Functional feed additive including the fermented of red ginseng extracts by lactic acid bacteria

Abstract

The present invention relates to a functional feed additive comprising a lactic acid bacteria culture cultured in a medium containing red ginseng extract, and a method for manufacturing the same. The functional feed additive according to the present invention has the effect of improving blood circulation and increasing antioxidant activity in animals, thereby not only improving the immunity and health of animals but also having an excellent effect on improving the quality of livestock products. Therefore, the functional feed additive and the method for manufacturing the same according to the present invention can be usefully utilized as functional feed and tonic nutritional supplements for companion animals and livestock.

Description

Technology Field

[0001] The present invention relates to a functional feed additive comprising a lactic acid bacteria culture cultured in a medium containing red ginseng extract, a feed composition comprising the same, and a method for manufacturing a functional feed additive comprising a lactic acid bacteria culture in a medium containing red ginseng extract. Background Technology

[0002] Although production efficiency in the livestock industry has significantly improved due to advancements such as genetic breeding and the development of superior feed, the actual increase in various metabolic diseases among animals is posing a major obstacle to the industry's competitiveness. As livestock production has shifted from confined spaces to commercialized mass-production systems, there is a growing trend of diseases associated with increased stress and immunodeficiency. Furthermore, the increased use of synthetic feed is acting as a cause for various diseases alongside weakened immunity, making countermeasures urgently needed. Meanwhile, while the related industry is booming due to the recent rise in households with companion animals, consumer demand for high-quality functional feed designed to promote pet health remains unmet.

[0003] Lactic acid bacteria are beneficial bacteria that have a positive effect on the health of humans and animals. They help prevent various diseases by inhibiting the proliferation of harmful bacteria in the intestines, thereby increasing immunity, and by producing useful substances involved in metabolism. Lactic acid bacteria are used as probiotics to prevent or cure diarrhea in weaned animals, and can be considered an effective alternative antibiotic for promoting metabolism and growth in various types of livestock.

[0004] Red ginseng is known to be effective for hematopoiesis, blood circulation improvement, blood sugar regulation, immune system strengthening, nutritional invigoration, liver function protection, physical strength enhancement, anticancer effects, skin beauty, anti-stress, hangover relief, and fatigue recovery, due to unique components not found in other types of ginseng such as fresh or white ginseng. Ginseng (Panax ginseng (CA Meyer) The saponins present in red ginseng are mainly malonyl ginsenosides (MG), which are triterpene oligoglycosides such as MG-Rb1, MG-Rb2, MG-Rc, and MG-Rd. During the heating process for manufacturing red ginseng, the malonic acid of the acidic saponin MG is detached, resulting in the specific presence of trace saponins such as G-Rg3, G-Rg2, G-Rh2, G-Rs1, G-Rs2, and Rh4 in red ginseng. Studies have reported that changes in these components demonstrate superior effects compared to ginseng, including improved blood circulation, inhibition of cancer development, and protection against infection. Furthermore, new efficacy of red ginseng regarding anti-allergic effects, memory improvement, and effects on erectile dysfunction has recently been reported. It is being suggested that these effects may be attributed to components such as G-Rg3, G-Rf, and G-Rh2, which are present only in red ginseng and not in ginseng.

[0005] Accordingly, while continuously conducting research to develop natural substances that can improve the health of livestock and companion animals and enhance production efficiency, the inventors completed the present invention by discovering that the use of lactic acid bacteria and red ginseng has effects such as improving blood circulation in animals, increasing antioxidant activity, and improving the quality of products. Prior art literature

[0006] Republic of Korea Registered Patent No. 10-0943017 Republic of Korea Registered Patent No. 10-1171942 The problem to be solved

[0007] One objective of the present invention is to provide a functional feed additive that promotes animal health and improves production efficiency.

[0008] Another objective of the present invention is to provide a functional feed composition that promotes animal health and improves production efficiency.

[0009] Another objective of the present invention is to provide a method for manufacturing a functional feed additive that promotes animal health and improves production efficiency. means of solving the problem

[0010] Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0012] To achieve one objective of the present invention, one aspect of the present invention provides a functional feed additive comprising a culture of lactic acid bacteria cultured in a medium containing red ginseng extract.

[0013] In this specification, the term "feed additive" refers to a substance added to a feed composition and may correspond to an auxiliary feed under the Feed Management Act. The said feed additive may be intended to improve the productivity or promote the health of animals or livestock.

[0014] The above red ginseng refers to light yellowish-brown ginseng obtained by selecting fresh ginseng, steaming it with the skin intact, and drying it. Red ginseng contains physiologically active substances such as polyacetylenes, saponins, phenolic compounds, acidic polysaccharides, peptides, gomisin, sesquiterpene compounds, and alkaloids, and its main components include ginsenosides Rg1, Rb1, Rb2, Rg3, Rc, Rd, and Re, which are components of triterpene saponins.

[0015] In the present invention, red ginseng extract refers to a substance extracted from red ginseng by any method, and is used to include, without limitation, the extract obtained in this way, the concentrate obtained therefrom, the dried product of the concentrate, and the powder. Various types of red ginseng may be used as the red ginseng used to manufacture the red ginseng extract, for example, heaven-grade ginseng, earth-grade ginseng, and / or good-grade ginseng, but are not limited thereto.

