JP6616631B2 - TNF-α production promoter - Google Patents

Description

 The present invention relates to a kale-derived fermentation composition having TNF-α secretion promoting action.

Tumor necrosis factor (TNF) is one kind of cytokine, and in a narrow sense, TNF-α, TNF-β (lymphotoxin (LT) -α) and LT-β. TNF-α is mainly produced by macrophages and was discovered as a cytokine that causes hemorrhagic necrosis for solid tumors. Speaking of tumor necrosis factor generally refers to TNF-α. Also in the present invention, the abbreviation TNF means TNF-α. TNF-α was isolated in 1975 as a factor that induces hemorrhagic necrosis in tumors transplanted into mice, and the gene was cloned in 1984. TNF-α is produced as a membrane-bound TNF-α (mTNF-α), which is a precursor protein with a molecular weight of 25 kDa, but is cleaved by the TNF-α converting enzyme (TACE) on the carboxyl side terminal domain existing outside the cell. To 17 kDa soluble TNF-α (sTNF-α) protein (157 amino acid residues). Both mTNF-α and sTNF-α have activity. Furthermore, TNF-α forms a 51 kDa homotrimer and circulates in the blood. In addition to being produced mainly by activated macrophages, TNF-α is also the source of production of monocytes, T cells, NK cells, smooth muscle cells, and adipocytes. Such pharmaceuticals and foods that promote secretion of TNF-α are known. If TNF-α production by living bodies and immune cells can be promoted, it is effective for improving immunity and treating infection.
Patent Document 1 describes that an enzyme degradation product of soybean protein has an immune promoting effect, and that a peptide having a specific sequence contained therein promotes TNF-α secretion.

On the other hand, vegetable beverages, which are squeezed vegetable juices, are rich in various vitamins and minerals, and therefore demand is increasing as consumers become more aware of vegetable shortages and health-conscious. In particular, consumption of beverages containing green vegetables typified by green juice, which is the juice of kale, is increasing year by year. In particular, kale green juice is known to have various physiological activities, and is drunk in anticipation of physiological effects. For example, as physiological actions related to Kale's immunity, interleukin 4 inhibitory action (Patent Document 2), bradykinin receptor antagonistic action (Patent Document 3), anti-allergy / anti-inflammatory action (Patent Document 4) and the like are known. .
Moreover, the method of preparing the food which gave the constipation improvement action by using together green juice, such as a kale, and lactic acid bacteria (patent document 5), the method of fermenting green juice with lactic acid bacteria, and improving a bad smell (patent document 6), etc. Kale processing technology is known.

Japanese Patent Application Laid-Open No. 08-073374 Japanese Patent Laying-Open No. 2005-082552 JP 2003-335688 A JP 2003-267880 A Japanese Patent Laid-Open No. 2004-081105 JP 2002-027955 A

  This invention makes it a subject to obtain the fermented material derived from Kale which has a TNF- (alpha) production promotion effect.

  The present inventor has found that the fermentation product of kale improves the TNF-α production ability of immune cells in the process of producing fermented foods using kale as a raw material, and proposes the present invention.

The main configuration of the present invention is as follows.
(1) A TNF-α production promoter comprising a precipitate obtained by fermenting a mixture of kale and saccharide and then centrifuging the obtained fermented product as an active ingredient.
(2) The TNF-α production promoter according to (1), wherein the final pH of fermentation is 4 or less.
(3) A method for producing a TNF-α production promoter comprising the following steps.
1) Step of washing the kale leaf with light so that resident bacteria remain 2) Step of cutting the kale leaf 3) Mixing with a saccharide having a mass corresponding to the water content of the kale leaf, aerobic to facultative anaerobic Step of fermenting under conditions 4) Step of recovering a filtrate having TNF-α production promoting activity by filtering and removing solid matter after completion of fermentation.
5) Step of collecting precipitates by centrifugation

  A new TNF-α production promoter is provided. This TNF-α production promoter can be used as a preventive or therapeutic agent for infectious diseases and cancer.

The result of having measured according to the fermentation period of the TNF- (alpha) production promotion activity of the fermented material which fermented the mixture of a kale and brown sugar is shown. The measurement result of TNF- (alpha) production promotion activity of the 1st month, 1.5 months, and 2 months from the fermentation start of the fermented material which fermented the mixture of a kale and brown sugar is shown. An operation procedure for concentrating TNF-α production promoting activity by centrifuging the fermented product in the second month from the start of fermentation of the fermented product obtained by fermenting a mixture of kale and brown sugar. The supernatant fraction obtained by centrifuging from the fermented product when the activity of the fermented extract (A) in the second month from the start of fermentation of the fermented product obtained by fermenting a mixture of kale and brown sugar is 100% B) shows the relative activity of the precipitate residue fraction (C) by centrifugation.

