KR101337406B1 - Fermentation medium composition containing rumen content and use of thereof - Google Patents

Abstract

The present invention relates to a medium composition containing rumen contents and its use, and more particularly, to a medium composition and a fermentation composition containing the rumen contents as a fermentation feed additive having excellent enzymatic activity.

Description

Medium composition containing rumen contents and its use {Fermentation medium composition containing rumen content and use of

The present invention relates to a medium composition containing rumen contents and its use, and more particularly, to a medium composition and a fermentation composition containing the rumen contents as a fermentation feed additive having excellent enzymatic activity.

The research for the present invention was supported by the RDA Bio Green 21 project, titled 'Development of high value-added material production technology utilizing slaughter by-products'.

In the domestic livestock industry, the weakness of the forage production base is recognized as a big problem. Most of the feed sources are dependent on imports, and thus, the burden of feed costs is very high and the prices are unstable. In addition, rice straw, which has been used as a major source of forage, has low nutrient content, utilization efficiency and palatability, and thus, did not sufficiently function as a fiber feed. In this situation, as the survey fee is imported in large quantities, production costs for cattle are being increased. Therefore, it is urgent to develop an economical and nutritional source of excellent nutritional value. Recently, as part of the development of new forage sources, there is a growing interest in slaughter by-products that can be used under the livestock law.

2,559 cattle are slaughtered every day at 168 slaughterhouses in Korea, and all the mid-cold contents of slaughter by-products are disposed of and discarded. The amount is 115 tons per day and 35,000 tons per year. The disposal cost of discarded rumen contents amounts to 6.2 billion won per year, which can cause serious environmental pollution due to high pollution load. Therefore, the recycling of rumen contents as slaughter by-products is very important for preventing pollution and developing new feed resources.

The rumen contents contain 21.1% crude protein, 20.4% crude fiber and 13.7% crude fat on a dry basis, which are similar to those of alcoholic liquor, which is frequently used as a protein source of ruminant extracts, and contains fibrin content such as alfalfa and beet pulp. This is similar. In addition, the rumen contents contain a large amount of microorganisms, digestive enzymes and unknown growth factors, etc. present in the rumen, and when used as a ruminant feed, it can have the stability and other various advantages of the rumen.

In ruminants, the addition of fibrinolytic enzymes such as cellulase and xylanase can increase productivity by improving nutrient utilization efficiency. Supplementary supplementation of fibrinolytic enzymes has been reported to increase fibrin utilization, enhance the synthesis of rumen microbial proteins and improve the rate of protein degradation. Supplementary supplementation of starch degrading enzymes improves starch digestion efficiency of feed fed and has been reported to increase milk yield and daily gain in beef cattle.

The present invention has been made by the necessity of the above, an object of the present invention is to provide a medium composition for the production of fermented products having enzymatic activity.

Another object of the present invention is to provide a method of using the rumen contents for the economic production of fermentation.

Still another object of the present invention is to provide a fermentation feed additive for ruminants, which has excellent enzyme activity and is effective in ruminant stability.

In order to achieve the above object, the present invention provides a medium composition comprising the rumen contents in the medium composition for microbial fermentation.

In the specification of the present invention Rumen contents refer to the contents contained in the first place of the slaughtered ruminant. The rumen contents used in the examples of the present invention were obtained through the cooperation of a slaughterhouse in Seoul, and the obtained rumen contents were stored in a sterile hermetic container at -70 ° C. and used for the experiment.

In one embodiment of the present invention, the rumen contents are preferably included in more than 20% to less than 40% by weight of the total medium composition, but is not limited thereto. Preferably, the media composition of the present invention is 20% rumen contents More preferably less than 40%, molasses 1-10%, corn starch 1-10%, soybean powder 1-10%, bran 20-40%, and chaff 1-10% (w / w). It is not limited to this.

The present invention also provides a fermentation composition with improved enzymatic activity by adding one or more strains selected from the group consisting of Bacillus and yeast strains to the medium composition of the present invention as a seed.

In the present invention, the improvement of enzyme activity means that it contains about 23% of rumen contents, which is an existing product, yeast powder 4%, chaff powder 8%, acidulant and buffer 2%, molasses 4%, rice bran 8%, beer foil Compared to the composition consisting of 31% and the other 20% of the other components, the starch enzyme activity is about 5 times improved, in the case of xylanase is about 40 times higher, but is not limited thereto.

In one embodiment of the present invention, the spawn is preferably Bacillus pumilis, Bacillus amyloliquifaciens, or Saccharomyces cerevisiae, and the enzyme Is preferably starch, fibrinase, or xylanase, but is not limited thereto.

