Lactobacillus casei zyhyy-005 probiotic preparation and its application in improving the overall health of dog hair

Abstract

This invention provides a postbiotic preparation of Lactobacillus casei ZYhyy-005 and its application in improving the overall health of canine hair. The Lactobacillus casei (… Lacticaseibacillus casei ZYhyy-005 was deposited on August 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31555. The metabiotic preparation of *Lactobacillus casei* ZYhyy-005 can achieve multi-dimensional effects targeting the structure and physiological characteristics of canine pet hair, specifically: increasing the brightness and color saturation of canine pet hair; increasing the peak tensile strength of canine pet hair, enhancing its tensile and breakage resistance; reducing the thickness of canine pet hair cuticles, improving the regularity and uniformity of canine pet hair, and optimizing the surface microstructure of canine pet hair; increasing the content of glutamic acid, cysteine, and proline in canine pet hair, and improving the keratin molecular composition of canine pet hair.

Description

Technical Field

[0001] This invention belongs to the field of pet nutrition and functional microbial preparations, and specifically relates to a postbiotic preparation of Lactobacillus casei ZYhyy-005 and its application in improving the overall health of canine hair. Background Technology

[0002] In recent years, with the increasing sophistication of pet care, healthy pet coat maintenance has become one of the core needs in the pet grooming industry. The luster, saturation, mechanical strength, and microstructural integrity of the coat not only directly affect a pet's appearance but are also closely related to its skin barrier function and overall health. Consequently, the market demand for professional coat-enhancing products for canines continues to grow. Currently, pet coat-enhancing products on the market are mainly topical conditioning agents and single-nutrient oral supplements. These products often only provide temporary, superficial improvements, have limited functionality, and some have issues such as insufficient ingredient safety and the potential for long-term side effects. They fail to achieve a systematic and lasting improvement in pet coat health from the internal level, and cannot meet the market's demand for efficient, safe, and comprehensive professional coat-enhancing solutions.

[0003] The development of modern microbial technology has provided a new path for improving pet hair health. Postbiotics, as a general term for inactivated bacteria and metabolites of probiotics, have the advantages of strong stability, high safety and diverse biological activities. The short-chain fatty acids, antioxidants, immunomodulatory factors and other active ingredients they contain can promote healthy hair growth from the endogenous level through multiple pathways such as regulating the balance of animal intestinal microecology, optimizing hair follicle nutrition metabolism and enhancing skin barrier function. They have become an important research and development direction for functional nutritional preparations for pets.

[0004] While some probiotics and their post-biotics have been used in pet coat care research, existing publicly available technologies still have significant limitations: (1) Most strains are general pet-type and have not been specifically developed for canine intestinal characteristics, hair structure and metabolic features, so their effects in dogs are limited. (2) Most of the published strains can only improve appearance indicators such as gloss and saturation, but cannot simultaneously improve hair mechanical strength, hair cuticle structure, hair regularity and uniformity and the content of multiple key amino acids; (3) Existing technologies lack specialized strains and preparations that can simultaneously enhance the three key amino acids of hair keratin: glutamic acid, cystine, and proline, making it difficult to achieve comprehensive and structural improvement of hair.

[0005] Therefore, developing targeted strains and their metabiotics based on the physiological characteristics of canine pets to achieve a comprehensive improvement in canine coat health is an urgent problem to be solved. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide a post-biotic preparation of Lactobacillus casei ZYhyy-005 and its application in improving the overall health of canine hair. The Lactobacillus casei ZYhyy-005 post-biotic preparation can improve the quality of pet dog hair from an endogenous systemic perspective, achieving a multi-dimensional hair beautification effect.

[0007] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides a Lacticaseibacillus casei ZYhyy-005 for comprehensive health improvement of canine pet hair. Lacticaseibacillus casei ZYhyy-005 was deposited at the China General Microbiological Culture Collection Center on August 5, 2024, with accession number CGMCC No. 31555.

[0008] The second aspect of this invention provides a post-biotic preparation of Lactobacillus casei ZYhyy-005 for comprehensive improvement of canine pet coat health. The post-biotic preparation is prepared by uniformly mixing post-biotic powder of Lactobacillus casei ZYhyy-005 described in the first aspect with glucose. The post-biotic powder is prepared by inactivating, concentrating, and freeze-drying Lactobacillus casei ZYhyy-005 bacterial solution.

