KR20190079116A - Method for preparing total mixed fermentation feed using lactic acid fermentation - Google Patents

Abstract

The present invention relates to a method for producing a total mixed fermented feed using lactic acid fermentation and, more specifically, to a method for producing a total mixed fermented feed using lactic acid fermentation, which is to produce total mixed fermented feed by inoculating lactic acid bacteria to a mixture including beer lees, performing fermentation to prepare raw materials for feed, and then mixing the same, thereby improving nutritional benefits of fermented feed by having high content of amino acid and increasing the meat grade of Korean native beef by having excellent antioxidative activities. According to the present invention, the method for producing total mixed fermented feed using lactic acid fermentation includes: a step of producing raw materials for fermented feed by inoculating lactic acid bacteria to a raw material mixture including beer lees, and fermenting the same with lactic acid; and a step of producing total mixed fermented feed by mixing the raw materials of fermented feed with total mixed ration (TMR) and aging the mixture.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a fully fermented fermented feedstuff using lactic acid fermentation,

The present invention relates to a method for producing a fully mixed fermented feed using lactic acid fermentation, and more particularly, to a method for producing a fully fermented fermented feed by fermenting lactic acid bacterium in a mixture containing beer, The present invention relates to a method for producing a fully mixed fermented feed using lactic acid fermentation which can improve the nutritional efficiency of a fermented feed and has an excellent antioxidant ability to enhance the meat quality of Korean beef cattle.

Total mixed ration (TMR) is a mixture of feed and concentrate (or commercial compound feed) in appropriate proportions to meet nutrient requirements and is made by adding various additives. Compared to feeds and concentrated feeds separately, The convenience of feeding the feed is improved.

TMF (total mixed fermentation) is a complete mixed feed with a proper amount of yeast or fermentation agent and water. It is a feed with a digestion rate of 30% higher than that of ordinary feed.

Korean Patent No. 10-1564886 (a fermented water control agent and a completely mixed fermented feed containing the same) and Korean Patent No. 10-1032654 (a method of producing a fully mixed fermented feed) disclose a technique for producing a fully mixed fermented feed In order to make a completely mixed fermented feed, the feed and the concentrated feed are blended and the water is supplemented and fermented so that the total moisture content is about 40%. In order to reduce the cost, distillation foil of soju, Industrial by-products.

However, the conventional fully mixed fermented feed has advantages such as improved digestion and absorption rate by fermentation, but the effect of the fermentation on the improvement of the quality of the feed, Research and development are still limited.

Korean Patent No. 10-1564886: fermented water control agent and fully mixed fermented feed containing the same Korean Patent No. 10-1032654: Method for producing a completely mixed fermented feed

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a fermented feedstuff containing fermented lactic acid bacterium, The present invention is intended to provide a method for producing a fully mixed fermented feed using lactic acid fermentation which can improve the nutritional efficiency of fermented feed and has an excellent antioxidant ability to enhance the meat quality of Korean beef cattle.

The present invention relates to a method for producing a fully mixed fermented feed using lactic acid fermentation, and more particularly, to a method for producing a fully fermented fermented feed by fermenting lactic acid bacterium in a mixture containing beer, The present invention relates to a method for producing a fully mixed fermented feed using lactic acid fermentation which can improve the nutritional efficiency of a fermented feed and has an excellent antioxidant ability to enhance the meat quality of Korean beef cattle.

A method of producing a fully mixed fermented feed using lactic acid fermentation according to the present invention comprises the steps of: fermenting fermented feedstock by fermenting lactic acid bacteria by inoculating lactic acid bacteria into a raw material mixture containing beer; and mixing the fermented feedstock with a completely mixed feed , Total Mixed Ration) to produce a completely mixed fermented feed.

In addition, the method of producing a fully mixed fermented feed using lactic acid fermentation according to the present invention is characterized in that the mixture containing the brewer's bean is mixed with the sugar beet, Fumix No. 1, corn flakes and wheat flour.

In addition, the method of producing a complete mixed fermented feed according to the present invention is characterized in that the mixture containing the brewing beverage comprises 30% by weight of beer, 43.5% by weight of sugar, 12.5% of Fermix 1, 1.5% of wheat flour 1.5% % Is mixed.

Also, the method for producing a fully mixed fermented feed using lactic acid fermentation according to the present invention is characterized in that the lactic acid bacteria include Lactobacillus casei KCTC3109 (Korea Research Institute of Bioscience and Biotechnology), Leuconostoc , pseudomesenteroides KCTC3652 (Korea Research Institute of Bioscience and Biotechnology), Lactobacillus plantarum KCTC 33131 (Korea Research Institute of Bioscience and Biotechnology), Leuconostoc pseudomesenteroides JLRI 01 and KCCM 11443P (Korean Microorganism Conservation Center).

In addition, the method of producing a fully mixed fermented feed using lactic acid fermentation according to the present invention is characterized in that lactic acid fermentation of fermented feed materials is carried out at a temperature of 20 to 30 ° C for 7 to 10 days under anaerobic conditions.

According to the above-described constitution, in the method for producing a fully mixed fermented feed using lactic acid fermentation according to the present invention, lactic acid bacterium is inoculated into a mixture containing brewers, fermented to produce a feedstuff, and the feedstuff is mixed to form a completely mixed fermented feedstock And thus the nutritional efficiency of the fermented feed can be improved and the antioxidant ability of the fermented feed can be enhanced to enhance the meat quality of the Hanwoo.

FIG. 1 is a photograph showing a container for lactic acid fermentation of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 2 is a photograph showing the state of fermented feed material lactic acid fermentation in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 3 is a graph showing changes in pH of fermented feed materials according to the fermentation period in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 4 is a graph showing changes in acidity of a fermented feed material according to a fermentation period in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 5 is a graph showing changes in sugar content according to the fermentation period of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 6 is a flowchart illustrating a process of a method for producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention.
FIG. 7 is a graph showing the content of polyphenols in fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 8 is a graph showing the Flavonoid content of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 9 is a graph showing the content of betaclucan in fermented feed materials in the method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
10 is a graph showing the lactic acid content of a fermented feed material in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
11 is a graph showing the DPPH content of a fermented feed material in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
12 is a graph showing the reduction power of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention
FIG. 13 is a graph showing the rating ratios for Hanwoo which is a completely mixed fermented feed containing fermented feed materials fermented by using Jeolla Province slaughtered Hanwoo, Naju slaughtered Hanwoo,

Hereinafter, a method for producing a fully mixed fermented feed material using lactic acid fermentation according to the present invention will be described in detail with reference to the drawings and Examples.

