KR102050658B1 - Probiotics comprising egg shell powder - Google Patents

Abstract

The present invention relates to a probiotic carrier comprising eggshell powder and soybean meal of a specific particle size, the probiotic carrier comprising an eggshell powder of a specific particle size of the microbial spawn of the probiotic compared to the soybean meal conventionally used as a carrier of the probiotic Improved viability and maintained a stable pH, in particular, the carrier containing the eggshell powder and soybean meal significantly improved the viability of the probiotic, it is possible to prepare a probiotic with improved storage properties, the prepared probiotic is weak alkaline As it passes through the stomach, it has the effect of relaxing the acidic environment and delivering probiotics more effectively.

Description

Probiotics containing eggshell powder {PROBIOTICS COMPRISING EGG SHELL POWDER}

The present invention relates to a probiotic comprising a eggshell powder as a carrier, and to a carrier for a probiotic comprising an eggshell powder and soybean meal of a specific particle size.

In the past, antibiotics for feeds were added to formulated feeds to prevent livestock epidemics and diseases and to promote growth. My antibiotic use has been discontinued entirely. As the use of feed additives has been discontinued, effective microorganisms have been cultivated and added to the feed as an alternative to antibiotics that can enhance livestock immunity.

Probiotics are common feed additives used in livestock and agriculture. However, reduced viability of probiotics can affect quality during storage. Encapsulation using emulsification and extrusion methods is used to increase probiotic preservation and resistance in harsh environments and coating raw materials include starch, fiber, alginate, chitosan, gum and milk. Domestic animal resource probiotics (probiotics) are mainly sold in the form of supplementary feed or veterinary medicine, and the scale of probiotics sold in the supplementary feed category is 32,000 tons (approximately 90 billion won) annually. In terms of volume, it accounts for 40-45% of feed supplements, accounting for the largest portion. In addition, the market size of probiotics sold in the form of veterinary drugs amounted to KRW 77 billion per year (estimated at 15% of order feed additives), and the amount of probiotics produced by 140 agricultural technology centers in Korea is 70 billion won. It is estimated to be around. In other words, the market size of animal resources probiotics is estimated at 250 billion won (as of 2014) and is expected to increase continuously.

The effects of the probiotic can be expected by feeding the livestock compound feed for the livestock added to cattle, pigs, chickens, dogs, etc. are as follows. Feeding cows has positive effects in terms of prevention of calf diarrhea, improved milk flow, improved milk quality, improved feed efficiency, improved manure odor, enhanced immune function and longer life span. For pigs, chickens, and dogs, increased pig production, growth and feed efficiency improvement, odor removal from manure, intestinal and disease prevention effects, spawning rate, egg quality improvement, digestion rate improvement, immune function enhancement, etc. Microbiology, History of Finite Culture, 2006, p59)

Although various microorganisms exist on earth, lactic acid bacteria and yeast bacteria are most commonly used as probiotics as useful microorganisms. The lactic acid bacteria live and reach the small intestine of the livestock to produce lactic acid, which lowers the acidity of the intestines to pH 3.0-4.0 to prevent contamination of various bacteria and produces hydrogen peroxide and natural antibiotics to inhibit the growth of pathogenic microorganisms. In addition, it produces a variety of vitamins, amino acids, nucleic acids, antibacterial substances, and anticancer substances as a fermentation product of lactic acid bacteria, which has positive effects such as stabilizing microbial flora in livestock, increasing feed efficiency, and increasing disease resistance. It is also effective in removing odors such as hydrogen sulfide gas. In the case of yeast, the cells themselves are used as excellent protein feed as fermented feed. Yeast is excellent in the composition of amino acids, fermentation products to produce natural flavors such as alcohol glutamic acid to enhance the palatability of the feed, and to produce acetic acid to provide a synthetic synthetic precursors. Yeast also has the ability to bind oxygen to help the intestinal anaerobic bacteria work (Park Seung Yong, Applied Livestock Microbiology, Finite Culture History, 2006, p54-55).

