阅读说明：本技术 发酵姜黄及其制备方法 (Fermented turmeric and its preparation method ) 是由 黄荣增 宋成武 金姝娜 张丽君 舒劲松 丁英平 韩永明 陈成 向星亮 于 2021-08-09 设计创作，主要内容包括：本发明涉及姜黄制备技术领域,具体而言,涉及发酵姜黄及其制备方法。发酵姜黄的制备方法包括：利用冠突散囊菌对姜黄原料进行发酵。通过该制备方法能够有效减少姜黄中含有的挥发油成分,大大减小姜黄的刺激性气味,增加姜黄中水溶性更高的姜黄素类物质,使姜黄活性成分整体生物利用度有效提高,继而发酵姜黄具有更好的调节糖脂代谢作用、抗肿瘤作用、抗风湿性关节炎作用、抗氧化应激作用和调节肠道菌群作用等。(The invention relates to the technical field of turmeric preparation, and particularly relates to fermented turmeric and a preparation method thereof. The preparation method of the fermented turmeric comprises: the curcuma longa raw material is fermented by using eurotium cristatum. The preparation method can effectively reduce volatile oil components contained in the turmeric, greatly reduce pungent smell of the turmeric, increase curcumin substances with higher water solubility in the turmeric, effectively improve the overall bioavailability of active ingredients of the turmeric, and then the fermented turmeric has better functions of regulating glycolipid metabolism, resisting tumors, resisting rheumatoid arthritis, resisting oxidative stress, regulating intestinal flora and the like.)

1. A method for preparing fermented turmeric, comprising: the curcuma longa raw material is fermented by using eurotium cristatum.

2. The method of producing fermented turmeric according to claim 1, comprising: activating eurotium cristatum strains to form eurotium cristatum spore suspension, mixing the turmeric raw material with an inorganic salt buffer solution to form a fermentation raw material, and then inoculating the eurotium cristatum spore suspension into the fermentation raw material for fermentation.

3. The method of claim 2, wherein the activating step comprises: inoculating the eurotium cristatum strain into an activation culture medium for activation culture, and then collecting mature colonies to form a eurotium cristatum spore suspension;

preferably, the conditions of the activation culture include: the temperature is 30-35 ℃, the humidity is 40-60%, and the time is 7-8 days;

preferably, the activation medium comprises, in mass percent: 10-20% of potato extract powder, 40-45% of glucose and 40-45% of agar.

4. The method of claim 2, wherein the concentration of the sporangium coronarium spore suspension is 1 x 10⁶-1×10⁷cfu/mL；

The ratio of the turmeric raw material to the eurotium cristatum spore suspension is 10-20 g: 10 mL.

5. The method for producing fermented turmeric according to claim 2, wherein the inorganic salt buffer is a solution;

the conditions under which the fermentation feedstock is formed include: the initial pH of the inorganic salt buffer solution is 5.0-6.0, and 0.7-0.8g of potassium dihydrogen phosphate and 0.05-0.1g of dipotassium hydrogen phosphate are added to 10g of the turmeric raw material.

6. The method of producing fermented turmeric according to any one of claims 1 to 5, wherein the fermentation conditions include: the temperature is 25-35 ℃, the humidity is 50% -80%, and the fermentation time is 7-8 days.

7. The method for producing fermented turmeric according to any one of claims 1 to 5, wherein the turmeric material is pretreated before fermentation.

8. The method of claim 7, wherein the pre-treatment comprises removing impurities from the turmeric material, powdering and sterilizing.

9. The method of producing fermented turmeric according to claim 8, wherein the sterilization is autoclaving;

preferably, the conditions of the autoclaving include: the temperature is 121 ℃ and 135 ℃, and the sterilization time is 20-40 minutes.

10. Fermented turmeric, characterized in that it is produced by the method for producing fermented turmeric according to any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of turmeric preparation, and particularly relates to fermented turmeric and a preparation method thereof.

