KR101869024B1

KR101869024B1 - Method for manufacturing pine needle probiotic and pine needle probiotic composition manufactured thereby

Info

Publication number: KR101869024B1
Application number: KR1020160026603A
Authority: KR
Inventors: 김삼철; 정윤섭; 주영호; 이혁준; 이성신
Original assignee: 하동군
Priority date: 2016-03-04
Filing date: 2016-03-04
Publication date: 2018-06-20
Also published as: KR20170103550A

Abstract

TECHNICAL FIELD The present invention relates to a method for producing a pine needle prophylactic agent, a pine needle prophylactic composition prepared thereby, and a method for breeding cattle containing the same. According to the present invention, an excellent useful microorganism can be selected and utilized in the pine pine pine needles existing in the pine needle leaf pine, which is being fermented by the fermentation mycelium, and can be utilized from the native pine from the pine needles, It is possible to secure. Further, by improving the seed culture method and the solid fermentation method which have been conventionally used, it is possible to smooth the growth of the seed and to obtain a sufficient number of viable cells.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a pine needle prophylactic and pine needle probiotic composition,

TECHNICAL FIELD The present invention relates to a method for producing a pine needle prophylactic agent, a pine needle prophylactic composition prepared thereby, and a method for breeding cattle containing the same.

Antibiotics have been used extensively in livestock specifications for the purpose of treating and promoting growth of livestock since the 1950s. The use of antibiotics in livestock feeds at low quasi-therapeutical levels resulted in improved productivity such as disease control, growth promotion, and feed efficiency improvements in livestock and contributed to the increase in the incomes of both farms. However, as the amount of antibiotics used increases throughout the world, resistant bacteria against antibiotics are generated, causing problems of environmental pollution. In particular, recently, antibiotics-resistant bacteria that may be harmful to the human body due to the use of antibiotics have been reported, and Korea has also banned the use of antibiotics for livestock feed since July 2011.

Therefore, the development and research of materials that can replace antibiotics are underway, among which living microbial supply sources are in the spotlight for probiotics that can improve intestinal microflora and livestock productivity, and demand for farm households is also surging.

Domestic probiotics market accounts for about 50 billion dollars, and the number and types of probiotics are increasing every year. For example, Korean Patent Registration No. 1197682 discloses a fermented probiotic as a feed additive. As a result, the number of probiotics producers has increased, and many probiotics have been manufactured and distributed. At the same time, many probiotics that have not been scientifically validated are on the market.

On the other hand, in Hadong County, Gyeongnam Province, the probiotics were manufactured using the microorganisms native to the pine needles, which are natural resources. However, since the conventional method for collecting the seeds was not selecting only the excellent strains but collecting all the bacteria present in the natural world, there was a concern about contamination by various kinds of bacteria or pathogenic microorganisms. The collected seeds were cultured using brown sugar, but not only the live cells were not grown, but the number of viable cells was less than the legal allowable value (10 ⁶ or more per g) There were disadvantages. Therefore, systematic and scientific technology development is urgently required for enhancing the brand value of the local excellent brand Korean pearl beef, and for promoting the Korean pearl producer, Chinese pearl farming, and Korean-related industries.

According to the present invention, it is possible to search a useful microorganism from pine needle indigenous microorganisms, to select an excellent strain through enzymatic vitality and antimicrobial activity test, to produce a high quality pine needle prodrug through liquid culture and solid fermentation, And a method of raising cattle comprising feeding a feed containing the pine needle probiotic agent.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

As a technical means for accomplishing the above technical object, the first aspect of the present invention provides a method for producing pine needle extract, Isolating lactic acid bacteria, Bacillus subtilis, and yeast strains having an antimicrobial activity against a fusarium species fungus having a cellulose hydrolysis activity from the pine leaf extract; Culturing the lactic acid bacteria, Bacillus subtilis and Yeast cells for 2 days to 6 days respectively; Adding a rice bran, molasses, water, bacillus subtilis and yeast bacteria to a solid fermentor and performing primary fermentation for 10 hours to 30 hours while stirring; And, after the first fermentation, further adding the lactic acid bacterium and performing secondary fermentation for 1 day to 6 days while stirring.

According to one embodiment of the present invention, the liquid culture of the lactic acid bacteria, Bacillus subtilis and Yeast cells may include culturing the lactic acid bacteria with Lactobacillus MRS broth, the Bacillus with LB broth, and the yeast with potato dextrose broth.

The second aspect of the present invention provides a pine needle procurement composition prepared by the method of the first aspect of the present application and having a number of microorganisms of 10 ⁷ to 10 ⁸ cfu / g.