Patents for feed additives such as probiotics, Patent No. 381726 (name: citrus fermentation broth as aquaculture feed additive) discloses a feed additive for aquaculture fish using a citrus fermentation broth, Patent Publication No. 1999-0046622 ( Name: Animal feed for animal husbandry and its manufacturing method) discloses a method for producing animal feed using fig leaves and berries, and Patent No. 336411 (name: high temperature anaerobic lactic fermentation mixed feed production method) 40 ~ A method for producing a feed by anaerobic lactic acid fermentation at a high temperature of 70 ℃ is disclosed.

Eggs, on the other hand, are rich in protein, vitamins and minerals, making them a popular and important food resource worldwide. The demand for eggs is increasing every year, producing around 6.5 * 10 ⁷ tonnes / year of eggs worldwide. Thus, as egg production increases, eggshell waste is also produced in large quantities, accounting for 3-12% of the total production. The resulting eggshell raises concerns about environmental pollution and potential risks as a habitat for pathogenic bacteria. In other words, it is important to apply and develop industrially to use eggshell as a value-added resource.

Thus, in the present invention, it was confirmed the possibility of using as a probiotic carrier for the added value of eggshell powder.

In the present invention, by confirming the use of eggshell powder as a probiotic carrier, by confirming the optimum mixing ratio with soybean meal that can improve the shelf life of the probiotic, by using a carrier containing an eggshell powder and soybean meal to produce a probiotic with improved shelf life For the purpose of

In order to achieve the above object, the present invention provides a probiotic carrier.

The present invention also provides a probiotic comprising the carrier of the present invention.

The present invention also provides a feed composition comprising the probiotic of the present invention.

In addition, the present invention provides a method for producing the probiotic of the present invention.

The probiotic carrier comprising the eggshell powder of the present invention improved the survival rate of the microbial spawn of the probiotics compared to the soybean meal conventionally used as a carrier of the probiotic, and maintained a stable pH, in particular, a carrier including both the eggshell powder and the soybean meal Has significantly improved the viability of the probiotics, so that probiotics with improved shelf life can be prepared, and the prepared probiotics maintain weak alkalinity, which can alleviate the acidic environment as they pass through the stomach to more effectively deliver probiotics. have.

1 is a diagram confirming the physical state, structure and probiotic attachment state of probiotics prepared using Lactobacillus strain and SBM, ESL, ESF, SBM + ESL or SBM + ESF carrier, respectively, by digital camera and SEM.
Figure 2 is a graph confirming the survival rate and pH change of probiotics prepared using Lactobacillus plantarum SK3494 and SBM, ESL, ESF, SBM + ESL or SBM + ESF carrier, respectively, at room temperature for 4 weeks.
Figure 3 is a graph showing the survival rate and pH for 4 weeks at 4 ℃ of the probiotic prepared with Lactobacillus plantarum SK3494 and SBM + ESF carrier.
4 is a graph showing the survival rate and pH for 4 weeks at 4 ° C. of probiotics prepared with Lactobacillus brevis SK1751 and SBM + ESF carriers.
5 is a graph showing the survival rate and pH for 4 weeks at 4 ° C. of probiotics prepared with Bacillus rickenformis SK3927 and SBM + ESF carrier.
6 is a graph showing the survival rate and pH for 4 weeks at 4 ° C. of probiotics prepared with Bacillus subtilis SK4082 and SBM + ESF carriers.
FIG. 7 is a graph showing the survival rate and pH for 4 weeks at 4 ° C. of probiotics prepared with Bacillus amiriquifaciens SK4079 and SBM + ESF carriers.
8 is Saccharomyces cerevisiae It is a graph showing the survival rate and pH of 4 weeks at 4 ℃ of the probiotic prepared with SK3587 and SBM + ESF carrier.
9 is a graph showing the survival rate and pH for 4 weeks at 30 ° C. of probiotics prepared with Bacillus rickenformis SK3927 and SBM + ESF carriers.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are presented by way of illustration of the present invention, and if it is determined that the detailed description of the well-known technology or construction well known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description thereof may be omitted. However, the present invention is not limited thereto. The invention is susceptible to various modifications and applications within the scope of the following claims and the equivalent scope thereof.

