KR102713904B1 - A feed additive comprising the fermented natural materials and a breeding method using the same - Google Patents

Abstract

The present invention relates to a feed additive comprising a fermented natural substance as an effective ingredient, and to a livestock breeding method using the same. According to the present invention, by using a fermented extract of pomegranate and ginkgo leaves or a fermented extract of pomegranate and citron peel as a feed additive for breeding broilers or piglets, the feed efficiency (gain/feed, G/F) can be improved, and as a result, a feed additive as an antibiotic substitute for broilers or piglets can be provided. In addition, by blending the feed additive as an antibiotic substitute for breeding pigs into the feed composition, a feed composition can be provided which can further improve the immunity and growth rate of broilers or piglets, and there is also an effect of providing a breeding method which can further improve the immunity and growth rate of pigs by feeding the feed composition to broilers or piglets during their growing season.

Description

Feed additive comprising the fermented natural materials as an effective ingredient and a livestock breeding method using the same {A feed additive comprising the fermented natural materials and a breeding method using the same}

The present invention relates to a feed additive containing a fermented natural substance as an effective ingredient and a livestock raising method using the same, and more specifically, to a feed additive composition for broilers or piglets containing a fermented extract of pomegranate and ginkgo leaves or citron peel as effective ingredients and a livestock raising method using the same.

With the rapid development of industry and economy, the national diet is changing toward a Westernized trend, and due to the increase in livestock product consumption, our livestock industry has been using antibiotics to prevent and treat livestock diseases, promote livestock growth, improve feed efficiency, and enhance livestock productivity.

As consumer concern about the safety of livestock products increases, the legitimacy of using antibiotics in livestock feed is being widely challenged, and the number of veterinary drugs that can be mixed in compound feed was reduced from 53 to 25 in accordance with the Ministry of Agriculture, Food and Rural Affairs’ Notice (2004), and then further reduced to 18 in December 2007. As continuous reduction is required due to animal resistance and cross-resistance with antibiotics used in humans, the use of growth promoting antibiotics (GPAs) except for anticoccidial agents has been completely banned since 2012.

Recently, research on alternatives to antibiotics using natural antibacterial substances has been diversified, but the results are minimal. There is an urgent need to explore substances that activate the immune function of livestock and develop agents that induce enhanced immunity against pathogens that invade from outside by comprehensively enhancing the body's immunity.

Recently, as the concept of Synbiotics has been established, the simple form of probiotic manufacturing has been changed to a method of drying and reusing the culture solution, a method of using excipients as culture materials, and further development of production methods such as adding functional substances and prebiotics, so Synbiotics probiotics are more effective than simple probiotics and can be applied to livestock from young to adult livestock. Probiotics with the concept of Synbiotics are becoming more widespread, and attempts are being made to develop probiotics with additional functionality that can replace antibiotics.

Recently, the consumption of active natural products has been increasing, and in the field of natural antibacterial agents, the use of plant extracts with fewer side effects and resistance has been increasing, and methods for safely and effectively extracting active substances from abundant biological resources are required.

Prebiotics are a general term for non-digestible food ingredients that selectively affect the growth and vitality of microorganisms residing in the intestines and have beneficial effects on the host. In particular, since the results were published (Paker, 1974) that the vitality of probiotics is increased by the synergistic effect induced by the use of probiotics and specific substrates together, and the health promotion effect of piglets is enhanced as a result, prebiotics, which are one of the substances that can cause a synergistic effect with probiotics, have been considered for use as feed additives. Prebiotics change the intestinal microbial flora by promoting the selective growth of specific strains, and research is being conducted on fructooligosaccharides (FOS) and inulin, among others.

However, antibiotics added to livestock feed are completely broken down and excreted after a long-term withdrawal period. If livestock are shipped and consumed before this period, there is a problem that residual antibiotics in the livestock may cause harm to humans who consume the meat products. Furthermore, if antibiotic-resistant bacteria are transferred to other bacteria through conjugation of plasmids, which are genetic material containing resistance to antibiotics, this poses a very fatal problem to public health and sanitation, such as causing the occurrence of salmonella and food poisoning bacteria. In addition, feed additives containing antibiotics in the past have led to the emergence of super bacteria that are resistant to antibiotics used on animals and humans.

Therefore, it has become very difficult to add antibiotics to livestock feed, and since such feed additives are not reliable in terms of safety, there is a need to develop new feed additives that can replace antibiotics but are also safe and environmentally friendly.

Representative feed additives include probiotics, acidifiers, enzymes, mineral supplements, and oligosaccharides. Recently, natural antimicrobial peptides (AMPs) extracted from animals (Zasloff, 1987; Skerlavaj et al., 1999; Henzler et al., 2003;) and plants (Taniguchi and Kubo, 1993; Broekaert et al., 1995;) have been discovered and are receiving much attention.

In addition, ascorbic acid, tocopherol, phenolic compounds, flavone derivatives, amino acids, etc. are known as representative natural antioxidants present in foods, but their use is limited because they are expensive and their antioxidant effects are limited.

Accordingly, the inventors of the present invention have continued to conduct research to develop a natural substance that is excellent in substitution effect as an antibiotic substitute, is inexpensive, has excellent antioxidant effect, and can be put to practical use as a feed additive. During this process, they discovered that a fermented extract of a mixed extract of pomegranate and ginkgo leaves or a mixed extract of pomegranate and citron peel can improve feed efficiency (gain/feed, G/F), and thus can be used as a feed additive as a substitute for antibiotics for broilers or piglets, thereby completing the present invention.

Therefore, the technical problem to be solved in the present invention is to provide a feed additive for use as an antibiotic substitute for livestock using natural substances.

In addition, another technical problem to be solved in the present invention is to provide a feed composition for livestock feed containing the feed additive.

In addition, another technical problem to be solved in the present invention is to provide a method for raising livestock by feeding the feed composition to livestock.

In order to solve the above-mentioned technical problem, the present invention provides a feed additive for use as an antibiotic substitute for livestock, which contains as an effective ingredient a fermented extract of a mixed extract of pomegranate and ginkgo leaves or a fermented extract of a mixed extract of pomegranate and citron peel.

In addition, in order to solve the other technical problems mentioned above, the present invention provides a feed composition for livestock breeding containing the feed additive.

In addition, in order to solve the above-mentioned further technical problem, the present invention provides a method for raising livestock, including a step of feeding the feed composition to livestock.

According to one embodiment of the present invention, the livestock is preferably a broiler or a piglet.

The pomegranate (Punica granatum L.), which is used as an effective ingredient of the feed additive of the present invention, is a plant native to Afghanistan and the northwestern region, and was used in religious ceremonies, art, and mythology in ancient Egypt and Greece. It is said that the name pomegranate is derived from the French word promergates, and the etymology of the word pomegranate is said to be a combination of the two words "granatus" meaning seed and "pomun" meaning apple (Jurenka, 2008). In Korea, it is cultivated in the form of an orchard in Gwangyang in the south. Domestically cultivated varieties are classified only into new pomegranate (sour and acidic) and sweet pomegranate (sweet and persimmon) according to their taste, and the size of the fruit is smaller than that of the Middle East due to the climate difference (Kim Hak-gyu et al., 1994). It has been cultivated in Goheung, Jeollanam-do since 2002, but the production volume is not large and the sugar content is low, so it is mainly used for medicinal purposes (Shim, 2001).

The efficacy of pomegranates includes the outer skin, which has the pharmacological effect of suppressing abnormal fermentation in the stomach, thus strengthening the intestines, and the volatile alkaloid components have the effect of eliminating parasites such as roundworms and tapeworms. In particular, the peel contains mucus and tannins that are hydrolyzed to produce ellagic acid (Sin, 2005), and is used as an astringent and for the treatment of diarrhea, dysentery (Seo Ja-myeong, 1998), and antioxidant (Jin, 2007). Pomegranate seeds are known to relieve thirst (Wynder, 1997), and the fruit contains organic acids such as malic acid and citric acid, and tannins that produce ellagic acid (Kim Seon-ho, 2005). Additionally, pomegranate extracts are known to be rich in flavonoids, including anthocyanins and phenolic compounds, and are reported to have anticancer, antioxidant, anti-inflammatory, antidiabetic, and antimicrobial activities (Kim, 2005).

Recently, the results of studies on various activities of pomegranate juice, peel, and seed oil have been reported. When polyphenolic antioxidant components contained in pomegranate juice were administered to hypercholesterolemic experimental mice, it significantly reduced the progression of atherosclerosis in human coronary artery endothelial cells (EC) (Nigris et al., 2005). Since flavonoids extracted from pomegranate juice showed a significant inhibitory effect on the development of atherosclerosis in arterial macrophages (Aviram et al., 2004), the effects of pomegranate juice consumption on cardiovascular disease in hypertensive patients are being studied (Aviram et al., 2001). In addition, since it was revealed that pomegranate extracts have an estrogen effect, there was a study result that reduced bone density in ovariectomized mice was restored to normal and bone volume and fiber distribution were increased. In addition, evidence has been confirmed that pomegranate extract has clinical effects on depression and bone loss in postmenopausal syndrome in women (Mori-Okamoto et al., 2004). Pomegranate peel extract has shown antioxidant effects by inhibiting oxidative enzymes such as catalase, peroxidase, and superoxide dismutase (SOD) and lipid peroxidation, and has a hepatoprotective effect in a CCI4-induced hepatotoxicity experimental rat model (Chidambara Murthy et al., 2002).

Ginkgo biloba L., used as an effective ingredient of the feed additive of the present invention, has existed on Earth for about 150 million years and is known as a living fossil, and has been used in China for asthma and bronchitis for about 50 million years (Kleijnen, 1992). It is a deciduous tree, and its leaves grow in groups of 3 to 5 at the ends of short branches, are fan-shaped, deeply or shallowly split in the middle, and have parallel veins. The flowers are monoecious and bloom from April to May, and usually produce spermatozoa and are fertilized in early September. The fruit is a spherical drupe. It matures in October and the outer testa is fleshy and is used for edible, medicinal, ornamental, and industrial purposes.

The main parts used in the Ginkgo tree are seeds, stems and leaves, seed coats, and bark, and they have been used medicinally for sedatives, tonics, boils, tuberculosis, insect repellents, astringents, hypertension, and diabetes (Park et al., 2002; Song et al., 1989). Figure 1 provides a description of the components contained in Ginkgo leaves. Flavonoids, diterpenes, sesquiterpene polyphenols, polysaccharides, catechins, tannins, and organic acids have been reported (Huh, 1990; Kim et al., 1995). In particular, flavonoid compounds account for approximately 50% of the extract of Ginkgo leaves (Agnoli, 1984). Flavonoid is a word derived from the Latin word flavus, meaning yellow, and refers to a yellow plant pigment with flavone as its basic structure.

More than 5,000 types of flavonoids have been discovered in nature, and they are usually divided into five groups based on their chemical structure: flavones, isoflavones, derivatives of flavans, anthocyanidins, and neoflavonoids. Anthoxanthin, a type of flavonoid, is the main cause of yellow flower petals and purple or red leaves in the fall.

Flavonoids become more vivid in acidic conditions where they remain stable, but their structure changes to dark yellow or brown in strong alkaline conditions. They also combine with metals such as copper and iron to form dark brown complexes. They have been used as vasodilators in traditional herbal medicine to promote blood circulation and oxygen consumption in body tissues, and it has been reported that numerous compounds with pharmacological effects in ginkgo leaves are gradually being discovered (Key, 1976).

Since standardization of ginkgo leaf extract is essential for safe and effective use, there is a research report that the main components of EGb 761 are approximately 24% flavonoid glycosides (kaempferol, quercetin, luteolin, etc.), 6% terpene trilactones (ginkgolide, a diterpenoid, and bilobalide, a sesquiterpene), and less than 5 ppm ginkgolic acid (Jacobs et al., 2000). It is widely used as a treatment for diseases such as decreased cerebral and peripheral blood flow, sensory nerve diseases, and decreased memory and cognitive function (DeFeudis, 1991), and recent studies have shown that it has an inhibitory effect on cell proliferation of colon cancer and breast cancer cells (Mutoh et al., 2000; Raso et al., 2001). A recent study showed that EGb 761 250 μg/ml administered to oral cancer cell lines inhibited cell proliferation, and that this inhibition of cell proliferation was due to apoptosis. The dose used in the above experiment was confirmed to be a relatively safe dose compared to the results of a stability test using rats (Kim et al., 2005).

Yuzu (Citrus junos Sieb), used as an effective ingredient of the feed additive of the present invention, is an evergreen tree belonging to the genus Citrus and the subgenus Metacitrus in taxonomy. Its main origin is the Sichuan and Yunnan provinces in the upper reaches of the Yangtze River in China (Oh et al., 1991). In Korea, it is cultivated in the warm climate areas of Goheung, Wando, and Geoje on the southern coast, where it has strong cold resistance. The cultivation conditions are an average annual temperature of 14–15°C, more than 2,400 hours of sunshine per year, and an average annual rainfall of 1,500 mm, making it a crop that requires a lot of water compared to other fruits (Kim et al., 2000; Shin et al., 2010; Byun et al., 1990). This is a paper studying the chemical components of yuzu (Citron junos) fruit. The main content is that nine compounds were isolated from yuzu fruit. Figure 3 shows that the structures were identified as 9-hydroxy-4-methoxypsoralen, Auraptene, Limonin, Deacetylnomilin, Cirsimaritin, Narirutin, Naringin, Hesperidin and Neohesperidin by physicochemical evidence (Cho, 2000).

Yuzu, unlike other citrus fruits, is a fruit that uses not only the pulp but also the peel, and has a strong sour taste and aroma. In addition, yuzu contains three times more vitamin C than lemon, which is good for colds and skin beauty, and is also rich in organic acids, which are effective in preventing aging and fatigue, and it also has a high content of vitamin B and minerals (Kim et al., 2006; Jeon et al., 2011). In addition, flavonoids contained in three forms in the peel of citrus fruits, including yuzu, have various physiological activities. That is, limonene, an essential oil component, has antibacterial effects (Lee et al., 2008; Kim et al., 2009), and naringin has been reported to have antibacterial, antioxidant, anti-inflammatory, antihypertensive, and blood lipid-lowering effects (Chae et al., 2008). Another component, hesperidine, has physiological functions such as lowering blood pressure, anti-allergy, reducing blood LDL cholesterol, and inhibiting carcinogenesis (Woo et al., 2000).

More than 70% of the production of yuja is used as a raw material for making yuja tea or yuja extract, and its superior physiological activity effects have been proven through various studies (Jeong Yo-han et al., 2010). (Yu Kyung-mi et al., 2004) reported that substances extracted from the peel and pulp of mature yuja have antioxidant and cancer cell death effects, and (Song Man-gang et al., 2004) suggested the need for the development of natural antioxidant substances because the antioxidant effect of yuja juice is higher than that of Vitamin C.

Limonene, which accounts for 74.4% of the fragrance of Korean yuzu, has immune function regulating activity and therefore needs to be developed as an alternative to antibiotics. Meanwhile, organic acids contained in large quantities in yuzu have been found to have excellent inhibitory abilities to the production of nitrosamines, which have the potential for mutagenesis, carcinogenicity and teratogenicity in living organisms (Shin et al., 2004). Yuzu has a strong sour taste and fragrance, so it has been widely used in beverages such as candied yuzu syrup, and unlike other citrus fruits that mainly use only the flesh, it is a fruit that uses both the flesh and the peel, so it has the advantage of being easier to consume the effective ingredients contained in the peel than in fruits such as tangerines or oranges (Yoo et al., 2005; Park et al., 2006). Therefore, yuzu is used to make yuzu tea, yuzu syrup, yuzu juice and yuzu flavor using the peel rather than the flesh.

Yuzu, a fragrant fruit, has been used as a traditional tea to prevent colds since ancient times. Recently, the physiological activity of yuzu extracts has also been actively evaluated, and many studies have reported on the reduction in cardiovascular disease incidence due to flavonoids (Calabro et al., 2004; Cha and Cho, 2001) and antioxidant and anticancer effects (Yoo and Hwang, 2004; Yoo et al., 2005). Yuzu is spherical or spherical, contains relatively few seeds, and is a white polyploid fruit with a strong sour taste, so it is rarely eaten raw, but is used in beverages such as tea. In the private sector, it is used as a bitter tonic, an expectorant, a cold medicine, and a headache medicine, and its pharmacological efficacy is already well known (Kang et al., 2006).