FIG. 1 is a photograph showing a container for lactic acid fermentation of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention. FIG. 3 is a photograph showing the fermented feed raw material lactic acid fermentation state in the method of manufacturing a fully mixed fermented feed using fermentation, and FIG. 3 is a photograph showing the fermented feed raw material fermented by the lactic acid fermentation method according to an embodiment of the present invention. FIG. 4 is a graph showing changes in pH of fermented feed materials according to the fermentation period in the method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention FIG. 5 is a graph showing the change in sugar content according to the fermentation period of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention. FIG. 6 is a flow chart illustrating a process for producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention. FIG. 7 is a graph showing the lactic acid fermentation according to an embodiment of the present invention. FIG. 8 is a graph showing the content of polyphenols in fermented feed materials in the method of producing fully mixed fermented feeds according to one embodiment of the present invention. FIG. FIG. 9 is a graph showing the content of beta-glucan in a fermented feed material in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention, and FIG. FIG. 11 is a graph showing the lactic acid content of a fermented feed material in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment, and FIG. FIG. 12 is a graph showing the DPPH content of a fermented feed material in a method for producing a fully mixed fermented feed using lactic acid fermentation. FIG. 12 is a graph showing the DPPH content of the fermented feed material in the method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention. And FIG. 13 is a graph showing the grade ratio of Hanwoo which is a completely mixed fermented feed containing fermented feed materials fermented by using Hanwoo, Naju slaughtered Hanwoo, and Ilwoo fermenting bacteria in Jeonnam slaughterhouse .

A method for producing a fully mixed fermented feed using lactic acid fermentation according to the present invention comprises feeding fermented lactic acid bacterium to a mixture containing brewer's grains to produce a feed material and mixing the feed material to prepare a completely mixed fermented feed, And the nutritional efficiency of the fermented feed is improved and excellent antioxidant power is obtained so that a fully mixed fermented feed can be produced which can enhance the meat quality of the Korean beef cattle.

Referring to FIG. 6 showing a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention, a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention includes: (S10) of fermenting fermented feedstuff by lactic acid fermentation by inoculating lactic acid bacteria into the raw material mixture, and mixing the fermented feedstock with TMR to produce a fully fermented fermented feed (Step S20).

In the step (S10) of producing the fermented feedstock, the mixture containing the brewer's bean is mixed with the sugar beet, Fumix No. 1, cornstarch and wheat flour. The blending ratio is 30% by weight, 43.5% 12.5% of No. 1 and 1.5% of wheat flour 1.5% are mixed.

As described above, the mixed mixture of brewers and the like is adjusted to have a content of 40 to 50% moisture while adding dry brewers, and then fermented by inoculating lactic acid bacteria. Lactobacillus casei KCTC3109, Leuconostoc pseudomesenteroides KCTC3652, Lactobacillus plantarum KCTC 33131, Leuconostoc pseudomesenteroides JLRI 01, KCCM 11443P (Korea Microorganisms Conservation Center). Lactic acid fermentation is carried out at 20 ~ 30 ℃ at anaerobic condition for 7 ~ 10 days.

≪ Example 1: Materials for Experiment >

- Brewer's beer: The beer used in the experiment for the practice of the present invention was produced from OB beer, and purchased beer in a refrigerator.

- Dumpling: The saccharide used in the experiment for the practice of the present invention was purchased from a subject, and stored in a refrigerator.

- Dry beer brew: Dried beer brew was purchased from OB, and the dried beer brew was purchased from the Ilwoo Farming Association and stored in the refrigerator.

- Firm Mix 1: Firm Mix 1 was purchased from the dominant feed as TMR (Total Mixed Ration) feed.

- Parfait and wheat bran: The wheat bran and wheat bran were purchased and used from the non - farming association.

- Reagents: MRS medium, Bacto agar

- Lactic acid bacteria: Lactobacillus casei KCTC3109, Leuconostoc pseudomesenteroides KCTC3652 and Lactobacillus plantarum KCTC 33131 were purchased from Korea Research Institute of Bioscience and Biotechnology, and inoculated on MRS selective medium for 48 hours at 37 ℃.

≪ Example 2: Fermentation of lactic acid &

- fermentation vessel

The fermentation vessel used in the test of the present invention was sealed with LDPE vinyl so as to be enclosed in a container as shown in FIG. 1 and used in a 25 × 15 cm container shown in FIG.

- Mixing ratio

The blending ratio setting for determining the optimum blending ratio is the same as that shown in Table 1 (blending ratio of raw material of fermented feed, unit: g). The effects of beer and beer on the quality of fermented diets were investigated by varying the concentrations of beer and dried beer. The general components of the fermented raw materials with different mixing ratios are shown in Table 2 (the general composition of the fermented feed raw materials with different mixing ratios, unit:%).

