CN112143782A - Method for monitoring stability of barley malting process - Google Patents

Abstract

The invention provides a method for monitoring the stability of a barley malting process, which belongs to the technical field of malting and adopts the technology of RT-PCR reverse transcription and capillary electrophoresis to monitor the expression quantity of related genes in the barley malting process so as to monitor the stability of barley in the malting process. The method provided by the invention can accurately evaluate the barley germination condition in the malting stage, and has guiding significance for controlling the barley quality from the source.

Description

Method for monitoring stability of barley malting process

Technical Field

The invention belongs to the technical field of malting, and particularly relates to a method for monitoring stability of a barley malting process.

Background

The various components of beer are derived from malt, hops and yeast. The quality of malt directly affects the quality of beer. The malt provides nutrient substances such as sugar, amino acid and the like for yeast fermentation, and simultaneously, the mellow and smooth mouthfeel of the beer is increased. The malt plays a significant role in the production, quality, cost and flavor stability of beer. The selection of high-quality malt becomes the key for producing high-quality beer for beer production enterprises.

The malting process is a process for preparing barley from raw barley, is the beginning of beer production, and has a decisive influence on the beer brewing process, the type of finished products and the quality of the finished products. During the malting process, the biological macromolecules such as protein, polysaccharide and the like of barley seeds are gradually dissolved, and nutrients such as sugar, amino acid and the like are provided for barley germination. The quality of the barley is essentially caused by the dissolution difference in the barley germination process, so that the quality control of the barley from the source has important significance.

Disclosure of Invention

The invention provides a method for monitoring the stability of a barley malting process, which can accurately evaluate the barley germination condition in a malting stage and has guiding significance for controlling the quality of barley from the source.

In order to achieve the above objects, the present invention provides a method for monitoring the stability of barley malting process, which employs RT-PCR reverse transcription and capillary electrophoresis technology in combination to monitor the stability of malt in malting process by monitoring the expression level of genes related to malt germination process.

Preferably, the method comprises the following steps:

screening related genes related to the malt hydrolase, and designing a multiplex primer according to the sequence of the related genes obtained by screening;

extracting to obtain total RNA of the malt cells;

taking total RNA of the malt cells as a template, and carrying out mRNA reverse transcription amplification on the total RNA by using a designed multiple primer to obtain an amplification product;

and quantitatively analyzing the expression quantity of the amplified product by utilizing capillary electrophoresis, and comparing the expression quantity with a gene expression map of a standard process to judge whether the malt is stable in the malting process.

Preferably, the related gene is selected from the group consisting of related genes LD, AMY1, AMY4, GLU, ASP, CYS, SERI, SERIII, MET, TRX, PDI, SEP, WRKY and internal control gene ACT. It is understood that the above genes are selected because they are involved in barley starch, proteolysis-related functions, and are closely related to barley solubilization during malting, thereby characterizing the germination state of barley during malting.

Preferably, the upstream primers of the multiplex primer designed according to the related gene are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 in sequence;

the downstream primers of the designed multiple primers are sequentially SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO. 28.

Preferably, the cell walls of the malt sample are disrupted by liquid nitrogen milling to extract malt total RNA.

Preferably, the reverse transcription and amplification of mRNA using the designed multiplex primer specifically comprises:

synthesizing a first cDNA chain by taking purified total RNA of the malt cells as a template and taking a downstream primer of a multiple primer designed according to related genes as a specific primer, wherein the reaction system is 10 mu L;

the first strand of the cDNA synthesized as described above was used as a template, and the upstream primer of the multiplex primer designed based on 13 related genes was used as a specific primer to perform RT-PCR amplification reaction.

Preferably, the reaction parameters for synthesizing the first strand of cDNA are specifically: 48 ℃ for 1 minute; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.

Preferably, the parameters of the RT-PCR amplification reaction are specifically as follows: pre-denaturation at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; the annealing temperature is 56 ℃ for 30 seconds; extension at 71 ℃ for 1min, and 35 cycles.