The present invention relates to a TNF-α production promoter containing a fermented product obtained by fermenting a mixture of kale and sugar as an active ingredient.
In the fermentation, aerobic to facultative anaerobic fermentation is performed. As a fermentation method, a technique disclosed in Japanese Patent Application Laid-Open No. 9-294560 as a primary fermentation method can be employed. In this prior art fermentation method, in order to perform facultative aerobic fermentation, the operation of adding brown sugar to the plant raw material is the same, but the prior art further adds yeast to ferment and further ferment. It is different from the method.
More specifically, the following method can be illustrated.
First, kale and saccharide of the same amount as the amount of water contained in kale are mixed. This mixture is allowed to stand for a while, and is naturally fermented under facultative aerobic conditions by microorganisms (or resident bacteria) resident in the kale while exuding various components such as moisture and protein in the kale.
As an operation procedure for fermenting, first, fully mature kale leaves are collected, and then as pretreatment, the harvested leaves are lightly washed with water after harvesting, drained, and wounds and spoiled parts are excised in advance. To do. The kale is cut into a size of about 1 to 5 cm using a cutter or a cutter. Kale leaves cut in this way are mixed with sugar in the same amount as the amount of water contained in the kale in the fermentation vessel, loaded with cobblestone, and passed through microorganisms (resident bacteria) resident in the kale at the beginning of fermentation. Allow aerobic fermentation to occur. The saccharide mixed with the kale is preferably a saccharide mainly composed of sucrose such as brown sugar, tri-warm sugar, refined sugar, granulated sugar, and particularly preferably brown sugar. The amount of water in the kale is measured in advance or mixed with the same amount of water (equivalent to about 90%) disclosed in the 5th Food Standard Composition Table. Various components and proteins contained in kale are extracted by osmotic pressure when mixed with sugars. It is considered that these components are converted into a composition having TNF-α production promoting action by fermentation. The amount of water based on Kale's 5th standard food ingredient list is 90.2 g per 100 g. Therefore, in the case where the amount of water in kale is not measured, saccharides may be added with the aim of 90% weight of kale.
In a fermentation container, in order to maintain facultative aerobic fermentation conditions, a weight (weight) is placed on the lid and left to stand. The weight of the weight is 0.2 to 2 kg per 100 square centimeters. At the start of fermentation, agitation is performed once to several times a day, and then aerobic fermentation is continued for 2 to 10 months while maintaining a temperature of 25 to 35 ° C. In this fermentation process, fermentation by microorganisms (resident bacteria) that are resident in kale progresses simultaneously, and the kale tissue undergoes enzymatic degradation during the fermentation process, resulting in a mixture of liquid and solid viscous kale fermented products. It becomes. By filtering this fermented kale product with a wire mesh or the like, a fermented kale product having an effect of promoting TNF-α production, which is an object of the present invention, is obtained.

The TNF-α production promoting activity of the present invention was measured over time using the production amount of TNF-α activity (TNF-α production amount) as an index. As a result, the TNF-α production amount increased after one month from the start of fermentation. It has been found from the following test examples that the total amount of activity reaches a peak after 2 months and gradually decreases until 9 months. Therefore, in order to produce the TNF-α production promoter most efficiently, it is preferable to set the fermentation period to 2 months as shown in the Examples.
The measurement of TNF-α activity can be easily performed by an enzyme immunoassay method using a commercially available TNF-α antibody.
In this measurement method, a mouse lymphocyte-derived cell line RAW264.7 (manufactured by ATTC), which is a cell that differentiates into macrophages, is added to Dulbecco's Modified Eagle Medium (DMEM, GIBCO BRL) containing 10% fetal bovine serum (FBS). ), Seeded on a 24-well plate (manufactured by Nunc) at a rate of 10 ⁵ / well, and a test sample added to the cultured cell culture system, the substance having a TNF-α production promoting action is RAW 264.7. Is differentiated into macrophage-like cells and TNF-α is produced in the culture medium. Then, the amount of TNF-α in the cell culture medium is measured by an enzyme immunoassay method using a TNF-α antibody as described above.