[0009] Furthermore, the preparation of the Lactobacillus casei ZYhyy-005 bacterial culture includes the following steps: (1) Take the frozen Lactobacillus casei ZYhyy-005 out of the -80℃ freezer and thaw it in a 37℃ water bath. Inoculate the strain into 5 mL of liquid MRS medium and culture it anaerobically at 37°C for 24-48 hours until the bacterial solution becomes turbid. (2) Take the turbid bacterial solution obtained in step (1) and inoculate it on MRS solid medium to activate it. Inoculate the activated bacteria into primary medium and obtain seed liquid through primary culture. Inoculate the seed liquid into secondary medium and obtain bacterial solution through secondary culture. (3) Centrifuge the bacterial culture at 4°C and 3000-5000 rpm for 15 min, collect the bacterial cells, wash with sterile physiological saline and resuspend to obtain a suspension. Adjust the concentration of the suspension to be greater than 100%. CFU / mL yields Lactobacillus casei ZYhyy-005 bacterial culture.

[0010] Furthermore, in step (1), the inoculation amount of the strain is 0.02~0.04%.

[0011] Furthermore, in step (2), both the primary culture medium and the secondary culture medium are MRS liquid culture medium.

[0012] Furthermore, in step (2), the inoculation amount of the seed liquid into the secondary culture medium is 1% to 2%.

[0013] Furthermore, in step (2), the conditions for primary culture are culturing at 36~38°C for 24~48 h; the conditions for secondary culture are culturing at 36~38°C for 24~48 h.

[0014] Furthermore, the inactivation conditions are sterilization at 121°C for more than 30 minutes, spreading the inactivated bacterial solution onto MRS plates, and anaerobic incubating at 37°C for more than 48 hours to ensure no colony growth.

[0015] The third aspect of this application provides the use of Lactobacillus casei ZYhyy-005 as described in the first aspect, or the Lactobacillus casei ZYhyy-005 postbiotic preparation as described in the second aspect, in the preparation of products that improve the coat health of canine pets.

[0016] Furthermore, the canine pets mentioned are domestic companion dogs, including Beagles, Poodles, and Golden Retrievers.

[0017] Furthermore, the products for improving the coat health of canine pets include canine-specific diet additives and canine-specific nutritional supplements.

[0018] Furthermore, the product can simultaneously achieve: (1) Enhance the brightness and color saturation of canine pet coats; (2) Improve the peak tensile strength of canine pet hair and enhance the tensile and fracture resistance of canine pet hair; (3) Reduce the thickness of the hair cuticles of canine pets, improve the regularity and uniformity of canine pet hair, and optimize the surface microstructure of canine pet hair; (4) Increase the content of glutamic acid, cystine and proline in the hair of canine pets and improve the molecular composition of keratin in the hair of canine pets.

[0019] Information on strain preservation: Lactobacillus casei ZYhyy-005, deposited at: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Deposit date: August 5, 2024; Accession number: CGMCC No. 31555; Classification: Lactobacillus casei.

[0020] The advantages of this invention compared to the prior art are as follows: 1. The Lactobacillus casei ZYhyy-005 used in this invention is a specific strain with the preservation number CGMCC No.31555. It is different from the existing general-purpose probiotic strains related to pet coat enhancement. Its postbiotic metabolite profile is more suitable for the intestinal microecological structure and nutrient absorption characteristics of canine pets. It can be efficiently utilized by the intestinal tract of canine pets, ensuring the coat enhancement effect from the source of absorption. This solves the technical pain point of low compatibility and reduced effectiveness of general-purpose preparations in canine pets. 2. The Lactobacillus casei ZYhyy-005 post-biotic formulation of this invention can simultaneously optimize the health of canine pet coats from four dimensions: appearance, mechanical properties, microstructure, and molecular composition. It not only significantly improves coat shine and color saturation, enhancing the coat's external appearance, but also effectively increases peak tensile strength, strengthens the coat's tensile and breakage resistance, and reduces hair loss. Simultaneously, it reduces cuticle thickness, improves hair regularity and uniformity, repairs damaged hair surface structures, and optimizes smoothness. Furthermore, it simultaneously increases the content of three key keratin amino acids—glutamic acid, cysteine, and proline—in the hair, improving the keratin composition at the molecular level and achieving a fundamental improvement in hair health. This multi-dimensional synergistic improvement effect is something that existing pet coat-enhancing formulations that only improve basic coat-enhancing indicators or regulate single amino acids cannot achieve. 3. This invention is the first to apply Lactobacillus casei ZYhyy-005 post-biotic preparation to the field of improving the coat health of canine pets. It specifically addresses the unique coat-enhancing needs of canine pets, such as coarse coat, large coat volume, and high mechanical strength requirements, filling the technological gap in microbial coat-enhancing preparations for canine pets. Compared with existing topical coat-enhancing agents and single nutrient supplements, the preparation of this invention has the advantages of high efficiency, safety, long-lasting effect, and multi-dimensionality, perfectly meeting the market demand for professional coat-enhancing products for canine pets, and has extremely high market application value and promotion prospects. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The hair scores for each group of dogs in Example 2; Figure 2 The values ​​for the brightness of the dog hair in each group in Example 2; Figure 3 The hair saturation of each group of dogs in Example 2; Figure 4 Here are scanning electron microscope images of canine hair from each group in Example 2; Figure 5 The thickness of the canine hair cuticles in each group in Example 2; Figure 6 The amino acid content (glutamic acid, cystine and proline) of each group of canine hair in Example 2. Detailed Implementation

