KR20190049043A - Microorganisms for fermentation of whole crop silage and Microbial additions including the same - Google Patents

Abstract

The present invention relates to microorganisms for whole crop silage fermentation and a microbial additive comprising the same. The present invention provides novel microorganisms, Cellulosilyticum sp. WCF-2 (KACC92181P) as microorganisms for whole crop silage fermentation. A microbial additive for producing whole crop silage comprises, as microorganisms for whole crop silage fermentation, cellulose decomposing microorganisms or mixed microorganisms including cellulose decomposing microorganisms and lactic acid bacteria. According to the present invention, dependency on import feed may be reduced, and silage feed, which has improved shelf life and digestibility compared to existing silage and can be used to feed pigs as well as ruminants, may be provided.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fermentation microorganism,

The present invention relates to a microorganism for fermentation of whole grain silage fermentation and a microorganism additive comprising the same, and more particularly, to a microorganism for fermentation of whole grain silage fermented by a total microorganism which can be applied not only to ruminant breeding but also to pig farming using microorganisms containing fibrinolytic bacteria or fibrin degrading bacteria and lactic acid bacteria And a microorganism additive comprising the microorganism.

Ruminants are important animal resources of humankind that do not compete with humans for food by using forage. Raw materials that are overproduced in the growing season are dried or fermented and fed to ruminants in the winter season. The fermented feed is called silage (dipping).

In other words, silage is a method of cutting grass and forage crops and putting them in a silo and maintaining low pH of pH 4.0 by lactic acid produced by lactic fermentation under airless anaerobic conditions, Quality veterinary expenses.

However, such a conventional method of producing silage relies on natural fermentation as in the case of conventional kimchi manufacturing, and there is a high possibility of abnormal fermentation and corruption, and it is difficult to obtain a uniform product.

Therefore, microorganisms are inoculated into plant silage in order to reduce the chance of growth of bad microorganisms and promote lactic acid fermentation. Such microorganisms are generally called inoculants, and the quality of silage is improved by adding this inoculant And productivity of livestock (yield and meat production) is improved.

It is a well-known fact that in Korea, 9,350 hectare pigs are being raised as of 2007, and most of them are being fed with pig-fed feeds, which is a concentrated cereal-based diet, which is in competition with human food resources. However, overfeeding only concentrated concentrates may result in overproduction of body fat, as well as weakening the digestive organs, which can interfere with the health and physiology of the breeding pigs. In addition, pregnant women have severe constipation before and after delivery. Since severe constipation can be associated with anemia (M.M.A), it is known that sufficient water intake and fiber-rich feed are needed.

Therefore, there are many studies on the possibility of application as feed for pigs by using silage, which is often used only in ruminant animals. However, the results of the study on microorganisms for silage fermentation suitable for pig farming are insufficient.

1. Korean Patent No. 10-0341796 " Strain suitable for plant silage fermentation with low sugar content and method for producing silage using the same " 2. Japanese Patent Registration No. P5931064 " Method for preparing new lactic acid bacteria and silage or fermented feed using the same "

1. Final Report of Agricultural and Rural Development Project (May 24, 2007): "Study on the Manufacture and Specification Technology of Whole-Paddy Pulse Silage Manufacturing"

It is an object of the present invention to provide a microorganism for fermentation of whole wheat silage, which is applicable not only to ruminants but also to pigs as well as to ruminants, by improving the low preservability of existing silages and increasing the digestibility thereof, and a microbial additive containing the same.

In order to accomplish the above object, the present invention relates to a microorganism for fermenting whole grain silage fermentation, which comprises a microorganism belonging to the genus Cellulosilyticum sp.) WCF-2 strain (KACC92181P).

In addition, the microbial additive for producing whole-grain pulp silage of the present invention is a microorganism for fermentation of whole-wheat silage fermentation, which contains a fibrinolytic fungus. The fibrinolytic fungus is a new microorganism Cellulosilyticum sp. WCF-2 strain (KACC92181P).

The microorganism additive may further include lactic acid bacteria, and the lactic acid bacteria may be Lactobacillus sp. Strain 5-1 (KACC 19351), which is a novel microorganism.

Another production method of the present inventors whole aneurysm silage is, comprising a plurality of the fermentation step for the entry into force of the whole aneurysm, and at least one fermentation step in the fermentation step in sila ET glutamicum Cellulofine (Cellulosilyticum sp.) WCF-2 strain (KACC92181P) or Cellulosilyticumum (KACC92181P) and 5-1 strain of Lactobacillus sp. (KACC 19351).

The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems.

According to the present invention, the reliance on imported feed is reduced, the preservability and digestibility of existing silages are improved, and silage feeds capable of raising pigs as well as ruminants can be provided.