Sample Beer) Dumb Dried beer Firm Mix 1 Wavefront Wheat flour One 265 400 200 125 0 0 2 265 400 185 125 15 0 3 300 435 100 125 15 15 4 200 535 70 125 30 30 5 0 600 235 125 15 15

Sample carbohydrate Crude fat Crude protein Crude fiber Views min moisture One 11.6 14.6 11.8 2.9 11.6 50.4 2 14.5 14.1 11.5 2.7 10.9 49 3 31.2 16.1 10.7 3 10.9 51.1 4 27 18.8 11.8 3.1 11.3 51.1 5 3.7 21.2 13.4 2.6 12.2 49.5

- Lactic acid fermentation

The fermentation broth was purchased from the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology case i KCTC 3109, Leuconostoc pseudomesenteroides KCTC 3652, Lactobacillus plantarum KCTC 33131 was cultured in a selective medium at 200 을 for 2 days at 37 캜. Lactic acid fermentation is inoculated with the blend samples shown in Table 1 Lactobacillus case i KCTC 3109 (Lc ), Leuconostoc pseudomesenteroides KCTC 3652 (Leup), Lactobaccillus plantarum KCTC 33131 (Lp), Lc + Leup, Leup + Lp, a Leuconostoc pseudomesenteroides JLRI 01, KCCM 11443P (Conservation Center Korea microorganisms) Japanese beef farming fermentation strains (Ilwoo) that is used in combination Corporation Was cultured at a concentration of 1 × 10 ⁹ cells / ml or more, added at 1% level of the sample, and cultured at room temperature. The mixture was agitated 1 to 2 times for mixing the seeds during the initial 3 days of fermentation, and then cultured as shown in Fig. After the fermentation for 10 days, the fermentation progress was analyzed.

≪ Example 3: Analysis and evaluation of feedstuff raw material composition and mixing ratio >

(1) Production of feed

- Analysis of feedstuffs in fermented feed

In order to analyze the fermentation process and to examine the optimum compounding ratio, a total of 25 samples were prepared using strains of Table 3 (strain for fermented feed production using beer), and the compounding ratio is shown in Table 4 (analysis of fermentation period and mixing ratio Composition).

Sample
Name Strain Con - Lc Lactobacillus casei Leup Leuconostoc pseudomesenteroides Lc + Lp Lactobacillus casei + Leuconostoc pseudomesenteroides Ilwoo Yeowo Farming Pressure Control Corporation Fermentation Agent

Name of sample Species Total amount 1,000g (100% / g) Beer Dumb Dried beer Firm Mix 1 Wavefront Wheat flour Seed dose

con1 Non-vaccination 265 400 200 125 0 0 10 con2 265 400 185 125 15 0 10 con3 300 435 100 125 15 15 10 con4 200 535 70 125 30 30 10 con5 0 600 235 125 15 15 10 Lc1 Lactobacillus casei KCTC3109 265 400 200 125 0 0 10 Lc2 265 400 185 125 15 0 10 Lc3 300 435 100 125 15 15 10 Lc4 200 535 70 125 30 30 10 Lc5 0 600 235 125 15 15 10 Lp1 Leuconostoc pseudomesenteroides KCTC3652 265 400 200 125 0 0 10 Lp2 265 400 185 125 15 0 10 Lp3 300 435 100 125 15 15 10 Lp4 200 535 70 125 30 30 10 Lp5 0 600 235 125 15 15 10 Lc + LP1 L. casei + L. pseudomesentemides mixed strain 265 400 200 125 0 0 10 Lc + LP2 265 400 185 125 15 0 10 Lc + LP3 300 435 100 125 15 15 10 Lc + LP4 200 535 70 125 30 30 10 Lc + LP5 0 600 235 125 15 15 10 ilwoo1 Conventional fermentation agent 265 400 200 125 0 0 10 ilwoo2 265 400 185 125 15 0 10 ilwoo3 300 435 100 125 15 15 10 ilwoo4 200 535 70 125 30 30 10 ilwoo5 0 600 235 125 15 15 10

(2) Analysis of the progress of fermentation

- Sugar analysis method

For the sugar content, 50 ml of the sample was centrifuged at 3200 rpm for 10 minutes using a centrifuge (Hanil, MF-80 General Purpose Centrifuge), and the supernatant was collected and analyzed using a digital sugar meter (PR-101 a, Atago. Was repeated three times, and the average value was calculated.

- Acid analysis method

20 mL of the fermentation broth was titrated with 0.1 N NaOH solution until the pH reached 8.3 by the AOAC method, and the required amount of 0.1 N NaOH was converted to the lactic acid content (%).

- pH analysis method

The pH of the sample solution was measured using a pH meter (Orion 3 star, Thermo, USA).

- pH and total acidity change

In order to prepare the fermented feed materials used in the method of producing the fully mixed fermented feed using lactic acid fermentation according to one embodiment of the present invention, pH and acidity change during the fermentation by varying the blending ratio and the different strains of beer and sugar, The results of the analysis are shown in FIG. 3 and FIG. In the case of pH change, it decreased to 4.5 level in most samples after 12 days fermentation at the initial 5.5 ~ 6.0. The effect of mixing ratio was not significant in the pH change, but the decrease was large in the test group with high brewer 's content. In the control without lactic acid bacteria, pH and acidity changes were relatively small, and Ilwoo strain (Ilwoo) showed the greatest variation. The fermentation of the control group without addition of lactic acid bacteria starter was progressed to some degree, which is thought to be due to lactic acid bacteria and acetic acid bacteria contained in beer. In the control group, acetic acid smell appeared to be stronger. Lactobacillus casei KCTC3109 and Leuconostoc Lc + Lp, a mixed strain of pseudomesenteroides KCTC3652, showed relatively good results. As a result, analysis of pH and acidity showed that fermentation proceeded normally.

- Change in sugar content

In the fermentation of fermented feedstuffs used in the method of producing fully mixed fermented feedstuffs using lactic acid fermentation according to one embodiment of the present invention, The results are shown in Fig. The initial sugar content was 3.7% in the test group which did not contain beer, and it was higher than 2.5% in the test group which had mixed beer. And decreased to 1% level after 6 days of fermentation. In the mixed strain of Ilwoo and Lc + Lp, the decrease was relatively high. This indicates that lactic acid fermentation was performed well with pH and acidity change.

(3) Development of optimum manufacturing process

In a fermented feedstuff preparation process for fermented feedstuffs, which is used in the method of producing a fully mixed fermented feedstuff by lactic acid fermentation according to an embodiment of the present invention, the beer is stabilized, and then the beer and its ingredients (glucose, fumix 1, After mixing for 1 hour, the dried beer is mixed for controlling the moisture, lactic acid bacteria are added and kept at room temperature and fermented under anaerobic conditions for about 2 weeks. FIG. 6 is a flowchart illustrating a process for producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention.

