Pet food for enhancing immunity and improving intestinal function comprising biogenics and method of manufacturing the same

Abstract

The present invention relates to a pet food for enhancing immunity and improving intestinal function in companion animals, and a method for manufacturing the same, comprising raw materials including tuna, chicken, fish, krill, and lamb bone extract; and a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria as an additive, vitamins, and taurine.

Description

Technology Field

[0001] The present invention relates to a pet food containing a lactic acid bacteria product for enhancing immunity and improving intestinal function in companion animals, and a method for manufacturing the same. More specifically, the invention relates to a pet food containing a lactic acid bacteria product for enhancing immunity and improving intestinal function in companion animals, which are often exposed to various diseases caused by abnormalities in the immune system, such as stomatitis, colds, and skin diseases, due to a decline in immunity, and a method for manufacturing the same. Background Technology

[0002] With the improvement of national income levels and the shift toward nuclear families, as well as the explosive growth of the pet market, pet-related products and services are emerging as a promising industry.

[0003] Although companion animals are raised under protection as members of the family rather than as animals kept outdoors, they are frequently exposed to polluted natural environments—such as air and water pollution—as well as processed foods and stress, just like humans. For these reasons, companion animals are currently susceptible to various diseases that commonly affect humans, such as weakened immunity, digestive disorders, skin conditions, and respiratory illnesses.

[0004] Generally, when a pet's immunity weakens, symptoms such as colds, stomatitis, and dermatitis appear in various forms. In such cases, moderate exercise and balanced nutrition are crucial for boosting immunity. While balanced nutrition is easily accessible through selective feeding management, exercise is relatively difficult to manage due to various factors. It is challenging to consistently maintain an appropriate amount of exercise depending on the owner's business trips, inclement weather, the pet's personality, and the condition of its joints and respiratory system. In such situations, providing the pet with immune-boosting substances—either separately or mixed with their food—allows them to maintain a healthy immune state regardless of time, place, or the pet's temperament, even without physical exercise.

[0005] Lactic acid bacteria, commonly known as probiotics, inhibit the proliferation of harmful bacteria in the human intestines. However, they do not directly inhibit harmful bacteria; instead, they suppress them by secreting substances or products that create an environment where harmful bacteria cannot survive. Lactic acid bacteria products are generally composed of small molecules of peptides or polypeptides. Unlike lactic acid bacteria cells, they are very stable against heat and acid because they have small molecular weights and very strong bonds.

[0006] When soybeans or black beans are fermented using lactic acid bacteria, glycosides are converted into non-glycoside forms, which improves absorption rates, and the bacteria secrete lactic acid products during the fermentation process. By feeding pets the lactic acid products produced through this method, an environment can be created to stably increase beneficial bacteria in the intestines without the need for consuming lactic acid bacteria. Furthermore, since the pet directly consumes the products released by lactic acid bacteria to improve the intestinal environment, the intestinal environment can be improved more quickly than by consuming lactic acid bacteria.

[0007] Prior art regarding pet food includes a method for manufacturing pet food using duck by-products (Registered Patent Publication No. 10-2292025), a health functional pet food with improved skin improvement functionality and a method for manufacturing the same (Registered Patent Publication No. 10-2376726), and a pet food containing Jeju bamboo grass and a method for manufacturing the same (Published Patent Publication No. 10-2022-0081413).

[0008] However, the aforementioned prior art does not disclose pet food and a method for manufacturing the same that can promote the enhancement of immunity and improvement of intestinal function in companion animals by including a lactic acid bacteria product. Prior art literature

[0009] Registered Patent Publication No. 10-2292025 Registered Patent Publication No. 10-2376726 Published Patent Publication No. 10-2022-0081413 The problem to be solved

[0010] As a result of conducting research to solve the problems of the prior art as described above, the inventors discovered that since the lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria maintains its effect even when used at high temperatures, it can be mixed with single feed ingredients such as tuna, chicken, lamb, or general fish, mixed with vitamin C, taurine, etc., placed in a can or pouch, and sterilized at high temperatures, thereby enabling long-term storage, and thus completed the present invention.

[0011] Therefore, the objective of the present invention is to provide a pet food for enhancing immunity and improving intestinal function in companion animals using a lactic acid bacteria product.

[0012] Another objective of the present invention is to provide a method for manufacturing pet food for enhancing immunity and improving intestinal function of companion animals using a lactic acid bacteria product. means of solving the problem

[0013] To achieve the above objectives, the present invention provides a pet food for enhancing immunity and improving intestinal function in companion animals, comprising raw materials including tuna, chicken, fish, krill, and lamb bone extract; and as additives, a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria, vitamins, and taurine.

[0014] As an embodiment of the present invention, the raw material comprises, with respect to 100 parts by weight of tuna, 40 to 60 parts by weight of chicken meat, 40 to 60 parts by weight of fish, 40 to 60 parts by weight of krill, and 1 to 10 parts by weight of lamb bone extract, and with respect to 100 parts by weight of the raw material, 0.01 to 5 parts by weight of the lactic acid bacteria producing substance, 0.01 to 0.5 parts by weight of the vitamin, and 0.05 to 5 parts by weight of the taurine.

[0015] As an embodiment of the present invention, the fish may be one or more selected from the group consisting of saury, mackerel, flounder, capelin, cod, herring, skipjack tuna, bass, bluegill, and salmon.

[0016] As an embodiment of the present invention, the pet food for improving immunity and intestinal function of the pet may further include 0.1 to 5 parts by weight of *Cheonsimnyeon* extract with respect to 100 parts by weight of the raw material.

