KR20100053271A - The making method of l. mesenterioids having increased immunity function - Google Patents

Abstract

PURPOSE: A method for producing Berberis koreana extract with enhanced immune activity is provided to prevent denaturation and damage of an ingredient, to improve extraction yield, and to release a novel material. CONSTITUTION: A method for producing Berberis koreana extract with enhanced immune activity comprises: a process of mixing sterilized Berberis koreana and distilled water in a ratio of 1:1; a process of inoculating lactobacillus to a homogenized sample; a process of fermenting under the anaerobic condition for 60-80 hours; a process of performing hot water extraction at 40-60 degree for 24 hours; and a process of extracting at 100-000bar for 5 to 15 minutes twice. The lactobacillus is B. longum B6, L. acidophius, L. brevis, L. casei, L. mesemferioids, L. paracasei, L. rhamnesus, or P. acidococcus.

Description

Production method of L. mesenterioids having increased immunity function

The present invention relates to an efficient extraction method of the functional components of the barberry, which is a natural product using a complex fermentation process, and an extract having useful physiological activity.

 Fermented foods are nutritionally and functionally superior to those that are not fermented. Alcohol and organic acids produced by fermentation are known to inhibit food storage, quality improvement, and growth of pathogens. When fermentation, the metabolism of microorganisms, the organic substances of the polymer are converted into low-molecular components to increase the digestive absorption rate when used as food or medicine. As described above, polysaccharides produced by microorganisms are widely used in food, pharmaceutical, and biological industries due to their physical properties and various functionalities.

In the present invention, by introducing a fermentation process to maximize the extraction of the useful active ingredient of the wood part of the bark of the barberry to increase the yield of the useful active ingredient and to increase the immune activity through the production of new substances. The fungi used for fermentation of barberry are B. longum B6, L. acidophius, L. brevis, L. casei, L. mesemferioids, L. paracasei, L. rhamnosus, and P. acidococcus . Among the 8 strains, excellent strains were selected for fermentation of barberry by evaluating the resolution and yield of the digested products, and the fermentation conditions of barberry were optimized through the fermentation process using the selected bacteria.

 In addition, in order to prevent the destruction and loss of useful physiologically active components of barberry and to increase the elution of the active ingredient in the wood part, which is difficult to obtain by the existing process, an extreme extraction process capable of improving the yield and enhancing the effective activity at low temperatures was introduced. Optimizing extraction conditions by introducing ultra-high pressure extraction process that can increase the yield of intracellular components and induce component denaturation through cell wall destruction, which impedes the extraction of wood-based components among plants by applying 500 bar and 1500 bar ultra high pressure, respectively. Was performed

 In addition, although tree resources have various physiological activities as natural products, there is almost no food or pharmacological use. Among them, barberry has been used as a medicine for pain relief, but modern research is insignificant, and there are no studies to verify its efficacy. In particular, in the existing extraction method, the wood resources are hard and the extraction yield is low, so the use of active materials is limited to bark. Therefore, there is a need to improve the development of useful bioactive materials derived from natural products from barberry. There is a need for a base technology process to identify the biological activity and use it as a functional food material.

Since useful components of natural products that are expected to be physiologically active exist in a chemically bonded state, they are usually pretreated by hot water extraction method, Soxhlet method, high temperature solvent extraction method or mechanical compression method. Extraction using these conventional methods is difficult to drastically increase the useful physiological activity due to heat instability, solvent toxicity, low solubility, etc. It has a limit of falling sharply.

The term probiotics is used against antibiotics and means' live microorganisms (lactic acid bacteria and other bacteria, or dried or which may have a beneficial effect on the health of the host by improving the host's intestinal flora). Yeast in yeast products). Lactic acid bacteria such as Lactobacillus, Bifidobacterium, Lactococcus are widely used as microorganisms that are used as probiotics, and Leuconostoc, Pediococcus, Bacillus, Propionibacterium and yeast are mainly used in animals. In particular, probiotic lactic acid bacteria have beneficial effects such as maintaining microbial flora of normal intestinal and urogenital organs, alleviating lactose intolerance, reducing blood cholesterol, anticancer activity, immune enhancement, and enhancing the nutritional value of food and microbial contamination of urogenital organs. Prevention, relief of constipation, prevention of traveler's diarrhea, prevention of infant diarrhea, relief of diarrhea derived from the use of antibiotics, prevention of elevated cholesterol in the blood, prevention of bowel cancer and bladder cancer, prevention of side effects of cirrhosis, effects of hyperacidity, prevention of osteoporosis It is known to have a therapeutic effect. In order for microorganisms to show useful functions as probiotics, a large number of ingested bacteria must pass through the human digestive system and adhere to the intestine. Therefore, in order to be used as probiotics, lactic acid bacteria are essential for the stability of stomach and bile acids of the human intestine. Must be retained. On the other hand, resistance to bile plays an important role in the survival and functionality of probiotics reaching the small intestine, which is a characteristic that must be used for probiotics. In addition, as the precondition for lactic acid bacteria to exhibit the function in the intestine, intestinal adhesion and proliferative capacity have been considered important.