In addition, yuzu is not only rich in vitamins A and C, but also known to have a wide range of uses, as limonene, a volatile component found in large quantities in the peel of yuzu, has antibacterial properties and has a fragrance (Kang et al., 2006). Studies on the physiological activities of yuzu have been reported, including studies on the antioxidant activity of yuzu extracts and the apoptotic effect on prostate cancer cells, as well as on the antioxidant activity and scavenging activity of nitrite, known as a precursor of carcinogenic nitrosamine (Shin et al., 2005; Yu and Hwang, 2004).

In the present invention, a mixed extract of pomegranate, ginkgo leaf or citron peel is used, and the pomegranate, ginkgo leaf or citron peel extract can be extracted using water or an organic solvent, and the extracted liquid can be used in liquid form or can be used after being concentrated and/or dried. The organic solvent is methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butylacetate, dichloromethane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, and the extraction can be performed at room temperature or under conditions where the effective ingredients of the extract are not destroyed or are minimized. In one specific embodiment, the pomegranate, ginkgo leaf or citron peel extract can be extracted using 50% ethanol at room temperature respectively and then mixed, or the pomegranate and ginkgo leaf or the pomegranate and citron peel can be mixed in advance and then extracted and used.

At this time, pomegranate and ginkgo leaves or pomegranate and yuzu peel can be mixed and used in a weight ratio of 5:1 to 2:1.

The above extraction method is not limited and includes, for example, cold extraction, ultrasonic extraction, reflux cooling extraction, etc.

The pomegranate, ginkgo leaf or citron extract of the present invention may be any extract, fraction, purified product, dilution, concentrate or dried product thereof obtained at each stage of extraction, fractionation or purification (separation, fractionation).

In the present invention, "fermented pomegranate and ginkgo leaf extract or fermented pomegranate and citron peel extract" refers to the pomegranate and ginkgo leaf extract or the pomegranate and citron peel extract, and at least one strain selected from the group consisting of Lactobacillus plantarum, Pediococcus acidilactici, Bacillus licheniformis, Bacillus subtilis, Saccharomyces cerevisiae and Saccharomyces boulardii , preferably Lactobacillus plantarum (lactic acid bacteria), Bacillus subtilis (spore bacteria) and Saccharomyces cerevisiae It means fermented with one or more strains selected from the group consisting of (yeast fungi).

In a preferred embodiment of the present invention, the fermented pomegranate and ginkgo leaf extract or the fermented pomegranate and citron peel extract can be prepared by inoculating the pomegranate and ginkgo leaf extract or the pomegranate and citron peel extract with one or more strains selected from the group consisting of Lactobacillus plantarum (lactic acid bacteria), Bacillus subtilis (spore bacteria), and Saccharomyces cerevisiae (yeast bacteria) in an amount of 0.1 to 10 wt% based on the weight of the extract and culturing for 10 to 60 hours.

In the present invention, the optimal strain combination of natural product fermentation probiotics is Lactobacillus plantarum (lactic acid bacteria), Bacillus subtilis (spore bacteria) and Saccharomyces cerevisiae (yeast) that has antibacterial effects against pathogenic bacteria and fungi and growth inhibition activity against these bacteria, and excellent antagonistic effects against intestinal pathogenic bacteria produced through the secretion of lactic acid and bacteriocin, were selected and the culture ability was evaluated, and the vitality was high at the level of ¹⁰⁸ to ¹⁰⁹ cfu/㎖.

According to one embodiment of the present invention, a natural fermented probiotic test product was evaluated for a mixing ratio of 30% pomegranate marmalade, 5% ginkgo leaf powder (5% green tea powder, 5% citron marmalade), 32% corn ethanol residue, 32% defatted rice bran, and 1% glucose, and a mixing ratio of 29% pomegranate marmalade, 10% ginkgo leaf powder (10% citron marmalade), 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose.

According to one embodiment of the present invention, in a productivity feeding test of broilers according to feeding pomegranate-ginkgo leaf fermented probiotic and pomegranate-green tea fermented probiotic, the weight gain of each treatment group during the entire test period (5 weeks) was compared, and the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group showed the highest weight gain of 2,021 g, showing a significant difference (P<0.05). In particular, the pomegranate-ginkgo leaf fermented probiotic 0.2% and 0.4% treatment groups showed significant improvements in weight gain and feed conversion ratio compared to the control group.

According to one embodiment of the present invention, in the evaluation of body composition of broilers according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-green tea fermented probiotic, the crude protein content of breast meat was the highest in the pomegranate-green tea fermented probiotic 0.4% treatment group at 25.14%.

According to one embodiment of the present invention, as a result of analyzing the cholesterol content of chicken meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-green tea fermented probiotic, the cholesterol content of the breast meat was compared, and the pomegranate-ginkgo leaf 0.2% fermented probiotic treatment group had the lowest at 92.47 mg/100g and the pomegranate-ginkgo leaf 0.4% fermented probiotic treatment group had the highest at 149.98 mg/100g, showing a significant difference between the treatment groups (P<0.05). The cholesterol content of the leg meat was the lowest at 100.98 mg/100g in the pomegranate-ginkgo leaf fermented probiotic treatment group and the highest at 182.73 mg/100g in the control group, showing a significant difference between the treatment groups (P<0.05).

According to one embodiment of the present invention, as a result of analyzing the fatty acid content of chicken meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-green tea fermented probiotic, the fatty acid content of the breast meat was analyzed. Myristic acid (C14:0) was the highest at 0.80% in the control group (P<0.05) and the lowest at 0.54% in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group, indicating a significant difference between treatment groups (P<0.05). γ-Linoleic acid (C18:3n6) was the highest at 0.46% in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group and the lowest at 0.10% in the 0.4% pomegranate-green tea fermented probiotic treatment group, indicating a significant difference between treatment groups (P<0.05).

According to one embodiment of the present invention, as a result of analyzing the fatty acid content of leg meat, Oleic acid (C18:1n9) was the highest at 41.52% in the 0.2% pomegranate-green tea fermented probiotic treatment group (P<0.05) and the lowest at 38.20% in the 0.2% ginkgo leaf powder treatment group, indicating a significant difference among treatment groups (P<0.05). γ-Linoleic acid (C18:3n6) was the highest at 0.43% in the 0.2% pomegranate-ginkgo leaf fermented probiotic treatment group (P<0.05) and the lowest at 0.30% in the 0.2% pomegranate-green tea fermented probiotic treatment group, indicating a significant difference among treatment groups (P<0.05).

According to one embodiment of the present invention, as a result of analyzing the blood immune components of broilers according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-green tea fermented probiotic, the blood immunoglobin IgA content was the highest in the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group at 15.49 mg/ml (P<0.05), and the lowest in the ginkgo leaf powder 0.2% treatment group at 0.91 mg/ml, showing a significant difference between treatment groups (P<0.05).

According to one embodiment of the present invention, the IgM content in blood was highest at 0.17 mg/ml in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group, and was low at 0.11 mg/ml in the control group, showing a significant difference between the treatment groups (P<0.05).

According to one embodiment of the present invention, when the weight gain, feed intake, and feed conversion ratio were compared among the treatment groups during the entire test period (5 weeks), the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the highest at 2,058 g, and the control group was the lowest at 1,830 g, showing a significant difference among the treatment groups (P<0.05). In the case of feed conversion ratio, the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group was the lowest at 1.48, showing a significant difference among the treatment groups (P<0.05). In particular, it was found that the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% and pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment groups showed significant improvements in weight gain and feed conversion ratio compared to the control group.

According to one embodiment of the present invention, as a result of analyzing the body composition of broilers according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic, in the case of breast meat of the carcass, the crude protein content was the highest in the control group at 24.95%, and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group was the lowest at 22.22%, showing a significant difference between treatment groups (P<0.05). The crude fat content was the highest in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group at 2.04% (P<0.05), and the control group was the lowest at 0.95%, showing a significant difference between treatment groups (P<0.05). The crude protein content of the leg meat of the conductor was the highest in the 0.4% pomegranate-yuzu fermented probiotic agent 1 (PCP-1) treatment group at 20.70% (P<0.05), and the lowest in the control group at 19.03%, showing a significant difference among the treatment groups (P<0.05). The crude fat content was the highest in the 0.4% pomegranate-yuzu fermented probiotic agent 1 (PCP-1) treatment group at 3.53% (P<0.05), and the lowest in the control group at 2.0%, showing a significant difference among the treatment groups (P<0.05).

According to one embodiment of the present invention, the results of analyzing the cholesterol content of chicken meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic were as follows: the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group showed the highest at 156.99 mg/100 g (P<0.05), and the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group showed the lowest at 120.99 mg/100 g, indicating a significant difference between the treatment groups (P<0.05).

According to one embodiment of the present invention, as a result of analyzing the fatty acid content of chicken meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic, the fatty acid content of breast meat was analyzed. Myristic acid (C14:0) was the highest at 2.0% in the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group (P<0.05), and the lowest at 0.93% in the control group, showing a significant difference between the treatment groups (P<0.05).

According to one embodiment of the present invention, Oleic acid (C18:1 n9) was highest in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group at 36.16% (P<0.05), and lowest in the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group at 32.95%, showing a significant difference between treatment groups (P<0.05).

According to one embodiment of the present invention, as a result of analyzing the fatty acid content of leg meat, Linoleic acid (C18:2n6) was found to be high at 18.22% in both the 0.4% pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) treatment group and the 0.4% pomegranate-yuzu fermented probiotic 2 (PCP-2) treatment group (P<0.05), and was the lowest at 16.70% in the 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment group, showing a significant difference between treatment groups (P<0.05).

According to one embodiment of the present invention, as a result of analyzing the blood immune components of broilers according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic, the blood immunoglobin IgG content was analyzed as the highest in the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group at 1.84 mg/ml, and the lowest in the control group at 1.20 mg/ml, showing a significant difference between the treatment groups (P<0.05). In addition, it was found that all treatment groups of the pomegranate-ginkgo leaf fermented probiotic and the pomegranate-yuzu fermented probiotic showed higher IgG contents than the control group.

According to one embodiment of the present invention, as a result of investigating the number of deaths and mortality rate of broilers fed with pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic after artificial infection with pathogenic bacteria, the number of deaths was the highest in the control group with 15, and the lowest in the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group with 3. The mortality rate by treatment group was 83.33% in the control group, 72.22% in the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group, 50% in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group, 22.22% in the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group, and 16.67% in the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group, showing that the survival rate was the highest in the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group.

According to one embodiment of the present invention, when the weight gain, feed intake, and feed conversion ratio were analyzed according to the feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic in piglets, the weight gain of each treatment group during the entire test period (3 weeks) was compared. The pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the highest at 20.40 kg, and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group was the lowest at 18.83 kg, showing a significant difference among the treatment groups (P<0.05). In the case of feed conversion ratio, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the lowest at 1.90.

According to one embodiment of the present invention, as a result of analyzing blood immune components according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic after artificial infection with pathogenic bacteria in piglets, the blood immunoglobin IgG content was analyzed to be the highest in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group at 1.84 mg/ml, and the lowest in the pomegranate-yuzu fermented probiotic 2 (PCP-2) at 1.76 mg/ml, showing a significant difference among treatment groups (P<0.05). As a result of analyzing blood immunoglobin IgM content, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was analyzed to be the highest at 2.01 mg/ml, showing a significant difference among treatment groups (P<0.05).

According to one embodiment of the present invention, after artificial infection with pathogenic bacteria in piglets, the blood cytokine analysis result according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic was the highest at 81.46 pg/ml in the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group, and the control group was the lowest at 59.56 pg/ml, showing a significant difference between the treatment groups (P<0.05). In addition, it was found that the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group showed higher blood cytokine contents compared to the control group.

According to a preferred embodiment of the present invention, the fermented pomegranate and ginkgo leaf extract or the pomegranate and citron extract can be added to the feed additive at 10 to 100%.

In the present invention, the feed additive can preferably be fed to broilers or piglets during their growing season, but is not limited thereto. By feeding the feed additive of the present invention to broilers or piglets during their growing season, there is an advantage in that the immunity and growth rate of broilers or piglets can be improved more efficiently.

In addition, the present invention provides a feed composition for raising broilers or piglets containing the feed additive.

In the present invention, the feed additive may be included in an amount of 0.05 to 80 wt% based on the total weight of the entire feed composition. By including the feed additive of the present invention in the above range, there is an advantage in that the growth of broilers or piglets can be promoted more efficiently.

In the present invention, the feed composition for raising broilers or piglets may, in addition to the feed additive, contain energy, amino acids, minerals, vitamins and water as essential nutrients that must be supplied from the feed for the maintenance and growth of broilers or piglets.

Energy is generally the most expensive nutrient in feed compositions for broilers or piglets due to its high demand, and the three sources of carbohydrates, fats, and proteins (amino acids) are mainly used as energy sources. In terms of energy sources for animals in general, carbohydrates are the most important energy source, followed by fats.

In the present invention, corn, wheat, oats, barley, etc. can be used as a carbohydrate source, and corn is most preferably used.

In the present invention, the corn can be used in either steamed or unsteamed form, and crushing and mixing it into the feed composition can facilitate the intake of livestock. As described above, corn is used as a carbohydrate source in the feed composition for growing pigs of the present invention. Accordingly, it is preferable that corn is included in an amount greater than an appropriate amount. In the present invention, corn can be included in an amount of 50 to 70 wt%, preferably based on the total weight of the entire feed composition.

In the present invention, a protein source can be used to supply energy and amino acids, and soybean meal, rapeseed meal, wheat bran, cottonseed meal, sesame meal, brewer's meal, etc. can be used.

In the present invention, soybean meal is used as a protein source of a feed composition for broilers or piglets. Soybean meal is a by-product of extracting oil from soybeans, and has a high crude protein content of about 44 to 50%, and also has a balanced amino acid content, supplementing the lack of protein and amino acid combination. In the present invention, soybean meal may preferably be included in an amount of 10 to 40 wt% based on the total weight of the entire feed composition.

In the present invention, rapeseed meal is a by-product obtained by removing the hull and oil from rapeseed seeds, and has relatively low palatability. As for nutritional properties, it has a crude protein content of about 35 to 40%, lower energy and lysine contents than soybean meal, and higher Ca (0.6%) and P (1.0%) contents. In the present invention, rapeseed meal may be preferably included in an amount of 1 to 5 wt% based on the total weight of the entire feed composition.

In the present invention, fat sources such as beef tallow, corn DDGS, and choice white grease can be used.

In the present invention, beef tallow is used as a fat source for a feed composition for raising broilers or piglets. Beef tallow refers to the solid fat around the waist and kidneys of cattle, sheep, and horses, and vegetable oil with similar characteristics is also called beef tallow. The main component is composed of glyceryl esters such as oleic acid, palmitic acid, and stearic acid. In the present invention, beef tallow may be preferably included in an amount of 1 to 5 wt% based on the total weight of the entire feed composition.

Minerals form the skeleton in the piglet's body and can be used as important nutrients during the metabolic process. The requirement for minerals can vary depending on the degree of distribution in the livestock's body. Usually, the requirement for Ca, P, Na, Cl, K, and Mg is high, and the requirement for Fe, Zn, I, Se, Mn, and Cu is known to be trace amounts. In piglet feed, K, Mg, Mn, and S are sufficiently contained in the feed so that there is no deficiency, but other minerals must be added to the feed.

In the present invention, defluorinated ore, calcium carbonate, mineral premix, limestone, and the like can be used as mineral supply sources.

In the present invention, a mineral premix means a mixture prepared in advance so that minerals necessary for broilers or piglets can be easily mixed with raw materials when compounding feed.

In the present invention, a commercially available product can be used as the mineral premix, and the appropriate mixing ratio of each mineral is known in the art. In one embodiment of the present invention, the mineral premix was used in an amount that provides Cu, 220 mg; Fe, 175 mg; Zn, 191 mg; Mn, 89 mg; I, 0.3 mg; Co, 0.5 mg; and Se, 0.4 mg per 1 kg of the entire feed. In the present invention, the mineral premix may preferably be included in an amount of 0.01 to 0.5 wt% based on the total weight of the entire feed composition.

In the present invention, the feed composition for raising broilers or piglets may additionally contain rice bran, molasses, salt, amino acids, choline chloride, and the like.

In addition, the present invention provides a method for raising broilers or piglets, comprising the step of feeding the feed composition to broilers or piglets.

As described above, the feed composition including the feed additive for antibiotic replacement of the present invention has an advantage in that it can be fed to broilers or piglets during the growing period to more efficiently improve the immunity and growth rate of piglets.

