CN113736775B - Application of xanthan gum pretreated by using expansin to reduce viscosity of xanthan gum - Google Patents

Abstract

The invention discloses an application method for reducing viscosity of xanthan gum by pretreatment of the xanthan gum through expansin, and belongs to the technical field of pretreatment for reducing viscosity of the xanthan gum. The invention uses the expansin to pretreat the xanthan gum for depolymerization, so as to promote the viscosity of the xanthan gum to be reduced. The method of the invention overcomes the defects of high equipment requirement, poor product quality and the like in the prior art when a large amount of alkali solution is used for treating xanthan gum by the traditional chemical method. Saving the cost, not causing environmental pollution and conforming to the environment-friendly type. Meanwhile, the method is simple and feasible, and is beneficial to industrial popularization and application.

Description

Application of pre-treating xanthan gum with dilatin to reduce its viscosity

Technical Field

The invention belongs to the technical field of pretreatment for reducing the viscosity of xanthan gum, and specifically relates to an application method of utilizing expansin to pretreat xanthan gum and reduce its viscosity.

Background Art

Xanthan gum is a microbial polysaccharide. It is a polysaccharide substance with β-(1,4) glucan as the main chain and a side chain formed by β-D-mannose, β-D-glucuronic acid, and α-D-mannose on a glucose group as the basic structural unit. Untreated xanthan gum molecules have abundant hydrogen bonds to form a complex secondary structure, resulting in a very high viscosity of its aqueous solution. Xanthan gum can only be pretreated with strong alkali, ionic liquid or high temperature to promote its viscosity reduction, and then hydrolyzed with hydrolase to generate xanthan gum oligosaccharides. The product xanthan gum oligosaccharides have the common properties of bioactive oligosaccharides such as chitosan oligosaccharides and seaweed oligosaccharides, such as the potential to scavenge free radicals in vitro and inhibit the growth of pathogenic bacteria. It has greater application value in the fields of medicine, food and health products, planting, etc. This makes reducing the viscosity of xanthan gum an important key step to improve its hydrolysis efficiency.

At present, the main method to reduce the viscosity of xanthan gum is to dissolve xanthan gum with a strong alkaline solvent under high temperature conditions to reduce its viscosity. Subsequently, the pH of the xanthan gum solution needs to be adjusted to neutral for enzymatic hydrolysis to generate xanthan gum oligosaccharides. This method requires high temperature in the production process and uses a strong alkaline solvent in the production process. It has high requirements for production equipment and is prone to environmental pollution. Therefore, finding a green and efficient method for pre-treating xanthan gum to reduce its viscosity has received increasing attention.

Expansin comes from bacteria and plants. It is a non-hydrolyzable auxiliary protein that has a destructive effect on substrates with polysaccharide network structures. It breaks the hydrogen bonds between polysaccharides without hydrolyzing them and opens the dense crystal structure of the polysaccharide substrate. This allows water molecules or hydrolases to enter the polysaccharide substrate and form a water-soluble form.

Summary of the invention

The present invention aims to provide a method for depolymerizing xanthan gum by pre-treating it with expansin, which aims to overcome the shortcomings of the prior art such as environmental pollution caused by chemical reagents and poor product quality during the hydrolysis of xanthan gum by traditional chemical methods, and to provide an economical, efficient and environmentally friendly enzymatic method for pre-treating xanthan gum.

A gene, the nucleotide sequence of which is shown as SEQ ID NO.1.

The amino acid sequence of the expansin encoded by the above gene is shown in SEQ ID NO.2.

A recombinant expression bacterium of the above expansion protein.

An application of using dilatin to pretreat xanthan gum to reduce its viscosity. The dilatin is added to the xanthan gum for pretreatment to reduce the viscosity of the xanthan gum.

Furthermore, the optimum temperature for pre-treating xanthan gum was 50°C and the reaction time was 72h.

Compared with the reported method for reducing the viscosity of xanthan gum, the present invention uses a biological method to efficiently hydrolyze the hydrogen bonds in the xanthan gum, destroy the dense grid structure of the xanthan gum to pre-treat the xanthan gum to depolymerize and reduce its viscosity. The method of the present invention overcomes the deficiencies in the prior art such as the use of a large amount of strong alkaline solution when treating xanthan gum by traditional chemical methods, high equipment requirements, and poor product quality. It saves costs, does not cause environmental pollution, and is environmentally friendly. At the same time, the method is simple and easy to implement, which is conducive to industrial promotion and application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an SDS-PAGE electrophoresis diagram of the purified expansin protein of the present invention; in the figure, the first lane: protein marker; the second lane: whole bacteria of empty bacteria pET-28a/BL21; the third lane: expression supernatant of empty bacteria pET-28a/BL21; the fourth lane: expression supernatant of recombinant bacteria pET-28a-HcEX/BL21.

FIG. 2 is the optimum temperature for depolymerization of xanthan gum pretreated with expansin according to the present invention.

