KR102084003B1 - Method for preparing nucleotides of lactic acid bacteria inclusion-complexed with low-molecular functional biomaterial and Cubisome prepared thereby - Google Patents

Abstract

The present invention relates to a method for simultaneously preparing a low-molecular functional biomaterial and a lactic acid bacteria nucleotide having utility in skin and cosmetics fields and, more specifically, to a method for preparing a lactic acid bacteria nucleotide (cubisome) inclusion-complexed to a low-molecular functional biomaterial, which is a 2-in-1 production method for simultaneously solving two problems of conversion of a high-molecular functional biomaterial to a low-molecular functional biomaterial and elution of lactic acid bacteria nucleotide components by a single cell crushing process. According to the present invention, a nucleotide derived from lactic acid bacteria, which effectively removes the intrinsic cytoplasmic protein and cell wall components of lactic acid bacteria affecting the production of fermentation odor of skin microbiome and raw materials and has increased stability by being inclusion-complexed with an exogenous low-molecular biomaterial, is produced at a high concentration, thereby being effectively used as a biomaterial in cosmetics, foods, and medicines.

Description

Method for preparing nucleotides of lactic acid bacteria inclusion-complexed with low-molecular functional biomaterial and Cubisome prepared according to the method for preparing lactic acid bacteria nucleotide encapsulated with a low molecular weight functional biomaterial

The present invention relates to a method for simultaneously preparing a low molecular weight functional biomaterial and a lactic acid bacterium nucleotide having utility in the field of skin and cosmetics, and more specifically, to a low molecular weight functional biomaterial from a high molecular weight functional biomaterial by one cell disruption process. And a method for producing a lactic acid bacterium nucleotide (cubisome) encapsulated with a low molecular weight functional biomaterial which simultaneously solves two problems of eluting bacterial nucleotide components (2-in-1 production method).

Skin can be divided into the epidermis, the epidermis, the dermis and the subcutaneous fat underneath, and have different functions for each layer. The epidermis functions include a permeation barrier, sensitizing immunity, protection against ultraviolet rays, wound recovery, and vitamin D synthesis. The dermal layer has functions such as inhibiting pathogen invasion, wound recovery, and maintaining skin structure and elasticity.

While the skin protects the body from physical and chemical stimuli from the outside, ultraviolet rays damage the DNA of skin cells and produce reactive oxygen species (ROS) to inhibit the synthesis of collagen in the skin and Promotes aging of the skin, creating wrinkles.

Ultraviolet rays are directly reflected at the boundary of the skin epidermis, or scattered after striking the cells and fibroblast particles in the skin tissue, or are absorbed and transmitted through the path. Only the absorbed ultraviolet rays cause a photochemical reaction to cause skin lesions. The degree of penetration of the skin varies depending on the wavelength. UVA, which has a long wavelength, penetrates deep into the dermis, but UVB and UVC have short wavelengths, causing severe skin lesions on the epidermis. Among them, UVB has a wavelength of 280-320 nm, but most of it is absorbed by the ozone layer. However, some of them reach the earth's surface and are shorter in intensity than UVA with high energy rays, causing a solar scar in a short time. Prolonged exposure to UVB has been identified as a cause of skin cancer.

There are statistically confirmed reports of differences in skin condition and type by region, race and age. Kompaore et al. (Skin Pharmacol., 1993; 6: 200-207) measured transdermal moisture loss in Asians, Caucasians, and blacks, and found that Asians and blacks were higher than whites. Man et al. (Skin Pharmacol. Physiol., 200; 22: 190-199) measured sebum levels of Chinese males and females according to age groups. Males tended to decrease, but showed similar trends in their 50s and then gradually decreased. Marrakchi et al. (Contact dermatitis, 2007; 57: 28-34) compared the moisture content and sebum content of young people aged 24 to 34 years old with those aged 66 to 83 years. Sebum volume was not significantly different between the young and the elderly.

According to the report on the establishment of skin characteristics banks by country, for Koreans, female skin types differed in the order of complex> dry> neutral> oily, and male skin types differed in the order of oily> complex> neutral> dry.

In the conventional statistical methods, there are differences according to race, region of living, gender, and age. However, there is no clear correlation between optical causes such as UVB causing skin aging such as transdermal moisture loss and skin wrinkles. It was difficult.

