阅读说明：本技术 采用β-葡萄糖苷酶抑制剂对草菇进行冷藏保鲜的方法 (Method for refrigerating and preserving straw mushrooms by adopting beta-glucosidase inhibitor ) 是由 龚明 邹根 万佳宁 鲍大鹏 黄天宇 李正鹏 唐利华 于 2021-07-27 设计创作，主要内容包括：本发明提供了采用草菇β-葡萄糖苷酶抑制剂对草菇进行冷藏的方法,所述的β-葡萄糖苷酶抑制剂的结构式依次如下所示：将草菇β-葡萄糖苷酶抑制剂Conduritol B epoxide(CB)和D-Glucono-1,5-lactone(DG)分别加水,使得β-葡萄糖苷酶抑制剂CB和DG的浓度为150μM和300μM,将蛋形期草菇浸泡在上述溶液中2min,然后在4℃进行储存。本发明发现浓度为150μM的草菇β-葡萄糖苷酶抑制剂CB和300μM的草菇β-葡萄糖苷酶抑制剂DG对子实体冷藏保鲜效果最佳。(The invention provides a method for refrigerating straw mushrooms by adopting a straw mushroom beta-glucosidase inhibitor, wherein the structural formula of the beta-glucosidase inhibitor is sequentially as follows: adding water into Volvariella volvacea beta-glucosidase inhibitor Conduritol B epoxide (CB) and D-Glucono-1,5-lactone (DG) respectively to make the concentration of the beta-glucosidase inhibitor CB and DG 150 μ M and 300 μ M, soaking Volvariella volvacea in the above solution for 2min, and storing at 4 deg.C. The invention discovers that the sub-entity refrigeration and fresh-keeping effects of the straw mushroom beta-glucosidase inhibitor CB with the concentration of 150 mu M and the straw mushroom beta-glucosidase inhibitor DG with the concentration of 300 mu M are optimal.)

1. The method for preserving the straw mushrooms by adopting the straw mushroom beta-glucosidase inhibitor is characterized in that the structural formula of the straw mushroom beta-glucosidase inhibitor is CB or DG type, and is as follows:

orThe method is characterized in that: adding water into the beta-glucosidase inhibitor, and if the beta-glucosidase inhibitor is CB type, adding water to enable the concentration of the beta-glucosidase inhibitor CB to be 150 mu M, soaking the straw mushrooms in the egg-shaped period for 2min, and then storing at 1-6 ℃; if the beta-glucosidase inhibitor is DG type, adding water to make the concentration of the beta-glucosidase inhibitor DG 300 μ M, soaking the egg-shaped period straw mushroom for 2min, and storing at 1-6 deg.C.

2. The method for cold preservation of straw mushrooms with β -glucosidase inhibitors as claimed in claim 1, wherein: storage was carried out at 4 ℃.

3. The beta-glucosidase inhibitor is used as a straw mushroom preservative, and the structural formula of the beta-glucosidase inhibitor is CB or DG type, and is shown as follows:

orThe concentrations of CB and DG were 150. mu.M and 300. mu.M, respectively.

Technical Field

The invention belongs to the field of food, relates to a food refrigeration and preservation technology, and particularly relates to a method for refrigerating and preserving straw mushrooms by adopting a beta-glucosidase inhibitor.

Background

Straw mushroom (Volvariella volvacea) is called "Chinese mushroom" and is an important edible fungus. The straw mushroom not only has delicious taste, but also has important medical health care functions, such as oxidation resistance, immunity regulation, tumor resistance and the like. The straw mushroom has delicious meat quality and short growth cycle (about 10 days), and is an edible mushroom with high economic benefit. However, the growth and fruiting of the hypha of volvariella volvacea require relatively high temperature (28-35 ℃), and especially the hypha and the fruiting body are not refrigerated, and even under the conventional refrigeration of 4 ℃, the hypha and the fruiting body can be softened, liquefied and even rotten in a short time, namely the so-called low-temperature autolysis. The uniqueness of the straw mushroom 'low-temperature autolysis' provides a preservation problem. Therefore, the shelf life of the straw mushrooms is obviously shorter than that of other mushrooms all the time, so that the cultivation and the popularization of the straw mushrooms are greatly limited, and the economic benefit of degrading the straws is indirectly influenced.