A third aspect of the present invention provides a method of raising cattle comprising feeding the pine needle probiotics produced by the method of the first aspect of the present invention in an amount of 0.5 to 5% by weight based on the feed and feeding.

According to the manufacturing method of the pine needle prophylactic agent of the present invention, superior beneficial microorganisms can be selected and utilized in the pine pine pine leaf which is being fermented by the fermentation mycelium, that is, the pine pine native microorganism existing in the pine needle leaf microbial pine. From the indigenous microorganism derived from pine needles, It is possible to secure a strain having excellent vitality.

In addition, there is an advantage that seed culture can be smoothly grown and sufficient viable cell counts can be obtained by improving the seed culture method and the solid fermentation method which have been conventionally used.

Furthermore, it is possible to improve the productivity of cattle by feeding the probiotics of the present invention including the above-mentioned excellent live bacteria to cattle, to suppress cattle intestinal harmful bacteria, to reduce odors of manure, and to provide cows having excellent meat quality.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a microorganism identification procedure carried out according to an embodiment of the method for producing a pine needle prophylactic agent of the present invention. FIG.
FIG. 2 is a photographic image showing the result of an experiment for verifying the antimicrobial activity performed according to an embodiment of the method for producing a procpholyte of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A, B, or A and B".

Hereinafter, a method for producing the pine needle prophylactic agent of the present invention, a pine needle prophylactic composition prepared by the method, and a method for raising cattle using the same will be described in detail with reference to embodiments, examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

According to a first aspect of the present invention, there is provided a method for producing pine needle extract, Isolating lactic acid bacteria, Bacillus subtilis, and yeast strains having an antimicrobial activity against a fusarium species fungus having a cellulose hydrolysis activity from the pine leaf extract; Culturing the lactic acid bacteria, Bacillus subtilis and Yeast cells for 2 days to 6 days respectively; Adding a rice bran, molasses, water, bacillus subtilis and yeast bacteria to a solid fermentor and performing primary fermentation for 10 hours to 30 hours while stirring; And, after the first fermentation, further adding the lactic acid bacterium and performing secondary fermentation for 1 day to 6 days while stirring.

For example, the step of culturing the lactic acid bacteria, Bacillus subtilis, and yeast cells may be performed for about 4 days, and the step of solid fermentation of the Bacillus subtilis and Yeast can be performed for about 24 hours, And the second fermentation step may be performed for about 4 days, but may not be limited thereto.

The lactic acid bacterium has an excellent ability to produce an organic acid, and Bacillus subtilis has a function to secrete various digestive enzymes. The yeast can be usefully used because it has a function of regulating the metabolic process in rumen rumen and providing nutritional components.

Since the step of culturing the lactic acid bacteria, Bacillus subtilis and Yeast cells for 2 days to 6 days is different from that required for the growth temperature, culture time, aerobic condition and culture medium of each microorganism, Can be performed to optimize the conditions and to eliminate the mutual inhibition phenomenon by the culture products.

In the solid fermentation step, rice bran and molasses which are easy to purchase raw materials, cheap and easy fermentation of microorganism can be used as a basic medium. For example, the base medium may be a weight ratio of raw water to water of 10: 1 to 10: 5, and the raw water may be used in an amount of about 50% to about 85% by weight based on the total weight of the medium. For example, molasses may be added thereto at a ratio of 0.1 to 5% by weight based on the raw steel.

The seed medium thus prepared can be inoculated in a liquid medium and then fermented. The liquid cultured seed bacterium may be added to the medium at the same ratio, for example, 0.1 to 10% by weight based on the basic medium, but may not be limited thereto.

According to one embodiment of the present invention, the liquid culture of the lactic acid bacteria, Bacillus subtilis and Yeast cells may include culturing the lactic acid bacteria with Lactobacillus MRS broth, Bacillus with LB broth, and yeast with potato dextrose broth, But may not be limited.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

1. Selection of excellent strains by searching pesticide-derived useful microorganism and verifying antibacterial activity

In this experiment, excellent microorganisms were selected from pine needle indigenous microorganisms and the characteristics of these microorganisms were studied.

For the selection of microorganisms, 10 paddy fields were randomly selected from the hills of Miyang-myeon, Hadong-gun, and the composted pine leaves were collected and mixed well. The well mixed materials and the sterilized distilled water were mixed at a ratio of 1: 9 and allowed to stand at room temperature for 1 hour and then filtered with a gauze to prepare an extract. Bacteria were firstly cultured using Lactobacilli MRS agar, LB agar and Potato Derxtrose agar to select lactic acid bacteria, Bacillus subtilis, and yeast strains. Lactic acid bacteria, Bacillus subtilis, and Yeast cells, which have the fastest growth rate in the first culture, were purified and then subjected to secondary culture. Then, enzyme activity and antimicrobial activity were measured using selected microorganisms.

