CN103173379A - Novel microbe for red ginseng fermentation, red ginseng fermentation liquid using the microbe and red ginseng fermentation drink - Google Patents
Novel microbe for red ginseng fermentation, red ginseng fermentation liquid using the microbe and red ginseng fermentation drink Download PDFInfo
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- CN103173379A CN103173379A CN2012105681132A CN201210568113A CN103173379A CN 103173379 A CN103173379 A CN 103173379A CN 2012105681132 A CN2012105681132 A CN 2012105681132A CN 201210568113 A CN201210568113 A CN 201210568113A CN 103173379 A CN103173379 A CN 103173379A
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- red ginseng
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/60—Sweeteners
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
Abstract
The invention relates to a novel microbe for red ginseng fermentation, a red ginseng fermentation liquid using the microbe and a red ginseng fermentation drink. The invention further relates to a microbe used for red ginseng fermentation and having a sequence number being 8.
Description
Technical field
The present invention relates to a kind of novel red ginseng fermentation and use microorganism, and utilize red ginseng fermented liquid manufacture method and the red ginseng fermented drink of this microorganism.
Background technology
The antifatigue of red ginseng saponin, strengthening immunity, improves the blood texts and be confirmed, and the result of study that continues shows also have anti-anti-ageing, liver function protecting always, improve blood circulation, improve the effects such as dementia and liver function.
especially, main saponin (the saponin) (Rg1 that is difficult to absorb in the saponin of red ginseng, Re, Rb1, Rc, Rb2 and Rd) through adding less important saponin (the saponin) (Rg3 of water decomposition generation, Rh2, Compound-K etc.) reported, has the transfer that hinders tumour cell in experimental mouse, hinder the invasion of tumour cell in vitro, bring out the suicide of cancer cells, the transfer of anticancer, reduce blood pressure, suppress the secretion of catecholamine hormone (catecholamine), activate the remarkable pharmacologically active such as analgesic agent and strengthening immunity.
Pharmacologically active along with the brilliance of less important saponin (saponin) is verified like this, generates very active that the research of less important saponin (saponin) carries out.Main method of attempting has acid to add water decomposition, thermal treatment, enzyme to add the methods such as water decomposition and organic synthesis.Wherein, enzyme adds the water decomposition method can be implemented in low temperature, easy and simple to handle, and because the medium characteristics byproduct of enzyme is considerably less, is to produce the most suitable method of less important saponin (saponin) thus.So far, the research of changing saponin by enzyme mainly concentrates on the microorganism that separates in intestinal bacterium, soil microorganisms and plant tissue.But these methods are not suitable for edible, so aspect practical, many difficulty are arranged still.
In addition, milk-acid bacteria (lactic acid bacteria) refers to generate in a large number the bacterium of lactic acid (lactic acid), utilizes the sugar (glucose) in food to generate lactic acid, not only improves the taste of food, also carry out the acidifying of food, hinder other harmful microorganism and grow up.And; milk-acid bacteria perches in the intestinal tube of various animals; the mucous membrane of protection in alimentary canal, improve the absorption etc. of the synthetic and calcium of abnormal fermentation in intestines, promoting digestion and absorption, promotion VITAMIN, so can use when the less important saponin of generation (saponin) from the main saponin (saponin) of red ginseng.
The cultivation of these milk-acid bacterias all can be trained base by the commercial Pei Ji of the typical milk-acid bacteria of usage comparison-MRS usually, but these commercial training bases are on its composition or chemical process treatment process, can't learn and all use which material, passed through which chemical treatment, so be not suitable for eating.As an example, the Korean registered patent has been put down in writing the microorganism culturing that comprises Tryptones, soya peptone, peptone etc. for No. 0680318 and has been trained the content of base with nutrition.But, the process of above-mentioned manufacturing Tryptones, soya peptone, peptone etc. is very complicated, and also having comprised the harmful chemical treating process of human body of processing the strong-base groups such as sodium hydroxide in manufacturing processed, the microorganism of cultivating in such training base can't be directly used in edible.
Therefore, for the main saponin (saponin) from red ginseng generates less important saponin (saponin), need the lactic bacterium strains of development of new and utilize this bacterial strain the red ginseng fermented liquid manufacture method and can be directly used in ediblely, adopt the milk-acid bacteria of harmless material to cultivate with the training base.
Summary of the invention
The object of the present invention is to provide a kind of novel red ginseng fermentation that promotes the efficiency of conversion of functional less important saponin (saponin) contained in the red ginseng extraction liquid to use microorganism.
The object of the present invention is to provide a kind of red ginseng fermented liquid manufacture method of utilizing mentioned microorganism.
And, the object of the present invention is to provide a kind of content of the functional less important saponin (saponin) of red ginseng extraction liquid that makes to maximize, have immunocompetence and antitumour activity, the red ginseng fermented drink that can take in safely.
Technical scheme
Use microorganism in order to realize above-mentioned purpose, the invention provides the red ginseng fermentation with sequence numbering 8 sequences.
According to an embodiment of the invention, mentioned microorganism presents, the Rg1 that red ginseng is contained, Re, Rb1, Rc, in Rb2 and Rd, more than one main saponin (saponin) converts F2 to, Rg3, the characteristic of the less important saponin more than (saponin) in Rh2 and Compound-K.
According to an embodiment of the invention, mentioned microorganism present with the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillus paracasei subsp. paracasei) homogeny of base sequence more than 99%.
According to an embodiment of the invention, mentioned microorganism has the sustenance numbering of KCTC12108BP.
The invention provides, add mentioned microorganism to red ginseng fermented liquid manufacture method that the red ginseng extraction liquid ferments.
According to an embodiment of the invention, also comprise, also add the stage that enzyme is processed after above-mentioned fermentation stage.
According to an embodiment of the invention, can add sucrose (sucrose) or salt (NaCl) during above-mentioned fermentation.
According to an embodiment of the invention, the microbial culture medium that uses during above-mentioned fermentation is that the more than one vegetables of selecting from the group that Chinese cabbage, radish, circle romaine lettuce, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, banana, potato and romaine lettuce form are made.
The invention provides, contain the immunostimulant red ginseng fermented drink of the red ginseng fermented liquid of above-mentioned manufacturing.
And, the invention provides, contain the alleviation cancer of red ginseng fermented liquid of above-mentioned manufacturing and the red ginseng fermented drink of preventing cancer.
Beneficial effect
Novel red ginseng fermentation of the present invention has with microorganism, converts the characteristic of less important saponin (saponin) to the very high efficient main saponin (saponin) that red ginseng is contained, improves thus the saponin assimilated efficiency in body.
And, red ginseng fermented liquid manufacture method of the present invention is optimized novel red ginseng fermentation of the present invention with the yeasting of microorganism, the efficiency of conversion of less important saponin (saponin) is promoted 2 ~ 3 times, and use edible microorganism culturing training base, guarantee food safety, save cost.And red ginseng fermented drink of the present invention has very excellent immunocompetence and antitumour activity (alleviating cancer and anticancer effect).
Description of drawings
Fig. 1 is the WJ-1 according to the inventive method screening, 2,4,6,23, the 16S DNA base sequence of 33 fermentation strains, Fig. 1 a are that WJ-1 bacterial strain, Fig. 1 b are that WJ-2 bacterial strain, Fig. 1 c are that WJ-4 bacterial strain, Fig. 1 d are that WJ-6 bacterial strain, Fig. 1 e are that WJ-23 bacterial strain, Fig. 1 f are the 16S rDNA base sequence of WJ-33 bacterial strain.
Fig. 2 is the TLC chromatography observations figure according to less important saponin (saponin) transfer capability of the fermentation strain of the inventive method screening.
Fig. 2 a is WJ-1, in the red ginseng extraction liquid of 2,4 and 6 bacterial strains, main saponin (saponin) is converted to design sketch (No. 1 swimming lane: WJ-1 of less important saponin (saponin), No. 2 swimming lane: WJ-2, No. 3 swimming lane: WJ-3, No. 4 swimming lane: WJ-4, No. 5 swimming lanes: standard, No. 6 swimming lane: WJ-5, No. 7 swimming lane: WJ-6, No. 8 swimming lane: WJ-8, No. 9 swimming lanes: WJ-9).
Fig. 2 b is WJ-23, in the red ginseng extraction liquid of WJ-33 bacterial strain, main saponin (saponin) is converted to design sketch (No. 1 swimming lane: WJ-32 of less important saponin (saponin), No. 2 swimming lane: WJ-33, No. 3 swimming lane: WJ-34, No. 4 swimming lane: WJ-35, No. 5 swimming lanes: standard, No. 6 swimming lanes: WJ-36).
Fig. 3 utilizes in the fermenting process of WJ-33 bacterial strain, be converted to (No. 1 swimming lane: WJ-32 of figure as a result of less important saponin (saponin) when adding respectively sucrose or salt, No. 2 swimming lane: WJ-33, No. 3 swimming lane: WJ-34, No. 4 swimming lane: WJ-35, No. 5 swimming lanes: standard, No. 6 swimming lanes: WJ-36).
Fig. 4 is for using simultaneously according to the fermentation strain of the inventive method screening and during as the sucrose (sucrose) of inducible factor or salt (NaCl), being converted to the efficiency of conversion observations of less important saponin (saponin).
When Fig. 4 a processes for together adding WJ-2 or WJ-4 bacterial strain and sucrose (sucrose) to the red ginseng extraction liquid, generate observation figure (No. 1 swimming lane: WJ-1 of less important saponin (saponin), No. 2 swimming lane: WJ-2, No. 3 swimming lane: WJ-3, No. 4 swimming lane: WJ-4, No. 5 swimming lanes: standard, No. 6 swimming lane: WJ-5, No. 7 swimming lane: WJ-6, No. 8 swimming lane: WJ-8, No. 9 swimming lanes: WJ-9).
When 4b processes for together adding WJ-2 or WJ-4 bacterial strain and salt (NaCl) to the red ginseng extraction liquid, generate observation figure (No. 1 swimming lane: WJ-1 of less important saponin (saponin), No. 3 swimming lane: WJ-3 of No. 2 swimming lane: WJ-2, No. 4 swimming lane: WJ-4, No. 5 swimming lanes: standard, No. 6 swimming lane: WJ-5, No. 7 swimming lane: WJ-6, No. 8 swimming lane: WJ-8, No. 9 swimming lanes: WJ-9).
Fig. 5 is for after utilizing the fermentation strain that screens according to the inventive method to ferment, and when together using the enzyme of sieving, the efficient that is converted to less important saponin (saponin) is observed figure (different swimming lanes adopt different % inoculate and react).
