A method for regulating malt PYF based on gene expression during malting and its application

Abstract

This invention proposes a method for regulating malt PYF based on gene expression during the malting process and its application, belonging to the field of bioengineering. It addresses the technical problems of existing malt PYF detection methods, such as limited detection targets, narrow applicability, and inability to guide subsequent malting processes. The technical solution includes steps such as gene screening, primer design, total RNA extraction, reverse transcription to prepare cDNA templates, multiplex PCR, PCR product electrophoresis, product fragment expression analysis, and prediction and improvement of malt PYF performance. This invention features short processing time, high detection efficiency, and high accuracy. It can accurately predict malt PYF performance during the malt production process and establish adjustment measures for the malting process based on the prediction results, ultimately achieving the goal of improving malt PYF performance. This invention can be applied to the improvement of malt PYF performance.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and in particular relates to a method for regulating malt PYF based on gene expression during the malting process and its application. Background Technology

[0002] Premature yeast flocculation (PYF) refers to the phenomenon where yeast flocculates and settles before sufficient fermentation time has been reached during beer fermentation. This slows down sugar degradation and acetaldehyde reduction during fermentation, resulting in poor maturity of the finished beer and affecting its fermentation quality, ultimately causing beer quality problems. PYF is a quality issue unique to the beer industry, caused by factors in the malt that induce premature yeast flocculation.

[0003] Currently, the detection of malt PYF (polyylated yeast fermentation) generally involves preparing wort from the malt, adding yeast for fermentation, and then detecting the number of suspended yeasts in the fermentation liquid. This method requires 40-48 hours of fermentation time, and with the wort preparation and yeast count detection, the total testing time is 3-4 days, which is quite long. Furthermore, this method detects the finished malt, lacking predictability for the malt production process and making timely adjustments to the malt production process impossible.

[0004] Therefore, based on the shortcomings of the prior art, this invention proposes a method for regulating malt PYF based on gene expression during the malting process. This method can accurately predict the PYF performance of malt from the malt production process, and can also establish adjustment measures for the malting process based on the prediction results, thereby achieving the goal of improving the PYF performance of malt. Summary of the Invention

[0005] This invention addresses the technical problems of existing malt PYF detection methods, such as limited detection targets, narrow applicability, and inability to guide subsequent malting processes. It proposes a malt PYF regulation method based on gene expression during the malting process. This method is characterized by short processing time, high detection efficiency, and high accuracy. It can accurately predict the PYF performance of malt from the malt production process and establish adjustment measures for the malting process based on the prediction results, ultimately achieving the goal of improving malt PYF performance.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for regulating malt PYF based on gene expression during the malting process includes the following steps:

[0008] By analyzing the gene expression differences between standard malt and PYF malt during the malting process, and combining the large-scale production process, tracking genes were selected. The tracking genes are AP1, WRKY70, OSM1, GA2ox3, thioedoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17 and WRKY5.

[0009] After designing relevant primers based on the tracked gene sequence, the following procedures were performed sequentially: total RNA extraction from the malt to be tested and standard malt; cDNA template preparation via reverse transcription; multiplex PCR; electrophoresis; and expression level analysis of the product fragments. The PYF performance of the malt to be tested was then predicted and improved according to the following criteria:

[0010] Calculate the principal component scores of the standard malt and the malt to be tested, and take the absolute value of the difference between the two.

[0011] When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%, the PYF prediction of the malt to be tested is normal, and there is no need to adjust the malting process.

[0012] When the absolute value of the difference between the two is greater than 0.5 and less than 5.0 or greater than 5.0, the PYF prediction of the malt to be tested is abnormal, and it is necessary to reduce the soaking temperature of the malt during the malting process or reduce the fresh air volume and increase the return air volume.

[0013] In one embodiment, the tracking gene is obtained by screening using the following method:

[0014] Transcriptome analysis was performed on the differences in gene expression during the germination process of standard malt and PYF malt during malting, and several genes with significant expression differences were selected as candidate genes.

[0015] The large-scale production process of the standard malt and PYF malt was tracked, and the candidate genes with the most obvious change trends were further selected as the tracking genes.