- Mixing ratio

Beer has high feed value and is highly available as a protein or supplementary feed for unit animals, but it contains a bitter taste of hops and is less favorable to livestock feed. In addition, the high water content and high perishability of the fermentation treatment and highly palatable feed should be treated to prevent pollution such as mold enhancement and palatability. In the present invention, five types of strains were inoculated and fermented in the five mixing ratios shown in Table 1, and the optimum mixing ratio was selected based on the fermentation process and the quality characteristics of the fermented feed materials. As a result, 30% of the fermented milk, 43.5% of the fermented milk, %, Wheat flour 1.5%, wheat flour 1.5% were selected as the optimum blending ratio of fermented feed materials.

- Moisture control

The moisture content of the blended raw materials is adjusted to the level of 40 to 50%.

- Lactic acid fermentation

Fermented by adding 1% of lactic acid bacteria culture to the water-regulated raw material. Fermentation conditions are fermentation at 20 ~ 30 ℃ for 7 ~ 10 days under anaerobic conditions.

- Fermented feed production using lactic acid bacteria

Fermented feedstuffs are prepared by mixing TMR feeds with fermented feedstocks rich in lactic acid bacteria and matured.

<Example 4: Analysis of components of fermented feed>

The ingredients for the fermented feed materials prepared in Examples 1 to 3 were analyzed.

(1) Free sugar analysis

- Analysis of free sugar was carried out by filtration with 0.45 μm membrane filter and Sep-pak C18 cartridge (Waters Associate, Milford, Mass., USA) to remove pigment and protein components and analyzed by HPLC. The flow rate was 1.0 mL / min and the injection volume was 10 μL. The flow rate was 1.0 mL / min and the flow rate was 1.0 mL / min. The column used was IonPac AS11-HS analytical (4 × 250 mm, 9 μm, Dionex Co., Ltd., Sunnyvale, CA, USA) , and the detector was an ELSD detector.

(2) Analysis of organic acids

Analysis of organic acids was carried out by filtration with a 0.45 μm membrane filter and a Sep-pak C18 cartridge (Waters Associate, Milford, MA, USA) to remove pigment and protein components and then analyzed by HPLC. The flow rate was 1.0 mL / min, the injection volume was 20 μL, and the injection volume was 20 μL. The column was loaded with Inertsil ODS-3V (4.6 × 250 mm, 5 μm, GL Sciences Inc., Tokyo, Japan), dihydrogenphosphate and phosphoric acid Detector used RI detector.

Lactic acid - containing organic acid is used as an additive in feed and feed. In particular, calves and piglets have lower acid production in the stomach and may cause gastrointestinal disturbances by decreasing the activity of protein digestive enzymes. In addition, acidification in the feed prevents bacterial contamination of the feed itself, and feed containing lactic acid can have the effect of adding antibiotics.

The lactic acid content of the fermented feed materials in the method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention is shown in FIG. Lactobacillus casei KCTC3109 and Leuconostoc 100 mg / Kg in the control without lactic acid bacterium and 5,500 mg / Kg of Lc + Lp, a mixed strain of pseudomesenteroides KCTC3652. Next, Leuconostoc KCTC3610 (Lc), and Lactobacillus casei KCTC3109 (Lc) showed about 1,500 mg / Kg for the fermentation of pseudomesenteroides KCTC3652 (Lp). Therefore, it was found that Lc + Lp mixed seeds were the best in terms of lactic acid production ability.

(3) free amino acids

For pretreatment for the extraction of free amino acids, 10 mL of the sample was added to 70% EtOH, sonicated for 1 hour, and then extracted at room temperature for 24 hours. The resultant was filtered with a 0.2 μ filter and a Sep-pak C18 cartridge to remove pigment and protein components and used as an analytical sample Respectively. HPLC analysis conditions were Dionex Ultimate 3000 and FL Detector (Emission 450nm, Excitation 340nm (OPA) Emission 305nm, Excitation 266nm (FMOC), UV Detector (338nm), Software used Chromeleon 6.8) as standard: 1 nmol / uL Amino The concentration of the standard product was diluted with the third distilled water and adjusted to 1000, 500, 100, 10 pmol / uL. Table 5 summarizes the analysis conditions of free amino acid.

Column VDSpher 100 C18-E (4.6mm x 150mm, 3.5um / VDS OptiLab, Germany) Mobile Phase A 40 mM sodium phosphate dibasic, pH 7 Mobile Phase B 3DW / Acetonitrile / Methanol (10: 45: 45 v / v%)

Injection Volume 0.5 uL Column Temperature 40 ℃ Sample Temperature 20 ℃ Reaction
protocol Pre-Amino acids are reacted automatically, and the reaction is terminated after stepwise mixing of sample with small amount of borate buffer, OPA / MPA, FMOC reagent. Reagent Samples: Amino acid standard (agilent 5061-3330, agilent 5062-2478)
Reagent A: Borate buffer (agilent 5061-3339)
Reagent B: OPA reagent (agilent 5061-3335)
Reagent C: FMOC solution (agilent 5061-3337)
Na2HPO4, Na2B4O7: Sigma
ACN. MeOH: HPLC grade (Merck) Injector program Draw 5uL from boratebuffer - Draw 1uL from sample - Draw 1uL from OPA - Draw 1uL from FMOC - Draw 32uL from water (Dilution) - Inject Reference Rapid, Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids' (Agilent Technologies)

- free amino acid analysis conditions

An amino acid is a compound having an amine group (-NH2) and a carboxyl group (-COOH) which are generated when a protein is hydrolyzed. There are more than 200 kinds of amino acids in living body, but the protein of feed is composed of 20 amino acids. Amino acids are divided into essential amino acids and non-essential amino acids. The protein synthesis in the body requires 20 kinds of amino acids. Most of them can be synthesized in the body. The amino acids that are not necessarily consumed are called non-essential amino acids. They can not be synthesized in the body or can not be synthesized even though they are synthesized. . Unlike other unit animals, ruminants have been recognized as being able to produce most amino acids by rumen microbes, so there is no need for supplementary feeding, but the amino acid requirement is insufficient only in the amino acids synthesized in the rumen. Arginine, histidine, isoleucine, , Methionine, phenylalanine, threonine, tryptophan, valine and other essential amino acids. Brewer's fermented feeds are supplied with high-quality protein from yeast and can be degraded into amino acid form during fermentation of lactic acid, which can help to promote growth.

