CN114181987A - Biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside - Google Patents
Biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside Download PDFInfo
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Abstract
The invention provides a biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside. The method comprises the following steps: (1) culturing Aspergillus niger cells or Aspergillus oryzae cells for growth and development, and preparing Aspergillus niger whole-cell catalyst or Aspergillus oryzae whole-cell catalyst; (2) dissolving the neohesperidin dihydrochalcone in distilled water, adding an Aspergillus niger whole-cell catalyst or an Aspergillus oryzae whole-cell catalyst, mixing uniformly to obtain a reaction system, biologically catalyzing the neohesperidin dihydrochalcone to perform hydrolysis reaction, centrifuging supernate after hydrolysis, performing analysis and identification, and simultaneously obtaining the hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside. Compared with the traditional chemical catalytic hydrolysis and enzyme preparation catalytic hydrolysis, the method has higher practicability and operability. The hydrolysate has better water solubility and better physiological activity than neohesperidin dihydrochalcone.
Description
Technical Field
The invention belongs to the technical field of food additives, and particularly relates to a biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
Background
In recent years, with the development of society, obesity has become a health problem that people need to solve urgently. The main cause of obesity is the lack of exercise and the great relationship with the individual's eating habits. It is known that sweetness plays an important role in people's daily diet, but if a high-sweetness substance is excessively taken, obesity, even diabetes and other chronic diseases are easily caused. Therefore, scientists have focused on developing a new sweetener to reduce calorie intake while maintaining the sweet taste of food, thereby ensuring human health while satisfying the normal diet of people.
The sweetener can be artificial synthetic sweetener and natural sweetener according to its source. Although the artificial synthetic sweetener is widely applied to the market at present, a great deal of research shows that excessive intake of the artificial synthetic sweetener can cause certain damage to human bodies. Therefore, natural sweeteners are the focus of current research. Natural sweeteners are natural high-sweetness, low-calorie, even non-caloric sweeteners extracted from plants. It has the characteristics of high sweetness, low calorie, no toxicity and the like, so that the sugar-free sweet potato is widely concerned by various social circles. Besides, partial natural sweeteners are found to have the effects of resisting oxidation, enhancing immunity, enhancing toughness of capillary vessels and the like, and are considered to be sweeteners with great potential. At present, the more common compounds comprise neohesperidin dihydrochalcone, stevioside, glycyrrhizin, mogroside and the like.
Neohesperidin dihydrochalcone is a natural sweetener from citrus, has wide raw material source, sweetness, low heat and strong stability, and is used in the production process of various foods at present. However, because the neohesperidin dihydrochalcone has the defects of poor water solubility, slow sweetness and the like, the neohesperidin dihydrochalcone needs to be compounded with other sweeteners in the using process, and the application prospect is hindered to a certain extent. Researches find that the neohesperidin dihydrochalcone is hydrolyzed to remove rhamnose bond or remove rhamnose bond and glucoside bond at the same time, the sweetness of hydrolysis products of the neohesperidin dihydrochalcone and the hesperetin dihydrochalcone-7-O-glucoside is 1500-fold and 1800 times of that of cane sugar, and the sweetness is similar to that of neohesperidin dihydrochalcone sold in the current market, and the sweetness is purer and the sweetness feedback is quicker. Meanwhile, compared with neohesperidin dihydrochalcone, the hydrolysis product hesperetin dihydrochalcone-7-O-glucoside and hesperetin dihydrochalcone have a certain degree of improvement in water solubility and a series of physiological activities. Therefore, the hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside are considered to be more capable of replacing cane sugar to participate in food application, and have immeasurable development prospect.
