CN117686446A - Evaluation method of wheat making performance based on plant hormone - Google Patents
Evaluation method of wheat making performance based on plant hormone Download PDFInfo
- Publication number
- CN117686446A CN117686446A CN202311691092.8A CN202311691092A CN117686446A CN 117686446 A CN117686446 A CN 117686446A CN 202311691092 A CN202311691092 A CN 202311691092A CN 117686446 A CN117686446 A CN 117686446A
- Authority
- CN
- China
- Prior art keywords
- barley
- wheat
- making performance
- plant hormone
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000209140 Triticum Species 0.000 title claims abstract description 40
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 40
- 239000003375 plant hormone Substances 0.000 title claims abstract description 21
- 238000011156 evaluation Methods 0.000 title claims abstract description 12
- 241000209219 Hordeum Species 0.000 claims abstract description 56
- 235000007340 Hordeum vulgare Nutrition 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 229930195732 phytohormone Natural products 0.000 claims abstract description 7
- ZNJFBWYDHIGLCU-HWKXXFMVSA-N jasmonic acid Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-HWKXXFMVSA-N 0.000 claims description 36
- JLIDBLDQVAYHNE-YKALOCIXSA-N abscisic acid group Chemical group C\C(\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C)=C\C(O)=O JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 25
- 238000012417 linear regression Methods 0.000 claims description 21
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 claims description 19
- ZNJFBWYDHIGLCU-UHFFFAOYSA-N jasmonic acid Natural products CCC=CCC1C(CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-UHFFFAOYSA-N 0.000 claims description 18
- 229930192334 Auxin Natural products 0.000 claims description 17
- 229930191978 Gibberellin Natural products 0.000 claims description 17
- 239000002363 auxin Substances 0.000 claims description 17
- 239000003448 gibberellin Substances 0.000 claims description 17
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 17
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 238000004890 malting Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000002965 ELISA Methods 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 238000010009 beating Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 abstract description 26
- 238000005516 engineering process Methods 0.000 abstract description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 38
- 102000004139 alpha-Amylases Human genes 0.000 description 22
- 229940024171 alpha-amylase Drugs 0.000 description 22
- 239000000243 solution Substances 0.000 description 21
- 238000002791 soaking Methods 0.000 description 20
- 239000000523 sample Substances 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 16
- 230000035784 germination Effects 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- 239000004382 Amylase Substances 0.000 description 9
- 102000013142 Amylases Human genes 0.000 description 8
- 108010065511 Amylases Proteins 0.000 description 8
- 235000019418 amylase Nutrition 0.000 description 8
- 238000009423 ventilation Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000011536 extraction buffer Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229920001353 Dextrin Polymers 0.000 description 5
- 239000004375 Dextrin Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 235000019425 dextrin Nutrition 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012089 stop solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000013024 dilution buffer Substances 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 230000007226 seed germination Effects 0.000 description 3
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 239000005980 Gibberellic acid Substances 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 108010019077 beta-Amylase Proteins 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 239000011976 maleic acid Substances 0.000 description 2
- 239000001630 malic acid Substances 0.000 description 2
- 235000011090 malic acid Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 229910000406 trisodium phosphate Inorganic materials 0.000 description 2
- 235000019801 trisodium phosphate Nutrition 0.000 description 2
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- JLIDBLDQVAYHNE-LXGGSRJLSA-N 2-cis-abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\C1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-LXGGSRJLSA-N 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 230000006578 abscission Effects 0.000 description 1
- 108010050181 aleurone Proteins 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- -1 bag rope Substances 0.000 description 1
- 229940098396 barley grain Drugs 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000020509 sex determination Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for evaluating wheat making performance based on plant hormone, and belongs to the technical field of malt performance evaluation. The evaluation method of the wheat making performance based on the plant hormone provided by the invention is characterized in that the wheat making performance score is judged, and the wheat making performance score is calculated based on the content of the influence factors of the plant hormone in the barley. The method can predict malt quality in advance by measuring the content of barley phytohormone, does not need to prepare malt, shortens detection time, is simple to operate, does not need complex instruments, and can meet the requirement of adjusting technology in the process of preparing wheat in time according to the prediction result.
Description
Technical Field
The invention belongs to the technical field of malt performance evaluation, and particularly relates to a method for evaluating wheat making performance based on plant hormone.
