JP6286128B2 - Fermentation and culture method, fermented eringi extract and blend thereof - Google Patents

Description

(the purpose)
Develop new materials that are not only novel but also highly appealing with regard to ingredients, manufacturing methods, effects, etc. and exhibit exceptional effects.

  Glycolipids of Pantoea agglomerans, a gram-negative bacterium, have oral and transdermal immunity regulating effects and are highly useful as functional food materials. Therefore, Pantoea agglomerans has a dietary experience (from the point of view of safety and security), is inexpensive, and is fermented and cultured in combination with food materials that image effects such as immune enhancement. There is a possibility that functional materials with high performance and effectiveness will be born.

  Edible materials that can be used for fermentation culture of Pantoea agglomerans may be vegetables, cereals, fruits, etc. (Patent Document 1), but mushrooms rich in β-glucan whose immunostimulatory action is already known are also candidates. . However, gram-negative bacteria fermented products based on mushroom-derived materials, especially fermented products using Pantoea agglomerans, have not been developed and are novel.

  Elingi, which is distributed as an edible mushroom, is popular with consumers as a food with a unique texture. Although the emphasis is on foods rather than functional ingredients, eringi is said to have a lifestyle-related disease prevention effect.

  By the way, about 30,000 tons of eringi are cultivated every year in Japan, but about 10% (3,000 tons of eringi (300 tons as dry powder)) is produced as a non-standard product for fresh food. Some are used as processed food, but some are discarded. Therefore, if this non-standard eringi can be used as a fermentation material for Pantoea agglomerans, it will become a highly functional material with a mushroom image, safety, security and low cost. In general, mushrooms have an image of effects such as prevention of liver dysfunction, elimination of constipation, osteoporosis, cancer prevention, prevention of hypertension, dieting, etc., in addition to an immune enhancing action. Therefore, it is fully considered that a new function is added to the function that has been reported so far in the Pantoea agglomerans fermentation product, or the function is synergistically increased, and if so, there is an inventive step.

International Publication No. 2005/0300938 JP 2012-082156 A International Publication No. 2007/102442

Krueger JM. Et al., J Physiol. 246: R994-9. (1984) Kimura M. et al., PsychiatryClin Neurosci. 53: 105-7. (1999) Van Dam AM. Et al., Neuroscience. 65: 815-26. (1995) Kimura M. et al., JNeuroscience, 20: 5544-5551. (2000)

  In this study, we examined whether eringi can be used for fermentation culture of Pantoea agglomerans.

  The fermentation and culturing method of the present invention is characterized in that eringi is fermented with pantoea agglomerans and the pantoea agglomerans are cultured at the same time.

  The fermented eringi extract of the present invention is obtained by the fermentation and culture method described above.

  The fermented eringi extract powder of the present invention is obtained from the fermented eringi extract.

  The fermented eringi extract composition of the present invention is characterized in that the fermented eringi extract or fermented eringi extract powder is blended.

  The fermented eringi extract blend is preferably a pharmaceutical, veterinary drug, quasi-drug, cosmetic, food, functional food, feed, or bath preparation.

  In addition, it is desirable that the fermented eringi extract composition is a sleep inducer.

It is a figure which shows the result of having confirmed culture | cultivation of Pantoea agglomerans in the culture medium which added eringi based on okara. It is a figure which shows the result which confirmed the effect of eringi addition to M9 culture medium. It is a figure which shows the result of having confirmed the pantoea agglomerans culture | cultivation by each eringi culture medium. It is a figure which shows the result of having confirmed eringi powder density | concentration and pantoea agglomerans culture | cultivation. It is a figure which shows the result of having confirmed eringi supernatant concentration and pantoea agglomerans culture | cultivation. It is a figure which shows the result of having confirmed the quantity of (beta) glucan in each eringi culture solution. It is a figure which shows the result of having confirmed the IL-1 (beta) induction from a THP-1 cell. It is a figure which shows the result of having confirmed the GM-CSF induction from THP-1 cells. It is a figure which shows the result of having confirmed the IL-1 (beta) induction from C8-B4 cell. It is a figure which shows the result of having confirmed the GM-CSF induction from C8-B4 cell.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings.