At present, hydrolysis aiming at flavonoid derivatives mainly comprises chemical hydrolysis and enzymatic hydrolysis. Zhang Yingzhen et al prepared hesperetin dihydrochalcone glucoside by hydrolysis of Yunnan Xiang glycosyl in acid solution (Zhang Yingzhen, Chaihong, New sweetener, synthesis research of hesperetin dihydrochalcone glucoside, fine chemical industry, 1988, 6: 39-41). Patent CN110950747A describes a method for obtaining hesperetin dihydrochalcone by hydrolyzing neohesperidin dihydrochalcone under acidic conditions, but the chemical hydrolysis has the defects of poor selectivity, difficult control of reaction, more byproducts, environmental pollution and the like. Liu et al used immobilized enzymes to hydrolyze neohesperidin dihydrochalcone (Aolu Liu, Baohua Huang, Lin Lei, et al.production of high antibiotic activity flavonoid from flavonoid peptides with immobilized a-L-rhodopsin in one step [ J ]. International Journal of Food Science and Technology,2019,54: 2854; 2862). However, the enzyme method has the disadvantages of complicated separation and purification of related enzymes, high cost and the like. Therefore, there is an urgent need to develop a new technology for efficiently hydrolyzing flavonoid derivatives. The whole-cell catalysis technology is considered as a novel and effective method for biologically catalyzing and hydrolyzing flavonoid derivatives. The whole-cell catalytic hydrolysis can hydrolyze the flavonoid derivatives according to specific hydrolase in cells. The method not only can effectively improve the disadvantages of a chemical method and an enzymatic method, but also has huge potential in industrial application due to low cost.
Disclosure of Invention
The invention aims to provide a biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside, wherein the hesperetin dihydrochalcone and the hesperetin dihydrochalcone-7-O-glucoside can be used as potential sweeteners and applied to foods, medicines and feeds.
A biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside takes neohesperidin dihydrochalcone as a starting material and is used for preparing the hesperetin dihydrochalcone and the hesperetin dihydrochalcone-7-O-glucoside through biocatalysis by an Aspergillus niger whole-cell catalyst or an Aspergillus oryzae whole-cell catalyst, and the method comprises the following specific steps:
(1) culturing Aspergillus niger cells or Aspergillus oryzae cells for growth and development, and preparing Aspergillus niger whole-cell catalyst or Aspergillus oryzae whole-cell catalyst;
(2) dissolving the neohesperidin dihydrochalcone in distilled water, adding an Aspergillus niger whole-cell catalyst or an Aspergillus oryzae whole-cell catalyst, mixing uniformly to obtain a reaction system, biologically catalyzing the neohesperidin dihydrochalcone to perform hydrolysis reaction, centrifuging supernate after hydrolysis, and analyzing and identifying to obtain the hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
Further, the Aspergillus niger cells in step (1) can be Aspergillus niger GDMCC 3.23, derived from Aspergillus niger van Tieghem CGMCC 3.939, or can be general-purpose Aspergillus niger cells; the Aspergillus oryzae cells described in step (1) may be Aspergillus oryzae GDMCC 3.451 derived from Aspergillus oryzae (Ahlburg) CohnCICC 2066, or may be general-purpose Aspergillus oryzae cells.
Further, the mass ratio of the neohesperidin dihydrochalcone in the step (2) to the aspergillus niger whole-cell catalyst or the aspergillus oryzae whole-cell catalyst is 1:1-1: 10.
Further, the volume ratio of the neohesperidin dihydrochalcone substance in the step (2) to distilled water is 1:2-6:1 mmol/L.
Further, the temperature of the hydrolysis reaction in the step (2) is 30-60 ℃, and the time of the hydrolysis reaction is 1-48 h.
Further, the rotating speed of the centrifugation in the step (2) is 8000rpm-16000rpm, and the centrifugation time is 1-10 min.
Further, the specific method for analyzing and identifying in the step (2) is as follows: and (3) diluting the chromatographic grade methanol, then filtering the diluted methanol by using an organic phase filter membrane, injecting a sample by using a high performance liquid chromatography autosampler, and carrying out chromatographic and spectral comparison with the standard substances of hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside to obtain the hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
Further, the specific method for analyzing and identifying in the step (2) is as follows: diluting with chromatographic grade methanol 1-50 times, filtering with 0.22 μm organic phase filter membrane, injecting 20 μ L sample with high performance liquid chromatography autosampler, and comparing with standard substance of hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside by chromatography and spectrum to obtain hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
Further, the aspergillus niger whole-cell catalyst or the aspergillus oryzae whole-cell catalyst biologically catalyzes neohesperidin dihydrochalcone to carry out hydrolysis reaction, disaccharide glycoside can be subjected to hydrolysis reaction to generate hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside, and the conversion rate is as high as 80% or more.
