KR101000364B1 - Double coating method for enhancing survival rate - Google Patents

Abstract

PURPOSE: A Lactobacillus particle which is double-coated with chitosan and poly-gamma-glutamic acid for enhancing viability of Lactobacillus is provided to improve stability and to prolong valid term of Lactobacillus product. CONSTITUTION: A Lactobacillus particle contains Lactobacillus; and chitosan-coated layer and poly-gamma-glutamic acid-coated layer. The weight ratio of the chitosan and poly-gamma-glutamic acid is 3:1~300:1. A composition as probiotics or antibiotics contains the Lactobacillus particle.

Description

Double-coating methods for enhancing viability}

The present invention relates to lactic acid bacteria particles and a method for producing the lactic acid bacteria to enhance the survival rate of the lactic acid bacteria by double coating with chitosan and poly gamma glutamic acid.

Lactobacillus is one of the longest widely used microorganisms in humans. It is a bacterium (bacteria) that breaks down carbohydrates such as glucose or lactose into lactic acid. A bacterium that does not produce harmful substances and has a beneficial effect. Lactic acid bacteria are used in the manufacture of foods such as fermented milk, health foods, functional foods, medicines, and probiotic preparations, and they promote animal growth, increase feed utilization efficiency, increase resistance to disease, inhibit the growth of harmful bacteria, and mortality. It is also used in the manufacture of feed for reducing and suppressing the production of toxic toxins. In addition, while inhabiting the human intestine, it inhibits the intestinal abnormal fermentation, activates the intestinal function to improve the abnormal function of the intestine and at the same time plays a role of smoothly maintaining. It is also known to have beneficial effects such as resistance to infection, anticancer effects, lowering blood cholesterol content and intestinal effects. Therefore, the improvement and development of lactic acid bacteria having excellent properties as probiotics, and the method of keeping the seed in good condition for a long time, have emerged as important problems.

Unlike other industrial microorganisms that mainly use fermented products, lactic acid bacteria use live bacteria themselves, so maintaining survival rate during distribution is a very important problem. However, because lactic acid bacteria use live bacteria themselves, their growth is inhibited by the harsh environment in the human body, or digestive enzymes and bile acids secreted by the gastrointestinal and small intestine, when the pH is lowered to 3 or less, and the number of live bacteria reaching the large intestine is reduced. It is pointed out that the intestinal fixation rate is lowered. In addition, although lactic acid bacteria are unstable and very beneficial in terms of their utility and value, they are subject to many limitations in their long-term storage and use.In addition, the survival rate of fermented milk products and probiotic products using them greatly affects the survival rate by oxygen or acid during the manufacturing and distribution periods. It is known to receive.

Lactic acid bacteria are usually used in liquid form, but may be prepared in powder form for convenience or long-term preservation. In general, lactic acid bacteria are prepared in powder form by a freeze drying method or spray drying method, and the freeze drying method is a method in which a microorganism is added to a freeze-drying solution, frozen and dried under reduced pressure. In the freeze-drying process, the crystal form of ice, salt concentration, incomplete dehydration, complete dehydration, free radical accumulation, and infiltration pressure shock in lactic acid bacteria decrease the survival rate of lactic acid bacteria. Therefore, many factors must be taken to keep the live lactobacillus stable for a long time. In particular, a protective substance (protective agent) capable of eliminating this cause is important for the stability of the lactic acid bacteria preparation. Currently, as a lyophilized protective agent, glucose, sugar, lactose, skim milk, albumin, polyethylene glycol and the like are used, but the effect is still weak.

Lyophilized live lactobacillus is very dry, so if it is stored for a long time in this process, it is easily hygroscopic and the strain is killed. In addition, because lactic acid bacteria are anaerobic and very sensitive to the surrounding environment, the powdered lactic acid bacteria have a low viability at low temperature and room temperature, thereby reducing the number of viable bacteria. Therefore, glucose and glucosamine are used to suppress the injury of lactic acid bacteria during this storage process.

In order to enhance the stability of lactic acid bacteria, a number of methods for producing capsules to protect the lactic acid bacteria with monosaccharides, gelatin, etc. have been tried, and many products are marketed in the pharmaceutical and food industries using this technology. Paul B. et al reported that Lactobacillus ashidophilus coated with monosaccharides had a low survival rate of 10% after 35 days at room temperature (Paul B. et al., Cryobiology, 2000, 41, 17). ~ 24). Claude PC et al also reported that gelatin-coated Lactobacillus rhamnosus stored at 20 ^° C. had a low survival rate of about 1% after 6 months and about 0.2% after 12 months (Claude PC et. al, Food Res. Int., 1996, 29, 555-562). Meanwhile, Korean Laid-Open Patent Publication No. 10-2009-0100033 discloses lactic acid bacteria particles coated with water-soluble chitosan after physically sealing the lactic acid bacteria in particles formed by dispersing lactic acid bacteria in an alginic acid solution and then solidifying the alginic acid. Lactobacillus Lactobacillus is physically encapsulated in the alginic acid matrix, not coated on its individual surface, providing a viable effect at low acidity, but related to increasing the shelf life of lyophilized powders used in the production of lactic acid bacteria products. It is not possible to increase chemical stability. As described above, in the related art, lactic acid bacteria are difficult to be stored at room temperature in a powder state, and have a problem of low survival rate even if they can be stored for a long time at low temperature.

Accordingly, the present invention is to provide a lactic acid bacteria particles and a method for preparing the coated lactic acid bacteria so that the survival rate of the lactic acid bacteria excellent even if the lactic acid bacteria are stored at room temperature in a lyophilized state.

The present invention also provides a food or medicament comprising the lactic acid bacteria particles.

The present invention is lactic acid bacteria; It provides a lactic acid bacteria particles comprising a chitosan coating layer and a poly gamma glutamic acid coating layer on the lactic acid bacteria.

