in amounts of about 10%–30% of dry matter. Fats provide energy, support healthy skin and fur, and aid in the absorption of fat-soluble vitamins.

[0041] Carbohydrates are typically obtained from rice, corn, barley, sweet potatoes, peas, and / or other grains or vegetables. Carbohydrates in cat food are preferably kept at low levels because cats have a limited capacity to efficiently digest them. Carbohydrates serve as an energy source but are not essential for cats.

[0042] Vitamins and minerals (such as taurine, vitamin A, vitamin D, calcium, phosphorus, etc.) are often added to cat food to support, for example, metabolic processes, bone health and immune function.

[0043] Moisture / water content is important for hydration, especially for wet foods with a higher moisture content than dry foods.

[0044] Examples of additives and preservatives that can be added to cat food include natural preservatives (such as vitamin E (tocopherol)) and / or synthetic preservatives to maintain freshness, as well as flavor enhancers or additional nutrients.

[0045] While the proportions of nutrients in pet food (such as cat food) can vary, a typical breakdown of appropriate nutrients in cat food is as follows (based on approximate percentages of dry matter): • Protein: at least 30%-50% • Fat: 10%-30% • Carbohydrates: Ideally less than 10% • Fiber: Approximately 2%-4% In addition to the basic components of protein, fat, carbohydrates, and fiber, pet food supplements and additives are typically included in commercial cat food and other pet foods to enhance nutritional value, improve health, or preserve the product. Some common supplements and additives are outlined below.

[0046] Adding supplements can enrich the nutritional profile of pet food or address specific health needs. Common supplements include: • Essential nutrients that are crucial for obligate carnivores such as cats, such as taurine, omega fatty acids (such as omega-3 and omega-6), vitamins (such as vitamin A, vitamin D and B vitamins (such as vitamin B12 and B6)) and minerals (such as calcium, phosphorus, magnesium, potassium, zinc, iron and selenium).

[0047] • Digestive health supplements, including probiotics (e.g., Lactobacillus acidophilus). Lactobacillus acidophilus ) and Enterococcus faecalis ( Enterococcus faecium Prebiotics (such as chicory root, fructooligosaccharides (FOS) and inulin).

[0048] • Fiber supplements, such as beet pulp, psyllium husk, or squash.

[0049] • Joint and bone supplements, especially for older cats, such as glucosamine, chondroitin, calcium, and phosphorus.

[0050] • Immune system enhancers, including antioxidants (such as vitamin C, vitamin E, beta-carotene, and selenium) and colostrum.

[0051] • Skin and fur health supplements, such as biotin, zinc, and fish oil or krill oil.

[0052] • Stress and anxiety management supplements, such as L-theanine, tryptophan, and valerian root or chamomile.

[0053] Additives are non-nutritive ingredients added for flavor, preservation, or texture. These are optional ingredients that can be added to play various important roles in pet food manufacturing.

[0054] Preservatives prevent spoilage and extend shelf life, and can be natural or synthetic. Examples of natural preservatives include vitamin E (tocopherol), vitamin C (ascorbic acid), and rosemary extract. Examples of synthetic preservatives include BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), and ethoxyquin.

[0055] Flavor enhancers can be added to make food more palatable to cats. These flavor enhancers include natural flavorings (such as chicken broth, liver powder, and fish meal) and artificial flavorings (such as synthetic meat or fish flavorings).

[0056] Coloring additives can be added to make food more appealing to humans (not cats, since they are mostly colorblind). These coloring additives include natural colorants (such as beet juice or turmeric) and artificial colorants.

[0057] Conditioners and binders can be added to improve texture and consistency, especially in wet or semi-wet foods. These conditioners and binders include gums and starches, such as guar gum, carrageenan, and xanthan gum.

[0058] Furthermore, the pet food compositions of the present invention may contain anti-hairball agents in addition to the proteases disclosed herein. Such agents can help reduce hairball formation by aiding digestion and fecal passage, and include, for example, added fiber (e.g., beet pulp, cellulose, or psyllium) and fatty acids (such as ω-3 or ω-6 fatty acids).

[0059] While the typical forms and ingredients of cat food have been described above, the methods and compositions of the present invention will also be suitable for feeding animals other than cats, particularly other pets (companion animals), such as rabbits, dogs, guinea pigs, or hamsters. Those skilled in the art will be familiar with pet food compositions and ingredients suitable for these or other animals, and will be able to readily practice these methods and prepare the compositions disclosed herein for feeding such other pets or other animals.