Fig. 6 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the NO growing amount of fermented red ginseng extraction liquid (" red ginseng ") confirm result.
Fig. 7 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the cells survival rate of fermented red ginseng extraction liquid (" red ginseng ") confirm figure as a result.
Fig. 8 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the TNF-α discovery amount of fermented red ginseng extraction liquid (" red ginseng ") confirm figure as a result.
Fig. 9 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the Sargassum fusiforme extract phagolysis of fermented red ginseng extraction liquid (" red ginseng ") confirm result.
(wild-type; DMEM substratum (10% FBS), P-CON; Zymosan (Zymosan),
I-CON; DMEM substratum (10% FBS)+inhibitor (2 μ M/24hr)+zymosan (zymosan),
Red ginseng; Fermented red ginseng extraction liquid+zymosan (zymosan) not,
Fermented red ginseng; Red ginseng fermented liquid+zymosan of the present invention (zymosan))
Figure 10 be oral red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the spleen cell multiplication capacity of the experimental mouse of fermented red ginseng extraction liquid (" red ginseng ") confirm figure as a result.
Figure 11 be oral red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the TNF-α in the experimental mouse peritoneal macrophage of fermented red ginseng extraction liquid (" red ginseng ") find to confirm figure as a result.
Figure 12 be oral red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the IL-1 β in the experimental mouse peritoneal macrophage of fermented red ginseng extraction liquid (" red ginseng ") find to confirm figure as a result.
Figure 13 be oral red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not in the experimental mouse peritoneal macrophage of fermented red ginseng extraction liquid (" red ginseng ") IL-6 find to confirm figure as a result.
Figure 14 be oral red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the Compound K content in the experimental mouse blood plasma of fermented red ginseng extraction liquid (" red ginseng ") confirm figure as a result.
Figure 15 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not the activity of the inhibition proliferation of lung cancer cells of fermented red ginseng extraction liquid (" red ginseng ") confirm figure as a result.
Figure 16 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not fermented red ginseng extraction liquid (" the red ginseng ") activity that suppresses proliferation of human gastric cancer cell confirm result.
Figure 17 be red ginseng fermented liquid of the present invention (" fermented red ginseng ") and not fermented red ginseng extraction liquid (" the red ginseng ") activity that suppresses colorectal cancer cells confirm figure as a result.
Figure 18 is for to add respectively in the training base of vegetalitas material by kind, cultivate the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) after, the measurement result figure of OD value.
Figure 19 adds respectively in the training base of sugar, the secondary cheese subspecies of cultivation lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) after, the measuring result figure of OD value.
In the training base of Figure 20 for the salinity that adds respectively different concns and nutritive ingredient, cultivate the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) after, the measuring result figure of OD value.
Figure 21 is for to add in the training base of vegetalitas material again, cultivate the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) after, the measuring result figure of OD value.
Figure 22 adds for mixing in the training base of vegetalitas material, the secondary cheese subspecies of cultivation lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) after 24 hours, the measuring result figure of OD value.
Embodiment
The below introduces the specific embodiment of the present invention in detail.
The present invention relates to a kind of novel microorganism and manufacture method and the red ginseng fermented drink that utilizes the red ginseng fermented liquid of this microorganism.
Novel microorganism of the present invention has, and main saponin (saponin) Efficient Conversion that red ginseng is contained is the characteristic of less important saponin (saponin), can separate from red ginseng rice wine.When using novel microbial fermentation red ginseng of the present invention to make beverage, be rich in less important saponin (saponin), have excellent immunostimulant and anticancer function.
Main saponin of the present invention (saponin) can be in Rg1, Re, Rb1, Rc, Rb2 and Rd more than one, but be not limited to this.Less important saponin of the present invention (saponin) can be in F2, Rg3, Rh2 and Compound-K more than one, but be not limited to this.
Novel microorganism of the present invention can be novel milk-acid bacteria, particularly, with Lactobacillus buchneri (
Lactobacillus buchneri), the class Lactobacillus buchneri (
Lactobacillus parabuchneri), the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillus paracasei subsp. paracasei),
Lactobacillus harbinensisOr yeast enzyme mother (
Saccharomyces cerevisiae CBS405) base sequence present homogeny more than 99%.And, with Lactobacillus buchneri (
Lactobacillus buchneri), the class Lactobacillus buchneri (
Lactobacillus parabuchneri), the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillus paracasei subsp. paracasei),
Lactobacillus harbinensisOr yeast enzyme mother (
Saccharomyces cerevisiae CBS405) compare, present the new bacterial strain of significant less important saponin (saponin) efficiency of conversion.In other words, novel microorganism of the present invention can be to present the characteristic that main saponin (saponin) more than in the Rg1 that red ginseng is contained, Re, Rb1, Rc, Rb2 and Rd is converted to the less important saponin more than (saponin) in F2, Rg3, Rh2 and Compound-K.Especially, novel microorganism of the present invention be with the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillus paracasei subsp. paracasei) present the new bacterial strain of 99% above homogeny.
Particularly, above-mentioned microorganism can be the some sequences that have in sequence numbering 3 to 8, preferably has sequence numbering 8 sequences.And microorganism of the present invention preferably has the sustenance numbering of KCTC12108BP.
And, the present invention relates to a kind of manufacture method of red ginseng fermented liquid, particularly, comprise and add above-mentioned novel microorganism to stage that the red ginseng extraction liquid ferments.According to an embodiment of the invention, above-mentioned microorganism is that the gross weight with the red ginseng extraction liquid is as the criterion with the ratio inoculation of 0.001 ~ 3 % (v/w).
And red ginseng fermented liquid manufacture method of the present invention is that the volume with the red ginseng fermented liquid is as the criterion, and adds sucrose (sucrose) or salt (NaCl) with the ratio of 0.001 ~ 3 % (v/v).
And, after utilizing novel microbial fermentation red ginseng of the present invention, for the transfer capability that makes saponin (saponin) doubles, can add again edible enzyme in above-mentioned red ginseng fermented liquid.Above-mentioned enzyme is not so long as edible getting final product has other restrictions.
On the one hand, the microbial culture medium that uses during above-mentioned fermentation can be the nutrient solution of making from edible material.Above-mentioned edible material can comprise vegetalitas material, sugared source and nutritive ingredient.
Vegetalitas material of the present invention can be in Chinese cabbage, radish, circle romaine lettuce, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, banana, potato and romaine lettuce more than one, the best results of radish wherein.And, as the vegetalitas material can also comprise in radish, Radix Dauci Sativae and banana more than one.The addition of above-mentioned vegetalitas material, microorganism culturing is as the criterion with the gross weight of training based component, and the weight ratio of interpolation 3 ~ 25% is better.This is because various vegetalitas materials mix, and uses as energy source.Above-mentioned vegetalitas material can be that form (type) or the powder morphology (type) with concentrated solution adds.
Sugared source in the present invention can be sucrose (sugar), poly-dextrose (polydextrose), fructose (fructose), in oligose (maltooligo), glucose (glucose), dextrin (dextrin), oligofructose (fructooligo) and lactose (lactose) more than one, relatively good more than in lactose and glucose especially.Above-mentioned sugared source is to be as the criterion with the gross weight of microorganism culturing with the training based component, adds 1 ~ 2.5% weight for well.This is that the growth of milk-acid bacteria is more active because when to a certain degree sugared source is arranged.
Nutritive ingredient in the present invention adopts the female extract of enzyme (yeast extract) for well.The female extract of above-mentioned enzyme is the pure natural additive with compositions such as amino acid and VITAMIN, can buy the female extract of commercial edible enzyme and use.Above-mentioned nutritive ingredient is to be as the criterion with training based component gross weight with microorganism culturing, adds 7 ~ 11% weight for well.
The composition that mentioned microorganism is cultivated with the training base can also comprise salinity.Above-mentioned salinity is Rhodiaphos DKP (Dipotassium phosphate preferably; K
2HPO
4).Above-mentioned salinity is to be as the criterion with the gross weight of microorganism culturing with the training based component, adds 0.01 ~ 0.03% weight for well.
Microorganism culturing of the present invention is to adopt vegetable matter with the training based component, is its feature so can directly eat.Especially, can be used for the cultivation of the microorganism that the manufacturing of leavened food uses.
The present invention relates to a kind of red ginseng fermented drink that contains the red ginseng fermented liquid of above-mentioned manufacturing.Red ginseng fermented drink of the present invention presents very strong enhancing immunity and the effect of alleviation and preventing cancer.
The record content that the manufacture method of above-mentioned red ginseng fermented drink is relevant all is applicable to red ginseng fermented drink of the present invention.
Introduce in detail the present invention below in conjunction with embodiment and experimental example.But following embodiment and experimental example are exemplary, and the present invention can not be confined to following embodiment and experimental example, and more modifications and changes can be arranged.
＜
The acquisition of novel red ginseng fermentation strain and same fixed
The acquisition of the fermentation strain that embodiment 1. is novel
(1) separation and cultivation milk-acid bacteria in rice wine
In order to isolate the required milk-acid bacteria of red ginseng fermentation, from loyal Nan Jin mountain, loyal Nanma field, wide area city, land for growing field crops collected the test portions such as ginseng, red ginseng rice wine and living rice wine (buying in Jin Shan market).After therefrom separating the milk-acid bacteria of bacterial strain more than 100, utilize the aqua sterilisa serial dilution to 10 of red ginseng rice wine
-1~ 10
-5, launch respectively (spreading) 100 in MRS agar plate after, 37 ℃ of culture medium culturing 48 hours.
(β-glucosidase) secretion capacity screens fermentation strain according to beta-glucosidase
Above-mentioned
(1) in the fermentation strain that separates in and cultivate, in order to filter out the bacterial strain with saponin transfer capability, utilization can be verified Vitamin C2 agar (Esculin agar) method of saponin transfer capability, the following beta-glucosidase (secretion of the β-glucosidase) bacterial strain that screened.
Many weeks, (secretion of β-glucosidase) bacterial strain is that (esculetin (structure of the β-glucose that comes off in Vitamin C2) and the ferric ammonium citrate (ferric ammonium citrate) of β-glucose) generate produce reaction, and the gathering ground (colony) on the training base forms the complex body (complex) of black on every side for the β-glucose of cutting Vitamin C2 to beta-glucosidase.That is, the milk-acid bacteria that forms these black complex bodys has the saponin transfer capability, so preferentially screened the beta-glucosidase (secretion of β-glucosidase) bacterial strain.In other words, produce the bacterial strain of black circle when having screened visual inspection around milk-acid bacteria, the name of the bacterial strain of screening is as table 1 like this.