The results of analysis of the amino acids of the fermented feed materials in the method of producing fully mixed fermented feeds using lactic acid fermentation according to one embodiment of the present invention are shown in Table 6 (free amino acid content of fermented feed materials) and Table 7 Amino acid content). Total amino acid content was determined by Leuconostoc The highest content of Lactobacillus casei KCTC3109 and Leuconostoc was 5,573.41 mg / Kg when fermented with pseudomesenteroides KCTC3652 (Lp) In the case of Lc + Lp, a mixed strain of pseudomesenteroides KCTC3652, 5,470.25 mg / Kg was almost similar. In the case of essential amino acids, Leu>Val>Ile>Phe>Thr>Tyr>Met>Lys> Arg. Of the non-essential amino acids, GABA is the most abundant, and Leuconostoc The highest value was found to be 1,164.86 mg / Kg when fermented with pseudomesenteroides KCTC3652 (Lp) strain and 1,046.23 mg / Kg with Lc + Lp mixed strain. Gamma Aminobutyric acid (GABA) is an inhibitory neurotransmitter that acts on the central nervous system of mammals. GABA plays a role in regulating nerve stimulation in the nervous system, and in humans, GABA is involved in directly regulating muscle condition. GABA is strictly speaking a kind of amino acid, but since it is not an alpha amino acid, GABA does not participate in protein composition. It is an inhibitory neurotransmitter that exists in the mammalian brain and blocks the secretion of several neurotransmitters in the central nervous system. It can easily be said to play a role in stabilizing the nerves. When stressed, extremely unstable, and hyperactive, GABA promotes brain alfaparine activity, inhibits stress, tranquilizing neurotransmission, normalizes brain function, stresses and tensions, nervous instability, tension-induced hypertension, Epilepsy, schizophrenia, hyperactivity, insomnia, depression and other symptoms are known to have various effects. It is thought that this will help the production of quality cattle by promoting mental and physical stability of breeding cattle and preventing diseases.

mg / Kg Asp Glu Ser Gln Gly Thr Arg Ala GABA Control 159.189 376.3076 208.0844 79.2506 190.7629 186.202 8.8281 956.2986 367.6702 LC-3 64.356 192.5429 126.6305 38.5662 268.0771 92.2589 84.1182 1173.934 777.9755 LP-3 14.902 78.3993 123.2001 40.5759 373.3125 79.583 4.1244 1552.908 1164.859 Lc + LP 31.167 142.9574 164.8443 27.6563 331.6392 137.2512 5.9208 1298.89 1046.227 ilwoo 67.092 155.3186 153.7619 45.046 344.6516 92.9583 6.6893 1350.335 1018.726

mg / Kg Tyr Val Met Phe Ile Leu Lys Pro Total Control 40.9792 388.4751 15.8325 199.687 238.8957 540.5551 22.1545 367.7707 4,346.95 LC-3 38.8886 446.5346 10.1231 182.701 301.8489 623.6804 61.3826 281.6847 4,765.31 LP-3 9.9861 544.9483 44.9794 166.912 392.6461 714.2385 4.0397 263.795 5,573.41 Lc + LP 9.1696 483.0004 15.3336 240.926 354.1424 653.5198 22.7895 504.8107 5,470.25 ilwoo 13.1713 504.4379 48.3911 155.8761 368.8892 706.5337 19.0647 290.6357 5,341.58

(4) Total polyphenol content

The total polyphenol content is determined by the Folin-Denis method (Folin and Denis 1912). That is, 2 mL of distilled water and 0.2 mL of Folin-ciocalteu's phenol reagent were added to 0.1 mL of the sample, and the mixture was reacted at room temperature for 5 minutes. To this, 1.5 mL of Na ₂ CO 3 solution (15%) was added, and the solution was allowed to stand for 2 hours and absorbance was measured at 765 nm. The total polyphenol content was determined from a standard curve prepared using gallic acid.

(5) Total flavonoid content

To measure the total flavonoid content, sample solution (1 mL), distilled water (4 mL), NaNO ₂ (5%) solution, and incubate at room temperature for 5 minutes. Add 0.3 mL of AlCl ₃ (10%) reagent. After standing for 6 minutes, add 1 M NaOH (2 mL), stop the reaction, and make the volume of the reaction solution to 10 mL with distilled water. The solution was measured for absorbance at 510 nm and the total flavonoid content was determined from the standard curve prepared using rutin.

(6) Analysis of β-Glucan

The content of beta-glucan in the fermented feed materials was quantified as follows using Mushroom and Yeast β-Glucan Kit. Fermented feed materials Beta glucan extract was pulverized and passed through 100 mesh to quantify 100 mg. Total glucan content was determined by adding 1.5 mL of 37% hydrochloric acid to each sample, followed by reaction at 30 ° C for 45 minutes. 10 mL of distilled water was added to the reaction mixture, and the mixture was heated at 100 ° C. for 2 hours, cooled, and adjusted to a volume of 100 mL using 10 mL of 2 N KOH and 200 mM sodium acetate buffer (pH 5.0). Β-glucosidase (100 U / mL) and β-glucosidase (20 U / mL) were dissolved in 200 mM sodium acetate buffer (pH 7.0) and the solution was centrifuged at 1,500 × g for 10 min. acetate buffer (pH 5.0)) was added and reacted at 40 ° C for 60 minutes. After the reaction was completed, 3 mL of GOPOD reagent was added, and the reaction was carried out at 40 ° C. for 20 minutes, and the absorbance at 510 nm was measured. The amount of α-glucan was determined by adding 2 mL of 2 M KOH to 100 mg of sample, reacting under cooling condition for 20 minutes, adding 8 mL of 1.2 M sodium acetate buffer (pH 3.8) and 0.2 mL of amyloglucosidase (1,630 U / mL) Lt; 0 &gt; C for 30 minutes. 0.1 mL of the supernatant obtained by centrifugation at 1,500 × g for 10 minutes was taken, and 0.1 mL of 200 mM sodium acetate buffer (pH 5.0) and 3.0 mL of GOPOD reagent were added thereto and reacted at 40 ° C. for 20 minutes. The absorbance of the reaction solution was measured at 510 nm, and the difference between the total glucan and the alpha glucan content was calculated as the content of beta glucan.

