CN112980813B

CN112980813B - Low-temperature modified exoinulase mutant MutS117G

Info

Publication number: CN112980813B
Application number: CN202110041587.0A
Authority: CN
Inventors: 周峻沛; 张蕊; 黄遵锡; 岑潇龙; 唐湘华; 许波; 李俊俊; 韩楠玉; 吴倩; 高艳秀
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2022-08-30
Anticipated expiration: 2041-01-13
Also published as: CN112980813A

Abstract

The invention discloses an exoinulase mutant MutS117G improved at low temperature, wherein the mutant MutS117G has an amino acid sequence shown as SEQ ID NO. 1. Compared with the wild enzyme InuAMN8, the mutant enzyme MutS117G has changed thermal activity and thermal stability, and the mutant enzyme MutS117G has higher low-temperature activity but lower thermal stability. The optimal temperature of the purified wild enzyme InuAMN8 is 35 ℃, and the optimal temperature of the mutant enzyme MutS117G is 25 ℃; after treatment at 50 ℃, the enzyme activity of the wild enzyme InuAMN8 is reduced from 88% to 81%, and the enzyme activity of the mutant enzyme MutS117G is reduced from 87% to 58%; after treatment at 55 ℃, 70% of the enzyme activity of the wild enzyme InuAMN8 is remained, and the mutant enzyme is completely inactivated. The mutant MutS117G can be applied to industries of food, brewing, washing and the like.

Description

Low-temperature modified exoinulase mutant MutS117G

Technical Field

The invention relates to an exoinulase mutant, in particular to an exoinulase mutant MutS117G improved at low temperature.

Background

The jerusalem artichoke is suitable for being planted on barren slope land and arid saline-alkaline non-ploughing marginal land, does not compete for farmland with crops, and has the advantages of high yield, cold resistance, barren tolerance, drought tolerance and the like. Inulin is the major ingredient constituting the tuber of jerusalem artichoke and may constitute 19% by wet weight or 70% by dry weight thereof.

The inulase can degrade inulin to generate fructose and a small part of glucose, and the fructose content in the fructose syrup can reach 95 percent of the total sugar content. The fructose is widely used in the industries of food, medicine, biological energy and the like. For example, fructose may be used as a natural sweetener in place of sucrose; the fructose metabolism is not restricted by insulin, and can be eaten by diabetic patients; the fructose can be used for producing bioethanol after being fermented by yeast and the like. Therefore, the inulase exonuclease can be applied to industries such as food, wine brewing and biological energy (Singh RS et al. International Journal of biological Macromolecules,2017,96: 312-.

In some practical applications, a low temperature environment is required, for example, food treatment at low temperature can prevent microbial contamination, nutrient loss and food quality degradation, fermentation of sake and wine, aquaculture environment, washing, sewage treatment are generally performed at low temperature; in addition, low temperature treatment can also reduce energy consumption (Cavicchiali et al. Microbiological Biotechnology,2011,4(4): 449-. Therefore, low temperature enzymes have important development value.

Low temperature exoinulinase can be used for fermentation of wine, pickling of food, decontamination, hydrolysis of inulin at low temperature to produce high concentration fructose syrup, etc. (Zhou et al, journal of bioscience and Bioengineering, 2015,119(3): 267-274). The low-temperature inulase exonuclease has unique advantages in practical application, such as high catalytic activity in a low-temperature environment, energy conservation, cost saving, enzyme inactivation by simple heat treatment, and simple, convenient and effective operation.

Therefore, the method for obtaining the exoinulase mutant which has higher activity in a low-temperature environment and is easier to heat treat, namely the exoinulase mutant with reduced optimal temperature and thermal stability is more beneficial to the practical application of the enzyme.

Disclosure of Invention

The invention aims to provide an exoinulase mutant MutS117G improved at low temperature, wherein the thermal activity and the thermal stability of the mutant MutS117G are changed, the low-temperature activity of the mutant MutS117G is high but the thermal stability is poor, and the mutant MutS117G can be applied to industries such as food, wine brewing, washing and the like.

In order to achieve the above object, the present invention provides a low temperature modified exoinulase mutant MutS117G, wherein the mutant MutS117G has an amino acid sequence as shown in SEQ ID NO. 1. In contrast to the exoinulinase sequence AGC01505(SEQ ID NO.3) recorded in GenBank, amino acid 117 of MutS117G is glycine and amino acid 117 of AGC01505 is serine.