Background

The main effective component of the turmeric is curcumin. Turmeric is used as a medicine-food homologous traditional Chinese medicine in China, and is not like food additives, coloring agents and the like which are commonly used in western countries. Because the natural main active substances in the turmeric, namely curcumin components, belong to fat-soluble compounds and are insoluble in water, the turmeric has low bioavailability, low oral absorption rate, short biological half-life and poor stability, and the wide clinical application is severely restricted. And because the volatile oil smell in the turmeric is extremely pungent, the development and popularization of turmeric related processed products in China are limited.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide fermented turmeric and a preparation method thereof. The preparation method can effectively reduce volatile oil components contained in the turmeric, greatly reduce pungent smell of the turmeric, increase curcumin substances with higher water solubility in the turmeric, effectively improve the overall bioavailability of active ingredients of the turmeric, and then the fermented turmeric has better functions of regulating glycolipid metabolism, resisting tumors, resisting rheumatoid arthritis, resisting oxidative stress, regulating intestinal flora and the like.

The invention is realized by the following steps:

in a first aspect, the present invention provides a method for preparing fermented turmeric, comprising: the curcuma longa raw material is fermented by using eurotium cristatum.

In an alternative embodiment, the method comprises the following steps: activating eurotium cristatum strains to form eurotium cristatum spore suspension, mixing the turmeric raw material with an inorganic salt buffer solution to form a fermentation raw material, and then inoculating the eurotium cristatum spore suspension into the fermentation raw material for fermentation.

In an alternative embodiment, the activating comprises: inoculating the eurotium cristatum strain into an activation culture medium for activation culture, and then collecting mature colonies to form a eurotium cristatum spore suspension;

preferably, the conditions of the activation culture include: the temperature is 30-35 ℃, the humidity is 40-60%, and the time is 7-8 days;

preferably, the activation medium comprises, in mass percent: 10-20% of potato extract powder, 40-45% of glucose and 40-45% of agar.

In an alternative embodiment, the concentration of the eurotium cristatum spore suspension is 1 × 10⁶-1×10⁷cfu/mL。

In an alternative embodiment, the ratio of turmeric material to the eurotium cristatum spore suspension is 10-20 g: 10 mL.

In an alternative embodiment, the inorganic salt buffer is a solution prepared from potassium dihydrogen phosphate and dipotassium hydrogen phosphate;

the conditions under which the fermentation feedstock is formed include: the initial pH of the inorganic salt buffer solution is 5.0-6.0, and 0.7-0.8g of potassium dihydrogen phosphate and 0.05-0.1g of dipotassium hydrogen phosphate are added to 10g of the turmeric raw material.

In alternative embodiments, the fermentation conditions include: the temperature is 25-35 ℃, the humidity is 50% -80%, and the fermentation time is 7-8 days.

In an alternative embodiment, the turmeric feedstock is pre-treated prior to fermentation.

In an alternative embodiment, the pretreatment comprises subjecting the turmeric material to impurity removal, powdering and sterilization.

In alternative embodiments, the sterilization is autoclaving;

preferably, the conditions of the autoclaving include: the temperature is 121 ℃ and 135 ℃, and the sterilization time is 20-40 minutes.

In a second aspect, the present invention provides a fermented turmeric prepared by the method of producing fermented turmeric according to any one of the above embodiments.

The invention has the following beneficial effects: according to the embodiment of the invention, the volatile oil component contained in the obtained fermented turmeric can be effectively reduced by fermenting the turmeric with eurotium cristatum, so that the pungent smell of the obtained fermented turmeric is greatly reduced; meanwhile, through a microbial conversion technology, the curcumin substances with higher water solubility in the fermented turmeric can be effectively increased, so that the overall bioavailability of the active ingredients of the fermented turmeric is effectively improved. The fermented turmeric has better functions of regulating glycolipid metabolism, resisting tumor, resisting rheumatoid arthritis, resisting oxidative stress, regulating intestinal flora and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing the results of detection of fermented turmeric in Experimental example 1 of the present invention;

FIG. 2 is a graph showing the results of measurement of fermented turmeric of comparative example 13 according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides a preparation method of fermented turmeric, which comprises the following steps:

s1, preprocessing turmeric raw materials;

removing impurities from Curcuma rhizome raw material, cleaning, oven drying, pulverizing to obtain Curcuma rhizome dry powder, and collecting in container for use.

The turmeric dry powder is sterilized, and the sterilization in the prior art is within the scope of the embodiments of the present invention, but is preferably an autoclave, wherein the conditions of the autoclave include: the temperature is 121 ℃ and 135 ℃, and the sterilization time is 20-40 minutes. The sterilization method and the sterilization conditions are more favorable for the sterilization effect of the turmeric, and the influence of sterilization on active ingredients in the turmeric is reduced as much as possible, thereby being favorable for subsequent fermentation.