In the present invention, the chitosan used in the chitosan coating layer may be oligochitosan. In one embodiment, the average molecular weight of the chitosan may be 200 to 30000. Chitosan having a molecular weight greater than 30000 is not suitable because it dissolves in weak acidic conditions and thus cannot effectively protect the lactic acid bacteria when the lactic acid particles pass through the gastrointestinal tract. On the other hand, chitosan having a molecular weight of less than 200 has a problem of poor coating power. Oligochitosan having a molecular weight of 200 to 30000 is harmless to the human body and is dissolved in an aqueous solution, and thus it is not necessary to use an organic solvent that is fatal to the stability of lactic acid bacteria. Therefore, the use of chitosan has the advantage of solving the problem of using an organic solvent when dissolving most polymer materials that have been used as a conventional lactic acid bacteria coating base. In addition, when the lactic acid bacteria are coated using a polymer material, the lactic acid bacteria must be treated at a high temperature in order to reduce the viscosity, so that the lactic acid bacteria lose their original activity or are killed. On the other hand, if chitosan is used to coat the lactic acid bacteria at room temperature, not only does not affect the activity of the lactic acid bacteria, chitosan is more favorable for the stabilization of lactic acid bacteria because it shows neutrality.

Lactobacillus particles coated with chitosan exhibit the effect of improving survival rate by itself, but when double coated with polygamma glutamic acid can be used to increase the survival rate of lactic acid bacteria more effectively. As can be seen in the examples below, polyglutamic acid exhibits an excellent coating effect. In addition to polygamma glutamic acid, polysaccharides such as hyaluronic acid and fucoidan may be used. However, when polygamma glutamic acid was used as a coating base through the comparison of the survival rate of lactic acid bacteria after the production of lactic acid bacteria particles, it showed the best efficiency. In addition, polygamma glutamic acid is known to exhibit various physiological activities by itself. Specifically, polygamma glutamic acid promotes absorption of minerals such as calcium, stimulates macrophages to induce the secretion of TNF-α and IL-1β, and enhances immune function, and IFN-γ in T-lymphocytes. It has been reported to promote the secretion of T cells and B cells, macrophages, NK cells and the like to inhibit the intracellular bacterial and viral infections and cancer cell killing. Therefore, lactic acid bacteria particles coated with polygamma glutamic acid can not only increase the survival rate of lactic acid bacteria but also have a beneficial effect on the living body when used as a food, medicine, feed, and the like.

In one embodiment, the average molecular weight of the poly gamma glutamic acid may be 300 to 20,000,000. When the molecular weight of polygamma glutamic acid is less than 300, there is a problem in that the efficiency of double coating is lowered, and when the molecular weight is 20,000,000 or more, there is a problem in that the yield of the manufacturing process is lowered due to a viscosity problem.

In one embodiment, the present invention provides a lactic acid bacteria particles having a chitosan coating layer and a poly gamma glutamic acid coating layer sequentially coated on the lactic acid bacteria.

Chitosan and polygammaglutamic acid are cationic and anionic substances in aqueous solution, respectively.The primary coating of weakly anionic lactic acid bacteria with chitosan having cationicity is followed by polygammaglutamic acid. By secondary coating, lactic acid bacteria can be stably double coated.

The type of lactic acid bacteria which can increase the survival rate by double coating with chitosan and polygammaglutamic acid is not particularly limited. Any of the known lactic acid bacteria can be enhanced by double coating with chitosan and polygammaglutamic acid according to the method of the present invention.

In one embodiment, the lactic acid bacteria are Lactobacillus sp. , Sporolactobacilus sp. , Streptococcus sp. , Lactococcus sp. , Luconosstock genus ( Leuconostoc sp. ), Pedioococcus sp. , Enterococcus sp. , And Bifidobacterium sp .

The invention also

Coating the lactic acid bacteria with chitosan and polygammaglutamic acid

Provided are methods for preparing lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid.

Referring to the production method of the lactic acid bacteria particles for example as follows.

First, lactic acid bacteria are added to a phosphate buffer solution to an appropriate concentration, for example, 1 × ^{10 8} to 1 × ^{10 12} CFU / ml, and chitosan aqueous solution is added thereto, followed by mixing.

Here, the chitosan aqueous solution is prepared at a concentration of 5 mg / ml chitosan, and added to the lactic acid bacteria solution of the phosphate buffer solution prepared at a concentration of 1x10 ⁸ to 1x10 ¹² CFU / ml so that the final concentration of chitosan is 0.05 to 1 mg / ml. can do. After coating of the lactic acid bacteria with chitosan, the polygamma glutamic acid aqueous solution is added to the mixture and mixed to perform the coating.

Herein, the polygamma glutamic acid aqueous solution is prepared at a concentration of 1 mg / ml polygamma glutamic acid, and the concentration of polygamma glutamic acid is 0.01 to 0.5 mg / l in a phosphate buffer solution prepared at a concentration of 1x10 ⁸ to 1x10 ¹² CFU / ml. It can be added to make a ml.

Coating with chitosan and polygammaglutamic acid may be performed sufficiently, but not limited to, about 5 to 20 minutes each.

On the other hand, the weight ratio of the chitosan and polygamma glutamic acid in the double coating is preferably used in excess of the positively charged chitosan compared to polyglutamic acid due to the weak negative charge of the surface of the lactic acid bacteria, more preferably 3: 1 to 300: 1 weight Can be ratio.

In one embodiment, the method for producing the lactic acid bacteria particles may further comprise the step of adding a freeze buffer solution to the double coated lactic acid bacteria particles and then lyophilized to obtain a lactic acid bacteria powder.

Lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid through the above method show a high survival rate for a long time despite being lyophilized as can be seen in the following examples. In addition, the production method of the lactic acid bacteria particles does not reach the survival rate or activity of the lactic acid bacteria because there is no process for increasing the temperature or using an organic solvent for lactic acid bacteria coating.

The invention also provides compositions comprising lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid. In one embodiment, the composition may be for use as a formal, probiotic or antimicrobial agent.

Here, the "formulation agent" refers to an agent that treats and ameliorates various symptoms caused by abnormal fermentation of the intestinal bacterial flora of humans or animals. The symptoms include, but are not limited to, infectious diarrhea caused by harmful microorganisms, gastroenteritis, inflammatory bowel disease, neuro enteritis syndrome, small intestine microbial hypergrowth and intestinal diarrhea.