[0060] As described above, another aspect of the invention relates to the use of a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1 for preventing or reducing hairball formation in animals or promoting hairball removal. Another aspect of the invention relates to a composition comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1, for use in preventing or reducing hairball formation in animals or promoting hairball removal. It will be apparent that in these respects, the use or composition may have any of the characteristics described above regarding the methods of the invention. In particular, the protease-active polypeptide may have any of the characteristics described above regarding the methods, and the composition (e.g., pet food (such as cat food), or pet food supplements or additives) may similarly have any of the characteristics described above regarding the methods.

[0061] Another aspect of the invention relates to the use of a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1 for reducing fecal odor in animals (e.g., pet animals such as cats, dogs, rabbits, guinea pigs, or hamsters). For example, fecal odor in cats or other pets may be caused or exacerbated by undigested proteins remaining in the later stages of the intestine, which can be a nutrient source for unwanted bacteria. Therefore, it is anticipated that a polypeptide having protease activity can digest undigested proteins, thereby preventing or reducing fecal odor.

[0062] Another aspect of the invention relates to the use of a polypeptide having protease activity and at least 70% sequence identity with SEQ ID NO: 1 for degrading antigenic proteins in animal food compositions (e.g., pet food, such as cat food or dog food) to prevent or reduce allergies; and a method for degrading antigenic proteins in animal food compositions (e.g., pet food, such as dog food or cat food), the method comprising treating the composition with or adding the polypeptide to the composition with a polypeptide having protease activity and at least 70% sequence identity with SEQ ID NO: 1. Pet food formulations typically contain a certain amount of plant protein, such as soy protein. It is known, for example, that soy protein includes specific proteins capable of triggering allergic reactions in some animals (e.g., dogs and, to a lesser extent, cats). Therefore, it is contemplated that proteins derived from, for example, soy can be degraded using a polypeptide having protease activity, thereby reducing the risk of allergic reactions in animals.

[0063] It is further anticipated that the protease peptides disclosed herein can be used to degrade meat proteins in pet foods. Cats are obligate carnivores, meaning they biologically require a diet primarily composed of meat, while dogs are facultative carnivores, meaning they can thrive on a diet including both meat and plant matter. However, both dog and cat foods typically contain a significant proportion of meat protein, and it may be desirable to improve the digestibility of meat proteins in, for example, cat or dog food. Therefore, another aspect of the invention relates to the use of a peptide having protease activity and at least 70% sequence identity with SEQ ID NO: 1 for degrading meat proteins in animal food compositions (particularly meat-containing pet foods, such as cat or dog food); and a method for degrading meat proteins in animal food compositions (particularly meat-containing pet foods, such as cat or dog food), the method comprising adding a peptide having protease activity and at least 70% sequence identity with SEQ ID NO: 1 to the composition.

[0064] On the other hand, the present invention relates to the use of a polypeptide having protease activity and at least 70% sequence identity with SEQ ID NO: 1 for the enzymatic hydrolysis of feather proteins, for example, for use in animal food compositions (e.g., pet food, such as cat food or dog food); and a method for performing enzymatic hydrolysis of feather proteins, for example, for use in animal food compositions (e.g., pet food, such as cat food or dog food), the method comprising performing enzymatic hydrolysis of feather proteins using a polypeptide having protease activity and at least 70% sequence identity with SEQ ID NO: 1. Feathers from poultry and other birds contain about 80%-90% keratin, thus making them a potential protein source for animal feed (such as pet food). However, this use is limited by the poor digestibility of feather proteins. Therefore, the protease of the present invention can be used by manufacturers of, for example, pet food as a processing aid to allow for more efficient extraction of protein hydrolysates.

[0065] The present invention is further illustrated by the following non-limiting examples.

[0066] Example Example 1: Proof-of-Concept Study on the Degradation of Cat Hairballs In this example, real cat hair was collected and used to evaluate the ability of a protease (available from Novonesis A / S under the trade names Targut® Digest or Yuchongxi®) to degrade hair in a simulated cat stomach environment.

[0067] method A hydrochloric acid solution with pH 2 was prepared by adding 100 ml of water to a 250 ml beaker, followed by adding approximately 1 ml of 1M HCl to adjust the pH to 2.0 ± 0.2 and mixing.