In another aspect, the present invention provides a feed additive composition comprising the fermentation composition of the present invention as an active ingredient.

In another aspect, the present invention is a microbial culture method, the contents of the rumen contents in the medium 20-40%, molasses 1-10%, corn starch 1-10%, soybean powder 1-10%, bran 20-40%, and chaff 1-10 It provides a microbial culture method comprising the step of culturing, including% (w / w).

In one embodiment of the present invention, the feed prepared by adding the rumen contents of the present invention is effective in improving the productivity of animals such as mammals, birds, reptiles, amphibians, fish, preferably mammals, more preferably ruminants It is preferred to have an effect on improving the productivity of the cow most preferably, but not always limited thereto.

The feed of the present invention can be made by appropriately blending the rumen contents fermentation composition of the present invention in a feed material. Examples of feed materials include cereal grains, crude steel, vegetable oils, animal feedstuffs, other feedstuffs, and refined products.

Examples of cereals include maize, wheat, barley, oats, rye, brown rice, buckwheat, buckwheat, sorghum, blood, corn, soybeans and the like.

Examples of the crude steel include rice bran, defatted rice bran, wheat bran, horse meal, wheat, embryo, barley bran, screening, pellets, corn bran, and corn embryo.

Examples of vegetable oils include soybean meal, soybean meal, flaxseed meal, cottonseed meal, groundnut meal meal, safflower meal, palm oil palm, palm oil, fomarak, sunflower oil, rapeseed meal, cowpea oil and mustard oil.

Examples of raw animal feeds include fish meal (bean mill, imported wheat, hall mill, coastal wheat), fish shavings, meat meal, fish meal, blood meal, Casein, dry whey, and the like.

Examples of other feed materials include plant foliage (alfalfa, heukube, alfalfa leaf, and similar acacia powder), corn processing industry by-products (conglutin, wheat, conglutinfeed, constay prima etc.), starch products, (Cassava, bean curd, bean curd, bean curd, malt bean curd, alcohol bean curd, jangyu bean curd), agricultural production byproducts (citrus processing, tofu, coffee, cocoa, Krill, spirulina, chlorella, minerals, etc.).

Examples of the purified product include proteins (casein, albumin, etc.), amino acids, saccharides (starch, cellulose, sucrose, glucose, etc.), minerals and vitamins.

The present invention also provides a ruminant feed additive composition comprising the rumen contents fermentation composition of the present invention as an active ingredient.

In another aspect, the present invention provides a fermentation medium composition using Bacillus and yeast as a medium composition and a fermentation strain that can improve the enzyme activity in the rumen contents fermentation.

In addition, the present invention is a method for culturing Bacillus and yeast, the contents of the rumen contents in the medium 30-40%, molasses 1-10%, corn starch 1-10%, soybean powder 1-10%, bran 20-40%, and chaff It provides a method for culturing Bacillus and yeast comprising 1 ~ 10% (w / w) fermentation.

Hereinafter, the present invention will be described.

First, the present invention investigated the fermentation conditions of rumen contents using Bacillus and yeast for the preparation of fermentation compositions containing rumen contents.

The effects of water, rumen contents, seed inoculation, molasses, corn starch, soybean powder, wheat bran, and NH4Cl on the fermentation of starch degrading enzymes, fibrinolytic enzymes, and xylanase activities were investigated.

Experimental design of factor analysis was set up using Taguchi method. After the normalization of responses obtained in each experiment, signal-to-noise ratio was calculated, and the magnitude of the effect was calculated using the average of signal-to-noise ratio for each factor level.

As a result of analyzing the signal-to-noise ratio of each factor, the water content was excellent at 40%, the amount of rumen contents added was 20%, the inoculum level was 5%, molasses level was 10%, corn starch level was 5 g / L, soybean. The powder was good at level 3 g / L, wheat vein 50 g / L, and NH 4 Cl at 0.1 g / L.

Through a series of results, fermentation medium is 40% water content, rumen contents 20%, molasses 5%, corn starch 5%, soybean powder 5%, wheat bran 40%, NH4Cl 0.1% Was prepared.

After inoculating Bacillus and yeast in the prepared fermentation medium, and cultured for 48 hours, amylazi activity was 198,410,000 U, fibrinase 39,680 U, and xylanase 41,080 U. Where U is 1 μmol of glucose formed through decomposition of the substrate for 1 minute in 1 g of sample.