FIG. 3 shows the optimal reaction time for depolymerization of xanthan gum pretreated with expansin according to the present invention.

FIG. 4 is a comparison of the viscosity of untreated xanthan gum and the depolymerized xanthan gum pretreated with the expansin of the present invention.

DETAILED DESCRIPTION

The method of the present invention is further described below by means of specific examples.

The present invention is further described below by way of examples, which are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all commercially available.

Example 1 Construction of recombinant bacteria of expansin

According to the nucleotide sequence shown in SEQ ID NO.1, a third party company was commissioned to synthesize, that is, the expansin gene sequence is shown in SEQ ID NO.1, and the coding region is 660bp long. The amino acid sequence encoded by the expansin gene is shown in SEQ ID NO.2. The synthetic SEQ ID NO.1 gene sequence was connected to the plasmid pET-28a to obtain the recombinant pET-28a-HcEX plasmid (synthetic gene of Jilin Province Kumei Biotechnology Co., Ltd.). Prepare Escherichia coli BL21 competent state, electrotransfer the recombinant pET-28a-HcEX plasmid into BL21, apply it on an LB plate containing 30μg/ml kanamycin for screening, pick multiple single colonies and culture them at 30°C and 200rpm in LB liquid culture medium containing 30μg/ml kanamycin for 12 hours, and use T7 universal primers for colony PCR verification. The result is an amplification product of the correct size, proving the correctly constructed recombinant expression bacteria, and the recombinant bacteria are named pET-28a-HcEX/BL21.

SEQ ID NO.1

GAAAACCGTGTTTCTGCGACTCACACCTCCGCCGCGCTGCATGAGGGTGAAGGTACTTACTACTTCTACAACGGCGGCGGCCATTGCAGCGTTCCGGTGCCGGCGATGTTCACTGCAGCGATGAACCAGACCGACTATAACGGTTCCCAGGCTTGTGGCGGTTGCGTTAAGGTGACCAACCGCAACAACGGCAAGTCCGTGGTGGCGCCGTTGACGACTCTTGTCCGGGCTGCAACCCGGGTGACGTGGATCTGACC GACGCCGCCTTCGCGCAGATTTCTCCACTGGAGGCGGGTCGCATTCCGATCAGCTGGGATTATGTTCCGTGC GATTATCCGTCTGTGCTGCTGTACTTCATGGAAGGTTCTAGCCAGTGGTGGACCGCCGTGCAAGTACGCGAACAGCGTTATCCGGTAAGCTCTCTGGCGTACCGTGAATCTGGTTCTACCGGCTCCTATCAGGAGATCGCCCGTGAAGACTACAACTACTTTGTTGAACGTTCCGGCATGGGTACCGGCCCGTTTGATTTTCGCATCACCGACATCTATGGTCATGTGCTGGAAGCAGGTAACATCACCCTGCAGTCTGGC GTTCCGATCAACACCCAGCAACAGTTTCCGTCTATGGGTACCTCCGGCGTTATTAACCAGGCAGACAAA

SEQ ID NO.2

Example 2 Expression of expansin in recombinant bacteria

The recombinant bacteria identified correctly in Example 1 were streaked with pET-28a-HcEX/BL21 on LB plates containing 30 μg/ml kanamycin; a single colony was picked in 5 mL of LB liquid containing 30 μg/ml kanamycin and cultured for 12 hours as a seed solution; the colony was inoculated into LB liquid medium containing 30 μg/ml kanamycin at a ratio of 1:50 (volume ratio) and cultured until OD600 = 1, IPTG was added to a final concentration of 0.6 mM, and cultured at 16°C and 200 rpm for 20 hours. The expression of expansin was detected by polyacrylamide gel electrophoresis, and the results are shown in Figure 1, and expansin was significantly expressed under the induction of IPTG.

Example 3 Study on the depolymerization conditions of xanthan gum pretreated with expansin

The expansin pET-28a-HcEX/BL21 obtained in Example 1 was used as a substrate to determine the depolymerization conditions of pre-treated xanthan gum, including the optimal temperature and reaction time.

(1) Determination of optimum temperature

The experimental group used 0.5wt% xanthan gum as the substrate, and the expansin was reacted at different temperatures (40℃, 50℃, 60℃), 200rpm, and 72 hours. The viscosity of the xanthan gum was measured using an NDJ-79A rotational viscometer (Shanghai Changji Geological Instrument Co., Ltd.). The 0.5wt% xanthan gum was the control group. The results are shown in Figure 2. When the reaction temperature was 50℃, the viscosity of the xanthan gum decreased from 214mPa.s to 150mPa.s, and the viscosity decrease was the most significant. Therefore, the optimal temperature of the expansin is 50℃.

(2) Determination of optimal reaction time

Under the above optimum temperature conditions, the same system was reacted at different times (12h, 24h, 36h, 48h, 72h), and the viscosity of xanthan gum was measured using a NDJ-79A rotational viscometer. The results are shown in Figure 3. The viscosity of xanthan gum has not changed at 84h. Considering the stability of the protein, the optimal reaction time for the extended protein effect on xanthan gum is 72h.