[0017] As an embodiment of the present invention, the starter culture comprises plant-based lactic acid bacteria, Bacillus, and yeast, and the plant-based lactic acid bacteria are plant-based lactic acid bacteria derived from vegetable or grain fermentation, and Lactobacillus fermentum JS deposited as KCCM10499 ( Lactobacillus fermentum JS may include one or more of Bacillus bacteria derived from fermented foods and yeast bacteria derived from fermented foods.

[0018] As an embodiment of the present invention, the plant-based lactic acid bacteria derived from vegetable or grain fermentation is Lactobacillus sakei ( L. sakei ), Lactobacillus brevis( L. brevis ), Lactobacillus casei( L. casei), Lactobacillus paracasei( L. paracasei ), Lactobacillus plantarum( L. plantarum ), Leuconostoc mesenteroides ( Leuc. mesenteroides ), Pediococcus Pentosaeus( Ped. pentosaceus ), Lactobacillus acidophilus( L. acidophilus ), Lactobacillus lactis( L. lactis ), Leuconostoc lactis ( Milk thistle ), Leuconostoc citreum ( Lemongrass ) and Lactobacillus rhamnosus ( L. rhamnosus One or more selected from ); and the Bacillus derived from the above fermented food is Bacillus subtilis ( B. subtillis ), Bacillus licheniformis( B. licheniformis ) and Bacillus coagulans( B. coagulans One or more selected from ); and the yeast derived from the above fermented food is Saccharomyces cerevisiae ( S. cerevisiae ), Peacha Cluyberry ( Pichia kluyveri ), Saccharomyces Pastorianus( St. pastorianus ), Saccharomyces intumdius( S. intermedia ) and Saccharomyces validus ( S. strong It may be one or more selected from ).

[0019] As an embodiment of the present invention, the lactic acid bacteria-producing substance may be an isoflavone glycoside including daidzein and genistein.

[0020] In addition, to achieve the above objectives, the present invention provides a method for manufacturing pet food for enhancing immunity and improving intestinal function in companion animals, comprising the steps of: (a) mixing and grinding raw materials including tuna, chicken, fish, and krill, and then adding a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria as an additive, vitamins, and taurine; (b) adding lamb bone extract to the mixture and filling it; (c) placing the filled mixture into a container, sealing it, and compressing it; and (d) primary sterilization and secondary sterilization of the compressed mixture.

[0021] As an embodiment of the present invention, a method for manufacturing pet food for enhancing immunity and improving intestinal function of companion animals comprises: (a) mixing and grinding raw materials comprising 40 to 60 parts by weight of chicken meat, 40 to 60 parts by weight of fish, and 40 to 60 parts by weight of krill, with respect to 100 parts by weight of tuna, and then adding and mixing 0.01 to 5 parts by weight of the lactic acid bacteria generating substance, 0.01 to 0.5 parts by weight of the vitamin, and 0.05 to 5 parts by weight of the taurine as additives with respect to 100 parts by weight of the raw materials; (b) filling by adding 1 to 10 parts by weight of the lamb bone extract with respect to 100 parts by weight of the tuna; and (c) placing the filled mixture into a pouch or can, and then sealing and compressing it. and (d) a step of first sterilizing the above-mentioned compressed mixture at 85~95℃ for 5~15 minutes, and then secondarily sterilizing at 121~125℃ for 20~30 minutes may be included.

[0022] As an embodiment of the present invention, in step (a), 0.1 to 5 parts by weight of *Cheonsimnyeon* extract may be further added and mixed with 100 parts by weight of the raw material.

[0023] As an embodiment of the present invention, the starter culture comprises plant-based lactic acid bacteria, Bacillus, and yeast; the plant-based lactic acid bacteria are plant-based lactic acid bacteria derived from vegetable or grain fermentation, and Lactobacillus fermentum JS deposited as KCCM10499 ( Lactobacillus fermentum JS), including one or more of Bacillus bacteria derived from fermented foods and yeast bacteria derived from fermented foods; the above plant-based lactic acid bacteria derived from vegetable or grain fermentation is Lactobacillus sakei ( L. sakei ), Lactobacillus brevis( L. brevis ), Lactobacillus casei( L. casei ), Lactobacillus paracasei( L. paracasei ), Lactobacillus plantarum( L. plantarum ), Leuconostoc mesenteroides ( Leuc. mesenteroides ), Pediococcus Pentosaeus( Ped. pentosaceus ), Lactobacillus acidophilus( L. acidophilus ), Lactobacillus lactis( L. lactis ), Leuconostoc lactis ( Milk thistle ), Leuconostoc citreum ( Lemongrass ) and Lactobacillus rhamnosus ( L. rhamnosus One or more selected from ); and the Bacillus derived from the above fermented food is Bacillus subtilis ( B. subtillis ), Bacillus licheniformis( B. licheniformis ) and Bacillus coagulans( B. coagulans One or more selected from ); and the yeast derived from the above fermented food is Saccharomyces cerevisiae ( S. cerevisiae ), Peacha Cluyberry ( Pichia kluyveri ), Saccharomyces Pastorianus( St. pastorianus ), Saccharomyces intumdius( S. intermedia ) and Saccharomyces validus ( S. strong It may be one or more selected from ).

[0024] As an embodiment of the present invention, the fish may be one or more selected from the group consisting of saury, mackerel, flounder, capelin, cod, herring, skipjack tuna, bass, bluegill, and salmon. Effects of the invention

[0025] The pet food according to the present invention is effective not only for pets with weakened immunity but also for strengthening immunity. Furthermore, by utilizing various single-ingredient feeds such as tuna, chicken, lamb, and various types of general fish as protein sources, and manufacturing it using a combination of vitamins and minerals, it has the advantage of strengthening immunity with just a snack, thereby preventing diseases that may occur due to weakened immunity, such as colds, atopic dermatitis, stomatitis, and dermatitis in pets.