Skin microbiome refers to the ecosystem of the flora of the genus flora distributed on the skin. Mainly, Staphylococcus genus and Propionibacterium genus maintain a healthy skin condition. The flora of the skin microbiome differs according to age group, women and men, race and region of residence. In particular, since skin dominant species by age group are analyzed by a statistical method to maintain skin health, the use of intestinal microorganisms on the skin can be said to have no scientific basis.

Recently, Patent No. 10-2017-0125755 discloses a fecal flora of mice that have been skin-damaged by UVB and an intestinal flora of non-photo-induced mice in a patent for its use of enteric microorganisms or endogenous metabolite markers for judging skin photodamage. This difference was suggested and the skin damage caused by UVB was indirectly interpreted as gut microbiome. However, this previous study showed that the intestinal flora of the intestinal microbiome according to diet, such as age, sex, and race is different, and allobaculum, Lachnoclostridium, Parvibacter, Lactobacillus, etc. may be reduced by other causes than skin damage. As the cause and solution for the skin flora, it showed its limitations. In addition, the intestinal flora is inoculated into the skin flora and the balance is maintained. The intestinal flora is mostly anaerobic and the skin flora is an aerobic or aerobic microorganism with different degrees of oxygen. Previous studies showing the effectiveness of using enteric Lactobacillus bacteria directly on the skin are unclear.

In addition, raw materials using lactic acid bacteria of the genus Bifidobacterium or Lactobacillus are currently on the market. Among them, Bifido fermentation concentrates and the like are the cell lysates as a main component. However, lipopolysaccharide (LPS), which is known to cause an immune reaction with the skin, is present in the cell wall. Excess of this component can cause skin problems such as atopy, erythema and rash.

One of the problems in the application of microbial materials for skin protection is that when the cell lysate is applied to the skin, the skin flora is a protein necessary for growth. Ohm's balance is broken. Since various microbial cell debris currently listed as international cosmetic raw materials contain many protein components that can affect human skin microbiome, this can minimize the protein components in the microbial cells and increase the functional active ingredients in the manufacturing process. Certain process strategies are required.

In addition, various biological materials including polysaccharides such as hyaluronic acid, beta glucan, and proteins such as collagen are used in the cosmetic field. For example, hyaluronic acid has a property of absorbing moisture, which is known to not only moisturize and elasticity of the skin, but also to inhibit inflammation and skin damage caused by ultraviolet rays. Beta-glucan not only exhibits antioxidant and anti-inflammatory effects, It is known to increase moisturizing and improve skin wrinkles. Collagen is known to improve skin wrinkles and increase skin elasticity as well as to prevent skin aging. However, since the biological material is made of a polymer, the skin is not absorbed well. Therefore, in recent years, in order to increase skin absorption of biological materials and increase skin beauty effects, processes for making these biological materials into low molecular weight have been separately developed.

The present invention overcomes limitations such as age, sex, race, living area, etc. in the previous studies and has completed the process for producing lactic acid bacterium nucleotides applicable to the skin microbiome. More specifically, by minimizing the protein component affecting the skin microbiome among the components of the lactic acid bacteria cells and optimizing the production method of lactic acid bacteria nucleotides to increase the concentration of nucleotides having excellent skin beauty effect, In skin application, the effects of lipopolysaccharide and protein, which are the source of skin problems, were minimized. In addition, in order to prepare lactic acid bacteria nucleotide components and skin-friendly biomaterials in one process, one cell crushing process solves two manufacturing problems: the conversion of polymer biomaterials to low molecular weight biomaterials and the leaching of lactic acid bacteria nucleotide components. By completing the -in-1 preparation method, the present invention was completed to prepare a lactic acid bacterium nucleotide raw material called cubisome.

KR 10-1426191 B KR 10-2008-0092090 A

Accordingly, the main object of the present invention is to provide a method for simultaneously producing a low molecular weight functional biomaterial and lactic acid bacteria nucleotide having utility in the field of skin care and cosmetics in one process.

Another object of the present invention is to provide a new bio material in the field of cosmetics, food and pharmaceuticals, called Cubisome, which is a lactic acid bacterium nucleotide encapsulated with a low molecular weight functional biomaterial prepared by the above-described manufacturing method.