[0016] In one embodiment, the screening criteria for the tracking gene are:

[0017] The change in gene expression is greater than or equal to 2.0 or less than 0.5;

[0018] The change in gene expression is defined as the ratio of the principal component score of PYF malt gene expression to the principal component score of standard malt gene expression.

[0019] In one embodiment, the primers designed based on the tracking gene sequence include a reverse transcription primer set and a multiplex PCR primer set.

[0020] In one embodiment, the reverse transcription primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, resulting in 13 primers as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, and SEQ ID NO.26.

[0021] In one embodiment, the multiplex PCR primer set was designed with 13 primers for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, and SEQ ID NO.25.

[0022] In one embodiment, the PYF performance of the malt to be tested is predicted using the following criteria:

[0023] Calculate the principal component scores of the standard malt and the malt to be tested, and take the absolute value of the difference between the two.

[0024] When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%, and the PYF prediction of the malt to be tested is normal.

[0025] When the absolute value of the difference between the two is greater than 0.5 and less than 5.0, the PYF value of the malt to be tested is 50-90%, the PYF prediction of the malt to be tested is abnormal, and its PYF value needs to be increased.

[0026] When the absolute value of the difference between the two is greater than 5.0, the PYF value of the malt to be tested is <50%, the PYF prediction of the malt to be tested is abnormal, and its PYF value needs to be increased.

[0027] In one embodiment, the prediction criteria for the PYF performance of the malt to be tested are obtained by the following method:

[0028] Gene expression analysis was performed on the tracking genes of different PYF malts to obtain the principal component scores of gene expression.

[0029] The principal component scores of gene expression are compared with those of standard malt to establish the PYF performance evaluation criteria for the malt.

[0030] In one embodiment, when the absolute value of the difference between the two is greater than 0.5 and less than 5.0 or greater than 5.0, the PYF prediction of the malt to be tested is abnormal. After adjusting the malting process by reducing the soaking temperature or reducing the fresh air volume and increasing the return air volume, gene expression detection and principal component score calculation are performed again until the absolute value of the difference between the two is less than 0.5.

[0031] The present invention also proposes an application of the malt PYF regulation method based on gene expression in the malting process described in any of the above embodiments in the improvement of PYF malt performance.

[0032] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0033] 1. This invention proposes a method for regulating malt PYF based on gene expression during the malting process. This method first selects tracking genes by comparing the gene expression differences between standard malt and PYF malt during the malting process and combining them with the large-scale production process. Then, by comparing the expression levels of standard malt and the malt to be tested with evaluation criteria, it achieves accurate prediction of malt PYF performance. Based on the prediction results, it is determined whether the PYF of the malt to be tested is abnormal. If the prediction is normal, no adjustment to the malting process is required. If it is abnormal, measures such as reducing the soaking temperature or reducing the fresh air volume and increasing the return air volume during the malting process are needed to increase the PYF value of the malt until the PYF of the malt is increased to the expected range. It can be seen that this method can accurately predict the PYF performance of malt from the malt production process and establish adjustment measures for the malting process based on the prediction results, ultimately achieving the goal of improving the PYF performance of malt.

[0034] 2. The present invention proposes a method for regulating malt PYF based on gene expression during the malting process. In order to improve the accuracy of the prediction results and thus provide more scientific guidance for subsequent malt production processes, the present invention establishes a dual standard including "tracking gene screening standard and malt PYF performance evaluation and improvement standard", which can achieve rapid and accurate analysis of malt PYF performance from the malt production process. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention provides a method for regulating malt PYF based on gene expression during the malting process, comprising the following steps:

[0037] S1. By analyzing the gene expression differences between standard malt and PYF malt during the malting process, and combining the large-scale production process, tracking genes were selected. The tracking genes are AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17 and WR KY5.

[0038] S2. After designing relevant primers based on the tracked gene sequence, the following steps were performed sequentially: total RNA extraction from the malt to be tested and standard malt, cDNA template preparation via reverse transcription, multiplex PCR, electrophoresis, and expression level analysis of the product fragments. The PYF performance of the malt to be tested was then predicted and improved according to the following criteria:

[0039] Calculate the principal component scores of the standard malt and the malt to be tested, and take the absolute value of the difference between the two.