[0026] In addition, the lactic acid bacteria-producing substance included in the pet food of the present invention is a substance that can be used at high temperatures, and can be sterilized and disinfected using a retort method, allowing for long-term preservation and the advantage of being able to be conveniently fed without being restricted by location. Brief explanation of the drawing

[0027] FIG. 1 shows a manufacturing process diagram of a pet food for improving immunity and intestinal function of companion animals containing a lactic acid bacteria product according to one embodiment of the present invention. Figure 2 shows the macrophage proliferation ability of a lactic acid bacteria-producing substance according to one embodiment of the present invention. Figure 3 shows the ability of a lactic acid bacteria-derived substance to secrete IL-6 into macrophages according to one embodiment of the present invention. Figure 4 shows the macrophage IL-1β secretion ability of a lactic acid bacteria product according to one embodiment of the present invention. Figure 5 shows the macrophage TNF-α secretion ability of a lactic acid bacteria-derived substance according to one embodiment of the present invention. Specific details for implementing the invention

[0028] The present invention will be described in detail below.

[0029] The first embodiment of the present invention relates to a pet food for enhancing immunity and improving intestinal function in companion animals, comprising raw materials including tuna, chicken, fish, krill, and lamb bone extract; and a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria as an additive, vitamins, and taurine.

[0030] Generally, lactic acid bacteria die at high temperatures, but the lactic acid bacteria product used in the present invention is a lactic acid bacteria product extracted by fermenting plant-based lactic acid bacteria using effective technology. Even when sterilized at high temperatures like tuna, chicken, lamb, or general fish, the lactic acid bacteria product remains unchanged. Therefore, even when placed in containers such as cans or pouches and retort sterilized, the peptides, amino acids, and organic acids produced during the culture of lactic acid bacteria are richly dissolved, and bacteriocin, a natural antibiotic, can play a role in helping beneficial bacteria eliminate harmful bacteria and toxins.

[0031] In addition, it contains a large amount of a cellular component called peptidoglycan, which exhibits excellent effects in suppressing malignant harmful bacteria entrenched in the intestines and activates immune cells and restores damaged intestinal cells, thereby enhancing the immune activity of pets.

[0032] Therefore, when providing pet food snacks containing high protein, vitamins, taurine, etc., as well as tuna, chicken, krill, and general fish used as the main ingredients of the pet food for improving immunity and intestinal function of the pet according to the present invention, it is possible to prevent diseases such as colds, atopic dermatitis, stomatitis, and dermatitis, which are representative diseases that occur when the pet's immunity is weakened.

[0033] In the present invention, examples of the pet animals include dogs, cats, birds, hamsters, rabbits, iguanas, snakes, or turtles, but are not limited thereto.

[0034] The above raw materials may comprise, for every 100 parts by weight of the tuna, 40 to 60 parts by weight of chicken meat, preferably 45 to 55 parts by weight, 40 to 60 parts by weight of fish, preferably 45 to 55 parts by weight, 40 to 60 parts by weight of krill, preferably 45 to 55 parts by weight, and 1 to 10 parts by weight of lamb bone extract, preferably 1.5 to 5 parts by weight, and for every 100 parts by weight of the above raw materials, 0.01 to 5 parts by weight of the lactic acid bacteria producing substance, preferably 0.05 to 0.5 parts by weight, the above vitamin, preferably 0.05 to 0.5 parts by weight, and 0.05 to 5 parts by weight of the above taurine, preferably 0.1 to 1 part by weight, but are not limited thereto.

[0035] The above fish species may be one or more selected from the group consisting of saury, mackerel, flounder, capelin, cod, herring, skipjack tuna, bass, bluegill, and salmon, preferably saury, mackerel, and salmon, but are not limited thereto.

[0036] For the above lamb bone extract, it is preferable to use reduced lamb bone obtained by adding purified water to the lamb bone extract and heating it at 80 to 85°C for about 30 minutes, but it is not limited thereto.

[0037] The pet food for enhancing immunity and improving intestinal function of pets according to the present invention is rich in peptides, amino acids, and organic acids produced while culturing lactic acid bacteria using lactic acid bacteria products, and contains bacteriocin, a natural antibiotic, which helps beneficial bacteria eliminate harmful bacteria and toxins, and has an excellent effect in suppressing malignant harmful bacteria established in the intestines, while simultaneously activating immune cells and restoring damaged intestinal cells. It can be provided by mixing it with tuna, chicken, lamb, fish, etc., to be administered to pets requiring immune function through a verified immune activity-enhancing effect using lactic acid bacteria products beneficial to the human body.

[0038] The pet food of the present invention for enhancing immunity and improving intestinal function of companion animals may further include 0.1 to 5 parts by weight, preferably 0.1 to 1 part by weight, of *Cheonsimnyeon* extract with respect to 100 parts by weight of the raw material, but is not limited thereto.

[0039] Cheonsimnyeon ( Andrographis paniculata It is a plant of the family Acanthaceae, with about 20 species found worldwide, and is known to be famous as an anti-inflammatory agent. It is a herb that grows throughout Asia, including Myanmar, India, the Indochina Peninsula, the Malay Peninsula, and the island of Kalimantan, and is also used in traditional medicine in China.

[0040] The above-mentioned *Cheonsimnyeon* is rich in andrographolide, and it is known that *Cheonsimnyeon* extract containing this andrographolide as an active ingredient not only exhibits an immune-improving effect but also has various other benefits. The above-mentioned andrographolide is effective in boosting important immunity by enhancing NK cells, a type of white blood cell, and helps activate immune function and strengthen the overall immune defense mechanism by improving the function of white blood cells called helper T cells.