(7) Polyphenol and Flavonoid Content

Polyphenol is a secondary metabolite that changes part of the sugar produced when a green plant plants photosynthesizes. It exists as a constituent of 8,000 structures in the plant system, and it is also referred to as a phenol complex. It is abundant in sugarcane, millet, barley, soybean, peanut, fruit, vegetables, tea, and is generally colored, bitter and bitter. These polyphenols consist of two flavonoids and non-flavonoids. Flavonoids are a generic term for substances that protect plant cells from free radicals. Both Flavonoids and Non-flavonoids have antioxidant activity and about 300 species. The types and contents of polyphenols, whose functions are widely known, are as follows. It regulates the balance of anthocyanin and female hormone which are rich in red wine, eggplant, blueberry which is good for the prevention of the heart disease, and isoflavone rich in soybean and soybean product, fatigue recovery, Is rich in cocoa and chocolate, is rich in cardamom polyphenols, strengthens capillaries, lowers blood pressure and improves memory, abundant roots in soba noodles and corn, excellent coronary artery sclerosis prevention, quercetin rich in onions and apple peels , Catechin which is abundant in tea such as green tea. Flavonoid is a water-soluble plant pigment belonging to polyphenol, which has antioxidant activity and strong capillary action. Flavonoids are generally referred to as vitamin P, which is a compound of a plant with a bright yellow pigment, a compound having a diphenylpropane structure in which two benzene rings in a chemical structure are linked to three carbon atoms.

The results of analyzing the content of polyphenol and flavonoid in the fermented feed materials in the method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention are shown in FIGS.

In general, plants contain secondary metabolites such as polyphenols and flavonoid compounds, which are aromatic compounds that donate electrons from the -OH group on the ring structure and become structurally stabilized by the phenol ring structure resonance and exhibit antioxidant activity. These polyphenols and flavonoids are involved in physiological activities such as antioxidation, antiinflammation, and anticancer. As shown in FIG. 7, when fermented with the Lc + Lp mixed strain and ilwoo strain, there was no significant difference in the level of 47 mg GAE / 100 g to 55 mg GAE / 100 g, but in the case of Lc and Lp alone strains, mg GAE / 100g, respectively. This result alone will not explain the reason. According to a study in the Korean Journal of Food Preservation Vol.20 No.4 pp.583-591, the content of polyphenols in the hydrothermal extracts and fermented extracts of L. bifidum were almost not reported. As shown in FIG. 8, the total plononoid content tended to decrease to 0.033-0.06 mg / 100g after lactic acid fermentation at 0.085 mg / 100g of the control without seed culture. In the Kwon et al. Study, It has been reported that flavonoid-based substances are reduced by fermentation.

(8) β-Glucan Content of Lactic Acid Fermented Diets

β-Glucan is a generic term for polysaccharides polymerized mainly by β-1,3 chemical bonds of glucose, and β-glucan derived from microorganisms produced by cell wall or extracellular polysaccharide of microorganisms such as mushroom, yeast, There is a vegetable β-glucan extracted from the same grains of dietary fiber. β-glucan is known to be a highly immunogenic substance and is widely used as a food additive with approval from the Food and Drug Administration (FDA) General Recognized as Safe (GRAS). 9, which is a graph showing the content of betaclucan in a fermented feed material in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention, The content of β-glucan in fermented mixed fermented diets was 42.55 mg / g, which was 3.5 times higher than that of control (12.45 mg / g). It is considered that the fully mixed fermented feed prepared by adding the fermented feed material in the method of producing the fully mixed fermented feed using the lactic acid fermentation according to an embodiment of the present invention may contribute to the prevention of the disease in the breeding stock.

&Lt; Example 5: Antioxidant property of fermented feed >

The antioxidative properties of the samples obtained in Examples 1 to 3 were examined.

(1) DPPH radical scavenging ability

In 0.2 mL of beer and sugar aliquots, add 0.8 mL of distilled water to make 1 mL, then make 1.5 × 10 4 DPPH in methanol and leave in a dark place for 2 hours. 40 μL of the sample was added to 160 μL of the DPPH solution, and the change in absorbance was measured 30 minutes later. The antioxidant capacity (AEAC, ascorbic acid equivalent, antioxidant capacity, mg%) of the sample was calculated after preparing a standard curve using ascorbic acid (1 mg / mL).

DPPH radical

(%) = (1-absorbance of sample added / absorbance of sample not added) x 100

Antioxidants are added into lipid systems to prevent the formation of various undesirable compounds due to oxidation. Various oxidation products produced by oxidation are known to damage DNA or cause cancer. BHT (butylated hydroxy anisol) and BHT (butylated hydroxy toluene), which are generally used as phenolic synthetic antioxidants, have been widely used because of their effectiveness and economical safety. However, excessive intake of toxic substances such as liver, gastric mucosa, lung, And it is necessary to develop a safe alternative antioxidant. The antioxidants of phenolic materials derived from plants are partly responsible for forming metal complexes, but their main function is their antioxidant activity. Therefore, natural antioxidants can be developed by searching radical scavenging function from plant extracts.

 As shown in FIG. 11 showing the graph of the DPPH content of the fermented feed materials in the method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention, the control (control) showed 85.88% LC of 85.97% LP 80.27%, but the difference of DPPH between LC + LP and ilwoo was 51.57 and 41.58%.