[0040] When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%, the PYF prediction of the malt to be tested is normal, and there is no need to adjust the malting process.

[0041] When the absolute value of the difference between the two is greater than 0.5 and less than 5.0 or greater than 5.0, the PYF prediction of the malt to be tested is abnormal, and it is necessary to reduce the soaking temperature of the malt during the malting process or reduce the fresh air volume and increase the return air volume.

[0042] The functions of the 13 genes mentioned in the above embodiments are as follows:

[0043]

[0044] Furthermore, in this invention, for malt with abnormal PYF prediction, the reason for lowering the soaking temperature or reducing the fresh air volume and increasing the return air volume during the malting process is that lowering the temperature during the soaking stage and reducing the fresh air volume and increasing the return air volume can inhibit the growth of microorganisms, reduce the rate of barley germination, thereby reducing the possibility of microbial contamination and thus reducing the generation of PYF factors.

[0045] In one specific embodiment, the tracking gene is obtained through screening using the following method:

[0046] Transcriptome analysis was performed on the differences in gene expression during the germination process of standard malt and PYF malt during malting, and several genes with significant expression differences were selected as candidate genes.

[0047] The large-scale production process of the standard malt and PYF malt was tracked, and the candidate genes with the most obvious change trends were further selected as the tracking genes.

[0048] In one specific implementation, the screening criteria for the tracking gene are:

[0049] The change in gene expression is greater than or equal to 2.0 or less than 0.5;

[0050] The change in gene expression is defined as the ratio of the principal component score of PYF malt gene expression to the principal component score of standard malt gene expression.

[0051] In the above implementation, the purpose of setting the screening criteria for tracking genes at a gene expression change of greater than or equal to 2.0 or less than 0.5 is mainly to identify genes with significant differences in expression before and after the change as characteristic genes. Generally, a change in expression greater than 2 times is considered a significant difference. This could be an increase of more than 2 times or a decrease to less than half of the original value, hence the standard of greater than or equal to 2.0 or less than 0.5.

[0052] In one specific embodiment, the primers designed based on the tracked gene sequence include a reverse transcription primer set and a multiplex PCR primer set.

[0053] In one specific embodiment, the reverse transcription primer set was designed for 13 tracking genes AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, resulting in 13 primers as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, and SEQ ID NO.26, respectively.

[0054] In one specific embodiment, the multiplex PCR primer set was designed with 13 primers for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, and SEQ ID NO.25, respectively.

[0055] In one specific implementation, the PYF performance of the malt under test is predicted using the following criteria:

[0056] Calculate the principal component scores of the standard malt and the malt to be tested, and take the absolute value of the difference between the two.

[0057] When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%, and the PYF prediction of the malt to be tested is normal.

[0058] When the absolute value of the difference between the two is greater than 0.5 and less than 5.0, the PYF value of the malt to be tested is 50-90%, the PYF prediction of the malt to be tested is abnormal, and its PYF value needs to be increased.

[0059] When the absolute value of the difference between the two is greater than 5.0, the PYF value of the malt to be tested is <50%, the PYF prediction of the malt to be tested is abnormal, and its PYF value needs to be increased.

[0060] In one specific embodiment, the prediction standard for the PYF performance of the malt to be tested is obtained by the following method:

[0061] (1) Gene expression analysis was performed on the tracking genes of different PYF malts to obtain the principal component scores of gene expression;

[0062] The different PYF malts mentioned in step (1) above refer to malts with different PYF values ​​(specifically including PYF-90% malt, PYF-50% malt and PYF-30% malt). By analyzing the gene expression of malts with different PYF values, the resulting principal component scores and the judgment criteria based on these scores are more accurate.

[0063] Furthermore, the reason why this invention selects the principal component score of gene expression as the analysis index is that, in order to comprehensively evaluate the expression of multiple genes, the principal component analysis method is used to reduce the dimensionality of the expression data of multiple genes, thereby obtaining a comprehensive quantitative score of multiple genes.

[0064] (2) The principal component score of gene expression is compared with the principal component score of gene expression of standard malt to establish the performance judgment standard of malt PYF.