Also, the terminology used herein is a term used to properly express a preferred embodiment of the present invention, which may vary depending on a user, an operator's intention, or customs in the field to which the present invention belongs. Therefore, the definitions of the terms should be made based on the contents throughout the specification. Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w)% solids / solids, (w / v)%, and unless otherwise noted, Liquid / liquid is (v / v)%.

In one aspect, the invention relates to a carrier for probiotic.

In one embodiment, the probiotic carrier of the present invention may be made of eggshell powder or may include eggshell powder and soybean meal together.

In one embodiment, when used alone as a probiotic carrier, the eggshell powder particles may be 3 to 5mm, when used as a mixed carrier of eggshell powder and soybean meal, the particles of the eggshell powder is 0.01 to 0.1mm and soybean meal The particle size of may be 0.3 to 2mm, the particle size of the eggshell powder is more preferably 0.03 to 0.05mm and the particle size of soybean meal is 0.71mm.

In one embodiment, the probiotic carrier of the present invention may include eggshell powder and soybean meal in a range of 1: 6 to 6: 1 (w / w), more preferably 2: 5 (w / w).

In one embodiment, the probiotic carrier of the present invention may be included in 50 to 90% (w / v) of the total dose of the probiotic, and more preferably in 70% (w / v). In one embodiment, the probiotic was prepared by mixing 21 g of the microbial cell pellet of the probiotic of the present invention with a carrier mixed with eggshell powder and soybean meal 2: 5 (w / w).

In one embodiment, the carrier of the present invention may be used as an excipient of the probiotic.

In one aspect, the invention provides a carrier comprising an eggshell powder and soybean meal; And a microbial seed including a lactic acid bacterium or a yeast bacterium.

In one embodiment, the carrier comprising eggshell powder and soybean meal may comprise 50 to 90% (W / V) of the total probiotic dose.

In one embodiment, the lactic acid bacterium may be a strain of the genus Lactobacillus, a strain of the genus Bacillus, and may be Lactobacillus plantarum, Lactobacillus brevis, Bacillus rickeniformis, Bacillus subtilis or Bacillus amiroliquifaciens, Lactobacillus plantarum SK3494, L. brevis SK1751, B. licheniformis SK3927, B. subtilis SK4082 and B. amyloliquefaciens ) SK4079 may be any one or more selected from the group consisting of.

In one embodiment, the yeast may be Saccharomyces cerevisiae, and Saccharomyces cerevisiae It may be SK3587.

In one embodiment, the microbial spawn adsorbed on the probiotic carrier of the present invention available as a probiotic is suitable for human or animal ingestion and can inhibit harmful pathogenic harmful bacteria or improve microbial balance in the mammalian intestine upon ingestion. Preference is given to those having probiotic activity. Examples of such probiotic microorganisms include yeasts, Aspergillus, Ryzopus, including Saccharomyces, Candida, Pichia, and Torulopsis. (Rhizopus), Muco, Penicillium, and other fungi such as Lactobacillus, Bifidobacterium, Clostridium, Leuconostoc, and Bacteroids Bacteroides, Staphylococcus, Lactococcus, Bacillus, Streptococcus, Fusobacterium, Propionibacterium, Propionibacterium, Enterococcus (Enterococcus) There are bacteria of the genus Pecocous and Micrococcus.

In one embodiment, it is possible to further enhance the effect of the probiotic of the present invention by further comprising a probiotic microbial mixed bacteria having excellent probiotic activity and excellent immune activity enhancement and anticancer activity.

In one embodiment, the probiotic of the present invention may be administered to a human or animal, and may be administered with a food, drug, animal drug, feed or dietary supplement.

In one embodiment, the probiotic of the present invention may be for livestock and may be solid.