[0016] The above red ginseng extract can be obtained by performing an extraction process that includes the step of extracting red ginseng, prepared by a conventional red ginseng manufacturing method of steaming and drying ginseng, with an extraction solvent selected from the group consisting of water, C1-C4 alcohols, and a mixed solvent of water and C1-C4 alcohols. Using water as the extraction solvent is more preferable because it can reduce environmental pollution and lower manufacturing costs.

[0017] The above extraction process can be performed by extracting with the above extraction solvent, preferably water, in an amount of about 1 to 20 times, preferably about 3 to 10 times, the weight of the red ginseng. Since the extraction is not significantly affected by temperature, it can be performed by methods such as cold maceration, hot water extraction, ultrasonic extraction, reflux cooling extraction, and pressurized extraction in various temperature ranges (e.g., 20°C to 100°C including room temperature). Additionally, although the extraction time varies depending on the extraction method, it can be performed once or multiple times within a range of about 1 hour to 10 hours.

[0018] The extract obtained by performing the above extraction can be obtained in a liquid form by filtering to remove impurities according to a conventional method, or the obtained liquid extract can be obtained in a powder form by concentrating under reduced pressure and / or drying according to a conventional method.

[0019] In the present invention, the lactic acid bacteria is Lactobacillus Plantarum Lactobacillus plantarum ), Lactobacillus bulgaricus( Lactobacillus bulgaricus ), Lactobacillus acidophilus( Lactobacillus acidophilus ), Lactobacillus lactis( Lactobacillus lactis ), Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Bifidobacterium bifidum ( Bifidobacterium bifidum It may be one or more strains selected from a group consisting of ) strains, but is not limited thereto, and any lactic acid bacterium used as a probiotic may be used in the present invention without limitation.

[0020] It is known that during the processing of red ginseng, the form or concentration of ginsenosides differs from those of fresh ginseng, and physical and chemical changes occur in polysaccharides, which account for about 60-70% of the composition, as well as in proteins, peptides, nitrogen-containing compounds, alkaloids, flavonoids, and fatty acids. However, it has been reported that converting one or more major saponins among ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd present in red ginseng into one or more minor saponins among F2, Rg3, Rh2, and Compound-K increases the absorption rate and pharmacological activity in the body. Accordingly, in order to eliminate differences in the efficacy and absorption rate of red ginseng caused by differences in the gut microbiome among individuals, the present invention attempts to overcome individual differences and enhance the absorption rate and pharmacological effects of red ginseng by pre-fermenting the red ginseng extract with lactic acid bacteria strains.

[0021] In addition, the above lactic acid bacteria are preferably Lactobacillus It may be a complex strain including Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus rhamnosus, and Bifidobacterium bifidum. In one embodiment, it was confirmed that the ability and time to decompose ginsenosides of red ginseng differed depending on the strain; accordingly, in order to increase the absorption rate of red ginseng and enhance its pharmacological effects, it may be desirable to ferment using all six strains. Furthermore, in one embodiment of the present invention, it was confirmed that a culture produced by fermenting six strains at once in a medium containing red ginseng extract exhibited superior antioxidant activity compared to culturing individual strains separately.

[0022] In the present invention, the culture medium for culturing the lactic acid bacteria comprises red ginseng extract. Thus, the red ginseng extract is fermented by the lactic acid bacteria, and the fermented product of the red ginseng extract can provide more beneficial pharmacological effects to animals. Furthermore, the red ginseng extract can promote the growth of lactic acid bacteria. In one embodiment, when red ginseng extract was not added to the lactic acid bacteria culture medium, a resting phase peak was recorded at 60 hours; however, when red ginseng extract was added, it was confirmed that all strains continued to grow without reaching a resting phase up to 72 hours.

[0023] The above red ginseng extract may be added to a lactic acid bacteria culture medium at a concentration of 0.5 to 10%, preferably at a concentration of 5%, but is not limited thereto. In one embodiment, the highest growth rate was observed when the red ginseng extract was added at 5%, and the growth rate tended to decrease when the concentration was lower or higher than 5%.

[0024] The above medium can be used without limitation on composition as long as it is a medium for culturing lactic acid bacteria, and any lactic acid bacteria culture medium known in the art can be used. For example, MRS medium or a medium containing 5 g / L of rice bran, 10 g / L of glucose, 1 g / L of ascorbic acid, and 10 g / L of soy peptone can be used.

[0025] The lactic acid bacteria culture included in the functional feed additive of the present invention may be a product obtained by culturing the lactic acid bacteria strain in a medium containing red ginseng extract at 37°C for 2 to 7 days. If cultured for less than 2 days, the fermentation of the red ginseng extract is not sufficiently carried out, and if cultured for 8 days or more, the useful efficacy of the culture, such as antioxidant activity, is reduced. In addition, if the culture temperature is not maintained at 37°C, the growth rate of the lactic acid bacteria is low, and fermentation does not proceed properly.

[0026] The above lactic acid bacteria culture refers to a product obtained by culturing lactic acid bacteria in a culture medium, and may include lactic acid bacteria cells, a culture solution (culture medium), a fermentation product of red ginseng extract by lactic acid bacteria, or a culture product of lactic acid bacteria; preferably, it may include lactic acid bacteria cells and a fermentation product of red ginseng extract. In one embodiment of the present invention, when a culture containing lactic acid bacteria cells and a fermentation product of red ginseng extract was prepared in powder form and fed to animals, it was confirmed that there were effects of improving blood circulation and enhancing antioxidant activity in rats, and that the egg quality of laying hens improved. This suggests that both the lactic acid bacteria cells and the fermentation product of red ginseng extract included in the feed additive of the present invention play a role in improving biochemical indicators in the animal's body and enhancing biological activity.