The mutant MutS117G has the optimum temperature of 25 ℃, and has 24 percent, 53 percent, 91 percent and 86 percent of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 35 ℃ respectively; after the treatment at 50 ℃ for 10-60 min, the enzyme activity of MutS117G is reduced from 87% to 58%; after treatment at 55 ℃ for 10min, MutS117G is completely inactivated; the enzyme can hydrolyze inulin to produce fructose.

Another objective of the invention is to provide the gene mutS117G encoding the mutant mutS 117G.

Preferably, the coding gene mutS117G has the nucleotide sequence shown in SEQ ID NO. 2.

Another object of the present invention is to provide a recombinant vector comprising said gene mutS 117G.

Another object of the present invention is to provide a recombinant bacterium comprising said encoding gene mutS 117G.

It is another object of the present invention to provide a method for preparing the mutant MutS117G, which comprises: connecting a wild exoinulase gene inuAMN8 with a nucleotide sequence shown as SEQ ID NO.4 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN 8; the plasmid pEasy-E1-inuAMN8 is taken as a template, and mutation primers of nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 are used for carrying out PCR amplification to obtain a recombinant expression plasmid pEasy-E1-mutS117G containing mutS 117G; transforming the recombinant expression plasmid pEasy-E1-mutS117G into Escherichia coli BL21(DE3) to obtain a recombinant strain containing mutS 117G; culturing the recombinant strain, and inducing the expression of the recombinant exoinulase mutant MutS117G to obtain the recombinant exoinulase mutant MutS 117G.

Preferably, the recombinant strain comprising mutS117G is prepared by digesting the recombinant expression plasmid pEasy-E1-mutS117G with DpnI enzyme using Mut

II Fast Mutagenesis Kit digested products were ligated and transformed into E.coli BL21(DE3) by heat shock.

Preferably, the induction is with IPTG.

Preferably, the product expressed by the recombinant exoinulase mutant MutS117G is subjected to affinity and purification by Nickel-NTAAgarose and 0-500 mM imidazole respectively to obtain the recombinant exoinulase mutant MutS 117G.

Another objective of the invention is to provide the application of the mutant MutS117G in food, wine brewing and washing.

The low-temperature improved exoinulase mutant MutS117G has the following advantages:

compared with the wild enzyme InuAMN8, the mutant enzyme MutS117G has changed thermal activity and thermal stability, the mutant enzyme MutS117G has higher low-temperature activity but worse thermal stability, lower optimal temperature and easier thermal denaturation, has higher catalytic activity in a low-temperature environment, and is favorable for controlling the catalytic reaction of the enzyme through temperature change. The optimal temperature of the purified wild enzyme InuAMN8 is 35 ℃, and the purified wild enzyme InuAMN8 has 15%, 32%, 58% and 74% of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 25 ℃, respectively, while the optimal temperature of the mutant enzyme MutS117G is 25 ℃, and the purified wild enzyme InuAMN8 has 24%, 53%, 91% and 86% of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 35 ℃, respectively; after being treated at 50 ℃ for 10-60 min, the enzyme activity of the wild enzyme InuAMN8 is reduced from 88% to 81%, and the enzyme activity of the mutant enzyme MutS117G is reduced from 87% to 58%; after the treatment of the temperature of 55 ℃ for 10min, 70 percent of the enzyme activity of the wild enzyme InuAMN8 is remained, and the mutant enzyme is completely inactivated. The low-temperature improved exoinulase mutant MutS117G can be applied to industries such as food, wine brewing, washing and the like.

Drawings

FIG. 1 is an SDS-PAGE analysis of the wild-type enzyme InuAMN8 and the mutant enzyme MutS 117G.

FIG. 2 shows the thermal activity of the purified wild enzyme InuAMN8 and the mutant enzyme MutS 117G.

FIG. 3 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutS117G at 50 ℃.

FIG. 4 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutS117G at 55 ℃.

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.

Experimental materials and reagents in the following examples:

1. bacterial strain and carrier

Escherichia coli BL21(DE3) and expression vector pEasy-E1 are commercially available from Beijing Quanyujin Biotechnology, Inc.; arthrobacter sp (Arthrobacter sp.) is provided by university of Master Yunnan and deposited at the Collection of microorganisms and research institute in Yunnan province under the number YMF 4.00006.

2. Enzymes and other biochemical reagents

Nickel-NTAAgarose was purchased from QIAGEN, DNA polymerase, dNTP and Mut

II Fast Mutagenesis Kit was purchased from Nanjing Novovin, inulin was purchased from Alfa Aesar, bacterial genomic DNA extraction Kit was purchased from Tiangen Biochemical technology (Beijing) Ltd, and the others were made of domestic reagents (all of which can be purchased from general Biochemical reagent Co.).