Since useful components of natural products that are expected to be physiologically active exist in a chemically bonded state, they are usually pretreated by hot water extraction method, Soxhlet method, high temperature solvent extraction method or mechanical compression method. Extraction using these conventional methods is difficult to drastically increase the useful physiological activity due to heat instability, solvent toxicity, low solubility, etc. It has a limit of falling sharply.

 The parallel treatment of cold extraction and ultrahigh pressure extraction can achieve the desired useful components without destroying or losing them. Ultra-high pressure extraction has been widely used for the use of useful ingredients in recent years because it is possible to elute without loss or denaturation of useful ingredients, flavors, pigments, essential oils, etc. in a variety of substances that are expected to be useful physiological activity, including natural products. Ultra-high pressure extraction typically increases the efficiency by breaking the cell walls and tissues by applying pressures of 100,000 psi or more, accelerating the dissolution of components and obtaining high elution in a short time, and weakening such as energy-bound hydrogen bonds, electrical bonds, and van der Waals bonds. The bonds separate to allow for new material elution.

Although tree resources have various physiological activities as natural products, there is almost no food or pharmacological use. Among them, barberry has been used as a medicine for pain relief, but modern research is insignificant, and there are no studies to verify its efficacy. In particular, in the existing extraction method, the wood resources are hard and the extraction yield is low, so the use of active materials is limited to bark. Therefore, there is a need to improve the development of useful bioactive materials derived from natural products from barberry. There is a need for a base technology process to identify the biological activity and use it as a functional food material.

The present invention by using the fermentation, ultra-high pressure extraction at the same time to obtain a high yield of useful bioactive substances of barberry compared to the conventional extraction and at the same time confirmed the effective function of the immune active part. The present invention provides a fermentation metabolic engineering design through the selection of strains suitable for fermentation of barberry and the use of appropriate strains among the bacteria currently recognized as GRAS (Generally Recognized As Safe) that can be used in food for the purpose of differentiation from simple fermentation process. We wanted to optimize it. To achieve the fermentation optimization of barberry extract and to increase the yield of the active ingredient to facilitate the removal of the bark by loosening the wood-based tissue was hard to elute the components through the fermentation process. In addition, it increases the shelf life of the extract through fermentation, and seems to be possible to improve the productivity of a new functional material that has not been obtained in the past.

  The ultra-high pressure extraction process solves the problems of the simple extraction process and at the same time has the advantage of showing a relatively high physiological activity through higher yield improvement and active ingredient elution, as well as the advantage of less change of components due to temperature because it proceeds at low temperature. The application of this extraction process is highly valuable in the extraction of functional food materials.

 Therefore, in the present invention, in order to overcome the limitation of decreasing the bioavailability of the conventional simple methods of extracting the biologically active substance, the introduction of low-temperature extraction, ultra-high pressure extraction and fermentation process technology as an efficient extraction process of immune active components of barberry It aims to improve competitiveness and industrialization by enhancing immune activity, improving functionality and enhancing biocompatibility.

 In order to achieve the above object, the method for preparing an extract of barberry extract with enhanced immune function of the present invention is a step of mixing and homogenizing the sterilized barberry and distilled water at a ratio of 1: 1, inoculating the lactic acid bacteria to the homogenized sample. Fermentation under 35-38 anaerobic conditions for 60-80 hours, and extracting the fermented product hot water at 40-60 for 24 hours, followed by ultra-high pressure extraction twice at 100 bar to 4000 bar for 5 minutes to 10 minutes. It is characterized by including.

In addition, the lactic acid bacteria in the present invention is characterized in that one strain selected from strains of B. longum B6, L. acidophius, L. brevis, L. casei, L. mesemferioids, L. paracasei, L. rhamnesus, and P. acidococcus Doing. In the present invention, by selecting excellent strains suitable for fermentation for maximizing the extraction and useful activity of the wood-based active ingredients through the barberry, the growth conditions and the active substances produced were evaluated and the genetic stability was evaluated. As a result, the fermentation process using the fungus with excellent screening function optimizes the fermentation conditions of the barberry, and the tissue is hard, so that the wood-based tissue that was difficult to elute was softened, thus increasing the yield of the active ingredient and extracting new substance. In this way, it was possible to produce a new functional material that has not been conventionally obtained from barberry.