[0027] The functional feed additive of the present invention may be manufactured in the form of powder or granules, and, if necessary, may additionally include one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acidic pyrophosphate, and polyphosphate; or natural antioxidants such as polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. The feed additive of the present application may be formulated in the form of conventional feed and may also include conventional feed ingredients.

[0028] The functional feed additive of the present invention may further include grains, for example, ground or crushed wheat, oats, barley, corn, and rice; plant protein feed, for example, feed with rapeseed, soybeans, and sunflower as main components; animal protein feed, for example, blood meal, meat meal, bone meal, and fish meal; and dried components consisting of sugars and dairy products, for example, various milk powders and whey powders, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0029] The functional feed additive composition of the present invention may contain, for example, preservatives, stabilizers, wetting agents or emulsifiers, solution accelerators, etc. The feed additive may be used by adding it to animal feed by soaking, spraying, or mixing.

[0030] The functional feed additive of the present invention can be applied to the diets of a number of animals, including mammals and poultry. As mammals, it can be used for pigs, cattle, sheep, goats, laboratory rodents, and as well as companion animals (e.g., dogs, cats), and as poultry, it can be used for chickens, turkeys, ducks, geese, pheasants, and quail.

[0032] In addition, for another purpose of the present invention, the present invention provides a feed composition comprising the functional feed additive.

[0033] In this specification, the term "feed composition" refers to food fed to animals. The said feed composition refers to a substance that supplies organic or inorganic nutrients necessary for maintaining the life of an animal or for producing meat, milk, etc. The said feed composition may include the feed additive of this application and may additionally include nutritional components necessary for maintaining the life of an animal or for producing meat, milk, etc.

[0034] The functional feed additive included in the feed composition of the present invention is as described above.

[0035] The content of the feed additive in the feed composition of the present invention can be appropriately adjusted according to the type and age of the animal to be applied, the form of application, the desired effect, etc., and may be used, for example, as 1 to 10% (w / w), 1 to 7% (w / w), 1 to 5% (w / w), 3 to 10% (w / w), or 5 to 10% (w / w).

[0037] In addition, for another purpose of the present invention, a method for manufacturing a functional feed additive comprising a lactic acid bacteria fermented product of red ginseng extract is provided, comprising the steps of: inoculating lactic acid bacteria into a medium containing red ginseng extract; fermenting the red ginseng extract by culturing the inoculated lactic acid bacteria at 37°C for 2 to 7 days; and obtaining the lactic acid bacteria culture.

[0038] In the method for manufacturing the above-mentioned functional feed additive, the lactic acid bacteria is Lactobacillus Plantarum Lactobacillus plantarum ), Lactobacillus bulgaricus( Lactobacillus bulgaricus ), Lactobacillus acidophilus( Lactobacillus acidophilus ), Lactobacillus lactis( Lactobacillus lactis ), Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Bifidobacterium bifidum ( Bifidobacterium bifidum It may be one or more strains selected from a group consisting of ) strains, but is not limited thereto, and any lactic acid bacterium used as a probiotic may be used in the present invention without limitation.

[0039] In addition, the above lactic acid bacteria are preferably Lactobacillus It may be a complex strain including Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus rhamnosus, and Bifidobacterium bifidum strains.

[0040] In the manufacturing method of the present invention, the functional feed additive is as described above.

[0041] The method for manufacturing a functional feed additive of the present invention may further include a step of preparing the obtained lactic acid bacteria culture in powder form after the step of obtaining the lactic acid bacteria culture. The step of preparing the powder form may involve drying and grinding the lactic acid bacteria culture, and any known method for drying and grinding may be used without limitation. For example, hot air drying, natural drying, or freeze-drying methods may be used as drying methods, and preferably, freeze-drying methods may be used, and the grinding process may be performed using a powder molding machine. Effects of the invention

[0042] The functional feed additive according to the present invention utilizes natural materials and has no side effects. It has the effect of improving blood circulation in animals and increasing antioxidant activity, thereby improving the immunity and health of animals, as well as improving the quality of livestock products, such as having a better effect on egg quality when fed to laying hens. Therefore, the functional feed additive of the present invention and the method of manufacturing the same can be usefully utilized in feed for companion animals and livestock, or as a tonic nutritional supplement. Brief explanation of the drawing