3. Culture medium

LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000 mL, natural pH (about 7). On the basis of the solid medium, 2.0% (w/v) agar was added.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

EXAMPLE 1 construction and transformation of expression vector for wild enzyme InuAMN8

(1) Extracting Arthrobacter genome DNA: centrifuging the liquid culture for 2d to obtain thallus, adding 1mL of lysozyme, treating at 37 deg.C for 60min, extracting Arthrobacter genome DNA according to the instruction of bacterial genome DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd.), and standing at-20 deg.C for use.

(2) Designing primers 5'-ATGAATTCATTGACGACGGC-3' (SEQ ID NO.5) and 5'-TCAACGGCCGACGACGTCGA-3' (SEQ ID NO.6) according to an inulinase exonuclease nucleotide sequence JQ863111(SEQ ID NO.4) recorded by GenBank, and carrying out PCR amplification by using Arthrobacter genome DNA as a template, wherein the PCR reaction parameters are as follows: denaturation at 95 deg.C for 5 min; then denaturation at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 1min for 30sec, and heat preservation at 72 ℃ for 5min after 30 cycles. The PCR result obtained the coding gene inuAMN8 of the wild exoinulase inuAMN 8. inuAMN8 can also be obtained by gene synthesis based on the inulinase nucleotide sequence JQ 863111.

(3) The exoinulase gene inuAMN8 and an expression vector pEasy-E1 are connected to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN 8.

(4) Coli BL21(DE3) was transformed by heat shock with pEasy-E1-inuAMN8 to obtain recombinant E.coli strain BL21(DE3)/inuAMN8 comprising inuAMN 8.

Example 2 construction and transformation of the expression vector for the mutant enzyme MutS117G

(1) Designing primers 5'-TTACACCGGTGCCTACAGTGACGCCGCGCCGC-3' (SEQ ID NO.7) and 5'-TGTAGGCACCGGTGTAAATGGCCACCAGCGGG-3' (SEQ ID NO.8), carrying out PCR amplification by taking a plasmid pEasy-E1-inuAMN8 as a template, wherein the PCR reaction parameters are as follows: denaturation at 95 ℃ for 30 sec; then denaturation at 95 ℃ for 15sec, annealing at 70 ℃ for 15sec, extension at 72 ℃ for 3min for 30sec, and heat preservation at 72 ℃ for 5min after 30 cycles. The PCR result gave the recombinant expression linearized plasmid pEasy-E1-mutS117G containing mut S117G (SEQ ID NO. 2). mut S117G and pEasy-E1-mutS117G can also be obtained by gene synthesis.

(2) mu.L of the PCR product of linearized plasmid pEasy-E1-mutS117G was digested with 1. mu.L of DpnI enzyme at 37 ℃ for 1 h.

(3) Using Mut

II Fast Mutagenesis Kit, the digestion product in step (2) was ligated for 30min at 37 ℃.

(4) The ligation product in step (3) was transformed into E.coli BL21(DE3) by heat shock to obtain recombinant strain BL21(DE3)/mutS117G comprising mutS 117G.

Example 3 preparation of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutS117G

(1) Recombinant strains BL21(DE3)/inuAMN8 and BL21(DE3)/mutS117G were inoculated in LB (containing 100. mu.g mL) at an inoculum size of 0.1% respectively ^-1 Amp) in the culture medium, the mixture was rapidly shaken at 37 ℃ for 16 hours.

(2) Then the activated bacteria liquid is inoculated with 1 percent of inoculum size respectivelySeed to fresh LB (100. mu.g mL) ^-1 Amp) was rapidly cultured with shaking for about 2 to 3 hours (OD600 reached 0.6 to 1.0), then IPTG was added to a final concentration of 0.7mM for induction, and shaking culture was continued at 20 ℃ for about 20 hours. Centrifuging at 12000 rpm for 5min, and collecting thallus. After the cells were suspended in an appropriate amount of McIlvaine buffer at pH7.0, the cells were disrupted by ultrasonic waves in a low-temperature water bath. After the crude enzyme solution concentrated in the above cells was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the objective protein was respectively subjected to affinity purification using Nickel-NTA Agarose and 0-500 mM imidazole.