In this way, by using the fermented extracts of pomegranate and ginkgo leaves or the fermented extracts of pomegranate and citron peel according to the present invention as a feed additive for raising broilers or piglets, the feed efficiency (gain/feed, G/F) can be improved, and as a result, a feed additive as an antibiotic substitute for broilers or piglets can be provided. In addition, by blending the feed additive as an antibiotic substitute for growing pigs into the feed composition, a feed composition can be provided which can further improve the immunity and growth rate of broilers or piglets, and there is also an effect of providing a breeding method which can further improve the immunity and growth rate of pigs by feeding the feed composition to the growing period of broilers or piglets.

Figure 1 shows the results of an experiment for strain selection.
Figure 2 shows photographs of natural substances before and after fermentation.

Hereinafter, in order to help understand the present invention, examples and the like will be described in detail. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

Example 1

A. Exploration and investigation of natural substances

The exploration and investigation of natural substances were conducted through comparative investigations using domestic and foreign literature, and the nutrient content analysis was performed using dried samples ground for 1 minute in a grinder (Hanil Electric, HMF-3150S). The content analysis was performed according to the AOAC (2000) method for moisture, crude protein, crude fat, and crude ash.

B. Search and selection of useful strains

In order to search for the optimal strain for fermentation of natural substances, the test strains were isolated from the intestines of animals and selected based on criteria such as producing antibiotics or microbial growth inhibitors and powerful digestive substances during growth. The culture conditions for useful strains are as shown in Table 1. In this study, MRS medium (Difco, Detroit, USA) was used as the culture medium for lactic acid bacteria, NB medium (Nutrient Broth, Difco) was used for spores, and YM broth medium (Difco) was used for yeast. The culture conditions were as follows: lactic acid bacteria were cultured at 37℃ for 48 hours with shaking at 150 rpm at 37℃ for 48 hours, and yeast was cultured at 30℃ for 24 hours.

Items Microbial strains Medium Culture
method Culture
time (hr) Lactic acid
bacteria Lactobacillus plantarum
Pediococcus acidilactici MRS Anaerobes culture 48 Bacillus Bacillus subtilis
Bacillus licheniformis NB Aerobic culture 48 Yeast Saccharomyces cerevisiae
Saccharomyces boulardii YM Anaerobes culture 24

D. For prototype manufacturing Fermentation agent Selection and natural product fermentation prototype manufacturing test

Fermentation excipients for the production of prototypes were made of defatted rice bran and corn ethanol dried grains, which are widely used as raw materials for animal feed, as fermentation materials, and six strains selected for the evaluation of fermentation ability were composed of two combinations, as shown in Table 2. In addition, for the production of prototypes of natural substance fermented probiotics, ginkgo leaves were directly collected from ginkgo trees in a nearby area, dried in the shade, and then crushed using a crusher. Pomegranate marmalade and other natural substances were used as byproducts from the production of beverages and extracts. Figure 1 shows the process of the natural substance fermented probiotic.

Treatment Proportion Inoculated microbial (X10 ⁷ cfu/㎖) T 1-1 DRB ¹⁾ 30% + DDGS ²⁾ 70% Lactobacillus plantarum
Bacillus subtilis
Saccharomyces cerevisiae T 1-2 DRB 30% + DDGS 70% Pediococcus acidilactici,
Bacillus licheniformis
Saccharomyces boulardii T 2-1 DRB 50% + DDGS 50% Lactobacillus plantarum
Bacillus subtilis
Saccharomyces cerevisiae T 2-2 DRB 50% + DDGS 50% Pediococcus acidilactici
Bacillus licheniformis
Saccharomyces boulardii T 3-1 DRB 70% + DDGS 30% Lactobacillus plantarum
Bacillus subtilis
Saccharomyces cerevisiae T 3-2 DRB 70% + DDGS 30% Pediococcus acidilactici
Bacillus licheniformis
Saccharomyces boulardii T 4-1 DRB 100% Lactobacillus plantarum
Bacillus subtilis
Saccharomyces cerevisiae T 4-2 DRB 100% Pediococcus acidilactici
Bacillus licheniformis
Saccharomyces boulardii T 5-1 DDGS 100% Lactobacillus plantarum
Bacillus subtilis
Saccharomyces cerevisiae T 5-2 DDGS 100% Pediococcus acidilactici
Bacillus licheniformis
Saccharomyces boulardii

¹⁾ DRB (Defatted Rice Bran): Defatted rice bran, ^{2 )} DDGS: Corn distillers' grains

Ra. Elucidation of the effect of adding natural fermented probiotics to feed on the productivity of broilers 1

This study was conducted to compare and evaluate the effects of feeding natural fermented probiotics (pomegranate and ginkgo leaf complex probiotics and pomegranate and green tea complex probiotics at 0.2 and 0.4%) and natural substances (ginkgo leaf complex probiotics at 0.2 and 0.4%) on the productivity of broilers.

1) Test animals and test design

In this study, 210 Ross Broiler chicks with uniform body weight were selected as test animals. The experimental design was conducted for 5 weeks with a total of 7 treatment groups, including the control group, the pomegranate-ginkgo leaf fermented probiotic treatment group with 0.2% and 0.4%, the pomegranate-green tea fermented probiotic treatment group with 0.2% and 0.4%, and the ginkgo leaf powder treatment group with 0.2% and 0.4%, respectively, with 5 replicates, and 6 chicks per replicate.

2) Test feed and feeding management

The test feed was mixed according to the NRC (1994) specification standard and used as the basal feed (control), and the basal feed mixing ratio is as shown in Table 3. The first basal feed contained 22% crude protein, 4% crude fat, and 3,100 kcal/kg of metabolizable energy, and the second basal feed contained 19% crude protein, 4.5% crude fat, and 3,150 kcal/kg of metabolizable energy. The test feeds for each test treatment group were mixed as the basal feed, the control feed, the basal feed with 0.2% pomegranate-ginkgo leaf fermented probiotic added, 0.4% added, the basal feed with 0.2% pomegranate-green tea fermented probiotic added, 0.4% added, and the basal feed with 0.2% ginkgo leaf powder added, 0.4% added. For feeding management, the growing temperature of the first hatchlings was maintained at 34–35°C and the humidity was maintained at 40–60% at the start of the feeding test, and the temperature was lowered by 1–2°C at weekly intervals. Feed and water were provided ad libitum. The test period was 5 weeks from November 10, 2014 to December 14, 2014, and the feeding test was conducted in a three-tiered iron cage at the livestock farm attached to the Department of Animal Resource Science, Soonchunhyang University. Analyses related to this experiment were conducted in the Animal Nutrition and Feed Laboratory.

Ingredient Starter Finisher Corn grain 57.58 60.64 Soybean meal 26.80 24.90 Corn gluten 5.00 3.50 Soybean oil 2.20 2.20 Animal fats 4.50 5.00 Salt 0.25 0.25 Dicalcium Phosphate 2.14 2.00 Limestone 0.92 0.88 Vit-Min. premix ¹⁾ 0.30 0.30 Choline 0.08 0.07 L-lysine 0.24 0.16 Methionine 0.20 0.10 Total 100.00 100.00 Chemical composition ME (kcal/kg) 3,100 3,150 Moisture (%) 12.07 13.08 Crude protein (%) 22.00 19.00 Crude fat (%) 4.00 4.50 Crude ash (%) 8.00 8.00 Crude fiber (%) 6.00 6.00 Methionine (%) 0.79 0.70 Ca (%) 0.80 0.75 P (%) 0.54 0.52

¹⁾ Vit-min. mix. provided following nutrients per kg of diet: Vitamin A, 9,000,000 IU; Vitamin D3, 2,100,000 IU; Vitamin E, 15,000 IU; Vitamin K, 2,000 mg; Vitamin B1, 1,500 mg ;Vitamin B2, 4,000 mg; Vitamin B6, 3,000 mg; Vitamin B12, 15 mg,; Pan-Acid-Ca, 8500 mg; Niacin, 20,000 mg; Biotin, 110 mg; Folic-Acid, 600 mg; Co, 300 mg; Cu, 3,500 mg; Mn, 55,000 mg; Zn, 40,000 mg; I, 600 mg; Se, 130 mg

3) Main research items

(1) Weight gain, feed intake and feed conversion ratio

Body weight was measured repeatedly at regular times from 2 PM to 5 PM every week from the start of the test to the end of the test, and the weight gain was calculated by subtracting the starting weight from the ending weight. Feed intake was calculated by measuring the remaining feed amount for each repetition just before the weekly weight measurement and deducting the remaining feed amount from the feed supply amount. Feed conversion ratio was calculated by dividing the feed intake by the weight gain.

(2) General conductor component content

The general components of the carcass were randomly selected from broilers close to the average body weight in each treatment group immediately after the end of the feeding test and used as samples. The selected broilers were divided into breast and leg meat and ground with a meat grinder, and moisture, crude protein, crude fat, and crude ash were analyzed according to the AOAC (2000) method.

(3) Conductor cholesterol content

The cholesterol content of the conductor was determined by adding a standard substance (5α-cholestane) to 1 g of the sample according to the method of King et al. (1998), adding 5 mL of 50% KOH (aq) and 22 mL of ethanol, saponifying at 23°C for 6 hours, and repeatedly extracting. The result was analyzed by gas chromatography (GC, DS 6200, Donam Co., Korea) under the conditions shown in Table 4.

Classification Condition Column HP-5 (J&W, 30 m Detector FID Carrier gas Nitrogen (1.0 ml/min) Make up gas H2 (30 /min) Temp. program 250℃(2 min) - 15/min -> 290℃ - 10/min -> 310℃ Detector temp. 280℃ Injector temp. 280℃ Split ratio 1:50 Injection volume 1

(4) Fatty acid content of conductor

For the analysis of fatty acid content of the conductor, 0.7 ml of 10 N KOH in water and 6.3 ml of methanol were mixed per 1 g of the sample and placed in a constant temperature water bath at 55℃ for separation of fatty acid methyl esters, and then heated. The sample was heated for 1 hour and 30 minutes, shaken vigorously every 30 minutes, cooled in prepared cold water for 1 to 2 minutes, added 0.58 ml of 24 N H ₂ SO ₄ in water, and then heated in a constant temperature water bath at 55℃ for 1 hour and 30 minutes, while again shaking vigorously once every 30 minutes. After heating, the sample was cooled in the prepared cold water, 3 mL of hexane was added, and centrifuged at 3,000 rpm for 5 minutes (HANIL, Combi-514R, KOR). After putting it into a vial using a Pasteur pipette, fatty acid analysis was performed using a gas chromatograph-flame ionization detector (Agilent, 7890 series, USA) under the following conditions. The injector was set to split mode with a split ratio of 25:1 at 250°C, and the detector was a flame ionization detector (FID) at 250°C. High-purity Air, high-purity H2, and high-purity He were used as carrier gases, and the flow rate was 40 mL/min for H2 and 400 mL/min for air. The column for analysis was DB-WAX (30 m X 0.25 um X 0.25 mm).

(5) Analysis of blood immune antibodies (IgG, IgM and IgA)

For blood analysis, 5 mL of blood was collected from the jugular vein of one animal per treatment group after the end of the experiment, placed in an SGS tube, and left at room temperature for 2 hours to coagulate the blood. The coagulated blood was centrifuged at 4°C and 3,000 rpm and measured using the enzyme-linked immunosorbent assay (ELISA). That is, the primary antibody (capture Ab) was diluted in PBS, 100 ㎕ was added to each plate, left overnight at 4°C, washed with washing solution (0.05% Tween 20/PBS), and blocked with block solution (1% BSA, 5% sucrose, 0.05% NaN3) for 2 hours. The culture supernatant was added, and after 3 hours, the plates were washed with washing solution and the secondary antibody (detection Ab) was added. After another 2 hours, the solution was washed with washing solution, and Streptavidin-HRP was added. After another hour, the solution was washed with washing solution, and the substrate (2, 2'-azino-bis, 0.1 M citric acid, H ₂ O ₂ ) was added to develop the color, and the absorbance was measured at 405 nm using a microplate reader and converted using a standard curve.

(6) Measurement of abdominal fat and organ weight in broilers

According to the method of Deaton (1974), the live weight of each individual was first measured for 7 animals per treatment group, and the weight of each organ and abdominal fat was measured by cutting the jugular vein of the testis and bleeding. After hair removal, the viscera were removed, and the fat accumulated around the breast and ribs, intestines, total excretory duct, and abdominal muscles was removed to measure the weight of the abdominal fat pad. In addition, the sac, heart, liver, intestines, proximal stomach, pancreas, cecum, kidney, small intestine, large intestine, spleen, abdominal fat, and spleen were each removed and measured for their weight.

(7) Microbiological examination of the cecum

Samples from the cecum were collected from each treatment group and the number of microorganisms was tested. 10 g of the sample was collected from each treatment group, diluted stepwise to 10 ¹¹ using sterilized saline solution, inoculated onto selective media, and cultured at 35℃ for 24–48 hours, and then the number of colonies was investigated. Figure 3 shows the process of analyzing microorganisms in the cecum of broilers used in the feeding test, and the selective media and culture conditions for five types (E. coli, Salmonella, lactic acid bacteria, yeast, and Bacillus) are as shown in Table 5.

Item Microbial Incubation time (hr) Incubation temperature(℃) MCS agar E. coli 28 37℃ SS ager Salmonella 28 37℃ MRS ager Lactobacillus spp. 48 37℃ PP ager Yeast 48 37℃ MYP ager Bacillus 48 37℃

Ma. Natural products Fermented probiotics Investigation of the effect of feed additives on broiler productivity 2

This study was conducted to compare and evaluate the effects of feeding natural fermented probiotics (pomegranate and ginkgo leaf complex probiotics and 0.4% pomegranate and yuzu complex probiotics) on the productivity of broilers.

1) Test animals and test design

In this study, 120 Ross Broiler chicks with uniform body weight were selected as test animals. The experimental design was a control group, a pomegranate-ginkgo leaf (containing 30% pomegranate and 5% ginkgo leaf powder) fermented probiotic 0.4% treatment group, a pomegranate-ginkgo leaf (containing 29% pomegranate and 10% ginkgo leaf powder) fermented probiotic 0.4% treatment group, a pomegranate-yuja (containing 30% pomegranate and 5% yuja) fermented probiotic 0.4% treatment group, and a pomegranate-yuja (containing 29% pomegranate and 10% yuja) fermented probiotic 0.4% treatment group. A total of 5 treatment groups, 4 replicates, 6 animals per replication, were randomly assigned and conducted for 5 weeks.

2) Test feed and test site

The test feed was mixed according to the NRC (1994) specification standard and used as the basal feed (control), and the basal feed mixing ratio was the same as the broiler productivity test 1 mentioned above. The test feeds for each test treatment group were mixed as the control feed, basal feed with 0.4% pomegranate-ginkgo leaf (containing 30% pomegranate and 5% ginkgo leaf powder) fermented probiotic, basal feed with 0.4% pomegranate-ginkgo leaf (containing 29% pomegranate and 10% ginkgo leaf powder) fermented probiotic, basal feed with 0.4% pomegranate-yuja (containing 30% pomegranate and 5% yuja) fermented probiotic, and basal feed with 0.4% pomegranate-yuja (containing 29% pomegranate and 10% yuja) fermented probiotic. The test was conducted for 5 weeks from March 25, 2015 to April 29, 2015, and the feeding test was conducted in a three-tiered iron cage at the livestock farm attached to the Department of Animal Resource Science, Soonchunhyang University. The analyses related to this experiment were conducted at the Animal Nutrition and Feed Laboratory.

3) Main research items

Weight gain, feed intake, general carcass composition, cholesterol content, fatty acid content, blood immune antibody analysis, and cecal microbiological examination were performed in the same manner as in the broiler productivity test 1 mentioned above.

B. Evaluation of the disease resistance of broilers by feeding natural fermented probiotics (Challenge Test)

This study was conducted to evaluate the anti-pathogenic activity of broilers fed with natural fermented probiotics (0.4% addition of pomegranate and ginkgo leaf complex probiotics and pomegranate and yuzu complex probiotics) against pathogenic bacteria.