[0022] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0025] Example 1: Preparation of Lactobacillus casei ZYhyy-005 postbiotic solid dosage form The specific method for preparing postbiotic solid dosage forms using Lactobacillus casei ZYhyy-005 in this embodiment is as follows: (1) Preparation of MRS liquid culture medium: 20.00 g glucose, 10.00 g peptone, 5.00 g yeast extract, 10.00 g beef extract, 1.00 g Tween 80, 5.00 g sodium acetate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate and 2.00 g dipotassium hydrogen phosphate were dissolved in 1 L deionized water and sterilized at 121°C for 15 min.

[0026] (2) Activate Lactobacillus casei ZYhyy-005 on MRS solid medium, inoculate the activated bacteria into MRS liquid medium, and culture at 37°C for 48 h to obtain seed culture; inoculate the seed culture into MRS liquid medium at an inoculation rate of 1%, and culture at 37°C for 48 h to obtain bacterial suspension; centrifuge the bacterial suspension, collect the bacterial cells, wash with sterile physiological saline, and resuspend to obtain suspension; adjust the concentration of the suspension to obtain... CFU / mL bacterial suspension.

[0027] (3) The bacterial suspension was sterilized at 121°C for 30 min. The inactivated bacterial solution was spread onto MRS plates and anaerobically cultured at 37°C for more than 48 hours. The sterilized bacterial suspension was freeze-dried to obtain postbiotic powder. Based on the viable count of the bacterial suspension, the postbiotic powder was mixed with glucose (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) to prepare a solution containing Lactobacillus casei ZYhyy-005 cells. CFU / g postbiotic solid dosage form.

[0028] Example 2: Effects of Lactobacillus casei ZYhyy-005 postbiotic solid dosage form on coat health in pet dogs To investigate the effects of the Lactobacillus casei ZYhyy-005 postbiotic solid formulation described in Example 1 on the coat health of canines, this example uses beagles as experimental subjects and conducts the following animal experiments: (1) Experimental grouping This experiment selected 24 Beagle dogs from the College of Veterinary Medicine, China Agricultural University, and randomly divided them into 3 groups of 8 dogs each: a negative control group, experimental group 1, and experimental group 2. The specific treatments were as follows: Negative control group: fed 400 g of basic dog food daily, without any test formulation added.

[0029] Experimental Group 1: Feed 400 g of basic dog food daily and mix in 0.05% (w / w) of Lactobacillus casei ZYhyy-005 postbiotic solid preparation prepared in Example 1 (i.e., add 0.2 g of postbiotic solid preparation per 400 g of food).

[0030] Experimental Group 2: Feed 400 g of basic dog food daily and mix in 0.1% (w / w) of Lactobacillus casei ZYhyy-005 postbiotic solid preparation prepared in Example 1 (i.e., add 0.4 g of postbiotic solid preparation per 400 g of food).

[0031] All dogs had completed their vaccinations and deworming before the experiment. The experimental environment operated on a 12-hour diurnal cycle, with the temperature controlled between 18-26°C and the relative humidity between 40%-70%. The kennels were cleaned and disinfected twice daily. Each dog was fed once a day with 400 grams of basal food, a baked staple food conforming to the nutritional standards of the Association of American Feed Control Officials (AAFCO, 2023) and the National Research Council (NRC, 2006). Free access to water was provided. The first week was an acclimatization period, followed by 42 days of the formal experimental period. Blood samples were collected from the dogs after the experiment for evaluation.

[0032] All data were statistically analyzed using GraphPad Prism 10.1.2. The significance level was set at ns, indicating no significant difference. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0033] This study strictly followed international guidelines for the use of animals and the "Regulations on the Management of Laboratory Animals in China," and was approved by the Animal Ethics Committee of China Agricultural University (Approval No.: AW11104202-5-2). All experiments were conducted with the aim of minimizing animal suffering.

[0034] (2) Hair score The pet dog coat scoring criteria are shown in Table 1.