(3) SDS-PAGE results (FIG. 1, M: protein Marker) showed that both recombinant InuAMN8 (SEQ ID NO.3) and MutS117G (SEQ ID NO.1) were purified and the product was a single band.

EXAMPLE 4 determination of the Properties of the purified recombinant wild-type enzymes InuAMN8 and the mutant enzyme MutS117G

1. Analysis of the Activity of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutS117G

The activity determination method adopts a 3, 5-dinitrosalicylic acid (DNS) method: dissolving the substrate inulin in a buffer solution to make the final concentration of the inulin be 0.5% (w/v); the reaction system contains 50 mu L of proper enzyme solution and 450 mu L of substrate; preheating a substrate at a reaction temperature for 5min, adding an enzyme solution, reacting for 10min, adding 750 mu L DNS to terminate the reaction, boiling in boiling water for 5min, cooling to room temperature, and measuring an OD value at a wavelength of 540 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down the substrate under the given conditions to produce 1. mu. mol reducing sugars (calculated as fructose) per minute.

2. Determination of the thermal Activity of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutS117G

The enzymatic reaction was carried out at 0-60 ℃ in a buffer at pH 7.0. Reacting for 10min by taking inulin as a substrate, and determining the enzymological properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutS 117G.

The results show that: the wild enzyme InuAMN8 has an optimum temperature of 35 ℃ and 15%, 32%, 58% and 74% of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 25 ℃ respectively, while the mutant enzyme MutS117G has an optimum temperature of 25 ℃ and 24%, 53%, 91% and 86% of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 35 ℃ respectively (FIG. 2).

3. Thermostability assay of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutS117G

The enzyme solutions with the same enzyme amount were treated at 50 deg.C and 55 deg.C for 10-60 min, and then the enzyme reaction was carried out at pH7.0 and 37 deg.C, with untreated enzyme solution as control. Reacting for 10min by taking inulin as a substrate, and determining the enzymological properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutS 117G.

The results show that: after treatment for 10-60 min at 50 ℃, the enzyme activity of the wild enzyme InuAMN8 is reduced from 88% to 81%, and the enzyme activity of the mutant enzyme MutS117G is reduced from 87% to 58% (figure 3). After 10min treatment at 55 ℃, 70% of the wild enzyme InuAMN8 remained in enzyme activity, while the mutant enzyme was completely inactivated (FIG. 4).

4. Analysis of the products of hydrolysis of inulin by the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutS117G

The product analysis reaction system contained 450. mu.L of 0.5% (w/v) inulin, 50. mu.L of an appropriate dilution of the enzyme solution (total 0.1U of the enzyme solution). The reaction was terminated after 4h of enzymatic reaction at pH7.0 and 37 ℃. The product analysis was carried out by thin layer chromatography (using high performance thin layer chromatography silica gel plate type G available from Qingdao ocean chemical Co., Ltd.).

The procedure for the above thin layer chromatography is as follows:

(1) preparing a developing solvent (20 mL of glacial acetic acid, 20mL of double distilled water and 40mL of n-butanol, uniformly mixing), pouring a proper amount of the developing solvent into a developing tank, and standing for about 30 min;

(2) activating the silica gel plate in a 110 deg.C oven for 30min, cooling, scribing, and spotting (0.5 μ L each time, blow drying, and spotting for 3 times);

(3) placing the silica gel plate at one end of the sample application downwards into an expansion tank, wherein the sample application point does not immerse a developing agent;

(4) when the developing agent is 1.5cm away from the upper edge of the silica gel plate, taking out the silica gel plate, drying and developing again;

(5) after the second unfolding, directly immersing the silica gel plate into a proper amount of color developing agent (1g of diphenylamine is dissolved in 50mL of acetone, 1mL of aniline and 5mL of 85% phosphoric acid are added after the dissolution, and the mixture is uniformly mixed and prepared on site;

(6) after a few seconds, the silica gel plate was immediately removed and placed in an oven at 90 ℃ for 10-15 min to develop the spots.