In one embodiment, the probiotic comprising the probiotic carrier of the present invention has an effect of maintaining the viability of the microorganism up to 4 weeks at room temperature, 4 ℃ and 30 ℃, it is excellent in preservation.

In one aspect, the present invention relates to a feed additive comprising the probiotic of the present invention.

In one embodiment, the feed additive may be added at 0.1 to 0.3% based on the total weight of the feed, but is not limited thereto.

In one aspect, the invention is a probiotic of the present invention; And it relates to a feed composition comprising a probiotic, including any one or more feed selected from the group consisting of forage, rich feed or flavor feed.

In the present invention, "feed" is a feed having a relatively high content of structural carbohydrates and a bulky feed, and having a high content of coarse fiber, a coarse and bulky one, and a low-cost, low-plasticizing nutrient. This includes coarse prey such as rice straw, raw and hay of grasses, raw and hay of wild grasses, and ensilage and pods made from green grass crops including grasses. The dry feed contains more than 18% crude fiber and has a low digestibility, but is important not only as a source of various nutrients but also for maintaining the normal functioning of the digestive system. In the present invention, rice straw, timothy, silage may be used as a feed material added to the feed composition, and such a feed material may be added in the range of 30 to 65% by weight based on the entire feed composition.

In the present invention, "rich feed" refers to a feed having a small volume and a low content of fiber, and a high content of plasticized nutrients such as soluble nitrogen, which can increase the growth efficiency of livestock and livestock products. Typically, grains such as corn, sorghum, wheat, and barley, and bark, bran, rice bran, barley bran, and bran, etc. left after squeezing vegetable oil, fish meal, or a compound feed made by mixing them in a certain ratio are included. In the present invention, the rich feed may preferably include components such as corn, wheat, protein skin, soybean meal, vitamins, minerals, and the like, and is not limited to these ingredients. Can be. Such a rich feed occupies a large weight in the feed composition of the present invention, it may be added in the range of 35 to 70% by weight relative to the total feed composition.

In one aspect, the present invention comprises the steps of culturing the microbial spawn; Separating the cells of the microbial spawn; Mixing with eggshell powder and soybean meal; And it relates to a method for producing a probiotic, comprising the step of drying.

In one embodiment, the eggshell powder comprises the steps of removing the eggshell membrane from the eggshell; Sterilization and drying; And it may be prepared by the step of preparing an eggshell powder by grinding to a particle size of 0.01 to 0.1mm.

In one embodiment, the drying may be dried so that the moisture content is less than 10%.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1 Eggshell Powder Preparation

1-1. Manufacture of large particle eggshell powder (ESL)

After the egg shell was dehydrated, it was heated at 150 ° C., dried and pulverized to 3 to 5 mm to prepare a large particle egg shell powder (ESL).

1-2. Small particle eggshell powder (ESF) manufacturing

The egg shell was immersed in water to remove the egg shell membrane, sterilized and dried at 100 ° C., and then ground to 0.03-0.05 mm to prepare small particle egg powder (ESF).

Example 2. Preparation of Probiotic Carrier

After mixing ESL of Example 1-1 or ESF of 1-2 with soybean meal (SBM) having a particle size of 0.71 mm, respectively 2: 2 (weight ratio), autoclaving at 121 ° C. for 15 minutes and then at 60 ° C. It was dried for 1 day to prepare a probiotic carrier.

Example 3. Probiotic Preparation

3-1. Probiotic Preparation Using a Single Carrier

The probiotics were prepared using the strains of Table 1 and the eggshell powder or soybean meal prepared in Example 1 above. Specifically, Lactobacillus plantarum SK3494 used in the study was isolated from Kimchi, Lactobacillus brevis SK1751 was isolated from sweet potatoes. Saccharomyces cerevisiae SK3587 was isolated from pig feces and Bacillus licheniformis SK3927 was isolated from fish gut and confirmed by 16S rRNA sequencing. Subsequently, strains of the genus Lactobacillus plantarum and Lactobacillus brevis of Table 1 are in MRS medium, strains of the genus Bacillus ( Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens ) in LB medium, and Saccharomyces cerevisiae. ( Saccharomyces cerevisiae ) were incubated at 37 ° C. for 24 hours in YM medium. After incubation, 50 ml of each strain culture was centrifuged at 5,000 g at 4 ° C. for 10 minutes to obtain cells, which were then washed three times with sterile DW. Cell pellets were resuspended in 9 ml of sterile DW and then mixed with the eggshell powder prepared in Example 1 or soybean meal of 0.71 mm in particle size, respectively, to prepare probiotics. The prepared probiotic was dried on a clean bench so that the moisture content was less than 10%.