1) Test animals and test design

The test animals for this experiment were 120 Ross Broiler chicks with uniform body weight. The test design consisted of a control group, a pomegranate-ginkgo leaf (containing 30% pomegranate and 5% ginkgo leaf powder) fermented probiotics 0.4% treatment group, a pomegranate-ginkgo leaf (containing 29% pomegranate and 10% ginkgo leaf powder) fermented probiotics 0.4% treatment group, a pomegranate-yuzu (containing 30% pomegranate and 5% yuzu) fermented probiotics 0.4% treatment group, and a pomegranate-yuzu (containing 29% pomegranate and 10% yuzu) fermented probiotics 0.4% treatment group. A total of 5 treatment groups, 4 replicates, 6 animals per replication, were randomly assigned and conducted for 1 week.

2) Challenge Test method and test location

In order to evaluate the anti-pathogenicity in this study, the pathogenic bacteria Salmonella enteritidis and E. coli were cultured separately and then mixed in a 1:1 ratio for use. In this test, 1 ml each of the culture solution containing 2.3 X 10 ⁷ cfu/ml of Salmonella enteritidis and 3.7 X 10 ⁷ of E. coli was orally inoculated using a micropipet tip. The test feed and water were provided ad libitum, and the feeding test was conducted in a three-tier iron cage at the animal resource science department of Soonchunhyang University.

3) Main research items

(1) Mortality rate survey

The number of dead individuals in each treatment group and repetition during the test period was determined, and the mortality rate was investigated by dividing the number of dead individuals in each treatment group by the total number in each treatment group and multiplying by 100.

(2) Analysis of microorganisms in the cecum

Samples from the cecum were collected from each treatment group and the number of microorganisms was tested. 10 g of the sample was collected from each treatment group and diluted stepwise to 10 ¹¹ using sterilized saline solution. After inoculating it onto selective media, it was cultured at 35°C for 24–48 hours and the number of colonies was investigated.

(3) Blood IgM antibody analysis

For blood analysis, 5 mL of blood was collected from the jugular vein of one animal per treatment group after the end of the experiment, placed in an SGS tube, and left at room temperature for 2 hours to coagulate the blood. The coagulated blood was centrifuged at 4°C and 3,000 rpm and measured using the enzyme-linked immunosorbent assay (ELISA). That is, the primary antibody (capture Ab) was diluted in PBS, 100 ㎕ was added to each plate, left overnight at 4°C, washed with washing solution (0.05% Tween 20/PBS), and blocked with block solution (1% BSA, 5% sucrose, 0.05% NaN3) for 2 hours. The culture supernatant was added, and after 3 hours, the plates were washed with washing solution and the secondary antibody (detection Ab) was added. After another 2 hours, the solution was washed with washing solution, and Streptavidin-HRP was added. After another hour, the solution was washed with washing solution, and the substrate (2, 2'-azino-bis, 0.1 M citric acid, H ₂ O ₂ ) was added to develop the color, and the absorbance was measured at 405 nm using a microplate reader and converted using a standard curve.

(4) Measurement of organ weight of broilers

According to the method of Deaton (1974), the live weight of each individual was first measured for 7 animals per treatment group, and the weight of each organ and abdominal fat was measured by cutting the jugular vein of the testis and bleeding. After hair removal, the viscera were removed, and the fat accumulated around the breast and ribs, intestines, total excretory duct, and abdominal muscles was removed to measure the weight of the abdominal fat pad. In addition, the sac, heart, liver, intestines, proximal stomach, pancreas, cecum, kidney, small intestine, large intestine, spleen, abdominal fat, and spleen were each removed and measured for their weight.

4) Statistical processing

The data obtained from the productivity and disease resistance evaluation of this test were analyzed for variance using the SAS Statical Package Program (SAS, Institute, 2003), and the significance of the treatment group mean was tested using Duncan's multiple test method.

Example 2

A. Analysis of changes in microbial and nutrient content according to the storage period of the product

To analyze the changes in microorganisms and nutrients due to long-term storage of natural product fermented probiotics, 12 samples (100 g each) of the initially manufactured prototypes were collected and stored at room temperature for 6 months (24 weeks) and used for analysis. Microbiological analysis was performed twice a month at 2-week intervals, and nutrient analysis was performed once a month at 4-week intervals. The medium specified for microbial analysis was MRS solid medium (Difco, Detroit, USA), and the culture condition was cultivating at 37℃ for 24 hours, and then the number of colonies was investigated. Nutrient content analysis was performed on crude protein, crude fat, and crude fiber according to the AOAC (2000) method.

B. Evaluation of the anti-pathogenicity of pigs (piggies) fed with natural fermented probiotics (Challenge Test)

This study was conducted to evaluate the anti-pathogenic effects of feeding natural fermented probiotics (pomegranate and ginkgo leaf complex probiotics and 0.4% pomegranate and yuzu complex probiotics) to piglets against pathogenic bacteria.

1) Test animals and test design

The test animals were 36 castrated piglets of uniform three-way crossbreed [(Landrace X yorkshire) X Duroc] with an average body weight of approximately 21.91 kg. The experimental design was a control group, a pomegranate-ginkgo leaf (containing 29% pomegranate and 10% ginkgo leaf powder) fermented probiotic 0.4% addition treatment group, and a pomegranate-yuzu (containing 29% pomegranate and 10% yuzu) fermented probiotic 0.4% addition treatment group, with three replicates, and four pigs per replicate, and the experiment was conducted for 3 weeks.

2) Test feed

The test feed was mixed according to the NRC (1994) specification standard and used as the basal feed (control), and the basal feed mixing ratio is as shown in Table 6. The basal feed contained 18% crude protein and 3,265 kcal/kg metabolizable energy. The test feeds for each test treatment group were mixed as the control feed, the basal feed with 0.4% added pomegranate-ginkgo leaf (containing 29% pomegranate and 10% ginkgo leaf powder) fermented probiotic, the basal feed with 0.4% added pomegranate-yuja (containing 29% pomegranate and 10% yuja) fermented probiotic.

3) Challenge Test method and test location

To evaluate the anti-pathogenicity in this study, pathogenic bacteria , Salmonella enteritidis and E. coli , were cultured separately and then mixed in a 1:1 ratio. 1 ml each of the culture solution containing 1.4 X 10 ⁸ cfu/ml of Salmonella enteritidis and 5.2 X 10 ⁷ of E. coli was orally inoculated using a Micropipet tip. In the second test, 1 ml each of the culture solution containing 5.0 X 10 ⁵ cfu/ml of Salmonella enteritidis and 7.0 X 10 ⁶ of E. coli was orally inoculated using a Micropipet tip. The feeding test was conducted in a piglet incubator in the animal farm affiliated with the Department of Animal Resource Science, Soonchunhyang University.

4) Main research items

(1) Weight gain, feed intake and feed conversion ratio

Body weight was measured repeatedly at a fixed time every week from the beginning of the test to the end of the test, and the weight gain was calculated by subtracting the starting weight from the ending weight. Feed intake was calculated by measuring the remaining feed amount repeatedly just before the weekly weight measurement. Feed conversion ratio was calculated by dividing the feed intake by the weight gain.

Ingredients Starter Yellow Corn 45.15 Wheat 23.00 Wheat bran 4.00 Soybean meal 18.00 Limestone 0.98 Calcium phosphate 1.10 Salt 0.25 Vit-min. premix ¹⁾ 0.55 Animal fat 2.50 Molasses 4.30 L-Lysine 0.17 Chemical composition ²⁾ ME (kcal/kg) 3,265.00 Crude Protein (%) 18.00 Ca (%) 0.70 Available. P (%) 0.55 Lysine (%) 0.95 Methionine (%) 0.30

¹⁾ Vit-min.mix provided following nutrients per kg of premix: vitmin A, 6,000IU; vitmin D ₃ , 800IU; vitmin E, 20IU; vitmin K ₃ , 2mg; thiamin, 2mg; riboflavin, 4mg; vitmin B ₆ , 2mg; vitmin B ₁₂ , 1mg; pantothenicacid, 11 mg; niacin, 10mg; biotin, 0.02mg; Cu (copper sulfate), 21 mg; Fe (ferrous sulfate), 100 mg; Zn (zinc sulfate), 60 mg; Mn (mznganese sulfate), 90 mg; I (calcium iodate), 1.0 mg; Co (cobalt nitrate), 0.3mg; Se (sodium selenite), 0.3 mg.

²⁾ Calculated value.

(2) Blood antibody analysis

For blood analysis, 5 mL of blood was collected from the jugular vein of one head per treatment group after the end of the experiment, placed in an SGS tube, and left at room temperature for 2 hours to coagulate the blood. The coagulated blood was centrifuged at 4°C and 3,000 rpm and measured using the enzyme-linked immunosorbent assay (ELISA). That is, the primary antibody (capture Ab) was diluted in PBS, 100 ㎕ was added to each plate, left overnight at 4°C, washed with washing solution (0.05% Tween 20/PBS), and blocked with block solution (1% BSA, 5% sucrose, 0.05% NaN3) for 2 hours. The culture supernatant was added, and after 3 hours, the plates were washed with washing solution and the secondary antibody (detection Ab) was added. After another 2 hours, the solution was washed with washing solution, and Streptavidin-HRP was added. After another hour, the solution was washed with washing solution, and the substrate (2, 2'-azino-bis, 0.1 M citric acid, H ₂ O ₂ ) was added to develop the color, and the absorbance was measured at 405 nm using a microplate reader and converted using a standard curve.

(3) Analysis of blood cytokines (TNF-α)

Analysis of cytokines in blood was performed using the ELISA test method, and was analyzed using Quantikine Porcine TNF-a/TNFSF2 Immunoassay (R&D, USA). The analyzer for the analysis was VERSA Max (Molecular device, USA).

(4) Analysis of microbial samples in feces

The excreted feces were collected from each treatment group for the same period of time and the number of microorganisms was tested. 10 g of the sample from each treatment group was mixed in 10 ml of saline solution for several minutes, 1 ml was taken, and the mixture was diluted stepwise to 10 ^{10 .} 1 ml was taken at a time and inoculated onto selective media. After culturing at 30℃ for 24-48 hours, the number of colonies was investigated.

5) Statistical processing

The data obtained from the productivity and disease resistance evaluation of this test were analyzed for variance using the SAS Statical Package Program (SAS, Institute, 2003), and the significance of the treatment group mean was tested using Duncan's multiple test method.

B. Search for useful strains

In order to develop a natural product fermentation probiotic, Enbiotech Co., Ltd. selected six strains as test strains, including two lactic acid bacteria, two Bacillus bacteria, and two yeast strains, out of 45 strains received from the Korean Collection for Type Culture (KCTC). The selected lactic acid bacteria are Lactobacillus plantarum and Pediococcus acidilactici , which produce starch, protein-decomposing enzymes and antibiotics (bacteriocins) and fermentation metabolites such as lactic acid, acetic acid, hydrogen peroxide, ethanol, and organic acids, and have the characteristics of being involved in the early and late fermentation and ripening of grains, fruits, and silage fermentation. The selected spore fungi, Bacillus licheniformis and Bacillus subtilis , have antibacterial effects on pathogenic bacteria and fungi and growth inhibition activity of these bacteria, and their antibacterial and antifungal activities are characterized by being stable at high temperatures. They are well known as major beneficial bacteria that destroy harmful bacteria and protect antioxidants such as nutrients. The selected yeast fungi, Saccharomyces cerevisiae and Saccharomyces boulardii , are beneficial strains that promote the decomposition of difficult-to-decompose substances such as cellulose, glucan, and pectin by secreting starch, protein, and fiber-decomposing enzymes, and produce natural flavoring substances such as alcohol and glutamic acid during fermentation, and produce yeast proteins, vitamin B complex, and supply unknown growth factors (UGF). In addition, the selected strains have an antagonistic effect on intestinal pathogenic bacteria produced through the secretion of lactic acid and bacteriocin, and have the effect of increasing immunity by preventing the establishment of pathogens such as Escherichia coli and Salmonella in the small intestine. These strains were analyzed to have excellent bacterial growth ability with more than ¹⁰⁹ cfu/ml among 45 strains when cultured in a single medium composition. Therefore, in this study, the above six beneficial strains were judged to be suitable as test strains for use in the production of natural product fermentation probiotics.

D. Evaluation of strain combination and fermentation agent mixing ratio for prototype manufacturing

For the development of a natural product fermentation probiotic, defatted rice bran (DRB) and distiller's dried grains solubles (DDGS), which are widely used as raw materials for animal feed, were used as fermentation excipients. After mixing at a set mixing ratio for each treatment group, the six selected strains were organized into two combinations, and the fungal cells and culture solution were evenly sprayed and inoculated onto the mixed surface of the natural product and excipients at a level of ¹⁰⁶ cfu/㎖ for each strain. The fermentation process was conducted for 5 days under anaerobic and aerobic conditions at 25–30°C. The results of the optimal mixing ratio of the fermentation excipient and the selection experiment for the appropriate fermentation strain are shown in Table 7.

Treatment Microbial Concentrations T 1-1 DRB ^{1 )} 30%
+
DDGS ^{2 )} 70% Lactobacillus plantarum 5.6 × 10 ⁸ cfu/g Bacillus subtilis 2.0 × 10 ⁸ cfu/g Saccharomyces cerevisiae 6.4 × 10 ⁷ cfu/g T 1-2 DRB 30%
+
DDGS 70% Pediococcus acidilactici 3.0 × 10 ⁸ cfu/g Bacillus licheniformis 1.4 × 10 ⁷ cfu/g Saccharomyces boulardii 2.8 × 10 ⁸ cfu/g T 2-1 DRB 50%
+
DDGS 50% Lactobacillus plantarum 2.4 × 10 ⁹ cfu/g Bacillus subtilis 1.8 × 10 ⁸ cfu/g Saccharomyces cerevisiae 4.0 × 10 ⁸ cfu/g T 2-2 DRB 50%
+
DDGS 50% Pediococcus acidilactici 2.2 × 10 ⁸ cfu/g Bacillus licheniformis 2.0 × 10 ⁷ cfu/g Saccharomyces boulardii 4.8 × 10 ⁸ cfu/g T 3-1 DRB 70%
+
DDGS 30% Lactobacillus plantarum 2.0 × 10 ⁸ cfu/g Bacillus subtilis 2.3 × 10 ⁷ cfu/g Saccharomyces cerevisiae 3.6 × 10 ⁷ cfu/g T 3-2 DRB 70%
+
DDGS 30% Pediococcus acidilactici 4.4 × 10 ⁷ cfu/g Bacillus licheniformis 3.2 × 10 ⁷ cfu/g Saccharomyces boulardii 4.1 × 10 ⁷ cfu/g T 4-1 DRB 100% Lactobacillus plantarum 2.4 × 10 ⁶ cfu/g Bacillus subtilis 3.2 × 10 ⁷ cfu/g Saccharomyces cerevisiae 1.8 × 10 ⁶ cfu/g T 4-2 DRB 100% Pediococcus acidilactici 2.2 × 10 ⁶ cfu/g Bacillus licheniformis 2.7 × 10 ⁷ cfu/g Saccharomyces boulardii 2.0 × 10 ⁷ cfu/g T 5-1 DDGS 100% Lactobacillus plantarum 3.5 × 10 ⁷ cfu/g Bacillus subtilis 3.2 × 10 ⁷ cfu/g Saccharomyces cerevisiae 2.6 × 10 ⁷ cfu/g T 5-2 DDGS 100% Pediococcus acidilactici 1.2 × 10 ⁷ cfu/g Bacillus licheniformis 3.0 × 10 ⁷ cfu/g Saccharomyces boulardii 2.8 × 10 ⁷ cfu/g

¹⁾ DRB (Defatted Rice Bran): Defatted rice bran, ^{2 )} DDGS: Corn distillers' grains

As a result of the experiment to select the optimal mixing ratio of fermentation agents and the appropriate fermentation strain combination, the density of the inoculated strain was the highest in the T 2-1 treatment group at 10 ⁸ to 10 ⁹ cfu/㎖ compared to the other treatment groups. T 2-1 was composed of a mixing ratio of 50% defatted rice bran and 50% corn distillers' dregs and a strain combination of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae. In this study, it was evaluated as an appropriate mixing ratio for manufacturing a prototype, and the combination of strains to be utilized was evaluated as the most efficient combination consisting of Lactobacillus plantarum (lactic acid bacteria), Bacillus subtilis (spore bacteria), and Saccharomyces cerevisiae (yeast bacteria).