The hesperetin dihydrochalcone-7-O-glucoside provided by the invention has a chemical structural formula
The hesperetin dihydrochalcone provided by the invention has a chemical structural formula
The sweetness of the hesperetin dihydrochalcone-7-O-glucoside and the hesperetin dihydrochalcone prepared by the invention is 1500-fold and 1800-fold of that of sucrose, is similar to that of neohesperidin dihydrochalcone, and can be widely applied to food, medicine and feed as a sweetening agent.
The present invention belongs to a food additive.
The existing process for hydrolyzing the neohesperidin dihydrochalcone is hydrolysis by a chemical method or hydrolysis by an enzymatic method. The chemical hydrolysis has the defects of poor selectivity, difficult control of reaction, more byproducts, environmental pollution and the like, and the enzymatic hydrolysis has the disadvantages of complex separation and purification of related enzymes, high cost and the like.
Compared with the prior art, the invention has the following advantages:
the method adopts aspergillus niger or aspergillus oryzae whole cell to catalyze the hydrolysis of the neohesperidin dihydrochalcone, avoids the defects of difficult control of a chemical method on reaction, environmental pollution and the like, simultaneously avoids the defects of high preparation cost, complex separation and purification and the like of related enzymes in an enzyme method, and has practicability and operability on the hydrolysis of the neohesperidin dihydrochalcone to generate the hesperetin dihydrochalcone-7-O-glucoside and the hesperetin dihydrochalcone. Meanwhile, the aspergillus niger whole-cell catalyst or the aspergillus oryzae whole-cell catalyst has the advantages of high substrate conversion rate, good reaction selectivity, mild reaction conditions, simple preparation, good stability, reusability, low price and the like.
Drawings
FIG. 1 is a high performance liquid chromatogram of a neohesperidin dihydrochalcone standard.
FIG. 2 is a high performance liquid chromatogram and a mass spectrum of a liquid chromatography-mass spectrometry combination for catalyzing the hydrolysis of neohesperidin dihydrochalcone to produce hesperetin dihydrochalcone-7-O-glucoside and hesperetin dihydrochalcone by using Aspergillus niger whole cells in example 1.
FIG. 3 is a high performance liquid chromatogram obtained in example 2 by separating and purifying hesperetin dihydrochalcone-7-O-glucoside through hydrolysis of neohesperidin dihydrochalcone catalyzed by Aspergillus oryzae whole cells.
FIG. 4 is a high performance liquid chromatogram obtained after separation and purification of hesperetin dihydrochalcone produced by using Aspergillus oryzae whole cell to catalyze neohesperidin dihydrochalcone to hydrolyze in example 2.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the scope of the invention as claimed is not limited to the following examples.
FIG. 1 is a high performance liquid chromatogram of the neohesperidin dihydrochalcone standard, and it can be observed from FIG. 1 that the retention time of the neohesperidin dihydrochalcone standard is 4.4 min.
Example 1
(1) Plate activation was performed 1 time (28 ℃, 60h) by inoculating 1-ring strain from the slant of the A.niger GDMCC 3.23 tube into PDA medium, the spores were dissolved in 5mL of sterile water, and the spore suspension (inoculum size 3%, v/v) was inoculated into the fermentation medium (500mL Erlenmeyer flask).
And after shaking culture for 48h, removing the culture solution by suction filtration to obtain wet thalli. The wet thallus is washed by distilled water or PBS buffer solution (pH 7.4) for 3 times to remove the culture medium remained by the thallus, and then is frozen and dried in vacuum at the temperature of minus 45 ℃ for 24 hours to obtain the freeze-dried thallus which is the aspergillus niger whole-cell catalyst.