[0041] In addition, diterpene lactone and flavonoids, which are the main components of *Cheonsimnyeon*, are known to help with antibacterial, anti-inflammatory, analgesic, antidiarrheal, hepatoprotective, and immune-boosting effects, and *Cheonsimnyeon* extract has been recognized by the Ministry of Food and Drug Safety as a functional ingredient for health functional foods.

[0042] The extraction method of the above-mentioned Cnidium monnieri extract included in the pet food of the present invention may include hot water extraction, organic solvent extraction, cold maceration extraction, ultrasonic extraction, or reflux cooling extraction methods, and while it is preferable to perform the extraction using the hot water extraction method, it is not limited thereto.

[0043] As a specific example, the hot water extraction can be performed by adding purified water to dried Xianxinlian and reacting at a high temperature, preferably 100 to 150°C, more preferably 110 to 130°C, for 20 minutes to 1 hour, preferably 20 minutes to 50 minutes, more preferably 20 minutes to 30 minutes.

[0044] As another specific example, the ultrasonic extraction can be performed on dried Xuanzhonglian powder using a 60% to 80% ethanol solution at room temperature for 24 to 72 hours. Preferably, it can be performed using a 65% to 75% ethanol solution at room temperature for 36 to 60 hours, and more preferably, using a 70% ethanol solution at room temperature for 48 hours.

[0045] If the value exceeds the lower limit of the above figure, the active ingredient of the present invention contained in Cheonshimryeon is not sufficiently extracted, and if the value exceeds the upper limit, there is no significant difference in the amount of active ingredient extracted, impurities other than the active ingredient are extracted, and the efficiency of the process decreases.

[0046] The above-mentioned extract of *Cheonsimnyeon* can be concentrated under reduced pressure using a vacuum vacuum concentrator or a vacuum rotary evaporator. Additionally, after reduced pressure concentration, it may be dried by freeze-drying, vacuum drying, vacuum drying, boiling drying, or spray drying, but is not limited thereto.

[0047] The above-mentioned Cnidium monnieri extract included in the pet food of the present invention can exhibit a synergistic effect in boosting immunity and improving intestinal function together with the above-mentioned lactic acid bacteria-producing substance.

[0048] The above starter culture includes plant-based lactic acid bacteria, Bacillus, and yeast, and the above-mentioned plant-based lactic acid bacteria are plant-based lactic acid bacteria derived from vegetable or grain fermentation, and Lactobacillus fermentum JS deposited as KCCM10499 ( Lactobacillus fermentum It may include one or more of Bacillus bacteria derived from fermented foods and yeast bacteria derived from fermented foods, but is not limited thereto.

[0049] The above-mentioned plant-based lactic acid bacteria derived from vegetable or grain fermentation is Lactobacillus sakei ( L. sakei ), Lactobacillus brevis( L. brevis ), Lactobacillus casei( L. casei ), Lactobacillus paracasei( L. paracasei ), Lactobacillus plantarum( L. plantarum ), Leuconostoc mesenteroides ( Leuc. mesenteroides ), Pediococcus Pentosaeus( Ped. pentosaceus ), Lactobacillus acidophilus( L. acidophilus ), Lactobacillus lactis( L. lactis ), Leuconostoc lactis ( Milk thistle ), Leuconostoc citreum ( Lemongrass ) and Lactobacillus rhamnosus ( L. rhamnosus It may be one or more selected from ), but is not limited thereto.

[0050] The Bacillus derived from the above fermented food is Bacillus subtilis ( B. subtillis ), Bacillus licheniformis( B. licheniformis ) and Bacillus coagulans( B. coagulansOne or more selected from ); and the yeast derived from the above fermented food is Saccharomyces cerevisiae ( S. cerevisiae ), Peacha Cluyberry ( Pichia kluyveri ), Saccharomyces Pastorianus( St. pastorianus ), Saccharomyces intumdius( S. intermedia ) and Saccharomyces validus ( S. strong It may be one or more selected from ), but is not limited thereto.

[0051] In addition, the above-mentioned lactic acid bacteria-producing substance may be an isoflavone glycoside including daidzein and genistein, but is not limited thereto.

[0052] A second embodiment of the present invention relates to a method for manufacturing pet food for enhancing immunity and improving intestinal function in companion animals, comprising the steps of: (a) mixing and grinding raw materials including tuna, chicken, fish, and krill, and then adding a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria as an additive, vitamins, and taurine to form a mixture; (b) adding lamb bone extract to the mixture and filling it; (c) placing the filled mixture into a container, sealing it, and then compressing it; and (d) primary sterilization and secondary sterilization of the compressed mixture.

[0053] The present invention will be described in more detail below with reference to FIG. 1, which is a manufacturing process diagram of a pet food for enhancing immunity and improving intestinal function of companion animals containing a lactic acid bacteria product according to one embodiment of the present invention.

[0054] The method for manufacturing the pet food for enhancing immunity and improving intestinal function of companion animals according to the present invention comprises: (a) a step of mixing by adding 40 to 60 parts by weight of chicken meat, preferably 45 to 55 parts by weight, 40 to 60 parts by weight of fish, preferably 45 to 55 parts by weight, and 40 to 60 parts by weight of krill, preferably 45 to 55 parts by weight, to 100 parts by weight of tuna; (b) a step of filling by adding 1 to 10 parts by weight of lamb bone extract, preferably 1.5 to 5 parts by weight, to 100 parts by weight of tuna; and (c) a step of placing the filled mixture into a pouch or can, and then sealing and compressing it. and (d) a step of first sterilizing the above-mentioned compressed mixture at 85 to 95°C, preferably 88 to 92°C for 5 to 15 minutes, preferably 8 to 12 minutes, and then secondarily sterilizing at 121 to 125°C, preferably 121°C for 20 to 30 minutes, preferably 25 minutes, may be included, but is not limited thereto.