[0035]

[0036] After 42 days of feeding, the dogs' coat condition was systematically assessed, and the scoring results were as follows: Figure 1 As shown in the figure, the coat health scores of experimental groups 1 and 2 were significantly higher than those of the negative control group, and the scores of each experimental group remained within the ideal range, with no adverse conditions such as oily coat. This indicates that continuous supplementation with Lactobacillus casei ZYhyy-005 post-natal vitamin solid preparation can effectively promote coat health in dogs and improve its luster and softness.

[0037] (3) Measurement of hair brightness and saturation values This experiment was conducted under standard indoor conditions (room temperature 15–23°C, light intensity 100–300 lux). A Meptop CR-400 colorimeter was used to measure the luminance and saturation values ​​of animal fur. Before measurement, the instrument underwent blank calibration, with the following parameters set: viewing angle 10°, D65 standard light source, and measuring aperture 10 mm. The animals were kept in a natural standing posture. Measurement sites included the head (midpoint of the line connecting the ears), back (where the forelegs cross the spine), and both hind legs (where the leg root connects to the torso). Areas with significant color difference were avoided at each site. Measurements were repeated three times, and the average value was recorded to ensure the systematic and accurate results.

[0038] like Figure 2 As shown, after feeding the *Lactobacillus casei* ZYhyy-005 post-biotic solid preparation described in Example 1, the hair luster value of experimental group 2 was significantly higher than that of the negative control group, and experimental group 1 was also significantly higher than that of the negative control group. The results indicate that this post-biotic solid preparation can effectively improve the luster of canine hair, and its effect may be related to regulating skin lipid metabolism and improving hair surface structure and reflectivity.

[0039] like Figure 3 As shown, the hair saturation in experimental group 2 was significantly higher than that in the negative control group, and the same was true for experimental group 1. This indicates that the Lactobacillus casei ZYhyy-005 post-biotic solid formulation described in Example 1 can further enhance the fullness and vibrancy of hair color. It is speculated that its mechanism may involve enhancing skin barrier function, optimizing hair follicle nutrient supply, and promoting pigmentation stability.

[0040] (4) Measurement of peak breaking force of hair Peak force of the hair was measured using a MARK-10 tension meter, which involved taking a single hair from the back of the dog and recording the instantaneous tension value at the moment of breakage.

[0041]

[0042] The effect of *Lactobacillus casei* ZYhyy-005 post-biotic solid formulation on the peak tensile force of canine hair is shown in Table 2. The results showed that the increases in peak tensile force in experimental groups 1 and 2 were 12.50 and 21.86, respectively, both significantly higher than the negative control group. This indicates that the post-biotic solid formulation can effectively enhance the mechanical properties of canine hair. Peak tensile force is a key indicator for evaluating hair toughness and structural strength. An increase in peak force means better tensile and breakage resistance, which is usually related to enhanced keratin disulfide bond cross-linking and a denser fiber structure. In practice, tougher, more stable hair not only has a lower shedding rate but also retains its surface luster and color for a longer period. Therefore, *Lactobacillus casei* ZYhyy-005 post-biotic solid formulation significantly improves the peak tensile force of hair, enhancing the health and overall quality of hair from a mechanical structural perspective, providing strong evidence for its practical application in pet hair care.

[0043] (5) Scanning electron microscopy evaluation of pet dog hair After 42 days of feeding, hair samples were collected from the back of each dog. During sampling, the hair in the desired area was gently shaved to minimize discomfort to the animals. The hair samples were fixed on a sample tray suitable for scanning electron microscopy (SEM). The hair diameter, cuticle length, and cuticle height of each sample were precisely measured using a Zeiss Sigma 300 / Oxford energy dispersive X-ray spectroscopy (EDS) microscope. High-resolution images of the hair surface were recorded for subsequent analysis. Changes in hair diameter, cuticle length, and cuticle thickness were compared between the control group and each experimental group. Statistical software was used to process the data, assessing the effects of different feeding formulations on hair microstructure. ImageJ was used to process the photographic results.

[0044] Figure 4 This study demonstrated the effect of Lactobacillus casei ZYhyy-005 postbiotic solid formulation on the surface structure of canine hair. The negative control group showed a rougher hair surface with obvious cracks and scale separation. In contrast, experimental groups 1 and 2 showed a smoother hair surface with more orderly scale arrangement and significantly reduced cracks. This indicates that the Lactobacillus casei ZYhyy-005 postbiotic solid formulation described in Example 1 has a significant effect on improving hair surface health and repairing damaged structures.