(2) Adding 10mmol neohesperidin dihydrochalcone and 20mL distilled water into a 50mL conical flask in sequence, oscillating and dissolving on a vortex oscillator, then adding 200mg aspergillus niger whole-cell catalyst, mixing uniformly, sealing the bottle mouth, and placing in a full-temperature shaking bed cabinet for reaction (30 ℃, 180 rpm). After sampling 50 μ L at 0h, 1h, 2h, 3h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h and 48h, centrifuging for 1min at 8000rpm, diluting by 50 times with chromatographic grade methanol, filtering with 0.22 μm organic phase filter membrane, and injecting 20 μ L with high performance liquid chromatography autosampler for HPLC analysis (detection wavelength 283nm, mobile phase: methanol (A)/pure water (B), 50%/50%, flow rate 1.0 mL/min). The products are identified as hesperetin dihydrochalcone-7-O-glucoside and hesperetin dihydrochalcone respectively. The HPLC chromatogram and the MS-MS spectrum of 48h of hydrolysis reaction are shown in FIG. 2, and it can be seen from (a) in FIG. 2 that the Aspergillus niger whole-cell catalyst has catalytic hydrolysis effect on neohesperidin dihydrochalcone. The mass spectrum of the liquid chromatography-mass spectrometry after 48h of hydrolysis reaction is shown in (b) in fig. 2, and it can be known from (b) in fig. 2 that the aspergillus niger whole-cell catalyst catalyzes the hydrolysis of neohesperidin dihydrochalcone to prepare hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
Example 2
(1) Plate activation was performed 1 time (28 ℃, 60h) by inoculating 1-ring strain from the tube slant of Aspergillus oryzae GDMCC 3.451 into PDA medium, dissolving spores in 5mL of sterile water, and inoculating the spore suspension (inoculum size 3%, v/v) into fermentation medium (500mL Erlenmeyer flask).
And after shaking culture for 48h, removing the culture solution by suction filtration to obtain wet thalli. Washing wet thallus with distilled water or PBS buffer solution (pH 7.4) for 3 times to remove culture medium residual in thallus, and vacuum freeze-drying at-45 deg.C for 24 hr to obtain freeze-dried thallus which is Aspergillus oryzae whole cell catalyst.
(2) Adding 180mmol (neohesperidin dihydrochalcone and 60mL distilled water in a 150mL conical flask in sequence, oscillating and dissolving on a vortex oscillator, adding 80mg of aspergillus oryzae whole-cell catalyst, mixing uniformly, sealing the bottle mouth, placing in a full-temperature shaking bed cabinet for reaction (45 ℃, 180rpm), sampling 50 mu L in 0h, 1h, 2h, 3h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h and 48h respectively, centrifuging at 12000rpm for 5min, diluted 50 times with chromatographic grade methanol, filtered through a 0.22 μm organic phase filter, and injected into a 20 μ L high performance liquid chromatography autosampler for HPLC analysis (detection wavelength 283nm, mobile phase: methanol (A)/pure water (B), 50%/50%, flow rate 1.0mL/min), identifying the products as hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside respectively.
FIG. 3 is a high performance liquid chromatogram obtained after separation and purification of example 2, which shows that the retention time of the separated and purified hesperetin dihydrochalcone-7-O-glucoside is 4.7min, wherein the hydrolysis of neohesperidin dihydrochalcone is catalyzed by aspergillus oryzae whole cells to generate hesperetin dihydrochalcone-7-O-glucoside. FIG. 4 is a high performance liquid chromatogram obtained after separation and purification of hesperetin dihydrochalcone produced by using Aspergillus oryzae whole cell to catalyze neohesperidin dihydrochalcone to hydrolyze in example 2, and it can be observed from FIG. 4 that the retention time of the hesperetin dihydrochalcone after separation and purification is 7.7 min.
Example 3
(1) Plate activation was performed 1 time (28 ℃, 60h) by inoculating 1-ring strain from the slant of the A.niger GDMCC 3.23 tube into PDA medium, the spores were dissolved in 5mL of sterile water, and the spore suspension (inoculum size 3%, v/v) was inoculated into the fermentation medium (500mL Erlenmeyer flask).
And after shaking culture for 48h, removing the culture solution by suction filtration to obtain wet thalli. The wet thallus is washed by distilled water or PBS buffer solution (pH 7.4) for 3 times to remove the culture medium remained by the thallus, and then is frozen and dried in vacuum at the temperature of minus 45 ℃ for 24 hours to obtain the freeze-dried thallus which is the aspergillus niger whole-cell catalyst.