In addition, the present invention improves the stability of the extraction component by extracting at a low temperature of 40 ~ 60, which is lower than the conventional simple hot water extraction and reduced the possibility of toxicity by using water as the extraction solvent. Then, ultra high pressure extraction was performed for 10 and 15 minutes at 500 bar and 1500 bar, respectively, using an ultra high pressure extraction device (Ilshin autoclave, Korea). Subsequently, the obtained extract was centrifuged to separate only the supernatant, fractionated with butanol, and only the material obtained in the water layer was used for verifying immune activity.

 Wood resources have a hard structure in the wood part, which limits their elution and extraction yield. In particular, the barberry tree shows excellent performance in pain relief, but no modern studies have been conducted. Extraction process establishment using complex fermentation technology through the present invention is to improve the extraction yield and extract stability by complementing the disadvantages such as denaturation and destruction of the components of the conventional extraction process, low yield, etc. By showing the effect of enhancing the immune activity through the useful ingredients and the dissolution of new substances, it is possible to create new added value as a functional food material using many activities of barberry.

 Hereinafter, the present invention will be described in detail by process, and will be described in detail by Examples and Experimental Examples.

Step 1: Homogenization Process]

For fermentation, homogenized by mixing the wood parts of the barberry sterilized with background bacteria and distilled water in a weight ratio of 1: 1.

[Step 2: Fermentation Process]

Probiotic Preferably, one of the strains of B. longum B6, L. acidophius, L. brevis, L. casei, L. mesemferioids, L. paracasei, L. rhamnesus, and P. acidococcus proceeds to the fermentation process. It was. Cultures of the lactic acid strains used in the experiment were cultured under 35-38 anaerobic conditions using medium containing 0.05% L-cysteineHCl in de Man, Rogosa and Sharpe (MRS) broth. Lactobacillus was inoculated on the barberry samples prepared in the second step and fermented under 37 anaerobic conditions for 60-80 hours.

[3rd process: low temperature extraction and ultra high pressure extraction process]

 The fermented product obtained in the second process is extracted with hot water at a temperature of 40 to 60 for 24 hours in a container for high pressure extraction, and then compressed at 100 bar to 4000 bar by compression of gas or liquid phase using an ultra high pressure extraction device (Ilshin autoclave, Korea). Extract ultra-high pressure twice in 5 ~ 15 minutes. In the present invention, water was used as a solvent in order to increase the stability of the extraction component and reduce the possibility of toxicity. In order to prevent the denaturation of the sample due to the heat generated during the ultra-high pressure extraction, it is recommended to use a liquid with a low compression ratio. Thereafter, the mixture was decompressed and the ultrahigh pressure-treated solvent and sample were taken out, and the mixture was separated by filtration and fractionation to obtain an extract.

[Step 4: Evaluation of Activity of Fermented Extract]

Antioxidative and anticancer activities were measured using fermented extracts according to strains to confirm the physiological activity of the barberry fermented extract.

Example 1 Selection of strains suitable for fermentation

 In order to optimize the fermentation conditions of barberry, excellent fermentation strains were selected for the fermentation of wood. Among Lactobacillus strains containing 8 kinds of Lactobacillus strains, Bifidobacterium longum B6, Lactobacillus acidophilus LA-2, Lactobacillus brevis, Lactobacillus casei, Leuconostoc mesenteroides, Lactobacillus paracasei, Lactobacillus rhamnosus GG and Pediococcus acidilactici. Growth and pH changes were measured to select strains.

To examine the growth, the initial probiotic bacteria were inoculated at a concentration of about 10 ⁵ CFU / mL, and then grown for 96 hours under 37 anaerobic conditions, the growth rate was measured using an MRS agar plate. In order to confirm the growth of inoculated strains after incubation, the extract pH of fermented barberry was measured.

 As culture medium of the lactic acid strain used in the experiment, MRS broth containing 0.05% L-cysteine-HCl added to MRS broth (Lactobacilli MRS, DifcoTM, Detroit, MI, USA) was used.

1 and 2 show the results of measuring the growth of strains for selection of fermented strains. All probiotic bacteria showed good growth under all conditions by the addition of barberry extract. Most strains showed high growth of 8 log CFU / mL after 96 hours of fermentation at the initial 5 log CFU / mL. The pH of all strains except L. brevis and L. mesenterioids were lowered, and the growth of the fermented strains was confirmed by lowering the pH of the fermentation extract from pH 5 to below 4.