On the other hand, 'probiotic' refers to a microorganism that has the effect of preventing or treating certain pathological conditions as a living microorganism, that is, a microorganism that can survive in the gastrointestinal tract when ingested by humans or animals. In general, probiotics are effective in treating and improving symptoms caused by abnormal fermentation of enterobacteriaceae, and when administered to humans and animals, they proliferate and settle in the gut wall of the gut, preventing harmful microorganisms from settling. It produces and lowers the intestinal pH to inhibit the growth of harmful microorganisms. The term 'antibacterial agent' refers to a formulation having an activity of inhibiting or inhibiting the growth of harmful microorganisms. Preferably, the harmful microorganism may be a microorganism present in the digestive tract in vivo and causing various diseases related to the digestive tract. The microorganisms include Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Enterocoxigenic E. coli, Listeria monocytosis. Listeria monocytogenes, Campylobacter jejuni, Clostridium perfringens, and combinations thereof, but are not limited thereto.

The composition may, for example, be included in food, medicine or feed to impart the functionality of lactic acid bacteria to these products. For example, the composition may be provided in the form of a food composition or a food additive composition. For example, the food composition according to the present invention may be formulated in the same manner as the following pharmaceutical composition to be used as a health functional food, or added to various foods. Accordingly, the present invention provides a food comprising a composition comprising lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid. Such foods include, for example, beverages, dairy products, vitamin complexes, and health functional foods.

For example, when the food composition of the present invention is prepared with beverages such as fermented milk, in addition to the double coated lactic acid bacteria particles of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, and various plant extracts may be further included.

The present invention also provides a dietary supplement comprising a composition comprising lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid. Health functional foods are foods prepared by adding any active ingredients to food materials such as beverages, teas, spices, gums, confectionery, or encapsulated, powdered, or suspensions. Unlike general medicines, it has the advantage that there are no side effects that can occur when taking long-term use of foods as raw materials. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. Currently, lactic acid bacteria are sold as health functional foods for eliminating toxic substances and decaying bacteria, intestinal action, antibacterial action and anticancer action.

Although the addition amount of the lactic acid bacterium particle of this invention in food cannot be prescribed | regulated uniformly according to the kind of food, what is necessary is just to add in the range which does not impair the original taste of food, Usually 0.01-50 weight% with respect to the target food. And preferably in the range of 0.1 to 20% by weight. In the case of granules, tablet or capsule form of a food, may be added in the range of from 0.5 to 80% by weight supposed to usually 0.1 to 100% by weight, preferably, in the case of a beverage in the form of food, usually 1x10 ⁸ to 1x10 ⁹ CFU / ml , Preferably in the range of ¹ × ^{10 8} to 1 × ^{10 10} CFU / ml.

In another embodiment, the present invention also provides a medicament comprising the composition. In one embodiment, the medicament may be a formal, probiotic or antimicrobial.

When the composition of the present invention is used for the manufacture of a medicament, it is preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid for administration. can do.

Pharmaceutical compositions may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants, which may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants, or flavoring agents, and the like. Can be.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops and the like. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and biocompatible, which include saline, sterile water, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. Can be used in combination, and other conventional additives such as antioxidants, buffers, bacteriostatics can be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into aqueous solutions, suspensions, emulsions and the like.

Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.

When used in a formal or probiotic, the lactic acid bacteria particles double-coated with the chitosan and polygammaglutamic acid of the present invention have a number of viable bacteria in the composition of 1 × ^{10 5} to 1 × ^{10 12} CFU / g, preferably once or several times a day for adults. May be administered to 1x10 ⁷ to 1x10 ¹² CFU / g. It will be apparent to those skilled in the art that the therapeutically effective amount and frequency of administration will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously.

In addition, the composition according to the invention may be provided in the form of a feed composition or a composition for feed addition. Feed containing the composition of the present invention is used as a substitute for the existing antibiotics to inhibit the growth of harmful intestinal bacteria and to maintain the intestinal flora in a stable state to improve the health of the livestock to improve the weight gain and meat quality of the livestock and the yield and immunity Can be increased. Feed composition of the present invention is not limited to this, but can be prepared in the form of fermented feed, compound feed, pellet form and silage (silage) and the like. For example, the blended feed may be prepared by mixing various kinds of general feed and lactic acid bacteria of the present invention, and the feed of the pellet form may be prepared by applying heat and pressure to the blended feed in a pellet machine.

The amount of the lactic acid bacteria particles of the present invention included in the feed may be administered to 1x10 ⁶ to 1x10 ¹⁰ CFU / g, preferably 1x10 ⁶ CFU / g to 1x10 ⁸ CFU / g.

In addition, the compositions according to the present invention are known to be suitable for ingestion by animals, including humans, together with the lactic acid bacteria particles of the present invention, known microorganisms having the activity of inhibiting the growth of harmful microorganisms and improving the balance of the intestinal flora when ingested. It may further comprise.

The lactic acid bacterium double coated with chitosan and polygammaglutamic acid according to the present invention improves the survival rate and stability of the lactic acid bacteria, thereby enabling long-term storage at low temperatures and room temperature, thereby extending the shelf life of the seed and the shelf life of the lactic acid bacteria products. In addition, even when stored in a lyophilized powder state, it is possible to keep the lactic acid bacteria in a long-term survival state, and can be manufactured by a simple method using safety-approved food ingredients, which is useful in the food, pharmaceutical and feed industries. Can be used.