Potassium dihydrogen phosphate and dipotassium hydrogen phosphate are mixed and the ratio of the two is adjusted so that the pH concentration of the formed inorganic salt buffer is 5.0-6.0. And mixing the turmeric dry powder with an inorganic salt buffer solution to form a fermentation raw material, wherein 0.7-0.8g of monopotassium phosphate and 0.05-0.1g of dipotassium phosphate are added to each 10g of turmeric raw material.

S2, activating the Eurotium cristatum strain;

inoculating the pure eurotium cristatum strain into an activation culture medium for activation culture, and then collecting mature colonies to form a eurotium cristatum spore suspension; wherein the conditions of the activation culture comprise: the temperature is 30-35 ℃, the humidity is 40-60%, and the time is 7-8 days; the activation medium comprises the following components in percentage by mass: 10-20% of potato extract powder, 40-45% of glucose and 40-45% of agar. The activation condition is favorable for obtaining eurotium cristatum spores with better performance, and is further favorable for subsequent fermentation of turmeric.

And the concentration of the obtained eurotium cristatum spore suspension is 1 multiplied by 10⁶-1×10⁷cfu/mL. Using the above concentrationsIs beneficial to subsequent fermentation.

S3, fermenting;

inoculating the bursin crown bursitis spore suspension into a fermentation raw material for fermentation, wherein the ratio of the turmeric dry powder to the bursin crown bursitis spore suspension is 10-20 g: 10 mL. The fermentation conditions include: the temperature is 25-35 ℃, the humidity is 50% -80%, and the fermentation time is 7-8 days.

The fermentation conditions are favorable for fermenting the turmeric, and the performance of the obtained fermented turmeric is favorably improved.

And (4) after the fermentation is finished, drying the fermented turmeric powder again, and then putting the turmeric powder into a sealing bag for vacuum storage to obtain a finished product.

The embodiment of the invention provides fermented turmeric, which is prepared by the preparation method of the fermented turmeric.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment of the invention provides a preparation method of fermented turmeric, which comprises the following steps:

s1, preprocessing turmeric raw materials;

removing impurities from Curcuma rhizome raw material, cleaning, oven drying, and pulverizing to obtain Curcuma rhizome dry powder. 15 g of dry turmeric powder were weighed and then autoclaved at 121 ℃ for 20 minutes.

Then 0.8g of monopotassium phosphate and 0.05g of dipotassium phosphate are taken and dissolved in 5mL of sterile water to prepare a phosphate buffer solution with the pH value of 5.0, and the phosphate buffer solution is added into the 15 g of turmeric powder and mixed evenly to prepare a fermentation raw material for later use.

S2, activating the Eurotium cristatum strain;

inoculating the pure eurotium cristatum strain into an activation culture medium for activation culture, and then collecting mature colonies to form a eurotium cristatum spore suspension; wherein the conditions of the activation culture comprise: the temperature is 30 ℃, the humidity is 50%, and the time is 7 days; the activation medium comprises: PDA culture medium containing 12% potato extract powder, 45% glucose and 43% agar to obtain Eurotium cristatum spore suspension with concentration of 4 × 10⁶cfu/mL。

S3, fermenting;

mixing 10g of fermentation raw material with 10ml of the eurotium cristatum spore suspension for fermentation, wherein the fermentation conditions comprise: the temperature was 30 ℃ and the humidity was 70% for 7 days.

Freeze drying fermented powder to obtain final product, and sealing for storage.

Test example 1

Identifying curcumin components based on time-of-flight LC-MS analysis:

preparation of a sample to be tested: about 100mg of the dried sample was taken, accurately weighed, added with 5mL of 80% methanol, ultrasonically extracted for 10min, and then centrifuged at 4000rpm, and the supernatant was taken. The steps are respectively and repeatedly extracted once by using 50 percent methanol and pure methanol, the three extracting solutions are combined, and a sample to be detected is obtained by filtering through a 0.22 mu m microporous filter membrane.

The instrument model is as follows: waters ACQUITY UPLC M-Class system.

Chromatographic conditions are as follows: a chromatographic column: waters ACQUITY UPLC BEH C18 reverse phase chromatography column (100X 2.1mm,1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 40 ℃; sample introduction amount: 2.0 mu L; mobile phase a was 0.1% formic acid water, mobile phase B was acetonitrile, chromatographic elution gradient: 0.01min, 20% B; 10min, 35% B; 30min, 55% B; 40min, 95% B; 43min, 95% B; 43.1min, 20% B; 46min, 20% B.