As can be seen through the present invention, the fermentation medium composition comprising the rumen contents obtained in the present invention activates the growth of Bacillus and yeast, and consequently aids in digestibility, starch, fibrinase, and xylanase It has the effect of improving the activity of ruminant can be used as a feed additive.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1. The medium components containing the rumen contents Bacillus and  Enzyme activity through the growth of yeast Bacillus  Influence on Growth and Yeast Growth

1. Experimental Method

1) Use strain and culture condition

In this embodiment, Bacillus pumilus , Bacillus excellent in starch, fibrinase and starch enzyme activity amyloliquifacien and the yeast strain Saccharomyces cerevisiae was used as a spawn. The spawn was cultured in LB (Difco, USA) for Bacillus, and YM (Difco, USA) for yeast. As the culture conditions, all the strains were used after shaking culture at a speed of 150 rpm for 20 hours at 30 ℃.

2) Screening of Media Components for Enzyme Production

Taguchi Design of Experiment was used in this experiment to investigate the factors affecting the enzyme activity of fermentation medium in solid fermentation including rumen extract. Taguchi Design of Experiment is a method devised by Japan's Taguchi Kenichi, and it is well known as a technique to search for the optimal condition of noise factor affecting the target system. The Taguchi technique is known for its robust design, which converts the magnitude of the resulting responses into a signal and noise ratio (SN ratio) to find the control factors that approximate the desired mean while reducing the spread (Sang-Bok et al. 2010). IE Interface 23: 257; Lim Pyo and Yang Kyun, 2006. Korean Society of Mechanical Engineers 5:59). In particular, it is advantageous to interpret the experimental results by dividing them into nominal-is-best, small-the-best, and large-the-best characteristics, depending on the purpose of the experiment. Journal of the Korean Society of Mechanical Engineers 5:59).

In this example, eight factors were set as factors that may affect solid fermentation including rumen extract. All of the factors were set to three levels except the two levels of water content, and the setting levels for each factor were as shown in Table 1. Each factor consisted of 18 experiments according to the orthogonal array method for each level, and the types of factor levels included in the experiments are shown in Table 2. For each enzyme activity obtained in each experiment, the SN ratio was calculated by applying the characteristic of the tower, and the formula used for analysis was as follows.

In the above formula, r is the total number of repetitions obtained for each experiment, y _i is the response value obtained for each iteration.

In this example, three enzyme activities were used as reaction values, and finally, an object of the present invention was to find factors exhibiting positive effects on all three enzyme activities. Therefore, it is necessary to analyze the effects of factors by applying the same reaction values of the three enzymes. The reaction values for the experiments of each enzyme activity were standard normalized, and the integrated SN ratio was calculated as a tower characteristic using the respective enzyme activities converted to the standard normal distribution. At this time, a value smaller than 0 was found in some of the values converted to the standard normal distribution, and was moved in parallel by + 2 to express it as a positive value. Finally, a distribution having an average of 2 and a variance of 1 was calculated. The magnitude of the effect was measured using the mean value of the SN ratio for each level of the finally obtained factors.

Where Z is the normalized form of X and X is the enzyme activity obtained in the experiment.

3) Culture condition

Fermentation medium was prepared according to the level of factors included in each experiment as shown in Table 2. The medium of each experiment was brought to a total amount of 200 g, and incubated in a 300 g aluminum container for 48 hours at 30 ℃. Stirring of the culture was performed once every 24 hours using sterile glass rods. All cultures were performed in three replicates.

4) Analysis of Enzyme Activity

The cultured cultures were used as a sample for enzyme activity analysis after sufficient stirring. The analysis of enzyme activity is as follows. As substrates, soluble starch (Difco) was used for starch degrading enzyme, Carboxymethyl cellulose (CMC, Sigma) was used for fibrinase measurement, and birchwood xylan (Sigma) was used as substrate. Substrate solution for starch and xylan degrading enzyme was used 10mM Tris-HCl (pH 7.0) containing 1% of each substrate, 0.5% CMC 10 mM Tris-HCl ( pH 7.0) was used. Enzyme reaction was performed by mixing 9 ml of the substrate solution to 1 g of the sample and reacting at 37 ° C. for 1 hour (150 rpm), followed by centrifugation (10,000 rpm 5 min) to separate the supernatant. 300 μL of the supernatant and 300 μL of the DNS solution (3,5-Dinitrosalicylic acid, 1%; sodium sulfate, 0.05%; sodium hydroxide, 1%) were mixed and reacted at 90 ° C. for 10 minutes. After the reaction solution was cooled to room temperature, 50 uL of 40% potassium sodium tartrate solution was mixed, and then the absorbance was measured at 575 nm after removing impurities with a 0.45 um filter. The back ground of the reducing sugar present in the culture was excluded using the absorbance of the solution reacted with 10 mM Tris-HCl (pH 7.0) solution containing no substrate. The concentration of sugar released through the enzymatic reaction was evaluated using a regression equation. Starch and fibrinase were used as glucose, and xylan degrading enzyme was used as standard for each enzyme. One unit of enzyme activity was expressed as 1 μM glucose or xylose produced from 1 g of sample per minute.