Background
Barley is a main raw material for beer brewing, various hydrolytic enzyme activities can be activated through making barley, and water-insoluble substances in malt are gradually hydrolyzed into small molecular water-soluble nutrient substances in the saccharification process, so that alcohol and various flavor substances can be produced through yeast fermentation. The indexes for evaluating malt performance mainly comprise: saccharification power, beta-glucan, alpha-amino nitrogen, enzyme activity (alpha-amylase, beta-amylase activity, limit dextrinase), pool value, viscosity, and the like. The conversion of starch into sugar during the malting process is mainly catalyzed by alpha-amylase, beta-amylase and limit dextrinase.
In the seed germination process, the plant seed germination agent is not only influenced by external environmental factors such as moisture, temperature, oxygen, light and the like, but also regulated and controlled by plant endogenous hormones. Phytohormone (phytohorone) refers to some trace amount of organic compounds produced in plants that can regulate (promote, inhibit) the physiological processes of themselves. The plant hormone has important regulation and control effects on the growth and development of plants, and participates in regulating and controlling cell division, elongation, differentiation, plant germination, rooting, flowering, fruit bearing, sex determination, dormancy, abscission and the like. The plant hormones Gibberellic Acid (GA) and abscisic acid (ABA) are the main hormones that antagonize and regulate barley germination. Research shows that gibberellin can induce the increase of aleurone layer alpha-amylase and abscisic acid blocks the reaction. It has also been studied that auxin (IAA) or Jasmonic Acid (JA) hormones can affect seed germination.
At present, the malt performance measuring method mainly prepares barley into malt through barley preparation and then measures related indexes, but the method needs longer time and special equipment and instruments, and can not predict the barley preparation performance in the barley stage, and has certain hysteresis, so that the adjustment of the barley preparation process can not be performed in time.
Disclosure of Invention
The invention provides a method for evaluating the wheat making performance based on plant hormone, which can predict the quality of malt in advance by measuring the content of the plant hormone in barley, does not need to make malt, shortens the detection time, is simple to operate, does not need a complex instrument, and can meet the requirement of adjusting the process in the wheat making process in time according to the prediction result.
In order to achieve the above purpose, the invention provides a method for evaluating the wheat making performance based on plant hormone, wherein the wheat making performance is evaluated through a wheat making performance score, and the wheat making performance score is calculated based on the content of the influence factors of the plant hormone in barley.
Preferably, the plant hormone influencing factors include positive influencing factors and negative influencing factors;
the positive influencing factors are gibberellin, jasmonic acid and auxin;
the negative influencing factor is abscisic acid.
Preferably, the malting performance score = positive influencing factor + negative influencing factor-8.44.
Preferably, the malting performance score=gibberellin 0.65+jasmonic acid 0.0022+auxin 0.38-abscisic acid 0.33-8.44.
Preferably, the evaluation criteria are:
when the wheat making performance score is more than 7, the evaluated barley is the barley of class I, and the wheat making performance is excellent;
when the wheat making performance score is more than or equal to 4 and less than or equal to 7, the evaluated barley is class II, and the wheat making performance is good;
when the malting performance score was <4, the barley evaluated was class III and malting performance was poor.
Preferably, the extraction method of the phytohormone in the barley comprises the following steps:
grinding barley with liquid nitrogen, weighing 0.1g of ground sample, adding 1mL of precooled PBS solution, shaking, mixing, extracting at 0-4deg.C for 1-2h, centrifuging at 10000-12000rpm for 10-20min, and collecting supernatant.
Preferably, the method for measuring the content of the phytohormone in the barley comprises the following steps:
standard substance holes, sample holes and blank holes are formed in the ELISA plate, 50ul of standard substances with different concentrations are respectively added in the standard substance holes, 50ul of samples to be detected are added in the sample holes, and 50ul of sample diluent is added in the blank holes; .
Adding 100ul of horseradish peroxidase-labeled gibberellin, abscisic acid, jasmonic acid or auxin detection antibody into each of the standard substance hole, the sample hole and the blank hole, covering a membrane plate, gently shaking and mixing, and incubating at 37 ℃ for 1 hour;
removing liquid in the holes, filling the washing liquid in each hole, oscillating for 30 seconds, removing the washing liquid, drying by beating with absorbent paper, and repeating the operation for a plurality of times;
adding substrate hydrogen peroxide and tetramethyl benzidine into each hole, mixing with gentle shaking, and incubating at 37deg.C for 15 min in dark place;
taking out the ELISA plate, and rapidly adding 50ul of 2M H 2 SO 4 Terminating solution, and immediately measuring the OD value of each hole at the wavelength of 450nm after adding the terminating solution;
and (3) taking the standard substance concentration as an abscissa and the corresponding OD value as an ordinate, drawing a standard substance linear regression curve, and calculating the concentration value of each sample according to a curve equation.