[0055] The method for manufacturing the pet food of the present invention may further add and mix 0.1 to 5 parts by weight, preferably 0.1 to 1 part by weight, of *Cheonsimnyeon* extract with respect to 100 parts by weight of the raw material in step (a).

[0056] In the method for manufacturing the pet food of the present invention, the starter culture, fish, lamb bone extract, and Cnidium monnieri extract, etc., have been described in detail in the first embodiment, so the details thereof are omitted.

[0057] The method for manufacturing the pet food according to the present invention has the advantage of enabling long-term storage by combining lamb bone extract with general single-ingredient feeds such as tuna, chicken, lamb, general fish, fruit, and grains, adding a lactic acid bacteria producing substance to the mixture, mixing in vitamin C, taurine, etc., filling it into a can or pouch, and performing retort sterilization and sterilization.

[0058] Hereinafter, to aid in understanding the present invention, it will be described in detail with reference to examples. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be interpreted as being limited to the following embodiments.

[0060] <Preparation Example> Preparation of a lactic acid bacteria product

[0061] 1. Culture of plant-based lactic acid bacteria starter cultures

[0062] Lactobacillus fermentum JS, a plant-based lactic acid bacterium Lactobacillus fermentum JS strain deposit number KCCM10499), Lactobacillus plantarum (L. plantarum ), Lactobacillus casei( L. casei ), Lactobacillus paracasei( L. paracasei ), Lactobacillus acidophilus( L. acidophilus 1×10⁶ ) each in de Mann Logosa and Sharp (MRS) media sterilized at 121°C for at least 15 minutes. 5 ~1×10 6 Inoculated at cfu / ml and cultured with shaking (100 rpm) at 40℃ for 16 hours.

[0063] The cultured plant-based lactic acid bacteria were concentrated using centrifugation and freeze-dried, and then sugars such as glucose and lactose were added to produce a high-concentration plant-based lactic acid bacteria powder according to conventional methods, at which time the number of plant-based lactic acid bacteria was 1.0–1.2 × 10⁶ 11 It was cfu / g.

[0064] At this time, the plant-derived lactic acid bacteria is a different plant-derived lactic acid bacteria other than the strain used in the above example, and is a plant-derived lactic acid bacteria derived from vegetable or grain fermentation, such as Lactobacillus sakei ( L. sakei ), Lactobacillus brevis( L. brevis ), Lactobacillus casei( L. casei ), Lactobacillus paracasei( L. paracasei ), Lactobacillus plantarum( L. plantarum ), Leuconostoc mesenteroides ( Leuc. mesenteroides ), Pediococcus Pentosaeus( Ped. pentosaceus ), Lactobacillus acidophilus ( L. acidophillus ), Lactobacillus lactis( L. lactis ), Leuconostoc lactis ( Leuc. lactis ), Leuconostoc citreum ( Leuc. citreum) , Lactobacillus rhamnosus ( L. rhamnosus You can use one or more of the selected options.

[0066] 2. Yeast starter culture

[0067] Saccharomyces cerevisiae S. cerevisiae 1×10⁶ ) in yeast malt broth medium sterilized at 121°C for at least 15 minutes 5 ~1×10 6 The yeast was inoculated at cfu / ml and cultured with shaking (100 rpm) at 25°C for 40 hours. After culture, the yeast was concentrated by centrifugation and freeze-dried, and then sugars such as glucose and lactose were added to prepare high-concentration yeast powder according to conventional methods, with a yeast colony count of 2.1 × 10⁶ 11 It was cfu / g.

[0068] At this time, the yeast is the Saccharomyces cerevisiae used above ( S. cerevisiae ) and other yeast strains derived from fermented foods, such as Peachia cloyberry ( Pichia kluyveri ), Saccharomyces Pastorianus (S. pastorianus ), Saccharomyces intumdius( S. intermdius ), Saccharomyces validus( S. validus You can use one or more of the selected options.

[0070] 3. Preparation of culture medium

[0071] Soybeans or black beans with a moisture content of 7% and dried lion's mane mushrooms with a moisture content of 6.8% were ground to 50 mesh or less using a pin mill, and a powder composition consisting of 50% soybean or black bean powder, 20% lion's mane mushroom powder, and 30% glucose was prepared using the powder, and distilled water was added tenfold. The impeller inside the fermentation tank was rotated at 80 rpm to suspend the mixture, and it was sterilized at 121°C for 30 minutes.

[0073] 4. Inoculation and Culture

[0074] Cool the sterilized culture medium to 38℃ and 1.0×10 8 Lactobacillus fermentum diluted to cfu / g ( L. fermentum ) JS (Strain deposit number KCCM10499) 1%, Lactobacillus plantarum( L. plantarum ) 1%, Lactobacillus( L. casei ) 1%, Lactobacillus paracasei( L. paracasei ) 1%, Lactobacillus acidophilus( L. acidophillus ) 1%, Bacillus subtilis( B. subtillis ) 5%, Saccharomyces cerevisiae 1% was inoculated and cultured at 38°C for 72 hours.

[0076] 5. Sterilization and Processing

[0077] The cultured lactic acid bacteria product was sterilized at 121°C for 30 minutes and then cooled to 30°C or below. The cooled product was placed in a vacuum hot air dryer and dried at 130°C for 48 hours until the moisture content was less than 3%. The dried product fragments were then ground to a size of 50 mesh or less using a pin mill to obtain the lactic acid bacteria product.