A. Manufacturing of prototypes of natural substance fermented probiotics

1) Manufacturing of first prototypes (2 types)

The prototype manufacturing of a natural substance fermented probiotic was manufactured by considering the optimal mixing conditions of the utilized strain combination and fermentation raw materials based on the results of the preliminary test. The mixing ratio of 50% defatted rice bran and 50% corn distillers' dregs, which were evaluated as the optimal conditions in the mixing conditions of the fermentation raw materials, and the strain combination consisting of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae were adjusted to contain 30% of natural pomegranate peel, 5% of ginkgo leaf powder, and 5% of green tea powder, thereby manufacturing two types of prototypes. The mixing ratio and strain combination of Prototype 1 were composed of 30% pomegranate extract, 5% ginkgo leaf powder, 32% corn ethanol extract, 32% defatted rice bran, and 1% glucose as a fermentation regulator. The useful strains were a combination of three strains, Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae, as the fermentation inoculum strains. The mixing ratio and strain combination of Prototype 2 were composed of 30% pomegranate extract, 5% green tea powder, 32% corn ethanol extract, 32% defatted rice bran, and 1% glucose as a fermentation regulator. The useful strains were the same as Prototype 1, Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae as the fermentation inoculum strains. The production of the prototype is done by mixing natural substances and excipients in a set mixing ratio using a solid fermenter, and then first adding lactic acid bacteria (Lactobacillus plantarum) at 10 ⁶ After inoculating the strain and culture together at the level of cfu/㎖ and maintaining the moisture content of the entire mixture at about 40%, the mixture was anaerobically fermented at 35℃ for 2 days. Then, Bacillus subtilis and Saccharomyces cerevisiae were each inoculated at the level of ¹⁰⁶ cfu/㎖ for the second time, and aerobic and anaerobic fermentation was performed at 35℃ for 2 days under the operating conditions of stirring (3 hours) and non-stirring (5 hours). After that, a pomegranate-ginkgo leaf fermented probiotic prototype was manufactured through a hot air drying process at 20℃ for 1 day. The pomegranate-green tea fermented probiotic prototype was manufactured through the same manufacturing process as the pomegranate-ginkgo leaf fermented probiotic prototype. The results of component analysis of the manufactured prototype are shown in Table 8. Moisture content was analyzed to be higher in the pomegranate-ginkgo leaf fermented probiotic product at 23.42% and in the pomegranate-green tea fermented probiotic product at 17.67%. The crude protein contents of the pomegranate-ginkgo leaf fermented probiotic product and the pomegranate-green tea fermented probiotic product were analyzed to be 10.98% and 11.86%, respectively. Crude fat was analyzed to be 2.41% in the pomegranate-ginkgo leaf fermented probiotic product and 2.15% in the pomegranate-green tea fermented probiotic product, and crude fiber was analyzed to be 9.83% and 11.66% in the pomegranate-ginkgo leaf fermented probiotic product and the pomegranate-green tea fermented probiotic product, respectively. Analysis of the microbial content of each sample product showed that the number of lactic acid bacteria (Lactobacillus plantarum) in the pomegranate-green tea fermented probiotic was slightly low at ^2.8X107 cfu/g, but both bacilli and yeast were analyzed at the level of ¹⁰⁸ cfu per g of sample product.

Treatment Natural materials
Probiotics 1 ¹ Natural materials
Probiotics 2 ² Chemical composition (%) Moisture 23.42 17.67 Crude protein 10.98 11.86 Crude fat 2.41 2.15 Crude fiber 9.83 11.66 Crude Ash 6.66 7.03 NFE 53.30 50.36 Kinds of microbes of Probiotics Lactobacillus plantarum ^1.5X108 cfu/g ^2.8X107 cfu/g Bacillus subtilis ^3.2X108 cfu/g ^2.0X108 cfu/g Saccharomyces cerevisiae ^1.7X108 cfu/g ^1.7X108 cfu/g

¹ Natural materials Probiotics 1: Pomegranate-Ginkgo Leaf Fermented Probiotics I (Pomegranate 30%, Ginkgo Leaf 5%)

² Natural materials Probiotics 2: Pomegranate-Green Tea Fermented Probiotics (Pomegranate 30%, Green Tea 5%)

2) Manufacturing of 2nd prototypes (3 types)

The second prototype was manufactured using a blending ratio of 50% defatted rice bran and 50% corn ethanol dried grains, which were previously evaluated as the optimal blending conditions for the fermentation raw materials, and a strain combination consisting of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae. The blending ratio was adjusted to contain 20-30% of natural products such as pomegranate peel, 10% of ginkgo leaf powder, and 5-10% of citron peel, thereby manufacturing three types of prototypes. The mixing ratio of prototype 3 was 29% pomegranate extract, 10% ginkgo leaf powder, 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator. The mixing ratio of prototype 4 was 30% pomegranate extract, 5% citron extract, 32% corn ethanol residue, 32% defatted rice bran, and 1% glucose as a fermentation regulator. The mixing ratio of prototype 5 was 29% pomegranate extract, 10% citron extract, 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator. The inoculation strain combination was the same combination of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae used in the manufacture of the first prototype. The manufacturing process of the prototype was the same as that of the manufacture of the first prototype. The results of component analysis of the manufactured prototype are shown in Table 9.

Treatment Natural materials
Probiotics 3 ¹ Natural materials
Probiotics 4 ² Natural materials
Probiotics 5 ³ Chemical composition (%) Moisture 26.12 30.62 29.80 Crude protein 15.13 14.84 15.72 Crude fat 2.07 1.72 1.51 Crude fiber 9.33 8.14 7.38 Crude Ash 6.49 5.60 5.52 NFE 40.86 39.08 40.07 Kinds of microbes of Probiotics Lactobacillus plantarum ^2.0X108 cfu/g ^1.4X109 cfu/g ^2.2X108 cfu/g Bacillus subtilis ^1.0X108 cfu/g ^1.2X108 cfu/g ^1.5X107 cfu/g Saccharomyces cerevisiae ^1.2X107 cfu/g ^1.5X108 cfu/g ^4.2X107 cfu/g

¹ Natural materials Probiotics 3: Pomegranate-Ginkgo Leaf Fermented Probiotics II (Pomegranate 29%, Ginkgo Leaf 10%)

² Natural materials Probiotics 4: Pomegranate-Yuja fermented probiotics I (pomegranate 30%, yuja peel 5%)

³ Natural materials Probiotics 5: Pomegranate-Yuja fermented probiotics Ⅱ (Pomegranate 29%, Yuja peel 10%)

Ma. Elucidation of the effect of adding natural fermented probiotics to feed on the productivity of broilers 1

In this study, the effects of feeding natural fermented probiotics (pomegranate and ginkgo leaf fermented probiotics and pomegranate and green tea fermented probiotics at 0.2 and 0.4%) and natural products (ginkgo leaf fermented probiotics at 0.2 and 0.4%) on the performance of broilers were evaluated.

1) Weight gain, feed intake and feed conversion ratio

The results on weight gain, feed intake, and feed conversion ratio when feeding pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder to Ross Broilers are shown in Table 10. As a result of the feeding test from the start of the test (week 0) to the 3rd week, the weight gain of each treatment group was the highest in the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group at 891 g, showing a significant difference (P<0.05). In the case of feed conversion ratio, the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group was the lowest at 1.48, showing a significant difference (P<0.05). As a result of the feeding test from week 3 to week 5, the weight gain was the highest in the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group at 1,129 g, showing a significant difference (P<0.05). When comparing the weight gain of each treatment group during the entire experimental period (5 weeks), the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group showed the highest at 2,021 g, showing a significant difference (P<0.05). In particular, it was found that the 0.2% and 0.4% pomegranate-ginkgo leaf fermented probiotic treatment groups showed significant improvements in weight gain and feed conversion ratio compared to the control group. Kim et al. (1988) reported that weight gain was improved by adding lactic acid bacteria ( Lactobacillus sporegenes ) to broiler feed, and Yeo and Kim (1997) reported that feeding lactic acid bacteria ( Lactobacillus casei ) to broilers improved daily weight gain for the first 3 weeks of the feeding test. Chiang and Hsieh (1995) reported that the addition of a complex probiotic containing Lactobacillus acidophilus and Streptococcus faecium to broiler diets increased the growth of broilers, and the pomegranate-ginkgo leaf fermented probiotic treatments of 0.2% and 0.4% in this study yielded similar results. However, the pomegranate-green tea fermented probiotic treatments of 0.2% and 0.4% showed lower results than the control group, suggesting that there was no effect on productivity improvement such as weight gain. Therefore, it is thought that the addition of 0.2% and 0.4% pomegranate-ginkgo leaf fermented probiotics to broiler diets can be utilized as a feed additive that can improve weight gain and feed efficiency.

Item Control PGLP ¹ PGTP ² GLP ³ SEM ⁴ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% 0-3 weeks Initial weight(g) 47 47 47 47 47 47. 47 0.23 Final weight(g) 868 ^b 897 ^ab 939 ^a 859 ^b 861 ^b 889 ^b 861 ^b 13.18 Weight gain(g) 821 ^b 850 ^ab 891 ^a 812 ^b 814 ^b 842 ^b 814 ^b 13.35 Feed intake(g) 1,298 1,327 1,317 1,270 1,290 1,305 1,297 15.52 FCR(Feed/Gain) 1.58 ^a 1.56 ^ab 1.48 ^b 1.56 ^ab 1.58 ^a 1.55 ^ab 1.59a 0.03 3-5 weeks Initial weight(g) 868b 897 ^ab 939 ^a 859 ^b 861 ^b 889 ^b 861 ^b 13.18 Final weight(g) 1,879 ^b 1,951 ^ab 2,068 ^a 1,909 ^b 1,922 ^b 1,889 ^b 1,858 ^b 41.14 Weight gain(g) 1,012 1,054 1,129 1,050 1,062 1,029 1,012 44.92 Feed intake(g) 2,031 ^a 1,785 ^b 2,006 ^a 1,876 ^ab 1,907 ^ab 1,794 ^b 1,827 ^b 46.03 FCR(Feed/Gain) 2.01 ^a 1.70 ^b 1.78 ^b 1.79 ^b 1.79 ^b 1.74 ^b 1.81 ^b 0.05 0-5 weeks Initial weight(g) 47 47 47 47 47 47 47 0.23 Final weight(g) 1,879 ^b 1,951 ^ab 2,068 ^a 1,909 ^b 1,922 ^b 1,889 ^b 1,858 ^b 41.14 Weight gain(g) 1,832 ^b 1,904 ^ab 2,021 ^a 1,863 ^b 1,875 ^b 1,842 ^b 1,811 ^b 41.08 Feed intake(g) 3,329 ^a 3,112 ^b 3,323 ^a 3,146 ^b 3,197 ^ab 3,099 ^b 3,124 ^b 50.27 FCR(Feed/Gain) 1.82 ^a 1.64 ^b 1.65 ^b 1.69 ^b 1.70 ^b 1.68 ^b 1.73 ^ab 0.03

^a,b Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics

² Fermentation pomegranate and green tea probiotics, ³ Ginkgo lef powder

⁴ SEM = standard error of the means

2) Analysis of general components (body composition) of conductors

The results of body composition analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder are shown in Table 11. In the case of breast meat of the carcass, the crude protein content was the highest in the pomegranate-green tea fermented probiotic 0.4% treatment group at 25.14%, and the crude fat content was the highest in the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group at 1.12%, but there was no significant difference among the treatment groups (P>0.05). The ash content was the highest in the pomegranate-green tea fermented probiotic 0.4% treatment group at 1.52%, and the lowest in the ginkgo leaf powder 0.2% treatment group at 1.26%, and there was a significant difference among the treatment groups (P<0.05). The crude protein content of the leg meat of the conductor was the highest in the 0.2% ginkgo leaf powder treatment group at 23.05%, and the lowest in the 0.4% pomegranate-green tea fermented probiotic treatment group at 21.34%, showing a significant difference among the treatment groups (P<0.05). The ash content was the highest in the 0.2% ginkgo leaf powder treatment group at 1.18%, and the lowest in the control group at 1.04%, showing a significant difference among the treatment groups (P<0.05).

Item Control PGLP ¹ PGTP ² GLP ³ SEM ⁴ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Breast meat Crude protein 24.73 23.72 23.42 24.24 25.14 24.84 24.95 0.46 Ether extract 0.96 0.99 1.12 1.06 0.72 1.03 0.94 0.18 Moisture 75.09 75.39 75.08 75.05 74.64 74.90 74.69 0.26 Crude ash 1.34 ^b 1.30 ^b 1.31 ^b 1.32 ^b 1.52 ^a 1.26 ^b 1.27 ^b 0.03 Thigh meat Crude protein 22.29 ^ab 22.18 ^ab 22.04 ^ab 21.98 ^ab 21.34 ^b 23.05 ^a 22.67 ^a 0.27 Ether extract 4.00 3.34 3.59 4.02 4.12 2.71 3.53 0.33 Moisture 75.17 ^abc 75.59 ^ab 74.61 ^abc 75.20 ^abc 73.91 ^c 75.79 ^a 74.17 ^bc 0.41 Crude ash 1.04 ^b 1.11 ^ab 1.13 ^a 1.15 ^a 1.14 ^a 1.18 ^a 1.11 ^ab 0.02

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics

² Fermentation pomegranate and green tea probiotics

³ Ginkgo leaf powder

⁴ SEM = standard error of the means

3) Analysis of cholesterol content in conductors

The results of cholesterol content analysis of chicken meat according to feeding pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder are shown in Table 12. When comparing the cholesterol content of the breast meat, the pomegranate-ginkgo leaf fermented probiotic 0.2% treatment group had the lowest at 92.47 mg/100g, and the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group had the highest at 149.98 mg/100g, showing a significant difference among the treatment groups (P<0.05). The cholesterol content of the leg meat was the lowest at 100.98 mg/100g in the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group and the highest at 182.73 mg/100g in the control group, showing a significant difference among the treatment groups (P<0.05). In laying hens, probiotics containing lactic acid bacteria increased egg weight and egg production (Nahashon et al., 1993) and decreased cholesterol in egg yolk (Haddadin et al., 1996). There are research results showing that adding Lactobacillus acidophilus to the diet increased egg production, feed efficiency, and decreased cholesterol in egg yolk (Abdulrahim et al., 1996).

Item Control PGLP ¹ PGTP ² GLP ³ SEM ⁴ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Breast 115.25 ^d 92.47 ^e 149.98 ^a 132.62 ^c 143.29 ^ab 130.31 ^c 137.38 ^bc 2.66 Thigh 182.73 ^a 165.19 ^b 100.98 ^c 168.62 ^ab 154.56 ^b 182.62 ^a 158.77 ^b 4.48

^a,b,c,d,e Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics

² Fermentation pomegranate and green tea probiotics

³ Ginkgo leaf powder

⁴ SEM = standard error of the means

4) Analysis of fatty acid content of conductors

The results of cholesterol content analysis of chicken meat fed pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder are shown in Tables 13 and 14. As a result of analyzing the fatty acid content of breast meat, myristic acid (C14:0) was the highest in the control group at 0.80% (P<0.05) and the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group at 0.54%, showing a significant difference among treatment groups (P<0.05). Pentadecanoic acid (C15:0) was the highest in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group at 3.47% (P<0.05) and the lowest in the 0.2% ginkgo leaf powder treatment group at 2.17%, showing a significant difference among treatment groups (P<0.05). γ-Linoleic acid (C18:3n6) was the highest in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group at 0.46%, and the lowest in the 0.4% pomegranate-green tea fermented probiotic treatment group at 0.10%, showing a significant difference among the treatment groups (P<0.05). ∑SFA was the highest in the 0.4% ginkgo leaf fermented probiotic treatment group at 36.06%, showing a significant difference (P<0.05), and ∑MUFA was the highest in the control group at 64.27%, showing a significant difference (P<0.05).