[0068] A simulated gastric juice solution was prepared by weighing 0.1 g of 2500 IU / ml pepsin and 0.3 g of sodium chloride, adding them to a hydrochloric acid solution, and dissolving them by stirring the contents of a beaker with a magnetic stirrer. The solution was then stored at room temperature.

[0069] Use a brush to collect cat hair from your pet cat and store it in a sealed bag.

[0070] The collected cat hair is rolled into balls by hand, each with a diameter of about 15 mm, and each ball weighs 0.1 g.

[0071] The hair was digested by adding tangled hairballs to a simulated gastric juice solution (one hairball per 40 ml) and shaking ten times to ensure the hair was completely submerged. The simulated gastric juice containing the hairballs was then heated in a 38°C water bath and manually stirred every 10 minutes for 2 hours to ensure the hair was completely submerged.

[0072] After 2 hours of pepsin reaction, the hair was rinsed in clean water, placed in a 250 ml beaker, and rinsed three times with 100 ml of water.

[0073] A pH 7.0 PBS buffer solution was prepared by weighing 10.90 g Na2HPO4·12H2O and 2.66 g NaH2PO4·2H2O and dissolving them in 1000 ml of deionized water (adjusting the pH to 7.0).

[0074] The enzyme solution was prepared by adding 40 ml of PBS buffer to a 50 ml centrifuge tube, weighing the required amount of the protease (SEQ ID NO: 1), and then adding it to the centrifuge tube. The solution was then shaken until the enzyme was completely dissolved. For this proof-of-concept study, enzyme concentrations ranging from 1% to 4% were tested, for example, adding 0.4 g of enzyme to 40 ml of buffer to obtain a 1% enzyme solution.

[0075] Cat hair was added to the enzyme solution and then placed in a 38°C water bath, stirred every 10 minutes with a glass rod for up to 60 hours. In this example, one hairball was used, but in other experiments, more than one hairball (up to eight) was tested.

[0076] Observe the tubes containing cat hairballs regularly for up to 60 hours. Hairball degradation is indicated by a darkening of the enzyme solution. In those cases, stir the hairballs with a glass rod to check for breakage. If breakage is observed, pour the enzyme solution from the tube into a 10 cm diameter petri dish for better observation of the hairballs.

[0077] The control group was treated in the same way as described above, using a simulated gastric juice solution and incubated in PBS buffer, but without the use of protease.

[0078] result The results of the study described above show that Figure 1 In the image, cat hairballs treated with the protease peptide of SEQ ID NO: 1 are shown on the right, while control cat hairballs not treated with the protease are shown on the left.

[0079] like Figure 1As seen in the image, treatment with the protease of SEQ ID NO: 1 resulted in complete degradation of the hairballs after 46 hours (right petri dish). In contrast, the control hairballs, which were not treated with the protease, remained intact after this time (left petri dish).

[0080] Example 2: Study on the Degradation of Cat Hairballs In this example, real cat hair was collected and used to evaluate the ability of the protease of the present invention (available from Novonesis A / S under the trade names Targut® Digest or Yuchongxi®) to degrade hair in a simulated cat stomach and intestinal environment compared to several commercially available proteases.

[0081] method A hydrochloric acid solution with a pH of 2 was prepared by adding approximately 5 ml of 1M HCl to a flask along with 500 ml of water to adjust the pH to 2.0 ± 0.2, and then mixing.

[0082] A simulated gastric juice solution was prepared by weighing 0.5 g of 2500 IU / ml pepsin and 1.5 g of NaCl, adding them to a flask along with hydrochloric acid solution, and dissolving them by stirring the contents of the flask with a magnetic stirrer. The solution was then stored at room temperature. The solution was aliquoted into 50 ml centrifuge tubes, 40 ml per tube.

[0083] Weigh the protease and add it to a centrifuge tube to prepare a 2% enzyme solution using 0.8 g of protease in 40 ml of simulated gastric juice solution. Shake the tube until the enzyme is completely dissolved, about 5 minutes, and adjust the pH to 2.0 ± 0.2 using 1M HCl and 1M NaOH.

[0084] Collect cat hair from your pet cat using a cat comb. Twist the collected cat hair into balls approximately 30 mm in diameter by hand, each ball weighing 0.4 g. Store the balls at room temperature.

[0085] Hair was digested by adding rolled-up hairballs to a simulated gastric juice solution (0.4 g hairballs per 40 ml) and shaking ten times to ensure the hair was completely submerged. The tubes containing the simulated gastric juice and hairballs were heated in a 38°C water bath and manually shaken 10 times every 10 minutes for 1 hour to ensure the hair remained completely submerged during the simulated gastric reaction.