Preferably, the quantitative analysis of the expression level of the amplification product by capillary electrophoresis specifically comprises:

obtaining electrophoresis peak of each gene by capillary electrophoresis, wherein the expression quantity of each gene is gene peak height H/internal control gene ACT peak height H_act。

Preferably, the step of judging whether the barley is stable in the production process by comparing the gene expression profile with that of a standard process is as follows:

sampling the malt in the 5 stages of the production process of the end of wheat soaking, the first day of germination, the second day of germination, the third day of germination and the fourth day of germination, and obtaining the gene expression quantity of 13 related genes in the 5 stages in total under the standard process, wherein 65 data points are counted;

sampling is carried out under a new production process by adopting the same sampling mode, and the gene expression quantity of 13 genes in 5 stages is obtained, wherein 65 data points are calculated;

performing dimensionality reduction on the two groups of data by using a Principal Component Analysis (PCA) method to obtain principal component scores of the two groups of data;

if the difference value of the principal component scores of the two groups of data is less than or equal to 0.5, the gene expression of the new production process is not greatly different from the standard process;

if the difference in the principal component scores of the two sets of data is >0.5, the gene expression of the new production process is considered to be significantly different from the standard process.

Compared with the prior art, the invention has the advantages and positive effects that:

according to the invention, related genes related to hydrolysis of proteins and polysaccharides in the barley germination process are selected, multiple primers are designed based on the related genes, the reverse transcription amplification RT-PCR technology is used in combination to detect the amplification of the related genes and the expression quantity of the related genes by capillary electrophoresis, so that the difference among different samples can be effectively judged, and whether the whole barley making process is stable or not is judged by comparing with a standard process map. Based on the above, by analyzing the weight of each gene expression in the principal component, it is also possible to confirm which production stage has a difference in gene expression, and further, to analyze in a targeted manner whether there is an abnormality in the production stage. Therefore, the method provided by the invention can accurately evaluate the barley germination condition in the malting stage, and has guiding significance for controlling the barley quality from the source.

Drawings

FIG. 1 is a gene expression profile of a standard process malting process provided in an embodiment of the present invention;

FIG. 2 is a heat map of gene expression during barley A malting according to an embodiment of the present invention;

FIG. 3 is a heat map of gene expression during barley malting B provided by an embodiment of the present invention;

FIG. 4 is a heat map of gene expression during barley C malting according to an embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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: evaluation of stability of newly purchased batch A barley malting Process

For a batch of barley a newly purchased in a factory, evaluation of malting stability was performed.

1) Green barley sampling:

during the process of preparing the green barley, the green barley which is germinated for 1 day, 2 days, 3 days and 3 days after the barley soaking is finished is taken. In order to ensure that the vital activity of green barley is not changed, the sample should be immediately subjected to RNA extraction or stored at-80 ℃.

2) Crushing green barley:

in order to ensure that RNA in green barley is not degraded, green barley was ground using liquid nitrogen.

3) RNA extraction:

the crushed green barley was subjected to RNA extraction by adding TRIZOL. TRIZOL is a total RNA extraction reagent, and can directly extract total RNA from cells or tissues.

50-100mg of sample was taken and 1ml of Trizol was added. After lysis by shaking, centrifugation was carried out at 12000g at 4 ℃ for 5min, and the supernatant was transferred to a Phasemarker tube and allowed to stand for 5 min. 0.2ml of chloroform was added, shaken manually for 15s, and left to stand for 10 min. Centrifuge at 12000g for 10min at 4 ℃. The supernatant (450. mu.l and 550. mu.l) was taken into an EP tube, 250. mu.l of 96% ethanol was added, and the mixture was mixed by a gun tip. The mixture was transferred to a silica gel tube, centrifuged at 12000g for 1min, and the effluent was removed. Add 700. mu.l WB1, 12000g and centrifuge for 1min to remove the waste liquid. Add 500. mu.l WB2, 12000g and centrifuge for 1min to remove the waste liquid. Add 500. mu.l WB2, centrifuge at 12000g for 1min and transfer the silica gel tube to a 1.5ml EP tube. Adding 50 μ l of nucleic-free water, standing for 1min, and centrifuging at 12000g for 1 min. The centrifuged RNA solution was collected and stored at-80 ℃.