The results show that: the products of hydrolysis of inulin by the wild enzyme InuAMN8 and the mutant enzyme MutS117G are almost all fructose.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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<120> Low temperature modified exoinulase mutant MutS117G

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ccgtgcgacg aacacgaggc catcttctcc ggctcggcgg tattcgatca gcacaacacc 300

agcggcctcg gcacagcggc caatcccccg ctggtggcca tttacaccag tgcctacagt 360

gacgccgcgc cgcttccggg ccggcaggcg cagtcgctcg cctacagcct cgacgaaggc 420

cggacctgga ccaagtacca cggcaatccc gtgctggacc gcgcgtccgc tgacttccgc 480

gatccaaagg ttttttggta cgacggcggc gccggaagtt actgggtgat ggtcgccgtc 540

gaggcggtgc agcgccaggt agtgctgtac aagtcggccg acctgaaggc gtgggaacac 600

ctgagcacct ttggccctgc caacgccacc ggcggcgtct gggaatgccc ggacctgttt 660

gagctgcccg tggacgggaa tccggaggac aaccggtggg tcctcattgt gaacatcaac 720

ccgggcggca ttgccggcgg ctccgcggga cagtacttcg tgggagagtt cgacggcgtg 780

gcgttccatt ccggatcgac tgtcaccgag ggcctccaga aggacagcag ccggatgcgg 840

gagtacggct ggctggactg ggggcgggac tactacgccg ccgtttcgtt cagcaacgtg 900

ccggacgggc gccggatcat gatcggctgg atgaacaact gggactacgc ccgcgagacg 960

cccaccggcg gctggcgcag cgccatgtcc ctgccgcggg aggtgtcgct gacccgggta 1020

gacgggaaag tgatgcttcg gcagcaagcc attgatccgt tgccggagcg ggaaacaggg 1080

cacgtccggc tggggccgca gcccttggcg tccggcgttc tggacgttcc ggccgccgca 1140

tccgtggcgc ggatcgacgt tgagctggag ccgggcgctg ccgcgggagt gggactggtg 1200

cttcgggcgg gggacgatga gcggacggtc ctccgctacg acacttcgga cgggatgctg 1260

cggctggacc gccgcgaatc cgggcaggtt gccttccacg aaaccttccc gtcgatcgaa 1320

gccatggccg tgcccttgca gggaggccgg ctgcgcctgc gggtctacct ggaccgctgc 1380

tcggtggagg ttttcgccca ggacgggctc gccacgctca ctgacctggt gttccccggg 1440

gaggcgagca cgggcctggc catcttcgcc gaaggtgagg gggcgcacct cgtggtgctc 1500

gacgtcgtcg gccgttga 1518

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgaattcat tgacgacggc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcaacggccg acgacgtcga 20

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttacaccggt gcctacagtg acgccgcgcc gc 32

<210> 8

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgtaggcacc ggtgtaaatg gccaccagcg gg 32

Claims

1. The low-temperature improved exoinulase mutant MutS117G is characterized in that the amino acid sequence of the mutant MutS117G is shown as SEQ ID number 1.

2. The mutant MutS117G according to claim 1, encoding gene mutS 117G.

3. The encoded gene mutS117G of claim 2, wherein the nucleotide sequence of the encoded gene mutS117G is shown in SEQ ID number 2.

4. A recombinant vector comprising the gene mutS117G according to claim 2 or 3.

5. Recombinant bacterium comprising the gene mutS117G according to claim 2 or 3.

6. A method for preparing the mutant MutS117G according to claim 1, comprising:

connecting a wild exoinulase gene inuAMN8 with a nucleotide sequence shown as SEQ ID number 4 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN 8;

the plasmid pEasy-E1-inuAMN8 is used as a template, and mutation primers of nucleotide sequences shown as SEQ ID number 7 and SEQ ID number 8 are used for carrying out PCR amplification to obtain a recombinant expression plasmid pEasy-E1-mutS117G containing mutS 117G;

transforming the recombinant expression plasmid pEasy-E1-mutS117G into Escherichia coli BL21(DE3) to obtain a recombinant strain containing mutS 117G;

the recombinant strain is cultured and the expression of the recombinant exoinulase mutant MutS117G is induced to obtain the recombinant exoinulase mutant MutS 117G.

7. The method according to claim 6, wherein the recombinant strain containing mutS117G is prepared by digesting the recombinant expression plasmid pEasy-E1-mutS117G with DpnI enzyme, ligating the digested products with Mut Express II Fast mutagenetics Kit, and transforming into Escherichia coli BL21(DE3) by heat shock.

8. The method of claim 6, wherein the induction is performed using IPTG.

9. The preparation method of claim 6, wherein the product expressed by the recombinant exoinulase mutant MutS117G is subjected to affinity by Nickel-NTA Agarose and then to elution and purification by 0-500 mM and not 0mM of imidazole to obtain the recombinant exoinulase mutant MutS 117G.

10. Use of the mutant MutS117G according to claim 1 in the food industry.

11. Use according to claim 10, characterized in that the food industry is the wine industry.