[0086] One hour after the gastric reaction, the pH was adjusted to 7.0 ± 0.2 using 1M HCl and 1M NaOH. The enzyme solution was then placed in a 38°C water bath for 45 hours to simulate an intestinal reaction, with stirring every 30 minutes using a glass rod, and the pH was adjusted to 7.0 ± 0.2 using 1M HCl and 1M NaOH.

[0087] 45 hours later, the solution was stirred with a glass rod to observe whether the cat hairballs broke apart, and the solution and cat hair were poured into a 10 cm diameter petri dish to observe the condition of the hair.

[0088] The following treatments were used in this study: 1. HT300 protease from Honest Corporation 2. Alkaline protease from VTR Biotech 3. Axtra® Pro protease from International Flavors & Fragrances (IFF) 4. Neutral protease from Longda Corporation 5. Papain P164463 from Aladdin Scientific. 6. Bromelain B4882 from Sigma-Aldrich 7. Control (without protease) 8. The protease of the present invention: SEQ ID NO: 1 result The results of the study described above show that Figure 2 In the study, it was observed that treatment with the protease of the present invention (SEQ ID NO:1; No. 8) (commercially available from Novonesis A / S under the trade names Targut® Digest or Yuchongxi® Intestinal Comfort) resulted in complete degradation of the hairballs. In contrast, no effect was observed on hairballs treated with six commercially available proteases (Nos. 1-6) or the control (no protease; No. 7) because these hairballs remained intact.

Claims

1. A method for preventing or reducing hairball formation in an animal or promoting hairball elimination, the method comprising administering to the animal a composition comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO:

1.

2. The method of claim 1, wherein the animal is a pet animal, such as a cat, dog, rabbit, guinea pig, or hamster.

3. The method of claim 2, wherein the animal is a cat.

4. The method as described in any of the preceding claims, wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

1.

5. The method as described in any of the preceding claims, wherein the composition is pet food, or a pet food supplement or additive.

6. The method of claim 5, wherein the composition is dry, wet, semi-wet, raw, or freeze-dried pet food.

7. The method of any of the preceding claims, wherein the composition is a pet food comprising the polypeptide in an amount of about 0.01 wt% to 2 wt%, about 0.05 wt% to 1 wt%, about 0.1 wt% to 0.5 wt%, or about 0.1 wt% to 0.2 wt% based on the total weight of the composition; or wherein the composition is a pet food supplement or additive comprising the polypeptide in an amount of up to about 12 wt%, typically up to about 10 wt%, such as about 1 wt% to 10 wt%, about 2 wt% to 10 wt%, or about 4 wt% to 8 wt% based on the total weight of the composition.

8. The use of a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1 for the prevention or reduction of hairball formation or for promoting hairball elimination in animals.

9. The use as described in claim 8, wherein the animal is a pet animal, such as a cat, dog, rabbit, guinea pig, or hamster.

10. The use as described in claim 9, wherein the animal is a cat.

11. The use according to any one of claims 8-10, wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

1.

12. The use as described in any one of claims 8-11, wherein the polypeptide is administered to the animal in pet food, or a pet food supplement or additive.

13. The use as described in any one of claims 8-12, wherein the polypeptide is administered to the animal in a pet food composition comprising an amount of the polypeptide based on the total weight of the composition of about 0.01 wt% to 2 wt%, about 0.05 wt% to 1 wt%, about 0.1 wt% to 0.5 wt%, or about 0.1 wt% to 0.2 wt%; or wherein the polypeptide is administered to the animal in a food supplement or additive comprising an amount of the polypeptide based on the total weight of the composition of up to about 12 wt%, typically up to about 10 wt%, for example about 1 wt% to 10 wt%, about 2 wt% to 10 wt%, or about 4 wt% to 8 wt%.

14. A composition comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1, the composition being used for preventing or reducing hairball formation in animals or promoting hairball elimination.

15. The composition of claim 14, wherein the composition is pet food, preferably cat food, or a pet food supplement or additive.

16. The use of a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1 for reducing fecal odor in animals, preferably wherein the animal is a pet animal, such as a cat, dog, rabbit, guinea pig or hamster.

17. A polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO: 1 for use in degrading antigenic proteins in pet food compositions for, for example, prevention or reduction of allergies.

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