[table 1]
Screen from red ginseng rice wine
Beta-glucosidase (β-glucosidase)The secretion bacterial strain
| Beta-glucosidase (β-glucosidase)The secretion bacterial strain |
| WJ-1 |
| WJ-2 |
| WJ-4 |
| WJ-6 |
| WJ-23 |
| WJ-33 |
The relatively acquisition of bacterial strain of comparative example 1.
Separate in above-mentioned embodiment 1. (1) and cultivate more than 100 in bacterial strain, (result of secretion capacity of β-glucosidase), (bacterial strain of β-glucosidase) has incorporated comparative example into to assessment beta-glucosidase in embodiment 1. (2) will not secrete beta-glucosidase.
The fermentation strain that experimental example 1. is novel same fixed
(1) extraction of DNA
The beta-glucosidase of above-mentioned embodiment 1 (β-glucosidase) extracted chromosomal DNA (chromosomal DNA) in the secretion microorganism.At this moment, adopted CoreOne bacterial DNA to reclaim test kit (Coretech. Co. Ltd. Korea).Particularly, in order to extract chromosomal DNA, the secretion microorganism has once been used in bacterial strain outstanding turbid washing in 1.5 test tubes of the upper pure culture of training base afterwards.Then, utilize 1% sepharose (agarose gel) to confirm by electrophoresis the DNA that extracts.
PCR amplification
For the microorganism to screening in above-mentioned embodiment 1 carries out with fixed, implemented to be take amplification 16S rDNA territory the PCR of purpose.For the PCR reaction, pour into respectively principal mode DNA (100 ng/) 1,9F (100 pmol) (sequence number 1) and 1512R (100 pmol) base fluid each 1, Taq polymerase (5 U/) 0.5, dNTP mix (10 mM each) 4,10 X reaction buffers 10, distilled water 82.5, made the reaction mixture of cumulative volume 100.
PCR reaction is carry out the first denaturation process of five minutes in 94 ℃ after, carry out carrying out in the sex change (denaturation), 60 ℃ of a minute in 94 ℃ one minute annealing (annealing), carry out extension (extension) process of a minute in 72 ℃ after, finally carried out extra amplification (extra-extension) implementation Process of seven minutes in 72 ℃.Afterwards, take care of the environment at 4 ℃ till being used for testing before.
Shown in the required 9F and 1512R base fluid following [table 3] of above-mentioned 16S rDNA territory amplification.After the PCR reaction, the 16S rDNA of amplification utilizes 1% agarose gel to confirm by electrophoresis.(seeing Table 2).
[table 2]
[table 3]
| Sequence | |
| 9F (sequence number 1) | 5'-GAGTTTGATCCTGGCTCAG-3' |
| 1512R (sequence number 2) | 5'-ACGGCTACCTTGTTACGACTT-3' |
(3) refinement of PCR product and sequencing
Above-mentioned PCR product is to adopt PCR purification kit (Bioneer, Korea) to refine DNA.The base sequence of the 16S rDNA of above-mentioned refinement is to adopt ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) and Automatic DNA sequencer (model 3700, Applied Biosystems) to analyze.
The analysis of the systematics of base sequence
Utilize the Blast of NCBI to determine near the WJ-1 that separates in rice wine, the family (strain) of 2,4,6,23 and No. 33 active bacterial strains.And, in order to draw the genealogical tree of isolated strains, utilization is registered in the sequence data (sequence data) of GenBank, investigation is positioned at the 16S rDNA base sequence of the various type strain institute's bacterium near, and utilize Bioedit program (Hall, 1999) and Clustal X program to base sequence carried out sorting (alignment) (Thompson et al., 1997).Follow the tracks of the operation of bacterial strain evolutionary process and adopted Kimura two-parameter model (Kimura, 1983), neighbor-joining method by MEGA 3 Program, determined the beta-glucosidase (position (Kumar et al., 2004) of the secretion bacterial strain of β-glucosidase) on systematics.
Its result, WJ-1 is by 1462bp, and WJ-2 is by 854bp, and WJ-4 is by 848bp, and WJ-6 is by 1478bp, and WJ-23 is by 1461bp, and WJ-33 is that the base by 1460bp consists of the (a-of Fig. 1
f), WJ-1 and Lactobacillus buchneri (Lactobacillus buchneri) present 99.9% homogeny, female (Saccharomyces cerevisiae) CBS405 of WJ-2 and WJ-4 and yeast enzyme presents 99%, WJ-6 and Lactobacillus buchneri (Lactobacillus buchneri) present 99.9% homogeny, and WJ-23 and WJ-33 present 99.4% and 99.6% homogeny (a to f of table 4 and Fig. 1) with the secondary cheese subspecies of lactobacillus paraceasi (Lactobacillus paracasei subsp. Paracasei) respectively.
[table 4]
| Bp | Similar bacterial strain | Homogeny | Sequence number | |
| WJ-1 | 1462bp | Lactobacillus buchneri | 99.9% | 3 |
| WJ-2 | 854bp | The yeast enzyme is female CBS405 | 99% | 4 |
| WJ-4 | 848bp | The yeast enzyme is female CBS405 | 99% | 5 |
| WJ-6 | 1478bp | Lactobacillus buchneri | 99.9% | 6 |
| WJ-23 | 1461bp | The secondary cheese subspecies of lactobacillus paraceasi | 99.4% | 7 |
| WJ-33 | 1460bp | The secondary cheese subspecies of lactobacillus paraceasi | 99.6% | 8 |
＜
Utilize manufacturing and the specificity analysis of the red ginseng fermented liquid of novel red ginseng fermentation strain
Embodiment 2-1. utilizes novel red ginseng fermentation strain to make red ginseng fermented liquid (not adding inducible factor)
After diluting from (strain) the sincere red ginseng concentrated solution of buying, the secretion beta-glucosidase of screening (six bacterial strains of β-glucosidase) in above-mentioned embodiment 1, inoculate respectively with the concentration of 0.001 ~ 3 % (v/w) in 500, then at 37 ℃ of bottom fermentations 6 ~ 72 hours.
Embodiment 2-2. utilizes novel red ginseng fermentation strain to make red ginseng fermented liquid (adding inducible factor (elicitor))
Active for the saponin conversion that promotes to greatest extent fermentation strain, added salt (NaCl) and sucrose (Sucrose) as inducible factor during fermentation.
That is, the fermentation condition of fermentation strain is identical with embodiment 2-1 with the TLC analytical procedure, and difference is just to have added salt (NaCl) or sucrose (Sucrose) when beginning to ferment.At this moment, salt (NaCl) is to add during with the fermentation of 1% concentration, and sucrose (Sucrose) is to add respectively above-mentioned bacterial strains to 1% concentration, and processes with interior at 24 hours.
Comparative example 2. is the manufacturing of fermented red ginseng extraction liquid not
Prepared the red ginseng extract without fermenting process in above-mentioned embodiment 2-1.The red ginseng concentrated solution is bought from (strain) is sincere.
The specificity analysis of experimental example 2. embodiment 2-1 red ginseng fermented liquids
(1) analysis is to the degree of conversion of less important saponin (saponin)
The saponin Rb1 that uses in this experiment purchases from material bank of ginseng gene institute of Kyung Hee University.
In order to analyze tunning, implemented the TLC tomographic map.In order to implement the TLC tomographic map, pour into the ratio of 1:1 and carry out in test tube carrying out centrifugation after vortex (vortex) adding the red ginseng concentrated solution of strain fermentation and bubble butanols in the red ginseng concentrated solution, separating butanol layer and red ginseng concentrate liquid layer, have obtained butyl alcohol extraction liquid.Particularly, use silica gel 60 F254 TLC plate, after butyl alcohol extraction liquid is titrated to TLC plate, adopted CHCl
3-MeOH-H
2O 65:35:10 v/v/v, the mixed solvent of lower phase extends.After spraying 10% sulfuric acid on the TLC plate after extension, striking has also been confirmed result.
Its result, as shown in Figure 2, the bacterial strain of screening all has main saponin (saponin) but is converted to the systemic Rg3 of donor, Rd, the effect of the brilliance of the less important saponin such as Rb1 (saponin).Particularly, WJ-1 and 6 bacterial strains conversion Rd and Rg3, WJ-2 and 4 conversion Rd, WJ-23 and 33 are that the ability of conversion Rg3 and F2 is especially excellent.(seeing Fig. 2)
(2) analysis of pH variation
Change in order to analyze red ginseng concentrated solution and each fermentation strain pH before and after fermenting, utilize pH meter (Orion-550A) to measure.
Its result, the pH value before fermentation is 4.44, though each bacterial strain has certain difference, has produced acidity slightly (seeing Table 5) along with what ferment.
[table 5]
The variation of pH value in the different fermentations time
| 2h | 4h | 8h | 12h | 16h | 20h | 24h | 28h | 36h | 44h | |
| WJ-1 | 4.4 | 4.35 | 4.34 | 4.37 | 4.34 | 4.31 | 4.31 | 4.32 | 4.23 | 4.15 |
| WJ-2 | 4.39 | 4.36 | 4.36 | 4.39 | 4.34 | 4.3 | 4.29 | 4.34 | 4.32 | 4.27 |
| WJ-4 | 4.39 | 4.36 | 4.35 | 4.35 | 4.35 | 4.32 | 4.27 | 4.34 | 4.29 | 4.28 |
| WJ-6 | 4.36 | 4.37 | 4.35 | 4.21 | 4.37 | 4.38 | 4.37 | 4.4 | 4.36 | 4.34 |
| WJ-23 | 4.35 | 4.33 | 4.36 | 4.35 | 4.4 | 4.4 | 4.36 | 4.41 | 4.35 | 4.37 |
| WJ-33 | 4.36 | 4.35 | 4.35 | 4.31 | 4.37 | 4.39 | 4.37 | 4.4 | 4.34 | 4.37 |
(3) analysis of pol variation
In order to analyze the variation of fermentation front and back Brix, utilize Brix meter (ATAGO-Rx-5000 α) to measure.
Its result, the pol before fermentation is 8.13Brix, though each bacterial strain difference to some extent, along with the carrying out of fermenting, pol has reduced some (seeing Table 6).