(2) Reducing power

The reducing power of the sample is measured using the principle that the degree of color development increases as the intensity of reducing iron ions from Fe ^{3 +} to Fe ^{2 +} increases. Take 1 mL of each sample solution, add 2.5 mL of phosphate buffer solution (0.2M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%, w / v), and mix at 50 ° C for 20 minutes. Add 2.5 mL of trichloroacetic acid (10%, w / v) to the reaction mixture, and centrifuge at 3,000 rpm for 10 minutes. Take 2.5 mL of the supernatant and place in a test tube, add 2.5 mL of distilled water and 0.5 mL of FeCl ₃ (0.1%, w / v) and measure absorbance at 700 nm.

The ability to convert to Fe ^{3 +} - Fe ^{2 +} was measured to determine the reducing power. Hydrogen is donated to stabilize free radicals and convert to Fe ^{2 +} . In the antioxidant activity experiments, the contents of total phenol contents were similar to those of DPPH and ABTS radical tests.

As shown in FIG. 12, which is a graph showing the reduction power of lactic acid fermented feed materials of fermented feed materials in a method of producing a fully mixed fermented feed using lactic acid fermentation according to an embodiment of the present invention, The highest OD value was 1.17 ± 0.02 in the test group, which was higher than the control value of 0.83 ± 0.03.

(3) ABTS radical scavenging activity assay method

The ABTS radical scavenging activity of fermented feed materials was measured by the method of Van den Berg et al. (16). 1.0 mM AAPH was mixed with 2.5 mM ABTS dissolved in 100 mM PBS buffer and reacted for 12 minutes at 68oC. The concentration of ABTS solution was determined to be 0.650 ± 10.2 at 734 nm. 20 μL of fermented feed materials and 980 μL ABTS solution were reacted in a 37 ° C water bath for 10 minutes while measuring the absorbance at 734 nm. The positive control group was Trolox® 1 mg / mL. ABTS radical scavenging ability of fermented feed materials was calculated according to the following equation.

ABTS radical = scavenging activity (%) (Blank O.D.-Sample O.D / Blank O.D.) 100

&Lt; Example 6: Influence of fermented feed diets on meat quality grade of fermented Hanwoo steers>

In order to improve the quality of beef, research on meat quality and carcass composition (Garcia et al., 2008; Gruber, et al., 2006), study on the chemical characteristics of beef (Gotoh, et al., 2009; et al., 2008; Schmidt, et al., 2010), sensory characteristics (Behrends, et al., 2007; Et al., 2008), and the correlation between physicochemical and sensory characteristics (Banoviæ, et al., 2009; Caine, et al., 2003; Destefanis, et al., 2008) (2004), and it has been reported that the beef quality is influenced by various factors such as muscle fat content, shear force, water retention, heating loss, pH, cooling method and aging (George-Evins et al., 2004; Lorenzen et al., 2003; Muchenje et al., 2009; Rhee et al., 2004). The results of this study suggest that meat quality and year promotion are the main factors affecting beef quality, but the food culture of beef is different in each country and different taste perceptions such as flavor, year and juiciness are different, In order to differentiate the quality of Korean beef meat, it is necessary to conduct scientific and systematic research for our food culture. Kim and Lee (2003), Kim et al. (2008), Moon et al. (2006), Song et al. (2010) and Yu et al. There are various factors such as breeding environment and age for improvement of meat quality grade, but the most important part is feed.

It is considered that the meat quality of beef is different by various feeds such as pasta, mixed feed, fermented feed and the like. According to one embodiment of the present invention, the complete mixed fermented feed prepared by the complete mixed fermented feed production method using lactic acid fermentation To investigate the improvement of the quality of Hanwoo steers, we analyzed the slaughter quality of Hanwoo steers from Jeollanam region (42,180 dwelling) and Naju area (3,162 dwelling) from January 2017 to November 2017. The total mixed fermented feedstuff containing the fermented feedstuffs was compared with the fermented Hanwoo steer (122 beef) in the method of producing the fully mixed fermented feedstuff using lactic acid fermentation according to an embodiment of the present invention. FIG. 13 is a graph showing the grade ratios for Korean cattle slaughtered in Jeollanam-do, Naju-slaughtered Hanwoo, and Hanwoo-fed Korean cattle diets. The fermented feedstuffs were used to produce fully mixed fermented feedstuffs in a fermented fermented lactic acid fermented lactic acid fermented according to an embodiment of the present invention. % In the Naju area, and 65% in the Naju area.

- Quality of fermented feed materials

According to the standards and specifications of feeds, the registered ingredients of the fiber fermented feeds are those that contain more than 15% (by dry weight) of the raw materials, However, 10% or more in case of large consumption meat stage) should be confirmed. Table 8 summarizes the standards and specifications of fermented feed.

designation Registered ingredient Remarks Minimal Maximum Other Fiber fermented feed Crude protein crude fiber moisture
Views min Fermentation strain Identify raw materials and fermentation treatment methods
Confirm that the crude fiber content is at least 15% (based on building), but not more than 10%

- Number of lactic acid bacteria

The fermented feed materials were prepared by inoculating lactic acid bacteria and then examined for changes in microbial content in fermented feed materials such as beer bacilli. The results are shown in Table 9 (lactic acid bacteria of fermented feed materials). Lactic acid bacteria counts were 1.0 × 10 ⁶ cells / g in the control, but 1.0 × 10 ⁹ cells / g in the Lp + Lc samples and 1.0 ~ 1.2 × 10 ⁸ cells / g in the rest. Srigopalram et al. (2015) reported that the number of lactic acid bacteria increased and that the number of yeast and mold decreased with the inoculation of lactic acid bacteria in rye. Similar results were obtained in the experiment for the verification of the present invention. The number of lactic acid bacteria, The number of lactic acid bacteria in silage seems to maintain a similar number, suggesting that lactic acid bacteria must be inoculated to produce high quality fermented feedstuffs. Table 10 summarizes the results of analyzing the fermented feed quality standards.