[0045] Figure 5The study demonstrated the effect of the Lactobacillus casei ZYhyy-005 postbiotic solid preparation described in Example 1 on the cuticle thickness of canine hair. The cuticle thickness of experimental groups 1 and 2 was significantly lower than that of the negative control group, indicating that the Lactobacillus casei ZYhyy-005 postbiotic solid preparation can effectively reduce cuticle thickness and improve hair smoothness.

[0046] (6) Determination of amino acid content in dog hair The hair samples collected in the above (5) pet dog hair scanning electron microscopy evaluation were analyzed using an automated amino acid analyzer (Biochrom30). + The content of glutamic acid, cystine and proline in hair was analyzed.

[0047] like Figure 6 As shown, after 42 days of feeding, significant changes occurred in the hair composition of the three groups of dogs. The glutamate content in experimental groups 1 and 2 was significantly higher than that in the negative control group. The increased glutamate content indicates improved polarity and hydration capacity of hair proteins, which is beneficial for enhancing hair's moisture retention, surface smoothness, and shine. The cysteine ​​content in experimental groups 1 and 2 was significantly higher than that in the negative control group. The significantly increased cysteine ​​content indicates enhanced disulfide bond cross-linking in hair keratin, which directly contributes to improving hair strength, toughness, and resistance to physical damage. The proline content in experimental group 2 was significantly higher than that in the negative control group, and the proline content in experimental group 1 was also significantly higher than that in the negative control group. The significant increase in proline content reflects a more complete keratin helical structure, which helps enhance hair's smoothness, elasticity, and morphological stability. These results indicate that post-natal solid preparations can systematically promote the synthesis and structural optimization of hair keratin at the molecular level. Keratin is a key component of hair structure, directly affecting hair's toughness, strength, and health. This indicates that Lactobacillus casei ZYhyy-005 postbiotic solid preparation may improve the absorption and metabolism of nutrients by regulating the intestinal microecology, promoting the synthesis and utilization of key components such as glutamic acid, cystine and proline, thereby enhancing keratin synthesis in hair, promoting healthy hair growth, and improving hair quality and strength.

[0048] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A *Lactobacillus casei* ZYhyy-005 formula for comprehensive improvement of canine pet coat health, characterized in that... Lacticaseibacillus casei ZYhyy-005 was deposited on August 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31555.

2. A postbiotic preparation of Lactobacillus casei ZYhyy-005 for comprehensive improvement of canine pet coat health, characterized in that, It is prepared by uniformly mixing the post-generic powder of Lactobacillus casei ZYhyy-005 as described in claim 1 with glucose. The post-generic powder is prepared by inactivating, concentrating and freeze-drying Lactobacillus casei ZYhyy-005 bacterial solution.

3. The Lactobacillus casei ZYhyy-005 postbiotic preparation according to claim 2, characterized in that, The preparation of the Lactobacillus casei ZYhyy-005 bacterial culture includes the following steps: (1) Take the frozen Lactobacillus casei ZYhyy-005 out of the -80℃ freezer and thaw it in a 37℃ water bath. Inoculate the strain into 5 mL of liquid MRS medium and culture it anaerobically at 37°C for 24-48 hours until the bacterial solution becomes turbid. (2) Take the turbid bacterial solution obtained in step (1) and inoculate it on MRS solid medium to activate it. Inoculate the activated bacteria into primary medium and obtain seed liquid through primary culture. The seed culture was inoculated into a secondary culture medium, and the bacterial culture was obtained through secondary culture. (3) Centrifuge the bacterial culture at 4°C and 3000-5000 rpm for 15 min, collect the bacterial cells, wash with sterile physiological saline and resuspend to obtain a suspension. Adjust the concentration of the suspension to be greater than 100%. CFU / mL yields Lactobacillus casei ZYhyy-005 bacterial culture.

4. The use of Lactobacillus casei ZYhyy-005 as described in claim 1, or the Lactobacillus casei ZYhyy-005 post-biotic preparation as described in claim 2 or 3, in the preparation of products that improve the coat health of canine pets.

5. The application according to claim 4, characterized in that, The canine pets mentioned are domestic companion dogs, including Beagles, Poodles, and Golden Retrievers.

6. The application according to claim 4, characterized in that, The products mentioned for improving the coat health of canine pets include canine-specific diet additives and canine-specific nutritional supplements.

7. The application according to claim 4 or 6, characterized in that, The product can simultaneously achieve: (1) Enhance the brightness and color saturation of canine pet coats; (2) Enhance the peak breaking force of canine pet hair; (3) Reduce the thickness of the hair cuticles in canine pets and improve the regularity and uniformity of canine pet hair; (4) Increase the content of glutamic acid, cystine and proline in the fur of canine pets.

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