In the specification of the present invention, 'functional biological material' means a substance derived from an organism having a functional function useful in the field of conventional cosmetics, foods and pharmaceuticals, and any functional biological material as long as the polymer can be degraded and differentiated And, for example, hyaluronic acid, polysaccharides such as betaglucan, proteins such as collagen, and the like. Such 'functional biomaterials' are not derived from the corresponding lactic acid bacteria to elute nucleotides, but are introduced from the outside (eg, derived from other microorganisms). In addition, the 'polymer' refers to the polymer state before the functional biological material is low molecular weight through the method of the present invention, the 'low molecule' refers to the state in which the polymer functional biomaterial is decomposed and low-differentiated through the method of the present invention it means. The 'polymer' and 'low molecule' are not limited to a specific molecular weight as a concept relative to each other. In addition, the term "inclusion" means that the low molecular weight functional biomaterial is complexed with or mixed with lactic acid bacteria nucleotides. Therefore, the low molecular weight functional biomaterial in the present invention serves as a carrier of the lactic acid bacteria nucleotides.

According to one aspect of the present invention, the present invention provides a method for preparing lactic acid bacteria nucleotides enclosed with a low molecular weight functional biomaterial comprising the following steps.

(a) pre-fermentation step of propagating lactic acid bacteria in nutrient medium;

(b) a post-fermentation step of separating the lactic acid bacteria grown in step (a) and culturing in a restriction medium containing a high molecular weight biological material;

(c) lysing the lactic acid bacteria nucleotides by lysing the lactic acid bacteria post-fermentation solution of step (b) under heat compression conditions;

(d) removing the cell wall and cytoplasmic protein components from the crushed fermentation broth of step (c) and recovering the lactic acid bacterium nucleotides in contact with the low molecular weight functional biomaterial.

In the present invention, the 'nutritive medium' of the step (a) means a medium containing carbon sources, nitrogen sources, growth factors such as vitamins, inorganic salts, nutrients necessary for the growth of microorganisms, for example, MRS liquid medium . It may include a medium such as BL Agar, Todd Hewitt liquid medium, MRS liquid medium was used in the embodiment of the present invention. In addition, the "limiting medium" of the step (b) means a medium that does not contain enough nutrients required for proliferation and the ingredients are limited. In the embodiment of the present invention, the polymer polysaccharide or collagen and glucose are dissolved in distilled water. Medium was used.

In the present invention, preferably, as a result of the post-fermentation of the step (b) and the heat compression conditions of the step (c), the high molecular weight functional biological material is characterized in that the low molecular weight of the functional biomaterial and lactic acid bacteria nucleotides included It provides a manufacturing method. According to the present invention, in the post-fermentation step of step (b), the nutrients of the restriction medium are not sufficient, so that the polymer biomaterial is more likely to be decomposed, and in the step (c) of the thermal compression conditions, by one step It is possible to prepare a 2-in-1 method that simultaneously solves two problems: the conversion from a polymeric biomaterial to a low molecular biomaterial and the dissolution of lactic acid bacteria nucleotide components.

In the present invention, the lactic acid bacteria of step (a) can be used in the cosmetic field and so on as long as nucleotides can be leaked by cell disruption, but any lactic acid bacteria can be used, preferably, Lactobacillus sp. Bifidobacterium sp., Streptococcus sp., Lactococcus sp, Enterococcus sp, Pediococcus sp, Weissella genus It provides a method for producing lactic acid bacteria nucleotides and low molecular weight functional biological material characterized in that at least one selected from the group consisting of sp).

In the present invention, the pre-fermentation culture is inoculated lactic acid bacteria to 1 ~ 10% (v / v) using MRS liquid medium and then incubated for 12 to 48 hours at 30 ~ 45 degrees. More preferably, 2-5% (v / v) of lactic acid bacteria were inoculated in MRS liquid medium and then incubated for 15 to 24 hours at 37 degrees. After incubation, the culture was centrifuged (4,000 rpm, 10 min), only the cells were recovered, washed twice with sterile distilled water, and suspended in final sterile distilled water.

In the present invention, the polymer functional biomaterial of step (b) may be any polymer functional biomaterial that can be used in the skin or cosmetic field, but is preferably a hyaluronic acid which is a polymer functional biomaterial not derived from the lactic acid bacteria. (hyaluronic acid), beta glucan (beta glucan), or collagen (collagen) is characterized in that at least one selected from the group consisting of. In an embodiment of the present invention, in order to maintain the stability of the nucleotides of lactic acid bacteria in the cell after the smooth elution and elution to maintain an environment similar to the environment in the cytoplasm, it was selected as the best carrier (carrier) in the microbial polymer material derived from microorganisms. .

