CN108828103B - Morchella HPCE fingerprint establishing method and standard fingerprint thereof - Google Patents
Morchella HPCE fingerprint establishing method and standard fingerprint thereof Download PDFInfo
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- CN108828103B CN108828103B CN201810955733.9A CN201810955733A CN108828103B CN 108828103 B CN108828103 B CN 108828103B CN 201810955733 A CN201810955733 A CN 201810955733A CN 108828103 B CN108828103 B CN 108828103B
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Abstract
The invention discloses a morchella HPCE fingerprint spectrum establishing method, which comprises the following steps: (1) oven drying Morchella esculenta mycelium to obtain powder, adding into water, ultrasonic treating, maintaining at 90-95 deg.C for 3.5-4.5 hr, filtering, and collecting supernatant; then, filtering with a water system filter membrane, collecting the subsequent filtrate, and adding the running buffer solution with the same volume as the subsequent filtrate to be used as a sample solution; (2) performing capillary electrophoresis on the sample solution, detecting wavelength at 214nm, separating voltage at 18-22kV, temperature at 20-25 deg.C, and pressure at 0.5psi for 8-12s, and running time at 30-40min to obtain standard fingerprint; and discloses a standard fingerprint spectrum thereof. The common characteristic peak of the morchella can be effectively distinguished from other edible fungi by comparing; the method has the advantages of simple sample treatment, easy realization of chromatographic conditions and no harm to the environment; the stability and the reproducibility are good; is suitable for identifying and controlling the authenticity and quality of the morchella.
Description
Technical Field
The invention relates to the field of component analysis, in particular to a morchella HPCE fingerprint establishing method and a standard fingerprint thereof.
Background
Ex per, Fr, belonging to ascomycota, Pezizomycotina of subphylum, Pezizomycotina of class campylobacter, Pezizomycetes of class campylobacter, Pezizales of order campylobacter, morchelaceae, is a precious edible and medicinal fungus because it is covered with irregular concave folds, as is known from Morchella. Morchella esculenta is distributed in Asia, Europe, North America and the like, and is mainly planted in a vegetation habitat of temperate deciduous broad-leaf forests, high-altitude dark-leaf coniferous forests and coniferous and broadleaf mixed forests in 3-5 months, and few species are planted in 7 months and 9-10 months.
Morchella esculenta is mainly divided into two main groups in China, namely more than 30 types of black morchella esculenta strains and yellow morchella esculenta strains, wherein only part of germplasm morchella esculenta can be artificially cultured. However, due to the lack of an effective strain identification method, strain confusion in morchella production is easily caused, and strain identification is an important prerequisite for artificial cultivation and production of morchella, so how to provide a scientific and effective morchella identification method is a problem that needs to be solved urgently by technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a morchella HPCE fingerprint establishing method and a morchella HPCE fingerprint standard, and morchella can be effectively identified through the HPCE fingerprint.
In order to achieve the purpose, the invention adopts the following technical scheme:
the toadstool HPCE fingerprint spectrum establishing method comprises the following steps:
(1) oven drying Morchella esculenta mycelium to obtain powder, adding into water, ultrasonic treating, maintaining at 90-95 deg.C for 3.5-4.5 hr, filtering, and collecting supernatant; then, filtering with a water system filter membrane, collecting the subsequent filtrate, and adding the running buffer solution with the same volume as the subsequent filtrate to be used as a sample solution;
(2) performing capillary electrophoresis on the sample solution, detecting wavelength at 214nm, separating voltage at 18-22kV, temperature at 20-25 deg.C, and pressure at 0.5psi for 8-12s, and running time at 30-40min to obtain fingerprint.
The sample processing method and the electrophoresis conditions ensure good separation degree of the sample, so that the peak shape is complete, no tailing exists, and the interference peak is small.
Wherein, through testing the detection wavelength in the range of 190-300nm, the invention has complete peak emergence of 12 characteristic peaks under the detection wavelength, small peak value of interference peak and best separation effect. In addition, the separation effect is better under 254nm and 280 nm.
The invention has complete peak shape under the separation voltage and the sample injection time, and has no peak output delay.
Preferably, the preparation method of the morchella powder in the step (1) is as follows:
1) activating and inoculating morchella in a potato liquid culture medium, and performing shake culture at 25 ℃ and 180r/min for 7 d;
2) filtering the fermentation liquid to obtain mycelium, washing the mycelium with sterile water for 3 times, drying at 60 deg.C, pulverizing, and sieving with 100 mesh sieve to obtain powder.
Preferably, the mass ratio of the bacterial powder to the water in the step (1) is 1:30, and the ultrasonic treatment is carried out for 30 min.
Preferably, the morchella esculenta powder is added into water for ultrasonic treatment, and the water bath kettle is kept warm for 4 hours at the temperature of 95 ℃.