[table 6]
The variation of different fermentations pol in the time
| 2h | 4h | 8h | 12h | 16h | 20h | 24h | 28h | 36h | 44h | |
| WJ-1 | 7.87 | 7.9 | 7.89 | 7.96 | 7.91 | 7.77 | 7.9 | 7.9 | 7.92 | 7.91 |
| WJ-2 | 7.9 | 7.87 | 7.86 | 7.82 | 7.64 | 6.76 | 6.67 | 6.62 | 6.6 | 6.43 |
| WJ-4 | 7.88 | 7.87 | 7.86 | 7.82 | 7.64 | 6.91 | 6.67 | 6.77 | 6.9 | 6.88 |
| WJ-6 | 7.9 | 7.9 | 7.93 | 7.96 | 7.93 | 7.89 | 7.92 | 7.9 | 7.91 | 7.96 |
| WJ-23 | 7.94 | 7.94 | 7.91 | 7.94 | 7.9 | 7.73 | 7.96 | 7.92 | 7.91 | 7.93 |
| WJ-33 | 7.91 | 7.9 | 7.9 | 7.93 | 7.92 | 7.94 | 7.95 | 7.9 | 7.91 | 7.94 |
The specificity analysis of experimental example 3. embodiment 2-2 fermented liquids (adding the effect of inducible factor)
Its result, as shown in Figure 3, the WJ-33 bacterial strain is compared when adding the salt timesharing than interpolation sucrose, has generated more Rg3 and F2 (seeing Fig. 3).
And the WJ-2 bacterial strain is when adding sucrose, and main saponin (saponin) Rb1 all decomposes, and has generated Rd and compound-K, and the WJ-4 bacterial strain has generated Rd and compound-K (seeing Fig. 4 a and 4b) adding the salt timesharing.And WJ-1 and 6 bacterial strains are example, compare during with interpolation sucrose, add the salt timesharing, and (β-glucosidase) activity is improved to have confirmed beta-glucosidase.
＜
Utilize the red ginseng fermented liquid that novel red ginseng fermentation strain makes functional confirmation (
In vitro)
In order to confirm red ginseng fermented liquid of the present invention specific absorption and immunizing power reinforced effects in vivo, utilize scavenger cell strain Raw 264.7 cell of experimental mouse, confirmed the discovery of the Immune interrelation factor.
After lactobacillus-fermented, in order to double the transfer capability of saponin (saponin), add edible enzyme in the red ginseng fermented liquid of making and process in above-mentioned embodiment 2-1.
At first, all enzymes with edible β-glucosidase activity of selling in market have been collected.
After the red ginseng concentrated solution is carried out lactobacillus-fermented, the enzyme of collecting is inoculated respectively 0.5 ~ 5%, carried out the reaction test of 10h ~ 72h in 45 ~ 60 ℃ of brooders.
Its result, in a lot of enzymes, the Viscozyme L of the Multifect pectinase FE of (strain) vision bio cam and Novozymes company is in the result of reacting under the condition of 55 ℃ of 40 ~ 48h with 3% ratio respectively, generate the unexistent compound-K of general red ginseng (Fig. 5), and made as stated above the red ginseng fermented liquid.
Experimental example 4. utilizes Raw 264.7 (mouse leukemic monocyte macrophage cell) to confirm Nitric Oxide growing amount
(1) confirm red ginseng fermented liquid (embodiment 3) and not Nitric Oxide (NO) generation of fermented red ginseng extraction liquid (comparative example 2)
Studies show that, nitrogen protoxide (NO) has cytotoxicity to various tumour cells or microorganism.In order to confirm whether NO is induced by red ginseng fermented liquid of the present invention, utilize griess reagent (Griess reagent) and reference material (sodium nitrite) to confirm, the oxide form NO of free NO in supernatant liquid in the cultivation that stimulation RAW264.7 cell strain obtains
2 -(nitrite) amount.
The cultivation of cell
Mouse macrophage cell RAW 264.7 cell purchase from Korea S cell strain bank (Korea of medical college of Seoul National University), carry out using after succeeding transfer culture in the research department.RAW 264.7 mouse macrophage cells are pasted the cell cultures dish, employing contains 1% antibacterial-antifungal solution (PAA of penicillin (penicillin) and Streptomycin sulphate (streptomycin), Canada) and add 10% FBS (PAA, Canada) DMEM (PAA, Canada), at 5% CO
2, cultivate in 37 ℃ of incubator.Nutrient solution was changed once at 2 ~ 3 days, during cell seeding, mixed with the ratio of 1:1 with training base and the Tryphan blue solution of cell mixing, utilized hemocytometer to count the cell that is not dyeed by tryphan blue.
2) NO assay
To 48 orifice plates, each 5 * 10 with RAW 264.7 cell plant division
4Cells/well.After 24 hours, with 1,10,100/concentration process red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) not, at 37 ℃ of 5% CO
2Cultivate in incubator.Test portion was processed after 24 hours, (the 1% sulfanilamide in 5% phosphoric acid+1% α-naphthylamide in H of supernatant liquid 50 and Griess reagent on mixed cell culture
2O) 50, after ten minutes, adopt ELISA reader to measure absorbancy in 96 orifice plate reactions under 520nm.Result is to use NO
2The drawing standard curve has calculated on the cell cultures NO value contained in supernatant liquid.
3) MTT analyzes
In order to confirm red ginseng fermented liquid (embodiment 3) and the cytotoxicity of fermented red ginseng extraction liquid (comparative example 2) not, utilize RAW 264.7 cells to confirm cell survival rate.To 48 orifice plates, each 5 * 10 with RAW 264.7 cell plant division
4Cells/well, after 24 hours with 1,10,100/concentration process red ginseng and fermented red ginseng test portion, at 37 ℃ of 5% CO
2Cultivate in incubator.Cultivate after 24 hours, MTT is added in each hole, and (10/well) reagent are cultivated after three hours in 37 ℃, are broken down into the amount of formazan in order to measure MTT, adopt LISA reader to measure absorbancy under 460nm.Each treatment group is respectively got 3 holes and is carried out the experiment repeatedly of three times, and the cell proliferation effect of red ginseng and fermented red ginseng is after getting the mean value of repeatedly testing for three times, to use the percentage between the control group of cultivating in the DMEM solution without any processing recently to represent.
Nitrogen protoxide (NO) has cytotoxicity to various tumour cells or microorganism.Therefore, for confirm NO whether by red ginseng fermented liquid (embodiment 3) and not fermented red ginseng extraction liquid (comparative example 2) induce, utilize griess reagent (Griess reagent) and reference material (sodium nitrite) to confirm its oxide form NO
2 -(nitrite) amount, in the cultivation that stimulation RAW264.7 cell strain for confirmation is obtained the supernatant liquid middle reaches from the amount of NO.
Confirm the generation result of nitrogen protoxide (NO)
Red ginseng fermented liquid (embodiment 3) and not fermented red ginseng extraction liquid (comparative example 2) nitrogen protoxide (NO) is generated institute's produce an effect investigation result, during an individual curing LPS (1 μ g/ml), the generation of NO has increased more than four times.This means that under the material LPS that causes inflammatory reaction, the reaction of scavenger cell has increased.Fermented red ginseng extraction liquid (comparative example 2) not, increase by 2.5 times during 100 μ g/ml concentration, red ginseng fermented liquid (embodiment 3), under 100 μ g/ml concentration, comparing with the undressed control group of test portion increases by 3.7 times, and the generation of NO has presented the increase (Fig. 6) of having a mind to.Red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) are not compared during individual curing inflammation incitant LPS, and the generation of NO has reduced.Such result shows, red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) is not although can cause inflammatory reaction, and the NO that increases scavenger cell generates, and helps immune response.And, red ginseng fermented liquid (embodiment 3) and not the thick saponin in fermented red ginseng extraction liquid (comparative example 2) be as the criterion, although the concentration of thick saponin is consistent, but the more minor saponin-of physiological active functions Compound is K, the content of F2, content in red ginseng fermented liquid (embodiment 3) is higher than fermented red ginseng extraction liquid (comparative example 2) not, and the judgement immune effect can be enhanced.
Experimental example 5. utilizes Raw 264.7 (mouse leukemic monocyte macrophage cell) to confirm the growing amount of TNF-α
1) confirmation red ginseng fermented liquid (embodiment 3) and the not method of the TNF-α generation of fermented red ginseng extraction liquid (comparative example 2)
Utilized RAW 264.7 cell measurements red ginseng fermented liquid (embodiment 3) that the generation of pro-inflammatory cytokine TNF-α is produced and the activity of fermented red ginseng extraction liquid (comparative example 2) not.To contain RAW 264.7 cells of cultivating in the DMEM of 10% FBS, with 2 x 10
6After the concentration of cell/ml is inoculated into 24 orifice plates, at 37 ℃, 5% CO
2Cultivated under condition 24 hours.Add LSP, red ginseng fermented liquid (embodiment 3) and not fermented red ginseng extraction liquid (comparative example 2) cultivated 24 hours, utilize the upper supernatant liquid of secreting in each cell, adopt the ELISA test kit to confirm the discovery amount of TNF-α.
Cell survival rate confirm result
For confirm red ginseng fermented liquid (embodiment 3) and not fermented red ginseng extraction liquid (comparative example 2) whether RAW 264.7 cells are had cytotoxicity, confirm the result that MTT analyzes, do not present cytotoxicity (Fig. 7) in red ginseng and fermented red ginseng extract.
Confirm red ginseng fermented liquid (embodiment 3) and the generation produce an effect of fermented red ginseng extraction liquid (comparative example 2) to TNF-α not
For confirm red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) whether the scavenger cell strain is produced active, confirm the result of the generation inducibility of cytokine TNF-α, the LPS that brings out macrophage activity is not processed, just with red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) when processing, to the few of difference of the generation of TNF-α not.But, use separately the LPS that brings out macrophage activity or with red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) when together processing not, compare with TNF-α growing amount 0.593 nM of the control group of be untreated LPS and test portion, during with the independent irritation cell of LPS of 1 μ g/ml concentration, TNF-α growing amount is that 0.866 nM has approximately increased by 46% left and right.Fermented red ginseng extraction liquid (comparative example 2) not, the growing amount under 100 μ g/ml concentration is 1.03 nM, and comparing with control group approximately increases by 70%, and comparing with positive controls LPS treatment group has increased by 18%.And, red ginseng fermented liquid (embodiment 3), the growing amount when 100 μ g/ml concentration is 1.166 nM, and comparing with control group approximately increases by 96%, and comparing with positive controls LPS treatment group approximately increases by 34%.Such result is, red ginseng fermented liquid (embodiment 3) is with fermented red ginseng extraction liquid (comparative example 2) relatively the time, also can activating macrophage, the generation of Immune interrelation factor TNF-α is increased more than 20%, become and enliven immunoreactive basis for estimation.(Fig. 8)
Experimental example 6. utilizes Raw 264.7 Cell
Confirm the phagolysis effect of scavenger cell
Use red ginseng fermented liquid (embodiment 3) and not fermented red ginseng extraction liquid (comparative example 2) process Raw 264.7 Cell, confirmed the phagolysis effect of scavenger cell.