Preferably, the standard substances are gibberellin, abscisic acid, jasmonic acid and auxin respectively.
Preferably, the gibberellin linear regression curve equation is
y＝0.0064x-0.0679，R 2 ＝0.9944；
The linear regression curve equation of the abscisic acid is y=0.0139 x-0.0546, R 2 ＝0.9978；
The linear regression curve equation of the jasmonic acid is y=0.0007x-0.0063, R 2 ＝0.9981；
The linear regression curve equation of the auxin is y= 0.1048x-0.0785, R 2 ＝0.9914。
Compared with the prior art, the invention has the advantages and positive effects that:
compared with the existing detection technology, the method for evaluating the barley making performance based on the plant hormone provided by the invention can predict the malt quality in advance by measuring the content of the barley plant hormone, does not need to make malt, shortens the detection time, is simple to operate, does not need a complex instrument, and can meet the requirement of adjusting the process in the barley making process in time according to the prediction result.
Drawings
FIG. 1 is a graph of linear regression of gibberellin provided in an embodiment of the present invention;
FIG. 2 is a linear regression graph of abscisic acid provided in an embodiment of the present invention;
FIG. 3 is a linear regression graph of jasmonic acid provided in an embodiment of the present invention;
FIG. 4 is a linear regression graph of auxin according to an embodiment of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
6 different varieties of barley are taken, and plant hormones in the different varieties of barley are respectively extracted by the following extraction method: grinding barley with liquid nitrogen, weighing 0.1g of ground sample, adding 1mL of precooled PBS solution, shaking, mixing, extracting at 4deg.C for 1h, centrifuging at 12000rpm for 10min, and collecting supernatant for the next content measurement.
The content of 6 barley phytohormones (gibberellin, abscisic acid, jasmonic acid, auxin) was measured separately as follows:
a) The required number of strips is determined according to the number of samples to be tested and the number of standard substances, each standard substance and blank hole are recommended to be used as a compound hole, each sample is determined according to the number of the sample, and the compound hole can be used as much as possible. The required strips were removed from the aluminium foil bag after 60min equilibration at room temperature and all reagents were thoroughly mixed before use. The liquid is not required to generate a large amount of foam, so that a large amount of bubbles are not added during sample addition, and errors in sample addition are avoided.
b) Setting a standard substance hole and a sample hole, wherein 50ul of standard substances with different concentrations are respectively added into the standard substance hole, 50ul of samples to be detected are added into the sample hole, 50ul of sample diluent is added into the blank hole, and the sample can be diluted by the sample diluent and then measured if the concentration of the sample is too high.
c) Adding 100ul of horseradish peroxidase-labeled gibberellin, abscisic acid, jasmonic acid or auxin detection antibody into each of the standard substance hole, the sample hole and the blank hole, covering a membrane plate, gently shaking and mixing, and incubating at 37 ℃ for 1 hour;
d) Removing liquid in the holes, filling the washing liquid in each hole, oscillating for 30 seconds, removing the washing liquid, drying by beating with absorbent paper, and repeating the operation for a plurality of times;
e) Substrate A, B (hydrogen peroxide, tetramethyl benzidine) was added 50ul each per well, mixed gently with shaking, incubated at 37℃for 15 min in the absence of light;
f) The ELISA plate was removed and 50ul of stop solution (2M H) was added rapidly 2 SO 4 ) Immediately after addition of the stop solution, the OD value of each well was measured at a wavelength of 450 nm;
g) And (3) taking the standard substance concentration as an abscissa and the corresponding OD value as an ordinate, drawing a standard substance linear regression curve, and calculating the concentration value of each sample according to a curve equation.