Item Control PGLP ² PGTP ³ GLP ⁴ SEM ⁵ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Myristic acid (C14:0) 0.80 ^a 0.75 ^ab 0.73 ^ab 0.66 ^b 0.54 ^b 0.78 ^ab 0.67 ^b 0.04 Pentadecanoic acid (C15:0) 2.23 ^b 2.42 ^b 2.41 ^b 2.88 ^ab 3.47 ^a 2.17 ^b 2.75 ^ab 0.28 Palmitic acid (C16:0) 20.58 19.89 21.08 20.09 20.80 20.61 19.78 0.54 Palmitoleic acid (C16:1n7) 4.02 3.58 4.33 3.60 3.33 4.07 3.48 0.42 Margaric acid (C17:0) 0.72 0.77 0.76 0.85 0.91 0.72 0.87 0.08 Heptadecenoic acid (C17:1) 0.54 0.58 0.58 0.70 0.59 0.60 0.77 0.09 Stearic acid (C18:0) 8.23 8.91 8.58 9.13 9.84 8.69 9.36 0.50 Oleic acid (C18:1n9) 36.06 35.14 36.00 34.84 31.11 34.92 33.93 1.23 Linoleic acid (C18:2n6) 16.94 17.20 15.99 16.62 17.05 17.04 16.88 0.38 Arachidic acid (C20:0) 0.65 ^a 0.60 ^ab 0.60 ^ab 0.37 ^b 0.37 ^b 0.64 ^a 0.56 ^ab 0.06 γ-Linoleic acid (C18:3n6) 0.41 ^a 0.44 ^a 0.46 ^a 0.24 ^b 0.10 ^b 0.43 ^a 0.42 ^a 0.04 Eicosenoic acid (C20:1n9) 0.43 0.51 0.49 0.51 0.59 0.54 0.62 0.06 Arachidonic acid (C20:4n6) 4.80 5.04 4.40 5.53 6.53 4.94 5.64 0.69 Tetracosaenoic acid (C24:1n9) 1.06 1.10 0.94 1.23 1.40 1.01 1.24 0.14 Total 97.47 96.92 97.35 97.27 96.91 97.13 96.99 0.24 ∑SFA ¹ 33.20 ^b 33.34 ^b 34.15 ^b 34.00 ^b 36.06 ^a 33.59 ^b 33.99 ^b 0.59 ∑MUFA ¹ 64.27 ^a 63.58 ^a 63.20 ^a 63.27 ^a 60.85 ^b 63.54 ^a 62.99 ^a 0.65 (MUFA+PUFA)/SFA 1.94 ^a 1.91 ^a 1.85 ^ab 1.86 ^ab 1.69 ^b 1.89 ^a 1.86 ^ab 0.05 Mono 42.11 40.91 42.34 40.88 37.18 41.13 40.06 1.32 poly 22.15 ^a 22.68 ^a 20.85 ^a 22.39 ^a 23.68 22.41 22.94 0.91 ∑PUFA ¹ 1.27 1.23 1.24 1.20 1.04 1.22 1.18 0.05 PUFA/SFA 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.00

^a,b Mean with different superscripts within the same row are significantly different (P<0.05)

¹ ∑SFA = saturated fatty acid; ∑MUFA = mono-unsaturated fatty acid;∑PUFA = poly-unsaturated fatty acid

² Fermentation pomegranate and ginkgo leaf probiotics

³ Fermentation pomegranate and green tea probiotics, ⁴ Ginkgo lef powder

⁵ SEM = standard error of the means

As a result of analyzing the fatty acid content of the thigh meat, pentadecanoic acid (15:0) was the highest in the 0.2% ginkgo leaf powder treatment group at 1.07% (P<0.05), and the lowest in the 0.4% pomegranate-green tea fermented probiotic treatment group at 0.58%, showing a significant difference among the treatment groups (P<0.05). Oleic acid (C18:1n9) was the highest in the 0.2% pomegranate-green tea fermented probiotic treatment group at 41.52% (P<0.05), and the lowest in the 0.2% ginkgo leaf powder treatment group at 38.20%, showing a significant difference among the treatment groups (P<0.05). γ-Linoleic acid (C18:3n6) was the highest in the 0.2% pomegranate-ginkgo leaf fermented probiotic treatment group at 0.43% (P<0.05) and the lowest in the 0.2% pomegranate-green tea fermented probiotic treatment group at 0.30%, showing a significant difference among treatment groups (P<0.05). Arachidonic acid (C20:4n6) was the highest in the 0.2% ginkgo leaf powder treatment group at 3.14% (P<0.05) and the lowest in the 0.4% pomegranate-green tea fermented probiotic treatment group at 1.68%, showing a significant difference among treatment groups (P<0.05). Tetracosaenoic acid (C24:1n9) was highest at 0.55% in the 0.2% ginkgo leaf powder treatment group (P<0.05), and lowest at 0.34% in the 0.4% pomegranate-green tea fermented probiotic treatment group, showing a significant difference between treatment groups (P<0.05).

Item Control PGLP ² PGTP ³ GLP ⁴ SEM ⁵ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Myristic acid (C14:0) 0.99 0.97 0.95 0.94 0.99 0.92 0.95 0.02 Pentadecanoic acid (15:0) 0.74 ^b 0.79 ^ab 0.71 ^b 0.68 ^b 0.58 ^b 1.07 ^a 0.87 ^ab 0.09 Palmitic acid (C16:0) 20.83 20.15 21.50 20.93 21.68 20.27 20.32 0.46 Palmitoleic acid (C16:1n7) 5.31 4.98 4.65 5.38 6.00 5.28 5.26 0.48 Margaric acid (C17:0) 0.27 0.30 0.26 0.25 0.21 0.37 0.33 0.03 Heptadecenoic acid (C17:1) 0.16 0.21 0.15 0.19 0.18 0.18 0.19 0.02 Stearic acid (C18:0) 6.70 6.98 6.45 6.69 6.45 7.26 6.73 0.22 Oleic acid (C18:1n9) 40.19 ^a 40.52 ^a 41.17 ^a 41.52 ^a 40.90 ^a 38.20 ^b 40.45 ^a 0.57 Linoleic acid (C18:2n6) 17.71 13.49 12.19 16.71 16.77 18.35 17.71 1.52 Arachidic acid (C20:0) 0.82 0.80 0.78 0.77 0.80 0.79 0.80 0.03 γ-Linoleic acid (C18:3n6) 0.40 ^ab 0.43 ^a 0.30 ^b 0.30 ^b 0.40 ^ab 0.37 ^ab 0.40 ^ab 0.03 Eicosenoic acid (C20:1n9) 0.20 ^ab 0.21 ^a 0.18 ^b 0.18 ^b 0.18 ^b 0.22 ^b 0.20 ^ab 0.01 Arachidonic acid (C20:4n6) 2.18b 2.31 ^ab 1.79 ^b 1.99 ^b 1.68 ^b 3.14 ^a 2.44 ^ab 0.27 Tetracosaenoic acid (C24:1n9) 0.44b 0.43 ^ab 0.36 ^bc 0.39 ^bc 0.34 ^c 0.55 ^a 0.51 ^ab 0.04 Total 96.94 92.58 91.44 96.93 97.15 96.97 97.15 3.79 ∑SFA ¹ 30.34 29.99 30.66 30.26 30.70 30.68 30.00 0.45 ∑MUFA ¹ 66.60 62.59 60.78 66.67 66.45 66.30 67.11 1.67 (MUFA+PUFA)/SFA 2.20 2.09 1.98 2.20 2.17 2.16 2.24 0.08 Mono 46.31 ^ab 46.36 ^ab 46.51 ^ab 47.66 ^a 47.61 ^a 44.44 ^b 46.56 ^ab 0.69 poly 20.29 16.23 14.27 19.00 18.85 21.86 20.55 1.74 ∑PUFA ¹ 1.53 1.55 1.52 1.58 1.55 1.45 1.55 0.03 PUFA/SFA 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.00

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ ∑SFA = saturated fatty acid; ∑MUFA = mono-unsaturated fatty acid;∑PUFA = poly-unsaturated fatty acid

² Fermentation pomegranate and ginkgo leaf probiotics

³ Fermentation pomegranate and green tea probiotics, ⁴ Ginkgo lef powder

⁵ SEM = standard error of the means

5) Analysis of blood immune antibodies (IgG, IgM and IgA)

The results of blood immune component analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder are shown in Table 15. The blood immunoglobin IgA content analysis results by test treatment group showed that the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group had the highest value at 15.49 mg/ml (P<0.05), and the ginkgo leaf powder 0.2% treatment group had the lowest value at 0.91 mg/ml, showing a significant difference among treatment groups (P<0.05).

For IgG content, it was the highest in the 0.2% ginkgo leaf powder treatment group at 0.49 mg/ml, and the lowest in the 0.4% pomegranate-green tea fermented probiotic treatment group at 0.43 mg/ml, showing a significant difference between treatment groups (P<0.05).

The blood IgM content was highest in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group at 0.17 mg/ml (P<0.05), while it was lower in the control group and the 0.2 and 0.4% ginkgo leaf powder treatment groups at 0.11 mg/ml, indicating a significant difference among the treatment groups (P<0.05).

It has been reported that when herbal ingredients are fed to livestock, the blood component values related to immunity increase due to the effects of herbal ingredients on immune cell activation and antibacterial action (Shon et al., 2004; Lee and Ha, 1994; Oh et al., 1998). Chen et al. (2003) reported that feeding Chinese herbs to broilers increases serum albumin, etc., thereby enhancing immunity, and Wang et al. (1998) reported that eugenol increases immunity by increasing IgG synthesis in the body and IgA synthesis in saliva.

Item Control PGLP ¹ PGTP ² GLP ³ SEM ⁴ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Immunoglobulins (mg/ml) IgA 1.54 ^ab 1.44 ^b 1.65 ^a 1.36 ^b 1.15 ^c 0.91 ^d 1.36 ^b 0.05 IgG 0.47 ^abc 0.48 ^ab 0.46 ^c 0.46 ^bc 0.43 ^d 0.49 ^a 0.46 ^c 0.01 IgM 0.11 ^cd 0.13 ^bc 0.17 ^a 0.14 ^b 0.12 ^cd 0.11 ^d 0.11 ^d 0.01

^a,b,c,d Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics

² Fermentation pomegranate and green tea probiotics

³ Ginkgo leaf powder

⁴ SEM = standard error of the means

6) Measurement of intra-abdominal fat and organ weight

The results of organ weight analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder are shown in Table 16.

The weight of the heart was analyzed to be the highest in the control group at 0.83%, and the lowest in the 0.4% pomegranate-green tea fermented probiotic treatment group at 0.55%, showing a significant difference among the treatment groups (P<0.05). The weight of the liver was analyzed to be the highest in the control group at 2.47%, and the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group at 2.11% and 2.02%, showing a significant difference among the treatment groups (P<0.05). The weight of the cecum was analyzed to be the highest in the control group at 0.58%, and the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group at 0.4%, showing a significant difference among the treatment groups (P<0.05). The weight of abdominal fat was analyzed to be the highest in the control group at 1.31%, and the lowest in the pomegranate-ginkgo leaf fermented probiotic 0.2% and ginkgo leaf powder 0.2% treatment groups at 0.97%, respectively, showing a significant difference among treatment groups (P<0.05). The weight of leg meat was the highest in the pomegranate-ginkgo leaf fermented probiotic 0.4% treatment group at 13.71%, and the lowest in the ginkgo leaf powder 0.4% treatment group at 11.94%, showing a significant difference among treatment groups (P<0.05). In the case of breast meat, the highest was 20.83% in the pomegranate-green tea fermented probiotic 0.2% treatment group at 20.83%, and the lowest was 18.79% in the ginkgo leaf powder 0.2% treatment group at 18.79%, showing a significant difference among treatment groups (P<0.05).

7) Analysis of cecal microorganisms

The results of the cecal microbial environment analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic, pomegranate-green tea fermented probiotic, and ginkgo leaf powder are shown in Table 16.

As a result of analysis of the density of lactic acid bacteria in the cecum, the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group had the highest at 9.09 cfu/ml, while the control group had the lowest at 8.54 cfu/ml, showing a significant difference among treatment groups (P<0.05). The density of yeast was the highest at 8.39 cfu/ml in the 0.2% ginkgo leaf powder treatment group, while the 0.4% pomegranate-ginkgo leaf fermented probiotic treatment group had the lowest at 8.15 cfu/ml, showing a significant difference among treatment groups (P<0.05). The density of Salmonella was the lowest at 7.21 cfu/ml in the 0.2% ginkgo leaf powder treatment group, showing a significant difference among treatment groups (P<0.05). The density of Escherichia coli ( E. coil ) was the highest in the 0.4% Ginkgo leaf powder treatment group at 8.65 cfu/ml, and the lowest in the 0.2% pomegranate-Ginkgo leaf fermented probiotic treatment group at 7.09 cfu/ml, showing a significant difference among the treatment groups (P<0.05). Guo et al. (2004) reported that herbal extracts (harb polysaccharide extract) increased the number of beneficial bacteria, bifidobacteria and lactbacilli , and decreased the number of harmful bacteria, Bacteroides spp. and E. coil , in the cecum. Wenk (2003) reported that harbs and essential oil had antibacterial, anticoccidial, and anthelmintic effects in monogastric animals. Hong et al. (2001) reported that it improved the small intestinal microbial flora and increased the serum IgG level in broilers.

Item Control PGLP ¹ PGTP ² GLP ³ SEM ⁴ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Crop wt./Live wt. 0.39 ^ab 0.26 ^cd 0.25 ^cd 0.29 ^bcd 0.35 ^abc 0.46 ^a 0.21 ^d 0.03 Heart wt./Live wt. 0.83 ^a 0.63 ^bc 0.62 ^bc 0.62 ^bc 0.55 ^c 0.69 ^b 0.62 ^bc 0.03 Liver wt./Live wt. 2.47 ^a 2.28 ^ab 2.11 ^c 2.31 ^ab 2.02 ^c 2.12 ^c 1.94 ^c 0.07 Gizzard wt./Live wt. 1.75 ^a 1.43 ^c 1.50 ^bc 1.72 ^a 1.61 ^abc 1.65 ^ab 1.51 ^bc 0.06 Pancreas wt./Live wt. 0.23 ^abc 0.22 ^bcd 0.21 ^cd 0.19 ^d 0.25 ^ab 0.26 ^a 0.26 ^a 0.01 Cecum wt./Live wt. 0.58 ^a 0.55 ^a 0.40 ^b 0.46 ^ab 0.48 ^ab 0.54 ^a 0.47 ^ab 0.04 Kidney wt./Live wt. 0.77 ^a 0.64 ^c 0.66 ^bc 0.62 ^c 0.70 ^b 0.66 ^bc 0.71 ^b 0.02 S. intestine wt./Live wt. 3.33 ^a 2.88 ^bc 2.71 ^c 3.14 ^ab 2.95 ^bc 2.79 ^c 2.67 ^c 0.10 L. intestine wt./Live wt. 0.16 0.18 0.16 0.18 0.18 0.16 0.19 0.01 Abdominalfat wt./Live wt. 1.31 ^a 0.97 ^b 1.21 ^ab 1.25 ^ab 1.10 ^ab 0.97 ^b 1.15 ^ab 0.09 Proventriculus wt./Live wt. 0.54 0.54 0.45 0.45 0.48 0.57 0.44 0.05 Spleen wt./Live wt. 0.08 0.07 0.07 0.09 0.08 0.07 0.08 0.01 Bursa wt./Live wt. 0.19 0.19 0.20 0.18 0.21 0.19 0.20 0.02 Gallbladder wt./Live wt. 0.11 0.12 0.11 0.09 0.10 0.10 0.11 0.01 Thigh wt./Live wt. 12.78 ^bc 12.93 ^ab 13.71 ^a 12.97 ^ab 12.88 ^ab 12.41 ^bc 11.94 ^c 0.24 Breast wt./Live wt. 19.51 ^bc 19.90 ^bc 20.53 ^ab 20.83 ^a 18.88 ^c 18.79 ^c 20.10 ^ab 0.32

^a,b,c,d Mean with different superscripts within the same row are significantly different (P<0.05)

Item Control PGLP ¹ PGTP ² GLP ³ SEM ⁴ 0.2% 0.4% 0.2% 0.4% 0.2% 0.4% Cecum microbiota (log10 (cfu/g)) Lactic acid bacteria 8.54 ^c 8.57 ^c 8.59 ^c 8.80 ^b 9.09 ^a 8.85 ^b 8.61 ^c 0.03 Yeast 8.36 ^bc 8.30 ^bc 8.15 ^c 8.53 ^bc 8.97 ^bc 8.39a 8.24 ^bc 0.07 Salmonella 8.22 ^a 8.35 ^a 8.31 ^a 8.67 ^a 8.63 ^a 7.21 ^b 8.44 ^a 0.15 E. coil 7.84 ^b 7.09 ^c 7.29 ^c 7.51 ^bc 7.55 ^bc 7.55 ^bc 8.65 ^a 0.12

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics

² Fermentation pomegranate and green tea probiotics

³ Ginkgo lef powder, ⁴ SEM = Standard error of the means

B. Natural products Fermented probiotics Investigation of the effect of feed additives on broiler productivity 2

In this study, the effect of feeding natural fermented probiotics (0.4% addition of pomegranate-ginkgo leaf fermented probiotics and pomegranate-yuzu fermented probiotics) on the performance of broilers was evaluated. The test fermented probiotic samples used in this feeding test were pomegranate-ginkgo leaf fermented probiotics mixed with 29% pomegranate marmalade, 10% ginkgo leaf powder, 30% corn ethanol dried residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator; pomegranate-yuzu fermented probiotics I mixed with 30% pomegranate marmalade, 5% yuzu marmalade, 32% corn ethanol dried residue, 32% defatted rice bran, and 1% glucose as a fermentation regulator; and pomegranate-yuzu fermented probiotics II mixed with 29% pomegranate marmalade, 10% yuzu marmalade, 30% corn ethanol dried residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator.