Researches on the straw mushroom preservation technology are developed, but the straw mushroom is preserved by acting on straw mushroom fruiting bodies. For example, the use of ethephon and 1-methylcyclopropene (1-MCP) on Volvariella volvacea in egg-shaped stage; the straw mushroom preservation is researched by adopting a chemical agent soaking method, such as chitosan composite membrane soaking treatment; the straw mushroom is preserved by adopting physical modes such as surface refrigeration air-drying treatment, microwave heating sterilization treatment and the like. The technologies are applied to the harvested straw mushroom fruiting bodies, and the problems of insignificant effect, influence on quality and the like generally exist. Moreover, the technologies all act on the study of low-temperature preservation of the straw mushroom fruiting body at 15 ℃, and the study of cold preservation at 4 ℃ is lacked.

Recent research proves that one type of straw mushroom ubiquitination synthase E2(a type of ubiquitin-conjugating E2 enzyme, UBEV2) is an effector for promoting low-temperature autolysis, and the low-temperature autolysis can be inhibited to a certain extent by adding a small-molecule inhibitor L345-0044 of UBEV2, so that the damage of cold pressing to straw mushrooms is reduced. On the basis, the gene and metabolite expression profiles of sporocarp treated by UBEV2 inhibitor (L345-0044) under the cold pressing action are analyzed by using absolute quantitative transcriptome and non-targeted metabolome combined omics to mine enzymes of UBEV 2-mediated downstream channels, so that the straw mushroom cold preservation and fresh-keeping technology is developed.

Disclosure of Invention

The invention aims to provide a method for refrigerating and preserving straw mushrooms by adopting a beta-glucosidase inhibitor, and the method for refrigerating and preserving straw mushrooms at low temperature aims to solve the problems that straw mushroom fruiting bodies cannot be refrigerated and preserved, the practicability is low, the food safety is high and the like in the prior art.

The invention provides a method for preserving straw mushroom by adopting a beta-glucosidase inhibitor, wherein the structural formula of the straw mushroom beta-glucosidase inhibitor is CB or DG type, and is as follows:

adding water into the beta-glucosidase inhibitor, and if the beta-glucosidase inhibitor is CB type, adding water to enable the concentration of the beta-glucosidase inhibitor CB to be 150 mu M, then soaking the straw mushrooms in the egg-shaped period for 2min, and then storing at 1-6 ℃; if the beta-glucosidase inhibitor is DG type, adding water to make the concentration of the beta-glucosidase inhibitor DG 300 μ M, soaking the egg-shaped period straw mushroom for 2min, and storing at 1-6 deg.C.

Specifically, CB and DG are Conduritol B epoxide (CAS No.:6090-95-5) and D-Glucono-1,5-lactone (CAS No.:90-80-2), respectively.

Further, storage was carried out at 4 ℃.

The invention also provides the application of the beta-glucosidase inhibitor as a straw mushroom preservative, wherein the structural formulas of the beta-glucosidase inhibitor are CB and DG types respectively, and are as follows:

the concentrations of CB and DG were 150. mu.M and 300. mu.M, respectively.

According to the invention, the beta-glucosidase inhibitor solutions with different concentrations and types are designed to carry out anti-freezing experimental verification on the straw mushrooms, so that an optimal scheme is provided for the research and development of the straw mushroom refrigeration and preservation technology. Based on the reported improvement effect of the UBEV2 inhibitor small molecular compound L345-0044 on the straw mushroom cold preservation, an experiment is designed to mine a target gene located in a UBEV 2-mediated downstream pathway, and a straw mushroom cold preservation technology with strong popularization and safety is further developed.

Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The method for refrigerating and preserving the straw mushroom by adopting the beta-glucosidase inhibitor solution has the following advantages:

(1) the beta-glucosidase is used as a target gene of a UBEV 2-mediated downstream pathway, and the aim of cold storage and fresh keeping of the straw mushrooms can be achieved by inhibiting the activity of the beta-glucosidase.