In the present invention, the limiting medium of step (b) may be any limiting medium in which the influence component is limited in addition to the polymeric functional biological material, but preferably, the low molecular weight functional material comprises only water and glucose in addition to the polymeric functional biological material. It provides a method for producing lactic acid bacteria nucleotides enclosed with a biological material. Specifically, the restriction medium may contain 0.1-10% (w / v) of the polymeric functional biomaterial and 0.1-10% (w / v) of glucose in distilled water.

In the present invention, the post-fermentation culture was incubated for 12-48 hours at 30-45 degrees after inoculating the suspended cells recovered through pre-fermentation in a restriction medium. More preferably, after inoculating the cells were incubated at 37 degrees for 15 to 24 hours.

In the present invention, the thermal compression conditions of the step (c) may be any condition that can break the lactic acid bacteria cells and decompose the polymer functional biological material, preferably a temperature of 80 to 130 degrees and a pressure of 0.01 to 0.30 MPa It provides a method for producing a low molecular weight functional biological material and lactic acid bacteria nucleotides, characterized in that the conditions.

In order to separate the conventional lactic acid bacterium nucleotides, the grown cell culture solution is centrifuged to discard the supernatant culture medium, and only the cells of the pellet are collected and crushed. It is done. As a result, it is possible to achieve low molecular weight and lactic acid bacteria nucleotide dissolution of biomaterials in one process, and to prepare a new biomaterial of lactic acid bacteria nucleotides complexed with a low molecular weight biomaterial.

In the present invention, removing the cell wall and the cytoplasmic protein in step (d) may be any method known in the prior art, but preferably, centrifugation to remove the pellet containing the cell wall, cooling and filtration Provided is a method for producing a low molecular weight functional biomaterial and lactic acid bacteria nucleotides clathrate to remove cytoplasmic protein. The centrifugation may use any centrifugation, such as 3,000 to 5,000 rpm, to remove the pellet containing the crushed cell wall, the cooling may be any temperature at which the denatured proteins can be aggregated or down by high temperature and high pressure, For example, 0 to +25 degrees may be used, and the filtration may separate cytoplasmic protein and nucleotides, which may be used, such as a filter of 0.20 to 20 um. By removing the cell wall and the cytoplasmic protein inherent in the lactic acid bacteria as described above, not only the production of fermentation odor and skin trouble of the raw material can be suppressed, but also the adverse effects on the skin microbiome can be prevented.

Specifically, denatured glycoprotein components are generated by a millard reaction, which is an irreversible reaction between sugar components and proteins in the cytoplasm, which are generated in the thermocompression process by crushing lactic acid to elute nucleotides, and cause abnormal fermentation odor. It has become a factor that impairs the sensory quality. However, according to the present invention, it is possible to effectively remove the cytoplasmic protein and cell wall components inherent in the lactic acid bacteria that are the quality inhibitors generated during the manufacturing process, and to increase the stability of the exogenous polymer by converting the carrier into low molecules.

According to another aspect of the present invention, the present invention provides a lactic acid bacterium nucleotide encapsulated with a low molecular weight functional biological material prepared by the method of the present invention. According to the present invention, not only the low molecular weight biomaterial useful in the field of cosmetics and the lactic acid bacterium encapsulated in complex can be provided at a time, but also the low molecular weight biomaterial naturally increases the stability by wrapping the lactic acid bacterium nucleotide. The lactic acid bacterium nucleotide encapsulated with the low molecular weight functional biomaterial of the present invention is referred to as Cubisome as a new biomaterial that has not existed before.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a production method based on the stable skin whitening effect of lactic acid bacteria-derived nucleotides and the removal of cell-derived proteins and cell wall components generated in the raw material preparation. More specifically, the present invention is to cancel the lactic acid bacteria unique cellular protein and cell wall components affecting the production of fermentation odor of skin microbiome and raw materials through the polymer polysaccharide latex effect tablet and high concentration of nucleotides derived from lactic acid bacteria having UV absorption effect The present invention relates to a method for producing a cubisome by encapsulating a polymer polysaccharide derived from microorganisms for the purpose of developing a bio-material which can be used in cosmetics, foods and medicines, thereby increasing its stability.