  Mushrooms are rich in β-glucan as a polysaccharide. However, it is generally estimated that gram-negative bacteria do not have the ability to degrade β-glucan into monosaccharides and cannot be used as carbon sources as they are. In addition, the mushroom component may contain a factor that inhibits bacterial growth. Therefore, in this study, the following experiment was conducted based on these conditions.

1. Is the eringi material resistant to pantoea agglomerans culture? Check with okara medium. Check with synthetic medium. Check the necessity of self-melting of eringi ^{* 1.}

2. Can pantoea agglomerans grow with eringi materials? ・ Examination with various processed eringi ・ Examination of the amount of eringi powder added (eringi powder is eringi fruit body (edible part) dried by warm air or freeze drying etc. (Crushed and powdered with a mixer etc.)

3. Measurement of β-glucan in fermentation culture In this experiment, as a preliminary experiment, all n = 1 (the number of tests performed under one condition. In other words, the number of tests performed under one condition is 1) I am considering it. The basic protocol is to prepare a medium in which eringi is added to a 50 ml tube, add pantoea agglomerans, and incubate overnight at 37 ° C. After that, collect the precipitate (pantoair agglomerans or okara or eringi solid residue) by centrifugation, add 1-5 ml of distilled water to this, treat at 90 ° C for 20 minutes and exceed 37 ° C for 15 minutes. Extract LPS (glycolipid) by sonication. Using the centrifuged supernatant as a sample, the amount of LPS is measured by Limulus activity (measured by Endspecy (Seikagaku Corporation)). Since there is a large error between experiments on the amount of proliferating cells depending on the culture conditions, this time, the standard medium LB (Luria broth) was used as the reference medium, and the limulus value of the LPS extracted sample of the cells obtained by culturing was 100 Displayed as%. Usually, when cultivated in LB medium, a limulus value of about 100 μg / ml is obtained. In addition, since there is a suspension, the amount of bacteria by absorbance is not measured.

* 1: Mushrooms contain β-glucan hydrolase. Therefore, mushrooms autolyze with their own enzymes and produce sugars that bacteria can use as nutrient sources.

(Influence of eringi on pantoea growth in okara medium)
Investigate the possibility that eringi inhibits Pantoea agglomerans culture. Consider with okara medium.

Exp. 1 Materials and methods
The composition of the medium per 100 ml is shown.
Formulation 1: Glucose (2 g) + M9 (Na2HPO4: 0.6 g, KH2 PO4: 0.3 g, NaCl: 0.05 g, NH4Cl: 0.1 g, 1 M MgSO4: 0.1 ml, 1 M CaCl2: 0.01 ml) ^{* 1}
Formula 2: Glucose (2g) + M9 + Okara (1g) ^{* 2}
Formula 3: Glucose (2g) + M9 + Okara (1g) + Eringi powder (1g) ^{* 3}
Formula 4: Glucose (2g) + M9 + Okara (1g) + Dried eringi (5g) extract ^{* 4}
Formula 5: Glucose (2g) + M9 + Okara (1g) + Fresh eringi (5g) extract ^{* 5}
LB medium: tryptone (1.0 g) + yeast extract (0.5 g), NaCl (0.5 g) ^{* 6}

Test Method 1 ml of Pantoea agglomerans pre-cultured in LB medium was added to each medium, and cultured with shaking at 35 ° C. for 24 hours. After incubation, the mixture was centrifuged at 3500 rpm for 10 minutes, collected, dissolved in 5 ml of distilled water, heated at 90 ° C. for 20 minutes, and then centrifuged at 3500 rpm for 10 minutes to prepare a supernatant. The amount of LPS in the supernatant was measured by endospecy.