1) Weight gain, feed intake and feed conversion ratio

The results of weight gain, feed intake, and feed conversion ratio when feeding pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic in Ross Broilers are shown in Table 18. As a result of the feeding test from the start of the test (week 0) to week 3, the weight gain in each treatment group was the highest in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group at 855 g, and the lowest in the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group at 759 g, showing a significant difference among the treatment groups (P<0.05). In the case of feed conversion ratio, the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group was the lowest at 1.35, but there was no significant difference among the treatment groups. As a result of the feeding test from 3 to 5 weeks, in terms of weight gain, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the highest at 1,242 g, and the pomegranate-citron fermented probiotic 1 (PCP-1) 0.4% treatment group was the lowest at 1,097 g, indicating a significant difference among the treatment groups (P<0.05). When comparing the weight gain of each treatment group during the entire test period (5 weeks), the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the highest at 2,058 g, and the control group was the lowest at 1,830 g, indicating a significant difference among the treatment groups (P<0.05). In the case of feed conversion ratio, the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group showed the lowest value at 1.48, and there was a significant difference among the treatment groups (P<0.05). In particular, it was found that the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% and pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment groups significantly improved weight gain and feed conversion ratio compared to the control group. It was reported that when plant extracts are fed to poultry feed, feed intake increases due to appetite enhancement, endogenous digestive enzyme secretion is improved, and fish quality is improved by antibacterial and antioxidant effects, and that feed conversion ratio is significantly improved when plant extracts containing carvacrol, cinnamaldehyde, and capsicin are fed to broilers (Jamroz et al., 2005). Cross et al. (2007) also reported that there was no difference in productivity when various types of herbal extracts, such as marjoram, oregano, rosemary, yallow, and thyme, were added to broiler feed. The results of this study were similar to the two studies above.

Therefore, it is judged that the 0.4% feeding level of fermented probiotics manufactured by mixing 29% pomegranate extract, 10% ginkgo leaf components, and 29% pomegranate extract, 10% citron components in broiler feed has high potential as a feed additive that can improve weight gain and feed efficiency in broilers.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% 0-3 weeks Initial weight(g) 39 39 39 39 39 0.02 Final weight(g) 764 ^c 759 ^c 855 ^a 798 ^b 763 ^c 7.41 Weight gain(g) 724 ^c 720 ^c 816 ^a 759 ^b 724 ^c 7.41 Feed intake(g) 1,013 ^b 1,027 ^b 1,167 ^a 1,025 ^b 998 ^b 19.18 FCR (Feed/Gain) 1.40 1.43 1.43 1.35 1.38 3-5 weeks Initial weight(g) 764 ^c 759 ^c 855 ^a 798 ^b 763 ^c 7.41 Final weight(g) 1,870 ^c 1,890 ^c 2,097 ^a 1,895 ^c 1,964 ^b 15.40 Weight gain(g) 1,106 ^bc 1,130 ^bc 1,242 ^a 1,097 ^c 1,201 ^ab 20.07 Feed intake(g) 1,786 ^c 1,896 ^ab 1,977 ^a 1,818 ^bc 1,860 ^bc 26.75 FCR (Feed/Gain) 1.61 ^ab 1.68 ^a 1.60 ^ab 1.66 ^a 1.55 ^b 0.03 0-5 weeks Initial weight(g) 39 39 39 39 39 0.01 Final weight(g) 1,870 ^c 1,890 ^c 2,097 ^a 1,895 ^c 1,964 ^b 10.89 Weight gain(g) 1,830 ^c 1,850 ^c 2,058 ^a 1,855 ^c 1,925 ^b 10.88 Feed intake(g) 2,800 ^d 2,924 ^b 3,144 ^a 2,844 ^cd 2,858 ^c 12.15 FCR(Feed/Gain) 1.53 ^b 1.58 ^a 1.53 ^b 1.53 ^b 1.48 ^c 0.01

^a,b,c,d Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

2) Analysis of general components (body composition) of conductors

The results of body composition analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic are shown in Table 19.

In the case of the breast meat of the carcass, the crude protein content was the highest in the control group at 24.95% and the lowest in the pomegranate-yuzu fermented probiotic agent 2 (PCP-2) 0.4% treatment group at 22.22%, showing a significant difference among the treatment groups (P<0.05). The crude fat content was the highest in the pomegranate-ginkgo leaf fermented probiotic agent 2 (PGLP-2) 0.4% treatment group at 2.04% (P<0.05) and the lowest in the control group at 0.95%, showing a significant difference among the treatment groups (P<0.05). The crude protein content of the leg meat of the carcass was the highest in the pomegranate-yuzu fermented probiotic agent 1 (PCP-1) 0.4% treatment group at 20.70% (P<0.05) and the lowest in the control group at 19.03%, showing a significant difference among the treatment groups (P<0.05). The crude fat content was highest in the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group at 3.53% (P<0.05), and lowest in the control group at 2.0%, showing a significant difference between the treatment groups (P<0.05).

According to Davis (1975), crude protein and crude fat in pork are inversely proportional, so that when the fat content is high, the protein content is low. In addition, according to Park and Yoo (2000) and Kim (2007), the addition of probiotics to broilers did not have a significant trend in the general components of the meat. Kim et al. (2002) reported that the crude fat content decreased when herbal medicine by-products and mugwort powder were fed.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% Breast meat Crude protein (%) 24.95 ^a 23.52 ^ab 22.80 ^b 23.71 ^ab 22.22 ^b 0.46 Crude fat (%) 0.95 ^d 1.05 ^cd 2.04 ^a 1.22 ^bc 1.43 ^b 0.08 Moisture (%) 74.35 ^ab 74.62 ^a 74.17 ^ab 73.85 ^b 74.60 ^a 0.21 Crude ash (%) 1.36 ^a 1.29 ^ab 1.21 ^b 1.40 ^a 1.29 ^ab 0.03 Thigh meat Crude protein (%) 19.03 ^c 19.34 ^bc 19.70 ^abc 20.70 ^a 20.45 ^ab 0.39 Crude fat (%) 2.00 ^b 2.90 ^a 3.37 ^a 3.53 ^a 3.01 ^a 0.28 Moisture (%) 75.35 74.34 74.51 74.43 75.12 0.37 Crude ash (%) 1.08 1.18 1.09 1.21 1.12 0.03

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

3) Analysis of cholesterol content in conductors

The results of the cholesterol content analysis of chicken meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic are as shown in Table 20.

When comparing the cholesterol content of breast meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic, the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group was the highest at 156.99 mg/100 g (P<0.05), and the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group was the lowest at 120.99 mg/100 g, showing a significant difference between treatment groups (P<0.05).

In terms of cholesterol content in leg meat, the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group had the highest at 183.27 mg/100 g (P<0.05), and the pomegranate-citron fermented probiotic 2 (PCP-2) 0.4% treatment group had the lowest at 133.41 mg/100 g, showing a significant difference among treatment groups (P<0.05). There is a research report (Abdulrahim et al., 1996) that adding Lactobacillus acidophilus to the feed increased laying rate, feed efficiency, and decreased cholesterol in egg yolk.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% Breast 140.89 ^ab 156.99 ^a 147.66 ^ab 120.99 ^b 122.16 ^b 5.53 Thigh 165.39 ^ab 183.27 ^a 146.14 ^ab 162.30 ^ab 133.41 ^b 8.85

^a,b Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

4) Analysis of fatty acid content of conductors

The results of fatty acid content analysis of chicken meat according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic are shown in Tables 21 and 22.

As a result of analyzing the fatty acid content of breast meat, myristic acid (C14:0) was the highest at 2.0% in the 0.4% pomegranate-yuzu fermented probiotic 1 (PCP-1) treatment group (P<0.05), and the lowest at 0.93% in the control group, showing a significant difference between the treatment groups (P<0.05).

Oleic acid (C18:1 n9) was highest in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group at 36.16% (P<0.05), and lowest in the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group at 32.95%, showing a significant difference between treatment groups (P<0.05).

∑SFA was highest at 35.35% in the 0.4% pomegranate-yuzu fermented probiotic 1 (PCP-1) treatment group, but there was no significant difference among the treatment groups. ∑MUFA was lowest at 41.35% in the control group, and there was a significant difference (P<0.05).

As a result of analyzing the fatty acid content of the leg meat, myristic acid (C14:0) was the highest in the control group at 1.10% (P<0.05) and the lowest in the pomegranate-yuzu fermented probiotic agent 1 (PCP-1) 0.4% treatment group at 1.02%, showing a significant difference among the treatment groups (P<0.05). Oleic acid (C18:1 n9) was the highest in the pomegranate-ginkgo leaf fermented probiotic agent 1 (PGLP-1) 0.4% treatment group at 40.35%, but there was no significant difference among the treatment groups. Linoleic acid (C18:2n6) was highest in both the 0.4% pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) and 0.4% pomegranate-yuzu fermented probiotic 2 (PCP-2) treatments, at 18.22% (P<0.05), and was lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment, at 16.70%, showing a significant difference among the treatment groups (P<0.05).

∑SFA was the highest in the control group at 31.46% and the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) treatment group at 30.25%, showing a significant difference among the treatment groups (P<0.05). ∑n-6 was the highest in the 0.4% pomegranate-yuzu fermented probiotic 2 (PCP-2) treatment group at 18.76% (P<0.05) and the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment group at 17.25%, showing a significant difference (P<0.05).

It has been reported that the fatty acid composition in the muscles of monogastric animals can be changed through feeding (Pascual et al. 2007). Oleic acid is a monounsaturated fatty acid that, when consumed in large quantities, reduces blood triglycerides and cholesterol, and is reported to have a beneficial effect on adult diseases such as arteriosclerosis (Grundy, 1986).

Items Control PGLP-1 ² PGLP-2 ³ PCP-1 ⁴ PCP-2 ⁵ SEM ⁶ 0.4% 0.4% 0.4% 0.4% Myristic acid(C14:0) 0.93 ^b 1.65 ^ab 1.60 ^ab 2.00 ^a 2.14 ^a 0.17 Pentadecanoic acid(C15:0) 2.24 ^a 2.28 ^a 1.09 ^b 0.96 ^b 0.97 ^b 0.13 Palmitic acid(C16:0) 21.11 20.45 19.92 20.23 19.96 0.34 Palmitoleic acid(C16:1n7) 4.38 4.15 4.39 4.03 3.76 0.23 Stearic acid(C18:0) 0.21 ^b 5.50 ^a 9.71 ^a 7.36 ^a 7.01 ^a 1.35 Oleic acid(C18:1 n9) 35.08 ^a 32.95 ^b 36.16 ^a 34.73 ^a 34.73 ^a 0.38 Linoleic acid(C18:2n6) 17.85 ^a 15.25 ^c 17.37 ^a 16.08 ^b 17.33 ^a 0.26 α-linolenic acid(C18:3n3) 0.43 ^a 0.34 ^b 0.45 ^a 0.47 ^a 0.44 ^a 0.02 Arachidic acid(C20:0) 0.74 ^a 0.48 ^b 0.77 ^a 0.64 ^a 0.71 ^a 0.04 Eicosaenoic acid(C20:1n9) 0.50 0.45 0.45 0.55 0.59 0.05 Eicosapentanoic acid(C20:5n3) 4.19 ^b 5.70 ^a 3.23 ^b 4.28 ^b 4.27 ^b 0.34 DGLA(C20:3n6) 1.99 1.72 1.75 2.11 2.01 0.14 Docosahexaenoic acid(C22:6n3) 1.12 ^b 2.78 ^a 1.31 ^b 1.94 ^ab 1.61 ^b 0.25 Tetracosaenoic acid(C24:1n9) 1.40 ^ab 1.54 ^ab 1.10 ^b 1.66 ^ab 1.91 ^a 0.16 ∑SFA ¹ 34.01 35.35 33.56 34.52 34.11 0.68 ∑MUFA ¹ 41.35 ^a 44.08 ^b 42.09 ^b 40.96 ^b 40.99 ^b 0.53 ∑PUFA ¹ 25.57 25.78 24.10 24.88 25.65 0.51 ∑n-3 ¹ 5.74 ^bc 8.81 ^a 4.98 ^c 6.69 ^bc 6.31 ^b 0.37 ∑n-6 ¹ 19.84 ^ab 16.96 ^c 19.11 ^a 18.20 ^b 19.34 ^ab 0.40 USFA/SFA 1.22 1.12 1.25 1.19 1.21 0.03 PUFA/SFA 0.75 0.74 0.72 0.72 0.75 0.02 n-6/n-3 3.53 ^ab 1.97 ^d 3.87 ^a 2.77 ^c 3.12 ^bc 0.18

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ ∑SFA = saturated fatty acid; ∑MUFA = mono-unsaturated fatty acid; ∑PUFA = poly-unsaturated fatty acid, ∑n-3 = total omega 3 fatty acid;∑n-6 = total omega 6 fatty acid

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

³ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁵ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁶ SEM = standard error of the means

Items Control PGLP-1 ² PGLP-2 ³ PCP-1 ⁴ PCP-2 ⁵ SEM ⁶ 0.4% 0.4% 0.4% 0.4% Myristic acid(C14:0) 1.10 ^a 1.03 ^ab 1.07 ^ab 1.02 ^b 1.07 ^ab 0.02 Myristoleic acid(C14:1 n5) 0.38 ^a 0.37 ^ab 0.34 ^b 0.35 ^b 0.34 ^b 0.01 Pentadecanoic(C15:0) 0.79 0.83 0.71 0.81 0.85 0.07 Palmitic acid(C16:0) 21.54 ^a 20.80 ^4b 20.06 ^c 21.18 ^ab 20.86 ^b 0.17 Palmitoleic acid(C16:1n7) 6.14 ^ab 6.75 ^a 5.52 ^b 5.84 ^b 5.57 ^b 0.23 Stearic acid(C18:0) 7.00 ^ab 6.66 ^b 7.42 ^a 7.12 ^ab 7.31 ^a 0.21 Oleic acid(C18:1 n9) 39.72 40.35 39.94 40.18 40.06 0.45 Linoleic acid(C18:2n6) 17.85 ^ab 16.70 ^c 18.22 ^a 17.21 ^bc 18.22 ^a 0.26 α-linolenic acid(C18:3n3) 0.57 0.58 0.61 0.61 0.58 0.02 Arachidic acid(C20:0) 1.03 ^ab 0.94 ^c 0.99 ^bc 0.99 ^bc 1.06 ^a 0.02 Eicosaenoic acid(C20:1n9) 0.21 ^b 0.21 ^b 0.25 ^a 0.23 ^ab 0.24 ^b 0.01 Eicosadienoic acid(C20:2 n6) 0.14 0.15 0.15 0.16 0.14 0.01 DGLA(C20:3n6) 0.37 0.40 0.37 0.46 0.41 0.04 Eicosapentanoic acid(C20:5n3) 2.15 3.07 2.19 2.70 2.23 0.36 Docosahexaenoic acid(C22:6n3) 0.88 0.52 0.36 0.99 0.83 0.19 ∑SFA ¹ 31.46 ^a 30.30 ^b 30.25 ^b 31.10 ^a 31.15 ^a 0.24 ∑MUFA ¹ 46.44 47.68 46.05 46.60 46.21 0.61 ∑PUFA ¹ 21.95 21.43 21.89 22.11 22.41 0.60 ∑n-3 ¹ 3.61 4.17 3.16 4.28 3.64 0.48 ∑n-6 ¹ 18.35 ^ab 17.25 ^c 18.73 ^a 17.83 ^bc 18.76 ^a 0.25 USFA/SFA 1.48 1.57 1.52 1.50 1.49 0.03 PUFA/SFA 0.70 0.71 0.73 0.71 0.72 0.02 n-6/n-3 5.44 ^b 4.50 ^b 12.77 ^a 4.44 ^b 5.18 ^b 1.46