The present inventors proceed with pre-fermentation to propagate lactic acid bacteria, fermentation odor and color, and in order to maintain the stability of the active ingredient when applying the skin to the post-fermentation by applying the technique of the polysaccharide derived from microorganisms and substitution culture and UV wavelength The present invention was completed by developing a method for preparing cubicsome raw materials in which intracellular nucleotides, which have the highest absorbance of C, were optimized for crushing conditions with temperature, pressure, and time variables.

The method for preparing a lactic acid bacterium nucleotide (cubisome) raw material enclosed with the low molecular weight biomaterial of the present invention includes the following steps.

(a) pre-fermentation step of propagating lactic acid cells;

(b) a post-fermentation step of propagating the lactic acid bacteria grown from step (a) in a medium containing a polymeric functional biomaterial;

(c) eluting the nucleotides of the lactobacillus post-fermentation solution of step (b) under heat pressure concentration conditions to produce lactic acid bacteria nucleotides in contact with low molecular weight biomaterials having high skin absorption and excellent preservation.

(a) Prefermentation stage of lactic acid bacteria:

Lactic acid bacteria pre-fermentation step was set to the fermentation conditions to maximize the growth of cells and minimize the metabolites that cause the fermentation odor. Pre-fermentation conditions excluded the fermentation odor from the sensory test by limiting the secondary variable intervals of viable cell number and pH as the cells in the exponential phase state on the microbial growth curve.

(b) Post-fermentation stage of lactic acid bacteria:

To ferment odor from cells recovered from pre-fermentation of lactic acid bacteria and to form a skin-friendly structure for continuous functioning in the skin epidermis, the microbial-derived polymer biomaterial was post-fermented.

The microorganism-derived polymer biomaterial is not particularly limited thereto, but may include not only polymer polysaccharides such as hyaluronic acid and beta glucan, but also proteins such as collagen, preferably hyaluronic acid and beta glucan, and more preferably. Refers to a post-fermentation medium composition containing hyaluronic acid.

(c) Cubisome production of functional lactic acid bacteria nucleotides entrapped in skin-friendly low molecular biomaterials:

The lactic acid bacteria after fermentation solution is optimized for two parameters of specific temperature and pressure, so that the polymer polysaccharide is converted into a low molecular weight polysaccharide which is easily absorbed by the skin, and the lactic acid bacteria elute nucleotides present in the cytoplasm out of the cytoplasm to be enclosed with the low molecular polysaccharide. Cotton was produced.

The microbial cell debris used as a cosmetic ingredient does not specify the action according to the composition, causing atopic dermatitis, erythema, rash, etc., which are the skin immune reactions of intracellular proteins and hosts. It can be used as a source of growth nitrogen in scabies and can be a potential threat to skin health. Therefore, in the present invention, the production process that can be obtained in a large amount of nucleotides in the cell utilizing the lactic acid bacteria from the results obtained in the Example [Fig. 1]. As the lactic acid bacteria ferment, the protein secreted out of the cell is removed through the post-fermentation process, and the polymer polysaccharide used as a medium component used in the post-fermentation process is converted into a low molecular polysaccharide in the process of eluting the lactobacillus nucleotide by applying heat and pressure. Not only can it be used as an easy preparation, it also shows the dual function of encapsulating lactic acid bacteria nucleotides to maintain its stability, and by inhibiting the production of protein in the medium excluding nitrogen sources in the post-fermentation process, it removes the fermentation odor from the final product. Sensory merchandise and quality were also improved compared to the process using cell wall components.

According to the present invention, high concentrations of nucleotides derived from lactic acid bacteria, which effectively removes cellular proteins and cell wall components unique to lactic acid bacteria that affect the production of fermentation odors of skin microbiomes and raw materials, and are enclosed with low molecular weight functional biological materials to increase their stability. It can be effectively used as a bio material in the cosmetic, food and pharmaceutical fields.

1 is a process chart showing a method for producing a high concentration of lactic acid bacteria nucleotides according to the present invention.
Figure 2 is a photograph showing the lactic acid bacteria nucleotide raw material prepared by the production method of the present invention.
Figure 3 is a photograph showing the lactic acid bacteria lysate raw material prepared by a conventional method.
Figure 4a is a photograph showing the UV wavelength absorbance of lactic acid bacteria nucleotides produced by pre-fermentation and post-fermentation (carrier oil).
Figure 4b is a photograph showing the UV wavelength absorbance of lactic acid bacteria nucleotides produced by pre-fermentation and post-fermentation (carrier free).
Figure 5 is a photograph showing the elution of lactic acid bacteria nucleotides through nucleic acid analysis. Figure 5a is a chromatogram of nucleic acid standard, Figure 5b is a chromatogram of lactic acid bacteria nucleotides.
6 is a photograph showing the low molecular weight of the polymeric biomaterial. FIG. 6A is a chromatogram of a polymeric biomaterial prior to low molecular weight, and FIG. 6B is a chromatogram of a biomolecule being low molecular weight.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Lactic Acid Bacteria Nucleotide (Cubisome) Entrapped with Low Molecular Weight Biomaterial (Haluronic Acid)