(2) In view of the fact that DG is widely applied to the food industry as a food additive (StandardNO: GB 7657) -2005, the food safety problem caused by the soaking of the inhibitor compound can be better solved by adopting DG to soak the straw mushroom fruiting body.

(3) Because the market cost of DG is low (the purity is 98 percent, and the price of 1kg is about 500 yuan), the problem of the cost of cold preservation and fresh keeping of the straw mushroom fruiting body soaked by the inhibitor compound solution is well solved.

(4) The concentration and dosage problems of the beta-glucosidase inhibitor solution for cold storage and fresh keeping of the straw mushrooms are solved well.

Drawings

FIG. 1 shows a 4 ℃ cold storage experiment of the immersed straw mushroom fruit body. 0H is the fruit body soaked in water for 2min, 24H is the fruit body soaked in water for 2min and refrigerated at 4 ℃ for 24H, and L24H is 150 μ M L345-0044 for soaking the fruit body soaked in water for 2min and refrigerated at 4 ℃ for 24H.

FIG. 2 analysis of the major components of the absolute quantitative transcriptome of Volvariella volvacea, and the numbers of the fruiting body samples are shown in the caption of FIG. 1.

FIG. 3 is a chromatogram of the base peak of the non-targeted metabolome of Volvariella volvacea, with the numbers of the fruiting body samples annotated with the title of FIG. 1.

FIG. 4 shows the correlation between the absolute quantitative transcriptome and metabolome derived significantly different genes, (| log2FoldChange | >1, P-value <0.05) and significantly different metabolites (P-value ≦ 0.05 and VIP ≧ 1).

Fig. 5 shows the correlation enzymes of significantly different genes and significantly different metabolites obtained for absolute quantitative transcriptome and non-targeted metabolome.

Figure 6 shows a KEGG pathway map (Ko00500) for starch and sucrose metabolism.

FIG. 7 shows a thermographic analysis of the gene expression of volvariella volvacea beta-glucosidase, h stands for hour. The numbers of the fruit body samples are annotated with the title of FIG. 1.

Figure 8 shows qPCR analysis of volvariella volvacea β -glucosidase. h represents hour, and the number of the fruit body sample is annotated with the title of FIG. 1.

FIG. 9 shows a western blot analysis of volvariella volvacea beta-glucosidase, h represents hour. The numbers of the fruit body samples are annotated with the title of FIG. 1.

FIG. 10 shows the enzymatic activity of straw mushroom β -glucosidase. The numbers of the fruit body samples are annotated with the title of FIG. 1.

Fig. 11 shows the structural formula of β -glucosidase inhibitor CB.

FIG. 12 shows a cold storage experiment of volvariella volvacea at 4 ℃ under treatment with β -glucosidase inhibitor CB (300 μ M). h represents hour.

FIG. 13 shows a cold storage experiment of volvariella volvacea at 4 ℃ under treatment with β -glucosidase inhibitor CB (150 μ M). h represents hour.

FIG. 14 shows the enzyme activity analysis of volvariella volvacea beta-glucosidase treated with beta-glucosidase inhibitor CB (300. mu.M). GC-case stands for β -glucosidase inhibitor CB treatment. h represents hour.

Fig. 15 shows the structural formula of the β -glucosidase inhibitor DG.

FIG. 16 shows a cold storage experiment of volvariella volvacea at 4 ℃ under treatment with a β -glucosidase inhibitor DG. DG treatment concentrations were 150. mu.M, 300. mu.M, 1000. mu.M, respectively. h represents hour.

Detailed Description

Example 1

(ii) experiment of refrigeration at 4 ℃ of immersed fruit body

In the reference (Wanjianing et al, "homology modeling of straw mushroom UBEV2 protein and screening of small molecule inhibitors", "Proc. edible mushrooms, 2020), L345-0044 (from Chemdiv compound database) is a UBEV2 inhibitor, 150 μ M of L345-0044 solution is used for soaking and treating straw mushroom fruiting bodies for 2min, and water treatment is adopted as a control, and then the straw mushroom fruiting bodies are respectively placed at 4 ℃ for 24h and 48h for refrigeration experiments. Wherein 0H is control treated fruiting body, 24H is water soaked fruiting body refrigerated at 4 deg.C for 24 hr, and L24H is 150 μ M fruiting body refrigerated at 4 deg.C for 24 hr soaked in L345-0044 (figure 1).