* 1: M9 and distilled water were added to glucose (2 g) to 90 ml to prepare a suspension, which was autoclaved to prepare a medium. However, MgSO4 was separately melted in 10 ml, and after autoclaving, it was mixed with the others to make 100 ml of glucose medium.
* 2: Okara powder (Okara powdered with warm air) (1 g), glucose (2 g), M9, and distilled water were treated in the same manner as * 1 to prepare 100 ml of okara medium.
* 3: Okara powder (1g), eringi powder (the edible portion of eringi dried with warm air and then powdered) (1g), glucose (2g) are mixed, M9 and distilled water are added, and this is * In the same manner as in 1, 100 ml of Eringi powder medium was prepared.
* 4: 5 g of dried eringi (dried edible part dried with warm air) was soaked in distilled water for 8 hours, allowed to absorb water, removed water, sterilized with 70% ethanol and then aseptically removed. Crush it finely, leave it at 37 ° C for 2 days, mix 1g of karako powder with the liquid obtained at 3500rpm x 10min, add M9 and distilled water, and operate it in the same way as * 1 to add 100ml of dry eringi extract medium. Created.
* 5: Mix 5g of raw eringi purchased at the supermarket with liquid obtained from 70% ethanol after * 4, add 1g of okara powder, add M9 and distilled water, and operate as in * 1 Then, 100 ml of fresh eringi extract medium was prepared.
* 6: LB: LB medium (Tryptone 1.0
g, yeast extract 0.5 g, NaCl 0.5 g) was added distilled water to 100 ml to prepare a suspension, and autoclaved to prepare 100 ml of LB medium.

Results As shown in FIG. 1, pantoea agglomerans culture was not inhibited even when 1% eringi powder was added to okara medium (formulation 2) (formulation 3). On the other hand, the growth of pantoea agglomerans in the okara medium was enhanced by the addition of dry eringi extract (formulation 4).

(Eringi effect on pantoea agglomerans growth on M9 and glucose media)
Since it was found that eringi did not adversely affect the okara medium, it was examined whether it could be cultured in M9 medium excluding okara.
Preliminary investigations were made as to whether eringi inhibits Pantoea agglomerans culture in M9 and glucose medium.

Exp. 2
The prescription per 100 ml is shown. The adjustment method conforms to Exp.1.
Formula 1: Glucose 2g + M9 + eringi powder (5g)
Formula 2: Glucose 2g + M9 + Dried Eringi (5g) Extract Formula 3: Glucose 2g + M9 + Fresh Eringi (5g) Extract Formula 4: Glucose 2g + M9
LB: LB medium

Test Method 1 ml of Pantoea agglomerans pre-cultured in LB medium was added to each medium, and cultured with shaking at 35 ° C. for 24 hours. After incubation, the mixture was centrifuged at 3500 rpm for 10 minutes, collected, dissolved in 5 ml of distilled water, heated at 90 ° C. for 20 minutes, and then centrifuged at 3500 rpm for 10 minutes to prepare a supernatant. The amount of LPS in the supernatant was measured by endospecy.

Results As shown in FIG. 2, both the ering powder (formulation 1), the dried eringi extract (formulation 2), and the raw eringi extract (formulation 3) showed a pantoea agglomerans culturing effect equivalent to or better than that of glucose + M9 medium (formulation 4). .

(Proliferation of pantoea agglomerans on eringi medium)
Since the addition of eringi did not inhibit even in M9 medium, it was examined whether it could be cultured in a medium with only eringi, nitrogen and metal ions (M9) added.

Exp. 3
The prescription per 100 ml is shown. The adjustment method conforms to Exp.1.
Formulation 1: Eringi powder (1g) + M9
Formula 2: Dry eringi (5g) extract + M9
Formula 3: Raw eringi (5g) extract + M9
Formula 4: Glucose (2g) + M9
LB: LB medium

Test Method 1 ml of Pantoea agglomerans pre-cultured in LB medium was added to each medium, and cultured with shaking at 35 ° C. for 24 hours. After incubation, the mixture was centrifuged at 3500 rpm for 10 minutes, collected, dissolved in 5 ml of distilled water, heated at 90 ° C. for 20 minutes, and then centrifuged at 3500 rpm for 10 minutes to prepare a supernatant. The amount of LPS in the supernatant was measured by endospecy.

