KR20140018444A - Microorganism for fermenting schisandra chinensis fruit byproduct and use of the fermented schisandra chinensis fruit byproduct - Google Patents

Abstract

The present invention relates to a microorganism for fermenting a schisandra chinensis fruit byproduct and a use of the fermented schisandra chinensis fruit byproduct. The microorganism is Wickerhamomyces anomalus SK1819(KACC91725P). The present invention relates to a method for obtaining polyphenol by extracting the fermented schisandra chinensis fruit byproduct using an organic solvent.

Description

[0001] The present invention relates to a microorganism for fermentation of Schizandra chinensis fruit byproduct and a fermented Schizandra chinensis fruit byproduct,

The present invention relates to a fermentation strain of Omija persimmon and its use.

Livestock industry is an industry for supplying high quality protein to mankind based on plant products. (1987), the Kellner feeding standard (1907), the Hanson feeding standard of Sweden (1915), the Morrison feeding standard of the United States (1915), the NRC (1955), the Armshy feeding standard (1925), and the UK's CNCPS (carbohydrate and protein system) programs have been used at the production site. However, since feed additives are not systematized, Many feed additive researches are needed until production and utilization program.

The plant contains various physiologically active substances such as antibiotic substitutes, antioxidants, immunostimulating substances and unknown growth factors for animals. However, the level of herbal feed additives and herbal feeds have not yet been established, and research on herb feed additives has been limited to some items. . Studies have been carried out on new physiologically active substances contained in plants that can be used as feed additives to provide safe food.

Antioxidant function plays an absolutely important role in the animal to maintain the normal life condition of the animal. SOD (superoxide dismutase), alpha-tocopherol, beta-carotene, f1avonoid, and beta-carotene, which are enzymatically treated with reactive oxygen species such as hydroxyl radicals and superoxide anion which are also disease causing substances such as tissue damage, cell senescence, inflammation phenomenon and hypertension, glutathione, vitamine C, and other non-enzymatic antioxidant mechanisms. Food preservatives such as butylatedhydroxy anisole (BHA), butylatedhydroxy toluene (BHT), propyl gallate (PG) and tert-butyl hydroquinone (TBHQ) Because of the anxiety about the possibility of inducing the natural antioxidant substitute materials are in the spotlight, and various studies have been conducted on this.

Effective July 1, 2011, the addition of antibiotics to feed is prohibited by law. It is expected that various problems will arise due to removal of antibiotics in feed. In order to solve these problems, researches on natural antibiotic substitutes and research on physiologically active substances for plants have been conducted actively (Greathead, H. 2003. Plants and plant extracts for improving animal productivity, Proc. Nutr. Soc. Yang, Y., P. Iji, and M. Choct. 2009. Dietary modulation of gut microflora in broiler chickens: a review of the role of six kinds of alternatives to in-feed antibiotics. 65: 97-114).

Korean Patent Publication No. 1020020036381 discloses a method for producing chicken feed using red ginseng. The method of producing chicken feed using extracts of red ginseng, red ginseng roots and Chinese medicine residue, A method for manufacturing a chicken feed using red ginseng to enhance tolerance and obtain high quality eggs is disclosed.

Korean Patent Laid-Open Publication No. 1020020008733 discloses a method for producing livestock products containing ginseng and the like, which contains many beneficial properties to humans, Which can be reused as a useful feed for the human body.

The present invention has been made in view of the above needs, and an object of the present invention is to provide a fermentation strain of Omija persimmon.

Another object of the present invention is to provide a complex functional feed additive using a fermented product to which a probiotic agent is added.

It is another object of the present invention to provide a method for producing a complex functional feed additive using a fermented product to which a probiotic agent is added.

In order to accomplish the above object, the present invention provides a strain of Wickerhamomyces which fermentes Schizandra chinensis.

In one embodiment of the present invention, the strain is preferably a strain of Wickerhamomyces anomalus SK1819 (KACC91725P), but is not limited thereto.

The present invention also provides a method for producing polyphenol by inoculating the strain of the present invention with the strain of the present invention and then fermenting the fermented omiza bark with an organic solvent.

The present invention also provides a method for obtaining a flavonoid or anthocyanin by inoculating the strain of the present invention with the strain of the present invention and then fermenting the fermented omiza foil with an organic solvent.

In addition, the present invention provides a method for obtaining an antibiotic active composition by inoculating a strain of the present invention to an ozaki leaf and fermenting the same, followed by extracting the fermented omza leaf with an organic solvent.

The present invention also provides a composition for feed comprising the fermented product of Schizophyllum sinensis inoculated with the strain of the present invention as an active ingredient.

Hereinafter, the present invention will be described.

In order to isolate the strains of the invention, the present invention is characterized in that the taxonomic properties of the isolates, the culture conditions and the characteristics of the strains, the viability of the isolates and the 18S rDNA analysis, The results were compared with the data of the query GenBank using MEGA 4 software.

The morphological characteristics of the strains were observed by microscopic observation of five strains SK1759, SK1760, SK1811, SK1812 and SK1819. SK189 and SK1760 strains were found in oval type, SK1811 and SK1812 in candida, and SK1819, which is ellipsoideus type and pastorianus type, were clustered into grape clusters . As a result of measurement of glycosylation by API 20C AUX Kit, SK1875 and SK1760 showed one sugar group, SK1812 and SK1819 had the same sugar potential, and SK1811 showed unique sugar resistance. 18S rDNA sequence data BLAST showed that SK1759 and SK1760 were 100% homologous to K azaxh stania viticola X99526 Type strain and SK1812 and SK1819 were compared with Wickerhamomyces anomalus AB126679 Type strain and was able to see that exhibit 99% homology,

The strain, SK1819, was deposited at the National Institute of Agricultural Science and Technology , National Academy of Agricultural Science, 88-20, Suwon-dong, Gwon, anomalus SK1819 (KACC91725P).

On the other hand, the growth curve of the five strains showed the highest absorbance at 36 ℃ in the YM broth medium at 25 ℃. The strains of SK1819 and SK1760 showed the greatest change with the pH near 3.0. Five strains isolated from natural fermented Omija pak were inoculated and incubated at 25 ℃ for ₁₀ log (cfu / ㎖) of Wickerhamomyces sp. The SK1819 strain showed the highest bacterial count of log ₁₀ (cfu / ㎖) value of 8.85 on day 7 and maintained the bacterial count of 8.26 until the 56th day of fermentation.

In addition, Wickerhamomyces (WFS) [ Wickerhamomyces (WFS)) which was inoculated with 1% of sp.SK1819 strain and fermented at 25 ° C for 28 days sp.SK1819-fermentation Schisandra antimicrobial activity, total flavonoid, total polyphenol, and anthocyanin content of non-fermented Schisandra chinensis fruit byproduct, which is not inoculated with chinensis fruit byproduct and yeast, Were measured for each physiological performance.

The general components of WFS were moisture 6.67%, crude protein 14.83%, crude fat 21.67%, crude fiber 16.48%, ash 1.80%, Ca 0.07% and P 0.33%. The total polyphenol content of the organic extracts was 0.24 ㎎ / ㎖ in the Eth-OH layer, 0.22 ㎎ / ㎖ in the CHCl ₃ layer, 0.19 ㎎ / ㎖ in the n-Hexan layer and Bu-OH layer, 0.18 ㎎ / ㎖. The extraction rate of Bu-OH layer was about twice as high as that of NFS. Total flavonoids were 604.7 ㎍ / ㎖ in CHCl ₃ layer and 22.82 times higher than NFS. The concentrations of flavonoids in the n-Hexan, CHCl ₃ , EtOAc, Bu-OH and aqueous layers of WFS are 90.35 ㎍ / ㎖, 235.64 ㎍ / ㎖, 71.89 ㎍ / ㎖ and 103.11 ㎍ / ㎖ respectively. The content of anthocyanin was 28.98 ㎎% for WFS and 11.26 ㎎% for NFS. In the DPPH free radical inhibition activity, EtAOc layer of WFS was about 8.26 times stronger than NFS, Bu-OH layer was about 3.05 times stronger than NFS, and CHCl ₃ layer was about 2.47 times stronger than NFS. The superoxide dismutase (SOD) -like activity of the CHCl ₃ layer was 68.89% for WFS and 22.42% higher than for NFS 46.47%. The Eth-OH layer was WFS 86.36%, 14.87% higher than NFS and 90.26% . In EtAOc layer, WFS was 76.66%, 8.69% higher than NFS and 90.36% higher than AFS in NFS.

The minimum inhibition concentration (MIC) for six pathogenic bacteria was WFS, Salmonella gallinarum ATCC 9184 strain was 12.50 ㎎ / ㎖ of n-Hexan, CHCl ₃ , EtAOc and aqueous layers, Haemopillus The Somnus strain was 3.13 ㎎ / ㎖ for the EtAOc layer and the Haemopillus parasuis strain was 6.25 ㎎ / ㎖ for the Bu-OH and aqueous layers, Enterotoxigenic E. coli Strain was 6.25 ㎎ / ㎖ in both Eth-OH and EtAOc layers, and Staphylococcus aureus strain was 6.25 ㎎ / ㎖ in the Eth-OH and EtAOc layers.