FIG. 1 shows colony-forming pictures of double coated Lactobacillus lactis particles prepared according to the molecular weight of various chitosan and the weight ratio condition of chitosan and polygammaglutamic acid [(A) Comparative Example 3, (B) Comparative Example 7, ( C) Comparative Example 16, (D) Example 1-1, (E) Example 3-2, (F) Example 10-1].
Figure 2 is a graph showing the survival rate of lactic acid bacteria compared to the untreated group of double coated Lactobacillus ashdophyllus particles prepared according to the molecular weight of various chitosan and the weight ratio condition of chitosan and polygamma glutamic acid.
Figure 3 is prepared by the double-coated Streptococcus thermophilus particles prepared according to the molecular weight of the various chitosan and the weight ratio condition of chitosan and polygamma glutamic acid, repeated five times the freeze-thawing process, each composition compared with the untreated group It is a graph showing the survival rate of lactic acid bacteria.
Figure 4 shows the survival rate of lactic acid bacteria in each composition compared to the untreated group after the production of lactobacillus luterri particles double coated with chitosan and polygamma glutamic acid, or chitosan and other anionic polymers to repeat the freeze-thawing process five times It is a graph.
FIG. 5 is a graph showing a change in the survival rate of lactic acid bacteria when Lactobacillus ashdophyllus coatings are prepared according to the composition of chitosan and polygammaglutamic acid, and stored at room temperature for a long time.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[Example]

Culture and obtaining of lactic acid bacteria

After dissolving the lactic acid bacteria culture MRS medium and sterilized by autoclaving at 121 ℃ for 15 minutes. 10 ml of sterilized lactic acid bacteria culture MRS medium was placed in a 50 ml tube, followed by 5% inoculation of a culture solution of Lactobacillus ashidophilic lactic acid bacteria, followed by incubation at 37 ° C. for 16 hours at 80 rpm to obtain lactic acid bacteria. .

The cultured lactic acid bacteria were collected by centrifugation at 3000 rpm for 10 minutes and washed twice with 1/10 of phosphate buffered saline (pH 7.4). At this time, the phosphate buffer solution was used by autoclaving at 121 ° C. for 15 minutes.

In the same manner as described above, Lactobacillus lactis ( Lactobacillus acidophilus ), Lactobacillus casei , Lactobacillus ruuteri ( Lactobacillus reuteri ), Lactobacillus brevis , Lactobacillus kefir used in the following examples kefir ), Streptococcus thermophilus and the like were cultured and obtained.

Preparation of Chitosan and Polygammaglutamic Acid Solution for Lactic Acid Bacteria Coating

In order to prepare lactic acid bacteria particles coated with chitosan and polygamma glutamic acid, chitosan solution of each molecular weight was prepared using a phosphate buffer solution at a concentration of 5 mg / ml, and the polygamma glutamic acid solution was phosphoric acid at a concentration of 1 mg / ml. It was prepared using a buffer solution. In this case, the used phosphate buffer solution was used by autoclaving at 121 ° C. for 15 minutes. After preparing this solution, it is easy to store at 4 degreeC at the time of storage.

Preparation of Double Coated Lactobacillus Particles

After adding the phosphate buffer solution so that the concentration of lactic acid bacteria was 10 ¹⁰ CFU / ml, it was stirred uniformly using a stirrer. Here, the aqueous solution of chitosan having a molecular weight corresponding to each composition of Examples 1-1 to 12-2 was added thereto, followed by primary coating at room temperature for 10 minutes, and then the aqueous solution of polygamma glutamic acid having a molecular weight according to each composition was further added to room temperature. Second coating for 10 minutes at. Thereafter, the resultant was centrifuged at 3000 rpm for 5 minutes to obtain double coated lactic acid bacteria particles.

Example Chitosan
Average molecular weight Polygamma Glutamic Acid Average Molecular Weight Chitosan: Polygamma glutamic acid
(Weight ratio) Lactobacillus species 1-1 450 3,000 50: 1 Lactobacillus lactis 1-2 450 3,000 100: 1 Lactobacillus lactis 2-1 450 8,500 5: 1 Lactobacillus ashdophyllus 2-2 450 8,500 20: 1 Lactobacillus ashdophyllus 3-1 4,000 3,000 30: 1 Lactobacillus lactis 3-2 4,000 3,000 10: 1 Lactobacillus lactis 4-1 4,000 8,500 70: 1 Lactobacillus ashdophyllus 4-2 4,000 8,500 150: 1 Lactobacillus ashdophyllus 5-1 4,000 1,000,000 50: 1 Lactobacillus rutheri 5-2 4,000 5,000,000 250: 1 Lactobacillus rutheri 6-1 7,500 3,000 50: 1 Streptococcus thermophilus 6-2 7,500 3,000 100: 1 Streptococcus thermophilus 7-1 7,500 30,000 70: 1 Streptococcus thermophilus 7-2 7,500 30,000 150: 1 Streptococcus thermophilus 8-1 7,500 60,000 30: 1 Lactobacillus ashdophyllus 8-2 7,500 60,000 200: 1 Lactobacillus ashdophyllus 9-1 7,500 60,000 50: 1 Streptococcus thermophilus 9-2 7,500 60,000 200: 3 Streptococcus thermophilus 10-1 15,000 3,000 70: 1 Lactobacillus lactis 10-2 15,000 3,000 150: 1 Lactobacillus lactis 11-1 15,000 1,000,000 70: 1 Lactobacillus ashdophyllus 11-2 15,000 5,000,000 300: 1 Lactobacillus ashdophyllus 12-1 4,000 1,000,000 30: 1 Lactobacillus rutheri 12-2 4,000 5,000,000 150: 1 Lactobacillus rutheri

Preparation of Lactic Acid Bacteria Particle Powder by Lyophilization

To the lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid obtained through the above process, autoclave freeze solution (skim oil stabilizer) was autoclaved at 121 ° C. for 15 minutes and then stirred uniformly using a stirrer. The lactic acid bacteria mixture thus prepared was lyophilized for 2 days to obtain a lactic acid bacteria powder double coated with chitosan and polygamma glutamic acid.

In this case, the cryobuffer solution includes a composition such as skim milk, glucose, glutamic acid, etc., which is widely used in the art.

Example 1-1. Lactobacillus lactis double coated with chitosan with an average molecular weight of 450 and polygammaglutamic acid with an average molecular weight of 3,000 ( L. lactis ) particle

According to the method described above, Lactobacillus lactis ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 450 and polygammaglutamic acid having an average molecular weight of 3,000 in a 50: 1 weight ratio.