1 is a diagram showing the molecular system of a fibrinolytic strain selected according to Example 1. (GenBank accession number in parentheses.)
2 is a diagram showing the molecular system of lactic acid bacterial strains selected according to Example 2. (GenBank accession number in parentheses indicates that CL is a commercial strain)
3 is a graph showing the degree of decomposition activity of the filter paper of the selective fibrinolytic bacterium (WCF-2) of Example 1. Fig.
Fig. 4 is a graph showing the degree of decomposition activity of the filter paper by pH of the selected fibrinolytic bacterium (WCF-2) of Example 1. Fig.
Figure 5 is a graph showing the results of the transgenic fungus Trichoderm reesei as in Example 1 starting cellulolytic bacteria (WCF-2) and the closely related species C. lentocelum of DSM 5427 ^T < / RTI > strain.
6 is a graph showing the growth rate on the MRS medium for the selected lactic acid bacteria of Example 2. FIG.
FIG. 7 is a graph showing the rate of pH drop on the MRS medium for the selected lactic acid bacteria of Example 2. FIG.
8 is a graph showing the growth rate of the selected lactic acid bacterium of Example 2 on a naked barley extract medium.
FIG. 9 is a graph showing the rate of pH drop on the naked barley extract medium of the starting lactic acid bacteria of Example 2. FIG.
To Figure 10 to determine the growth inhibition of silage represents bupaegyun targeting the selected lactic acid bacteria of Example 2, Fusarium graminearum And the time taken for KACC 41040 to reach OD ₆₀₀ = 0.5.
11 is a graph showing the growth inhibition of silage representative rot fungus in the selected lactic acid bacteria of Example 2. Aspergillus fumigatus And the time taken for KACC 41016 to reach OD ₆₀₀ = 0.5.
12 is a graph showing the degree of inhibition of the growth of representative silage rot fungi in the selected lactic acid bacteria of Example 2, glabrata KCTC 7219 is measured by measuring the time taken to reach OD ₆₀₀ = 0.5.
FIG. 13 is a view showing the silage prepared by inoculating the selected strain of the present invention. FIG.
FIG. 14 is a graph showing pH decay rate measured in the production of silage according to the selected strain of the present invention. FIG.
FIG. 15 is a graph showing the amount of gas generated when a supernatant of a pig body is inoculated into a silage prepared by inoculating the selected strain of the present invention. FIG.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

The inventors of the present invention have conducted several studies to extend the possibility of using silage which was used only as feed for ruminant animals, and as a feed for pig farming, it has been found out that microorganisms 2 for silage fermentation which can increase the digestibility, One of which belongs to the genus Cellulosilyticum sp. And the other belongs to the genus Lactobacillus sp.

As a result, the WCF-2 strain was applied as a microorganism for silage fermentation of whole grains according to one aspect of the present invention. As a result, the 16S rRNA gene sequence a novel of silanol ET glutamicum in (Cellulosilyticum sp.) bar, cellulose belonging to the results, the starter strain WCF-2 is cellulose close to the strain of the Silas ET glutamicum in (Cellulosilyticum sp.) closely related confirming the molecular schematic It turned out to be a strain.

Therefore, the present invention named the above-mentioned strain as Cellulosilyticum sp. WCF-2 strain, which was deposited with the National Institute of Agricultural Science and Technology of the National Institute of Agricultural Science and Technology on June 13, KACC92181P, which was confirmed to consist of the 1,453 bp nucleotide sequence described in SEQ ID NO: 1.

The 5-1 strain, which is another isolate, is closely related to the strain of Lactobacillus sp. As a result of confirming the molecular system based on the 16S rRNA gene sequence. In the present invention, the 5-1 strain belongs to the genus Lactobacillus ( Lactobacillus sp.) 5-1. This strain was deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, and received the accession number KACC 19351 on June 20, 2017. This strain was named 1,489 bp As shown in Fig. The 5-1 strain has been found to be able to obtain higher efficacy than the WCF-2 strain when it is used together with the WCF-2 strain, because it enhances the silage fermentation speed and increases the silage preservation.

Accordingly, the microorganism additive for producing whole-grain vinegar silage of the present invention is a microorganism for fermentation of whole-grain vinegar silage, which contains a fibrinolytic microorganism. Preferably, the cellulolytic microorganism is Cellulosilyticum sp. WCF -2 strain (KACC92181P), or the microorganism additive further comprises lactic acid bacteria. Preferably, the lactic acid bacteria is a novel microorganism, Lactobacillus sp. 5-1 strain (KACC 19351) It is best to use.

A method of manufacturing a total barley silage according to one aspect of the present invention includes a plurality of fermentation steps for fermentation of whole pulp, at least one fermentation step of the fermentation step is a cellulosilyticum sp. The WCF-2 strain (KACC92181P) or the Cellulosilyticum genus sp.) WCF-2 strain (KACC92181P) and Lactobacillus sp. 5-1 strain (KACC 19351).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

<Example 1> Isolation of cellulose-degrading strains and confirmation of molecular hierarchy

1) Strain isolation and culture method

Anaerobic bacteria with high fibrinolytic ability were purified.