[0079] <Experimental Example 1> In vivo Evaluation of the immune-enhancing effects of lactic acid bacteria products in a system

[0080] 1. Materials and Methods

[0081] (1). Test substance

[0082] The test substance and positive control substance used were lactic acid bacteria products provided by Wellbeing LS Co., Ltd.

[0084] (2). Rearing of experimental animals and administration of test substances

[0085] BALB / c mice, commonly used in immunology tests, were used as experimental animals. Four-week-old male BALB / c mice were purchased from Orient Bio Co., Ltd. and housed in an SPF (Specific Pathogen Free) facility set to a temperature of 23 ± 3℃, relative humidity of 50 ± 10%, ventilation rate of 10-15 times / hour, lighting time of 12 hours (08:00 - 20:00), and illuminance of 150 - 300 Lux. During a one-week adaptation period, the animals were allowed to freely consume solid feed for laboratory animals (Cargill Agri Purina, Gunsan, Korea) and drinking water.

[0086] After a one-week quarantine and adaptation period, healthy animals without body weight loss were selected and classified into seven experimental groups (Table 1 below) based on a randomized block design (10 animals per group), and the test substance was fed. After 4 weeks of feeding, spleens were extracted from 5 animals per group, and the splenic cell proliferation capacity and cytokine secretion capacity of the splenic tissue were measured.

[0087] The remaining 5 animals in each group 4 days before sacrifice thioglycolate (BD Bioscience ® , Sparks, MD, USA) solution was injected into the abdominal cavity of experimental animals to induce peritoneal inflammation, and the proliferative ability and cytokine secretion ability of abdominal macrophages treated with the test substance were investigated.

[0088] During the experiment, experimental animals consumed the AIN-93G diet (Research Diets, Inc., New Brunswick, NJ, USA) mixed with the test substance at various concentrations. All groups had free access to drinking water, and the lactic acid bacteria group was 1.0 × 10 7Free intake was provided daily at a concentration of cfu / mL. Body weight and food intake were measured at a fixed time once a week.

[0089] All animal experiments in this study were conducted under the approval of the Hallym University Animal Ethics Committee (Hallym R2015-14), and in Table 1 below, the control group refers to the group that received no treatment.

[0090]

[0092] (3). Blood sampling and organ weight measurement

[0093] Experimental animals were fasted for 12 hours prior to sacrifice, and blood was collected via the angular vein after anesthesia. The blood was placed in a serum separate tube (Becton Dickinson, USA), left at room temperature for 30 minutes, and centrifuged at 3,000 rpm for 20 minutes to separate the serum; the samples were stored at -70°C until analysis. After blood collection, the abdomen was opened to immediately remove the liver, spleen, and thymus. These organs were washed with cold physiological saline, excess moisture was removed using filter paper, and their weights were measured.

[0095] (4). Isolation of abdominal macrophages and measurement of proliferation capacity

[0096] The method for isolating intraperitoneal macrophages is to use 3 mL of thioglycolate (BD Bioscience) in a 5 mL syringe without introducing air bubbles. ® The solution was injected into the abdominal cavity of mice. Starting from day 0, the mice were left to accumulate abdominal macrophages for 4 days, after which they were sacrificed by cervical dislocation and the abdominal macrophages were recovered.

[0097] Minimum Essential Medium (MEM, Gibco ®,1 mL of culture medium (Grand Island, NY, USA) was injected into the peritoneal cavity, and the peritoneal cavity was massaged. The outer skin was cut off, and 7 mL of MEM culture medium was injected into the peritoneal cavity. Since 8 mL was injected, a similar amount was recovered immediately upon injection and placed in a 15 mL tube. The recovered culture medium was centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the sample was washed twice with a culture medium containing 10% FBS added to MEM before being used for the next experiment.

[0098] The proliferative capacity of intraperitoneal macrophages is CellTiter 96 ® AQ ueous Measurements were performed using the ONE Solution Assay Kit (Promega). 3 × 10 macrophages isolated from the peritoneal cavity 4 Cells were seeded into 96-well plates at a rate of cells / well and cultured for 48 hours in MEM culture medium or MEM culture medium supplemented with 10 μg / mL LPS and 2.5 μg / mL Con A in a humidified CO2 incubator (5% CO2, 95% air) at 37°C.

[0099] After 48 hours of incubation, 20 μL of ONE solution was added to each well and incubated for an additional 2 hours, after which the lymphocyte proliferation ability was measured by measuring the absorbance at 490 nm using a SpectraMaxM2 Microplate reader (Molecular Devices).

[0101] (5). Measurement of cytokines produced and secreted by peritoneal macrophages

[0102] IL-6, IL-1β, and TNF-α produced and secreted by intraperitoneal macrophages were measured using their respective ELISA Kits (R&D Systems) according to the method provided by the manufacturer.

[0103] 1 × 10 macrophages isolated from the abdominal cavity 5Cells were seeded into a 48-well plate at a rate of cells / well and cultured for 24 hours in MEM culture medium, MEM culture medium supplemented with 10 μg / mL LPS, and MEM culture medium supplemented with 2.5 μg / mL Con A in a humidified CO2 incubator (5% CO2 / 95% air) at 37°C. After 24 hours of culture, the cell culture medium was collected and measured.

[0104]

[0105] (6). Statistical processing

[0106] All analysis values ​​were expressed as mean ± SEM. Statistical analysis of the experimental data was performed using GraphPad Prism 4.0 (GraphPad software, San Diego, CA, USA), and one-way analysis of variance (ANOVA) was used to determine the significance between the means of each experimental group. A P-value of less than 0.05 was deemed statistically significant.

[0108] 2. Experimental Results

[0109] (1). Body weight, food intake, and organ weight of experimental animals

[0110] The results of measuring the body weight (g), food intake (g), and organ weight (g) of the experimental animals are shown in Table 2 (body weight), Table 3 (food intake), and Table 4 (organ weight), respectively.