Utilize Raw 264.7Cell
Confirm the phagolysis effect of scavenger cell
With RAW 264.7 cells with 5 * 10
4The cells/well plant division is to 48 orifice plates, after 24 hours with 100/concentration process red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) not, at 37 ℃ of 5% CO
2Cultivate in incubator.Cultivate after 24 hours, use phagocytosis assay kit to confirm the phagolysis effect of scavenger cell.
The confirmation result
Most of scavenger cells are all the cells that does not move, and stay and organize inner filtration and destroy foreign matter.But the scavenger cell that a part comes off is gone around in the space between the recycle system or cell.These scavenger cells are the indispensable key elements of immune response, and the cytophagy of scavenger cell makes the surface molecular of foreign matter (antigen) expression out, stimulates lymphocytic reaction, and the formation of antibody is the phagolysis of stimulating expression of macrophage greatly again.Therefore, the inventor utilizes red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) not, has confirmed the level of activity (Fig. 9) of macrophage phagocytic effect.
In vitroThe upper phagocytotic result of confirming scavenger cell by phagocytosis assay, the wild-type of extract that is untreated and for suppressing the treated negative control group of phagocytotic inhibitor of scavenger cell, (P-con) compares with positive controls, and the phagolysis of scavenger cell has obviously reduced.And when processing with the not fermented red ginseng extraction liquid (comparative example 2) of 100 μ g/ml concentration, its level of activity is similar to positive controls.On the contrary, when processing with the red ginseng fermented liquid (embodiment 3) of 100 μ g/ml concentration, compare with positive controls, the phagolysis level of activity of scavenger cell has increased more than 16%, but there is no to find effect intentionally.
＜
Confirm to utilize red ginseng fermented liquid that novel red ginseng fermentation strain makes functional (
In vivo)
In order to confirm red ginseng fermented liquid of the present invention specific absorption in vivo and the effect of strengthening immunity, adopt the experimental mouse animal, confirmed that red ginseng and fermented red ginseng strengthen the effect of immunity.
Experimental example 7. is confirmed the multiplication capacity of immunocyte
Three all oral red ginseng fermented liquids (embodiment 3) and not after fermented red ginseng extraction liquid (comparative example 2), the spleen cell of separating experiment mouse is confirmed the multiplication capacity of spleen cell, has confirmed that test portion strengthens the effect of immunity.
Confirm the method for immune cell propagation ability
Get spleen under sterile state, put into the container that appropriate aseptic HBSS solution is housed, chop up with tweezers, make the single cell suspension liquid.Wash three times with HBSS liquid after using Filter to filter.When each washing, carry out centrifugation with the speed of 1,000 rpm.With cell swim in dye in complete culture solution 2 after, calculate the cell count (95% above survival rate) of survival.Cell concn is adjusted to 2 * 10
6Cell/.
With cell suspension 24 orifice plates of packing into, each hole fills 1, then adds the factor-LPS that stimulates the B cell and the factor that stimulates the T cell-Con A solution (be equivalent to 5 /).Together with control group, at CO
2Cultivated 72 hours in 5%, 37 ℃ of substratum.Cultivate to finish before four hours, 0.7 upper supernatant liquid is all removed in each hole, when adding the RPMI RPMI-1640 0.7 that does not contain FBS, adds MTT liquid (5 ㎎/) 50/hole, then continues to cultivate in four hours.After cultivating end, put into 1 acid isopropyl alcohol mixing in each hole, until the purple crystal body dissolves fully.Then, be divided in 96 orifice plates.After the 3-6 hole is assigned in each hole, under 570 nm wavelength, adopt ELISA (enzyme linked immunosorbent assay) reader to measure.
Confirm the result of immune cell propagation ability
Spleen is the important second immunisation organ that participates in cellular immunity and body fluid immunity, bears the initial stage immune response in organism.Therefore, the inventor is in order to confirm oral red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) not, on the impact of experimental mouse spleen cell multiplication capacity, as the index multiplication capacity of spleen cell that adopted the MTT analysis to measure.Red ginseng fermented liquid (embodiment 3) and the affect result of fermented red ginseng extraction liquid (comparative example 2) on the spleen cell multiplication capacity not are presented at Figure 10.
Oral three all red ginseng fermented liquids (embodiment 3) and not after fermented red ginseng extraction liquid (comparative example 2), the separating spleen cell, in the situation that do not add the mitogen that activates T cell or B cell, cell proliferation rate does not all have on cell proliferation to produce impact at oral ginseng fermented liquid (embodiment 3) and not in the experimental group of fermented red ginseng extraction liquid (comparative example 2).And the Concanavalin A (Con A, 5 μ g/ml) that in cellular immunity, selectivity is increased the T cell in the experimental group with the oral red ginseng fermented liquid of the concentration of 200mg/kg (embodiment 3), has approximately increased more than 38% when processing.Such presentation of results, even compare with fermented red ginseng extraction liquid (comparative example 2) not, proliferation rate also exceeds more than 15%.But in the body fluid immunity, the lipopolysaccharides (LPS, 5 μ g/ml) that selectivity is increased the B cell is when processing, and the difference of cell proliferation rate is all little in all groups.
This shows, red ginseng fermented liquid of the present invention (embodiment 3) activates the immune response of T cell, helps to promote the immunity of human body.
Experimental example 8. is confirmed the Cytokine secretion capacity of peritoneal macrophage
Three all oral red ginseng fermented liquids (embodiment 3) and do not separate peritoneal macrophage in the experimental mouse of fermented red ginseng extraction liquid (comparative example 2), the TNF-α that secretes in the confirmation peritoneal macrophage and the discovery amount of IL-6 have confirmed that test portion strengthens the effect of immunity.
The confirmation method of secretion by peritoneal macrophages Cytokine ability
Three all oral red ginseng fermented liquids (embodiment 3) and not after fermented red ginseng extraction liquid (comparative example 2) inject the RPMI 1640 training bases of 20ml for the experimental mouse abdominal cavity of anesthesia with syringe, then massaged 10 minutes.Afterwards, use transfer pipet again to reclaim the training base, move into the 50ml test tube, carry out the centrifugation of ten minutes with the speed of 1500rpm under the condition of 4 ℃.After using RPMI 1640 training base washing three times, use the RPMI 1640 training bases that contain 10% FBS, be diluted to 1 * 106 cell/.With the dilution enchylema 100 μ l plant division to 96 orifice plates, at 5% CO
2, cultivate in 37 ℃ of substratum and adhered in 2 hours.After the cell that removal is swum, the scavenger cell that usage quantity is adhered to.
Measure respectively cytokine (IL-1 β, the TNF-α) secretory volume of secreting in supernatant liquid in the cultivation that the peritoneal macrophage of culture of isolated produces.Remove non-attached cells, after only staying attached cells, after adding the mitogen LPS and Pei Ji 100 μ L that activates 10%-FBS RPMI 1640,900 μ L and scavenger cell, at 37 ℃, cultivated 48 hours in 5% CO2 incubator (Sanyo).Supernatant liquid in separation uses ELISA cytokine test kit (R﹠amp; D system, USA) measured the IL-1 β that accumulates in the nutrient solution, the amount of TNF-α.
Measure the activity of natural killer cell (NK cell)
The spleen cell that separates the laboratory of oral red ginseng and fermented red ginseng is cultivated together with target cell YAC-1 cell, confirms the activity of Natural Killer cell, has confirmed that test portion strengthens the effect of immunity.
The cultivation of target cell and effect cell
Cultivated target cell (YAC-1 cell) before testing 24 hours.Clean three times with HBSS solution before using, use the RPMI-1640 nutrient solution, cell concn is adjusted into 1 * 10
5Cells/.
Three all oral red ginseng fermented liquids (embodiment 3) and not in the experimental mouse of fermented red ginseng extraction liquid (comparative example 2) after the separating spleen cell, use the RPMI-1640 nutrient solution that cell concn is adjusted into 1 * 10
5Cells/.
The confirmation result of cytokine secretion capacity
-. utilize peritoneal macrophage to confirm the discovery amount of TNF-α
Tumor necrosis factor (TNF) is mainly by scavenger cell secretion, and the cytokine that also can generate by T cell, NK cell and the mastocyte of antigenic stimulation is the glycoprotein of 17kD size.TNF is along with the sensitization of scavenger cell, with the IFN-γ of T cell and NK Hemapoiesis effect of increasing together.Especially, induced reaction nitrogen metabolism thing NO makes scavenger cell have wider anti-microbial effect.Therefore, the inventor has confirmed oral red ginseng fermented liquid (embodiment 3) and the not impact of fermented red ginseng extraction liquid (comparative example 2) TNF-α discovery amount of experimental mouse peritoneal macrophage after three weeks.
Confirm in peritoneal macrophage that the TNF-α under the LPS stimulation generates the result of inducibility, in the undressed control group of LPS and test portion, the growing amount of TNF-α is 4571 pg/ml.In the positive controls of only LPS being processed, the growing amount of TNF-α is 6293 pg/ml, has increased by 37% than non-processor group.
Oral 200mg/kg concentration is not in the experimental group of fermented red ginseng extraction liquid (comparative example 2), the growing amount of TNF-α is 6312 pg/ml, comparing with the non-processor control group has approximately increased by 38% left and right, presents similar growing amount with the positive controls of processing LPS.But in the experimental group of oral 200mg/kg concentration red ginseng fermented liquid (embodiment 3), the growing amount of TNF-α is 6976 pg/ml, and comparing with the non-processor control group has approximately increased by 52% left and right.And, compare with fermented red ginseng extraction liquid (comparative example 2) not, also increased more than 14%.(Figure 11).
And, without the stimulation of LPS, when individual curing red ginseng and fermented red ginseng extract, to compare with the control group of the test portion that is untreated, the growing amount of TNF-α has increase, but there is no difference intentionally between test portion.
This shows, has potential immunocompetence after taking in red ginseng fermented liquid of the present invention (embodiment 3), when in exterior materials (antigenic substance) invasion body, red ginseng fermented liquid (embodiment 3) acts on the sensitization of immunocyte, affects immunologic function.