Specifically, in June 2015, nine samples of oyster samples and nine samples of silage samples collected from Youngdong, Boeun, Cheongju, Icheon, Chuncheon, Wanju and Pohang were inoculated on CM3 medium containing cellulose powder as the sole carbon source, The volume of gas produced during anaerobic cultivation was measured and 14 strains with excellent gas production were selected. Of these, only WCF-2 strain was identified as Whatman No. 1 strain. 1 filter paper was completely disassembled. Therefore, the study was conducted using only this strain.

The composition of CM3 medium is as follows (g / L): (NH 4) SO 4 1.3, KH 2 PO 4 1.5, K 2 HPO 4 2.21, MgCl 2 6H 2 O 0.2, CaCl 2 0.057, FeSO 4 7H 2 O 0.0013, resazurin 0.0005, Yeast extract 2, L-cystein-HCl 0.45, Na ₂ CO ₃ 2, trance mineral solution 10 ml, pH 6.0,

The trace mineral solution has the following composition (g / L): nitrilotriacetic acid 1.5, MgSO ₄ 7H ₂ O 3, MnSO ₄ H ₂ O 0.45, NaCl 1, FeSO ₄ 7H ₂ O 0.1, CoCl ₂ 0.1, CaCl 2 2H 2 O 0.1, ₄ 7H ₂ O 0.18, CuSO ₄ 5H ₂ O 0.01, K ₂ Al ₂ (SO ₄ ) ₄ 24H ₂ O 0.09, H ₃ BO ₃ 0.01, NaMoO ₄ 2H ₂ O 0.025, NiSO ₄ 6H ₂ O 0.024.

2) Identification of molecular system

Based on the 16S rRNA gene sequence analysis of the fungal standard strain, the molecular structure of the isolated fibrinolytic strain (WCF-2) was confirmed by a neighboring joining tree (Fig. 1: parentheses GenBank accession number in.)

3) Results

As a result of the above experiment, it was confirmed that the new strain selected in Example 1 was identified as a final new microorganism strain Cellulosilyticum sp., And as described in the attached deposit certificate, the cellulosilicateum Genus Cellulosilyticum This strain was deposited with the Institute of Agricultural Biotechnology and received the accession number KACC92181P on June 13, 2017.

Example 2 Isolation and Identification of Lactic Acid Bacterium Strains

1) Strain isolation and culture method

In order to increase the fermentation speed of silage, 16 kinds of lactic acid bacterial strains were isolated as shown in Table 1 by obtaining silage, cow milk,

Specifically, in June 2015, we visited a barn at Chungju, Icheon, Chuncheon, Sacheon, Boeun, and Yeongdong, sampled from the bottom of the barn and feeding silage, placed in a zipper bag and transferred to the laboratory Respectively. The silage and cow milk samples were appropriately diluted in phosphate buffer and applied to deMan Rogosa Sharpe medium (MRS, pH 6.5, Difco) and Lactobacillus Selection medium (LBS, pH 5.5, Difco).

(DG250, Don Whitley Scientific) at 28 ℃, followed by subculture in the same medium with fast growth rate and purified. The commercially available commercially available lactic acid bacteria (C strain) (CL strain) was used as a comparative strain Respectively.

Strain badge Separation area Separation source 14-1 LBS Chungbuk Cheongju Rice straw silage 5-1 MRS Kangwon Chuncheon Rice straw silage 3-1 MRS Gyeongcheon Rice straw silage 11-1 MRS Kangwon Chuncheon Rye silage 6-2-1 MRS Kangwon Chuncheon Corn silage 17-1 LBS Kangwon Chuncheon Rice straw silage 13-1 MRS Kangwon Chuncheon Ubuntu KM-2 MRS Gyeongnam Sacheon Culture solution 16-2 LBS Chungbuk Cheongju Rice straw silage 13-2 MRS Kangwon Chuncheon Ubuntu KL-1-1 LBS Gyeongnam Sacheon Culture solution 27-1 MRS Gyeongcheon Rice straw silage 32-2 LBS Chungbuk Cheongju Ubuntu 31-2 LBS Gyeongcheon Ubuntu 29-1 MRS Boeun Chungbuk Ubuntu 24-1 LBS Gyeongcheon Rice straw silage

2) Identification of molecular system

Based on the 16S rRNA gene sequencing analysis of the standard strains of the isolated lactic acid bacteria as shown in Table 1 above, the molecular schematics created by the neighboring joining tree were confirmed (Fig. 2: GenBank accession in parentheses number. CL indicates a commercial strain).