Results As shown in FIG. 3, it was revealed that pantoea agglomerans can be cultured only with the medium (formulations 1 to 3) added with each eringi and M9. In particular, ering powder (formulation 1) has a high culture effect. The above results indicate that eringi can be used as a carbon source.

(Examination of the amount of eringi powder in Pantoea agglomerans culture)
From the experimental results so far, it was found that eringi can be used without inhibiting the Pantoea agglomerans culture. Furthermore, pantoea agglomerans could be cultured with eringi and M9. Therefore, in order to increase the usefulness as a culture material, the amount of ering powder added was examined in order to prepare a sample containing an immunostimulating substance (first expected is β-glucan) from ering powder.

Exp. 4
The prescription per 100 ml is shown. The adjustment method conforms to Exp.1.
Formulation 1: Eringi powder (1g) + M9
Formula 2: Eringi powder (2g) + M9
Formula 3: Eringi powder (4g) + M9
LB: LB medium

Test Method 1 ml of Pantoea agglomerans pre-cultured in LB medium was added to each medium, and cultured with shaking at 35 ° C. for 24 hours. After incubation, the mixture was centrifuged at 3500 rpm for 10 minutes, collected, dissolved in 5 ml of distilled water, heated at 90 ° C. for 20 minutes, and then centrifuged at 3500 rpm for 10 minutes to prepare a supernatant. The amount of LPS in the supernatant was measured by endospecy.

Results As shown in FIG. 4, eringi powder (formulations 1 to 3) is not changed significantly by addition of 1 to 4% (eringi powder (1 g) corresponds to a concentration of 1%) and is equivalent to LB medium (formulation 4). Culture was possible.

(Pantoeia agglomerans culture study of eringi powder extract (C))
Since it may be difficult to cultivate industrially if powder enters Pantoea agglomerans culture, cultivation with eringi powder extract was studied.

Exp. 5
The prescription per 100 ml is shown. A suspension was prepared by suspending 5 g of eringi powder in 100 ml of distilled water. This was autoclaved at 120 ° C./20 minutes. The suspension was centrifuged at 3500 rpm for 10 minutes, and the supernatant was collected to obtain 5 g / 100 ml (5%) eringi powder extract. To 20ml, 40ml or 80ml, add salt to 10ml of distilled water (M9, Na2HPO4: 0.6g, KH2PO4: 0.3g, NaCl: 0.05g, NH4Cl: 0.1g, 1M CaCl2: 0.01ml) and autoclave Then, 1 ml of 0.1 M MgSO4 autoclaved separately was added, and autoclaved distilled water was added thereto to adjust the total volume to 100 ml, thereby preparing 100 ml of eringi powder extract medium of each concentration.
Formulation 1: Elingi powder (1g) extract + M9
Formula 2: Eringi powder (2g) extract + M9
Formula 3: Elingi powder (4g) extract + M9
LB: LB medium

Test Method 1 ml of Pantoea agglomerans pre-cultured in LB medium was added to each medium, and cultured with shaking at 35 ° C. for 24 hours. After incubation, the mixture was centrifuged at 3500 rpm for 10 minutes, collected, dissolved in 5 ml of distilled water, heated at 90 ° C. for 20 minutes, and then centrifuged at 3500 rpm for 10 minutes to prepare a supernatant. The amount of LPS in the supernatant was measured by endospecy.

Results As shown in FIG. 5, pantoea agglomerans growth with relatively good addition of ering powder (4 g) extract (formulation 3) was observed.