Mass spectrum conditions: an electrospray ion source; MS (Mass Spectrometry)^eMode collection of fragments dissociated by primary and secondary mass spectra; ESI ion source temperature: 100.0 ℃; electrospray voltage: 3 kV; drying gas: flow rate 600 L.h^-1At a temperature of 250 ℃; taper hole air flow rate: 50 L.h^-1(ii) a First-order collision dissociation energy: 10V; secondary collision dissociation energy: 30V; the first-stage and second-stage full-scan mass number ranges are: m/z 100-1000.

Test example 2

In vitro measurement of pharmacological activity index of sample:

(1) in vitro alpha glucosidase inhibition rate measurements:

preparation of a sample to be tested: the same as in test example 1.

Sample testingDetermining: first, the concentration of the mixture was adjusted to 0.1 mol. L^-1Phosphate Buffered Saline (PBS) at pH 6.8 was used as a solvent in the measurement process. 50 μ L of the sample was mixed with 200 μ L of LPBS and 50 μ L of alpha-glucosidase (1U. mL)^-1) Mix and incubate at 37 ℃ for 15 min. Then 150 mu L of p-nitrophenol-beta-glucuronide (p-NPG, 5 mmol. L^-1) Added as a substrate to the reaction system and reacted at 37 ℃ for 20 min. Finally 300. mu.L of sodium carbonate (0.2 mol. L) is added^-1) The reaction was terminated. The mixed system without the test sample added served as a negative control. The absorbance of the sample was measured at a wavelength of 405nm using a microplate reader, and the inhibition rate was calculated.

Calculating the formula:

wherein A is₁Is the absorbance of the reacted solution added with the test sample, A₀Absorbance of the negative control sample.

(2) In vitro free radical clearance measurement:

preparation of a sample to be tested: the same as in test example 1.

And (3) sample determination: 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) was prepared to a concentration of 0.1 mmol.L using 95% methanol as a solvent in the measurement^-1The solution of (1). 50. mu.L of the sample and 250. mu.L of DPPH solution were reacted at room temperature in the dark for 1 hour and then measured. The mixed system without the test sample added served as a negative control. The absorbance of the sample was measured at a wavelength of 515nm using a microplate reader, and the clearance was calculated.

Calculating the formula:

wherein A is₁Is the absorbance of the reacted solution added with the test sample, A₂Absorbance of the test solution, A₀Absorbance of the negative control sample.

Test example 3

Detecting various indexes of blood fat of Kunming mice:

preparation of a sample to be tested: performing intraperitoneal injection anesthesia on the target mouse with sodium pentobarbital, taking blood from eyeball, standing for 0.5h at 5000 r.min^-1Centrifuging for 10min, and collecting 1mL of serum at-80 deg.C.

(1) Determination of serum Total Cholesterol (TC):

the determination of serum total cholesterol levels was performed according to the instructions of the total cholesterol (T-CHO) test kit, as follows:

TC content determination operation table:

the TC content was calculated from the absorbance values measured for each well.

Calculating the formula:

wherein A1 is sample well absorbance, A2 is calibration well absorbance, A0 is blank well absorbance, and C is calibrator concentration (mmol. L)^-1)。

(2) Measurement of Low Density lipoprotein Cholesterol (LDL-C):

the measurement of serum low-density lipoprotein cholesterol level was performed according to the instructions of the low-density lipoprotein cholesterol (LDL-C) test kit, and the specific procedures were as follows:

LDL-C assay protocol:

LDL-C content was calculated from the absorbance values measured for each well.

Calculating the formula:

wherein A1 is sample well absorbance, A2 is calibration well absorbance, A0 is blank well absorbance, and C is calibrator concentration (mmol. L)^-1)。

(3) Determination of high Density lipoprotein Cholesterol (HDL-C):

the measurement of serum high density lipoprotein cholesterol content was performed according to the specification of the high density lipoprotein cholesterol (HDL-C) test kit, and specifically as follows:

HDL-C assay protocol:

HDL-C content was calculated from the absorbance values determined for each well.