4) RT-PCR reverse transcription:

using the purified barley cell RNA as a template, a genome Lab of Beckmann Coulter was used^TMThe GeXP start kit utilizes the downstream primer of the designed multiple primers as a specific primer, synthesizes a first cDNA chain by taking the total RNA of barley cells as a template, and has a reaction system of 10 mu L. The first strand cDNA synthesis reaction parameters were set as follows: 48 ℃ for 1 minute; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.

Then, DNA polymerase and genome Lab from Beckmann Coulter were used^TMGeXP start kit. RT-PCR amplification reaction is carried out by using the first strand of the synthesized cDNA as a template and the upstream primer of the 13 multiplex primers as a specific primer. The RT-PCR amplification parameters were set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds; the annealing temperature is 56 ℃ and 30 seconds; extending at 71 ℃ for 1 minute, circulating for 35 times, and finally obtaining a reverse transcription amplification product of the expression gene.

5) Analysis of gene expression level:

and (3) carrying out quantitative analysis on the reverse transcription product by utilizing capillary electrophoresis. Mu.l of the PCR multiplex product was added to the wells of the upper plate containing 39. mu.l of a mixture of 95% deionized formamide (SLS) and 400bpMarker, mixed well with a pipette and covered with a drop of paraffin oil. Additionally 250. mu.l of separation buffer was added to each well of the buffer plate. After all the preparations are finished, the capillary electrophoresis is carried out on a machine.

After electrophoresis is finished, the expression quantity of different genes at different stages is obtained by the ratio of the electrophoresis peak height of the related gene to the electrophoresis peak height of the reference gene ACT, and the figure 1 and the figure 2 are expression quantity heat maps of different stages of specific genes respectively.

6) Evaluation of malting stability

PCA (principal component analysis) is carried out on the gene expression data of the two samples to obtain the PCA of the principal component score of the standard malting process_{Standard of merit}2.5, main component score PCA of barley A malting process_{Barley A}The difference between the two was less than 0.5 when it was 2.9, so barley a was considered to be similar to the standard process with little difference.

Example 2: evaluation of stability of newly purchased batch B barley malting Process

For a batch of barley B newly purchased in a factory, evaluation of malting stability was performed.

1) Green barley sampling:

during the process of preparing the green barley, the green barley which is germinated for 1 day, 2 days, 3 days and 3 days after the barley soaking is finished is taken. In order to ensure that the vital activity of green barley is not changed, the sample should be immediately subjected to RNA extraction or stored at-80 ℃.

2) Crushing green barley:

in order to ensure that RNA in green barley is not degraded, green barley was ground using liquid nitrogen.

3) RNA extraction:

the crushed green barley was subjected to RNA extraction by adding TRIZOL. TRIZOL is a total RNA extraction reagent, and can directly extract total RNA from cells or tissues.

50-100mg of sample was taken and 1ml of Trizol was added. After lysis by shaking, centrifugation was carried out at 12000g at 4 ℃ for 5min, and the supernatant was transferred to a Phasemarker tube and allowed to stand for 5 min. 0.2ml of chloroform was added, shaken manually for 15s, and left to stand for 10 min. Centrifuge at 12000g for 10min at 4 ℃. The supernatant (450. mu.l and 550. mu.l) was taken into an EP tube, 250. mu.l of 96% ethanol was added, and the mixture was mixed by a gun tip. The mixture was transferred to a silica gel tube, centrifuged at 12000g for 1min, and the effluent was removed. Add 700. mu.l WB1, 12000g and centrifuge for 1min to remove the waste liquid. Add 500. mu.l WB2, 12000g and centrifuge for 1min to remove the waste liquid. Add 500. mu.l WB2, centrifuge at 12000g for 1min and transfer the silica gel tube to a 1.5ml EP tube. Adding 50 μ l of nucleic-free water, standing for 1min, and centrifuging at 12000g for 1 min. The centrifuged RNA solution was collected and stored at-80 ℃.