[0065] In one specific implementation, when the absolute value of the difference between the two is greater than 0.5 and less than 5.0 or greater than 5.0, the PYF prediction of the malt to be tested is abnormal. After adjusting the malting process by reducing the soaking temperature or reducing the fresh air volume and increasing the return air volume, gene expression detection and principal component score calculation are performed again until the absolute value of the difference between the two is less than 0.5.

[0066] The present invention also provides an application of the malt PYF regulation method based on gene expression in the malting process described in any of the above embodiments in improving the performance of PYF malt.

[0067] To more clearly and in detail introduce the method for regulating malt PYF based on gene expression in the malting process and its application provided by the embodiments of the present invention, the following description will be based on specific embodiments.

[0068] Example 1

[0069] This embodiment provides a screening process for candidate genes and tracking genes in a method for predicting malt PYF performance based on barley gene expression during the malting process, specifically as follows:

[0070] (1) Studies have shown that the problem of PYF malt production is related to the stress response of barley caused by external stimuli during planting and germination. Therefore, this invention analyzed the gene expression differences between standard malt and PYF malt during the malting process, especially during seed germination, through transcriptome analysis. Several genes with large expression differences were found, and 13 genes with the most obvious differences were selected as monitoring genes.

[0071] (2) Based on the results of step (1), by tracking the large-scale production process (i.e., during the malting process, green malts after soaking are taken. To ensure that the vitality of green malts does not change, the samples taken should be immediately subjected to RNA extraction or stored at -80℃), 13 genes with the most obvious change trends are selected as tracking genes. The screening criteria for tracking genes are: the gene expression change range is greater than or equal to 2.0 or less than 0.5, and the gene expression change range is the ratio of the gene amount of PYF malt to the gene expression amount of standard malt (see Table 1 for relevant data).

[0072] Table 1. Statistical analysis of gene expression in standard malt and PYF malt.

[0073]

[0074]

[0075] Example 2

[0076] This embodiment provides a process for determining the malt PYF performance evaluation criteria in a malt PYF performance prediction method based on barley gene expression during the malting process, specifically as follows:

[0077] Gene expression was analyzed and detected in three different PYF malts (PYF-90% malt, PYF-50% malt, and PYF-30% malt), and principal component scores of gene expression were obtained. The relevant standards were determined by comparing the scores with those of standard malt (see Table 2 for relevant data).

[0078] Table 2 Principal component scores of standard malt and PYF malt

[0079]

[0080]

[0081] As shown in the table above, by comparing the principal component scores of gene expression in malts with different PYF values ​​with those in standard malts, it was found that the absolute value of the difference between the principal component scores of gene expression in PYF-90% malt and standard malt was less than 0.5, the absolute value of the difference between the principal component scores of gene expression in PYF-50% malt and standard malt was greater than 0.5 but less than 5, and the absolute value of the difference between the principal component scores of gene expression in PYF-30% malt and standard malt was greater than 5.

[0082] Example 3

[0083] This embodiment provides a method for regulating malt PYF based on gene expression during the malting process. Specifically, this embodiment predicts and improves the PYF performance of newly purchased batch A barley, as follows:

[0084] (1) Selection of monitoring genes: Thirteen genes including AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17 and WRKY5 were selected as monitoring genes.

[0085] (2) Sample collection: During the wheat processing, take the green malt after soaking (Note: In order to ensure that the vitality of the green malt does not change, the sample taken should be extracted for RNA immediately or stored at -80℃).

[0086] (3) Green malt grinding: To ensure that the RNA in green malt is not degraded, green malt is ground and pulverized using liquid nitrogen;

[0087] (4) RNA extraction:

[0088] The crushed green malt was then subjected to RNA extraction by adding Trizol (Note: Trizol is a total RNA extraction reagent that can directly extract total RNA from cells or tissues).