Calculating the formula:

wherein A1 is sample well absorbance, A2 is calibration well absorbance, A0 is blank well absorbance, and C is calibrator concentration (mmol. L)^-1)。

Identification

The number of components of turmeric dry powder (unfermented) and fermented turmeric in example 1, as well as the number of different water-soluble substances, were determined using chromatographic conditions in time-of-flight LC-MS analysis, as described in test example 1, with the following table and FIG. 1:

note: compounds that peaked earlier than 30% acetonitrile were defined as highly water soluble, compounds that peaked at 30% acetonitrile and 45% acetonitrile as moderately water soluble, and compounds that peaked later than 45% acetonitrile as poorly water soluble, according to the mobile phase gradient used for time-of-flight LC/MS analysis. From the above table, it can be seen that the curcuminoid components are classified into a high water-soluble group, a medium water-soluble group and a low water-soluble group according to the retention time based on the chromatographic conditions in the time-of-flight LC-MS analysis. The sample was found to have a significant increase in the content of 7 components in the highly water soluble group after fermentation.

Examples 2 to 3

Fermented turmeric was prepared according to the preparation method provided in example 1, except that the temperature for activating and culturing the Eurotium cristatum strain was different, as specifically shown in the following table:

as can be seen from the above table, the Eurotium cristatum strain grows best at a temperature of 30-33 ℃. Wherein the diameter of the colony is the largest and the number of colonies is the largest at 30 ℃.

Examples 4 to 8

Fermented turmeric was prepared according to the preparation method provided in example 1, with the only difference that the humidity of the activated cultured Eurotium cristatum strain was different, see in particular the following table:

according to the above table, it can be seen that the growth vigor of the Eurotium cristatum strain is not obviously different when the humidity is 40% -60%. Wherein at 50 ℃, the diameter of the colony is the largest, and the number of the colony is large.

Examples 9 to 10

Fermented turmeric was prepared according to the preparation method provided in example 1, except that the time for activating the cultured bursa species was varied, as specifically shown in the following table:

as can be seen from the above table, the Eurotium cristatum strain gradually grows to the mature stage within 7 days, wherein the 7 th day can be judged as the optimal collection time according to the colony diameter and color.

Example 11 example 14

Fermented turmeric was prepared according to the preparation method provided in example 1, differing only in the fermentation conditions, see test example 2, specifically the mass ratio of fermentation feedstock to spore suspension, see table below:

according to the table, the quality of the turmeric fermentation raw material is within 10g-20g, the eurotium cristatum can keep higher conversion capability and has higher alpha glucosidase inhibition rate and free radical clearance rate by using the method for fermentation.

Example 15 example 17

Fermented turmeric was prepared according to the preparation method provided in example 1, differing only in the fermentation conditions, see test example 2, specifically the fermentation temperature, see in particular the following table:

according to the table, the Eurotium cristatum can keep higher conversion capability and has higher alpha glucosidase inhibition rate and free radical clearance rate in the fermentation temperature range of 25-35 ℃.

Examples 18 to 20

Fermented turmeric was prepared according to the preparation method provided in example 1, differing only in the fermentation conditions, see test example 2, specifically the humidity of the fermentation, see in particular the following table:

according to the table, the Eurotium cristatum can keep higher conversion capability and has higher alpha glucosidase inhibition rate and free radical clearance rate in the fermentation humidity range of 50% -80%.

Comparative example 13: fermented turmeric was prepared according to the preparation method provided in example 1, except that eurotium cristatum was replaced with monascus, and the number of active ingredients in the obtained fermented turmeric was as shown in fig. 2.

The fermented turmeric of comparative example 13 and example 1 were tested for alpha glucosidase inhibition and free radical scavenging, respectively, as described in test example 2, and the results are shown in the following table:

according to the above table, it can be seen that the eurotium cristatum fermented turmeric has higher alpha glucosidase inhibition rate and free radical scavenging rate than those of monascus fermented turmeric and unfermented turmeric, and reflects that the eurotium cristatum fermented turmeric has better pharmacological activity of regulating blood sugar and scavenging free radicals to a certain extent.

The fermented curcuma longa obtained in comparative example 13 and example 1 was tested for pharmacological activity on regulation of blood lipid metabolism in hyperlipidemic mice, respectively, as described in test example 3, and the results are shown in the following table:

according to the above table, it can be seen that the eurotium cristatum fermented turmeric can significantly regulate serum Total Cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) of the hyperlipidemic mouse, compared with the monascus fermented turmeric and the unfermented turmeric, which indicates that the eurotium cristatum fermented turmeric has a better regulating effect on the hyperlipidemic mouse.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.