4) RT-PCR reverse transcription:

using the purified barley cell RNA as a template, a genome Lab of Beckmann Coulter was used^TMThe GeXP start kit utilizes the downstream primer of the designed multiple primers as a specific primer, synthesizes a first cDNA chain by taking the total RNA of barley cells as a template, and has a reaction system of 10 mu L. The first strand cDNA synthesis reaction parameters were set as follows: 48 ℃ for 1 minute; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.

Then, DNA polymerase and genome Lab from Beckmann Coulter were used^TMGeXP start kit. RT-PCR amplification reaction is carried out by using the first strand of the synthesized cDNA as a template and the upstream primer of the 13 multiplex primers as a specific primer. The RT-PCR amplification parameters were set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds; the annealing temperature is 56 ℃ and 30 seconds; extending at 71 ℃ for 1 minute, circulating for 35 times, and finally obtaining a reverse transcription amplification product of the expression gene.

5) Analysis of gene expression level:

and (3) carrying out quantitative analysis on the reverse transcription product by utilizing capillary electrophoresis. Mu.l of the PCR multiplex product was added to the wells of the upper plate containing 39. mu.l of a mixture of 95% deionized formamide (SLS) and 400bpMarker, mixed well with a pipette and covered with a drop of paraffin oil. Additionally 250. mu.l of separation buffer was added to each well of the buffer plate. After all the preparations are finished, the capillary electrophoresis is carried out on a machine.

And after the electrophoresis is finished, obtaining the expression quantity of the unused genes at different stages by the ratio of the electrophoresis peak height of the related genes to the electrophoresis peak height of the reference gene ACT. FIGS. 1 and 3 are heat maps of expression levels of specific genes at different stages, respectively.

6) Evaluation of malting stability

By aligning two samplesPerforming Principal Component Analysis (PCA) on the gene expression data of the product to obtain PCA of the principal component score of the standard malting process_{Standard of merit}1.9, barley A Process principal Components score PCA_{Barley B}The difference between the two is more than 0.5 when the barley B is 1.2, so that the barley B malting process is considered to have larger difference from the standard process, the amylase and protease related gene expression quantity difference between the first and second days of germination is larger through weight analysis, and the investigation reason is caused by certain fluctuation of the malting process at the initial stage of germination.

Example 3: evaluation of stability of newly purchased batch C barley malting Process

For a batch of C barley newly purchased in a factory, evaluation of malting stability was performed at the micro-malting stage.

1) Green barley sampling:

during the process of preparing the green barley, the green barley which is germinated for 1 day, 2 days, 3 days and 3 days after the barley soaking is finished is taken. In order to ensure that the vital activity of green barley is not changed, the sample should be immediately subjected to RNA extraction or stored at-80 ℃.

2) Crushing green barley:

in order to ensure that RNA in green barley is not degraded, green barley was ground using liquid nitrogen.

3) RNA extraction:

the crushed green barley was subjected to RNA extraction by adding TRIZOL. TRIZOL is a total RNA extraction reagent, and can directly extract total RNA from cells or tissues.