The linear regression curve equation of each standard is shown in fig. 1-4, and specifically comprises the following steps:
the linear regression curve equation of gibberellin is y=0.0064x-0.0679, r 2 ＝0.9944；
The linear regression curve equation of the abscisic acid is y=0.0139 x-0.0546, R 2 ＝0.9978；
The linear regression curve equation of the jasmonic acid is y=0.0007x-0.0063, R 2 ＝0.9981；
The linear regression curve equation of the auxin is y= 0.1048x-0.0785, R 2 ＝0.9914。
According to the linear regression curve equation of each standard, the contents of gibberellin, abscisic acid, jasmonic acid and auxin of 6 different varieties of barley are calculated, and the calculated scores are shown in Table 1.
TABLE 1
| 1 | 2 | 3 | 4 | 5 | 6 | |
| Gibberellin content (pmol/ml) | 34.94 | 44.55 | 28.33 | 20.21 | 58.61 | 30.03 |
| Abscisic acid content (ng/ml) | 25.47 | 43.55 | 33.09 | 22.41 | 113.10 | 46.81 |
| Jasmonic acid (pmol/l) | 1424 | 757 | 1207 | 1768 | 3288 | 2077 |
| Auxin (ng/ml) | 1.86 | 2.28 | 9.37 | 12.35 | 10.09 | 6.73 |
| Scoring of | 9.70 | 8.68 | 5.27 | 5.88 | 3.40 | 2.76 |
| Grading of wheat making performance | Excellent (excellent) | Excellent (excellent) | Good grade (good) | Good grade (good) | Difference of difference | Difference of difference |
Method verification
The 6 different varieties of barley were subjected to barley making, index measurement and classification according to the indexes by the conventional method, and the test results are shown in table 2.
Wherein:
1. the traditional wheat making method comprises the following specific steps:
malt preparation:
wheat selection: selecting barley with full seeds and 99% germination rate;
pretreatment of barley: cleaning barley, and removing wheat straw, bag rope, wood block, iron nail, screw, metal wire, stone, miscellaneous wheat grain, crushed barley grain, etc.; screening and grading, and reserving barley with uniform size and length of 2.5-2.8 mm;
wheat soaking: adding barley soaking water into a barley soaking tank, then discharging barley to the barley soaking tank, collecting floating barley and impurities of which the small part floats to the liquid surface through a collecting port, and finishing the operation of floating barley; after finishing the wheat floating, changing the water quality, soaking the wheat by adopting a three-soaking two-breaking multi-stage soaking water-breaking method, controlling the water temperature of the wheat soaking at 16-17 ℃, adopting continuous ventilation and oxygen supply in the wheat soaking process, and sequentially comprising a first soaking stage (soaking for 4 hours), a first dry soaking stage (soaking for 7 hours), a second soaking stage (soaking for 5 hours), a second dry soaking stage (soaking for 5 hours) and a third soaking stage (soaking for 1 hour), wherein the process is adjusted according to varieties, and the final wheat soaking degree reaches 39%;
sprouting: the germination process is carried out in ventilated type germination equipment, the first stage of germination is the 0 th to 24 th hours, and the germination temperature is controlled to be 16 to 17 ℃; the second stage of germination is 24-72 h, and the germination temperature is controlled at 15-16 ℃; 72 to 96 hours is a third stage of germination, and the germination temperature is controlled between 17 and 18 ℃; in the germination process, stirring and wheat turning operation is carried out every 8 hours; after germination for 96 hours, the water content of the prepared green malt is 42%, the average leaf bud length is 2/5-5/5 of the wheat grain length, the total malt proportion is 80%, and the germination rate reaches 99%;
drying green malt: comprising the following five successive stages:
drying the first stage: delivering the green malt obtained in the above steps into a drying furnace for ventilation and moisture removal, wherein the ventilation capacity of each ton of green malt is 100m 3 The difference between the height of the wheat layer in the drying furnace is kept within 6cm during ventilation for/min, and the moisture content of the green malt is reduced to 11.8% after moisture is removed for 6 h;
and (3) a second stage of drying: maintaining ventilation, heating to 79.5 ℃ at a constant speed of 1.5 ℃/h, and baking at constant temperature for 1h;
and a third stage of drying: maintaining ventilation, heating to 87.5 ℃ at a constant speed of 2.5 ℃/h, and baking at constant temperature for 1h;
and a fourth stage of drying: maintaining ventilation, naturally cooling to 82 ℃, and baking at constant temperature for 2h;
and a fifth stage of drying: maintaining ventilation, heating to 87.5 ℃ at a constant speed of 4.5 ℃/h, and baking at constant temperature for 0.5h;
root removal: and removing roots from the dried malt in 12 hours to obtain finished malt, and crushing the malt to be used for measuring the alpha-amylase and the limit dextrinase serving as indexes of the subsequent malt.