Survey item

Identification of microorganisms: The selected useful microorganisms were identified and classified through Maldi-TOF / TOF. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a procedure for identifying a microorganism to be performed in the method for producing a procpholyte of the present invention. FIG.

Measurement of various digestive enzymes activity of microorganisms: The enzymatic activity evaluation was performed on the activity of cellulose hydrolase and estrase in the medium. Each isolate was inoculated into each enzyme - active medium and evaluated after 5 days of incubation in a 28 ° C incubator.

Measurement of antimicrobial activity of microorganisms: Animal nutrition laboratory 17 kinds of livestock feed added microorganisms were inoculated on PDK medium and cultured. Two days after the culture, Fusarium moniliforme was inoculated. The antimicrobial activity was evaluated by measuring mycelial growth inhibition ability at 3, 5 and 7 days after inoculation.

Measurement result

The results of analysis of the activities of the cellulose hydrolytic enzymes and the estase enzymes of the microorganisms are shown in Table 1 below.

As a result of evaluating the activity of cellulose hydrolytic enzymes and the activity of esterases of 17 kinds of isolates (13 species of Bacillus subtilis, 2 kinds of lactic acid bacteria and 2 kinds of yeast), 6 kinds of cellulolytic enzymes were secreted Species, and 1 kind of lactic acid bacteria) were detected. However, strains having the activity of an estase enzyme that efficiently degrades lignin of plant cells were not detected (see Table 1). In Table 1, " high " represents Bacillus, " oil " represents lactic acid bacteria, and " oil " represents yeast strain.

FIG. 2 shows the antimicrobial activity against Fusarium moniliforme, a fungus toxin producing fungus (13 species), lactic acid bacteria (2 species) and yeast fungi (2 species) among the microorganisms isolated from the pine needle leaf fungus Table 2 shows the results of the antimicrobial activity analysis. The results are shown in Table 2. < tb > < TABLE >

Among the two lactic acid bacteria, Yu 1-2 (1) (8-1) showed mycelial growth inhibition ability against Fusarium moniliforme. For yeast, the antimicrobial activity was not verified (see Table 2).

Research summary

In Experiment 1, Bacillus subtilis was selected as 'Ganoderma 4-2 ② (6-1)', lactic acid bacillus was identified as 'Yu 1-2 (8-1)' and yeast was identified as 'Hyo 4-1 ①'.

2. Establishment of liquid culture condition of selected useful microorganisms

Experiments to establish optimal liquid fermentation conditions using the selected useful microorganisms were carried out as follows.

Selection of seedlings: In Experiment 1, lactic acid bacteria, Bacillus subtilis, and Yeast strains, which were found to have excellent enzymatic activity and antimicrobial activity, were selected and used as seeds.

Conventional seed culture method: After the seeds were prepared, they were sprayed in a place where the pine needles were abundant in the wild mountain of Hyodang-myeon, Hadong-gun and left for about 5 days. This allowed them to inoculate the microorganisms present in the composted pine leaves. After the incubation for 5 days, the collected rice was collected and mixed with brown sugar and water at a ratio of 1: 1, sealed for about 1 week, and the cultured liquid was used as a seed.

New seed culture method: As described in Experiment 1 above, the pine needles that were composted were collected in the same place as the existing seed collection sites, and the first and second cultures were obtained to obtain excellent strains. Lactobacillus MRS broth, lactobacillus LB broth and yeast were cultured in a 39 ° C incubator for 4 days using Potato Derextrose broth at intervals of 1 day, And the number of bacteria was examined.

Survey item

Investigation of Optimum Liquid Fermentation Conditions: The medium components of the selected strains were mixed in a 2 L container in a laboratory, sterilized at 121 ° C for 15 minutes through a high pressure sterilizer, and inoculated with the selected bacteria in the sterilized medium, And the number of bacteria was measured.

Microbial counts: Lactobacillus was cultured on MRS agar, LB agar and PDA agar, and lactic acid bacteria, Bacillus subtilis, Yeast fungus and Fungi were analyzed. The cultured lactic acid bacteria were cultured in a CO ₂ incubator at 39 ° C for 1 to 8 days. Bacillus, yeast and fungi were cultured in an aerobic incubator at 39 ° C for 1 to 8 days.

Results

The changes in the number of bacteria according to the culture period are shown in Table 3 below. Lactic acid bacteria, Bacillus subtilis and Yeast cells showed the highest number of bacteria about 4 days after the cultivation. Therefore, it was confirmed that fermentation of the seed culture was most effective for about 4 days. As a result, the bacterium was grown in the liquid culture by the conventional bacterium culture method, but the bacterium grew smoothly when the new culture method was applied, and the optimum culture period was confirmed to be about 4 days.