Preferably, the pore diameter of the water-based filter membrane in the step (1) is 0.22 μm.
Preferably, the running buffer is 10mmol/LpH 9.21.21 borax buffer.
By screening various buffer systems such as phosphate buffer solution and the like, the type, concentration and pH value of the running buffer solution are obtained, and the good separation degree and peak shape of a sample are ensured.
Preferably, the electrophoresis conditions in step (2) are: an uncoated quartz capillary tube of 50 μm × 57cm, with an effective detection length of 48 cm; the running buffer solution is 10mmol/L borax buffer solution with pH of 9.21; the detection wavelength is 214 nm; the separation voltage is 20 kV; the temperature is 25 ℃; the sample was injected at 0.5psi for 10s and run time 30 min.
The toadstool HPCE standard fingerprint comprises 12 characteristic peaks, wherein the retention time of the characteristic peaks from number 1 to number 12 is 6.237 to 6.554min, 7.183 to 7.696min, 7.675 to 8.454min, 10.146 to 10.613min, 11.512 to 12.567min, 13.35 to 14.829min, 14.363 to 16.179min, 14.663 to 16.604min, 14.942 to 17.235min, 16.512 to 18.45min, 17.829 to 20.579min and 18.788 to 21.954min respectively.
Preferably, the characteristic peak of the black morchella strain No. 4 is a single peak, and the characteristic peak of the yellow morchella strain No. 4 is a double peak.
According to the technical scheme, compared with the prior art, the invention discloses the establishment method of the Morchella esculenta HPCE fingerprint and the standard fingerprint thereof, and common characteristic peaks of the Morchella esculenta can be effectively distinguished from other edible fungi by comparison; the method has the advantages of simple sample treatment, easy realization of chromatographic conditions, no use of toxic and harmful solvents and no harm to the environment; the stability and the reproducibility are good; is suitable for identifying and controlling the authenticity and quality of the morchella.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is the standard fingerprint of Morchella esculenta HPCE.
FIG. 2 is the fingerprint of morchella esculenta treated by borax buffers with different concentrations.
FIG. 3 is finger print of Morchella esculenta under different separation voltages.
FIG. 4 is finger print of Morchella esculenta at different injection time.
FIG. 5 is the fingerprint of Morchella Mel-10.
FIG. 6 is the fingerprint of Morchella Mel-6.
FIG. 7 is the fingerprint of Morchella Mel-7.
FIG. 8 shows fingerprint of Morchella esculenta 201.
FIG. 9 is the fingerprint of Morchella Mes-9.
FIG. 10 is the Mes-6 fingerprint of Morchella esculenta.
FIG. 11 is the fingerprint of Morchella Mes-24.
FIG. 12 is the fingerprint of Cordyceps militaris.
FIG. 13 is the fingerprint of Phellinus linteus.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The toadstool HPCE fingerprint spectrum establishing method comprises the following steps:
(1) activating and inoculating morchella in a potato liquid culture medium, and performing shake culture at 25 ℃ and 180r/min for 7 d; filtering the fermentation liquid to obtain mycelium, washing the mycelium with sterile water for 3 times, drying at 60 deg.C, pulverizing, and sieving with 100 mesh sieve to obtain powder. Adding the bacterial powder into water, wherein the mass ratio of the bacterial powder to the water is 1:30, carrying out ultrasonic treatment for 30min, carrying out heat preservation in a water bath kettle at 95 ℃ for 4h, and filtering to obtain a supernatant; then filtering with 0.22 μm water system filter membrane, collecting the subsequent filtrate and adding borax buffer solution with the same volume of 10mmol/LpH 9.21.21 as sample solution.
(2) Carrying out capillary electrophoresis on the sample solution, wherein the electrophoresis conditions are as follows: an uncoated quartz capillary tube of 50 μm × 57cm, with an effective detection length of 48 cm; the detection wavelength is 214 nm; the separation voltage is 20 kV; the temperature is 25 ℃; the sample was injected at 0.5psi for 10s and run time 30 min.
The standard fingerprint of HPCE prepared by using black morchella as test material is shown in figure 1, and comprises 12 characteristic peaks. The retention time of characteristic peaks No. 1-12 is 6.471min, 7.583min, 8.279min, 10.521min, 12.313min, 14.488min, 15.800min, 16.175min, 16.896min, 18.025min, 19.988min and 21.196min respectively.
Example 2
The separation of yellow morchella samples was performed using 10, 20, 50, 75mmol/LpH 9.21.21 borax buffers, respectively, and the separation effect of different buffers was compared, except that the conditions were the same as in example 1. As shown in FIG. 2, the profiles obtained from the treatment of 10mmol/L borax buffer solution, 20 mmol/L borax buffer solution, 50 mmol/L borax buffer solution, 75mmol/L borax buffer solution, 10mmol/L borax buffer solution with pH 9.21, were selected as the running buffer solution, wherein the separation effect was the best.
Example 3
The conditions were the same as in example 1 except that the conditions for migration and separation were compared at separation voltages of 15, 20 and 30 kV. As shown in FIG. 3, the top-down spectra are respectively the spectra corresponding to the separation voltages of 15kV, 20kV and 30kV, wherein the baseline is poor at 15kV, the peak is delayed at 30kV, and the peak shape is trailing, so 20kV is finally selected as the running voltage of the electrophoresis measurement.
And (3) measuring the sample injection time of 5s, 10s and 20s, wherein as shown in fig. 4, the sample injection time is respectively a map corresponding to the sample injection time of 5s, 10s and 20s from top to bottom, wherein the sample injection 5s has incomplete peak shape, the 20s concentration is too high, the peak connection condition is increased, and the sample injection time of 10s has good effect.
Example 4
Performing fingerprint analysis on different varieties of morchella esculenta, wherein experimental samples comprise Mel-10, Mel-6, Mel-7, 201, Mes-9, Mes-6 and Mes-24, wherein the Mel-10, Mel-6, Mel-7 and 201 are black morchella esculenta, and the black morchella esculenta is collected from Sichuan places, Wuhan places and the like and can be artificially cultured; mes-9, Mes-6 and Mes-24 are yellow morchella, collected from Sichuan and Liaoning, and can not be cultivated artificially.
The fingerprint of 7 morchella is shown in fig. 5-11, and the 7 morchella have 12 characteristic peaks. Wherein the characteristic peak of the black morchella branch system No. 4 is a single peak, and the characteristic peak of the yellow morchella branch system No. 4 is a double peak.
Example 5
The fingerprint spectrums of cordyceps militaris and phellinus igniarius are respectively measured by using the method of the embodiment 1, and as shown in figures 12-13, the fingerprint spectrums of cordyceps militaris and phellinus igniarius are obviously different from the fingerprint spectrum of morchella esculenta of the invention.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. The toadstool HPCE fingerprint spectrum establishing method is characterized by comprising the following steps:
(1) drying morchella mycelium to obtain powder, adding into water, performing ultrasonic treatment, keeping the temperature of a water bath kettle at 90-95 ℃ for 3.5-4.5h, and filtering to obtain supernatant; then, filtering with a water system filter membrane, collecting the subsequent filtrate, and adding the running buffer solution with the same volume as the subsequent filtrate to be used as a sample solution;
(2) carrying out capillary electrophoresis on the sample solution, wherein the electrophoresis conditions are as follows: an uncoated quartz capillary tube of 50 μm × 57cm, with an effective detection length of 48 cm; the running buffer solution is 10mmol/L borax buffer solution with pH of 9.21; the detection wavelength is 214 nm; the separation voltage is 20 kV; the temperature is 25 ℃; injecting sample under 0.5psi for 10s, and running time is 30min to obtain fingerprint.
2. The method for establishing the HPCE fingerprint spectrum of morel according to claim 1, wherein the preparation method of morel powder in the step (1) is as follows:
1) activating and inoculating morchella in a potato liquid culture medium, and performing shake culture at 25 ℃ and 180r/min for 7 d;
2) filtering the fermentation liquid to obtain mycelium, washing the mycelium with sterile water for 3 times, drying at 60 deg.C, pulverizing, and sieving with 100 mesh sieve to obtain powder.
3. The method for establishing the HPCE fingerprint spectrum of morchella esculenta according to claim 1, wherein the mass ratio of the bacteria powder to water in the step (1) is 1:30, and the ultrasonic treatment is carried out for 30 min.
4. The method for establishing the HPCE fingerprint of morchella esculenta according to claim 1, wherein the pore size of the aqueous filter membrane in the step (1) is 0.22 μm.
5. Use of the fingerprint establishment method for Morchella esculenta HPCE according to any one of claims 1 to 4 in Morchella esculenta identification, wherein the standard fingerprint of Morchella esculenta HPCE comprises 12 characteristic peaks, the retention time of characteristic peaks 1 to 12 is 6.237-6.554min, 7.183-7.696min, 7.675-8.454min, 10.146-10.613min, 11.512-12.567min, 13.35-14.829min, 14.363-16.179min, 14.663-16.604min, 14.942-17.235min, 16.512-18.45min, 17.829-20.579min, and 18.788-21.954min, respectively.
6. The use of claim 5, wherein the characteristic peak of the black Morchella esculenta strain No. 4 is a single peak, and the characteristic peak of the yellow Morchella esculenta strain No. 4 is a double peak.
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