[0089] Take 50-100 mg of sample, add 1 ml of Trizol, lyse by shaking, and centrifuge at 12000 g for 5 min at 4 °C. Transfer the supernatant to a Phasemarker tube and let stand for 5 min. Add 0.2 ml of chloroform, shake manually for 15 s, and let stand for 10 min. Centrifuge at 12000 g for 10 min at 4 °C. Take the supernatant (450-550 μL) to an EP tube, add 250 μL of 96% ethanol, and mix with a pipette tip. Transfer the mixture to a silicone tube, centrifuge at 12000 g for 1 min, and remove the waste liquid. Add 700 μL of WB1, centrifuge at 12000 g for 1 min, and remove the waste liquid. Add 500 μL of WB2, centrifuge at 12000g for 1 min, and remove the runoff waste liquid; add 500 μL of WB2, centrifuge at 12000g for 1 min, and transfer the silicone tube to a 1.5 ml EP tube; add 50 μL of nuclease-free water, let stand for 1 min, and centrifuge at 12000g for 1 min; collect the centrifuged RNA solution and store it at -80℃;

[0090] (5) RT-PCR reverse transcription:

[0091] Using the purified barley cell RNA obtained in step (4) as a template, the first strand of cDNA was synthesized using the Beckman Coulter GenomeLab™ GeXP starter kit with downstream primers of the designed multiple primers as specific primers (the sequences of which are shown in Table 3) and total RNA from barley cells as a template. The reaction system was 10 μL, and the parameters for the first strand of cDNA synthesis were set as follows: 48℃ for 1 minute; 42℃ for 60 minutes; 95℃ for 5 minutes.

[0092] Then, the DNA polymerase from Beckman Coulter and the GenomeLab™ GeXP starter kit were used. Using the synthesized first-strand cDNA as a template and the upstream primers of the 13 multiplex primers as specific primers (their sequences are shown in Table 3), RT-PCR amplification was performed. The RT-PCR amplification parameters were set as follows: 95℃ pre-denaturation for 10 minutes; 94℃ denaturation for 30 seconds; annealing at 56℃ for 30 seconds; extension at 71℃ for 1 minute, for 35 cycles, finally obtaining the reverse transcription amplified product of the expressed gene.

[0093] Table 3 Upstream and downstream primer sequences

[0094]

[0095]

[0096] (6) Gene expression level analysis:

[0097] The reverse transcription products were quantitatively analyzed using capillary electrophoresis. 1 μl of the PCR multiplex product was added to the well of a plate containing 39 μl of a mixture of 95% deionized formamide (SLS) and 400 bp marker, mixed with a pipette, and then covered with a drop of paraffin oil.

[0098] In addition, 250 μl of separation buffer was added to each well of the buffer plate. After all preparations were completed, capillary electrophoresis was performed. After electrophoresis, the expression levels of different genes at different stages were obtained.

[0099] (7) PYF performance prediction:

[0100] PCA analysis was performed on the gene expression of sample A and standard barley (the results are shown in Table 4):

[0101] Table 4. PCA analysis of gene expression in batch A malt and standard malt.

[0102] serial number Gene Standard malt gene expression Batch A malt gene expression 1 AP1 0.39 0.33 2 WRKY70 0.66 0.58 3 OSM1 0.48 0.34 4 GA2ox3 0.92 0.67 5 thioredoxin 0.4 0.51 6 serpin 0.38 0.42 7 RPP13 0.79 0.68 8 RbohE 0.73 0.78 9 RbohB2 0.55 0.66 10 A2 0.86 1.02 11 IAA16 1.1 0.98 12 BGU17 2.3 2.18 13 WRKY5 9.19 10.01

[0103] Principal component analysis showed that the principal component score of standard malt was -1.563, and the principal component score of malt A was -1.628. The absolute value of the difference between the two was 0.065, which is less than 0.5. The PYF value of malt A was 99%, indicating that the PYF prediction of malt was normal and no process adjustment was required.

[0104] Example 4

[0105] This embodiment provides a method for regulating malt PYF based on gene expression during the malting process. Specifically, this embodiment predicts and improves the PYF performance of newly purchased batch B barley, as follows:

[0106] The specific prediction method is the same as in Example 3, the only difference being the batch of malt.

[0107] PCA analysis was performed on the gene expression of batch B and standard barley (the results are shown in Table 5):

[0108] Table 5. PCA analysis of gene expression in batch B malt and standard malt.

[0109]

[0110]

[0111] Based on the data in the table above, through principal component analysis, the principal component score of standard malt is -1.385, and the principal component score of barley B is 0.181. The absolute value of the difference between the two is 1.566, which is greater than 0.5 and less than 5. The PYF value of batch B barley is 78%, which is low. Process adjustments are needed to improve the PYF value.