[0043] Figure 1 is a graph showing the results of confirming the growth curves (6-72 hr) of each of the six types of lactic acid bacteria cultured in MRS medium and production medium with added red ginseng extract. Figure 2A shows six types of lactic acid bacteria cultured in MRS medium containing 5% red ginseng extract. This is a graph showing each growth curve, and Fig. 2B shows the ginsenosides according to culture time in lactic acid bacteria cultures cultured in the control group (red ginseng extract and MRS medium containing red ginseng extract) and MRS medium containing 5% red ginseng extract. This is the result of analyzing the change using thin-layer chromatography. * Lane 1, Red ginseng extract; Lane 2, MRS medium containing red ginseng extract; Lane 3, Lactobacillus Plantarum culture; Lane 4, Bifidobacterium bifidum culture; Lane 5, Lactobacillus lactis culture; Lane 6, Lactobacillus acidophilus culture; Lane 7, Lactobacillus bulgaricus culture; Lane 8, Lactobacillus Ramnosus Culture medium. Figure 3 is a graph showing the change in total sugar content of the medium after 72 hours of fermentation of each of the six types of lactic acid bacteria in MRS medium (A) and MRS medium containing 5% red ginseng extract (B). Figure 4 is a graph showing the antioxidant activity of the medium after 72 hours of fermenting each of the six types of lactic acid bacteria in MRS medium (A) and MRS medium containing 5% red ginseng extract (B). Fig. 5 is This shows the change in body weight during the feeding period of Sprague Dawley rats administered a feed (G1) containing a culture of six types of lactic acid bacteria cultured in MRS medium and a feed (G2) containing a culture of six types of lactic acid bacteria cultured in MRS medium containing 5% red ginseng extract. Fig. 6 is These are the results of an analysis of changes in blood lipid levels in the serum of Sprague Dawley rats fed G1 and G2 feed powders. (A) Total cholesterol; (B) High-density cholesterol (HDL); (C) Low-density cholesterol (LDL); (D) Triglycerides. Fig. 7 is This is the result of analyzing the effects of each feed powder on liver function in the serum of Sprague Dawley rats fed G1 and G2 feed powders. (A) Alanine aminotransferase (ALT); (B) Aspartate aminotransferase (AST); (C) Alkaline phosphatase (ALP); (D) Insulin-like growth factor (IGF-1). Fig. 8 is This is the result showing the quantitative and qualitative changes in egg weight and yolk after feeding G2 feed powder containing a culture of 6 types of lactic acid bacteria cultured in MRS medium containing 5% red ginseng extract. (A) Changes in eggshell and yolk color; (B) Changes in egg weight and yolk weight; (C) Changes in yolk height (albumin height) and egg freshness (HU). Specific details for implementing the invention

[0044] The present invention will be described in detail below by way of examples. However, these examples are specific examples of the present invention, and the scope of the present invention is not limited by these examples.

[0046] <Example 1> Preparation of Lactic Acid Bacteria and Red Ginseng Extract

[0047] To confirm the effects of the functional feed additive of the present invention, a total of 6 types of lactic acid bacteria (Lactobacillus) Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus rhamnosus, and Bifidobacterium bifidum were prepared. Lactobacillus Plantarum and Lactobacillus bulgaricus Strains were isolated from regular kimchi and yogurt, respectively, and used after identifying the 16S rRNA gene sequences by genomic DNA extraction. Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus rhamnosus, and Bifidobacterium bifidum strains were obtained from Mediogen Co., Ltd. (Jecheon, Korea).

[0048] For the culture of lactic acid bacteria, MRS broth medium (10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 5 g / L sodium acetate, 3 g / L diammonium citrate, 1 mL Tween 80, 2 g / L K2HPO4, 0.2 g / L MgSO47H2O, 0.2 g / L MnSO47H2O, pH 6.5), a complex medium specialized for the growth of lactic acid bacteria, was mainly used. In addition, the production medium was an optimized medium obtained by adding 10 g / L of 1% soy peptone to industrial medium (5 g / L rice bran, 10 g / L glucose, 1 g / L ascorbic acid) and comparing the growth efficiency with that of MRS medium. Meanwhile, for use in animal experiments, 5% v / v red ginseng extract was added to each of the above media, followed by inoculation with lactic acid bacteria and culture for 72 hours, then bacterial cells (~2 × 10⁶ 9 An equal amount of oxtail powder (Korea Goksan, Seoul, Korea) was mixed with a culture containing cells / mL, dried, and then mixed into the basic medium for use in animal experiments.

[0049] Red ginseng extract (Brix 2, pH 5.0) was purchased from Gaon F&B Co., Ltd. and used. Specifically, the red ginseng extract was prepared by mixing 5 to 6 times the amount of purified water with the red ginseng raw material and pressurizing the extraction for 7 to 8 hours while maintaining the extraction container at 110℃. After the extraction was completed, the extract was heated and sterilized, and impurities were separated and removed by a centrifuge.

[0051] <Example 2> Confirmation of the effect of red ginseng extract on the growth of lactic acid bacteria

[0052] Each frozen lactic acid bacterium was released into 10 ml of MRS medium and cultured overnight at 37°C to activate it. Afterward, it was transferred to a new medium for reactivation, and the growth rate was measured at different times from 12 to 72 hours. Specifically, 250 ml of MRS or production medium was placed in a 1 L baffle flask, sterilized, and inoculated with 2% inoculum. Samples were collected at different times (6-72 hr) while stirring at 200 rpm at 30°C, and the absorbance was measured at 600 nm using a spectrophotomer to construct growth curves for each strain. To determine the optimal concentration of red ginseng extract (Brix 2, pH 5.0), the red ginseng extract was added at concentrations of 0.5-10% v / v using the same method as above, and the growth rates of each strain were compared.

[0053] In this experiment, six types of lactic acid bacteria with different biological effects—namely, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium bifidum—were used to measure growth rates in complex media and production media. After fermenting the lactic acid bacteria in a medium supplemented with red ginseng extract, powders were prepared using the culture medium and bacterial cells, and biological activity was investigated through animal experiments.