Also, Wickerhamomyces sp. The experiment was carried out to investigate the effect of SK1819 non - fermented Omiza bean on the productivity of laying hens and laying hens by feeding 0.25% and 0.50% of dry non - fermented Omiza bean, respectively. The treatments consisted of a control diet fed general diet, NFS25 supplemented with 0.25% non - fermented omiza paste, NFS50 supplemented with 0.50% non - fermented omiza paste, and Wickerhamomyces The WFS25 treated with 0.25% FFS and the WFS50 treated with 0.50% were tested for 28 days with 9 replicates per 5 treatments. In terms of productivity, productivity was high at 90.83% ( P <0.05) in WFS25 treatment at 1 ~ 2 weeks and 88.69% and 91.63% in WFS50 at 3 ~ 4th week respectively. Egg mass was significantly higher ( P <0.05) in WFS25 treatment at 55.46 g / day than at 1 and 2 weeks. Egg color was highest at 43.66 ( P <0.05) in WFS25 treatment at 3 ~ 4 weeks, but no consistent change was observed in egg yolk color. The fatty acid composition of egg yolk was significantly higher ( P <0.05) in WFS25 treated group, with 9.23% of stearate, 49.09% of oleate, 49.19% of total MUFA and 2.47% of arachidonate. There was no significant difference in the weight of liver, spleen and abdominal fat and the ratio of body weight to liver, spleen and abdominal fat weight. Total bacterial counts and E. coli were significantly lower in WFS50 ( P <0.05). The number of yeast was significantly higher in NFS25 and WFS25 treatments ( P <0.05). Cecal microflora in the total number of bacteria and E. coli showed no significant difference in treatments like the intestine. Dietary salary of fermentation Schisandra nights in the present invention showed that the hens I have a positive impact on improving productivity

As can be seen from the present invention, the following three experiments were carried out in order to examine the effects of preparing a fermented product of Omija powder by using a strain native to Omija foil and feeding the fermented Omija foil to the laying hens .

In the present invention, identification of strains, culturing conditions and culture characteristics, and viability tests of the isolated strains were conducted in order to search for strains harvested from natural fermented Omija foil. The five isolates were divided into three groups, of which SK1819 strain was Wickerhamomyces sp. The inoculation of non-fermented omija powder resulted in the best strain to grow at 25 ℃.

The morphological characteristics of the strains showed the highest growth at 25 ℃ with morphological observations of five strains SK1759, SK1760, SK1811, SK1812 and SK1819. The reason why the five yeast strains were found in Omiza Park is that the other strains can not survive and the yeast strains can survive due to strong antimicrobial activity against other strains except for yeast and fungi.

Microscopic observations and API 20C AUX Kit showed that the two strains SK1759 and SK1760 were the first group; SK1812 strain and SK1819 strain were the second group; And SK1811 strains were found to have the same sugar availability, respectively. In this group, the strain SK1819 maintained the highest growth curve at 25 ℃ after five inoculum strains.

18S rDNA sequence data BLST search The SK1819 strain has 99% homology with Wickerhamomyces anomalus AB126679 Type strain and its name is Wickerhamomyces sp. SK1819. Wickerhamomyces sp. The fermentation temperature was determined to be room temperature, which is easy to apply in the field. Fermentation was carried out for 30 days considering the number of viable cells and proper fermentation period.

In addition, the Wickerhamomyces sp. Schisandra chinensis fruit byproduct (WFS) fermented with SK1819 and non-fermented Schisandra The physiological activity of chinensis fruit byproduct (NFS) was measured.

The total polyphenol content of WFS is 0.08 ㎎ / ㎖ higher in the Bu-OH layer than that of the omija bush. In the n-hexane layer, the total polyphenol concentration of NFS was 0.19 mg / ml which was 1.26 times higher than that of 0.15 mg / ml.

The concentration of total flavonoids in the CHCl ₃ layer of WFS was about 23 times higher than that of NFS and the anthocyanin concentration was about 2.6 higher than that of NFS. The DPFH free radical inhibition activity was about 8 times higher in the EtAOc layer than in the NFS, Layer was about 3 times, and the CHCl ₃ layer was about 2.5 times stronger. Superoxide dismutase (SOD) Like activity was higher in the CHCl ₃ layer, Eth-OH layer, n-Hexan layer, EtAOc layer and Aqueous layer than NFS. The physiological activity of fermented omija foil was confirmed to be higher in the biological activity of WFS from the hydrophobic to hydrophilic organic solvent for extraction.

The minimum inhibition concentration (MIC) for six pathogens ranged from 3.13 to 100 ㎎ / ㎖. WFS is Salmonella The gallinarum ATCC 9184 strain contained 12.50 ㎎ / ㎖ of n-Hexan, CHCl ₃ , EtAOc and Aqueous layers, and Haemopillus The somnus strain was 3.13 ㎎ / ㎖ in the EtAOc layer, and Haemopillus The parasuis strain was 3.13 mg / ml in the Bu-OH layer and the aqueous layer, and Enterotoxigenic E. coli Strain was 6.25 ㎎ / ㎖ in both Eth-OH and EtAOc layers, and Staphylococcus aureus strain was 3.13 ㎎ / ㎖ in the Eth-OH and EtAOc layers.

Wickerhamomyces sp. Experiments were carried out to investigate the effects of feeding of SK1819 strain on the productivity of laying hens and laying hens by 0.25% and 0.50% of the non - fermented. The treatments consisted of control, which fed general diet, NFS25 treated with 0.25% non - fermented omija, NFS50 treated with 0.50% non - fermented omija, and Wickerhamomyces sp.SK1819 The WFS25 treated with 0.25% fermented Omija foil and the WFS50 treated with 0.50% were tested for 28 days with 9 replicates per 5 treatments. Feed intake and mortality were not significantly different between treatments. The productivity of WFS25 was 90.83% ( p <0.05) at 1 ~ 2 weeks, 88.69% of the control and 88.03% of the WFS25. Egg Mass was significantly higher ( P <0.05) as WFS25 treatment was 55.46 g / day at the 1st and 2nd week. Egg quality was highest at 43.66 ( P <0.05) in WFS25 treatment at 3 ~ 4 weeks. The fatty acid composition of egg yolk was significantly higher ( P <0.05) in WFS25 treated group, which was 9.23% in stearate, 49.09% in oleate, 49.19% in total MUFA and 2.47% in arachidonate. There was no significant difference in the weight of liver, spleen and abdominal fat and the ratio of body weight to liver, spleen and abdominal fat weight. Total number of bacteria and E. coli were significantly lower ( P <0.05). The number of yeast was significantly different between NFS25 and WFS25, and log ₁₀ (cfu / g) values were 2.55 and 2.51, respectively ( P <0.05). In the cecum, the total number of microorganisms and E. coli showed no significant difference in the treatment interval as in the small intestine

Wickerhamomyces sp. The addition of the fermented omija powder inoculated with SK1819 strain to the laying hens showed a positive effect on the productivity improvement of laying hens. Therefore, it seems to be useful for the reduction of the processing cost due to the by-product reuse and the environmental preservation effect and the healthy food production.

FIG. 1 is a photograph (magnification: x 1,000) of SK1759, SK1760, SK1811, SK1812, and SK1819 strain by a phase contrast microscope. Arrows indicate the emergence of yeast. The cells were cultured in YM agar for 24 hours at 25 ° C.
Figure 2 shows the 18S rDNA sequence alignment between K1759 and SK1760.
Figure 3 shows the 18S rDNA sequence alignment between SK1812 and SK1819.
4 Kazachstania sp. Figure 1 shows the evolutionary relationships of the strains and the Evolutionary relationships of SK1759 and SK1760 strains.
FIG. 5 is a graphical representation of Wickerhamomyces sp. Figure shows the evolutionary correlation between strains and SK1812 and SK1819 strains.
FIG. 6 is a graph (n = 5) showing the growth curves of SK1759, SK1760, SK1811, SK1812 and SK1819 according to the incubation temperature.
FIG. 7 is a graph showing the pH change of Schizandra chinensis during 25 ° C incubation (n = 3).
8 is Kazachstania sp. SK1759, Kazachstania sp. SK1759, Candida sp. SK1811, Wickerhamomyces sp. SK1812, and Wickerhamomyces sp. Figure 5 shows the number of 5 yeast strains from Schizandra chinensis in SK1819 fermentation (n = 5).
Fig. 9 is a schematic view of organic solvent extraction from non-fermented or fermented Omija foil
Fig. 10 is a graph showing the results of non-fermented Omija foil and Wickerhamomyces sp. SK1819-fermented omiza bark (n = 3).
Fig. 11 is a graph showing the results of non-fermented Omija foil and Wickerhamomyces sp. SK1819-fig showing the amount of flavonoid of fermented omiza bark (n = 3).
Fig. 12 shows the results of a comparison between the non-fermented Omija foil and Wickerhamomyces sp. SK1819-fermented oyster mushroom (n = 3).
Fig. 13 shows the results of a comparison between the non-fermented Omija foil and Wickerhamomyces sp. SK1819-DPPH IC ₅₀ of ethanol extract of fermented Omiza bean (n = 3).
Fig. 14 is a graph showing the results of non- sp. SK1819-SOD-like activity of fermented omiza bark (n = 3).