[0112] This embodiment reduces the soaking temperature during the barley processing from 18℃ to 16℃, then re-detects gene expression and calculates principal component scores. The score is -1.393, with an absolute difference of 0.008, less than 0.5. At this point, the PYF value of the malt is 91%. (Note: Previous studies have shown that PYF problems are related to external factors affecting barley during the barley processing, such as microbial contamination. Lowering the soaking temperature can reduce the growth rate of contaminating microorganisms and slow down barley germination, thus alleviating PYF problems and increasing PYF values.)

[0113] Example 5

[0114] This embodiment provides a method for regulating malt PYF based on gene expression during the malting process. Specifically, this embodiment predicts and improves the PYF performance of newly purchased batches of C barley, as follows:

[0115] The specific prediction method is the same as in Example 3, the only difference being the batch of malt.

[0116] PCA analysis was performed on the gene expression of batch C and standard barley (the results are shown in Table 6):

[0117] Table 6. PCA analysis of gene expression in batches C malt and standard malt.

[0118]

[0119] Based on the data in the table above, principal component analysis showed that the principal component score of standard malt was -1.385, while that of batch C was 4.241. The absolute value of the difference between the two was 5.627, which is greater than 5. The PYF value of malt C was 43%, indicating that the PYF of this batch of malt was low and process adjustments were needed to improve the PYF value.

[0120] This embodiment reduces the fresh air volume from 100% to 50% and increases the return air volume to 50% by decreasing the fresh air volume and increasing the return air volume. Gene expression detection is then performed again, followed by principal component analysis (PCA) calculation. The PCA score is -0.359, and the absolute difference between the two is 1.026, which is greater than 0.5 and less than 5. The PYF value of batch C malt is increased to 82%. (Note: Studies have shown that PYF problems are related to external factors affecting barley during the malting process, such as microbial contamination. Reducing the fresh air volume and increasing the return air volume can reduce the growth of contaminating microorganisms and decrease barley growth, thus alleviating PYF problems and improving PYF values.)

[0121] Verification test

[0122] To further verify the accuracy of the malt PYF regulation method based on gene expression during the malting process, the malt PYF performance prediction method was verified (Verification Experiment 1), and the malt PYF performance improvement method was further verified (Verification Experiment 1), as detailed below:

[0123] 1. Verification Experiment 1: The results were verified using traditional evaluation methods, as detailed below:

[0124] The traditional evaluation method (which involves preparing malt into wort, adding yeast for fermentation, and then detecting the number of suspended yeast cells in the fermentation broth) was used, and the results were compared with those of Examples 3-5. The results are as follows:

[0125] The traditional evaluation methods are as follows:

[0126] (1) Preparation of wort: Take a certain amount of malt, crush it, add water for saccharification, and then filter to obtain saccharified wort;

[0127] (2) Yeast inoculation and fermentation: Take a certain amount of yeast mud and place it in sterilized wort, shake to aerate, and then ferment at 20℃ for 48 hours;

[0128] (3) Yeast counting: Take a certain amount of fermentation broth and count the yeast using a yeast counter. Obtain the yeast concentration in the fermentation broth;

[0129] (4) PYF value calculation: PYF value = number of yeast cells in the sample / number of yeast cells in the standard sample × 100%.

[0130] Table 7 Comparison of results from traditional evaluation methods and Examples 3-5

[0131]

[0132] As shown in Table 7, compared with traditional evaluation methods, the method of the present invention can accurately predict the performance of malt PYF by comparing the gene expression differences between standard malt and PYF malt during the malting process, selecting tracking genes in the large-scale production process, and then comparing the expression levels of standard malt and the test malt in combination with evaluation standards.

[0133] 2. Verification Experiment 2

[0134] This experiment verified the results of batches B and C of malt after adjusting the malting process in Examples 4-5. The experiment also used traditional evaluation methods for result verification, with the specific operating steps being the same as in Verification Experiment 1. The only difference was that the malt used was batch B malt and batch C malt after adjusting the process. The experimental results are as follows:

[0135] Table 8 Comparison of results from traditional evaluation methods and Examples 4-5

[0136]

[0137] A comprehensive analysis of the improved test results in Table 8 and the results before improvement in Table 7 shows that the improvement method provided in Examples 4-5 of this invention can establish adjustment measures for the malting process based on the prediction results, ultimately achieving the goal of improving the performance of malt PYF.