3) Results

As a result of the above-mentioned experiment, the new strains selected in Example 2 were selected from strains of Lactobacillus sp. Or Enterococcus sp.) or it was found that the way Cellar in (Weissella sp.) identified as strain, the final fermentation rate as compared to Figure treat growth inhibition in their growth rate per medium, using pH drop rate, carbohydrate also, silage bupaegyun Strains exhibiting a high degree of inhibition against the silage rot fungus were selected to be the highest (backed by Examples 3 to 5 below).

The strain 5-1 was finally selected and identified as a Lactobacillus sp. Strain. As described in the deposit document attached thereto, Lactobacillus sp. Strain 5-1 This strain was deposited with the Institute of Agricultural Biotechnology and received grant number KACC 19351 on June 20, 2017.

EXPERIMENTAL EXAMPLE 1 Physiochemical Characterization of the Selective Fibrinolytic Bacterium (WCF-2) of Example 1

1. Experimental Method

Based on the molecular diagram of Fig. 1, the selective fibrinolytic bacterium (WCF-2) of Example 1 and C. lentocelum The physiochemical properties of DSM 5427 ^T strain were compared.

Specifically, all experiments were carried out under anaerobic conditions, unless otherwise noted, and anaerobic conditions were established using nitrogen gas or anaerobic packs (AnaeroGen, Oxoid). Changes in temperature, salinity, and pH were measured using a 96-well plate. 5 μl of the culture medium, which had been cultured at 28 ° C for 1 day in CM3 (a medium containing Celobiose as a sole carbon source), was added to 100 μl of CM3 medium, which had been divided into 96-well plates, While absorbance was measured with Spectra Max M2 (Molecular Device).

 The effect of salinity was determined by inoculating and culturing medium containing 0 to 7% of NaCl in CM3 medium and the pH of CM3 medium in the range of 3 to 10 with HCl and NaOH, Was fixed at 28 ° C. Carbohydrate availability was analyzed using API 50A (bioMerieux). The organic acid was cultured in CM3 medium containing filter paper as the only carbon source for 28 days and 16 days, and the filtrate was analyzed by HPLC.

2. Experimental results

The results are shown in Table 2 below.

Growth range (optimum) WCF-2 DSM 5427 ^T    pH 6-10 (8) 6-10 (6-7)    NaCl (%) 0-1 (0) 0-2 (0-1)    Temperature (° C) 20-45 (30) 20-35 (30) Growth on    L-tryptophane - -    Urea - -    D-glucose w +    D-mannitol - w    D-lactose - w    D-saccharose - +    D-maltose + +    Salicin - -    D-xylose - -    L-arabinose - -    Gelatin - -    Esculin + -    Glycerol - w    D-cellobise + +    D-mannose - -    D-melezitose - -    D-raffinose - -    D-sorbitol - -    D-rhamnose - -    D-trehalose - - Fermentation product (g / L)    Citric acid 0.01 0.03    Lactic acid 0.26 0.02    Formic acid 1.09 1.01    Acetic acid 0.74 0.71    Ethanol 0.87 0.57

As shown in Table 2, it was confirmed that there is a difference in the physiochemical characteristics exhibited even when belonging to the same species, which shows that there is also a difference in the degree of decomposition activity of the filter pater (related to the degradation of pulverization).

Experimental Example 2 Determination of Degree of Degradation Activity of Filter Paper of First Fibrous Degraded Bacterium (WCF-2) of Example 1

1. Experimental Method

Based on the molecular diagram of Fig. 1, the selective fibrinolytic bacterium (WCF-2) of Example 1 and C. lentocelum The degree of decomposition activity of filter paper with DSM 5427 ^T strain was compared.

The degree of decomposition activity of the filter paper shown here is for predicting the degradation degree of silage ripening due to the introduction of the strain, and it is possible to predict the ripening degree of the silage production through the degree of decomposition activity of the filter paper, Can be predicted. In other words, unlike ruminants, which contain cows, pigs do not have digestive function through rumination when consumed. Therefore, when the existing ruminant silage is applied to pig farming, the digestibility is lowered, . Therefore, when the conventional silage is applied as feed for pigs, it is possible to increase the fibrin digestibility of the silage itself. Thus, it is possible to predict whether the feed can be provided as feed for pigs.

Detailed explanations of this experiment are as follows. 1 ml of pH buffer solution containing 1 filter paper (1 x 6 cm) was inoculated with 1 ml of cellulolytic bacterial culture filtrate, and cultured at 30 ° C for 24 hours. The amount of glucose produced was calculated by dividing the amount of glucose by the total amount of protein in the culture filtrate. The fibrinolytic bacterium culture broth was obtained from Whatman No. 1. 1 filter paper (1 x 6 cm) as the sole carbon source and cultured at 28 ° C for 2 weeks until filter paper was completely decomposed. The solution was filtered with a 0.2 μm filter.