Hereinafter, the present invention will be described more specifically with reference to non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  One: Omiza Park  Identification and Inoculation of Fermentation Strain Survivability

Materials and Usage Medium Conditions

This study was conducted in August, 2010 in the green general food of Gongpyeong-dong, Myeong-buk, Gyeongsangbuk-do. Omija usually harvests in the middle of September, 120 to 125 days after flowering, and dries the water to less than 25%. It is a by-product after extracting dried Omiza berries in hot water for 24 hours.

For the isolation of the strains, 1 ml of YM (1% glucose, 0.5% peptone, 0.3% malt extract, 0.3% yeast extract, 2.0% agar (Becton, Dickinson and Company, USA) (90 × 150 ㎜, SPL, Gyeongki-do, Korea) and dried at room temperature for 16 hours or more. The plate was then sterilized by using a plate for bacterial isolation Respectively.

Pure isolation and preservation of yeast strain

1 g of spontaneously fermented Schizosaccharomyces pomaceii was homogenized in 9 ml of sterile physiological saline (0.85% w / v NaCI) solution and homogenized with a voltex (G-560, Scientific Industries, USA) and left for 10 minutes. After sequentially diluted sample ^{1 × 10 -5 ~ 1 × 10} - to ^18-3 concentration samples the YM plate and smeared on the medium 30 ℃ incubator (Scientific VS-1203P , Vision, Korea) to 25 hours of incubation were isolated strains . The colony of isolated strains was subcultured three times by streaking in YM plate medium to isolate single yeast strains.

The pure strains were inoculated 1% in sterilized YM broth and cultured in a shaking incubator (J-SIL-R, Jisico Co., Ltd, Korea) at 30 ° C for 12 to 24 hours. 100 μl of the culture was added to a sterilized 1 ml and 900 μl of DMSO (Bio Basic Inc, Ontario, Canada) was added to the vial. After homogenization, the cells were stored at -70 ° C deep freezer (MDF-U53V, Sanyo electric Co., Ltd., Japan).

Identification of the strain

Based on Kreger-Van R (1984) and Barnett JA (2000), the taxonomic position of the isolated strains was investigated according to their morphological, physiological and biochemical characteristics. (1952) and Yarrow D (1998), respectively. The morphological separation method of the isolated strains was observed by observing the shape, color and size of the colony grown in the YM solid plate medium and observing the shape of the single cell with and without the ascospores in an optical microscope (CX-40, Olympus, Japan).

Investigation of general characteristics of yeast

The colony characteristics of the YM solid plate culture broth, YM broth culture characteristics, optimum temperature, etc., were classified according to Lodder J (1952) and Yarrow D (1998).

Party Harmony  exam

Using the API 20C Aux Kit (bioM? Rieux? Sa, France), the presence or absence of glycosylation of the isolates was determined (Lodder and Kreger-van Rij, 1952). D-glucose, glycerol, calcium 2-keto-gluconate, L-arabinose, D-xylose, adonitol, xylitol, D-galactose, inositol, D-sorbitol, D-maltose, D-saccharose (sucrose), D-trehalose, D-melezitose and D-raffinose.

One platinum strain was inoculated into 5 ml of sterilized YM broth and cultured for 24 to 48 hours in a shaking incubator (J-SIL-R, JISCO Co., LTD, Korea). The isolate culture broth was incubated with C 20 medium (ammonium sulphate 5 g / l, monopotassium phosphate 0.31 g / l, dipotassium phosphate 0.45 g / l, disodium phosphate 0.92 g / l, sodium chloride 0.1 g / trace element 10 ml / l, calcium phosphate 0.05 g / l, magnesium sulphate 0.2 g / l, histidine 0.005 g / l, tryptophane 0.02 g / l, methionine 0.02 g / , final pH: 6.4-6.8). The suspension medium (Denineralized water, 2 ml) enclosed in a kit box was supplemented with mcFariand standard 1 # (BaSO4, 2.4 × 10-5 mol / Lt; / RTI &gt; solution.

The mixture was poured into API 20C Aux Kit to prevent air bubbles from entering and finished with mineral oil (125 mL, bioM? Rieux? Sa, France) to block oxygen. Culture of the strains injected into the API 20C Aux Kit was shown in the results based on the incubation results for 48 hours and 72 hours in a 25 ° C incubator (Scientific VS-1203P, Vision, Korea).

Growth characteristics of cells

The cells were cultured on YM flat plate for 48 hours and inoculated into 2% glucose-yeast extract-peptone water (glucose 20 g, yeast extract 5 g, peptone 10 g, DW 1 L) (CX-40, Olympus, Japan). All of them were observed at the same magnification × 1,000, and the differences among the individuals were classified based on observation of the presence or absence of budding and differences in the appearance of the budding.

Comparison of Growth Performance of Isolated Strains

YM broth was used for the growth of SK1759, SK1760, SK1811, SK1812 and SK1819 strains in the solid medium and the MRS solid medium. YM Broth Inoculated with five strains isolated on two different mediums and incubated at six temperatures of 20, 25, 30, 37, 40 and 45 ℃ (Scientific VS-1203P, Vision, Korea ) Were used to measure the growth of strains of 0, 12, 24, 36 and 48 hours, respectively. The absorbance of the strain was measured using a 96 well plate (SPL, Korea). The actual number of bacteria was calculated based on the absorbance and the result of coating on the actual plate.

DNA  detach

Chromosomal DNA isolation from isolated strains was performed using GenEx ™ genomicsx genomic DNA extraction kit (General Biosystel 11, Korea) and isolated genomic DNA was used as a template for 18S rDNA sequencing.

Primer

For the identification of yeast strains isolated in this experiment, forward primer; NS1 (5'-GTAGTCATATGCTTGTCTC-3 '), NS2 (5'-CTCTTTATTGATTGAGTTGG AATTTCAT GAT-3'), reverse primer; NS3 (5'-GCAAGTCTGGTGCCAGCAGCC-3 ') NS4 (5'-TTCCATCAATTCCTTTAAG-3') was amplified and analyzed (White et al., 1990).

PCR Amplification by

PCR (polymerase chain reaction) was performed using 5U of Taq polyl11erase, 10mM dNTP, 10mM Tris-HCI (pH 8.3) and 1.51M MgCb using a PCR apparatus (9600 therrnocycler, PE Applied Biosysterns, Washin- PCR mixtures were used. PCR was performed with denaturation at 95 ° C for 60 seconds, annealing at 55 ° C for 45 seconds, and extension at 72 ° C for 5 minutes (Innis et al., 1990).

18S rDNA Nucleotide Sequence Analysis

The amplified PCR product was electrophoresed on 1.0% agarose gel and DNA fragments were recovered. The nucleotide sequence of the determined 18S rDNA was determined by GeneBank (Roche Diagnostics, St. Louis, MO, USA) using the ABI PRISM @ 3100-avant genetic analysis (Applied Biosystel 11s, USA) The nucleotide sequence and homology of 18S rDNA already registered with GeneBank were analyzed using the BLAST program of GeneBank, and compared with the GeneBank database of NCBI (National Center for Biotechnology inforrnation).

The results of the above embodiment are as follows.

From Omiza Park  Common strain Survivability

Three strains of Bacillus subtillis SK878, Lactobacillus brevis SK1304 and Saccharomyces cerevisiae SK1708, which are commonly used as probiotics in animal husbandry, were inoculated with Omija powder to investigate the strains that could be used for Omija bacillus. Bacillus subtilis SK878, Lactobacillus brevis SK1304, and Saccharomyces cerevisiae SK1708 were inoculated at a dose of 1% on the basis of dry weight of omija, and the number of bacillus subtilis SK878 bacteria was 5 × 10 ⁷ cfu / g, The number of bacteria of Lactobacillus brevis SK1304 and Saccharomyces cerevisiae SK1708 was 3 × 10 ⁷ cfu / g.

As a result of the incubation of the three different strains of the above-mentioned three strains, the microorganisms were completely killed at the day of inoculation at 30 ° C. The strong antimicrobial activity of Bacillus subtilis SK878, Lactobacillus brevis SK1304 and Saccharomyces It is believed that three strains of S. cerevisiae SK1708 die.

Table 1 shows the number of surviving cells of three kinds of probiotics for the fermentation of Schizandra chinensis.

Characteristics of isolated strains

Morphological feature

Microscopic observations of the morphology of the SK 1759, SK 1760, SK 1811, SK 1812 and SK 1819 strains grown in the YM solid medium are shown in Figure 1. The formation of budding daughter cells, a typical breeding characteristic of yeast, . Based on this, five strains of SK 1759, SK 1760, SK 1811, SK 1812 and SK 1819 are mainly yeast strains.