[0054] As shown in Figure 1, although there were no significant differences in growth rates among the strains in the MRS complex medium and production medium with red ginseng extract (5%) added, Lactobacillus plantarum showed a slightly higher growth rate than the other strains, while Lactobacillus lactis showed the lowest growth rate. In addition, unlike the medium without red ginseng extract (resting peak at 60 hr, data not shown), all strains continued to grow without reaching a resting phase up to 72 hours. It is presumed that the growth was influenced by sugars and other components present in the red ginseng extract, and it is expected that the existing major saponins were converted into minor saponins that are relatively well absorbed and have increased efficacy. When comparing growth rates by treating with red ginseng extract at different concentrations (0.5-10% v / v), the highest growth rate was observed when the red ginseng extract was added at a concentration of 5% v / v, and the growth rate tended to decrease slightly as the concentration decreased or increased compared to 5% (data not shown). This appears to be because, while increasing the concentration of red ginseng extract provides some nutrient sources for the growth of lactic acid bacteria, higher concentrations than the optimal concentration may result in decomposition products that inhibit bacterial growth or the influence of lactic acid produced by bacterial growth.

[0056] <Example 3> Analysis of changes in ginsenoside derivatives caused by lactic acid bacteria

[0057] Thin layer chromatography was used to measure changes in ginsenoside derivatives produced by each lactic acid bacteria strain in a medium supplemented with red ginseng extract. 250 ml of MRS or production medium was placed in a 1 L baffle flask and sterilized. Red ginseng extract was added at a concentration of 5%, and each activated strain was inoculated at 2% inoculum. Samples were collected at intervals (6-72 hr) while stirring at 200 rpm at 30°C to obtain the culture medium. 100 µl of culture solution was taken and a solid pellet obtained by freeze-drying was suspended by adding 100 µl of methanol, then an amount corresponding to red ginseng extract was spotted and developed with a developing solvent (chloroform:methanol:distilled water = 65:35:10), then the silica gel plate was thoroughly dried, a colorimetric reagent (ethanol + 30% sulfuric acid) was evenly sprayed and dried again, and then the color was developed at 110°C for about 10 minutes to compare and confirm the changes of the ginsenoside derivatives.

[0058] As a result, as shown in Figure 2, major saponins present in red ginseng, such as Rb1, Rc, Rd, Re, and Rf, were observed in the undiluted red ginseng extract, and significant differences in degradation ability were observed depending on the strain. In the case of Lactobacillus plantarum, it appears to be almost unable to degrade Rb1 or Rc, which are major saponins of the protopanaxadiol family with large molecular weights, and also Lactobacillus In the case of Lactobacillus bulgaricus, the degradation efficiency was slightly lower, but it was generally observed to degrade over time. The remaining strains (Lactobacillus acidophilus, Lactobacillus lactis, Bifidobacterium bifidum, and Lactobacillus rhamnosus) were found to completely degrade ginsenosides Rb1 or Rc after 36 hours of culture. Therefore, it is believed that Rb1, which is hardly absorbed in the body, is degraded by lactic acid bacteria and converted into Compound K, thereby enhancing its pharmacological effects. Furthermore, regarding the relatively small molecular weight protopanaxadiol-based Rd and protopanaxatriol-based Re and Rf, although there were differences in the degree of degradation among strains, they were generally found to be almost completely degraded as the culture time increased. This is attributed to differences in the activity and types of degrading enzymes, including β-glucosidase, β-xylocidase, α-arabinopyranosidase, and α-rhamnosidase, among the various lactic acid bacteria. Therefore, it is reported that to exhibit effective pharmacological effects in vivo, large molecular weight ginsenosides (Rb1, Rb2, Rc, Re, etc.) have low intestinal permeability and bioavailability; thus, they must be broken down by lactic acid bacteria into smaller molecular weight ginsenosides (F2, Rg3, Rh2, compound K, etc.) to increase absorption and achieve effective pharmacological effects. Accordingly, after measuring total sugar content and antioxidant activity using lactic acid bacteria grown in MRS medium containing red ginseng extract and the fermented liquid, a powder was prepared, and physiological changes and effects were investigated in Sprague Dawley rats and laying hens (Hy-line brown).

[0060] <Example 4> Analysis of changes in total sugar content and antioxidant activity of lactic acid bacteria culture supplemented with red ginseng extract

[0061] 4.1. Analysis of Changes in Total Sugar Content of Lactic Acid Bacteria Cultures

[0062] Total sugar content is Dubois et alAnalysis was performed according to the method of (Anal Chem, 1956, 28, 350-358). Specifically, 5% phenol was added to 0.5 mL of a (1:10) culture solution diluted with distilled water, and 2.5 mL of 100% H2SO4 was added and mixed well. The mixture was allowed to stand at room temperature for 30 minutes, then cooled on ice. The absorbance was measured at 490 nm using a spectrophotometer and compared with a glucose standard curve, after which the results were graphed.