Utilize peritoneal macrophage to confirm the discovery amount of IL-1 β
IL-1 β is the medium of inflammatory reaction most crucial in cytokine, and the growing amount of scavenger cell is maximum.When being subject to specific inflammatory stimulus, as the result generation of its reaction.And IL-1 β plays important effect in the cytokine networks such as sensitization of T cell.When such IL-1 β reduces, weaken the resistibility to infectious diseases, so the important elements in immune response.
Confirm that the IL-1 β in peritoneal macrophage generates the result of inducibility, in the undressed control group of test portion, IL-1 β growing amount is 198 pg/ml, and when processing LPS as the stimulus of scavenger cell, IL-1 β growing amount is 337 pg/ml, has approximately increased by 70%.
On the contrary, in the experimental group of fermented red ginseng extraction liquid (comparative example 2), IL-1 β growing amount is not 280 pg/ml to oral 200mg/kg concentration, shows that the growing amount of IL-1 β has increased by 40% than control group.And in the experimental group of oral 200mg/kg concentration red ginseng fermented liquid (embodiment 3), IL-1 β growing amount is that 348 pg/ml have approximately increased by 73% left and right.(Figure 12)
But, the generation of IL-1 β, the LPS that uses as positive controls compares, and the usefulness of red ginseng and fermented red ginseng extract does not have difference intentionally.
Utilize peritoneal macrophage to confirm IL-6 discovery amount
IL-6 has the immunological activity degree characteristics such as inflammatory, metabolic, physiological, haematopoietic.IL-6 is originally the differentiation factor of B cell, increases the protein that B cell antibody generates.Up-to-date studies show that, IL-6 plays the cytokine of central action in the host defense mechanisms such as immune response and hematopoiesis adjusting, be regarded as the important elements in immune response.
Three all oral red ginseng fermented liquids (embodiment 3) and not in the peritoneal macrophage of the experimental mouse of fermented red ginseng extraction liquid (comparative example 2), confirm the result of the generation inducibility of IL-6, in the undressed control group of test portion, the growing amount of IL-6 is 107 pg/ml, when processing LPS for stimulating expression of macrophage, the growing amount of IL-6 is 193 pg/ml, and the growing amount of IL-6 has approximately increased by 180% left and right.
Fermented red ginseng extraction liquid (comparative example 2) not, in the experimental group of oral 200mg/kg concentration, the growing amount of IL-6 is 208 pg/ml.On the contrary, red ginseng fermented liquid (embodiment 3), in the experimental group of oral 200mg/kg concentration, the growing amount 227pg/ml of IL-6, comparing with control group has approximately increased by 212% left and right (Figure 13).And red ginseng fermented liquid (embodiment 3) is even compare with fermented red ginseng extraction liquid (comparative example 2) not, and the IL-6 growing amount has more more than 18%.
Such result shows, has potential immunocompetence after taking in red ginseng fermented liquid (embodiment 3), when exterior materials (antigenic substance) intrudes in body, the peritoneal macrophage of red ginseng fermented liquid (embodiment 3) activation experiment mouse promotes the generation of IL-6 to strengthen immunologic function.
Experimental example 9. is confirmed and fermented red ginseng specific absorption in vivo
Oral red ginseng fermented liquid (embodiment 3) and not after fermented red ginseng extraction liquid (comparative example 2), take the blood of experimental mouse, separated plasma from blood, be regarded as red ginseng fermented liquid (embodiment 3) and the Compound K content of the functional ingredient of fermented red ginseng extraction liquid (comparative example 2) not in analysed for plasma, confirmed test portion specific absorption in vivo.
Analytical equipment and instrument
- Mass spectrometry: API 4000 Q-trap Mass Spectrometer
- LC system: Agilent 1100 series
- Peak Simple Chromatography Data System: Analyst 1.4.2 (Applied Biosystems)
Analysis condition
As shown in table 7 below.
[table 7]
3) drafting of calibration curve
Compound K standard substance are dissolved in methyl alcohol, after making the concentration of compound K reach 1 mg/mL, then are dissolved in mouse plasma, making respectively concentration is 1,2,5,10, the standard blood plasma test portion of 100,400,800,1000 ng/mL.After adding respectively each 100 μ L of 10 mM potassium phosphate buffer (pH 7.4) in each 100 μ L test portion, pour ethyl acetate 900 μ L into and carry out vortex, carried out the centrifugation of ten minutes in 2,500g.After the upper supernatant liquid of evaporation 300 μ L under 30 ℃ of conditions concentrate, be dissolved as mobile phase 100 μ L, after then pouring internal standard material 10 μ L into and carrying out vortex, be injected into LC-MS/MS and analyze.In the specimen analyzing process, after at first measuring calibration curve and drawing test portion, the QC test portion before the Measurement and analysis test portion, be confirmed whether to enter theoretical value ± 15% in.
The processing of blood plasma test portion
Room temperature is melted and carry out vortex with certainly being placed at the mouse of-20 ° of C plasma.Get in right amount carry out pre-treatment by the identical method of the method for drafting of calibration curve after, be injected into LC-MS/MS.
The calculating of blood level
Try to achieve the peak area of internal standard material and the peak area ratio of compound K from the tomographic map that obtains, tried to achieve the concentration of compound K in the blood plasma test portion from the calibration curve of prior drafting.
Red ginseng fermented liquid (embodiment 3) and fermented red ginseng extraction liquid (comparative example 2) the confirmation result of specific absorption in vivo not
Oral disposition red ginseng fermented liquid (embodiment 3) and after the experimental mouse of fermented red ginseng extraction liquid (comparative example 2) is not taken a blood sample, separated plasma in blood, be regarded as red ginseng fermented liquid (embodiment 3) and the content of the Compound K of the functional ingredient of fermented red ginseng extraction liquid (comparative example 2) not in analysed for plasma, confirmed test portion specific absorption (Figure 14 and Figure 15) in vivo.
Its result, in the experimental mouse blood plasma of control group and oral not fermented red ginseng extraction liquid (comparative example 2), the content of Compound K is below 1ng/ml.Such result fails to enter in the theoretical value measured when drawing calibration curve ± 15%, and judgement absorbs in vivo.
But, confirming 6ng/ml in the experimental mouse blood plasma of oral 200mg/kg concentration red ginseng fermented liquid (embodiment 3), judgement Compound K is absorbed in the experimental mouse body.Such result shows, absorbs in the functional ingredient Compound K body of red ginseng fermented liquid (embodiment 3), can strengthen immunity.
The inhibition of red ginseng fermented liquid of the present invention to cancer cell multiplication 〉
The present inventor uses lung cancer cell line A549-1 cell, stomach cancer cell line AGS-1 cell, colorectal cancer cell lines HT 29 cells, has confirmed red ginseng fermented liquid (embodiment 3) that the propagation of each cancer cells is produced and the inhibition of fermented red ginseng extraction liquid (comparative example 2) not.
Experimental example 10. confirms that red ginseng fermented liquid of the present invention is to the inhibition of proliferation of lung cancer cells
When processing red ginseng fermented liquid (embodiment 3) with the concentration of 100 μ g/ml, the survival rate of lung cancer cell line A549-2 cell is 20% left and right, as seen the inhibition of proliferation of lung cancer cells rate is had a mind to.On the contrary, when processing not fermented red ginseng extraction liquid (comparative example 2) with the concentration of 100 μ g/ml, survival rate reaches more than 80%, and is visible to the few of restraining effect of the proliferation rate of lung carcinoma cell (Figure 15).
Experimental example 11. confirms that red ginseng fermented liquid of the present invention is to the inhibition of proliferation of human gastric cancer cell
When processing red ginseng fermented liquid (embodiment 3) with the concentration of 100 μ g/ml, the survival rate of stomach cancer cell line AGS-1 cell is 25% left and right, as seen the inhibition of proliferation of human gastric cancer cell rate is had a mind to.On the contrary, when processing not fermented red ginseng extraction liquid (comparative example 2) with the concentration of 100 μ g/ml, cell survival rate reaches more than 80%, and is visible to the few of inhibition of the proliferation rate of stomach cancer cell (Figure 16).
Experimental example 11. confirms that red ginseng fermented liquid of the present invention is to the inhibition of proliferation of colorectal cancer cells
When processing red ginseng fermented liquid (embodiment 3) with the concentration of 100 μ g/ml, the survival rate of colorectal cancer cell lines HT 29 cells is 40% left and right, as seen the inhibition of proliferation of colorectal cancer cells is had meaning.On the contrary, when processing not fermented red ginseng extraction liquid (comparative example 2) with the concentration of 100 μ g/ml, cell survival rate reaches more than 70%, as seen fails effectively to restrain the proliferation rate (Figure 17) of colorectal cancer cells.
＜
Utilization contains the training base manufacturing red ginseng fermented liquid of vegetalitas material
Experiment material
1. experiment material
The material that uses in this experiment is the foodstuffs material of the world FL of food mfg company (strain) supply.
2. the milk-acid bacteria of using in the experiment
Adopt bear Tianjin food (strain) place on the secondary cheese subspecies of the patent application milk-acid bacteria (KCTC 12108BP) at Korea S's microorganism keeping center-lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) test.
The construction experiment of the basic training base of experimental example 12.
(1) microorganism culturing of different vegetalitas material kinds experiment
Add respectively in the training base of the material (material) can participate in the milk-acid bacteria fertility-Radix Dauci Sativae (Carrot), banana (Banana), radish (Radish) and circle romaine lettuce (Lettuce), cultivate the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei), measured respectively the OD value of milk-acid bacteria.Its result sees Table 8 and Figure 18.
[table 8]
| 1% | 5% | 10% | 20% | 30% | |
| Radix Dauci Sativae (Carrot) | 0.131 | 0.492 | 0.354 | 0.078 | 0.091 |
| Banana (Banana) | 0.031 | 0.383 | 0.417 | 0.329 | 0.305 |
| Radish (Radish) | 0.046 | 0.211 | 0.551 | 0.884 | 0.748 |
| Circle romaine lettuce (Lettuce) | 0.018 | 0.065 | 0.104 | 0.13 | 0.211 |
As table 8 and shown in Figure 180, be suitable for the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) fertility be followed successively by radish, Radix Dauci Sativae, banana and circle romaine lettuce, wherein most suitable is radish.
(2) microorganism culturing of different sugar kind experiment
Add respectively in the training base of the spendable sugared source-sucrose of milk-acid bacteria (sugar), poly-dextrose (polydextrose), fructose (fructose), oligose (maltooligo), glucose (glucose), dextrin (dextrin), oligofructose (fructooligo) and lactose (lactose), the secondary cheese subspecies of cultivation lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei), measured respectively the OD value of milk-acid bacteria.Its result sees Table 9 and Figure 19.