3. Establishment of technology for manufacturing pine needle probiotics using liquid cultures of selected useful microorganisms

In order to establish the technology for the production of pine needle probiotics, solid fermentation was carried out using a selected strain after liquid culture.

The solid fermentation method was compared with the conventional method and the new method, and the blending ratio of the raw materials is shown in Table 4 below.

In the conventional culture method, all raw materials are put into a solid fermenter and cultured at 39 ° C for 6 days. During the incubation period, the stirrer was stopped for 2 hours and then stirred for 30 minutes.

In the new culture method 1, rice bran, molasses, water, Bacillus subtilis, yeast and lactobacillus were all added to a solid fermenter and cultured at 39 ° C for 6 days. During the incubation period, the agitator was set under the same conditions as the conventional culture method.

In the new culture method 2, rice bran, molasses, water, Bacillus subtilis, and yeast were mixed in a solid fermenter and primary fermentation was performed at 39 ° C for 1 day. At this time, stirring of the stirrer was repeated for 30 minutes after stopping for 1 hour. On the second day, the lactic acid bacteria were mixed and then the second fermentation was carried out. In this case, the stirrer was stirred for 30 minutes after stopping for 2 hours and fermented for a total of 6 days. That is, the first fermentation was performed using aerobic microorganisms such as Bacillus subtilis and yeast bacteria, and the agitation of the anaerobic microorganism was mixed during the second fermentation after the first fermentation and the operation time of the stirrer was reduced Reduced supply of oxygen.

Samples were collected at 1, 2, 4, and 6 days during solid fermentation, and used for analysis. Microbial counts were performed in the same manner as described in Experiment 2.

Results

The results of investigating the changes in viable cell counts according to the fermentation conditions and the incubation period are shown in Table 5 below.

In the change of the number of bacteria according to the fermentation period, the existing culture method showed the highest number of bacteria on the 4th day of fermentation and the new culture methods 1 and 2 showed the highest number of bacteria on the 2nd day of fermentation. Changes in the number of bacteria according to the fermentation conditions showed that lactic acid bacteria, Bacillus subtilis and Yeast cells were more than 10 ⁴ to 10 ⁵ on the ^4th day of fermentation, whereas the new culture method 1 was more than 10 ⁷ on the 2nd day of fermentation. However, the new culture method 2 was more than 10 ⁸ on the 2nd day of fermentation and was higher than other culture methods.

In conclusion, it was confirmed that the optimum culture method for the solid fermentation, that is, the preparation of the pine needle prodrug, after the liquid culture of the selected strain was the new culture method 2 and the fermentation period was 2 days. In other words, it was confirmed that culturing for 1 day using Bacillus subtilis and yeast, aerobic bacteria for solid fermentation, and cultivation for 1 additional day using lactic acid bacteria were the best.

That is, according to the present invention, it is possible to obtain a strain having excellent enzyme activity and antimicrobial activity from indigenous fungus derived from pine needle, securing a liquid culture technique using a secured excellent strain, and producing a high quality pine needle probiotic preparation using liquid culture Technology can be established. In addition, from the economic and industrial point of view, it is possible to enhance the brand value of livestock products in Hadong County through the production of high quality pine needle probiotics, and it can be expected to increase the income of farm households by improving productivity of livestock through high quality pine needle probiotics.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims

Preparing a pine needle extract from the pine needle leaf mallow;
Isolating lactic acid bacteria, Bacillus subtilis, and yeast strains having an antimicrobial activity against a fusarium species fungus having a cellulose hydrolysis activity from the pine leaf extract;
Culturing the lactic acid bacteria, Bacillus subtilis and Yeast cells for 2 days to 6 days respectively;
Adding a rice bran, molasses, water, bacillus subtilis and yeast bacteria to a solid fermentor and performing primary fermentation for 10 hours to 30 hours while stirring; And
Further comprising the step of adding the lactic acid bacterium after the first fermentation and performing secondary fermentation for 1 to 6 days while stirring.

The method according to claim 1,
Wherein the liquid culture of the lactic acid bacteria, Bacillus subtilis and yeast cells comprises culturing lactic acid bacteria with Lactobacillus MRS broth, Bacillus anthracis with LB broth, and yeast strain with potato dextrose broth.

A pine needle prophylactic agent composition produced by the method of claim 1 or 2, wherein the number of microorganisms is 10 ⁷ to 10 ⁸ cfu / g.

A method for breeding cows, which comprises feeding 0.5 to 5% by weight of the pine needle probiotics produced by the method of claim 1 or 2 to the feed.