[0111]

[0112]

[0113]

[0114] As shown in Tables 2 to 4 above, there were no significant results regarding weekly changes in body weight, dietary intake, and organ weight compared to the control group when lactic acid bacteria products were administered.

[0116] (2). Proliferative capacity of macrophages

[0117] As shown in Table 5 and Figure 2 below, in the case of macrophage proliferation ability, the lactic acid bacteria product did not have a significant effect compared to the control group.

[0118]

[0120] (3). Macrophage cytokine secretion ability

[0121] ① Secretory capacity of IL-6 and IL-1β

[0122] As shown in Table 6 and Figures 3 and 4 below, when the secretion ability of IL-6 secreted by macrophages was confirmed, the lactic acid bacteria product had no significant effect compared to the control group.

[0123] In addition, when the secretion ability of IL-1β secreted by macrophages was confirmed, the lactic acid bacteria product showed a 20.2% increase compared to the control group.

[0124]

[0126] ② TNF-α secretory ability

[0127] As shown in Table 7 and Figure 5 below, when the secretion capacity of TNF-α secreted by macrophages was confirmed, the secretion capacity of the lactic acid bacteria product increased by 26% compared to the control group.

[0128]

[0130] 3. Conclusion

[0131] When assessed comprehensively, since lactic acid bacteria products are judged to have an immune-enhancing effect, it is determined that it is possible to maintain a stable increase in immunity without side effects.

[0133] <Examples 1-4 and Comparative Examples 1-2> Preparation of Pet Food>

[0134] As raw materials, tuna, chicken, fish such as saury, mackerel, and salmon mixed in equal weight ratios, and krill were mixed and ground, and then the lactic acid bacteria product prepared in the above preparation example, vitamin C, taurine, and Chinese ginseng extract were added as additives to form a mixture.

[0135] Lamb bone extract was added to the above-described mixture and filled, and the filled mixture was placed into a pouch or can, sealed, and then compressed. The compressed mixture was first sterilized in a retort manner at 90°C for 10 minutes, then secondarily sterilized at 121°C for 25 minutes, and then cooled to 25°C to produce pet food.

[0136] The content of the above components is the same as the composition of Table 8 below (unit: parts by weight).

[0137] division Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 tuna 100 100 100 100 100 100 Tuna white meat 50 50 50 50 50 50 Tuna blood meat 50 50 50 50 50 50 chicken 50 50 50 50 50 50 fish 50 50 50 50 50 50 krill 50 50 50 50 50 50 Lactic acid bacteria products 0.05 0.05 0.1 0.1 - - Vitamin C 0.08 0.08 0.08 0.08 0.08 0.08 Taurine 0.5 0.5 0.5 0.5 0.5 0.5 Chinese quince extract - 0.5 - 0.5 - 0.5 Lamb bone extract 1.7 1.7 1.7 1.7 1.7 1.7

[0139] <Experimental Example 2> Evaluation of Intestinal Function Improvement

[0140] In order to determine whether the intestinal function of pet dogs could be improved, 10 dogs in the experimental group were fed 100g of the pet food prepared in Example 1 above for 3 days, and the number of diarrhea, dehydration, and daily weight loss decreases were observed for 10 days, and the results are shown in Table 9 below. At this time, as the control group, 10 dogs were fed 100g of the pet food prepared in Comparative Example 1 above for 3 days.

[0141] division Clinical observation results diarrhea dehydration Daily weight gain experimental group 0 / 10 0 / 10 0 / 10 control group 3 / 10 2 / 10 5 / 10

[0142] As shown in Table 9 above, it can be confirmed that there are significant differences in diarrhea, dehydration, and daily weight gain in the experimental group compared to the control group, and it can be seen that this can improve intestinal function.

[0144] <Experimental Example 3> Evaluation of Storage Safety

[0145] The pet food prepared in the above Examples 1 and 2 and Comparative Examples 1 and 2 was sterilized by retort at a temperature of 121°C for 25 minutes, and then stored for 12 hours under accelerated conditions of a temperature of 45°C and a relative humidity of 70%. The results of evaluating the storage stability of each pet food are shown in Table 10 below.

[0146] At this time, the above storage stability evaluation was evaluated through appearance and smell, using a 5-point scale from 1 point (very poor) to 5 points (very good).

[0147] division Example 1 Example 2 Comparative Example 1 Comparative Example 2 appearance 4.4 4.7 3.1 3.5 smell 4.3 4.6 3.0 3.4

[0148] As shown in Table 10 above, it can be seen that the pet food according to Example 1 and Example 2 of the present invention has storage stability compared to the pet food according to Comparative Example 1 and Comparative Example 2. In particular, it can be seen that the storage stability of the pet food according to Example 2, which contains both a lactic acid bacteria product and a Cnidium monnieri extract, is excellent.

[0149] From these results, it can be confirmed that storage stability is improved by including both lactic acid bacteria products and *Cheonsimnyeon* extract.

[0151] As specific parts of the content of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention.

[0152] Accordingly, the substantial scope of the present invention shall be defined by the appended claims and their equivalents. Simple variations or modifications of the present invention may be readily utilized by those skilled in the art, and all such variations or modifications shall be deemed to be included within the scope of the present invention.