50-100mg of sample was taken and 1ml of Trizol was added. After lysis by shaking, centrifugation was carried out at 12000g at 4 ℃ for 5min, and the supernatant was transferred to a Phasemarker tube and allowed to stand for 5 min. 0.2ml of chloroform was added, shaken manually for 15s, and left to stand for 10 min. Centrifuge at 12000g for 10min at 4 ℃. The supernatant (450. mu.l and 550. mu.l) was taken into an EP tube, 250. mu.l of 96% ethanol was added, and the mixture was mixed by a gun tip. The mixture was transferred to a silica gel tube, centrifuged at 12000g for 1min, and the effluent was removed. Add 700. mu.l WB1, 12000g and centrifuge for 1min to remove the waste liquid. Add 500. mu.l WB2, 12000g and centrifuge for 1min to remove the waste liquid. Add 500. mu.l WB2, centrifuge at 12000g for 1min and transfer the silica gel tube to a 1.5ml EP tube. Adding 50 μ l of nucleic-free water, standing for 1min, and centrifuging at 12000g for 1 min. The centrifuged RNA solution was collected and stored at-80 ℃.

4) RT-PCR reverse transcription:

using the purified barley cell RNA as a template, a genome Lab of Beckmann Coulter was used^TMThe GeXP start kit utilizes the downstream primer of the designed multiple primers as a specific primer, synthesizes a first cDNA chain by taking the total RNA of barley cells as a template, and has a reaction system of 10 mu L. The first strand cDNA synthesis reaction parameters were set as follows: 48 ℃ for 1 minute; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.

Then, DNA polymerase and genome Lab from Beckmann Coulter were used^TMGeXP start kit. RT-PCR amplification reaction is carried out by using the first strand of the synthesized cDNA as a template and the upstream primer of the 13 multiplex primers as a specific primer. The RT-PCR amplification parameters were set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds; the annealing temperature is 56 ℃ and 30 seconds; extending at 71 ℃ for 1 minute, circulating for 35 times, and finally obtaining a reverse transcription amplification product of the expression gene.

5) Analysis of gene expression level:

and (3) carrying out quantitative analysis on the reverse transcription product by utilizing capillary electrophoresis. Mu.l of the PCR multiplex product was added to the wells of the upper plate containing 39. mu.l of a mixture of 95% deionized formamide (SLS) and 400bpMarker, mixed well with a pipette and covered with a drop of paraffin oil. Additionally 250. mu.l of separation buffer was added to each well of the buffer plate. After all the preparations are finished, the capillary electrophoresis is carried out on a machine.

And after the electrophoresis is finished, obtaining the expression quantity of the unused genes at different stages by the ratio of the electrophoresis peak height of the related genes to the electrophoresis peak height of the reference gene ACT. FIGS. 1 and 4 are heat maps of expression levels of specific genes at different stages, respectively.

6) Evaluation of malting stability

PCA (principal component analysis) is carried out on the gene expression data of the two samples to obtain the PCA of the principal component score of the standard malting process_{Standard of merit}3.2, principal Components score PCA of barley C malting Process_{Barley C}The difference between the two is more than 0.5 when the value is 4.3, so that the barley C malting process is considered to have larger difference from the standard process. Due to the micro-wheat process controlTo be precise, it is therefore considered that varieties of barley C and standard malt differ greatly, resulting in large differences in gene expression.