Name of sample Analytical component Remarks Fermented feedstock raw material Developed products (CFU / g) standard goal Fermented feedstock (control) 1.210 ⁶ none More than 10 ⁸ Fermented feedstock LC 1.210 ⁸ Fermented feed ingredients LP 1.010 ⁸ Fermented feed material LC + LP 1.010 ⁹ ilwoo 1.010 ⁸

Name of sample Analytical component Remarks Fermented feed Breeding (breeding) Developed products standard goal moisture 35.85 35.2 Building basis crude fiber 15% or more
Less than 40% moisture Crude protein 10.6 9 Crude fiber 15.02 16.56 Views min 7.82 2.13 ADF 21.22 21.77 NDF 31.5 36.7 Large Consumption Electricity (nurturing) moisture 30.74 33.5 Crude protein 12.75 11.82 Crude fiber 13.06 14.2 Views min 7.06 4.78 ADF 21.99 19.63 NDF 33.94 32.83 Large Consumption Meat Meal (fattening) moisture 33.5 34.53 Crude protein 11.82 9.94 Crude fiber 14.2 12.74 Views min 4.78 1.15 ADF 19.63 16.66 NDF 32.83 29.26

- conclusion

The fermentation period was monitored by lactic acid bacteria inoculation of fermented feedstuffs such as beer to find fermentation agent to increase the decay and shelf life of fermented feed in lactic acid fermentation according to the present invention. The pH ranged from 4.26 to 4.74 and the total acidity increased from 0.8 to 2.3%. During the fermentation period, organic acids, lactic acid and other acids are produced during the fermentation period to reduce the contamination of other microorganisms, thereby affecting safe fermentation. In the case of sugarcane, it was maintained at a high level in the early stage and decreased to 1% level mostly by the proliferation of lactic acid bacteria. It was found that the lactic acid fermentation proceeded well with pH and acidity change. Lactic acid bacteria counts were 1.0 × 10 ⁶ cells / g in the control, but 1.0 × 10 ⁹ cells / g in the Lp + Lc samples and 1.0 ~ 1.2 × 10 ⁸ cells / g in the rest.

Analysis of physicochemical components of fermented feedstuffs showed that 100 mg / Kg of organic acid was used for control, and Lactobacillus casei KCTC3109 and Leuconostoc and 5,500 mg / Kg of Lc + Lp, a mixed strain of pseudomesenteroides KCTC3652. And about 1,500 mg / Kg for Lactobacillus casei KCTC3109 (Lc) and for the yeast feedstuff (ilwoo). Therefore, it was found that Lc + Lp mixed seeds were the best in terms of lactic acid production ability. Total amino acid content was determined by Leuconostoc The Lc + Lp was 5,470.25 mg / Kg in the case of the mixed strain of Lactobacillus casei KCTC3109 and Leuconostoc pseudomesenteroides KCTC3652, which was almost similar to that of 5,470.25 mg / Kg in the case of fermentation with pseudomesenteroides KCTC3652 (Lp). In the case of essential amino acids, Leu>Val>Ile>Phe>Thr>Tyr>Met>Lys> Arg. GABA plays a role in regulating neurotoxicity in the nervous system and is an inhibitory neurotransmitter that exists in the mammalian brain and blocks the secretion of several neurotransmitters in the central nervous system. It can easily be said to play a role in stabilizing the nerves. The content of polyphenols and flavonoids decreased from 0.085 to 0.033 to 0.06 mg / 100g after lactic acid fermentation, and the content of β-glucan, which is known to be highly immunosuppressive, was 42.55 mg / g.

In order to verify the superiority of fermented feed materials, antioxidant experiments of fermented feed materials were conducted. DPPH radical was 41.58 ~ 85.88%, and the reducing power was 0.83 ~ 1.17%, which was similar or slightly better than the control.

Fermented diets were prepared using fermented feedstuffs of high quality and confirmed to have the effect on the meat quality of Hanwoo steers.

The method for producing a fully mixed fermented feed using lactic acid fermentation according to the present invention is characterized in that fermentation is performed by inoculating lactic acid bacterium mixed with brewing liquor to produce a functional fermented feed having higher nutritional quality of the fermented feed than that of commercial fermented feed, It is judged that the production is possible.

The method of manufacturing a fully mixed fermented feed using the lactic acid fermentation described above and the above-described lactic acid fermentation method is only one embodiment for carrying out the present invention, and should not be construed as limiting the technical idea of the present invention. The scope of protection of the present invention is defined only by the matters set forth in the following claims, and the embodiments improved and changed without departing from the gist of the present invention are obvious to those having ordinary skill in the art to which the present invention belongs It will be understood that the invention is not limited thereto.

Claims (5)

Lactic acid bacteria are inoculated in a raw material mixture containing brewer's grains, fermenting lactic acid to produce a fermented feed material,
And fermenting the fermented feedstock to a complete mixed fermentation feed (TMR, Total Mixed Ration) to produce a completely mixed fermented feedstuff. The method according to claim 1,
Wherein the mixture containing the brewer's beans is mixed with sugar, fumix No. 1, corn flakes and wheat flour. 3. The method of claim 2,
Wherein the mixture containing the brewing beverage is a mixture of 30% of beer, 43.5% of sugar beet, 12.5% of Fermix 1 and 1.5% of wheat flour 1.5% . 4. The method according to any one of claims 1 to 3,
The lactic acid bacteria include Lactobacillus casei KCTC3109 (Korea Research Institute of Bioscience and Biotechnology), Leuconostoc wherein at least one of pseudomesenteroides KCTC3652 ( Lactobacillus plantarum KCTC 33131), Leuconostoc pseudomesenteroides JLRI 01, and KCCM 11443P (Korean microorganism preservation center) is selected. A method for producing a fully mixed fermented feed using fermentation. 5. The method of claim 4,
Wherein lactic acid fermentation of fermented feed materials is carried out at a temperature of 20 to 30 DEG C for 7 to 10 days under anaerobic conditions.

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