Glucose concentration was analyzed by Nelson-Somogyi (NS) reducing sugar assay and protein concentration was analyzed using Qubit protein assay kit from Molecular probe. All cultures were carried out under anaerobic conditions.

2. Experimental results

As a result of the above-mentioned experiment, as shown in Fig. 3, the homologous strain C. lentocelum It was confirmed that the degradation rate of the selective fibrinolytic bacterium (WCF-2) of Example 1 was faster than that of the DSM 5427 ^T strain.

As shown in FIG. 4, the decomposition activity of WCF-2 and C. lentocelum DSM 5427 ^{T in} the pH range of 3 to 7 was 1.6 to 4.1 times higher than that of DSM 5427 ^T Respectively.

As shown in FIG. 5, it was confirmed that the preferred fibrinolytic bacterium (WCF-2) of Example 1 corresponds to 45% of the decomposition activity of Trichoderma reesei strain, which is a strain that has been commercialized as a fibrinolytic microorganism.

In other words, Trichoderm reesei Although the strain is known to have excellent fibrinolytic ability, the strain itself is an aerobic fungus and thus can not be used for fermenting the silage required for swine farming. In the case of the selective fibrinolytic bacterium (WCF-2) of Example 1 of the present invention, It is well suited for fiber - degrading bacteria when silage fermentation is required for pig farming.

<Experimental Example 3> Comparison of growth rate and pH drop rate of the selected lactic acid bacteria of Example 2

1. Experimental Method

In order to isolate the lactic acid bacteria having a high fermentation speed, the growth rate and the rate of pH decrease were measured for a total of 16 lactic acid bacteria selected in Example 2. In other words, the lactic acid bacteria are added to increase the fermentation speed of the silage used for feeding the existing ruminant, so that the growth speed of the bacteria must be high in a short time in order to increase the fermentation speed, In the case of silage, it is important to prevent corruption during storage, and it is also important to lower the pH in a short time.

In this experiment, all experiments were carried out under anaerobic conditions and anaerobic conditions were established using nitrogen gas or anaerobic pack (AnaeroGen, Oxoid).

In order to evaluate the growth ability of selected strains, MRS broth was cultured at 28 ° C and 150 rpm for 24 hours, and then inoculated to the same medium with an absorbance (OD600) of about 0.1. The absorbance and pH were measured while culturing for 48 hours to draw a growth curve. In addition, a barley-extracted medium (180 g of 900 g of distilled water was added to 900 g of barley harvested in 2016, and the supernatant was filtered through a 0.45 μm filter, followed by heating at 50 ° C. for 2 hours. I painted it. Changes in temperature, salinity, and pH were measured using a 96-well plate. The temperature-dependent growth was determined by adding 5 μl of the cultured MRS broth for 1 day at 28 ° C to 100 μl of MRS broth in a 96-well plate and incubating at various temperatures. The absorbance was measured with Spectra Max M2 (Molecular Device) Were measured and analyzed.

The effect of salinity was determined by inoculating and culturing the media in which 0-12% NaCl was added to MRS broth and the pH was adjusted to 1 ~ 13 with HCl and NaOH in the pH of MRS broth. Was fixed at 28 ° C. Carbohydrate availability was analyzed using API 50 CHL (bioMerieux). L-lactic acid kit (R-Biopharm) was used for the determination of the isomerism of the lactic acid. Gas production was considered to be positive when gas was generated more than 1 ml after culturing in 10 ml of MRS broth for 24 hours.

2. Experimental results

6, 7, 8, and 9, the Lactobacillus sp. Strains 5-1, 14-1, and 3-1 among the 16 lactic acid bacteria in both the MRS medium and the naked barley extract medium Growth rate and pH rate were the highest. Especially, the growth rate and pH rate of Lactobacillus sp. Strain 5-1 were the highest in the barley extract medium.

G1 G2 G3 G4 G5 CL 14-1 5-1 3-1 11-1 6-2-1 17-1 13-1 KM-2 D-arabinose - - - - - - - - w L-arabinose + + + + + + + - + D-xylose - - - - + + + - + D-adonitol - - - - - - - - - D-galactose + + + + + + w + + D-mannose + + + + - + + + + Rhamnose + - - - - - - - - Inositol - - - - - - - - - Manitol + + + + - - - + - Sorbitol + + + + - - - - - alpha- methyl-Dmannoside - + + - - - - - - alpha- methyl-D-glucoside - - - - w + + - + Amygdalin + + + + - + + + + Arbutin + + + + - + - + + Esculin + + + + - + + + + Salicin + + + + - + - + + Cellobiose + + + + + + - + + Lactose + + + + + + + + w Melibiose + + + + + + + + + Sucrose + + + + - + + + + Trehalose + + + + - + + + + Inulin - - - + - - - - - Melezitose + + + + - + + - - Raffinose + + + + - + - + - Starch - - - - - - - - w Gentiobiose + + + + - + + + + D-turanose + + + - - - w - - D-tagatose - - - - - - - - + D-arabitol w - - - - - - - - Gluconate + + + + w + + - + 2-keto-gluconate - - + - + - + - w 5-keto-gluconate - - - - - - + - - Number of + / w 21 20 21 19 8 18 17 14 20