Example 4: Pretreatment of xanthan gum with dilatin to reduce viscosity

15 mg of the expansin pET-28a-HcEX/BL21 obtained in Example 1 was used to act on 20 ml of 0.5 wt% xanthan gum, and the reaction was carried out at 50°C and 160 rpm for 72 h, and the viscosity change of the xanthan gum was measured using a NDJ-79A rotational viscometer. The control group was 20 ml of 0.5 wt% xanthan gum, and the reaction was carried out at 50°C and 200 rpm for 72 h, and the viscosity change of the xanthan gum was measured using a NDJ-79A rotational viscometer. The viscosity of the xanthan gum after the reaction was compared with the viscosity of the xanthan gum before the reaction to obtain a relative viscosity ratio. As shown in FIG4 , the relative viscosity ratio of the xanthan gum in the control group did not change, while the viscosity of the xanthan gum after the expansin pretreatment was reduced by 30%.

SEQUENCE LISTING

<110> Dalian University of Technology

<120> Application of pre-treating xanthan gum with dilatin to reduce its viscosity

<130> 2021

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 660

<212> DNA

<213> Artificial Sequence

<400> 1

gaaaaccgtg tttctgcgac tcacacctcc gccgcgctgc atgagggtga aggtacttac 60

tacttctaca acggcggcgg ccattgcagc gttccggtgc cggcgatgtt cactgcagcg 120

atgaaccaga ccgactataa cggttcccag gcttgtggcg gttgcgttaa ggtgaccaac 180

cgcaacaacg gcaagtccgt ggtggcgcgc gttgacgact cttgtccggg ctgcaacccg 240

ggtgacgtgg atctgaccga cgccgccttc gcgcagattt ctccactgga ggcgggtcgc 300

attccgatca gctgggatta tgttccgtgc gattatccgt ctgtgctgct gtacttcatg 360

gaaggttcta gccagtggtg gaccgccgtg caagtacgcg aacagcgtta tccggtaagc 420

tctctggcgt accgtgaatc tggttctacc ggctcctatc aggagatcgc ccgtgaagac 480

tacaactact ttgttgaacg ttccggcatg ggtaccggcc cgtttgattt tcgcatcacc 540

gacatctatg gtcatgtgct ggaagcaggt aacatcaccc tgcagtctgg cgttccgatc 600

aacacccagc aacagtttcc gtctatgggt acctccggcg ttattaacca ggcagacaaa 660

<210> 2

<211> 220

<212> PRT

<213> Artificial Sequence

<400> 2

Glu Asn Arg Val Ser Ala Thr His Thr Ser Ala Ala Leu His Glu Gly

1 5 10 15

Glu Gly Thr Tyr Tyr Phe Tyr Asn Gly Gly Gly His Cys Ser Val Pro

20 25 30

Val Pro Ala Met Phe Thr Ala Ala Met Asn Gln Thr Asp Tyr Asn Gly

35 40 45

Ser Gln Ala Cys Gly Gly Cys Val Lys Val Thr Asn Arg Asn Asn Gly

50 55 60

Lys Ser Val Val Ala Arg Val Asp Asp Ser Cys Pro Gly Cys Asn Pro

65 70 75 80

Gly Asp Val Asp Leu Thr Asp Ala Ala Phe Ala Gln Ile Ser Pro Leu

85 90 95

Glu Ala Gly Arg Ile Pro Ile Ser Trp Asp Tyr Val Pro Cys Asp Tyr

100 105 110

Pro Ser Val Leu Leu Tyr Phe Met Glu Gly Ser Ser Gln Trp Trp Thr

115 120 125

Ala Val Gln Val Arg Glu Gln Arg Tyr Pro Val Ser Ser Leu Ala Tyr

130 135 140

Arg Glu Ser Gly Ser Thr Gly Ser Tyr Gln Glu Ile Ala Arg Glu Asp

145 150 155 160

Tyr Asn Tyr Phe Val Glu Arg Ser Gly Met Gly Thr Gly Pro Phe Asp

165 170 175

Phe Arg Ile Thr Asp Ile Tyr Gly His Val Leu Glu Ala Gly Asn Ile

180 185 190

Thr Leu Gln Ser Gly Val Pro Ile Asn Thr Gln Gln Gln Phe Pro Ser

195 200 205

Met Gly Thr Ser Gly Val Ile Asn Gln Ala Asp Lys

210 215 220

Claims (2)

1. An application of pre-treating xanthan gum with an expansin to reduce its viscosity, characterized in that the viscosity of the xanthan gum can be reduced by adding the expansin to the xanthan gum for pre-treatment; The amino acid sequence of the expansin is shown in SEQ ID NO.2. 2. The use according to claim 1, characterized in that the optimum temperature for pretreating xanthan gum is 50°C and the reaction time is 72h.

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