[0063] Using the culture solutions (G1) of each of the six types of lactic acid bacteria grown in MRS medium and the culture solution (G2) after fermentation of the six types of lactic acid bacteria grown in MRS medium with 5% red ginseng extract added, Dubois et al Total sugar content was measured according to the method. As can be seen in Figure 3A, the total sugar content was found to decrease as fermentation of lactic acid bacteria grown in MRS medium progressed; specifically, the total sugar content decreased from a concentration of 28.8–30.2 mg / ml before culture to 15–18.2 mg / ml after 72 hours of culture, depending on the strain, as fermentation time progressed. The residual total sugar content after fermentation varied depending on the strain, Lactobacillus In the case of *Bulgaricus*, it is 50.9%, and *Lactobacillus* In the case of Lactis, the figure was 60.3%, indicating that the difference in sugar utilization affects the growth rate. As shown in Figure 3B, after 72 hours of culture of lactic acid bacteria grown in MRS medium supplemented with 5% red ginseng extract, the ratio of residual total sugar content to the initial total sugar content was found to be 43.5% for Lactobacillus bulgaricus, 43.5% for Bifidobacterium bifidum, and 43.8% for Lactobacillus plantarum. This appears to be associated with a relatively slightly higher growth rate compared to other strains. In contrast, Lactobacillus acidophilus, Lactobacillus lactis, and Lactobacillus rhamnosus, which showed relatively lower growth rates, exhibited residual total sugar contents of 56%, 57.7%, and 61.4%, respectively. Therefore, the residual total sugar content after fermentation was found to be inversely proportional to the growth rate. When comparing Figures 3A and 3B, it was found that there were no factors inhibiting the growth rate of lactic acid bacteria when red ginseng extract was added.

[0064] 4.2. Measurement of Antioxidant Activity of Lactic Acid Bacteria Cultures

[0065] The antioxidant activity of the culture medium was 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + The degree of radical ion scavenging was measured and analyzed for each culture medium using ) Specifically, 7 mM ABTS + After mixing with 2.45 mM potassium persulfate in a 1:1 ratio, react in a dark room for 16 hours, and then OD 734 It was diluted with distilled water to a concentration of 0.7 and used in the reaction. 900 µl of the prepared ABTS+ solution and 100 µl of culture medium were mixed for 45 seconds, and then reacted at room temperature for 30 minutes, after which the OD 734 The absorbance was measured, and the antioxidant activity of the culture medium was calculated as follows.

[0066] * Activity(%) = (AB)A × 100 (A: ABTS + Solution, B: ABTS + Solution + Culture Solution)

[0067] Antioxidant activity was measured according to the method of Emad and Sanna (2013, J. Indian Geo-Marine Sciences. 42: 556-564) using the post-fermentation cultures of six types of lactic acid bacteria grown in MRS medium and six types of lactic acid bacteria grown in MRS medium supplemented with red ginseng extract. ABTS + Radical [2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS +The antioxidant activity measurement method is quite stable, does not require an oxidation promoter, and is a feasible measurement method that can be applied to both organic and aqueous phases. It is also a method for measuring antioxidant capacity through a process in which the color becomes lighter due to the electron-donating ability of the antioxidant substance, although it initially appears dark blue-green due to the loss of electrons by potassium persulfate. In all strains grown in MRS medium, antioxidant activity increased 2.7 to 6.1 times after 72 hours of fermentation compared to before the start of fermentation, and in particular, Bifidobacterium bifidum, Lactobacillus plantarum, and Lactobacillus rhamnosus showed high increases of 6.1, 4.9, and 4.8 times, respectively. In the case of lactic acid bacteria cultures containing red ginseng extract, the activity also increased 2.7 to 6.3 times, and Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium bifidum showed high increases of 6.3, 4.8, and 4.0 times, respectively (Fig. 4). When red ginseng extract was added, although there were differences depending on the strain, when comparing the MRS medium cultures of each strain with the MRS medium cultures with added red ginseng extract (fermented liquid) after 72 hours of culture, it was confirmed that the antioxidant activity increased by approximately 25-34% in the medium with added red ginseng extract. In addition, when comparing the MRS medium cultures containing red ginseng extracts of individual strains with the MRS medium cultures containing red ginseng extracts of six mixed strains, it was confirmed that the antioxidant activity of the six mixed strain cultures was approximately twice as high (data not shown). The difference in ABTS radical scavenging activity caused by fermentation by these lactic acid bacteria may be attributed to the oxidation of natural antioxidants such as polyphenol compounds and vitamins C and E during the fermentation process, as well as a decrease in activity due to differences in secreted enzymes among the strains. Meanwhile, in the animal experiments below, the experiment was conducted using the six mixed strain cultures containing red ginseng extracts, which showed the highest antioxidant activity.

[0069] <Example 5> Confirmation of biological activity of lactic acid bacteria culture supplemented with red ginseng extract in an animal model

[0070] 5.1. Preparation of Experimental Animals and Administration of Lactic Acid Bacteria Culture

[0071] The experimental animals used in this experiment were 8-week-old male Sprague Dawley rats obtained from Hyochang Science (Daegu, Korea) and acclimatized for one week in a rearing room maintained at constant temperature and humidity (23±2℃, 60±2% RH). Commercially available solid rat feed (Purina, Seongnam, Korea) was used as the base diet. The feed for the experimental group consisted of six types of lactic acid mycelial cells (2x10⁶) cultured in a production medium. 9 6 types of lactic acid mycelial cells (2x10) cultured in a production medium supplemented with cells / g of wheatsort) and its culture medium and 5% red ginseng 9 The study was prepared by adding a powder of cells / g of wheatsort and its culture medium to the basic feed at a concentration of 5%. Drinking water and feed were provided free of charge, and the animal experiments were conducted with approval (DUIACC-2023-2) in accordance with the regulations of the Animal Ethics Committee of Daegu University. Eight animals were selected for each of the control and experimental groups in two replicates. Body weight was measured weekly to monitor trends in body weight changes, and blood was collected from the tails of rats every two weeks, placed in heparin-containing tubes, left at room temperature for approximately 30 minutes, and then centrifuged to collect the blood for analysis. Changes in total cholesterol, triglycerides, HDL, LDL, ALT, AST, ALP, and IGF-1 were compared and analyzed using serum isolated from each group.