G6 G7 G8 G9 16-2 13-2 KL-1-1 27-1 32-2 31-2 29-1 24-1 D-arabinose - - - - - - - - L-arabinose - - - + - - - + D-xylose - - - - - - - - D-adonitol + + - - - - - - D-galactose + + + + + + + + D-mannose + + + + + + + + Rhamnose - - - + - - - - Inositol w - - - - - - - Manitol + + + + - - - w Sorbitol + + + - - - - + alpha- methyl-Dmannoside - - - + - - - - alpha- methyl-D-glucoside w w + + - - - + Amygdalin - + + + + + + + Arbutin - + + + + + + + Esculin + + + + + + + + Salicin + + + + + + + + Cellobiose - + + + + + + + Lactose - - - + + + + + Melibiose - - - - + + + + Sucrose + + + - + + + + Trehalose + + + + + + + + Inulin - - - - - - - - Melezitose + + + - - - - + Raffinose - - - - - - - + Starch - - - - - - - - Gentiobiose - - - + + + + + D-turanose + + + - - - - + D-tagatose + + + + - - + - D-arabitol - - - - - - - - Gluconate + + + - - - - + 2-keto-gluconate - - - - - - - + 5-keto-gluconate - - - - - - - - Number of + / w 15 17 16 16 12 12 13 21

(+, Positive; -, negative;, w, weak positive)

G1 G2 G3 G4 G5 CL 14-1 5-1 3-1 11-1 6-2-1 17-1 13-1 KM-2 pH 4-12
(6-8) 3-12
(5-10) 3-12
(6-10) 3-12
(6-10) 4-11
(6-7) 4-10
(5-7) 4-9
(6-7) 4-11
(6-8) 4-9
(6-8) Temp. (° C) 8-40
(30-35) 8-40
(25-30) 8-40
(30-35) 8-40
(30-40) 15-40
(30-40) 20-40
(35-40) 25-40
(35) 15-40
(30-35) 15-40
(35-40) NaCl 0-9
(0-1) 0-10
(1-3) 0-11
(0-1) 0-10
(0-2) 0-9
(0) 0-8
(0-2) 0-8
(0-1) 0-8
(0-2) 0-7
(0) Form of lactic acid isomer DL DL DL DL DL DL DL DL L Gas generation - - - - + + + - +

G6 G7 G8 G9 16-2 13-2 KL-1-1 27-1 32-2 31-2 29-1 24-1 pH 4-11
(6-9) 4-11
(7-9) 4-11
(6-8) 4-11
(6-8) 6-13
(12) 5-13
(12) 6-13
(12) 4-12
(6-9) Temp.
(° C) 15-40
(35) 8-40
(35-40) 8-40
(30-35) 15-40
(35-40) 20-45
(35-40) 15-45
(35-40) 15-45
(35) 15-40
(30-35) NaCl 0-8
(0) 0-9
(0) 0-9
(0-1) 0-8
(0-1) 0-8
(0-1) 0-8
(0-1) 0-8
(0-1) 0-7
(0-1) Form of lactic acid isomer L L L L L L L D Gas generation - - - - - - - +

In addition, as shown in Tables 3, 4, 5 and 6, among the 16 kinds of lactic acid bacteria, Lactobacillus sp. Strain 5-1, 14-1 and 3-1 use relatively many kinds of sugars , PH, temperature and salinity of the widest range.

<Experimental Example 4> The inhibition of the growth of the first lactic acid bacteria of Example 2 against the silage decay bacteria was also confirmed

1. Experimental Method

Silage is generally a sage to be fed to livestock by cutting and fermenting pasture or forage crops for long-term storage. It is also important that the lactic acid bacteria introduced during fermentation increase the fermentation speed. Above all, it is very important to suppress the propagation of defective bacteria so that they can be stored for a long time.

Thus, in this experiment, the growth inhibition degree against the silage rot fungus was also confirmed in the lactic acid bacteria selected in Example 2 above.