Sequence listing

<110> Qingdao beer Ltd

<120> method for monitoring stability of barley malting process

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 38

<212> RNA

<213> Forward primer LD

<400> 1

AGGTGACACTATAGAATATGCTCTTCAAAGCCTTACAA 38

<210> 2

<211> 38

<212> RNA

<213> Forward primer AMY1

<400> 2

AGGTGACACTATAGAATAAAGCAGAGCGGCGGGTGGTA 38

<210> 3

<211> 38

<212> RNA

<213> Forward primer AMY4

<400> 3

AGGTGACACTATAGAATATACATCTTGTTCTGGTGGGA 38

<210> 4

<211> 38

<212> RNA

<213> forward primer GLU

<400> 4

AGGTGACACTATAGAATAACTATGCCGAGTTCTGCTTC 38

<210> 5

<211> 38

<212> RNA

<213> Forward primer ASP

<400> 5

AGGTGACACTATAGAATAGCTACCTCCACTCACGCTAC 38

<210> 6

<211> 38

<212> RNA

<213> Forward primer CYS

<400> 6

AGGTGACACTATAGAATAGTTGCGATGCTAACAAGAAA 38

<210> 7

<211> 37

<212> RNA

<213> Forward primer SER I

<400> 7

AGGTGACACTATAGAATACGACGGATTCGCATACTTT 37

<210> 8

<211> 36

<212> RNA

<213> Forward primer SER III

<400> 8

AGGTGACACTATAGAATAATGTCGCTCGTCTGGAAC 36

<210> 9

<211> 37

<212> RNA

<213> Forward primer MET

<400> 9

AGGTGACACTATAGAATATTTCTGGGACCTCTATTGC 37

<210> 10

<211> 38

<212> RNA

<213> Forward primer TRX

<400> 10

AGGTGACACTATAGAATAAACTTCAGTGCTTCGTGGTG 38

<210> 11

<211> 37

<212> RNA

<213> Forward primer PDI

<400> 11

AGGTGACACTATAGAATAGACCAGGCACCACTTATCC 37

<210> 12

<211> 36

<212> RNA

<213> Forward primer SEP

<400> 12

AGGTGACACTATAGAATAAATCTGAGGTCCAGTCCG 36

<210> 13

<211> 37

<212> RNA

<213> Forward primer WRKY

<400> 13

AGGTGACACTATAGAATACCACTACAGCAAGCACCAT 37

<210> 14

<211> 36

<212> RNA

<213> internal control Gene ACT

<400> 14

AGGTGACACTATAGAATAATGGTCAAGGCTGGTTTC 36

<210> 15

<211> 37

<212> RNA

<213> reverse primer LD

<400> 15

GTACGACTCACTATAGGGAAAACCCATTCGGCTCTAA 37

<210> 16

<211> 38

<212> RNA

<213> reverse primer AMY1

<400> 16

GTACGACTCACTATAGGGATTCGTTGGAGACGGAGTGC 38

<210> 17

<211> 41

<212> RNA

<213> reverse primer AMY4

<400> 17

GTACGACTCACTATAGGGATCTGCCTATGTTTATCCTGATT 41

<210> 18

<211> 39

<212> RNA

<213> reverse primer GLU

<400> 18

GTACGACTCACTATAGGGATGGAAGTCCCTCGCCCTCTG 39

<210> 19

<211> 40

<212> RNA

<213> reverse primer ASP

<400> 19

GTACGACTCACTATAGGGAAGTAATACCTGGCTCCTTTGT 40

<210> 20

<211> 38

<212> RNA

<213> reverse primer CYS

<400> 20

GTACGACTCACTATAGGGACCGTGAAGATACCCGATTT 38

<210> 21

<211> 38

<212> RNA

<213> reverse primer SER I

<400> 21

GTACGACTCACTATAGGGATCCTTGTATCCCTTTGACG 38

<210> 22

<211> 41

<212> RNA

<213> reverse primer SER III

<400> 22

GTACGACTCACTATAGGGACTGTATAGGCTTTGTATTGGAT 41

<210> 23

<211> 37

<212> RNA

<213> reverse primer MET

<400> 23

GTACGACTCACTATAGGGAAGACCTTCGCCACTGATT 37

<210> 24

<211> 37

<212> RNA

<213> reverse primer TRX

<400> 24

GTACGACTCACTATAGGGAGGATGTCCCATGTTGAGC 37

<210> 25

<211> 37

<212> RNA

<213> reverse primer PDI

<400> 25

AGGTGACACTATAGAATAGACCAGGCACCACTTATCC 37

<210> 26

<211> 39

<212> RNA

<213> reverse primer SEP

<400> 26

GTACGACTCACTATAGGGACATTAAGAAGGTGGAAATCG 39

<210> 27

<211> 37

<212> RNA

<213> reverse primer WRKY

<400> 27

GTACGACTCACTATAGGGAAATACGGCCATCAGGACA 37

<210> 28

<211> 37

<212> RNA

<213> internal control Gene ACT

<400> 28

GTACGACTCACTATAGGGAATGTCATCCCAGTTGCTT 37

Claims (10)