② Absolute quantitative transcriptome analysis of immersion treated samples at 4 ℃ refrigeration

Putting the soaked sample into a refrigeration experiment at 4 ℃ for 24h, extracting a corresponding sample and carrying out absolute quantitative transcriptome analysis, and mainly comprising the following steps: after RNA extraction, quantification, purification and library construction, the second Generation Sequencing technology (NGS) is adopted to perform double-ended (PE) Sequencing on the libraries based on an Illumina Sequencing platform. And filtering reads obtained by sequencing, and then comparing the filtered reads with a reference genome to obtain gene expression data so as to analyze the expression quantity among samples. The results of the principal component analysis of the obtained gene expression data showed that the L345-0044 treated samples were clearly separated from the control samples (fig. 2), indicating that the expression profiles of the L345-0044 treated samples and the water-treated samples were clearly different in cold pressing.

Analysis of non-target metabolic components of immersion-treated samples at 4 ℃ cold storage

The metabolites of 18 sub-entities (6 each of 0H, 24H and L24H) were detected by LC-MS/MS, the main steps being: the metabolite of the sample is extracted, part of the sample is mixed for quality control, and then the rest of the sample is processed by an LC-MS detection machine. Adopting a Thermo Ultimate 3000 system to be equipped with ACQUITYHSST 3 (150X 2.1mm,1.8 μm, Waters) was chromatographed. By using Thermo Q active HF-XAnd (4) mass spectrum identification. From the Base Peak Chromatography (BPC), it can be seen that the three treated samples have different differences in ESI positive and negative ion modes after being treated with L345-0044 (FIG. 3), which indicates that the metabolic profiles of the L345-0044 treated sample and the water treated sample are obviously different in the refrigeration at 4 ℃.

Example 2

Analysis of correlation between absolutely quantitative transcriptome and non-targeted metabolome

Correlation analysis of differentially expressed metabolites in 24H _ vs _ L24H with genes resulted in correlated differential genes (>3) and differential metabolites (fig. 4), such as glucose 1-phosphate (C00103) and cellobiose (C00185). EC annotation of these genes showed that β -glucosidase (3.2.1.21) is the second largest number of enzymes (fig. 5). The KEGG pathway (Ko00500) shows that glucose 1-phosphate and cellobiose are involved in starch and sucrose metabolism, with β -glucosidase responsible for the breakdown of cellobiose to D-glucose (fig. 6).

② qPCR and western blot analysis of straw mushroom beta-glucosidase

Thermographic analysis of β -glucosidase expression showed that L345-0044 solution treatment down-regulated β -glucosidase expression (fig. 7), suggesting that β -glucosidase down-regulation is essential for straw mushroom antifreeze. The heatmap analysis also showed that 3 β -glucosidase genes were up-regulated in 24H. qPCR experiments confirmed that they were up-regulated in 24H expression, in particular VVO — 08574 was abnormally high expressed in 24H (fig. 8). The probability of VVO-08574 having a signal peptide was predicted to be 93.193% (signal peptide type: Sec/SPI) using SignalP 5.0, suggesting that VVO-08574 is likely to belong to an extracellular enzyme. Western blot experiments with β -glucosidase showed the disappearance of the band at about 70kDa at 24H (FIG. 9). The molecular weight of VVO _08574 was 71.35kDa, which confirmed that VVO _08574 belongs to an extracellular enzyme. These results indicate that cold pressing induces secretion of extracellular β -glucosidase.

③ analysis of enzyme activity of straw mushroom beta-glucosidase

The beta-glycosidase activity verification result shows that the 24H enzyme activity value is the highest (figure 10). The activity of β -glucosidase in 24H exudate was measured and the measured Δ a (fluorescence reading) averaged 0.2809(s.d. ═ 0.003), confirming that cold pressing induced extracellular β -glucosidase secretion. Considering that L24H had no exudate and 24H had exudate, this indicates that cold-pressed 24H had a higher beta-glucosidase enzyme activity than actually tested.