  When the amount of TNF-α production increases with the fermentation and reaches the expected value, the fermentation is terminated. The target fermentation period is about 2 months at 25 ° C. After the fermentation, the fermented kale obtained by removing coarse solids by filtration may be subjected to solid-liquid separation as it is or further by centrifugation, and the resulting precipitate portion may be used as a TNF-α production promoter. it can. Moreover, in order to make it a TNF- (alpha) production promoter, it is preferable on handling that a fermented material is dried by various methods, and is set as a dried material. Since the TNF-α production promoting activity in the fermented product is mainly present in the solid part that precipitates by centrifugation, the liquid part is removed by microfiltration of the water in advance or by centrifugation, and the liquid part is removed. It can be set as the powder which has TNF- (alpha) production promotion activity more efficiently by making it dry. Any drying method can be employed for drying, but low temperature freeze drying is particularly preferred.

The powder having TNF-α production promoting activity obtained above may be made into a powder having a TNF-α production promoting activity and easy to handle by granulating starch as an excipient and a binder. it can. Specific granulation methods include fluidized bed granulation, stirring granulation, extrusion granulation, and the like. The apparatus normally used for these granulation methods can be used for this invention.
Among them, fluidized bed granulation is preferably used in the present invention. Fluidized bed granulation is preferable because a granulated product can be produced easily and in large quantities. Furthermore, according to fluidized bed granulation, the granulation operation can be carried out at a relatively low temperature, so that the destruction of nutrients can be suppressed, and a granulated product having a low bulk density is obtained. The flavor and texture can be made soft, and a granulated product with good water solubility is obtained, which is preferable.
This granulated product can be eaten and eaten in the same manner as the so-called green juice powder produced by squeezing kale leaves. For example, it may be eaten in addition to curry, stew, miso soup, steamed bread, hot cake, waffle, crepe and the like. It can also be taken with beverages such as water, hot water, milk, fruit juice, and soy milk.
Furthermore, it can be mixed with excipients, lubricants and the like, and tableted to form tablets. It can also be filled into gelatin or hydroxymethylcellulose capsules to form capsules.

Hereinafter, although an Example and a test example are shown and manufacture of the TNF- (alpha) production promoter of this invention is demonstrated, this invention is not limited to these Examples.
Test 1. Method for Measuring TNF-α Production Promoting Activity Measured by the following operation procedure (1) In a DMEM medium (hereinafter, DMEM medium) containing 10% FBS, 10,000 Unit / mL Penicillin and 10,000 μg / mL Streptomycin, 200 mM glutamic acid Murine leukemia cell line RAW264.7 (manufactured by ATCC) is cultured under conditions of 37 ° C. and 5% CO ₂ .
(2) RAW264.7 is peeled off with a solution containing 0.25% Trypsin and 1 mM EDTA · 4Na, and centrifuged (3000 rpm, 5 min) to collect cells.
(3) DMEM medium is added and the cell density is adjusted to 5.0 × 10 ⁵ cells / mL.
(4) 400 μL of cells are seeded in a 24 microwell plate and incubated at 37 ° C. in a 5% CO ₂ environment for 24 hours.
(5) After incubation, aspirate the DMEM medium in each well.
(6) Next, 495 μL of RPMI1640 medium containing 10,000 Unit / mL Penicillin and 10,000 Unit / mL Streptomycin and 5 μL of each measurement sample were added to each well, and incubated at 37 ° C. in a 5% CO ₂ environment for 24 hours. To do.
(7) The supernatant is collected, and the amount of TNF-α produced is measured using a TNF-α ELISA kit (manufactured by Thermo Scientific).
(8) The TNF-α production increased compared to the control (no sample added) compared to the TNF-α production amount of the cells without the test sample added (control) is defined as the TNF-α production promoting activity.

Test 2. Kale fermentation method Kale fermentation was carried out as follows.
(1) Wash 5.1 kg of kale leaves with water and drain thoroughly.
(2) Cut to about 5 cm with a cutter.
(3) Add 4.6 kg of brown sugar and mix well.
(4) Put in a plastic container (pickled barrel), put a lid, place 6 kg of weight on the lid, let stand at 25 ° C and let it ferment naturally.