[0072] 5.2. Changes in Body Weight in Sprague Dawley Rat

[0073] The feed for the experimental group was prepared by adding 5% of each of the following to the basic feed: a dried powder of six types of lactic acid mycelia and culture solution grown in MRS medium (G1 powder), and a dried powder of six types of lactic acid mycelia and culture solution grown in MRS medium containing 5% red ginseng extract (G2 powder). The resulting dietary powder was then placed in a formulation machine. Eight 8-week-old Sprague Dawley rats were placed in cages per group and acclimatized for one week. Afterward, the rats were freely fed the basic feed (control group) and the prepared feed (experimental group), and their body weight was measured weekly. As shown in Figure 5, there was no significant difference until the 4th week, but starting from the 5th week of administration, body weight began to increase slightly compared to the control group. By the 8th week, the G1 (6 types of lactic acid bacteria) administration group showed an 8.4% increase compared to the control group, while the G2 (6 types of lactic acid bacteria grown in medium containing red ginseng extract) administration group showed an 8.9% increase. This is presumed to be due to increased digestion and absorption and improved intestinal metabolic activity resulting from the addition of lactic acid bacteria and beneficial metabolites from the fermentation liquid.

[0074] 5.3. Changes in blood levels in Sprague Dawley rats

[0075] Blood samples were collected every two weeks from the control and experimental groups administered rat feed, and changes in total cholesterol, triglycerides, HDL, LDL, ALT, AST, ALP, and IGF-1 were compared and analyzed. As shown in Figures 7, 8, and 9, while there was a slight increase in total cholesterol levels during the growth process after 8 weeks for both the control and experimental groups, there appeared to be no significant difference between the groups. However, HDL levels were found to increase by 5.4% in the G1 administration group and 6.6% in the G2 administration group after 8 weeks, while LDL levels were found to decrease significantly by 23.6% in the G1 administration group and 25.1% in the G2 administration group after 8 weeks. This suggests that lactic acid bacteria improve digestive function, preventing the absorption of cholesterol by fibers such as dietary fiber, and help excrete bad cholesterol (LDL) from the body by increasing metabolic rate. Furthermore, it has been reported that lactic acid bacteria are effective in lowering cholesterol levels by inhibiting the process of cholesterol production in the liver. Furthermore, in the case of triglycerides, blood levels gradually decreased as the feed administration period increased, showing a decreasing trend of 7.1% in the G1 administration group and 8.7% in the G2 administration group after 8 weeks (Fig. 6D). This result is similar to previous reports indicating that lactic acid bacteria influence intestinal microbial activity to lower hepatic fat and triglycerides. In addition, lactic acid bacteria It has been reported that Lactobacillus plantarum Q18 inhibits the activity of lipase, a fat-degrading enzyme, thereby preventing the intestinal absorption of triglycerides, and furthermore, increases fatty acid oxidation in the liver to inhibit the synthesis of triglycerides, thus reducing their levels. Therefore, lactic acid bacteria improve digestion and promote the absorption of nutrients, and help prevent cardiovascular disease by normalizing triglyceride levels.

[0076] To investigate the effects of feed supplementation containing lactic acid bacteria and fermented liquid on liver function in Sprague Dawley rats, ALT (GPT, Alanine aminotransferase), AST (GOT, Aspartate aminotransferase), and ALP (Alkaline phosphatase) were measured using kits. As shown in Figure 7, there was no significant difference between the control and experimental groups, and it was determined that the added feed powder and red ginseng extract had almost no effect on liver function. Furthermore, regarding ALP, in the G1 group supplemented with dried powder of six types of lactic acid mycelia cultured in MRS medium, ALP levels increased by 4.2-36.5% from 2 to 8 weeks. Similarly, in the G2 group supplemented with dried powder of six types of lactic acid mycelia cultured in MRS medium containing red ginseng extract, ALP levels also increased by 3.8-45.8% (Figure 7C). These results suggest that ALP is related to in vivo function. In other words, it is a protein produced in vivo in the liver, bones, intestines, and kidneys, and secreted by the placenta during pregnancy; since it catalyzes chemical reactions and is involved in the transport of nutrients and enzymes, cell growth and differentiation, the transport of calcium phosphate, the transport of fatty acids, and energy storage, an increase in ALP levels in rats appears to be associated with signaling pathways for metabolism and growth promotion in animals. However, blood BUN (Blood urea nitrogen) levels were found to be almost unchanged (data not shown).

[0077] Insulin-like growth factor 1 (IGF-1), which plays an important role in animal growth, differentiation, and metabolism, is produced in the liver and its protein production is promoted by growth hormone. It is expressed by the IGF-1 gene, consists of 70 amino acids, and has a molecular weight of 7,640 daltons. As a secondary messenger of growth hormone, it plays a major role in promoting growth and metabolism by transmitting growth-related signals to various tissues, bones, and mammary glands. Therefore, when IGF-1 levels were measured using an IGF-1 analysis kit with serum from the control group and the experimental group (G1 and G2 administration groups), as shown in Fig. 6H, IGF-1 levels began to increase in the G1 and G2 experimental groups starting from week 4, and by week 8, the G1 group showed a 4.1% increase and the G2 group showed a 5.5% increase compared to the control group. This indicates that the administration of lactic acid bacteria grown in MRS medium and lactic acid bacteria grown in MRS medium supplemented with red ginseng extract resulted in an increase in the level of IGF-1, a secondary messenger that affects rat growth, and these results appear to be consistent with the increase in body weight and improved results in various biochemical indicators.