3-2. Probiotic Preparation Using Mixed Carriers

Probiotics were prepared using the strains of Table 1 and the mixed carrier ESL + SBM or ESF + SBM of the present invention prepared in Example 2. Specifically, among the strains of Table 1, strains of the genus Lactobacillus ( Lactobacillus plantarum and Lactobacillus brevis ) are in MRS medium, strains of the genus Bacillus ( Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens ) in LB medium, Saccharomyces Cervage was incubated at 37 ° C. for 24 h in YM medium each. After incubation, 50 ml of each strain culture was centrifuged at 5,000 g at 4 ° C. for 10 minutes to obtain cells, which were then washed three times with sterile DW. Cell pellets were resuspended in 9 ml of sterile DW and then mixed with 21 g of the mixed carrier ESL + SBM or ESF + SBM of the present invention prepared in Example 2 to prepare probiotics. The prepared probiotic was dried on a clean bench so that the moisture content was less than 10%.

Example 4 Confirmation of Proliferation of Probiotics

The physical state, structure and probiotic attachment state of probiotics prepared using Lactobacillus strains and SBM, ESL, ESF, SBM + ESL or SBM + ESF carriers, respectively, were confirmed using a digital camera and SEM (H 3000N; Hitachi). . Specifically, Lactobacillus plantarum SK3494 and carrier SBM, ESL, ESF, SBM + ESL or SBM +, which are mainly selected for investigating the effect of ES carrier on cell viability at room temperature (21 ± 3 ° C.) Probiotics prepared using ESF were fixed at 1.0 × 1.0 cm for 24 hours at 4 ° C. with Karnovsky fixative, followed by secondary fixation by adding osmic acid (1%, w / v). Then, dehydrated with ethanol at 50%, 70%, 85%, 95%, and 100% concentration for 1 hour, and washed three times for 10 minutes with 0.05 M cacodylate buffer. The solution was replaced with 50% and 100% isoamyl acetate and then dried with a critical point dryer (HCP 2, Hitachi). An ion sputter coater (Hitachi) was used to coat 120 seconds at an acceleration voltage of 14.3 kV.18 kV, followed by imaging.

According to the digital camera image, the probiotic mixed and dried with the cells was observed to be in a coagulated state. As a result of confirming the microstructure of the carrier by SEM, Lactobacillus strains were well attached to each carrier ( 1).

Example 5. Probiotic Viability and pH Verification

5-1. Check viability and pH at room temperature

Probiotics prepared using Lactobacillus plantarum SK3494 and SBM, ESL, ESF, SBM + ESL or SBM + ESF carriers were stored at room temperature for 4 weeks, and the survival rate of the probiotic according to each carrier was measured by the CFU counting method. Each pH was measured using a pH meter (pH / ISE Meter 735P, Istek Co., Ltd, Seoul, Korea).

As a result, the viability of the probiotic using ESL, SBM + ESL or SBM + ESF as a carrier was significantly higher than the viability of the probiotic using SBM or ESF as a carrier from 2 weeks after storage at room temperature (FIG. 2). In addition, probiotics using SBM + ESL and SBM as carriers were pH 6.5 and pH 6.0, respectively, and probiotics using ESF carriers showed higher pH than 9.0. Probiotics using ESL or SBM + ESF as carriers were pH 8.0 PH was stable with little change (FIG. 2).