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ ∑SFA = saturated fatty acid; ∑MUFA = mono-unsaturated fatty acid; ∑PUFA = poly-unsaturated fatty acid, ∑n-3 = total omega 3 fatty acid;∑n-6 = total omega 6 fatty acid

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

³ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁵ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁶ SEM = standard error of the means

5) Blood Immune Antibody (IgG) Analysis

The results of blood immune component analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic are shown in Table 23. As a result of analyzing the blood immunoglobin IgG content by test treatment group, the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group was analyzed to be the highest at 1.84 mg/ml, and the control group was analyzed to be the lowest at 1.20 mg/ml, showing a significant difference among the treatment groups (P<0.05). In addition, it was found that all treatment groups of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic showed higher IgG contents than the control group. Woo Kyung-Cheon et al. (2007) reported that when herbs and plant extracts were added to broilers, the IgG concentration significantly increased, and Hong Seong-Jin et al. (2001) reported that when herbal medicines were fed to broilers, the serum IgG concentration was significantly higher than in the control group. The results of this study showed similar trends.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% IgG (mg/ml) 1.20 ^c 1.84 ^a 1.51 ^ab 1.32 ^b 1.34 ^b 0.22

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

6) Analysis of microbial environment in the cecum

The results of the cecal microbial environment analysis of broilers fed pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic are shown in Table 24. The results of the cecal lactic acid bacteria density analysis showed that the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group had the lowest value at 6.79 log ¹⁰ cfu/g, and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group had the highest value at 8.01 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups. The density of yeast was the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment group at 6.90 log ¹⁰ cfu/g and the highest in the 0.4% pomegranate-yuzu fermented probiotic 2 (PCP-2) treatment group at 7.62 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups. The density of Escherichia coli ( E. coil ) was the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment group at 4.73 log ¹⁰ cfu/g and the highest in the 0.4% pomegranate-yuzu fermented probiotic 2 (PCP-2) treatment group at 8.55 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% Cecum microbiota (log ¹⁰ cfu/g) Lactic acid bacteria 7.33 6.79 7.60 7.39 8.01 0.35 Yeast 7.36 6.90 7.41 7.22 7.62 0.29 E. coli 6.49 4.73 7.64 6.59 8.55 0.53 pH 5.85 6.12 6.20 5.72 5.59 0.30

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

4. Natural products Fermented probiotic Evaluation of disease resistance in broilers by feeding (Challenge Test)

In this study, to evaluate the anti-pathogenicity of feeding natural fermented probiotics (pomegranate and ginkgo leaf complex probiotics and 0.4% pomegranate and yuzu complex probiotics) to broilers against pathogenic bacteria, Salmonella enteritidis and E. coli were cultured in SS broth and MCS broth, respectively, for 2 days (37℃). The density of pathogens in the culture solution was 2.35 X 10 ⁷ cfu/ml for Salmonella enteritidis and 3.70 X 10 ⁷ cfu/ml for E. coli . The two culture solutions were mixed 1:1, and 1 ml was forcibly inoculated into the oral cavity of the test animals in each treatment group using a micropipet tip to induce artificial infection. Then, this experiment was conducted.

The test fermentation probiotic prototypes used in the specification test were pomegranate-ginkgo leaf fermentation probiotic mixed with 29% pomegranate marmalade, 10% ginkgo leaf powder, 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator; pomegranate-yuzu fermentation probiotic I mixed with 30% pomegranate marmalade, 5% citron marmalade, 32% corn ethanol residue, 32% defatted rice bran, and 1% glucose as a fermentation regulator; and pomegranate-yuzu fermentation probiotic II mixed with 29% pomegranate marmalade, 10% citron marmalade, 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator.

1) Investigation of mortality and mortality rate by treatment area

After artificial infection with pathogenic bacteria, the results of the investigation of the number of deaths and mortality rate in each treatment group are as shown in Table 25. The number of deaths in each treatment group during the entire test period was the highest in the control group with 15, and the lowest in the pomegranate-yuzu fermented probiotic agent 2 (PCP-2) 0.4% treatment group with 3. The mortality rate by treatment group was 83.33% in the control group, 72.22% in the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group, 50% in the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group, 22.22% in the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group, and 16.67% in the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group, showing that the survival rate was the highest in the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ Total 0.4% 0.4% 0.4% 0.4% Total birds 18 18 18 18 18 90 Dead birds 15 13 9 4 3 44 Live birds 3 5 9 14 15 46 Mortality (%) 83.33 72.22 50 22.22 16.67 48.89

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

2) Analysis of the microbial environment in the cecum

The results of the analysis of the microbial environment in the cecum of the test animals in each treatment group after artificial infection with pathogenic bacteria are shown in Table 26. The density of lactic acid bacteria in the cecum of each treatment group was the highest in the 0.4% pomegranate-yuzu fermented probiotic 2 (PCP-2) treatment group at 9.21 log ¹⁰ cfu/g (P<0.05), and the density of yeast was the highest in the control group at 8.59 log ¹⁰ cfu/g, and the 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment group was the lowest at 6.34 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups. The density of Escherichia coli ( E. coil ) was highest in the control and 0.4% pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) treatment groups at 8.05 and 8.38 log ¹⁰ cfu/g, respectively, and the lowest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) treatment group at 5.57 log ¹⁰ cfu/g, showing a significant difference among treatment groups (P<0.05). In the case of Salmonella , the highest was 8.15 log ¹⁰ cfu/g in the control group and the lowest was 6.0 log ¹⁰ cfu/g in the 0.4% pomegranate-yuzu fermented probiotic 1 (PCP-1) treatment group, but there was no statistically significant difference among treatment groups.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% Cecal microbiota (log ¹⁰ cfu/g) Lactic acid bacteria 8.59 ^b 8.57 ^b 8.46 ^b 8.32 ^b 9.21 ^a 0.14 Yeast 8.59 6.34 7.25 8.21 8.35 0.69 Escherichia coli 8.05 ^a 8.38 ^a 5.57 ^c 6.13 ^bc 6.81 ^b 0.79 Salmonella enteritidis 8.15 6.16 6.05 6.00 6.77 0.82 pH 6.37 6.05 6.90 6.13 6.21 0.01

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

3) Analysis of blood immune antibodies (IgG, IgM and IgA)

The results of the blood immune component analysis of broilers fed pomegranate-ginkgo leaf fermented probiotics and pomegranate-yuzu fermented probiotics after artificial infection with pathogenic fungi are shown in Table 27.

In case of IgM content, it was analyzed to be high at 0.12 and 0.11 mg/ml in the 0.4% pomegranate-yuzu fermented probiotic 1 (PCP-1) and pomegranate-yuzu fermented probiotic 2 (PCP-2) treatment groups, respectively, and low at 0.07 mg/ml in the 0.4% pomegranate-ginkgo leaf fermented probiotic 1, 2 (PGLP-1, 2) treatment groups, showing a significant difference between the treatment groups (P<0.05).

In the case of IgA content, the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group had the highest at 0.61 mg/ml, and the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group had the lowest at 0.49 mg/ml, showing a significant difference between treatment groups (P<0.05).

In the case of IgG content, the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group was the highest at 1.07 mg/ml, and the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the lowest at 0.92 mg/ml, showing a significant difference between treatment groups (P<0.05).

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ 0.4% 0.4% 0.4% 0.4% Immunoglobulins IgM (mg/ml) 0.08 ^b 0.07 ^b 0.07 ^b 0.12 ^a 0.11 ^a 0.01 IgA (mg/ml) 0.60 ^ab 0.49 ^c 0.60 ^ab 0.61 ^a 0.53 ^bc 0.02 IgG (mg/ml) 0.96 ^b 0.94 ^b 0.92 ^b 0.96 ^b 1.07 ^a 0.02

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

4) Long-term weight measurement

The results of analysis of intraperitoneal fat and organ weights in broilers fed pomegranate-ginkgo leaf fermented probiotics and pomegranate-yuzu fermented probiotics after artificial infection with pathogenic fungi are shown in Table 28.

Items Control PGLP-1 ¹ PGLP-2 ² PCP-1 ³ PCP-2 ⁴ SEM ⁵ T1 T2 T3 T4 Liver 4.42 4.39 5.19 3.76 5.40 0.46 Heart 0.99 ^ab 0.91 ^ab 1.15 ^ab 0.72 ^b 0.96 ^ab 0.07 Gizzard 6.95 ^ab 5.83 ^b 8.67 ^b 4.99 ^b 6.46 ^ab 0.64 Ceca 0.80 0.77 0.83 0.98 1.00 0.15 S. intestine 9.54 7.77 9.19 9.35 8.10 0.59 L. intestine 0.46 ^C 0.43 ^C 0.79 ^a 0.43 ^C 0.50 ^b 0.05 Proventriculus 1.35 1.58 1.77 1.21 1.24 0.16 Spleen 0.13 0.09 0.12 0.11 0.13 0.03 Kidney 1.16 1.01 1.09 1.01 1.29 0.12 Bursa 0.19 0.16 0.16 0.19 0.23 0.02 Pancreas 0.61 0.67 0.54 0.61 0.55 0.06

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 30%, ginkgo leaf 5%)

² Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

³ Fermentation pomegranate and citron probiotics(pomegranate 30%, citron 5%)

⁴ Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

⁵ SEM = standard error of the means

In the case of heart weight by treatment group, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the highest at 1.15%, and the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group was the lowest at 0.72%, showing a significant difference among treatment groups (P<0.05). In the case of seedling pockets, the control group was the highest at 6.95%, and the pomegranate-yuzu fermented probiotic 1 (PCP-1) 0.4% treatment group was the lowest at 4.99%, showing a significant difference among treatment groups (P<0.05). In the case of the large intestine, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group showed the highest at 0.79%, and the pomegranate-ginkgo leaf fermented probiotic 1 (PGLP-1) 0.4% treatment group showed the lowest at 0.43%, showing a significant difference between treatment groups (P<0.05).

Example 3

A. Evaluation of the disease resistance of piglets fed with natural fermented probiotics (Challenge Test)

In this study, to evaluate the anti-pathogenicity of feeding natural fermented probiotics (pomegranate and ginkgo leaf complex probiotics and pomegranate and yuzu complex probiotics with 0.4%) to piglets against pathogenic bacteria, Salmonella enteritidis and E. coli were cultured in SS broth and MCS broth, respectively, for 2 days (37℃). The density of pathogens in the culture solution was analyzed to be 1.4 X 10 ⁸ cfu/ml for Salmonella enteritidis and 5.2 X 10 ⁷ cfu/ml for E. coli. The two culture solutions were mixed 1:1, and 1 ml was forcibly inoculated into the oral cavity of the test animals in each treatment group using a micropipet tip to induce artificial infection, and then the present experiment was conducted. The test fermentation probiotic prototypes used in this specification test were a pomegranate-ginkgo leaf fermentation probiotic mixed with 29% pomegranate marmalade, 10% ginkgo leaf powder, 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator, and a pomegranate-yuzu fermentation probiotic mixed with 29% pomegranate marmalade, 10% yuzu residue, 30% corn ethanol residue, 30% defatted rice bran, and 1% glucose as a fermentation regulator.

1) Weight gain, feed intake and feed conversion ratio

The results on weight gain, feed intake, and feed conversion ratio according to the feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic after artificial infection with pathogenic fungi in piglets are shown in Table 29. When the weight gain of each treatment group was compared during the entire experimental period (3 weeks), the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the highest at 20.40 kg, and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group was the lowest at 18.83 kg, showing a significant difference among the treatment groups (P<0.05). In the case of feed conversion ratio, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was the lowest at 1.90.

Items Control PGLP-2 ¹ PCP-2 ² SEM ³ 0.4% 0.4% 0∼3 Weeks Initial weight (kg) 21.70 21.77 22.29 0.26 Final weight (kg) 40.98 42.17 41.12 0.53 Weight gain (kg) 19.28 ^b 20.40 ^a 18.83 ^c 0.66 Feed intake (kg) 38.95 38.71 38.67 0.12 FCR (feed/gain) 2.02 1.90 2.05 0.06

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

² Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

³ SEM = standard error of the means

2) Blood antibody analysis

The results of blood immune component analysis according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic after artificial infection with pathogenic bacteria in piglets are as shown in Table 30.

As a result of analyzing the blood immunoglobin IgA content by treatment group, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was analyzed to be the highest at 1.16 mg/ml, and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group was analyzed to be the lowest at 0.98 mg/ml, but there was no significant difference among treatment groups. As a result of analyzing the blood immunoglobin IgG content, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was analyzed to be the highest at 1.84 mg/ml, and the pomegranate-yuzu fermented probiotic 2 (PCP-2) was analyzed to be the lowest at 1.76 mg/ml, and there was a significant difference among treatment groups (P<0.05). As a result of analysis of blood immunoglobin IgM content, the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group was analyzed to have the highest level at 2.01 mg/ml, showing a significant difference among the treatment groups (P<0.05).

Item Control PGLP-2 ¹ PCP-2 ² SEM ³ 0.4% 0.4% Immunoglobulins IgA (mg/ml) 1.00 1.16 0.98 0.08 IgG (mg/ml) 1.81 ^b 1.84 ^a 1.76 ^c 0.03 IgM (mg/ml) 1.76 ^b 2.01 ^a 1.75 ^b 0.12

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

² Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

³ SEM = standard error of the means

3) Analysis of blood cytokines (TNF-α)

The results of blood cytokine analysis according to feeding of pomegranate-ginkgo leaf fermented probiotic and pomegranate-yuzu fermented probiotic after artificial infection with pathogenic bacteria in piglets are shown in Table 31.

As a result of analyzing the blood cytokine TNF-α content by treatment group, the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group had the highest analysis at 81.46 pg/ml, and the control group had the lowest analysis at 59.56 pg/ml, showing a significant difference among the treatment groups (P<0.05). In addition, it was found that the pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) 0.4% treatment group and the pomegranate-yuzu fermented probiotic 2 (PCP-2) 0.4% treatment group showed higher blood cytokine contents compared to the control group.

Item Control PGLP-2 ¹ PCP-2 ² SEM ³ 0.4% 0.4% TNF-α (pg/ml) 59.56 ^c 80.77 ^b 81.46 ^a 10.17

^a,b,c Mean with different superscripts within the same row are significantly different (P<0.05)

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

² Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

³ SEM = standard error of the means

4) Analysis of microbial environment in feces

After artificial infection with pathogenic bacteria, the results of the microbial environment analysis in the feces of the test animals in each treatment group are shown in Table 32. The density of lactic acid bacteria in the feces of each treatment group was analyzed to be the highest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) treatment group at 8.48 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups. The density of yeast was analyzed to be the highest in the 0.4% pomegranate-ginkgo leaf fermented probiotic 2 (PGLP-2) treatment group at 6.93 log ¹⁰ cfu/g, and the lowest in the control group at 6.55 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups. The density of Escherichia coli ( E. coil ) was analyzed to be the highest in the control group at 4.97 log ¹⁰ cfu/g, and the lowest in the pomegranate-yuzu fermented probiotic agent 2 (PCP-2) 0.4% treatment group at 4.42 log ¹⁰ cfu/g, but there was no significant difference among the treatment groups.

Items Control PGLP-2 ¹ PCP-2 ² SEM ³ 0.4% 0.4% Fecal microbiota (log ¹⁰ cfu/g) Lactic acid bacteria 8.37 8.48 8.29 0.08 Yeast 6.55 6.93 6.86 0.16 E. coli 4.97 4.71 4.42 0.22

¹ Fermentation pomegranate and ginkgo leaf probiotics(pomegranate 29%, ginkgo leaf 10%)

² Fermentation pomegranate and citron probiotics(pomegranate 29%, citron 10%)

³ SEM = standard error of the means

Claims (1)

A feed additive for use as an antibiotic substitute for livestock, comprising as an effective ingredient a fermented extract of a mixture of pomegranate and ginkgo leaves or a fermented extract of a mixture of pomegranate and citron peel,
The above pomegranate and ginkgo leaves, or pomegranate and citron peel, are characterized by being mixed in a weight ratio of 5:1 to 2:1.
A feed additive for use as an antibiotic substitute for livestock, characterized in that the fermentation is carried out for 10 to 60 hours by inoculating a strain consisting of Lactobacillus plantarum , Bacillus subtilis , and Saccharomyces cerevisiae in an amount of 0.1 to 10 wt% based on the weight of the extract.