The strains isolated from natural fermented Omiza pak were divided into three groups. SK1759 and SK1760 strains were found to be one group. SK1811 and SK1812 strains were similar to each other. SK1819 strains were different from the strains of the two groups. The strains SK1759 and SK1760 were oval - shaped and distributed in the medium. SK1811 strain and SK1812 strain were able to observe the survival pattern of the hyphae of the hyphae in the culture medium with the candida - like cells forming the pseudonycelium due to mutual connection of the individuals. The SK1819 strain is a mixture of ellipsoideus and pastorianus, and the breeders are composed of grapevine clusters of several tens to several hundreds. Most of the cells are oval, and most of the cells outside the cluster are sausage type Which is considered to be a characteristic of the fungus with different shapes depending on the size of the fungus.

Per se of the isolate Harmony

Five strains grown in the YM medium were tested for their ability to inhibit glycosylation of 19 sugars by the API 20C AUX Kit (Table 2). The strains SK1759 and SK1760 were glycerol, calcium 2 -keto-gluconate and N-acetyl-glucosamine, and negative responses to the remaining 16 sugars. D-maltose, D-saccharose, D-galactose, D-galactose, D-sorbitol, N-acetylglucosamine, D-saccharose, trehalose and D-melezitose, and negative reaction was observed for the remaining sugars. D-glucose, glycerol, methyl-α-D-glucopyranoside, D-celiobiose, D-maltose, D-saccharose (sucrose) and D-melezitose were the most effective sugars in the SK1811 strain.

The morphological properties of the 19 strains of SK1759, SK1760, SK1811, SK1812 and SK1819 were identical to those of the three groups: SK1759 and SK1760 were oval, SK1811 And SK1812 were divided into Candida type group, SK1819 Ellipsoideus type and Pastorianus type mixed group, and in this experiment, one type of the two types of magnetization SK1759 and SK1760, another group of SK1811 strain and SK1812 strain, and SK1819 strain Respectively.

Table 2 shows the biochemical characteristics of SK1759, SK1760, SK1811, SK1812 and SK1819 strains.

Antibiotic resistance of isolated strains

The antimicrobial resistance of five strain SKs isolated from natural fermented omija pak was measured. The method of isolating pure isolates using antibiotics is a very effective method for isolating different microorganisms simply by isolating and identifying microorganisms.

In order to distinguish the homology between five strains of SK1759, SK1760, SK1811, SK1812 and SK1819 isolated from natural fermented omija, six different antibiotics diluted at each concentration were added with different concentration of ampicillin 50, 100 ㎍ / ㎖, The viability of the strains was determined by adding carbenicillin 5, 10 ㎍ / ㎖, chloramphenicol 5, 10 ㎍ / ㎖, gentamicin 12.5, 25 ㎍ / ㎖, kanamycin 10, 20 ㎍ / ㎖, rifampicin 10 and 20 ㎍ / Respectively.

The antibiotic resistance results of five strains isolated from pure fermented Omija pak were positive in all antibiotics except ampi-cillin 100 ㎍ / ㎖. These characteristics are seen in yeast and mold. Five strains of SK1759, SK1760, SK1811, SK1812 and SK1819 isolated from natural fermented Omija pak were judged to be yeast or mold.

 Table 3 shows antibiotic resistance test results of strains isolated from Schizandra chinensis.

 18S rDNA  Identification of strains by analysis

18S rDNA  Comparison of nucleotide sequences

The results of comparative analysis of the nucleotide sequences of SK1759 and SK1760 are shown in Figure 2. The comparison results of the nucleotide sequences of the four strains SK1812 and SK1819 are shown in Figure 3. Based on the result of each nucleotide sequence, As a result of comparative analysis using sbjct sequence as SK1760 strain, the query sequence / sbjctsequence = 1081/1091 and the homology of the homology was 99.08%. The gap between them was 10/1091, which was 0.92% In the nucleotides 132, 257 and 1968 in the sbjctsequence and 561, 562, 1076, 1090, 1096, 1100 and 1103 bases. In comparison with the SK1812 strain as the query sequence and the sbjctsequence as the query sequence, the query sequence / sbjct sequence = 1095/1102 and the homology was 99.36%. The gap, which was shown to be different from each other, was 7/1102 and showed a 0.63% phase difference. The positions were found at positions 132, 221, 1029 and 1102 in 39, 1067 and 1077 base and sbjct sequence in the query sequence.

Based on these results, SK1759, SK1760, SK1812 and SK1819 were divided into two groups, respectively. In each group, homology was 99.08% and 99.36% on 18S rDNA, respectively.

MEGA 4  Used Bacterial week Homology  compare

The BLAST search results for 18S rDNA of four SK1759, SK1760, SK1812, and SK1819 isolates isolated from natural fermented Omiza mats were shown in FIG. 4 and FIG. SK1811 was not shown in the figure because the search result type strain was not searched in the query GenBank.

Compared with the same type of strain confirmed the results of the strain SK1759, and SK1760 comparison of the two strain analysis on the GenBank (http://www.ncbi.nlm.nih.gov) using the software MEGA 4 is Kazachstania and 100% homology with viticola X99526 type strain. Based on this, SK1759 strain and SK1760 strain were identified as K azachst ania sp. SK1759 and Kazachstania sp. SK1760. The genus Kazachstania is the acacia separated from grapes fermented in Kazakhstan, Saccharomycetaceae family.

Comparison of SK1812 and SK1819 strains using MEGA 4 software revealed that Wickerhamomyces anomalus And 99% homology with AB126679 type strain. Therefore, these two strains were named Wickerhamomyces sp.

SK1811  Strain BLAST  Search

GenBank sequence data and alignment results using BLAST as the 18S rDNA sequence data of SK1811 strain are shown in Table 4. As compared with the GenBank sequence data using the MEGA 4 software, the SK1811 strain did not form any strain class. In BLAST search results, the accession numbers representing 99% max identity were AM279680 and AY518522, respectively. Candidakrissii JCM 9454 and Candida oleophila JCM 1620 strain. Max identity 98%, respectively; Bispora sp., Candida sp., Dedaryomyces sp., Marine sp., Pichia sp. And Taphrina sp. Respectively.

Table 4 is a homology search chart of SK1811.

Optimal culture temperature of isolate

The results of absorbance at 550 nm were plotted in Fig. 6 in order to investigate the optimum growth temperature range of five strains SK1759, SK1760, SK1811, SK1812 and SK1819 isolated from natural fermented omija. In YM broth, the growth curves of five strains showed almost identical growth curves, except for some strains by temperature. In the culture broth of YM broth at 25 ℃, the absorbance of O.D., SK1812 and SK1760 was 1.82 and 1.48, respectively, at 20, 30, 35, 40 and 45 ℃ treatment at 36 hours. There was a tendency for all five strains to grow uniformly at 20 ℃ and 35 ℃, but the absorbance was lower than 1.5 at the other temperature treatments and the maximum value of bacterial growth was found at 48 hours. Growth pattern.

The culture temperature of 25 ℃ was the most suitable growth temperature for all 5 isolates.

Omiza Park  Upon fermentation pH  change

The pH changes of 0, 2, 3, 7, 15, 21, and 28 days after inoculation of 5 strains isolated from natural fermented omija pak were shown in Fig. The pH of the control was not significantly changed near 3.0, but SK1819 and SK1760 showed a large pH change compared with the other three strains. The pH of omiza was 3.6, which is similar to the results of this experiment. Omija was originally strong in acidity and maintained low acidity, and some microbial species such as bacteria were too low to grow, so we could confirm that yeast species that are less affected by acidity can grow. After inoculation, it was confirmed that pH was kept in a low range of 3.0 to 4.5.

Omiza Park  Growth curve of strain inoculated with powder

Fig. 8 shows changes in the number of viable cell counts of viable bacteria at 25 占 폚 after inoculation of 5 strains isolated from natural fermented Omija pomaceae. The log ₁₀ (cfu / ml) value of the strain after the inoculation of the 0 day old strain culture solution sp. SK1759 strain and Kazachstania sp. SK1760 showed 8.11 and 8.03, respectively, and Wickerhamomyces sp. The log ₁₀ (cfu / ml) value of SK1819 strain was 7.26, and Pichia sp. SK1811, Wickerhamomyces sp. SK1812 showed 4.94 and 5.23, respectively. Wickerhamomyces sp. The SK1819 strain showed the highest log ₁₀ (cfu / ㎖) value of 8.85 on day 7 and the strain number of 8.26 was maintained up to 56 days of fermentation. Wickerhamomyces sp . The strain SK1812 exhibited a maximum of 7.82 on the 7th day of fermentation and showed 6.30 on the 56th day of fermentation. Kazachstania sp. SK1759 strain and Kazachstania sp. Treatment with SK1760 strains gradually decreased the number of bacilli to 2.14 and 0.77 on the 56th day of fermentation, respectively. Wickerhamomyces sp. The treatments inoculated with SK1811 strain rapidly dropped to 0.42 from the 3rd day of fermentation, and the number of bacteria did not increase again until 56 days of fermentation.

The pH of the fermented omija foil is 5.0 or less, and at a generally low pH, some microbial species such as bacteria do not grow well. The extracts of Omija seed were Lactobacillus plantarum (KCCM 11322), Bacillus subtilis (KCCM 11314), Escherichia coli (ATCC 10536), Staphylococcus aureus (KCCM 12103), Salmonella typhimurium (KCCM 11806), Penicillium citrirum (KCCM 11663) was strongly antimicrobial (Jung et al., 2000). In this study, the inoculated Kazachstania sp. SK1759 strain, Kazachstania sp. SK1760 strain and Candida sp. The reason why the SK1811 strain can not adapt and the number of bacteria is decreased seems to be the reason of breaking the omiza pak.