Example 1-2. Lactobacillus lactis double coated with chitosan with an average molecular weight of 450 and polygammaglutamic acid with an average molecular weight of 3,000 ( L. lactis ) particle

According to the method described above, Lactobacillus lactis ¹ × ^{10 10} CFU / ml was coated with 100: 1 weight ratio of chitosan having an average molecular weight of 450 and polygammaglutamic acid having an average molecular weight of 3,000.

Example 2-1. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 450 and polygammaglutamic acid with an average molecular weight of 8,500 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 450 and polygammaglutamic acid having an average molecular weight of 8.500 in a 5: 1 weight ratio.

Example 2-2. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 450 and polygammaglutamic acid with an average molecular weight of 8,500 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 450 and polygammaglutamic acid having an average molecular weight of 8.500 in a 20: 1 weight ratio.

Example 3-1. Lactobacillus lactis double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 3,000 ( L. lactis ) particle

According to the method described above, Lactobacillus lactis ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 3,000 in a 30: 1 weight ratio.

Example 3-2. Lactobacillus lactis double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 3,000 ( L. lactis ) particle

According to the method described above, Lactobacillus lactis ¹ × ^{10 10} CFU / ml was coated with 10: 1 weight ratio of chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 3,000.

Example 4-1. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 8,500 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with a 70: 1 weight ratio of chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 8,500.

Example 4-2. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 8,500 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with a chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 8,500 in a 150: 1 weight ratio.

Example 5-1. Lactobacillus luteri double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 1,000,000 ( L. reuteri ) particle

According to the method described above, Lactobacillus luteri 1 × ^{10 10} CFU / ml was coated in a 50: 1 weight ratio of chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 1,000,000.

Example 5-2. Lactobacillus luteri double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 5,000,000 ( L. reuteri ) particle

According to the method described above, Lactobacillus luteri 1 × ^{10 10} CFU / ml was coated with a 250: 1 weight ratio of chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 1,000,000 .

Example 6-1. Streptococcus thermophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 3,000 ( S. thermophilus ) particle

According to the method described above, Streptococcus thermophilus 1 × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 7,500 and polygammaglutamic acid having an average molecular weight of 3,000 in a 50: 1 weight ratio.

Example 6-2. Streptococcus thermophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 3,000 ( S. thermophilus ) particle

According to the method described above, Streptococcus thermophilus 1 × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 7,500 and polygammaglutamic acid having an average molecular weight of 3,000 in a 100: 1 weight ratio.

Example 7-1. Streptococcus thermophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 30,000 ( S. thermophilus ) particle

According to the method described above, Streptococcus thermophilus 1 × ^{10 10} CFU / ml was coated with a 70: 1 weight ratio of chitosan having an average molecular weight of 7,500 and polygammaglutamic acid having an average molecular weight of 30,000.

Example 7-2. Streptococcus thermophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 30,000 ( S. thermophilus ) particle

According to the method described above, Streptococcus thermophilus 1X10 ¹⁰ CFU / ml was prepared using chitosan having an average molecular weight of 7,500 and an average molecular weight of 30,000. Polygamma glutamic acid was coated in a 150: 1 weight ratio.

Example 8-1. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 60,000 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 7,500 and polygammaglutamic acid having an average molecular weight of 60,000 in a 30: 1 w / w weight ratio.

Example 8-2. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 60,000 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with 200: 1 weight ratio of chitosan having an average molecular weight of 7,500 and polygammaglutamic acid having an average molecular weight of 60,000.

Example 9-1. Streptococcus thermophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 60,000 ( S. thermophilus ) particle

According to the above method, Streptococcus thermophilus 1 × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 7,500 and polygammaglutamic acid having an average molecular weight of 60,000 in a 50: 1 weight ratio.

Example 9-2. Streptococcus thermophilus double coated with chitosan with an average molecular weight of 7,500 and polygammaglutamic acid with an average molecular weight of 60,000 ( S. thermophilus ) particle

According to the method described above, Streptococcus thermophilus 1X10 ¹⁰ CFU / ml was prepared with chitosan having an average molecular weight of 7,500 and an average molecular weight of 60,000. Polygamma glutamic acid was coated in a 200: 3 weight ratio.

Example 10-1. Lactobacillus lactis double coated with chitosan with an average molecular weight of 15,000 and polygammaglutamic acid with an average molecular weight of 3,000 ( L. lactis ) particle

According to the above method, Lactobacillus lactis ¹ × ^{10 10} CFU / ml was coated with a 70: 1 weight ratio of chitosan having an average molecular weight of 15,000 and polygammaglutamic acid having an average molecular weight of 3,000.

Example 10-2. Lactobacillus lactis double coated with chitosan with an average molecular weight of 15,000 and polygammaglutamic acid with an average molecular weight of 3,000 ( L. lactis ) particle

According to the method described above, Lactobacillus lactis ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 15,000 and polygammaglutamic acid having an average molecular weight of 3,000 in a 150: 1 weight ratio.

Example 11-1. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 15,000 and polygammaglutamic acid with an average molecular weight of 1,000,000 ( L. acidophilus ) particle

According to the method described above, Lactobacillus ashidophilus 1X10 ¹⁰ CFU / ml was prepared using chitosan having an average molecular weight of 15,000 and an average molecular weight of 1,000,000. Polygamma glutamic acid was coated in a 70: 1 weight ratio.

Example 11-2. Lactobacillus ashidophilus double coated with chitosan with an average molecular weight of 15,000 and polygammaglutamic acid with an average molecular weight of 5,000,000 ( L. acidophilus ) particle

According to the above method, Lactobacillus ashidophilus ¹ × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 15,000 and polygammaglutamic acid having an average molecular weight of 5,000,000 in a 300: 1 weight ratio.

Example 12-1. Lactobacillus luteri double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 1,000,000 ( L. reuteri ) particle

According to the above method, Lactobacillus luteri 1 × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 1,000,000 in a 30: 1 weight ratio.

Example 12-2. Lactobacillus luteri double coated with chitosan with an average molecular weight of 4,000 and polygammaglutamic acid with an average molecular weight of 5,000,000 ( L. reuteri ) particle

According to the method described above, Lactobacillus luteri 1 × ^{10 10} CFU / ml was coated with chitosan having an average molecular weight of 4,000 and polygammaglutamic acid having an average molecular weight of 5,000,000 in a 150: 1 weight ratio.