(Quantitative determination of β-glucan in eringi / pantoea culture medium)
The hot water extract after pantoea agglomerans culture in eringi medium contains β-glucan as a new immune activation component compared to the conventional pantoea agglomerans culture methods (wheat, soybean, rice, etc.) Be expected. Therefore, the amount of β-glucan in the hot water extract of eringi culture Pantoea agglomerans was quantified. (Ajinomoto intravenous lentinan is used as a standard substance)

Exp. 6-A
The sample used for the measurement is shown.
Formulation 1: Elingi powder (4g) + M9 culture extract (same as Pantoea agglomerans extract cultivated using Formula 3 of Exp.4)
Formula 2: Elingi powder (4g) extract + extract after M9 culture (same as Pantoea agglomerans extract cultivated using Formula 3 of Exp. 5)
LB (same as Pantoea agglomerans extract cultured with Exp.5 LB)
SL-100: Fermented wheat extract (a liquid obtained by culturing pantoea agglomerans with wheat as a medium component, extracted with hot water and containing 1% glycolipid)

Exp. 6-B
5% EP suspension: Elingi powder (4g) + M9 medium
5% EP supernatant: eringi powder (4g) extract + M9 medium

Test method The amount of β-glucan was measured by Fungitec G (Seikagaku Corporation).

Results As shown in FIG. 6, SL-100 (wheat fermentation extract) and LB culture hot water extract hardly contain β-glucan (FIG. 6-A). On the other hand, 114 mg / ml β-glucan in terms of lentinan was contained in the eringi powder-added medium (FIG. 6-B 5% EP suspension).

(Discussion)
Pantoea agglomerans showed growth equivalent to or better than that of LB medium even in a medium in which eringi was mixed with M9 (Fig. 3), and the glycolipid material cultured in a medium prepared from eringi powder had a high concentration (66 mg / ml). ) Β-glucan (FIG. 6-A Formula 1). That is, both the LPS (glycolipid) and β-glucan immunostimulatory substances exist in the hot water extract of Pantoea agglomerans culture using eringi as a culture material. So far, Pantoea agglomerans has been cultivated with flour, rice bran, okara, etc., but materials containing β-glucan have not been made into materials. In addition, it has been confirmed that the synergistic effect of β-glucan and LPS (glycolipid) is 2.5 times higher than the additive effect obtained independently (patent document 2, not disclosed on priority date). Based on the above, pantoea agglomerans cultures using eringi are novel, and when used as a fermentation substrate for pantoea agglomerans, an unanticipated effect such as a 10-fold increase in pantoair amount was observed, and progress was also made in production methods. It is clear that there is sex. Therefore, it was judged that it could be a candidate for a new material that has a high appeal for ingredients and production methods, and that exhibits exceptional effects. For this reason, it is considered that the ering fermented extract obtained by the described fermentation and culture method can induce health maintenance actions such as analgesia, sleep induction, infection prevention, antiallergy, anticancer, lifestyle-related disease prevention and the like. . In addition to humans, it can be applied to pets.

(Sleep-inducing activity of eringi / pantoea culture solution)
It has recently been shown that macrophages are present in the brain and have a nerve maintenance function. It is also known that a plurality of sleep inducers are produced from macrophages. For example, IL-1β, PGD2, which induces non-rapid eye movement (Non-REM) sleep, GM-CSF related to REM sleep and Non-REM sleep therapy, and the like have been suggested. In order to evaluate the sleep-inducing action of the hot water extract after pantoea agglomerans culture in a medium prepared from eringi powder at the cellular level, as a cultured cell of macrophages in the brain, C8-B4 cells of mouse microglia cell line and human As a macrophage model, highly versatile THP-1 cells were used to measure IL-1β and GM-CSF production inducing ability.

Exp. 7
Human macrophage cell line THP-1 cells were used.
As a control, purified glycolipid (LPSp) derived from Pantoea agglomerans was used. LPSp was set at 4 doses of 1 ng / ml, 10 ng / ml, 100 ng / ml and 1000 ng / ml at the LPSp concentration measured by ELISA (Patent Document 3). The hot water extract (fermented eringi extract) after pantoea agglomerans culture in eringi medium is diluted with RPMI-1640 medium (Wako Pure Chemical Industries, Ltd.) with LPSp concentration of 251 μg / ml measured by ELISA method. The LPSp concentrations were 1 ng / ml, 10 ng / ml, 100 ng / ml and 1000 ng / ml, respectively. Eringi powder is a RPMI containing 10% fetal calf serum (Hana-Nesco) at the same dilution rate as the Eringi fermentation extract by preparing an aqueous suspension of the same concentration as the Eringi powder concentration contained in the Eringi fermentation extract. Diluted with -1640 medium and used for testing.