Claims

1. A method for regulating malt PYF based on gene expression during malting, characterized in that, Includes the following steps: By analyzing the gene expression differences between standard malt and PYF malt during the malting process, and combining the large-scale production process, tracking genes were selected. The tracking genes are AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17 and WRKY5. After designing relevant primers based on the tracked gene sequence, the following procedures were performed sequentially: total RNA extraction from the malt to be tested and standard malt; cDNA template preparation via reverse transcription; multiplex PCR; electrophoresis; and expression level analysis of the product fragments. The PYF performance of the malt to be tested was then predicted and improved according to the following criteria: Gene expression analysis was performed on the tracking genes of standard malt and malts with different PYF values. Principal component analysis was used to obtain the principal component scores of gene expression. The different PYF malts refer to malts with different PYF values, specifically including PYF-90% malt, PYF-50% malt and PYF-30% malt. Calculate the principal component scores of the standard malt and the malt to be tested, and take the absolute value of the difference between the two. When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%, the PYF prediction of the malt to be tested is normal, and there is no need to adjust the malting process. When the absolute value of the difference between the two is greater than 0.5 and less than 5.0 or greater than 5.0, the PYF prediction of the malt under test is abnormal, and it is necessary to reduce the soaking temperature of the malt during the malting process or reduce the fresh air volume and increase the return air volume.

2. The method for regulating malt PYF based on gene expression during the malting process according to claim 1, characterized in that, The tracking gene was obtained through screening using the following method: Transcriptome analysis was performed on the differences in gene expression during the germination process of standard malt and PYF malt during malting, and several genes with significant expression differences were selected as candidate genes. The large-scale production process of the standard malt and PYF malt was tracked, and the candidate genes with the most obvious change trends were further selected as the tracking genes.

3. The method for regulating malt PYF based on gene expression during the malting process according to claim 2, characterized in that, The screening criteria for the tracking genes are as follows: The change in gene expression is greater than or equal to 2.0 or less than 0.5; The change in gene expression is defined as the ratio of the principal component score of PYF malt gene expression to the principal component score of standard malt gene expression.

4. The method for regulating malt PYF based on gene expression during the malting process according to claim 1, characterized in that, The primers designed based on the tracked gene sequence include a reverse transcription primer set and a multiplex PCR primer set.

5. The method for regulating malt PYF based on gene expression during the malting process according to claim 4, characterized in that, The reverse transcription primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5. Thirteen primers were designed and are shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, and SEQ ID NO.26, respectively.

6. The method for regulating malt PYF based on gene expression during the malting process according to claim 5, characterized in that, The multiplex PCR primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5. Thirteen primers were designed and are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, and SEQ ID NO.25, respectively.

7. The method for regulating malt PYF based on gene expression during malting according to claim 1, characterized in that, The PYF performance of the malt under test was predicted using the following criteria: Calculate the principal component scores of the standard malt and the malt to be tested, and take the absolute value of the difference between the two. When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%, and the PYF prediction of the malt to be tested is normal. When the absolute value of the difference between the two is greater than 0.5 and less than 5.0, the PYF value of the malt to be tested is 50-90%, the PYF prediction of the malt to be tested is abnormal, and its PYF value needs to be increased. When the absolute value of the difference between the two is greater than 5.0, the PYF value of the malt to be tested is <50%, the PYF prediction of the malt to be tested is abnormal, and its PYF value needs to be increased.

8. The method for regulating malt PYF based on gene expression during the malting process according to claim 1, characterized in that, When the absolute value of the difference between the two is greater than 0.5 and less than 5.0 or greater than 5.0, the PYF prediction of the malt under test is abnormal. After adjusting the malting process by reducing the soaking temperature or reducing the fresh air volume and increasing the return air volume, gene expression detection and principal component score calculation are performed again until the absolute value of the difference between the two is less than 0.

5.

9. The application of the malt PYF regulation method based on gene expression in the malting process according to any one of claims 1-8 in improving the performance of PYF malt.

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