Comparative Example 1. Lactobacillus ashdophilus in phosphate buffer solution ( L. acidophilus )

As a comparative example for the double coated Lactobacillus ashidophilus particles prepared according to Examples 2, 4 and 11, Lactobacillus ashidophilus was obtained from a lactic acid bacterium culture solution, and 1 × ^{10 10} CFU / ml. Phosphoric acid buffer solution (pH 7.4) was added thereto.

Comparative Example 2. Streptococcus thermophilus in phosphate buffer solution S. thermophilus )

As a comparative example for the double coated Streptococcus thermophilus particles prepared according to Examples 6, 7 and 9, Streptococcus thermophilus was obtained from a lactic acid bacterium culture solution, such that ¹ × ^{10 10} CFU / ml. Phosphoric acid buffer solution was added.

Comparative Example 3. Lactobacillus lactis on phosphate buffer solution ( L. lactis )

As a comparative example for the double coated Lactobacillus lactis particles prepared according to Example 1, Example 3 and Example 10, Lactobacillus lactis is obtained from the lactic acid bacteria culture medium, phosphate buffered to 1X10 ¹⁰ CFU / ml The solution was added.

Comparative Example 4. Lactobacillus ashidophilus on Cryo buffer Solution L. acidophilus )

As a comparative example for the double coated Lactobacillus ashidophilus particles prepared according to Examples 2, 4 and 11, Lactobacillus ashidophilus was obtained from a lactic acid bacterium culture solution, and 1 × ^{10 10} CFU / ml. Cryobuffer solution was added to this. Cryo buffer solution was prepared using a composition of 10% by weight skim milk, 2% by weight glucose, 0.1% by weight glutamic acid.

Comparative Example 5. Streptococcus thermophilus on cryobuffer solution ( S. thermophilus )

As a comparative example for the double coated Streptococcus thermophilus prepared according to Examples 6, 7 and 9, Streptococcus thermophilus was obtained from a lactic acid bacteria culture medium, and frozen to ¹ × ^{10 10} CFU / ml. Buffer was added.

Comparative Example 6. Lactobacillus Lusteri on Cryo buffer Solution L. reuteri )

As a comparative example for the double-coated Lactobacillus lutery prepared according to Example 5 and Example 12, Lactobacillus lutery was obtained by lactic acid bacteria from the lactic acid bacteria culture medium, cryobuffer solution was added to 1X10 ¹⁰ CFU / ml.

Comparative Example 7. Lactobacillus lactis on cryobuffer solution ( L. lactis)

As a comparative example for the double coated Lactobacillus lactis prepared according to Example 1, Example 3 and Example 10, Lactobacillus lactis was obtained from the lactic acid bacteria culture solution, cryobuffer solution to be 1X10 ¹⁰ CFU / ml Was added.

Comparative Example 8. Lactobacillus ashidophilus coated with chitosan having a molecular weight of 300-600 ( L. acidophilus ) particle

Lactobacillus ashidophilus was obtained from the lactic acid bacteria culture medium, and an aqueous solution in which chitosan having a molecular weight of 300-600 was dissolved so as to be ¹ × ^{10 10} CFU / ml was added. The final concentration of chitosan is 1 mg / ml.

Comparative Example 9. Lactobacillus ashidophilus coated with chitosan having a molecular weight of 3000 to 5000 ( L. acidophilus ) particle

Lactobacillus ashdophyllus was obtained from the lactic acid bacteria culture medium, and an aqueous solution in which chitosan having an average molecular weight of 3000 to 5000 was dissolved to ¹ × ^{10 10} CFU / ml was added. The final concentration of chitosan is 0.5 mg / ml.

Comparative Example 10 Lactobacillus ashidophilus coated with chitosan having a molecular weight of 10000 to 20000 ( L. acidophilus ) particle

Lactobacillus ashidophilus was obtained from the lactic acid bacteria culture medium, and an aqueous solution in which chitosan having a molecular weight of 10000 to 20000 was dissolved to ¹ × ^{10 10} CFU / ml was added. The final concentration of chitosan is 0.1 mg / ml.

Comparative Example 11. Streptococcus thermophilus coated with polygammaglutamic acid having a molecular weight of 750 to 5000 ( S. thermophilus ) particle

Streptococcus thermophilus was obtained from the lactic acid bacteria culture medium, and an aqueous solution in which polygamma glutamic acid having a molecular weight of 750 to 5000 was added to ¹ × ^{10 10} CFU / ml was added. The final polygamma glutamic acid concentration at this time is 0.5 mg / ml.

Comparative Example 12 Streptococcus thermophilus coated with polygammaglutamic acid having a molecular weight of 20000 to 40000 ( S. thermophilus ) particle

Streptococcus thermophilus was obtained from the lactic acid bacteria culture medium, and an aqueous solution in which polygammaglutamic acid having a molecular weight of 20000 to 40000 was added to ¹ × ^{10 10} CFU / ml was added. In this case, the final polygamma glutamic acid concentration is 0.1 mg / ml.

Comparative Example 13. Streptococcus thermophilus coated with polygammaglutamic acid having a molecular weight of 50,000 to 70,000 ( S. thermophilus ) particle

Streptococcus thermophilus was obtained from the lactic acid bacteria culture medium, and an aqueous solution in which polygammaglutamic acid having a molecular weight of 50,000 to 70,000 was dissolved was added to ¹ × ^{10 10} CFU / ml. In this case, the final polygamma glutamic acid concentration is 0.5 mg / ml.

Comparative Example 14. Lactobacillus ruteri double coated with chitosan having a molecular weight of 3,000 to 5,000 and fucoidan as an anionic polymer ( L. reuteri ) particle

Lactobacillus ruteri was obtained from the lactic acid bacteria culture medium, and firstly coated with an aqueous solution of chitosan having a molecular weight of 3,000 to 5,000 so as to have a ¹ × ^{10 10} CFU / ml, followed by secondary coating by adding an aqueous solution of fucoidan to the mixture. The ratio of chitosan and fucoidan used at this time is 50: 1 w / w.