1. A method for monitoring the stability of malt malting process is characterized in that RT-PCR reverse transcription and capillary electrophoresis technology are used together to monitor the expression quantity of relative genes in malt malting process so as to monitor the stability of malt in malting process.

2. The method of claim 1, comprising the steps of:

screening related genes related to the malt hydrolase, and designing a multiplex primer according to the sequence of the related genes obtained by screening;

extracting to obtain total RNA of the malt cells;

taking total RNA of the malt cells as a template, and carrying out mRNA reverse transcription amplification on the total RNA by using a designed multiple primer to obtain an amplification product;

and quantitatively analyzing the expression quantity of the amplified product by utilizing capillary electrophoresis, and comparing the expression quantity with a gene expression map of a standard process to judge whether the malt is stable in the malting process.

3. The method of claim 2, wherein the related gene is selected from the group consisting of related genes LD, AMY1, AMY4, GLU, ASP, CYS, SERI, SERIII, MET, TRX, PDI, SEP, WRKY, and Intro-Control gene ACT.

4. The method of claim 3, wherein the upstream primers of the multiplex primers designed based on the above-mentioned related genes are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, in that order;

the downstream primers of the designed multiple primers are sequentially SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO. 28.

5. The method according to claim 2, wherein the cell walls of the malt sample are disrupted by liquid nitrogen milling to extract total RNA from malt.

6. The method of claim 4, wherein the reverse transcription amplification of mRNA using the designed multiplex primers specifically comprises:

synthesizing a first cDNA chain by taking purified total RNA of the malt cells as a template and taking a downstream primer of a multiple primer designed according to related genes as a specific primer, wherein the reaction system is 10 mu L;

the first strand of the cDNA synthesized as described above was used as a template, and the upstream primer of the multiplex primer designed based on 13 related genes was used as a specific primer to perform RT-PCR amplification reaction.

7. The method of claim 6, wherein the reaction parameters for the first strand synthesis of cDNA are: 48 ℃ for 1 minute; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.

8. The method according to claim 6, wherein the parameters of the RT-PCR amplification reaction are specifically: pre-denaturation at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; the annealing temperature is 56 ℃ for 30 seconds; extension at 71 ℃ for 1min, and 35 cycles.

9. The method according to claim 2, wherein the quantitative analysis of the expression level of the amplification product by capillary electrophoresis comprises:

obtaining electrophoresis peak of each gene by capillary electrophoresis, wherein the expression quantity of each gene is gene peak height H/internal control gene ACT peak height H_act。

10. The method according to claim 2, wherein the determination of the stability of malt during the production process by comparison with the gene expression profile of the standard process is specifically:

sampling the malt in the 5 stages of the production process of the end of wheat soaking, the first day of germination, the second day of germination, the third day of germination and the fourth day of germination, and obtaining the gene expression quantity of 13 related genes in the 5 stages in total under the standard process, wherein 65 data points are counted;

sampling is carried out under a new production process by adopting the same sampling mode, and the gene expression quantity of 13 genes in 5 stages is obtained, wherein 65 data points are calculated;

performing dimensionality reduction on the two groups of data by using a Principal Component Analysis (PCA) method to obtain principal component scores of the two groups of data;

if the difference value of the principal component scores of the two groups of data is less than or equal to 0.5, the gene expression of the new production process is not greatly different from the standard process;

if the difference in the principal component scores of the two sets of data is >0.5, the gene expression of the new production process is considered to be significantly different from the standard process.

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