Example  2: Fermentation Omiza Park  Experiments on organic solvent extracts

The object of the present invention is to provide a method for the fermentation of &lt; RTI ID = 0.0 &gt; The organic solvent extracts of Omiza japonica fermented with the sp.SK1819 strain were used as the biosurfactant in the six fractional layers of the two kinds of omnivores including Eth-OH, n-Hexan, CHCl ₃ , EtOAc, Bu-OH and Aqueous Were compared.

Fermentation of Omiza Pak  Manufacturing and general ingredients

Fifteen kilograms of omega - starch powder for fermentation were ground with a blender (HR2860, Philips, Netherland) and filtered through a standard sieve (16 ^# , Chunggye sang gong sa, Seoul, Korea) Compassionate Wickermamomyces sp. SK1819 strain was selected. (J-SIL-R, JISICO, Japan) was inoculated in 5 ml of YM broth (1% glucose, 0.5% peptone, 0.3% malt extract, 0.3% yeast extract, 2.0% agar, Becton, Dickinson and Company, USA) Co., Ltd., Korea) for 48 hours. The culture was inoculated 1% in 100 ml of YM broth and cultured in a shaking incubator at 30 ° C for 36 hours. Inoculation was inoculated evenly with a sterile small sprayer (700 ml, Loving Home, Korea). Starter yeast for fermentation Wickerhamomyces sp. After the inoculation of SK1819 strain, the powder of Omija was fermented at room temperature for 30 days. Yeast cells inoculated with Starter were examined for the number of live yeast cells by flat plate solid smear method at 0, 3, 7, Actual measured values of live cells were expressed in terms of Log ₁₀ (cfu / g).

The general components of Omija pak and fermented omiza pak were analyzed according to the A.O.A.C method (Cunniff, 1997).

Organic solvent extraction

(1 ^# , Whatman, England) with 2 l of extractive ethanol (Merck, Darmstadt, Germany) and shaking 2 times a day for 7 days at room temperature and extracting 1 ㎏ of non fermented Omija and fermented Omija The filtrate from which the impurities were removed was subjected to ethanol extraction with a vacuum concentrator (NVC-2000, Eyela, Tokyo, Japan). Hexane (Sigma Co., St. Louis, Mo., USA) (100 ㎖ three times) and chloroform (Sigma Co., St. Louis, MO) were added to the distilled water-soluble extract layer and diluted with 200 ml of distilled water. (Sigma Co., St. Louis, Mo., USA) (3 times by 100 ml each) and butanol (Sigma Co., St. Louis, Mo., USA) (1 × 100 ml). The mixture was concentrated for more than 10 hours. The n-hexane, chloroform, ethyl acetate, butanol, and Aqueos layers were concentrated using a vacuum concentrator (FIG.

Antioxidant activity

gun polyphenol  Content measurement

In order to measure the total phenol content of non-fermented omiza mats and fermented omiza mats, folin-ciocalteau reagent and 10% Na ₂ CO ₃ were added to 1 ml of various organic solvent extracts based on the folin-denis method (Gutfinger, 1981) (G-560, Scientific Industries, USA). After incubation for 60 minutes at room temperature, the absorbance at 700 nm (Optical Density, OD) was measured with a UV spectrophotometer (UV1650PC, Shimadzu, Tokyo, Japan) ) Were measured. The absorbance was measured at 25, 50, 75 and 100 ㎍ / ㎖ of caffeic acid (Sigma Co., St. Louis, MO, USA) Standard calibration curve was prepared. The total phenol content of the sample extracts was calculated by comparing the absorbance of the standardized calibration curve and the sample extracts of the disclosed non - fermented omiza pulp and fermented omiza pak.

gun flavonoid  Content measurement

The total flavonoid content of non-fermented Omiza bean and fermented Omiza bean was measured (Moreno et al., 2000). 0.1 ml of 10% aluminum nitrate (Sigma Co., St. Louis, MO, USA) and 0.1 ml of 1 M potassium acetate (Sigma Co., St. Louis, Mo., USA) and 4.3 ml of ethanol were added to 0.5 ml of the measurement sample After mixing for 40 min at room temperature, the absorbance was measured at 415 nm using UV spectrophotometer. The absorbance of non-fermented Omija pak and fermented Omiza pak extract was measured by using a standard calibration curve obtained from quercetin (Sigma Co., St. Louis, MO, USA) at a concentration of 0, 25, 50, 75 and 100 ㎍ / The total flavonoid contents of fermented omija and fermented omija were estimated.

DPPH radical For Electron donating ability  Measure

The electron donating ability (EDA) of non-fermented Schisandra chinensis and fermented Schisandra chinensis extract was measured by measuring the reducing power of each sample by the electron donating effect of DPPH (α, α-diphenyl-picrylhydrazyl) on each extract sample. (Blais, 1958). 0.5 ml of a 0.5 mM DPPH (Sigma Co., St. Louis, MO, USA) solution (Abs. EtOH soln.) was added to a test tube in which 1 ml of ethanol, 10 μl of the sample and 990 μl of 100 mM sodium acetate buffer After incubation in a dark room for 5 minutes, the absorbance was measured at 517 nm using UV spectrophotometer (Lee and Lee, 2008). The electron donating ability was expressed by the following equation, and calculated by substituting the measured absorbance values of the treatment SA and the control CA.

DRSA: DPPH-Radicals Scavenging Activity

SA: Sample Absorbance

CA: Control Absorbance

SOD Similar activity  Measure

The superoxide dismutase (SOD) -like activity of fermented organic solvent extracts was determined by measuring the amount of pyrogallol that catalyzes the conversion of reactive oxygen species to hydrogen peroxide (H ₂ O ₂ ) (Marklund and Marklund, 1974) Indicating the degree of SOD-like activity in the sample. 2.6 ml of tris-HCl buffer (Sigma Co., St. Louis, Mo., USA) (50 mM tris [hydroxymethyl] amino-methane, 10 mM EDTA, pH 8.5) After adding 0.2 ml of 7.2 mM pyrogallol, the reaction was stopped by adding 0.1 ml of 1 N HCl (Sigma Co., St. Louis, MO, USA) to the reaction mixture at room temperature for 10 minutes. The amount of oxidized pyrogallol in the reaction solution was measured by using a spectrophotometer at 420 ㎚. SOD - like activity was expressed as a percentage of absorbance difference between the control sample and the control.

The concentration of the control of ascorbic acid is 100㎍ / ㎖, fermented and non-fermented Schisandra Chinensis foil layer foil Eth-OH, n-Hexan layer, CHCl ₃ Layer, the EtAOc layer, the Bu-OH layer, and the Aqueous layer were measured at 500 μg / ml, respectively.

SODA: Superoxide dismutase (SOD) activity

As: Absorbance after 10 minutes of 7.2 mM pyrogallol added sample

Ac: absorbance of control group

Antimicrobial Activity

To determine the antimicrobial activity of the organic extracts of non-fermented Omija and fermented Omija foams, Salmonella gallinarum ATCC 9184, Haemopillus Somnus , Haemopillus parasuis , Mannheimiahaemolytica , Enterotoxigenic E. coli Staphylococcus strains and Gram positive bacteria aureus strains were used.

LB (Luria-Bertani) broth, a medium for culture of harmful strains, was sterilized at 121 ° C for 12 to 15 minutes using an auto clave (MAC-501, Eyela, Tokyo, Japan) The minimum inhibition concentration (MIC) was determined by diluting the non-fermented Omija mackerel and fermented Omija organic solvent extract with 1/2 dilution on a 96-well plate (90096, SPL, Korea) (Breeuwer et al., 1996; Oyaizu, 1986).

Statistical processing

In vitro The statistical significance of the results of the experiments was analyzed using a statistical analysis system (SAS release version 9.2, SAS Inc., North Carolina, USA) (Barr and Goodnight, 1971) The test results were tested at 95% confidence interval using multiple range-test (Duncan, 1955). Each experiment was carried out in three replicates using the same three samples

The results of the above embodiment are as follows.

General ingredient content

The results of the analysis of the general components of non-fermented Omija bean and fermented Omija bean are shown in Table 5. From the results of the general composition analysis, fermented omijaek showed lower contents than other components except water and Ca. The general composition of fermented omija was 6.67% in moisture, 14.83% in crude protein, 21.67% in crude fat, 16.48% in crude fiber, 1.80% in ash, 0.07% in Ca and 0.33% in P. Non Fermented Omiza Pak and Wickerhamomyces sp. The moisture content of fermented omija foams was 1.5% higher than that of omija foams, crude protein was 1.46% lower and crude fat was 1.85% lower than that of SK1811 inoculated fermented omija foams. The crude fiber was also found to be 17.73% higher in the oyster mushroom, less than 0.08% in the fermented oyster mash, and the total P was higher by 0.01%. The Ca content was analyzed to be 0.07% in both cases. The content of common ingredients of Omiza fruit was 57.5% moisture, 18.8% crude fat, 12.6% carbohydrate, 11.1% crude protein, 5.4% crude fiber and 4.9% ash (Kim and Choi, And fermented omija foams.