After inoculating Lactobacillus to 3% (v / v) using MRS liquid medium, centrifugation (4,000 rpm, 10 min) after 15-24 hours pre-cultivation at 37 ° C, and only cells were recovered and sterilized distilled water. Wash twice with and suspend in final sterile distilled water. The collected suspended cells were post-incubated at 37 ° C. for 15-24 hours after inoculation into the restriction medium to which glucose 1% (w / v) and hyaluronic acid 1% (w / v) were added to distilled water. After incubation, cells were crushed at 121 degrees, 0.15 MPa and 30 minutes, and the lactic acid bacteria lysate was centrifuged (4,000 rpm, 10 min) to recover only the final supernatant. The recovered supernatant was finally filtered through a 0.45 um filter after chilling at 4 degrees. After filtration and storage for 1 week in refrigeration was confirmed the state. Figure 2 is a photograph showing the lactic acid bacteria nucleotide (cubisome) prepared by the production method of the present invention.

Example 2: Preparation of Lactic Acid Bacteria Nucleotide (Cubisome) Enclosed with Small Molecular Biomaterial (Betaglucan)

After inoculating Lactobacillus to 3% (v / v) using MRS liquid medium, centrifugation (4,000 rpm, 10 min) after 15-24 hours pre-cultivation at 37 ° C, and only cells were recovered and sterilized distilled water. Wash twice with and suspend in final sterile distilled water. The recovered suspended cells were post-incubated at 37 degrees for 15-24 hours after inoculation into the restriction medium to which glucose 1% (w / v) and beta glucan 1% (w / v) were added to distilled water. After incubation, cells were crushed at 121 degrees, 0.15 MPa and 30 minutes, and the lactic acid bacteria lysate was centrifuged (4,000 rpm, 10 min) to recover only the final supernatant. The recovered supernatant was finally filtered through a 0.45 um filter after chilling at 4 degrees. After filtration and storage for 1 week in refrigeration was confirmed the state.

Example 3: Preparation of Lactic Acid Bacteria Nucleotide (Cubisome) Enclosed with Small Molecular Biomaterial (Collagen)

After inoculating Lactobacillus to 3% (v / v) using MRS liquid medium, centrifugation (4,000 rpm, 10 min) after 15-24 hours pre-cultivation at 37 ° C, and only cells were recovered and sterilized distilled water. Wash twice with and suspend in final sterile distilled water. The recovered suspended cells were post-incubated at 37 ° C. for 15-24 hours after inoculation into the restriction medium to which glucose 1% (w / v) and collagen 1% (w / v) (Example 3) were added to distilled water. After incubation, cells were crushed at 121 degrees, 0.15 MPa and 30 minutes, and the lactic acid bacteria lysate was centrifuged (4,000 rpm, 10 min) to recover only the final supernatant. The recovered supernatant was finally filtered through a 0.45 um filter after chilling at 4 degrees. After filtration and storage for 1 week in refrigeration was confirmed the state.

Comparative Example 1: Preparation of Lactic Acid Bacteria Crushed Material

Inoculate the intestinal lactic acid bacteria to 3% (v / v) using MRS liquid medium, incubate at 37 ° C for 15 ~ 24 hours, centrifuge (4,000 rpm, 10 min), and recover only the cells and wash them twice with sterile distilled water. were washed and suspended in final sterile distilled water. Lactic acid bacteria suspension was 121 ℃, 0.15 MPa, cells were disrupted for 30 minutes and the lactic acid bacteria lysate was centrifuged (4,000 rpm, 10 min) to recover only the supernatant. Figure 3 is a photograph showing the lactic acid bacteria lysate raw material prepared by a conventional method.