Test 3. Changes in fermentation period and TNF-α production promoting activity After 1 month, 2 months, 6 months and 9 months from the start of fermentation, a liquid part was collected from the solid-liquid mixture of the fermented product, and this was used as a test sample. The TNF-α activity was measured by the method described in 1 above, and this was converted into the amount of TNF-α produced per 1 mL of the test sample to obtain TNF-α promoting activity. Changes in the TNF-α production promoting activity amount are shown in FIG.

  The amount of TNF-α production promoting activity was low (244 pg / mL) in the first month of fermentation, but rapidly increased (1768 pg / mL) in the second month. After that, it gradually decreased and became about 1512 pg / mL after 9 months from the start of fermentation.

Test 4. Test for Confirmation of Optimum Fermentation Condition Test 3 revealed that the time when the TNF-α production promoting activity was most enhanced during the fermentation period was the second month from the start of fermentation. During this period, in order to confirm what kind of change the fermentation environment shows, a fermented material is prepared in the same manner as in Test 2, and at the 3rd time point of the first month, the first month, the second month, and the second month, TNF-α production promoting activity amount, pH change, and microbiota change were observed. Changes in the microflora were determined by measuring changes in the number of general bacteria, lactic acid bacteria, and fungi (yeast) per 1 mL of fermented product using a standard agar medium, BCP-added plate count agar medium, and potato dextrose agar medium.
Changes in TNF-α production promoting activity are shown in FIG. Table 1 below collectively shows the measurement results of TNF-α production promoting activity, pH change, and microflora.

From FIG. 2, it was confirmed that the TNF-α production promoting activity increased three-fold between 1.5 months and 2 months from the start of fermentation. Therefore, in order to obtain a TNF-α production promoter, it became clear that it was necessary to ferment a mixture of kale and saccharides for at least 2 months.
On the other hand, it was found that the pH was lowered from pH 4.17 to pH 3.89. The microflora maintained the bacterial flora of the first month immediately after the start of fermentation for 1.5 months and 2 months, but the number of bacteria decreased. The decrease in the number of bacteria is presumed to be due to the autolysis of the cells accompanying fermentation.
From this test, it was predicted that the control of the fermentation period and pH was important for the TNF-α production promoting activity.

Test 5. Confirmation test of existence state of TNF-α production promoting activity in fermented product The following test was conducted to confirm the existence state of TNF-α production promoting activity present in the fermented product after 2 months of fermentation obtained in Test 4 Went.
The contents were taken out from the container 2 months after the start of fermentation and filtered using a fine wire mesh to collect the filtrate. A test was conducted using this solution. In addition, the filtered fermentation filtrate is called a fermentation extract (A) in the following tests.
1.0 mL of the fermented extract was centrifuged at 10,000 rpm for 10 minutes, and separated into two layers of a supernatant (supernatant) and a precipitate. The supernatant was then collected with a pipette. The collected supernatant was 0.984 mL.
Subsequently, the supernatant was microfiltered using a 0.45 μm Millipore filter, and the TNF-α production activity was measured by the method of Test 1 using the microfiltrate (B) as a sample.
In addition, the fraction of precipitate residue precipitated by centrifugation was collected separately. And purified water was added so that it might become 0.984 mL, and this was made into the TNF- (alpha) production activity measurement sample (C) similarly.
The above operation procedure is shown in FIG. 3 as a schematic diagram.
The TNF-α production activity measurement results of the microfiltrate (B) and the precipitation residue (B) were expressed as relative values with the activity of the fermentation extract (A) as 100%. The results are shown in FIG.
From the results of FIG. 4, it was confirmed that the TNF-α production activity in the fermented product was hardly present in the supernatant (B) but present in the precipitate residue fraction (C). The TNF-α production promoting activity was expected to be present in the fermented product in an insoluble state in water.

Claims (3)

  A TNF-α production promoter comprising a precipitate obtained by fermenting a mixture of kale and saccharide and then centrifuging the obtained fermented product as an active ingredient. The TNF-α production promoter according to claim 1, wherein the final pH of fermentation is 4 or less. The manufacturing method of the TNF- (alpha) production promoter which consists of the following process.
1) Step of washing the kale leaf with light so that resident bacteria remain 2) Step of cutting the kale leaf 3) Mixing with a saccharide having a mass corresponding to the water content of the kale leaf, aerobic to facultative anaerobic Step of fermenting under conditions 4) Step of recovering a filtrate having TNF-α production promoting activity by filtering and removing solid matter after completion of fermentation.
5) Step of collecting precipitates by centrifugation

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