Table 5 shows the results of the non-fermented Omiza gum by- product and Wickerhamomyces sp. Table showing the results of component analysis of SK1819 fermented omija ash by-product

Physiologically active substance content

gun polyphenol  content

The total polyphenol content of the organic solvent extracts of fermented omiza bacillus and non-fermented omiza bacillus was shown in Figure 10. The total polyphenol content in the five extracts except the EtOAc extract layer of the fermented organic solvent extracts of the fermented omiga- The total polyphenol content of the organic solvent extract was higher than that of the organic solvent extract. In the aqueous layer, 0.18 ㎎ / ㎖ was found for the Eth-OH layer, 0.22 ㎎ / ㎖ for the CHCl ₃ layer, 0.19 ㎎ / ㎖ for the n-Hexan layer and the Bu-OH layer, Ml. &Lt; / RTI &gt; The main anthocyanin of Omija was poenidin-3-glucoside (Yang et al., 1982).

When the total polyphenol content of fermented omija foams is higher than that of Omija foams, the order of concentration is 0.08 ㎎ / ㎖ higher in Bu-OH layer, about 1.7 times higher. n-Hexan layer was about 1.26 times higher than the total polyphenol concentration of 0.15 mg / ml of non-fermented Schizosaccharum. However, the total polyphenol content in the non - fermented Omiza - Pak EtOAc layer was 0.17 ㎎ / ㎖ higher than the 0.12 ㎎ / ㎖ of the fermented Omija organic solvent extract EtOAc layer. The total phenol content of white ginseng was 0.379% (Choi et al., 2006).

Although most phenol substances are effective for free radicals, they act selectively depending on the free radical substrate. Flavonoids change their free radical scavenging ability according to the substituent position, and the methoxyl group binds to the B-ring Free radical scavenging ability decreases.

Pu-Erh Tea (Tea) Streptomyces bacillaris (Jeng et al., 2007). It was reported that the contents of statin, gamma-aminobutyric acid (GABA) and total polyphenols were increased when fermenting Streptomyces cinereus . In addition, it has been reported that the analysis of various polyphenol components by natural fermentation by immersing black olives in brine (8% NaCl, 0.4% acetic acid) is greatly different (Romero et al., 2004). The main reaction during the fermentation process was acid hydrolysis of glucosides and aglycons, which was the most important phenol compound with 12 mM of hydroxytyrosol in the final fermentation product. As anthocyanins of fresh olives, cyanidin 3-rutinoside and cyanidin 3-glucoside were not detected after natural fermentation.

According to the experiment, the layer with higher total polyphenol content tends to become higher toward the hydrophilic organic solvent layer, which is in contrast to the higher total polyphenol content in the oil soluble organic solvent layer. Omiza gum powder is Wickerhamomyces sp. It is suggested that polyphenol is changed from lipid soluble to water soluble by fermentation through SK1819 strain.

gun Flavonoid  content

The flavonoid contents of the organic solvent extracts of the non-fermented Omija foil and the fermented Omija foil are shown in Figure 11. The flavonoid concentrations of the organic solvent extracts of Omija foil and fermented Omija foil were higher than those of the n-hexane extract layer except for the Eth- The content of flavonoids in the CHCl ₃ extract layer, the EtOAc extract layer, the Bu-OH extract layer and the aqueous layer was higher than that of the non-fermented Schizosaccharomyces sp. In particular, CHCI ₃ The flavonoid content of the extract layer is 604.7 ㎍ / ㎖, which is 22.82 times that of 26.5 ㎍ / ㎖ of Omija. The organic solvent extract flavonoid concentrations of the n-Hexan, CHCl ₃ , EtOAc, Bu-OH, and aqueous layers of fermented omiga paste were 90.35 ㎍ / ㎖, 604.67 ㎍ / ㎖, 235.64 ㎍ / 71.11 / / ㎖, 103.11 / / ㎖, respectively. The non-fermented omiza bacillus was 32.15 ㎍ / ㎖, 153.11 ㎍ / ㎖, 20.37 ㎍ / ㎖ and 78.20 ㎍ / ㎖. The flavonoid concentrations of organic solvent extracts of fermented omijaek were 2.81, 1.54, 3.52 and 1.32 times higher than those of non - fermented omija, respectively.

In this experiment, flavonoid extract was higher than that of non - fermented Schizosaccharomyces pombe which was fermented. It is considered that the fermentation process results in an increase in the extraction efficiency of materials that enhance the physiological performance such as antioxidant activity.

Anthocyanin  Content analysis

The results of measurement of anthocyanin extract concentration of non - fermented Omiza bean and fermented Omiza bean are shown in Figure 12. The anthocyanin content of fermented Omiza bean was 28.98 ㎎% The content of anthocyanin in Schizandra chinensis was 11.26 ㎎%. These results suggest that Wickerhamomyces The extracting rate of anthocyanin in fermented oyster mushroom by inoculation of sp.SK1819 strain was about 2.6 times higher than that of the anthocyanin extract in the absence of fermentation. It is thought that this is due to the decomposition of yeast and the effect of increasing the solubility of anthocyanin in the oyster shell due to the production of Eth-OH in the yeast.

The anthocyanin pigment, which is a functional substance, is known to have antioxidant activity, cholesterol lowering and improvement of vision, anticancer effect, inflammation, vascular protection, arteriosclerosis, prevention of heart disease, prevention of diabetes and protection against ultraviolet rays. Especially, The inhibitory effect of malondiadehyde (MDA) production is 12 times higher than tocopherol (Yi and Kim, 2010). The anthocyanin content of the North Korean semi-dried omija was 2.0 ㎎% (Cho et al., 2003), which was about 15 times higher in this study.

D PPH free electron donating ability of the radical

The results of measurement of the electron donating ability of DPPH free radicals of the five organic solvent extract samples of the disclosed Schizandra chinensis and fermented Schizosaccharomyces pomace are shown in Figure 13. DPPH free radicals shown in CHCl ₃ , EtAOc, Bu-OH and Aqueous layer extracts The electron donating ability of the solution showed a tendency to increase gradually.

DPPH shows the maximum absorbance at around 517 ㎚ with free radicals. When the electron is received, the absorbance decreases and the free radical is canceled due to its high antioxidative activity. As a measure of delayed aging.

The strong electron donating effect on the DPPH free radical of the Aqueous layer of fermented omija was shown to be about 186 times higher than that of unfermented Schizosaccharomyces pombe. The EtAOc layer extract of fermented Omiza mushroom exhibited about 8.26 times greater potency than the Omiza mushroom extract. The extract of fermented Omiza mushroom Bu-OH layer showed about 3.05 times higher effect than that of Omiza mushroom, and the CHCl ₃ layer The DPPH free radical scavenging ability of the extract was 2.47 times higher than that of Schizandra chinensis.

SOD Similar activity

The results of measurement of the SOD-like activity of the organic solvent extracts of the fermented omiza bacillus and the non-fermented omiza bacillus were shown in Figure 14. The similar activities of the organic extracts of the fermented omiza pomace were as follows: The organic extracts showed higher activity than the similar activities. In the whole organic solvent extract layer, the layer having the largest difference between the fermented omija and nonfermented omija foams was CHCl ₃ layer, and the fermented omija was 68.89% and the non-fermented omija was 46.47%. In the Eth-OH layer, 86.36% of the activity of fermented omija was 1.2 times higher than the activity of 71.49% of non-fermented omija. In the n-Hexan layer, 90.26% of the activity of fermented omija foil was about 1.1 times higher than the activity of non-fermented omija foil 82.7%. In the EtAOc layer, 76.66% of the fermented omiza pulp was active at a rate of about 1.1 times higher than that of the non-fermented omiza pulp at 67.37%, and the aqueous layer was about 1.2 times higher than that of the non-fermented omiza pulp .

The activity of hydrothermal extract of Omija was 28.0% at 1 mg / ㎖ and 83.2% at 10 ㎎ / ㎖, which was lower than that of the aqueous layer of 90 ㎍ / ㎖ concentration of 500 ㎍ / ㎖. As a result, SOD - like activity was 46.47 ~ 93.74% in non - fermented oyster mushroom and 68.89 ~ 90.39% in fermented oyster mushroom. The activity of SOD was similar to that of fruit, vegetable and medicinal herb. It was confirmed that the similar bow performance was very high.