Therefore, it can be confirmed that both lactic acid bacteria are suitable for barberry fermentation, it is preferable to use the eight kinds of lactic acid bacteria verified in the experiment in the fermentation process.

Example 2 (Preparation of plum tree extract of the present invention).

The barberry tree part sterilized at 121 for 30 minutes and distilled water was mixed and homogenized in a weight ratio of 1: 1. Homogenized barberry samples were inoculated with B. longum B6, L. acidophius, L. brevis, L. casei, L. mesemferioids, L. paracasei, L. rhamnesus, and P. acidococcus. Fermentation was carried out for 72 hours under miracle conditions. The obtained fermented product was extracted with hot water for 24 hours at a temperature of 40 to 60 and then placed in a container for high pressure extraction, using a high pressure extraction device (Ilshin autoclave, Korea) to compress the gas or liquid phase continuously at 500 bar and 1500 bar, respectively. Apply pressure for 15 minutes.

Experimental Example 1.

In order to observe the internal structure of the barberry samples, EF-TEM (Transmission Electron Microscipy) is used. To investigate the characteristics of the ultra high pressure treated material which shows the effect of increasing the extraction yield and the activity of the activity, staining the barberry samples through the ultra high pressure treatment process by negative staining and constructing with EF-TEM (Transmission Electron Microscipy) And shape were observed. By the method of Example 2, the bark of the plum bark treated with the ultra-high pressure of the bark of plum and the bark of the extract of hot water at 60 ° C. for 24 hours are dyed in a solution of phosphotunhstic acid (pH 6.8, 0.1%) for 1 hour. And thinly placed on a copper grid coated with a foam (formvar) and a carbon film (carbon film) (dry) for 24 hours to remove all moisture. After EF-TEM, the shape was observed at 120kV.

The organization observation results of the barberry samples through the ultra-high pressure treatment process using the TEM (electron transmission microscope) are shown in FIG. As a result, it can be seen that the tissue of barberry samples (a) extracted from hot water for 60 24 hours still remains large. On the other hand, the bark samples treated with ultra high pressure can be found to be broken into small pieces. This is due to the change in the structure and structure of the cell wall of the barberry samples through the ultra-high pressure treatment, it is believed that the increase in extraction yield and the elution of the active substance was made.

Experimental Example 2.

1,1-diphenyl-2-picryl-hydrazyl (DPPH), used for measuring electron donating ability, is a stable free radical, exhibiting a maximum absorption near 517 due to its non-covalent electrons, and absorbance near 517 upon receipt of electrons or hydrogen. In addition, if each extract has a high ability to reduce or cancel such radicals, high antioxidant activity and scavenging activity against other radicals including active oxygen can be expected and can be used as a measure of inhibiting aging by active radicals in the human body. . The results of comparing the DPPH scavenging activity of the barberry fermentation extract of Example 2 by concentration are shown in FIG. 4. All extracts showed activity, and extracts fermented by B. longum B6 and L. paracasei showed relatively high activity.

Experimental Example 3.

SRB method for the growth of cells according to the addition of five barberry fermentation extracts of 20, 40, 60, 80, 100 g / L using the human lung cancer cell line A549 to determine the cancer cell growth inhibitory activity of the barberry extract of Example 2 Measured through and the results are shown in FIG. As can be seen from the results, all the extracts showed the inhibitory effect on the cancer cells, especially the extracts from the fermentation strains of B. longum B6, L. brevis, P. acidococcus showed very high anticancer activity.

1 is a graph measuring the growth of fermented strains through fermentation (37, 96 hours) of barberry extract.

Figure 2 is a graph of pH change measurement during fermentation strains of barberry extract (37, 96 hours).

Figure 3 is a tissue TEM observation picture of the barberry samples according to the extreme treatment process.

Figure 4 is a graph of the electron donating ability for DPPH radical according to the addition of barberry fermentation extract.

Figure 5 is a graph of the anticancer activity measurement of human lung cancer cells (A549) of the barberry fermentation extract by fermentation strains.

Claims (2)

  Mixing and homogenizing the sterilized barberry and distilled water at a ratio of 1: 1 to ferment the lactic acid bacteria to the homogenized sample and fermenting for 60-80 hours under anaerobic conditions at 35-38; After extracting hot water at 40-60 for 24 hours, a method for producing an immune function-enhanced barberry extract, comprising the step of ultra-high pressure extraction twice in 5 minutes to 15 minutes at 100bar ~ 4000bar. The method of claim 1, wherein the lactic acid bacteria is one strain selected from strains of B. longum B6, L. acidophius, L. brevis, L. casei, L. mesemferioids, L. paracasei, L. rhamnesus, and P. acidococcus Method of producing a barberry extract with enhanced immune function.

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