[0079] <Example 6> Confirmation of biological activity of lactic acid bacteria culture supplemented with red ginseng extract in laying hens

[0080] The laying hen experiment was conducted at a laying hen farm (Daegu University Farm) equipped with an automatic watering system. 100 80-week-old Hy-line Brown hens were randomly assigned to each of the two treatment groups, and the experiment was carried out in two replicates for 6 weeks by adding the above-prepared powder to the feed at a concentration of 1%. During the feeding period, 30 eggs were randomly selected weekly, and eggshell color, yolk color, egg weight, yolk weight, albumen height, yolk height, and Haugh Unit (HU) were qualitatively and quantitatively measured alongside the control group. This feeding experiment was conducted in accordance with the regulations of the Daegu University Animal Ethics Committee (DUIACC- 2020-14-0901-001).

[0081] Six strains of lactic acid bacteria grown in MRS medium supplemented with red ginseng extract (2 x 10 strains per strain) 9cells / ml) and culture powder (G2 powder) were added to the feed at a concentration of 1%. Water and feed were freely supplied to 100 laying hens (Hy-line brown, 80 weeks old) per group, and the results were administered in two replicates for 6 weeks. Thirty eggs were collected randomly each week, and the eggshell color, egg color, egg weight and yolk weight, albumen height, yolk height, and haugh unit (HU) were measured. As a result, as shown in Figure 8a, the eggshell and yolk colors began to darken starting 2 weeks after administration, and the eggshell remained dark brown and the yolk remained dark yellow until 6 weeks later. This indicates a color change similar to the results of the iron protein administration experiment. Although not analyzed using CIE color coordinates (L*a*b*), this was qualitatively confirmed, indirectly suggesting that the administration of lactic acid bacteria improves marketability and eggshell and yolk quality without compromising them, even though the hens were 80 weeks old and in the late laying stage. As shown in Fig. 8b, the egg weight and yolk weight were measured, and after 6 weeks, the egg weight increased by 4.5% compared to before administration, while the yolk weight increased by 5.7%. These results are consistent with reports indicating that the administration of lactic acid bacteria increases egg weight and laying rate, and that administering two or more strains of lactic acid bacteria yields better effects than administering a single strain. Albumen thickness and yolk thickness increased by 3.6% and 5.7%, respectively, after 6 weeks, appearing to increase proportionally with the increase in egg weight and yolk weight; egg freshness (HU) also increased by approximately 1% compared to before administration (Fig. 8c). Therefore, 6 types of lactic acid bacteria grown in MRS medium supplemented with red ginseng (2 x 10 9 It is determined that feeding cells / ml / strain and culture powder has a more positive effect on egg quality, as the color of the eggshell and yolk deepens, the egg weight and yolk weight increase, and the albumen thickness, yolk thickness, and haugh unit improve.

[0082] In light of these experimental results, it was assessed that the ability to produce high-quality eggs is due to the beneficial components of complex lactic acid bacteria and their fermentation products (fermented red ginseng extract) being fed to laying hens and transferred into the eggs.

Claims

Claim 1 A method for manufacturing a functional feed additive comprising a lactic acid bacteria fermented product of red ginseng extract, wherein the manufacturing method comprises the steps of: inoculating lactic acid bacteria into a medium containing red ginseng extract at a concentration of 5%; fermenting the red ginseng extract by culturing the inoculated lactic acid bacteria at 37°C for 2 to 7 days; obtaining a lactic acid bacteria fermented product of red ginseng extract comprising the lactic acid bacteria and culture solution produced in the fermentation step; and preparing the obtained lactic acid bacteria fermented product of red ginseng extract in powder form, wherein in the lactic acid bacteria inoculation step, the lactic acid bacteria is Lactobacillus A method for manufacturing a functional feed additive comprising a lactic acid fermented product of red ginseng extract, characterized by being a complex strain consisting of Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus rhamnosus, and Bifidobacterium bifidum. Claim 2 A method for manufacturing a functional feed additive comprising a lactic acid bacteria fermented product of red ginseng extract, wherein, in claim 1, the medium is an MRS medium or a medium containing 5 g / L of rice bran, 10 g / L of glucose, 1 g / L of ascorbic acid, and 10 g / L of 1% soy peptone. Claim 3 A method for manufacturing a functional feed additive comprising a lactic acid bacteria fermented product of red ginseng extract, wherein, in claim 1, the red ginseng extract is prepared by mixing 5 to 6 times the amount of purified water with red ginseng raw material and pressurizing the extraction at 110°C for 7 to 8 hours. Claim 4 A method for manufacturing a functional feed additive comprising a lactic acid bacteria fermented product of red ginseng extract, wherein, in claim 1, the step of preparing the lactic acid bacteria fermented product of the obtained red ginseng extract in powder form is characterized by mixing an equal amount of oxalate powder with the lactic acid bacteria fermented product and drying it. Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete

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