Fermentation Omiza's  Antimicrobial activity

The results of the MIC antimicrobial activity of the yeast inoculated fermented omiza mushroom are shown in Table 6. Non-fermented Omisadak organic extract and Wickerhamomyces sp. The antimicrobial activity of the organic extract of non-fermented Schizandra chinensis extract and the fermented Schizandra chinensis extract was diluted with 1/2 dilution method to give the antimicrobial activity of the organic extract of Schizandra chinensis 100, 50, 25, 12.5, 6.25 and 3.13 mg / ml. The concentration of antimicrobial activity of each extract was in the range of 3.13-100 ㎎ / ㎖. Salmonella among five gram-positive bacteria gallinarum The strains showed antimicrobial activity from 12.50 mg / ㎖ of n-Hexan layer of non-fermented Omija foil and n-Hexan, CHCl ₃ , EtAOc and aqueous layer of fermented Omija foil, and Haemopillus Somnus The strain showed 3.13 ㎎ / ㎖ in the EtAOc layer of non - fermented Omija foil and showed antimicrobial effect in the EtAOc layer of fermented Omija foil at 6.25 ㎎ / ㎖. Haemopillus parasuis The strains were found to be 6.25 ㎎ / ㎖ in the EtAOc layer and aqueous layer of non-fermented Schizandra chinensis, Bu-OH layer and aqueous layer of fermented Schizandra chinensis. Enterotoxigenic E. coli The antimicrobial inhibitory effect of the non - fermented Omija pak organic extracts on the strains was high.

The antioxidant activity of the non - fermented Schizosaccharomyces rugosum extract was observed at the concentration of 3.13 ㎎ / ㎖ in the EtAOc layer. Gram-positive Staphylococcus aureus The antimicrobial activity of non - fermented Schizosaccharomyces pombe was observed from 12.5 ㎎ / ㎖ in EtAOc layer. However, the fermented Schizosaccharomyces pombe showed antimicrobial activity in both Eth - OH and EtAOc layers at 6.25 ㎎ / ㎖. The activity of non - fermented omija pak organic extracts was higher than that of fermented omija pak organic extracts in antifungal activity performance of six livestock - causing harmful strains. However, the aerobic performance of the fermented Omija mushroom in the aqueous layer was higher than that of the fermented mushroom.

To determine the antimicrobial activity of the ethanol extract of Omija, antimicrobial activity against 12 microorganisms including bacteria, yeast and fungi was measured. The extract inhibited the growth of bacteria but did not inhibit the growth of yeast and fungi. The minimum lethal concentration of B. subtilis and S. aureus was 1.6 ~ 3.2 ㎎ / ㎖, and the minimum lethal concentration of gram negative bacteria including E. coli was 6.3-12.5 ㎎ / ㎖.

Example  3: In-feed fermentation Omiza Park  Effect of supplementation on productivity of laying hens

In this experiment, we used Wickerhamomyces sp. In order to investigate the effect of fermented omija (fermented soybean oil) fermented with SK1819 strain on the productivity of laying hens during the addition of 0.25% and 0.50% Proceeded for the purpose

Fermentation Omiza Park  Manufacturing and experimental feed

The fermented omiga paste used in the experiment was added to 2 kg of non-fermented omiga paste powder, Wickerhamo- myces sp. SK1819 strain YM culture was inoculated at 1%, and fermented at room temperature for 30 days and then exposed.

Experimental laying hens were purchased from Seoul diets and corn - soybean meal - based diets were used. Experimental diets were fed with corn-soybean meal as basic feedstuffs with 2,780 ㎉ / ㎏ of ME, 16.50% of CP, 0.21% of phosphates, and 3.86% of calcium, based on NRC (1994) ).

Experimental animal and experimental design

In this test, the number of 45-week Hy-Line Brown 180 was used as an experimental animal. From April 23rd to May 13th, 2011, the experiment was conducted for 5 weeks. After the feed adaptation period of 1 week, the fermented omija nut was tested for 4 weeks.

NFS50 (normal feed + non fermented omija foil 0.25%), NFS50 (general feed + non fermented omija foil 0.5%), WFS25 (general feed + Wickerhamomyces) were used as the control. sp. &Lt; / RTI &gt; 0.25%) and WFS50 (general feed + Wickerhamomyces sp. SK1819 strain fermented omiza pak (0.5%)), 5 treatments, 4 replicates, and 9 replicates per batch.

Spec Management and Processing

The breeding space was divided into three groups of 90 cage, 90 cage and 90 ㎝. Drinking water was freely collected through an automatic watering nipple installed per one of three laying hens. Nipple and feed trough numbers were installed in the same way.

The lighting management was performed using the automatic lighting controller at 16L: 8D, and other breeding management methods were conducted based on the Hy-Line specification management guide (Hy-Line, 2006). The cumulative number of rearing per treatment was calculated by applying to the feed intake and egg productivity based on the date of our company 's death.

This experiment was carried out and sacrificed according to the regulations of the Experimental Animal Ethics Committee of Konkuk University.

Survey items and methods

Feed intake and egg productivity and Our company

Feed intake and egg productivity were investigated in order to investigate the effect of supplementation of fermented omiza mats on the productivity of laying hens. The amount of feed was divided into daily treatments, and the amount of feed was considered as the actual amount of feed. The remaining amount in the feed feed and the amount of feed from the bottom of the feed in the actual feed amount and the amount of feed lost in the bottom were calculated, The feed intake was calculated by subtracting the mortality from the total number of eggs in the experimental system.

The egg production rate was confirmed at 14 o'clock every day during the specimen run. The eggs produced by the treatments were divided into the total number of eggs laid by total number of eggs combined with the number of eggs, the number of eggs, the number of eggs, and the number of bluegrass. Average egg weight was measured by weighing eggs of each treatments at the same day and the average value of the total weight of the eggs divided by the number of eggs was indicated in the results.

Ovary and I horny

The eggshell strength, egg thickness, and crustal quality were investigated in order to investigate the effects of feed intake on the egg quality, Fujihara Industry Co., Ltd., Japan). The eggshell thickness was determined by measuring the egg shell thickness at the central part of the egg after the measurement of egg shell strength using an average thickness of 20 eggs measured by an egg shell (FHK Peacock, Fujihara Industry Co., Ltd., Japan). The Haugh unit of the eggs was calculated by comparing the egg height to the egg weight (FHK, Fujihara Industry Co., Ltd., Japan).

Haugh unit = 100 log (H + 7.75 - 1.7W 0 .37)

H = height of egg white (mm)

W = lunar (g)

The egg yolk color and egg shell color comparison were visually compared with the colorimeter to indicate the chromaticity. The egg color was measured using an egg shell colorimeter (QCM ⁺ , Technical Services and suplies, York Ltd., England). The egg color was compared using a Roche egg yolk color fan (Roche, Switzerland).

Long-term weight and Enterobacteriaceae  Census

In order to determine the relative weights of the organs of the livestock, the weight of the liver and spleen was measured by 8 digits of treatment distinction.

Tenth of the manure in the small intestine and the cecum by taking 1 g per day dilution method In order to examine the intestinal microflora of about 10 ^-1 to about 10 ^- each processing distinguished intestinal E. coil, Yeast, the total number of bacteria was diluted with ⁷ log ₁₀ concentration (cfu / g).

Statistical processing

in vivo Statistical analysis was performed using a statistical analysis system (SAS release version 9.2, SAS Inc., North Carolina, USA) (Barr and Goodnight, 1971) and Abrahamse, 1969). The significance of the treatment interval was tested using the Duncan's multiple range-test (Duncan, 1955). The Q-test (Muranaka, 1999) before the data of each treatment was processed by SAS, and the values outside the 95% confidence interval were discarded. Each experiment was carried out in 4 replicates, 9 replicates per treatment.

The results of the above embodiment are as follows.

Feed intake and mortality

Table 8 shows the effects of 0.25% and 0.50% addition of fermented omiza bacon and nonfermented omiza bacon on feed intake and mortality of laying hens, respectively. In this experiment, there was no significant difference in feed intake between treatments according to supplement level. Feed intake was highest in the order of WFS50, control, WFS25 and NFS25 in the order of NFS50 supplemented with 0.50% of unfermented Omija bark.

In the whole experimental group, there was no significant difference in the treatments between the WFS50 treatment on the 12th day of the experiment, 1 of the NFS25 treatment on the 14th day and 1 case of the WFS25 treatment on the 16th day of the experiment. In a total of 35 days of experiment, including 7 days of adaptation period, 3 out of 180 specimens resulted in mortality of about 1.67% of the total test area, which is lower than the average of 3% of other studies (Choi and Son, 2006)

I am productive

Table 9 shows the effect of 0.25% and 0.50% addition of fermented omiza bacon and nonfermented omiza bacon on egg production in laying hens. In this experiment, the productivity of eggs was compared between 1 ~ 2 weeks and 3 ~ 4 weeks. I am productive at 1-2 weeks in general commercial feed Wickerhamomyces sp. The productivity of WFS25 treated with 0.25% of Omiza pomaceus cultured inoculated with SK1819 strain was 90.83%, which showed significant difference ( P < 0.05). The next highest productivity was 0.50% of fermented Omija The productivity of the WFS50 was 87.63%. In the 3rd and 4th weeks, productivity of WFS50 reached 91.63%, which was significantly higher than that of control (88.69%) and WFS25 (88.03%). The yield of NFS50 was 86.30, which was slightly higher than that of NFS25 treated with 0.25% non - fermented omija.

The egg production rate was 73.4%, 73.9% and 74.2%, respectively, but the difference was not significant compared to the control (71.7%) (Kim et al., 2006). In addition, the egg production rate of 0.1, 0.2% addition treatment of crude herbaceous herb which contains Angelica japonica, Rhizopus spp., Glycyrrhizae spp., Chungcheongcho, Dongguk, Donggol and Omija were significantly decreased in laying hens in high temperature stress environment. However, 0.05% Showed similar productivity (Min et al., 2004). In this experiment, the productivity of the basic diet, fermented omija bacon and non-fermented omija bacillus was higher than that of other herbal medicines mixed with other herbal medicines such as fermented bacon and nonfermented bacon. .