The higher pH of the eggshell carrier can reduce cell viability because more calcium carbonate is released, and the weak alkalinity can mitigate the acidic environment as the probiotics pass through the stomach. , SBM + ESF was determined to be the most stable to maintain the viability of the strain, was selected as the optimal carrier of the probiotic.

5-2. Viability and pH Check at 4 ℃

Table 1 Lactobacillus plantarum SK3494, Lactobacillus brevis SK1751, B. licheniformis SK3927, B. subtilis SK4082, Bacillus amyloriquifaciens ( B. amyloliquefaciens ) SK4079 or Saccharomyces cerevisiae After probiotics were prepared using the SK3587 strain and the SBM + ESF carrier selected as the optimum carrier in Example 5-1, the viability and pH change at each 4 ° C were confirmed.

As a result, Lactobacillus plantarum SK3494 showed little change in survival rate at 4 ° C. for 4 weeks, and maintained about pH7 (FIG. 3). In addition, Bacillus brevis also showed little change in survival rate for 4 weeks at 4 ° C., and was shown to maintain pH 7-8 (FIG. 4). In addition, Bacillus rickeniformis, Bacillus subtilis and Bacillus amiriquiquiciens also showed little change in survival for 4 weeks at 4 ° C. and were maintained at about pH8 (FIGS. 5-7). In addition, Saccharomyces cerevisiae SK3587 showed a stable survival rate at 4 ℃, pH was 6.5 to 8.0 (Fig. 8).

5-3. Viability and pH Check at 30 ℃

After the probiotic was prepared using Bacillus rickenformis SK3927 strain and SBM + ESF carrier selected as the optimum carrier in Example 5-1, viability and pH change at 30 ° C. were confirmed for 4 weeks.

As a result, Lactobacillus rikenimoformis SK3927 showed stable viability with little change in viability for 4 weeks at 30 ° C., and showed a constant value of pH 7 to 8 (FIG. 9).

From the above results, it was found that the carrier (carrier) type is an important parameter for the preservation of the probiotic, and the use of SBM + ESF as the carrier of the probiotic improves the viability of the probiotics and improves the preservation.

Example 6 Statistical Analysis

Statistical analysis was performed using the IBM SPSS statistics 24 on Windows (IBM, New York, US) and the values are expressed as mean ± standard deviation. The significance test of the treatment period using Duncan's multiple rage test was performed at the significance level p <0.05 and statistically significant when the p value was less than 0.05.

Claims (11)

A probiotic carrier comprising a eggshell powder pulverized to a particle size of 0.01 to 0.1mm and soybean meal with a particle size of 0.3 to 2mm in a mixing ratio of 1: 6 to 6: 1 (w / w). delete delete delete A carrier comprising a eggshell powder pulverized to a particle size of 0.01 to 0.1 mm and soybean meal having a particle size of 0.3 to 2 mm in a mixing ratio of 1: 6 to 6: 1 (w / w); And
A probiotic comprising a microbial seed comprising a lactic acid bacterium or a yeast, wherein the carrier comprises 50 to 90% (W / V) of the total probiotic dose. delete The probiotic of claim 5, wherein the microbial spawn is a strain of the genus Lactobacillus, a strain of the genus Bacillus or Saccharomyces cerevisiae. Probiotic of claim 5; And feed composition comprising a probiotic, any one or more feed selected from the group consisting of forage, rich feed or flavor feed. Culturing the microbial spawn;
Separating the cells of the microbial spawn;
Mixing the eggshell powder ground to a particle size of 0.01 to 0.1 mm and a soybean meal having a particle size of 0.3 to 2 mm with a carrier mixed at a mixing ratio of 1: 6 to 6: 1 (w / w); And
Producing a probiotic, comprising the step of drying. The method of claim 9, wherein the eggshell powder is
Removing the egg shell from the egg shell;
Sterilization and drying; And
The method of producing a probiotic, which is prepared by grinding to a particle size of 0.01 to 0.1mm to produce an eggshell powder. 10. The method of claim 9, wherein the drying is such that the moisture content is less than 10%.

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