Claims

Claim 1 A pet food for enhancing immunity and improving intestinal function in companion animals, comprising raw materials including tuna, chicken, fish, krill, and lamb bone extract; and as additives, a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria, vitamins, and taurine, wherein the fish comprises one or more types selected from the group consisting of Pacific saury, mackerel, flounder, capelin, cod, herring, skipjack tuna, bass, bluegill, and salmon; and wherein the lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria is prepared by inoculating the starter culture into a culture solution obtained by adding distilled water 10 times to a powder composition composed of 50% by weight of soybean or black bean powder, 20% by weight of Sparassis crispa mushroom powder, and 30% by weight of glucose, culturing at 38°C for 72 hours, and then sterilizing, cooling, drying, and grinding to a size of 50 mesh or less, wherein the starter culture comprises 1.0 × 10⁻³ relative to the total volume of the culture solution. 8 1 volume%, 1.0×10⁻⁶ Lactobacillus fermentum JS (strain deposit number KCCM10499) diluted to cfu / g 8 1 vol%, 1.0×10⁻⁶ Lactobacillus plantarum diluted to cfu / g 8 1 volume%, 1.0×10⁻⁶ Lactobacillus casei diluted to cfu / g 8 1 vol%, 1.0×10⁻⁶ Lactobacillus paracasei diluted to cfu / g 8 1 volume%, 1.0×10⁻⁶ Lactobacillus acidophilus diluted to cfu / g 8 5 volume% of Bacillus subtilis diluted to cfu / g, and 1.0×10 8 Pet food for boosting immunity and improving intestinal function in companion animals, containing 1 volume% of Saccharomyces cerevisiae diluted to cfu / g. Claim 2 A pet food for enhancing immunity and improving intestinal function of companion animals according to claim 1, wherein the raw material comprises, with respect to 100 parts by weight of tuna, 40 to 60 parts by weight of chicken meat, 40 to 60 parts by weight of fish, 40 to 60 parts by weight of krill, and 1 to 10 parts by weight of lamb bone extract, and with respect to 100 parts by weight of the raw material, 0.01 to 5 parts by weight of the lactic acid bacteria producing substance, 0.01 to 0.5 parts by weight of the vitamin, and 0.05 to 5 parts by weight of the taurine. Claim 3 delete Claim 4 A pet food for enhancing immunity and improving intestinal function of companion animals, characterized in that, in claim 1, it further comprises 0.1 to 5 parts by weight of *Cheonsimnyeon* extract per 100 parts by weight of the raw material. Claim 5 delete Claim 6 delete Claim 7 A pet food for enhancing immunity and improving intestinal function of companion animals, characterized in that, in claim 1, the lactic acid bacteria-producing substance is an isoflavone glycoside including daidzein and genistein. Claim 8 (a) a step of mixing and grinding raw materials including tuna, chicken, fish, and krill, and then adding a lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria as an additive, vitamins, and taurine to form a mixture; (b) a step of adding lamb bone extract to the mixture and filling it; (c) a step of placing the filled mixture into a container, sealing it, and then compressing it; A method for manufacturing pet food for enhancing immunity and improving intestinal function in companion animals, comprising the steps of: (d) primary sterilization and secondary sterilization of the above-described mixture, wherein the fish is one or more selected from the group consisting of Pacific saury, mackerel, flounder, capelin, cod, herring, skipjack tuna, bass, bluegill, and salmon; and the lactic acid bacteria product extracted by fermenting a starter culture containing plant-based lactic acid bacteria is prepared by inoculating the starter culture into a culture solution in which distilled water is added 10 times to a powder composition composed of 50% by weight of soybean or black bean powder, 20% by weight of Sparassis crispa mushroom powder, and 30% by weight of glucose, and then culturing at 38°C for 72 hours, followed by sterilization, cooling, drying, and grinding to a size of 50 mesh or less, wherein the starter culture is 1.0 × 10⁻⁶ relative to the total volume of the culture solution 8 1 volume%, 1.0×10⁻⁶ Lactobacillus fermentum JS (strain deposit number KCCM10499) diluted to cfu / g 8 1 vol%, 1.0×10⁻⁶ Lactobacillus plantarum diluted to cfu / g 8 1 volume%, 1.0×10⁻⁶ Lactobacillus casei diluted to cfu / g 8 1 vol%, 1.0×10⁻⁶ Lactobacillus paracasei diluted to cfu / g 8 1 volume%, 1.0×10⁻⁶ Lactobacillus acidophilus diluted to cfu / g 8 5 volume% of Bacillus subtilis diluted to cfu / g, and 1.0×10 8 A method for manufacturing pet food for enhancing immunity and improving intestinal function in companion animals, containing 1 volume% of Saccharomyces cerevisiae diluted to cfu / g. Claim 9 In claim 8, (a) a step of mixing and grinding raw materials comprising 40 to 60 parts by weight of chicken meat, 40 to 60 parts by weight of fish, and 40 to 60 parts by weight of krill, with respect to 100 parts by weight of tuna, and then adding and blending 0.01 to 5 parts by weight of the lactic acid bacteria producing substance, 0.01 to 0.5 parts by weight of the vitamin, and 0.05 to 5 parts by weight of the taurine as additives with respect to 100 parts by weight of the raw materials; (b) a step of filling by adding 1 to 10 parts by weight of the lamb bone extract with respect to 100 parts by weight of the tuna; (c) a step of placing the filled mixture into a pouch or can, and then sealing and compressing it. and (d) a step of first sterilizing the above-mentioned mixture at 85~95℃ for 5~15 minutes, and then secondarily sterilizing it at 121~125℃ for 20~30 minutes, characterized by a method for manufacturing pet food for enhancing immunity and improving intestinal function of companion animals. Claim 10 A method for manufacturing pet food for enhancing immunity and improving intestinal function of companion animals, characterized in that, in step (a) above, 0.1 to 5 parts by weight of *Cheonsimnyeon* extract are further added and mixed with 100 parts by weight of the raw material. Claim 11 delete Claim 12 delete

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