Comparative Example 2: Preparation of Lactobacillus Nucleotide

Inoculate the intestinal lactic acid bacteria to 3% (v / v) using MRS liquid medium, incubate at 37 ° C for 15 ~ 24 hours, centrifuge (4,000 rpm, 10 min), and recover only the cells and wash them twice with sterile distilled water. were washed and suspended in final sterile distilled water. The recovered suspended cells were post-cultured at 37 ° C. for 15-24 hours using a limiting medium made with only 1% glucose (w / v). After incubation, cells were crushed at 121 degrees, 0.15 MPa and 30 minutes, and the lactic acid bacteria lysate was centrifuged (4,000 rpm, 10 min) to recover only the final supernatant. The recovered supernatant was finally filtered through a 0.45 um filter after chilling at 4 degrees. After filtration and storage for 1 week in refrigeration was confirmed the state.

Experimental Example 1 Selection of Lactic Acid Bacterial Nucleotide Inclusion Optimal Carrier

In order to maintain the stability of the nucleotides of the lactic acid bacteria in the cell after elution and elution to maintain an environment similar to the environment in the cytoplasm was selected as the optimum carrier (carrier) in the polymer functional biological material derived from microorganisms. For this purpose, collagen was used as a carrier based on hyaluronic acid, beta glucan, and protein as the polymer polysaccharide-based carrier, and the abnormal odor (odor) generated during low molecular conversion in the polymer under conditions that elute the lactic acid bacteria nucleotides was used. It was evaluated as a sensory element [Table 1]. In the process of separating nucleotides of lactic acid bacteria, heat and pressure under certain conditions are applied, and the polymer polysaccharide component reacts with the structural protein constituting the cell wall of lactic acid bacteria, resulting in a millard reaction that becomes a caramel sugar, resulting in a dark or irreversible color of the fermentation broth. Glycoprotein (denatured protein), etc. due to the opaque and cloudy phenomena, which causes abnormal fermentation odor. Therefore, the cooling process was observed for one week after the thermo-pressure process in the carrier to confirm that the group was not cloudy and kept transparent and that no abnormal fermentation odor occurred was a stable carrier that did not induce glycoprotein components. It is indicated as +++ when it becomes cloudy in the colloidal form and has an abnormal fermentation odor, and a glycoprotein precipitate is formed and sinks to the bottom, and a precipitate is formed and sinks to the bottom and has an abnormal fermentation odor. Glycoprotein precipitate did not occur, was transparent, and no abnormal fermentation odor was indicated by +. The culturing process was performed in the same manner as in <Example 1>, and the polymer polysaccharide-based solution was used as the final suspension in the step of lactic acid bacteria cell disruption, and the analysis was then performed in the same manner.

Polymer Biomaterials Denatured protein
Creation Fermentation odor generation
Whether Example 1 (hyaluronic acid) + + Example 2 (betaglucan) + + Example 3 Collagen + ++ Comparative Example 1 +++ +++

Experimental Example 2: Evaluation of the Stability of Lactic Acid Bacteria Nucleotide Due to Inclusion Carrier

In the functional biological material used in Experimental Example 1, hyaluronic acid, which is skin-friendly and favorable to the manufacturing process, was used to evaluate the safety of nucleotides of lactic acid bacteria due to a carrier. According to Example 1, produced by pre-fermentation and post-fermentation (carrier oil). UV wavelength absorption of the lactic acid bacteria nucleotides was evaluated UV absorption of the lactic acid bacteria nucleotides produced by pre-fermentation and post-fermentation (carrier free) according to Comparative Example 2. As a result, as shown in Figure 4a, b, it was confirmed that the UV wavelength absorbance of Example 1 is higher than the UV wavelength absorbance of Comparative Example 2. This indicates that the carrier does not degrade the lactic acid bacteria nucleotides and remains stable for a long time.

Experimental Example 3: Nucleic Acid Analysis of Lactobacillus Nucleotide Entrapped with Carrier