Comparative Example 15 Lactobacillus Luteri Double-Coated with Chitosan with a Molecular Weight of 3,000 to 5,000 and Hyaluronic Acid, an Anionic Polymer ( L. reuteri ) particle

Lactobacillus ruteri was obtained from the lactic acid bacteria culture medium, followed by primary coating by adding an aqueous solution of chitosan having a molecular weight of 3,000 to 5,000 to ¹ × ^{10 10} CFU / ml, followed by secondary coating by adding an aqueous solution of hyaluronic acid to the mixture. . The ratio of chitosan and hyaluronic acid used at this time is 70: 1 w / w.

Comparative Example 16 Lactobacillus lactis double coated with a polymer chitosan having a molecular weight of 600,000 or more and a polygammaglutamic acid having a molecular weight of 750 to 5,000 ( L.lactis ) particle

Lactobacillus lactis was obtained from a lactic acid bacterium culture solution, and firstly coated with an aqueous solution in which molecular weight of chitosan having a molecular weight of 600,000 or more was added to 1 × ^{10 10} CFU / ml, and an aqueous solution in which polygammaglutamic acid having an average molecular weight of 750 to 5,000 was dissolved in the mixture. Was added to the secondary coating. The ratio of chitosan and polygamma glutamic acid used at this time is 100: 1 w / w composition.

Comparative Example 17 Lactobacillus ashdophyllus comprising a composition comprising a polymer chitosan having a molecular weight of 600,000 or more and a polygamma glutamic acid having an average molecular weight of 2,000 to 150,00 ( L. acidophilus ) particle

Lactobacillus lactis is obtained from the lactic acid bacteria culture medium, the polymer having a molecular weight of 600,000 or more to 1X10 ¹⁰ CFU / ml An aqueous solution in which chitosan was dissolved was added to the first coating, and an aqueous solution in which polygamma glutamic acid having an average molecular weight of 2,000 to 150,00 was added to the mixture, followed by second coating. The ratio of chitosan and polygamma glutamic acid used at this time is 50: 1 w / w.

[Experimental Example]

Experimental Example 1.Comparison of survival rate of Lactobacillus lactis according to double coating with chitosan and polygammaglutamic acid

Lactobacillus prepared using Lactobacillus lactis 1X10 ¹⁰ CFU prepared by double coating with chitosan and polygammaglutamic acid and lyophilized for 2 days according to Example 1-1 and Example 3-2, Example 10-1 All of the coated lyophilized powder was dissolved in 1 ml of sterile distilled water to prepare a lactic acid bacteria solution. In order to compare the survival rate of Lactobacillus lactis according to the composition of each example, the lactic acid bacteria solution was prepared by the same process using the lactic acid bacteria lyophilized powder according to the composition of Comparative Example 3, Comparative Example 7 and Comparative Example 16.

1.5% agar powder was added to MRS medium, autoclaved at 121 ^o C for 15 minutes, and the medium was cooled down. After 10 ml aliquots were added to a petri dish, it was completely hardened at room temperature. Prepared. Each lactic acid bacteria solution was diluted to 1/10, 1/100, 1/1000, 1/10000, 1/100000 in MRS agar medium, and 100 L of each solution was smeared. Colonies were counted after incubation at 37 ^o C for 24 h.

FIG. 1 shows colony-forming pictures of double coated Lactobacillus lactis particles prepared according to the molecular weight of various chitosan and the weight ratio condition of chitosan and polygammaglutamic acid [(A) Comparative Example 3, (B) Comparative Example 7, ( C) Comparative Example 16, (D) Example 1-1, (E) Example 3-2, (F) Example 10-1].

Figure 1A is Comparative Example 3, by lyophilizing the untreated lactic acid bacteria, when the untreated lactic acid bacteria can be seen that the survival rate is very low, less than 5%. FIG. 1B is Comparative Example 7, when lyophilized with the addition of a freeze buffer solution, the survival rate was higher than that without any treatment, and as shown in FIGS. 1D, 1E, and 1F, coated with chitosan and polygammaglutamic acid. In case of lyophilization, the survival rate of lactic acid bacteria was higher than 80%. Although there is no significant difference in the lactic acid bacteria survival rate according to the molecular weight of the chitosan in a certain range, as shown in FIG. 1C showing the result of Comparative Example 16 using the polymer chitosan having a molecular weight of 600000 or more, a large protective effect in the freeze-drying process rather than in the chitosan of too large molecular weight Did not look.

Experimental Example 2. Comparison of survival rate of Lactobacillus ashdophyllus by monocoating with chitosan

According to Example 2-1, Example 4-2, and Example 11-1, Lactobacillus ashdophyllus lyophilized powder prepared by double coating with chitosan and polygammaglutamic acid and lyophilized for 3 days was sterile distilled water 1 It was dissolved in ml to prepare a lactic acid bacteria solution. In order to compare the survival rate of Lactobacillus ashdophyllus according to the composition of each example, lactic acid bacteria solution was prepared by using the lactic acid bacteria lyophilized powder according to the composition of Comparative Example 1 and Comparative Example 4. In Comparative Example 8, Comparative Example 9, and Comparative Example 10, as a group treated only with chitosan by molecular weight, when only the cationic polymer was mixed and when the lactic acid bacteria were coated due to the electrostatic attraction between the cationic polymer and the anionic polymer, Compared.

In the same manner as in Experiment 1, the lactic acid bacteria solution was smeared on MRS medium, and the number of colonies was counted to calculate the survival rate compared with the untreated group. The untreated group was obtained by lactic acid bacteria and inoculated in MRS agar medium in the state before lyophilization to measure the number of colonies.

FIG. 2 is a graph showing the survival rate of lactic acid bacteria of double coated Lactobacillus ashidophilus particles prepared according to the molecular weight of various chitosan and the weight ratio condition of chitosan and polygammaglutamic acid.