(2) Adding 30mmol of neohesperidin dihydrochalcone and 5mL of distilled water into a 10mL conical flask in sequence, oscillating and dissolving on a vortex oscillator, then adding 20mg of Aspergillus niger whole-cell catalyst, mixing uniformly, sealing the bottle mouth, and placing in a full-temperature shaking bed cabinet for reaction (60 ℃, 180 rpm). After sampling 50 μ L at 0h, 1h, 2h, 3h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h and 48h, centrifuging for 10min at 16000rpm, diluting by 50 times with chromatographic grade methanol, filtering with 0.22 μm organic phase filter membrane, and introducing 20 μ L with high performance liquid chromatography autosampler for HPLC analysis (detection wavelength 283nm, mobile phase: methanol (A)/pure water (B), 50%/50%, flow rate 1.0 mL/min). The products are identified as hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside respectively.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A biocatalysis method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside is characterized in that neohesperidin dihydrochalcone is used as a starting raw material and is biocatalyzed by an Aspergillus niger whole-cell catalyst or an Aspergillus oryzae whole-cell catalyst to simultaneously prepare the hesperetin dihydrochalcone and the hesperetin dihydrochalcone-7-O-glucoside, and the method comprises the following specific steps:
(1) culturing Aspergillus niger cells or Aspergillus oryzae cells for growth and development, and preparing Aspergillus niger whole-cell catalyst or Aspergillus oryzae whole-cell catalyst;
(2) dissolving the neohesperidin dihydrochalcone in distilled water, adding an Aspergillus niger whole-cell catalyst or an Aspergillus oryzae whole-cell catalyst, mixing uniformly to obtain a reaction system, biologically catalyzing the neohesperidin dihydrochalcone to perform hydrolysis reaction, centrifuging supernate after hydrolysis, and analyzing and identifying to obtain the hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
2. The biocatalytic process for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, wherein the Aspergillus niger cells of step (1) can be Aspergillus niger GDMCC 3.23, source Aspergillus niger van tieghem gmcc 3.939; the Aspergillus oryzae cells of step (1) may be Aspergillus oryzae GDMCC 3.451, derived from Aspergillus oryzae (Ahlburg) CohnCICC 2066.
3. The biocatalytic method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, characterized in that the mass ratio of the neohesperidin dihydrochalcone in the step (2) to the Aspergillus niger whole-cell catalyst or the Aspergillus oryzae whole-cell catalyst is 1:1-1: 10.
4. The biocatalytic method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, characterized in that the volume ratio of the neohesperidin dihydrochalcone substance in step (2) to distilled water is 1:2-6:1 mmol/L.
5. The biocatalytic method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, characterized in that the temperature of the hydrolysis reaction in step (2) is 30-60 ℃, and the time of the hydrolysis reaction is 1-48 h.
6. The biocatalytic method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, characterized in that the rotation speed of the centrifugation in step (2) is 8000rpm to 16000rpm, and the time of the centrifugation is 1 to 10 min.
7. The biocatalytic method for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, characterized in that the specific method for analysis and identification in step (2) is as follows: and (3) diluting the chromatographic grade methanol, then filtering the diluted methanol by using an organic phase filter membrane, injecting a sample by using a high performance liquid chromatography autosampler, and carrying out chromatographic and spectral comparison with the standard substances of hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside to obtain the hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside.
8. The biocatalysis method for simultaneously preparing the hesperetin dihydrochalcone and the hesperetin dihydrochalcone-7-O-glucoside according to claim 1, wherein the Aspergillus niger whole-cell catalyst or the Aspergillus oryzae whole-cell catalyst biologically catalyzes a hydrolysis reaction of the neohesperidin dihydrochalcone to enable the disaccharide glycoside to undergo a hydrolysis reaction to generate the hesperetin dihydrochalcone and the hesperetin dihydrochalcone-7-O-glucoside, and the conversion rate is up to 80% or more.
9. The biocatalytic process for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to claim 1, wherein the hesperetin dihydrochalcone has a chemical structural formula of
10. The biocatalytic process for simultaneously preparing hesperetin dihydrochalcone and hesperetin dihydrochalcone-7-O-glucoside according to any of claims 1-9, characterized in that the hesperetin dihydrochalcone-7-O-glucoside has the chemical structural formula
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