Nucleic acid of the lactic acid bacterium nucleotide contained in the hyaluronic acid which is skin-friendly and favorable to a manufacturing process was analyzed among the polymer biological material derived from microorganisms selected from Experimental Example 1. Take 5 ml of the sample, add 1 ml of 5 mM ZnCl2 and 1.5 ml of 50 mM sodium acetate (pH5.5), add 100 μl of Nuclease P1 (136 ppm in 0.1 M ammonium acetate pH 4.50), and then heat the enzyme at 60 ° C for 2 hours. After disassembly and cooling. Thereafter, the resultant was filtered with a 0.45 μm filter and used as a test solution. DAMP, dCMP, dGMP, dTMP were identified using HPLC equipped with a Luna C18 (3 μm, 4.6 mm * 150 mm) column. Mobile phase using solvent A (DW 1000ml + TBA-OH (40%) 5ml + H3PO4 (85%) 1ml) and solvent B: 80% MeOH + TBA-OH (40%) 5ml + H3PO4 (85%) 1ml) The detector was a UV detector. Figure 5 is a photograph showing the elution of lactic acid bacteria nucleotides through nucleic acid analysis. Figure 5a is a chromatogram of nucleic acid standard, Figure 5b is a chromatogram of lactic acid bacteria nucleotides.

Experimental Example 4: Confirmation of Low Molecular Weight of Polymeric Biomaterials through Post-fermentation

Based on the polysaccharide hyaluronic acid among the polymer biomaterials selected from Experimental Example 1, the low molecular weight of the polymer biopolymers was confirmed. The molecular weight of hyaluronic acid was measured using a high molecular weight hyaluronic acid raw material (FIG. 6A) and a low molecular weight hyaluronic acid and a lactic acid bacterium entrapped (FIG. 6B) as a sample, which were the result of Example 1 including the post-fermentation process. . Low molecular weight: TOSOH G3000PWXL, polymer: molecular weight was measured using GPC (gel permeation chromatography) equipped with Viscotek GMPWxl column. The solvent was a low molecular weight: DW: Acetic acid (3930: 70), a polymer: 200mM NaCl (in 0.1% Sodium Azide), RI detector was used. 6 is a photograph showing the low molecular weight of the polymeric biomaterial. FIG. 6A is a chromatogram of a polymeric biomaterial prior to low molecular weight, and FIG. 6B is a chromatogram of a biomolecule being low molecular weight. The molecular weight (Mw) of the high molecular weight hyaluronic acid before the low molecular weight was 1.385 x 10 ⁶ Daltons, it can be seen from the present invention that the molecular weight (Mw) of the low molecular weight hyaluronic acid is 422 daltons.

Although the present invention has been described and described with reference to the above embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. . Therefore, the true technical protection scope of the present invention will be defined by the technical details of the appended claims.

Claims (8)

A method of preparing a complex of low molecular weight functional biomaterials and lactic acid bacteria nucleotides selected from the group consisting of hyaluronic acid, beta glucan, or collagen, comprising the following steps:
(a) a pre-fermentation step of propagating lactic acid bacteria in a nutrient medium containing a carbon source, nitrogen source, vitamins and inorganic salts;
(B) the post-fermentation step of separating the lactic acid bacteria grown in the step (a), incubated in a limited medium consisting of glucose and water with a polymeric functional biomaterial selected from the group consisting of hyaluronic acid, beta glucan, or collagen;
(c) subjecting the lactic acid bacteria post-fermentation solution of step (b) to thermal compression conditions so that the lactic acid bacteria cells are crushed to elute the lactic acid bacteria nucleotides;
(d) removing the cell wall and cytosolic protein components from the resultant fermentation broth obtained in step (c), and recovering a complex of a low molecular weight functional biomaterial and a lactic acid bacterium nucleotide having a lower molecular weight than the polymer functional biomaterial of step (b). Steps.
delete According to claim 1, wherein the lactic acid bacteria of step (a) is Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp. , Enterococcus sp, Pediococcus sp, Bisella sp. (Weissella sp) is a method for producing a complex of low molecular weight functional biomaterials and lactic acid bacteria nucleotides, characterized in that at least one selected from the group consisting of.
delete delete According to claim 1, wherein the thermal compression conditions of step (c) is a low molecular weight functional biomaterial and the production of a complex of lactic acid bacteria nucleotides, characterized in that the temperature of 80-130 degrees ( ^o C) and pressure conditions of 0.01 ~ 0.3MPa Way.
According to claim 1, wherein the step (d) in the step of centrifugation to remove the pellet containing the cell wall and the cytoplasmic protein by cooling and filtration characterized in that the manufacturing method of the complex of low molecular weight functional biomaterials and lactic acid bacteria nucleotides. .
A low molecular weight functional biomaterial selected from the group consisting of hyaluronic acid (hyaluronic acid), beta glucan, or collagen (collagen) prepared by the method according to claim 1, a complex of lactic acid bacteria nucleotides.

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