[table 9]
| Sugar type (Carbon source) | OD 600 | Sugar type (Carbon source) | OD 600 |
| Control | 0.363 | Glucose (glucose) | 0.71 |
| Sucrose (sugar) | 0.69 | Dextrin (dextrin) | 0.692 |
| Poly-dextrose (polydextrose) | 0.663 | Oligofructose (fructooligo) | 0.695 |
| Fructose (fructose) | 0.681 | Lactose (lactose) | 0.745 |
| Oligose (maltooligo) | 0.66 |
As table 9 and shown in Figure 19, lactose and glucose be suitable for the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) fertility.
The microorganism culturing experiment of different salinities and nutritive ingredient kind
Table 10 and Figure 20 show, the experimental result of the available salinity of milk-acid bacteria and nutritive ingredient.As table 10 and shown in Figure 20, Rhodiaphos DKP (Dipotassium phosphate; K
2HPO
4) concentration 0.1 be as the criterion, when adding the female extract of enzyme (yeast extract) with 0.5 to 5 concentration, be suitable for the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) fertility.
[table 10]
| Salt | OD 600 | Salt | OD 600 | Salt | OD 600 |
| K 0.1 Y 0.1 | 0.52 | K 0.5 Y 0.1 | 0.319 | K 1 Y 0.1 | 0.326 |
| K 0.1 Y 0.5 | 0.821 | K 0.5 Y 0.5 | 0.442 | K 1 Y 0.5 | 0.392 |
| K 0.1 |
0.952 | K 0.5 |
0.493 | K 1 |
0.491 |
| K 0.1 |
0.984 | K 0.5 |
0.581 | K 1 |
0.564 |
In table 10 and Figure 20, K represents Rhodiaphos DKP (Dipotassium phosphate; K
2HPO
4), Y represents the female extract (yeast extract) of enzyme.
Based on the above results, the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp.Paracasei) fertility, the weight of all training based component is as the criterion, radish and lactose 0.2 weight ratio, glucose 0.2 weight ratio and Rhodiaphos DKP (Dipotassium phosphate; K
2HPO
4) female extract (yeast extract) content of enzyme of 0.1 weight ratio and 5 weight ratios is best proportionings.
Experimental example 13. is the construction experiment of additive again
(1) add the relevant microorganism culturing experiment of vegetalitas material
Based on the result of above-mentioned experimental example 12, training based component gross weight is as the criterion, radish and lactose 0.2 % by weight, glucose 0.2 % by weight and Rhodiaphos DKP (Dipotassium phosphate; K
2HPO
4) in the training based component that forms of the female extract of 0.1 % by weight and enzyme (yeast extract) 5 % by weight, as the vegetalitas material respectively with 0.1 % by weight, 0.5 % by weight, the ratio of 1 % by weight has added Chinese cabbage (Cabbage), Radix Dauci Sativae (Carrot), banana (Banana), circle romaine lettuce (Lettuce) etc., with the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) cultivate after 24 hours, measure the OD value, be presented at respectively table 11 and Figure 21.
[table 11]
| Material (concentration) | OD 600 | Material (concentration) | OD 600 |
| Chinese cabbage (Cabbage) 0.1% | 0.988 | Radix Dauci Sativae (Carrot) 0.1% | 0.789 |
| Chinese cabbage (Cabbage) 0.5% | 0.609 | Radix Dauci Sativae (Carrot) 0.5% | 0.852 |
| Chinese cabbage (Cabbage) 1% | 0.569 | Radix Dauci Sativae (Carrot) 1% | 1.052 |
| Material (concentration) | OD 600 | Material (concentration) | OD 600 |
| Banana (Banana) 0.1% | 1.053 | Circle romaine lettuce (Lettuce) 0.1% | 0.835 |
| Banana (Banana) 0.5% | 1.06 | Circle romaine lettuce (Lettuce) 0.5% | 1.043 |
| Banana (Banana) 1% | 1.088 | Circle romaine lettuce (Lettuce) 1% | 1.059 |
Its result shows, when adding Radix Dauci Sativae and banana with 1% weight ratio respectively, has presented best culture effect.
Mix and add the relevant microorganism culturing experiment of vegetalitas material
Based on the result of above-mentioned experimental example 12, training based component gross weight is as the criterion, and contains radish and lactose 0.2 % by weight, glucose 0.2 % by weight and Rhodiaphos DKP (Dipotassium phosphate; K
2HPO
4) in the training based component of female extract (yeast extract) 5 % by weight of 0.1 % by weight and enzyme, as shown in table 5, mix and add the vegetalitas material, with the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillusparacasei subsp. paracasei) carry out the cultivation of 24 hours after, measure the OD value, be presented at respectively in table 12 and Figure 22.
[table 12]
Its result shows, is as the criterion with the gross weight of composition, mixes when adding Radix Dauci Sativae and banana with 1 % by weight, presented best culture effect.
SEQUENCE LISTING
<110> WOONGJIN FOODS CO., LTD
＜120〉microorganism is used in novel red ginseng fermentation, and utilizes red ginseng fermented liquid and the red ginseng fermented drink of this microorganism
<130> HY111398
<160> 8
<170> KopatentIn 1.71
<210> 1
<211> 19
<212> DNA
＜213〉artificial sequence (Artificial Sequence)
<220>
＜223〉be used for the forward primer (Forward primer for PCR) (16S rDNA) of PCR
<400> 1
<210> 2
<211> 21
<212> DNA
＜213〉artificial sequence (Artificial Sequence)
<220>
<223> Reward primer for PCR(16S rDNA)
<400> 2
acggctacct tgttacgact t 21
<210> 3
<211> 1462
<212> DNA
＜213〉Lactobacillus buchneri (Lactobacillus buchneri)
<400> 3
ggctagacgc atagattgat ctgactcgga gccgtataag gtagccgtat gcagttgaaa 60
catttccatt gagacgagtg gcgaactggt gagtaacacg tgggtaacct gcccttgaag 120
taggggataa cacttggaaa caggtgctaa taccgtataa caaccaaaac cacctggttt 180
tggtttaaaa gacggcttcg gctgtcactt taggatggac ccgcggcgta ttagcttgtt 240
ggtaaggtaa cggcctacca aggcgatgat acgtagccga cctgagaggg taatcggcca 300
cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca 360
atggacgaaa gtctgatgga gcaacgccgc gtgagtgatg aagggtttcg gctcgtaaaa 420
ctctgttgtt ggagaagaac aggtgtcaga gtaactgttg acatcttgac ggtatccaac 480
cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 540
tccggattta ttgggcgtaa agcgagcgca ggcggttttt taggtctgat gtgaaagcct 600
tcggcttaac cggagaagtg catcggaaac cgggagactt gagtgcagaa gaggacagtg 660
gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720
gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780
accctggtag tccatgccgt aaacgatgag tgctaagtgt tggagggttt ccgcccttca 840
gtgctgcagc taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc 900
aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gatgctacgc 960
gaagaacctt accaggtctt gacatcttct gccaacctaa gagattaggc gttcccttcg 1020
gggacagaat gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1080
agtcccgcaa cgagcgcaac ccttattgtt agttgccagc attcagttgg gcactctagc 1140
aagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1200
tgacctgggc tacacacgtg ctacaatgga cggtacaacg agtcgcgaaa ccgcgaggtc 1260
aagctaatct cttaaagccg ttctcagttc ggattgtagg ctgcaactcg cctacatgaa 1320
gttggaatcg ctagtaatcg tggatcagca tgccacggtg aatacgttcc cgggccttgt 1380
acacaccgcc cgtcacacca tgagagtctg ttctgagaaa atccgtgacg aacagtcgaa 1440
acaagcgcct acgaactcct aa 1462
<210> 4
<211> 854
<212> DNA
＜213〉yeast enzyme female (Saccharomyces cerevisiae)
<400> 4
gccttctttt cggtgggggg tctgggaagg atcattaaag aaatttaata attttgaaaa 60
tggatttttt ttgttttggc aagagcatga gagcttttac tgggcaagaa gacaagagat 120
ggagagtcca gccgggcctg cgcttaagtg cgcggtcttg ctaggcttgt aagtttcttt 180
cttgctattc caaacggtga gagatttctg tgcttttgtt ataggacaat taaaaccgtt 240
tcaatacaac acactgtgga gttttcatat ctttgcaact ttttctttgg gcattcgagc 300
aatcggggcc cagaggtaac aaacacaaac aattttattt attcattaaa tttttgtcaa 360
aaacaagaat tttcgtaact ggaaatttta aaatattaaa aactttcaac aacggatctc 420
ttggttctcg catcgatgaa gaacgcagcg aaatgcgata cgtaatgtga attgcagaat 480
tccgtgaatc atcgaatctt tgaacgcaca ttgcgcccct tggtattcca gggggcatgc 540
ctgtttgagc gtcatttcct tctcaaacat tctgtttggt agtgagtgat actctttgga 600
gttaacttga aattgctggc cttttcattg gatgtttttt tttccaaaga gaggtttctc 660
tgcgtgcttg aggtataatg caagtacggt cgttttaggt tttaccaact gcggctaatc 720
ttttttatac tgagcgtatt ggaacgttat cgataagaag agagcgtcta ggcgaacaat 780
gttcttaaag tttgacctca aatcaggtag gagtacccgc tgaacttaag cattcataag 840
cccggagaaa aagg 854
<210> 5
<211> 848
<212> DNA
＜213〉yeast enzyme female (Saccharomyces cerevisiae)
<400> 5
ctttctttcg gggggggtac tgggaaggat cattaaagaa atttaataat tttgaaaatg 60
gatttttttt gttttggcaa gagcatgaga gcttttactg ggcaagaaga caagagatgg 120
agagtccagc cgggcctgcg cttaagtgcg cggtcttgct aggcttgtaa gtttctttct 180
tgctattcca aacggtgaga gatttctgtg cttttgttat aggacaatta aaaccgtttc 240
aatacaacac actgtggagt tttcatatct ttgcaacttt ttctttgggc attcgagcaa 300
tcggggccca gaggtaacaa acacaaacaa ttttatttat tcattaaatt tttgtcaaaa 360
acaagaattt tcgtaactgg aaattttaaa atattaaaaa ctttcaacaa cggatctctt 420
ggttctcgca tcgatgaaga acgcagcgaa atgcgatacg taatgtgaat tgcagaattc 480
cgtgaatcat cgaatctttg aacgcacatt gcgccccttg gtattccagg gggcatgcct 540
gtttgagcgt catttccttc tcaaacattc tgtttggtag tgagtgatac tctttggagt 600
taacttgaaa ttgctggcct tttcattgga tgtttttttt tccaaagaga ggtttctctg 660
cgtgcttgag gtataatgca agtacggtcg ttttaggttt taccaactgc ggctaatctt 720
ttttatactg agcgtattgg aacgttatcg ataagaagag agcgtctagg cgaacaatgt 780
tcttaaagtt tgacctcaaa tcaggtagga gtacccgctg aacttaagca tatctaancc 840
ggaagaaa 848
<210> 6
<211> 1474
<212> DNA
＜213〉Lactobacillus buchneri (Lactobacillus buchneri)