Example 3

Freezing experiment of fruit body under soaking treatment at 4 deg.C

The results of example 2 suggest that cold pressing stimulates β -glucosidase activity, resulting in low temperature autolysis. Adding water into beta-glucosidase inhibitor CB (figure 11) and DG respectively to make the concentration of the beta-glucosidase inhibitor CB and DG 150 μ M and 300 μ M in sequence, soaking the egg-shaped period straw mushroom for 2min respectively, treating with water as a control, and then placing at 4 ℃ for 24h and 48h for refrigeration experiments respectively. The frost resistance test shows that the egg-shaped fruiting bodies of the straw mushroom, which are respectively impregnated with 150 mu M CB and 300 mu M DG, have full appearance, bright color and hard hardness under the cold pressure treatment, and are obviously superior to the control treatment (figure 12 and figure 16). For example, 24h after cold pressing treatment, 150 μ M CB and 300 μ M DG solution soaked the treated straw mushroom fruit body, the texture was full, the color was bright, and the surface was dry (FIGS. 12 and 16). In contrast, after 24H of cold pressing, H₂And (4) softening the fruiting body of the straw mushroom in the egg-shaped period after the soaking in the step (O), and partially browning the fruiting body. After 24H of cold pressing treatment, only H is obtained₂The O treatment forms the exudate, which is not the case with the CB and DG treatments. Compared with the water treatment, the CB and DG solution treatment maintained the shape of the fruit body better after 48 hours of cold pressing treatment, but the fruit body became soft and partially browned, forming a exudate (fig. 12 and 16).

② enzyme activity test of the immersed fruit body in cold storage at 4 ℃

The verification result of the beta-glucosidase activity shows that the CB treatment obviously reduces the beta-glucosidase activity of the straw mushroom in 24-hour cold storage, which is similar to H₂The O-treated volvaria volvacea β -glucosidase activity was increased differently (fig. 13). The results show that the antifreezing effect of the straw mushroom is improved by reducing the activity of the beta-glucosidase inhibitor after the soaking of the beta-glucosidase inhibitor, and the cold storage and fresh-keeping time of the straw mushroom is prolonged.

Comparative example 1

(1) The experiment was performed by soaking for 2 minutes using water as a control treatment.

(2) The results show that: the fruiting body after water treatment is refrigerated and stored for 24 hours, and the mushroom body is shriveled, collapsed and partially browned. During 48 hours of refrigerated storage, the mushroom shape had been severely deformed, collapsing (fig. 12).

Comparative example 2

(1) The soaking experiment was performed for 2 minutes using 300. mu.M CB solution as a control.

(2) The results show that: the fruiting body treated by 300 μ M CB solution is preserved in 24 hours with refrigeration, the mushroom shape is kept intact, but the surface is wet; the cut section shows slight browning of the fruiting body. During 48 hours of cold storage, the mushroom body had deformed, partially collapsed, and dissolved (FIG. 14).

Comparative example 3

(1) The fruiting body immersion experiment was carried out for 2 minutes using 150. mu.M DG (FIG. 15) solution as a control.

(2) The results show that: the fruiting body treated with 150 μ M solution is kept intact in 24 hr cold storage, but the surface of mushroom body is wet. During 48 hours of refrigerated storage, the mushroom bodies had partially deformed, collapsed and dissolved, with a large amount of exudate present (FIG. 16).

Comparative example 4

(1) The fruiting body was immersed for 2 minutes using 1000. mu.M DG (FIG. 16) solution as a control.

(2) The results show that: the fruiting body treated with 1000 μ M solution can be preserved in 24 hr under refrigeration, with the mushroom shape intact, but the top of the mushroom slightly collapses. The cut section shows slight browning of the fruiting body. During 48 hours of refrigerated storage, the mushroom bodies had partially deformed, collapsed and dissolved, with a large amount of exudate present (FIG. 16).

It will be apparent to those skilled in the art that other various changes and modifications may be made based on the above-described embodiments, and all such changes and modifications are intended to fall within the scope of the appended claims.