The negative control was medium only. THP-1 cells were adjusted to 8 × 10 ⁴ cells / 100 μl / well and plated on 96-well flat bottom plates. 100 μl of the diluted sample was added to THP-1 cells and cultured in a CO ₂ incubator for 24 hours. 150 μl of the culture supernatant was collected and stored frozen at −80 ° C. until measurement. IL-1β and G-CSF in the culture supernatant were measured by a commercially available ELISA (Bioligend).

result
When LPSp and fermented eringi extract were added, a dose-dependent increase in IL-1β in the culture solution was observed (FIG. 7). Induction of IL-1β was not observed with eringi powder alone. The fermented eringi extract showed higher IL-1β values at all measured doses of 1-1000 ng / ml compared to LPSp.

  In the case of GM-CSF, the same result as IL-1β was obtained. When LPSp and fermented eringi extract were added, a dose-dependent increase in GM-CSF in the culture medium was observed (FIG. 8). In the case of eringi powder alone, GM-CSF was below the detection limit as in the negative control, and no induction was observed. In the fermented eringi extract, an excellent and unexpected effect was observed in which the GM-CSF value was extremely low compared to LPSp in a smaller amount than that of LPSp.

Exp. 8
Mouse microglia cell line C8-B4 cells were used.
The test operation was performed in the same manner as Exp.7. IL-1β and G-CSF in the culture supernatant were measured by a commercially available ELISA (Bioligend).

result
When LPSp, ering powder and fermented ering extract were added, a dose-dependent increase in IL-1β in the culture medium was observed (FIG. 9). The fermented eringi extract showed a higher IL-1β value than that of LPSp at the same concentration as LPSp 1000 ng / ml. This suggested that other components coexisting with LPSp acted additively and synergistically in the production of IL-1β. In the case of GM-CSF, the same result as IL-1β was obtained. When LPSp and fermented eringi extract were added, a dose-dependent increase in GM-CSF was observed in each culture medium (FIG. 10). In the case of eringi powder alone, GM-CSF was below the detection limit as in the negative control, and no induction was observed. The fermented eringi extract had an unexpected and excellent effect that, depending on the dose, it showed a very high GM-CSF value compared to LPSp in a smaller amount than LPSp.

Consideration
IL-1β has been reported to induce transient non-REM sleep by intravenous administration or intracerebroventricular administration to rabbits (Non-patent Document 1). In addition, it has been reported that GM-CSF induces both REM sleep and non-REM sleep by intraventricular administration (Non-patent Document 2). This time, the fermented eringi extract induced IL-1β and GM-CSF from microglia, indicating that the fermented eringi extract promotes the induction of non-REM sleep and REM sleep.

  LPS is known to induce IL-1β of microglia in the cerebral cortex, hippocampus, hypothalamus, etc. by intravenous injection or intraperitoneal injection into rats (Non-patent Document 3). Orally, there is a possibility that IL-1β and GM-CSF may be induced from microglia.

Although the relationship between sleep and growth hormone is known (Non-Patent Document 4), GM-CSF induces growth hormone by inducing somatostatin in the pituitary gland. The possibility of acting on promotion is also suggested.

Claims (5)

  A fermentation and culturing method comprising fermenting eringi with Pantoea agglomerans and simultaneously culturing the pantoea agglomerans.   An eringi fermented extract obtained by the fermentation and culture method according to claim 1.   A fermented eryngii extract powder obtained from the fermented eringi extract according to claim 2.   A fermented eringi extract composition comprising the fermented eringi extract according to claim 2 or the fermented eringi extract powder according to claim 3. The fermented eringi extract composition according to claim 4, wherein the fermented eringi extract composition is a pharmaceutical, veterinary drug, quasi-drug, cosmetic, food, functional food, feed, or bath preparation.

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