Fermented omiza mats and fermented omija mats treated with 0.25% and 0.50% non - fermented omiza mats were significantly higher than those of the control ( P < 0.05 ). The highest NFS25 treatment was 61.56 g, followed by NFS50 treated 61.40 g, WFS50 treated 61.10 g and WFS25 treated 61.04 g, respectively. In the third and fourth weeks of experiment, NFS25 treatment was 57.59 g, NFS50 treatment was 57.62 g, WFS50 treatment was 57.52 g and WFS25 treatment was 56.82 g. There was no enemy difference.

The egg weight was 60.47 g and 60.27 g in the crude herb treatment at 4 weeks after the initiation of the test when the crude herb medicine was added to the laying hens in the high temperature stress environment at a certain ratio of 0.1, 0.2% (Min et al., 2004).

Egg mass, which accounts for the daily egg productivity, is expressed in Wickerhamomyces sp. ( P < 0.05) compared with control group (50.26 g / day) in WFS25 treated with 0.25% of fermented omiza bacillus inoculated with SK1819 strain. WFS50 treatment 53.55 g / day, NFP50 treatment 52.99 g / day and NFS 25 treatment 61.56 g / day. The egg weight of 3 ~ 4 weeks of the experimental diets was not significantly different from that of the experimental diets. The dietary supplementation of 0.25% fermented omija was shown to be good productivity, and the productivity of non - fermented omija was increased There was no phenomenon.

The results of the analysis on the egg quality of the 1 st to 2 nd and 3 nd to 4 th males in the laying hens with fermented omija and non fermented omija were shown in Table 10. The eggshell color was measured ( P < 0.05), which was higher than that of the control (25.58), in the WFS25 treated group (0.25%). Egg yolk showed the highest color intensity at 7.97. Anthocyanin was thought to have affected the egg shell color without affecting the yolk color affected by fat - soluble pigment as a water - soluble substance.

In this experiment, the extraction effect of anthocyanin, a useful pigment of fermented omija, was 257.37% higher than that of unfermented omija, but it was considered that the effect was affected to some extent. In WFS50 treated with 0.50% . Egg shell thickness of 1-2 37.35 of the control to the parking ㎜ / ₁₀₀ and NFS25 treatment 37.60 ㎜ / ₁₀₀ was higher than the other groups egg shell thickness of the eggs produced between the test proceeds 3-4 cycles NFS50 treatment is 36.90 ㎜ / ₁₀₀ in Results showed significant difference ( P < 0.05) between treatments than control (34.85). WFS50 and WFS25 treatments were slightly higher, but there was no significant difference.

In order to investigate the effects of mixed herb extracts such as omija and aruguswort on the productivity, egg quality, blood characteristics and egg shelter of laying hens, Haugh unit was 100.7 ( P < 0.05), but did not show significant differences in egg yolk color, egg shell color, egg shell thickness, egg shell strength.

Fatty acids in eggs

In order to investigate the effects of supplemented fermented omiza bacon and nonfermented omiza bacon on the composition of egg yolk ingredient contents, the content of fatty acid components in the 4th egg yolk of the end of the experiment was analyzed and the results are shown in Table 11. Wickerhamomyces sp. Stearate (C18: 0), oleate (C18: 1n9), total MUFA and arachidonate (C20: 4ω6) were significantly higher in WFS25 treated with 0.25% ( P < 0.05). stearate is 8.47% of control to 9.23%, oleate is 43.75% of control by 49.09%, total MUFA represents a compared to 2.35% in the control group to 49.19% in the control 48.02% and arachidonate 2.47% high difference significantly (P <0.05) . Wickerhamomyces sp. In the WFS50 supplemented with 0.50% non-fermented omiza paste fermented with SK1819 strain, the total SFA was 34.74%, which was significantly higher than the control 34.43%. In the NFS25 supplemented with 0.25% ( P < 0.05). Total PUFAs were 19.98% and total PUFA / SFA was 0.59, which was the highest among all treatments ( P < 0.05). In the NFS50 treated with 0.50% non - fermented omiza pork, the total ω6 / ω3 value was 19.91.

Palmitate and oleate account for a large proportion of egg yolk in the egg yolk (Koo et al., 2002). In this study, oleate was the highest content in WFS25 and palmitate was the highest in the treatments. Unsaturated fatty acids are considered to be beneficial to human health as compared to saturated fatty acids because they are considered to be less likely to cause symptoms such as hyperlipemia after being ingested into the body because the chain of fatty acids is fluid. Palmitate and oleate constitute monounsaturated fatty acids, linolenate, arachidonate, EPA and DHA constitute polyunsaturated fatty acids. Wickerhamomyces sp. The contents of oleate, total MUFA, and arachidonate were significantly higher in WFS25 supplemented with 0.25% of omza - zoo fermented with SK1819 strain, but omega - 3 fatty acids were not detected. When diets supplemented with fermented or unfermented Omija bamboo were fed to the laying hens, the fatty acid compositions of the egg yolks varied according to the types of fatty acids, and there was no consistent change depending on the kinds and contents of the additives, It is difficult to interpret the difference in the fixation of fatty acid in the treatment zone as the influence of the additive.

With organ  Abdominal cavity  Fat

The effects of dietary supplementation on the liver, spleen, and abdominal fat of the fermented omiza bacillus and nonfermentous omiza bacillus were shown in Table 12 as percentages by weight.

The ratio of body weight of liver, spleen and abdominal fat to weight of liver, spleen, and abdominal fat in control group and control group was higher than that of control group in 0, 0.25, 0.50% There was no significant difference in the interval. In order to investigate the effect of Omiza extract on the growth of obesity-related intestinal bacteria, the initial weights of 227.4 g and 239.3 g of the 0.5% addition of 0.5% The final body weight was 409.8 g and 408.7 g, indicating no significant difference in the treatment interval (Jeong et al., 2009). This indicates that the self-physiologically active substance was lower Therefore, it is considered that the short-term consumption of Omija component has little effect on organ and abdominal fat.

Total of intestines and appendixes Number of bacteria

The number of intestinal and cecal microflora was investigated in order to investigate the difference of total bacterial counts between 0.25% and 0.50% fed fermented and nonfermented omija foams in laying hens. The results are shown in Table 13.

Total bacterial counts were obtained from E. coli using mac conkey agar using nutrient agar. The total number of bacteria and E. coli were significantly lower ( P < 0.05) in WFS25 treated with 0.25% fermented omija foil and WFS50 treated with 0.50% fermented omija foil. WFS25 and WFS50 treatments were 4.65 and 4.64, respectively, and significantly lower than those of control (5.00). As the values shown in the table are converted to log ₁₀ (cfu / g), the actual number of bacteria is about 3 to 4 times larger than that of the control. The yeast number in the small intestine was measured using a YM agar medium, NFS25 and WFS25 treatments were significantly higher ( P < 0.05). And 2.55 and 2.51, respectively, which were significantly different from the control (2.05) ( P < 0.05).

In the cecal yeast, WFS25 treated with 0.25% fermented omija foil and WFS50 treated with 0.50% fermented omija foil showed 2.75 and 2.75, respectively, which were significantly higher than those of control (2.20 and P < 0.05). In the YM agar medium in the cecum, there was no significant difference in the bacterial counts of E. coli and E. coli between the treatments as the small intestine .

The viable cell counts of Lactobacillus sp . In broiler chicks fed 10 - day - old chickens were significantly higher than those in broiler chicks fed 10 - day - old chickens . It was reported that lactic acid bacilli increased in the intestine when 10 ⁹ cfu / g of culture was fed (Jin, 1996). Lactobacillus culture was fed to broiler chickens and the weight of experimental broiler chickens during 6 weeks of experiment was significantly (Kalavathy et al., 2008), showing a significant difference ( P < 0.05) for 1700.33 g of control at 42 days of age.

In this experiment, the difference in microflora between the small intestine and the cecum seems to be due to the microorganisms of Schizandra chinensis and Wickerhamomyces sp.

Agricultural Biotechnology Research Institute KACC91725 20120703

Claims (6)

Wickerhamomyces strain fermenting Schisandra chinensis. The strain according to claim 1, wherein the strain is Wickerhamomyces anomalus SK1819 (KACC91725P) strain. A method of obtaining polyphenol by inoculating fermented Schisandra chinensis seed according to claim 1 or 2, and then extracting the fermented Schisandra chinensis with an organic solvent. After inoculating fermented strains of claim 1 or 2 in Schisandra chinensis, the fermented Schisandra chinensis extract is extracted with an organic solvent to obtain flavonoids or anthocyanins. A method of obtaining antimicrobial composition by inoculating fermented Schisandra chinensis seed according to claim 1 or 2, and then extracting the fermented Schisandra chinensis with organic solvent. Feed composition comprising a Schisandra chinensis fermented with the strain of claim 1 or 2 as an active ingredient.

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