5) Other analysis

The pH of the culture was measured by suspending 1 g of the sample in 9 mL of distilled water. The amount of cells contained in the culture was measured by colony forming unit obtained by the dilution smear method. The approximate method is as follows. 1 g of the culture sample was suspended at 150 rpm at room temperature for 30 minutes after suspending 9 mL of sterile 0.8% NaCl solution, and then diluted sequentially 10-fold in sterilized 0.8% NaCl solution. The diluted suspension was plated on LB and YM plates and incubated in a 30 ° C. incubator for 24 hours to determine the number of colonies formed. The measured values were then used after conversion to log ₁₀ for ease of calculation.

6) Statistical Analysis

Taguchi's experimental design for factor evaluation on fermentation was performed using MINITAB (version 14.0, Minitab Inc., USA).

2. Experimental results

The mean values of signal-to-noise ratios for each level of media components are shown in Table 3. For moisture, the second level, 40%, was excellent. In rumen contents, the second level was 20%. The inoculation amount was excellent as the level was increased, the third level was the highest. Since, except for NH ₄ Cl, the third level was excellent in all of corn starch, soybean powder, and wheat bran.

However, in the case of molasses, the difference between the second level and the third level was not so large that the second level was selected as the optimal medium composition. In addition, in case of molasses, the viscosity is strong, and when the amount of use increases, the aggregation of media raw materials increases, and the second level is selected as the optimum medium composition.

As a result, it contains the rumen contents and media composition to improve the enzyme activity, water 25.9%, rumen contents 20%, inoculation 1%, molasses 5%, corn starch 5%, soybean powder 5%, wheat bran 34.7%, NH4Cl 0.1% and 4% of the remaining weight was determined to use chaff powder having low availability as a substrate as a filler.

Comparative Example 2: Effects of rumen contents on different levels and comparison with existing products

The rumen contents not only act as a substrate of fermentation process for the production of enzymes, but are also fed to ruminants after being produced and can play various roles in the rumen. Therefore, it is necessary to maximize the amount of use through the determination of the exact amount of addition. Therefore, in the present comparative example, the amount of rumen contents was determined to be 20%, 30%, 40%, and the level of addition was determined. In addition, it was intended to demonstrate the superiority of the present invention by comparing the efficacy of the feed additive (product name: lumen bio, manufacturer: Nature and Technology Co., Ltd.) containing the rumen contents commercialized previously.

1. Test Method

1) Experimental Design

The contents of the rumen were gradually increased to 20%, 30%, and 40%, and the excess fraction of the weight ratio according to the increase of the contents of the rumen was offset by decreasing the moisture. In the case of rumen contents, the solid content is about 20% and the remaining 80% is made up of water. Therefore, in the experiment of the present comparative example, the decrease in moisture content according to the increase of the rumen contents did not show a significant difference as ± 1.6%.

The product containing the rumen contents used in this comparative example contains about 23% rumen contents, yeast powder 4%, chaff powder 8%, acidulant and buffer 2%, molasses 4%, rice bran 8%, It consists of 31% beer foil and about 20% of the other ingredients.

2) Fermentation Conditions and Analysis Items

Cultures prepared according to the level of rumen contents were cultured in the same manner as in Example 1, and the enzyme activity was also evaluated in the same manner.

2. Test result

As a result of analyzing the effects of the rumen contents, the treatment group 1 containing 20% of the rumen contents was the best. On the other hand, fibrinase and xylanase showed no significant difference in the rumen contents up to 30% but significantly decreased in the 40% treatment group (P <0.05). The results showed that the addition of rumen contents was possible up to 30%.

As a result of comparing the activity of the existing product with the developed fermentation product, starch dehydrogenase activity was improved by 5 times compared with the existing product. Fibrinase activity was not significantly different from the existing product. However, xylanase showed about 40 times higher fermentation products than existing products. As a result, in the case of the product of the present invention, the enzyme activity was very excellent compared to the existing product, and the microbial concentration was also very excellent.

Claims (9)

delete delete delete delete delete delete delete Water 25.9% (w / w), bovine rumen liquid 20% (w / w), strain 1% (w / w), molasses 5% (w / w), corn starch 5% (w / w), soybean powder Starch dehydrogenase activity of fermentation products by fermenting the strains in a medium composition comprising 5% (w / w), 34.7% (w / w) wheat flour, and 0.1% (w / w) NH ₄ Cl as an active ingredient. A method for enhancing xylanase activity. The method according to claim 8, wherein the strain is at least one microorganism selected from the group consisting of Bacillus and yeast.