2. Method for measuring alpha-amylase (U/g) and limit dextrinase (mU/g):
method for measuring alpha-amylase (U/g)
Preparing the following reagents:
amylase extraction buffer: weighing 5.8g of Maleic acid (Sigma M0375;100 mM), dissolving in 400mL of double distilled water, adjusting pH to 5.5, fixing volume to 500mL, and preserving at 4 ℃ for 6 months;
alpha-amylase dilution mother liquor: 67.05g of Malic acid (Malic acid, sigma M0875), 35g of Sodium hydroxide (Sodium hydroxide), 29.2g of Sodium chloride (Sodium chloride), 2.95g of Calcium chloride (Calcium chloride. 2H) 2 O) in 400ml ddH 2 O, adjusting the pH to 5.4, then fixing the volume to 500ml, and preserving for 6 months at 4 ℃;
alpha-amylase dilution buffer: weighing 50ml of alpha-amylase dilution mother solution, fixing the volume to 1L, and storing for 6 months at 4 ℃ as alpha-amylase dilution buffer solution;
alpha-amylase stop solution: 10g of trisodium phosphate (tri-sodium phosphate), or 23.2gof Na, was weighed out 3 PO 4 ·12H 2 O, dissolving in 1L double distilled water, adjusting pH to 11, and preserving at 4deg.C for 6 months;
alpha-amylase transferase substrate: purchased from Megazyme (K-CERA), the alpha-amylase substrate bottle was filled with 10ml of sterile water, 1ml was packed into 1.5ml centrifuge tubes, and frozen for preservation;
300mg (+ -25 mg) of malt powder was weighed into a 10ml EP tube and the exact powder mass was recorded. 5ml of amylase extraction buffer is added, mixed well and placed on a metal bath, and vibrated for 16h at 20 ℃. Centrifuging at 12000rpm for 10min, and collecting supernatant as amylase extractive solution.
The amylase extract was diluted 250-fold with alpha-amylase dilution buffer. Adding 50ul of amylase diluent into a 2ml centrifuge tube, carrying out warm bath at 40 ℃ for 2min, adding 50ul of alpha-amylase substrate, carrying out 40 ℃ reaction for 10min, adding 750ul of alpha-amylase stopping solution, mixing uniformly, and measuring the absorbance at 405nm (OD 405) to obtain the alpha-amylase OD. The blank sample is sequentially added with the enzyme extracting solution, the reaction stopping solution and the substrate with the same volume, and the reaction is carried out for 10min at 40 ℃. Preferably, the reaction tube is a flat bottom centrifuge tube; in the reaction process, the rotating speed of the shaking period is 450rpm, so as to effectively mix the enzyme and the substrate; when a plurality of reaction systems are adopted, alpha-amylase substrates are added in a certain sequence according to the same interval, and at the same time, reaction stopping solution is added in the same time interval and time sequence when the reaction is finished, so as to ensure that the reaction time of each tube is 10min.
Calculation ofα-Amylase activity.
Determination method of Limit dextrinase (mU/g):
preparing the following reagents:
amylase extraction buffer: weighing 5.8g of Maleic acid (Sigma M0375;100 mM), dissolving in 400mL of double distilled water, adjusting pH to 5.5, fixing volume to 500mL, and preserving at 4 ℃ for 6 months;
total limit dextrinase extraction buffer: weighing 0.1g of DTT, adding into 25ml of amylase extraction buffer solution, uniformly mixing, and extracting total limit dextrin enzyme buffer solution, wherein the solution is prepared at present;
limit dextrinase stopping solution: weighing 10g Trizma base solution, and dissolving to 1L;
limit dextrinase specific substrates: available from Megazyme (T-LDZ 200), the substrate was in the form of granules, ground into powder, and stored at room temperature.
300mg (+ -25 mg) of malt powder was weighed into a 10ml EP tube and the exact powder mass was recorded. For measuring total limit dextrinase activity.
Adding 5ml total limit dextrin enzyme extraction buffer solution, mixing, placing on a metal bath, and shaking at 20 ℃ for 16h. The rotation speed is set to 12000rpm for centrifugation for 10min, and the supernatant is the total limit dextrinase extract.