In Comparative Example 4, Comparative Example 8, Comparative Example 9, and 10, the number of lactic acid bacteria colonies was similar, and when the chitosan aqueous solution was simply mixed, it can be seen that there is no great effect during lyophilization. In Comparative Example 17, when lactic acid bacteria were coated using chitosan having a high molecular weight, the survival rate was lower than that when only the cryo buffer solution was treated. Example 2-1, Example 4-2 and Example 11-1 show similar survival rates to the untreated group even after lyophilization. This suggests that recoating chitosan with polygammaglutamic acid, an anionic polymer, is superior to lactic acid bacteria rather than coating with chitosan alone.

Experimental Example 3. Comparison of Survival Rate of Streptococcus Thermophilus with Single Coating with Polygammaglutamic Acid

Incubating Streptococcus thermophilus in the same manner as in Experimental Example and preparing lactic acid bacteria particles with compositions according to Examples 6-2, 7-2 and 9-1, freezing and thawing After freezing and thawing was repeated five times to freeze-dried. This process created a harsh environment for the lactic acid bacteria to survive, and tested the protective effect of the double coated lactic acid bacteria particles. Figure 3 is prepared by the double-coated Streptococcus thermophilus particles prepared according to the molecular weight of the various chitosan and the weight ratio condition of chitosan and polygamma glutamic acid, repeated five times the freeze-thawing process, each composition compared with the untreated group It is a graph showing the survival rate of lactic acid bacteria. As can be seen in Figure 3, Comparative Example 5, despite the treatment of the cryo buffer solution, it can be seen that the survival rate of lactic acid bacteria during the freeze thawing process as low as 20%, Comparative Example 11, only treated with polygamma glutamic acid by molecular weight The compositions of Example 12 and Comparative Example 13 also show similar results. However, in Example 6-2, Example 7-2 and Example 9-1 composition according to the present invention shows a survival rate of 60% or more lactic acid bacteria, in the freeze-thawing process repeated double coating with chitosan and poly gamma glutamic acid It can be seen that plays a role in protecting the lactic acid bacteria.

Experimental Example 4. Comparison of Survival Rate of Lactobacillus Luterry According to Modification of Anionic Polymer

Lactobacillus luteri was cultured in the same manner as in Experimental Example to obtain lactic acid bacteria, and lactic acid bacteria particles were prepared for each composition of Example 5-1, Example 12-2, Comparative Example 6, Comparative Example 14 and Comparative Example 15. By repeating the freeze-thawing process five times, to prepare a powder, it was dissolved in 1 ml of sterile distilled water to make a lactic acid bacteria solution. Lactobacillus solution was inoculated in MRS agar medium to count the number of colonies to compare the effect on the survival rate of lactic acid bacteria according to the type of anionic polymer. Figure 4 shows the survival rate of lactic acid bacteria in each composition compared to the untreated group after the production of lactobacillus luterri particles double coated with chitosan and polygamma glutamic acid, or chitosan and other anionic polymers to repeat the freeze-thawing process five times It is a graph. As can be seen from Figure 4, when the coating using a cationic polymer and an anionic polymer it can be seen that the lactic acid bacteria are more stabilized during the freezing and thawing process. In particular, the use of poly-gamma glutamic acid for the same cationic polymer than other anionic polymers can be seen that the lactic acid bacteria protection effect is excellent.

Experimental Example 5. Measurement of long-term storage rate of Lactobacillus ashdophyllus by double coating with chitosan and polygammaglutamic acid

By culturing Lactobacillus ashdophyllus in the same process as above, lactic acid bacteria were obtained, and lactic acid bacteria particles were prepared according to Examples 4-1, 8-1, Comparative Example 1, and Comparative Example 4, which were then used for 2 days. Lyophilization to prepare a lactic acid bacteria coating powder. Then, when stored at room temperature for a long time, the survival rate of the lactic acid bacteria over time was measured. As a result, as shown in Figure 5, the first lactic acid bacteria colony number was the same as 1X10 ¹⁰ CFU, but showed a difference in the survival rate of lactic acid bacteria by composition over time.

Claims (16)

Lactobacillus; And lactic acid bacteria particles comprising a chitosan coating layer and a polygamma glutamic acid coating layer on the lactic acid bacteria.
The method of claim 1,
Lactic acid bacteria particles having an average molecular weight of the chitosan is 200 to 30,000.
The method of claim 1,
Lactic acid bacteria particles having an average molecular weight of the poly gamma glutamic acid is 300 to 20,000,000.
The method of claim 1,
Chitosan coating layer and the polygamma glutamic acid coating layer on the lactic acid bacteria, lactic acid bacteria particles are sequentially coated.
The method of claim 1,
The lactic acid bacteria are Lactobacillus genus (Lactobacillus sp.), Lactobacillus genus (Sporolactobacilus sp.), Streptococcus genus (Streptococcus sp.), Lactococcus genus (Lactococcus sp.), Lou Kono Stock in (Leuconostoc sp.) In Spokane , Lactic acid bacteria particles which are one or more strains selected from the group consisting of Pedioococcus sp. , Enterococcus sp. And Bifidobacterium sp .
The method of claim 1,
The lactic acid bacteria particles are lactic acid bacteria particles that are present in the lyophilized powder state.
Coating the lactic acid bacteria with chitosan and polygammaglutamic acid
Method for producing lactic acid bacteria particles double coated with chitosan and polygammaglutamic acid.
The method of claim 7, wherein
The weight ratio of the chitosan and poly gamma glutamic acid is 3: 1 to 300: 1 method for producing lactic acid bacteria particles.
The method of claim 7, wherein
After adding the cryo buffer solution to the lactic acid bacteria particles lyophilized method of producing a lactic acid bacteria particles further comprising obtaining a lactic acid bacteria powder.
A composition comprising the lactic acid bacteria particles of any one of claims 1 to 6.
The method of claim 10,
The composition is for use as a formal, probiotic or antimicrobial agent.
A food comprising the composition of claim 10.
The method of claim 12,
The food is a food or beverage or health functional food.
A medicament comprising the composition of claim 10.
The method of claim 14,
The medicine is a drug or probiotics.
A feed comprising the composition of claim 10.

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