This experimental method will be described in more detail. Based on the previous studies, the fungus Fusarium graminearum KACC 41040, Aspergillus fumigatus KACC 41016, Penicillium roqueforti KACC 47196, yeast Candida glabrata KCTC 7219, Pichia anomala KCTC 17625, and anaerobic bacterium Clostridium tyrobutyricum KCTC 5387 were selected. CL strains were cultured in broth medium at 28 ℃, pH 7.0, and pH of 6.5. The cultures were treated with modified MRS (mMRS, peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, K ₂ HPO ₂ , pH 6.5) After incubation for 48 hours under anaerobic conditions, the cells were filtered with a 0.2 μm sterile filter. Polysorbate 80, sodium acetate, ammonium citrate, magnesium sulfate, and manganese sulfate contained in MRS were excluded because they could inhibit the growth of fungi and yeast.

After mixing 80 μl of this culture with 100 μl of Reinforced Clostridium Medium (RCM, pH 6.8, C. tyrobutyricum ) or Potato Dextrose Broth (PDB, pH 4.5, remaining microorganism) % NaCl solution was inoculated with 20 [mu] l of the suspension.

The incubation was continued at 28 ° C in a 96-well plate while observing the absorbance. C. tyrobutyricum was incubated with AnaeroGen (Oxoid) in a 96-well plate in an anaerobic jar (MGC), and other microorganisms were cultured aerobically. The time taken for the absorbance to be 0.5 was used as a measure for the degree of inhibition and calculated using the two points closest to OD600 = 0.5. The significance of the difference between the inhibition times was determined using the MS Excel 2010 t-test.

2. Experimental results

As a result of the experiment, as shown in Figs. 10, 11 and 12, the representative silage rot fungus Fusarium graminearum , Aspergillus fumigatus , Candida glabrata It was confirmed that all 3-1, 5-1 and 14-1 strains showed the inhibition of growth, and 5-1 strains showed the highest growth inhibition.

In accordance with the present invention, lactic acid bacteria ( Lactobacillus sp . ), Which exhibits the highest fermentation rate when applied to silage fermentation among various lactic acid bacteria and exhibit high inhibition of growth of the silage decoy bacteria, 5-1 were finally selected and applied to the following silage production.

<Experimental Example 5> Silage production and quality evaluation of the present invention

1. Silage production method

As shown in FIG. 13, after the Kumtang wheat harvested on May 30, 2017 was crushed to a size of 1 cm or less, WCF-2 (1 × 10 ⁶ cells / kg), which is the fibrinolytic bacterium isolated in Example 1, (3 × 10 ⁸ cells / kg) of lactic acid bacteria isolated in Example 2 were sealed together with WCF-2 (1 × 10 ⁶ cells / kg) and sealed in a thermostatic chamber at 28 ° C. for 72 days The silage was preserved.

In addition, as a comparative example, only the lactic acid bacterium 5-1 (3 × 10 ⁸ cells / kg) isolated in Example 2 was inoculated to prepare a silage, and a silage prepared by fermentation without a strain was prepared as a control.

2. Silage quality evaluation method

In order to evaluate the quality of the silages prepared according to the above method, the pH drop rate was measured. In this case, 1 g of each silage was taken in each time zone, added to a 50 ml centrifuge tube, and then 20 ml of distilled water was added. The pH of the supernatant after 20 minutes of vortexing was measured using a pH meter (Fisher scientific, accumet XL200).

3. Experimental Results

As shown in FIG. 14, when the WCF-2 strain, which is a fibrinolytic bacterium isolated in Example 1, was inoculated, the rate of pH decrease was higher than that of the control without treatment. The strain 5-1 was inoculated with the strain WCF-2, and the pH of the isolate was higher than that of the WCF-2 strain alone. Thus, it was confirmed that the preservation of the silage could be enhanced through the isolates of the present invention.

<Experimental Example 6> Confirmation of suitability of the swine silage of the present invention

1. Experimental Method

In the last experiment, the in vitro total digestibility was measured in order to confirm whether the silage used only in the existing ruminants could be used as feed for pigs. The silage prepared for 72 days was opened and 0.25 g was taken as the treatment type and added to the serum bottle. Pig 50 g were collected in acid livestock farms and _{nutrient buffer (NaHCO 3 9.24 g /} L; Na2HPO 4 12H 2 O 7.125 g / L; NaCl 0.47 g / L; KCl 0.45 g / L; Na 2 SO 4 0.1 g / L 400 ml of trace mineral solution (10 ml) was mixed with 0.047 g / L of CaCl _2, 0.047 g / L of MgCl _2, 0.4 g / L of urea and 25 ml of the gauze for surgical gauze was added to the upper serum bottle . This serum bottle was put into anaerobic condition by injecting CO ₂ + N ₂ (20:80) mixed gas, sealed and incubated at 35 ° C. The generated gas volume was measured using a gauge gas generation amount measuring instrument.