Taking 11.5-12.5mg in a 2ml centrifuge tube, carrying out water bath at 40 ℃ for 2min, accurately taking 100ul of total limit dextrin enzyme extract, adding the total limit dextrin enzyme extract into the centrifuge tube with a substrate, accurately reacting for 10min at 40 ℃, adding 1ml of limit dextrin enzyme stop solution, centrifuging at 12000rpm for 40min at normal temperature, sucking out the supernatant, and measuring the absorbance value at 595nm (OD 595). The total OD of the limit dextrinase is determined by the total limit dextrinase extracting solution. Sequentially adding a substrate, a reaction stopping solution with the same volume and an enzyme extracting solution into a blank control sample; and reacting for 10min at 40 ℃, and obtaining supernatant after centrifugation, namely the blank control. Preferably, the reaction tube is a flat bottom tube; in the reaction process, the rotating speed of the shaking period is 450rpm, so as to effectively mix the enzyme and the substrate; and adding enzyme extracting solutions in a certain sequence at the same intervals when a plurality of reaction systems are used, and adding reaction stopping solutions at the same time intervals and time sequence when the reaction is finished, so as to ensure that the reaction time of each tube is 10min. And calculating the limit dextrinase content.
3. According to the judgment standard of alpha-amylase and limit dextrinase:
when alpha-amylase is more than 130 and limit amylase is more than 300, the barley is evaluated to be superior;
when 120< α -amylase <130, and 200< limit dextrinase <300, the barley evaluated is good;
when the α -amylase content is <120 and the limit dextrinase is <200, the barley is evaluated as poor;
TABLE 2
From the above, the classification result of the method for classifying 6 varieties of barley is consistent with the classification result of the traditional barley, which indicates that the evaluation method is accurate. In addition, the evaluation method only needs to analyze the content of phytohormone in the barley, does not need large-scale equipment, and takes a short time (1-2 days). Whereas the conventional method requires preparing barley into malt, requires special equipment for preparing the barley (equipment is expensive), and takes a long time (8 days for preparing the barley and 2 days for measuring the enzyme activity). Therefore, compared with the traditional mode, the method has great advantages.
Claims (9)
1. The method for evaluating the wheat making performance based on the plant hormone is characterized in that the wheat making performance is evaluated through a wheat making performance score, and the wheat making performance score is calculated based on the content of the influence factors of the plant hormone in barley.
2. The method of evaluation according to claim 1, wherein the influence factors of the plant hormone include a positive influence factor and a negative influence factor;
the positive influencing factors are gibberellin, jasmonic acid and auxin;
the negative influencing factor is abscisic acid.
3. The method of evaluation according to claim 1 or 2, wherein the malting performance score = positive influencing factor + negative influencing factor-8.44.
4. The method of claim 3, wherein the malting performance score = gibberellin 0.65+ jasmonic acid 0.0022+ auxin 0.38-abscisic acid 0.33-8.44.
5. The method according to any one of claims 1 to 4, wherein the evaluation criterion is:
when the wheat making performance score is more than 7, the evaluated barley is the barley of class I, and the wheat making performance is excellent;
when the wheat making performance score is more than or equal to 4 and less than or equal to 7, the evaluated barley is class II, and the wheat making performance is good;
when the malting performance score was <4, the barley evaluated was class III and malting performance was poor.
6. The method for evaluating according to any one of claims 1 to 4, wherein the method for extracting phytohormone from barley comprises the steps of:
grinding barley with liquid nitrogen, weighing 0.1g of ground sample, adding 1mL of precooled PBS solution, shaking, mixing, extracting at 0-4deg.C for 1-2h, centrifuging at 10000-12000rpm for 10-20min, and collecting supernatant.
7. The method for evaluating the plant hormone according to any one of claims 1 to 4, wherein the method for measuring the content of the plant hormone in barley comprises the steps of:
standard substance holes, sample holes and blank holes are formed in the ELISA plate, 50ul of standard substances with different concentrations are respectively added in the standard substance holes, 50ul of samples to be detected are added in the sample holes, and 50ul of sample diluent is added in the blank holes; .