<400> 6
cccccagcgc ggctatctgc agtcgacgcg tctccgttat gattttaggt gcttgcactt 60
gaaagattta acattgagac gagtggcgaa ctggtgagta acacgtgggt aacctgccct 120
tgaagtaggg gataacactt ggaaacaggt gctaataccg tataacaacc aaaaccacct 180
ggttttggtt taaaagacgg cttcggctgt cactttagga tggacccgcg gcgtattagc 240
ttgttggtaa ggtaacggcc taccaaggcg atgatacgta gccgacctga gagggtaatc 300
ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360
ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgatgaaggg tttcggctcg 420
taaaactctg ttgttggaga agaacaggtg tcagagtaac tgttgacatc ttgacggtat 480
ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540
cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaggt ctgatgtgaa 600
agccttcggc ttaaccggag aagtgcatcg gaaaccggga gacttgagtg cagaagagga 660
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720
aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780
tagataccct ggtagtccat gccgtaaacg atgagtgcta agtgttggag ggtttccgcc 840
cttcagtgct gcagctaacg cattaagcac tccgcctggg gagtacgacc gcaaggttga 900
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 960
tacgcgaaga accttaccag gtcttgacat cttctgccaa cctaagagat taggcgttcc 1020
cttcggggac agaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta ttgttagttg ccagcattca gttgggcact 1140
ctagcaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200
ccttatgacc tgggctacac acgtgctaca atggacggta caacgagtcg cgaaaccgcg 1260
aggtcaagct aatctcttaa agccgttctc agttcggatt gtaggctgca actcgcctac 1320
atgaagttgg aatcgctagt aatcgtggat cagcatgcca cggtgaatac gttcccgggc 1380
cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagccg gtgaggtacc 1440
ttcggggacc agccgtctaa gtagatcacg ctgg 1474
<210> 7
<211> 1461
<212> DNA
＜213〉the secondary cheese subspecies (Lactobacillus paracasei subsp. paracasei) of lactobacillus paraceasi
<400> 7
cccgcaggcg caatctgcag tcgacgagtt ctcgttgatg atcggtgctt gcaccgagat 60
tcaacatgga acgagtggcg gacgggtgag taacacgtgg gtaacctgcc cttaagtggg 120
ggataacatt tggaaacaga tgctaatacc gcatagatcc aagaaccgca tggttcttgg 180
ctgaaagatg gcgtaagcta tcgcttttgg atggacccgc ggcgtattag ctagttggtg 240
aggtaacggc tcaccaaggc gatgatacgt agccgaactg agaggttgat cggccacatt 300
gggactgaga cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg 360
acgcaagtct gatggagcaa cgccgcgtga gtgaagaagg ctttcgggtc gtaaaactct 420
gttgttggag aagaatggtc ggcagagtaa ctgttgtcgg cgtgacggta tccaaccaga 480
aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg 540
gatttattgg gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga aagccctcgg 600
cttaaccgag gaagcgcatc ggaaactggg aaacttgagt gcagaagagg acagtggaac 660
tccatgtgta gcggtgaaat gcgtagatat atggaagaac accagtggcg aaggcggctg 720
tctggtctgt aactgacgct gaggctcgaa agcatgggta gcgaacagga ttagataccc 780
tggtagtcca tgccgtaaac gatgaatgct aggtgttgga gggtttccgc ccttcagtgc 840
cgcagctaac gcattaagca ttccgcctgg ggagtacgac cgcaaggttg aaactcaaag 900
gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag 960
aaccttacca ggtcttgaca tcttttgatc acctgagaga tcaggtttcc ccttcggggg 1020
caaaatgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080
ccgcaacgag cgcaaccctt atgactagtt gccagcattt agttgggcac tctagtaaga 1140
ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac 1200
ctgggctaca cacgtgctac aatggatggt acaacgagtt gcgagaccgc gaggtcaagc 1260
taatctctta aagccattct cagttcggac tgtaggctgc aactcgccta cacgaagtcg 1320
gaatcgctag taatcgcgga tcagcacgcc gcggtgaata cgttcccggg ccttgtacac 1380
accgcccgtc acaccatgag agtttgtaac acccgaagcc ggtggcgtaa ccctttagga 1440
gcgagcctct agtgcatgtt t 1461
<210> 8
<211> 1460
<212> DNA
＜213〉the secondary cheese subspecies (Lactobacillus paracasei subsp. paracasei) of lactobacillus paraceasi
<400> 8
ttttcggatg gcgcaatctg cagtcgacga gtctcgttga tgattggtgc ttgcccgaga 60
ttcaacatgg aacgagtggc ggacgggtga gtaacacgtg ggtaacctgc ccttaagtgg 120
gggataacat ttggaaacag atgctaatac cgcatagatc caagaaccgc atggttcttg 180
gctgaaagat ggcgtaagct atcgcttttg gatggacccg cggcgtatta gctagttggt 240
gaggtaacgg ctcaccaagg cgatgatacg tagccgaact gagaggttga tcggccacat 300
tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg 360
gacgcaagtc tgatggagca acgccgcgtg agtgaagaag gctttcgggt cgtaaaactc 420
tgttgttgga gaagaatggt cggcagagta actgttgtcg gcgtgacggt atccaaccag 480
aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc 540
ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg aaagccctcg 600
gcttaaccga ggaagcgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa 660
ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 720
gtctggtctg taactgacgc tgaggctcga aagcatgggt agcgaacagg attagatacc 780
ctggtagtcc atgccgtaaa cgatgaatgc taggtgttgg agggtttccg cccttcagtg 840
ccgcagctaa cgcattaagc attccgcctg gggagtacga ccgcaaggtt gaaactcaaa 900
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960
gaaccttacc aggtcttgac atcttttgat cacctgagag atcaggtttc cccttcgggg 1020
gcaaaatgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080
cccgcaacga gcgcaaccct tatgactagt tgccagcatt tagttgggca ctctagtaag 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtgcta caatggatgg tacaacgagt tgcgagaccg cgaggtcaag 1260
ctaatctctt aaagccattc tcagttcgga ctgtaggctg caactcgcct acacgaagtc 1320
ggaatcgcta gtaatcgcgg atcagcacgc cgcggtgaat acgttcccgg gccttgtaca 1380
caccgcccgt cacaccatga gagtttgtaa cacccgaagc cggggcgtaa ccctttagga 1440
gcagcgtcta tgagacggtt 1460
Claims (10)
1. microorganism is used in the red ginseng fermentation with sequence numbering 8 sequences.
2. microorganism is used in red ginseng fermentation according to claim 1, it is characterized in that, the main saponin more than in Rg1, Re, Rb1, Rc, Rb2 and Rd that mentioned microorganism can be contained with red ginseng converts the less important saponin more than in F2, Rg3, Rh2 and Compound-K to.
3. microorganism is used in red ginseng fermentation according to claim 1, it is characterized in that,
Mentioned microorganism present the secondary cheese subspecies of lactobacillus paraceasi (
Lactobacillus paracasei subsp. paracasei) homogeny of base sequence more than 99%.
4. microorganism is used in red ginseng fermentation according to claim 1, it is characterized in that, mentioned microorganism has the sustenance numbering of KCTC12108BP.
5. the manufacture method of a red ginseng fermented liquid, is characterized in that, comprises, adds claim 1 to a certain microorganism in claim 4 to stage that the red ginseng extraction liquid ferments.
6. red ginseng fermented liquid manufacture method according to claim 5, is characterized in that, also comprises, after above-mentioned fermentation stage, adds the stage that enzyme is processed.
7. red ginseng fermented liquid manufacture method according to claim 5, is characterized in that, during above-mentioned fermentation, adds sucrose or salt.
8. the manufacture method of red ginseng fermented liquid according to claim 5, it is characterized in that, the microbial culture medium that uses during above-mentioned fermentation is making more than of selecting from the group that Chinese cabbage, radish, circle romaine lettuce, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, rubber, potato and romaine lettuce form.
9. a red ginseng fermented drink that strengthens immune use, is characterized in that, comprises the red ginseng fermented liquid of making according to the manufacture method of claim 5.
10. a red ginseng fermented drink of alleviating cancer and preventing cancer, is characterized in that, comprises the red ginseng fermented liquid of making according to the manufacture method of claim 5.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104738636A (en) * | 2015-04-19 | 2015-07-01 | 通化青山实业集团有限公司 | Health oral liquid of fermented red ginseng |
| CN106498018A (en) * | 2016-11-07 | 2017-03-15 | 江南大学 | A kind of method that compound bacteria prepares the rare anticancer saponin Compound K of Radix Ginseng |
| CN109182441A (en) * | 2018-08-31 | 2019-01-11 | 吉林农业大学 | The preparation method and applications of ginsenoside Rb1's fermentation liquid |
| CN110591952A (en) * | 2019-09-24 | 2019-12-20 | 扬州大学 | Lactobacillus paracasei with capability of decomposing oil and fat and application thereof |
| CN113841888A (en) * | 2021-09-24 | 2021-12-28 | 安敏燮 | Red ginseng fermentation liquid and red ginseng fermentation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102147103B1 (en) * | 2018-08-24 | 2020-08-24 | 주식회사 진켐 | A pharmaceutical composition for immunity enhancement comprising Red Ginseng and sialyllactose |
| KR102175436B1 (en) * | 2018-09-03 | 2020-11-06 | 연세대학교 산학협력단 | A pharmaceutical composition for functional recovery of cord blood endothelial progenitor cells and treat of preeclampsia by Ginseng including Rg3 |
| KR102587892B1 (en) * | 2021-07-21 | 2023-10-12 | 주식회사 에치와이 | Lactobacillus Paracasei HY7017 With Enhanced Functional Characteristics and Enhanced Immunity Function by Using Red Ginseng as a Nutrient Source, and Use Thereof |
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| KR101295444B1 (en) | 2013-08-09 |
| KR20130079227A (en) | 2013-07-10 |
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