2. Experimental results

As shown in FIG. 15, in the case of the WCF-2 strain, which is a fibrinolytic bacterium isolated in Example 1, and the 5-1 strain of lactic acid bacteria isolated in Example 2, the amount of gas accumulated was the highest , And also showed a high gas accumulation amount even in the case of the WCF-2 strain alone isolated as the fibrinolytic bacterium isolated in Example 1. In the case of 5-1 strain inoculation, the rate of pH decrease was higher than that of the control without treatment And it was confirmed that a high digestibility can be obtained by applying the isolates of the present invention to swine farming. As a result, it was found that the silage could be applied not only to ruminants but also to feeds for pigs.

Based on the above results, it can be seen that the present invention reduces the dependency on imported feed, improves the preservability and digestibility of the existing silage, and provides silage feeds for raising pigs as well as ruminants.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be possible. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC92181P

Checked on: 20170613

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC 19351

Checked on: 20170620

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microorganism for fermentation of whole crop silage and Microbial          additions including the same <130> p2017-0269 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1453 <212> DNA <213> Cellulosilyticum sp. WCF-2 <400> 1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagtgatag gagcttgctc 60 ctagaactta gtggcggacg ggtgagtaac gcgtgggtaa cctgccctat gcagggggat 120 aacgtttgga aacgaacgct aataccgcat aaactatggg caatcgcatg attgttatag 180 taaagatttt atcggcatag gatggacccg cgttggatta gctagttggt gagataacag 240 cccaccaagg cgacgatcca tagccggcct gagagggtga acggccacat tgggactgag 300 acacggccca aactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc 360 tgatgcagcg acgccgcgtg aaggaagaag gtcttcggat tgtaaacttc tatcagcagg 420 gaagaaaaaa tgacggtacc tgactaagaa gctccggcta actacgtgcc agcagccgcg 480 gt; ttgttaagtc agaagtgaaa tttaggggct caacctctaa gctgcttctg aaactgatga 600 actagagtgt gggagaggaa agtggaattc cgagtgtagc ggtgaaatgc gtagagattc 660 ggaggaacac cagtagcgaa ggcggctttc tggaccataa ctgacgctga ggcacgaaag 720 cgtggggagc aaacaggatt agataccctg gtagtccacg ctgtaaacga tgaatgctag 780 gtgtcggggc ttacgggtct cggtgccgaa gttaacacat taagcattcc acctgggaag 840 tacgatcgca agattgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat 900 gtggtttaat tcgaagcaac gcgaagaacc ttacctaaac ttgacatcct tttgactaga 960 gggtaatgcc tctttctcta gcttgctaga acataagtga caggtggtgc atggttgtcg 1020 tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctatctttag 1080 tagccagcat tragttgggc actctagaga gactgccagg gataacttgg aggaaggtgg 1140 ggatgacgtc aaatcatcat gccccttatg tttagggcta cacacgtgct acaatggctg 1200 ctacaaaggg aagcgatctc gcgagagtca gcaaacctca aaaaagcagt cccagttcgg 1260 attgtagtct gcaactcgac tacatgaagt tggaatcgct agtaatcgcg aatcagaatg 1320 tcgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg ggagttgggg 1380 gggcccaacg ccggtgaccc aacccttcgg ggagggagcc gtctaaggca aaaccaataa 1440 ctggggtgaa gtc 1453 <210> 2 <211> 1489 <212> DNA <213> Lactobacillus sp. 5-1 <400> 2 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60 gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120 tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180 cgcatggtcc gagtttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240 ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300 taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420 gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480 ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 780 aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 840 ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 900 gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 1020 gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 1140 gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 1260 tcgcgagagt aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 1320 cctacatgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440 gtaacctttt aggaaccagc cgcctaaggt gggacagatg attagggtg 1489

Claims (6)

As a new microorganism for whole-wheat silage fermentation,
Cellulosylticumum sp.) WCF-2 strain (KACC92181P).
Whole wheat silage Microorganisms for fermentation, including fibrinolytic bacteria,
Microbial additive for making whole wheat silage.
3. The method of claim 2,
The fibrinolytic microorganism is a microorganism belonging to the genus Cellulosilyticum sp.) WCF-2 strain (KACC92181P).
Microbial additive for making whole wheat silage.
3. The method of claim 2,
Wherein the microbial additive further comprises lactic acid bacteria.
Microbial additive for making whole wheat silage.
5. The method of claim 4,
Wherein the lactic acid bacterium is a novel microorganism Lactobacillus sp. 5-1 strain (KACC 19351)
Microbial additive for making whole wheat silage.
Comprising a plurality of the fermentation step for the entry into force of the whole aneurysm, and at least one fermentation stage of the fermentation step is a cellulose sila ET glutamicum in (Cellulosilyticum A mixed microorganism of WCF-2 strain (KACC92181P) or Cellulosilyticum sp. WCF-2 strain (KACC92181P) and Lactobacillus sp. 5-1 strain (KACC 19351) &Lt; / RTI &gt;
Method of making whole wheat silage.

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