Adding 100ul of horseradish peroxidase-labeled gibberellin, abscisic acid, jasmonic acid or auxin detection antibody into each of the standard substance hole, the sample hole and the blank hole, covering a membrane plate, gently shaking and mixing, and incubating at 37 ℃ for 1 hour;
removing liquid in the holes, filling the washing liquid in each hole, oscillating for 30 seconds, removing the washing liquid, drying by beating with absorbent paper, and repeating the operation for a plurality of times;
adding substrate hydrogen peroxide and tetramethyl benzidine into each hole, mixing with gentle shaking, and incubating at 37deg.C for 15 min in dark place;
taking out the ELISA plate, and rapidly adding 50ul of 2M H 2 SO 4 Terminating solution, and immediately measuring the OD value of each hole at the wavelength of 450nm after adding the terminating solution;
and (3) taking the standard substance concentration as an abscissa and the corresponding OD value as an ordinate, drawing a standard substance linear regression curve, and calculating the concentration value of each sample according to a curve equation.
8. The method according to claim 7, wherein the standard substances are gibberellin, abscisic acid, jasmonic acid, and auxin, respectively.
9. The evaluation method according to claim 8The gibberellin linear regression curve equation is y=0.0064x-0.0679, R 2 ＝0.9944；
The linear regression curve equation of the abscisic acid is y=0.0139 x-0.0546, R 2 ＝0.9978；
The linear regression curve equation of the jasmonic acid is y=0.0007x-0.0063, R 2 ＝0.9981；
The linear regression curve equation of the auxin is y= 0.1048x-0.0785, R 2 ＝0.9914。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311691092.8A CN117686446A (en) | 2023-12-11 | 2023-12-11 | Evaluation method of wheat making performance based on plant hormone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311691092.8A CN117686446A (en) | 2023-12-11 | 2023-12-11 | Evaluation method of wheat making performance based on plant hormone |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117686446A true CN117686446A (en) | 2024-03-12 |
Family
ID=90127926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311691092.8A Pending CN117686446A (en) | 2023-12-11 | 2023-12-11 | Evaluation method of wheat making performance based on plant hormone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117686446A (en) |
-
2023
- 2023-12-11 CN CN202311691092.8A patent/CN117686446A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109477048A (en) | The beverage based on cereal of purification | |
| Deme et al. | Evaluation of malting potential of different barley varieties | |
| WO2019165758A1 (en) | Method for rapidly determining activities of enzymes in cereals and predicting optimum temperature of enzymes | |
| Briggs | Malt modification—a century of evolving views | |
| Reid | The effect of light on the accumulation of ascorbic acid in young cowpea plants | |
| US20030148012A1 (en) | Treatment of germinating malting grain | |
| CN117686446A (en) | Evaluation method of wheat making performance based on plant hormone | |
| CN105316206A (en) | Brewing method of pueraria lobata vinegar | |
| Cozzolino et al. | Relationships between fatty acid contents of barley grain, malt, and wort with malt quality measurements | |
| Andrews et al. | Dormancy studies in seed of Avena fatua. 6. Germinability of the immature caryopsis | |
| Iwouno et al. | Effects of experimental variables on the malting quality of Nigerian yellow maize (Zea mays), farz 27 variety | |
| Perata et al. | Ethanol production and toxicity in suspension-cultured carrot cells and embryos | |
| Kefale et al. | Assessment of malt quality attributes of barley genotypes grown in Bekoji, Holeta and Ankober, Ethiopia | |
| WO2020073350A1 (en) | Processing technology of barley malt | |
| Nicholls | Influence of temperature during grain growth and ripening of barley on the subsequent response to exogenous gibberellic acid | |
| US3085945A (en) | Malting process | |
| Van Onckelen et al. | Relationship of ribonucleic acid metabolism in embryo and aleurone to α-amylase synthesis in barley | |
| CN110603928B (en) | Treatment method for improving germination rate of pakchoi seeds | |
| WO2006133496A1 (en) | Malting process | |
| Mis et al. | Wheat grain hardness modified by the laboratory sprouting test | |
| CN116466029B (en) | Method for rapidly evaluating variety characteristics of beer barley | |
| US2903399A (en) | Process for the production of acidified malt | |
| CN117686445A (en) | Barley germination performance evaluation method based on hardness index and water absorption protein content | |
| Zhao et al. | Germination vigour difference of superior and inferior rice grains revealed by physiological and gene expression studies | |
| Bojovic | The effects of temperature, length of storage and plant growth regulators on germination of wheat (Triticum aestivum L.) and triticale seeds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |