CN111100903B - Method for quantitatively detecting iota-carrageenan by enzyme method - Google Patents

Abstract

The invention relates to the technical fields of biotechnology and biochemical detection, in particular to a method for quantitatively detecting ι-carrageenan by enzymatic method. Utilize ι-carrageenanase to specifically degrade ι-carrageenan into reducing sugar, after inactivating the enzyme, add p-hydroxybenzoic hydrazide solution to carry out color reaction, measure supernatant at 400-420nm after centrifugation Absorbance value is compared with standard curve to obtain ι-carrageenan content. The detection method of the invention has good linear range and high accuracy, and can be popularized and used in the market.

Description

A method for enzymatic quantitative detection of iota-carrageenan

technical field

The invention relates to the technical fields of biotechnology and biochemical detection, in particular to a method for quantitatively detecting iota-carrageenan by enzymatic method.

Background technique

Carrageenan (carrageenan), also known as carrageenan or carrageenan, is a natural anionic sulfate linear polysaccharide extracted from the cell walls of red algae such as carrageen, Eucheuma, and firella. According to the sulfate group on galactose in the structure There are many types of carrageenans depending on the number and linking position and the content of 3,6-anhydrogalactose. ι-carrageenan is one of three common types of carrageenan (κ-, ι-, and λ-carrageenan), which consist of alternating sulfated-α-D-3,6-anhydrogalactose and sulfated-β- Composed of D-galactose residues, due to its wide range of sources and diverse functions, it is used in many industrial productions such as food, medicine, cosmetics, etc., as a coagulant, binder, stabilizer, emulsifier, suspending agent, thickener And so on.

The quantitative detection of ι-carrageenan is the basic link in the quality control, function research and product development of ι-carrageenan. At present, the commonly used quantitative detection methods for iota-carrageenan are phenol-sulfuric acid method and liquid chromatography-tandem mass spectrometry. The phenol-sulfuric acid method has high sensitivity and does not require expensive instruments, but it is easily affected by other polysaccharides in the sample, and it is time-consuming and consumes more samples and reagents. The liquid chromatography-tandem mass spectrometry method hydrolyzes, derivatizes and pretreats the sample, and obtains the content of iota-carrageenan by measuring the total content of monosaccharides composed of iota-carrageenan. This method has high sensitivity and good accuracy, but the cost of the instrument is high , The operation is cumbersome.

p-Hydroxybenzohydrazide (pHBH) is an aromatic hydrazide compound, which can form a yellow product with β-diketone compounds under strong alkaline conditions. Studies have shown that pHBH can react with reducing sugars such as glucose to produce similar yellow substances under high temperature and alkaline conditions. The color depth after the reaction is proportional to the concentration of reducing sugars, and the sensitivity of the reaction is high. This provides feasibility for enzymatic quantitative detection of iota-carrageenan.

In summary, it is of great significance to find a quantitative detection method for ι-carrageenan with high sensitivity, good accuracy and easy operation.

Contents of the invention

The technical problem to be solved in the present invention is that the commonly used iota-carrageenan quantitative detection methods are phenol-sulfuric acid method and liquid chromatography-tandem mass spectrometry, but these two methods have the disadvantages of poor reproducibility and complex operation, time-consuming and labor-intensive respectively, The detection with high sensitivity, good accuracy and easy operation cannot be realized.

In order to solve the above problems, the present invention provides a method for enzymatic quantitative detection of iota-carrageenan, using specific iota-carrageenase to specifically degrade iota-carrageenan into reducing sugars, and using p-hydroxybenzoyl The color reaction of hydrazine (pHBH), by measuring the absorbance value at 400-420nm place, measures the content of iota-carrageenan.

In order to achieve the above object, the present invention is achieved through the following technical solutions: a method for quantitatively detecting ι-carrageenan by enzymatic method, using ι-carrageenan enzyme to specifically degrade ι-carrageenan into reducing sugar, and after the enzyme is inactivated , adding p-hydroxybenzoic hydrazide solution to carry out color reaction, after centrifugation, measure the absorbance value of the supernatant at 400-420nm, and compare the standard curve to obtain the iota-carrageenan content.

Further, the ι-carrageenase is ι-carrageenase Cgi1_Wf, and its amino acid sequence is SEQ ID NO.1. Because at present in the research of GH82 family ι-carrageenase, most of the enzymes have poor stability and fast inactivation, which have great limitations in practical application, and the degradation of ι-carrageenan is not completely uncontrollable, which will lead to larger experiment error.

SEQ ID NO.1:

NEIEQEITANNQQEFNLSREAKTGITSTGYNSTNYFQPPTNLPTKNFTGSTSTQLQTLIN NSSTGAIIKIPKKTYNWGEIKLKSKIQLEIESGTIIKPSNNNIKRIFSIGSSGNGTRVTDVSIIGV GGKFTIDLSATANLNQNMAVIKMGRVSNFKISNFIIKDRRTSLASILLNYIPSNSDNEPYPKN GVIEKINQTGISHTGYGLIQAYSASNVLFKNLYCKGGVTLRLETDDKTMKDAVKNGGKLFG LRNIYADMIKCTSGLCPIMFSPHFTENGKITARNITATGCAFAVRVEHGFIEVFDTNKTYALTS SGGNQFKNFIAGKISGTGNSSKFIGNQYKRANGTQWAIRLSDASINGSLDPYITNQIGYLKN GSFESTTIENVTTIYKPTNAKLKQSFLPFIPCNDWTSKIKNPTDTGMGNGFEYYGPSLGERF DNTNGTNSNGNYIINVNGTTTRFSTVRNILYNTPTACTSNAYGTIPTTSNSPGL

Further, the optimal reaction temperature of the enzyme is 25°C, and it can still maintain more than 80% of its activity at room temperature, and its optimal reaction pH value is 8.0, and it is basically stable in the pH range of pH 5.0-9.0. The enzyme has good storage stability, can be stored stably for at least 30 days at 4°C, and can still maintain 80% of its activity after being placed at 25°C for 10 days.

The highest sequence similarity between this enzyme and other known enzymes is 41% (the closest sequence is CgiA produced by Zobelligalactanivorans DsijT). Using MEGA6 to construct a phylogenetic tree with Cgi1_Wf and all ι-carrageenase sequences annotated as GH82 family, the result is shown in Figure 3: it can be seen that ι-carrageenase Cgi1_Wf is in the phylogenetic development of GH82 family ι-carrageenase in the tree. Therefore, Cgi1_Wf is a new member of the ι-carrageenase GH82 family. Blast analysis was performed on the amino acid sequence of ι-carrageenase Cgi1_Wf, and multiple sequence alignments were performed between Cgi1_Wf and 2 ι-carrageenase sequences of the GH82 family whose properties had been studied using ClustalX2. Except for the strictly conserved site, other sites show different degrees of specificity, and Blast confirmed that Cgi1_Wf only has the highest similarity of 41% compared with the CgiA sequence produced by Zobelligalactanivorans DsijT, indicating that Cgi1_Wf is a novel gene in the GH82 family. iota-carrageenase.

The above-mentioned ι-carrageenan enzyme Cgi1_Wf has high activity for ι-carrageenan, but has no degradative effect on other marine polysaccharides, such as agar, algin, κ-carrageenan, etc. Its optimum reaction temperature is 25°C, and it can still maintain more than 80% of its activity after being placed at room temperature for 10 days. Its optimum reaction pH value is 8.0, and it is basically stable in the pH range of pH 5.0-9.0. The enzyme kinetic constant Km is 2.73mg/mL, Kcat is 560.75s ^-1 , and Km/Kcat is 501.72μM ^-1 s ^-1 . As can be seen from the above, compared with other ι-carrageenases, the ι-carrageenase Cgi1_Wf of the present invention has excellent enzymatic properties, good stability, easy storage, strong specificity for substrate binding, and fast enzymolysis rate, which is a good choice. Ideal enzyme for quantitative detection of iota-carrageenan.

Further, a gene encoding the above-mentioned ι-carrageenase, its nucleotide sequence is SEQ ID NO.2 or can translate all genes of SEQ ID NO.1.

SEQ ID NO.2:

AACGAGATTGAACAAGAAATCACCGCAAACAATCAACAAGAATTTAACCTCTCTA GAGAAGCTAAAACCGGAATAACTTCAACAGGATATAATTCTACAAACTATTTTCAACCCC CAACCAACCTACCCACAAAAAATTTCACTGGAAGTACAAGTACACAACTTCAAACACTT ATTAATAATAGCAGCACTGGAGCCATTATTAAAATCCCTAAAAAAACGTATAACTGGGGA GAAATCAAATTAAAATCCAAAATACAATTAGAAATAGAAAGTGGAACTATTATTAAACCA TCTAACAATAATATTAAACGCATTTTCAGTATTGGTAGTTCTGGTAATGGTACTAGAGTTA CTGATGTTAGTATTATAGGTGTAGGCGGAAAATTTACAATAGACCTATCTGCCACCGCTA ACCTAAACCAAAACATGGCAGTGATAAAAATGGGAAGAGTATCTAATTTTAAAATCTCT AATTTTATTATTAAAGACAGAAGAACTTCACTAGCATCAATCTTACTAAATTACATCCCAT CAAACTCTGACAATGAACCTTATCCTAAAAATGGTGTCATCGAAAAAATTAACCAAACA GGAATTTCACACACAGGTTATGGGTTAATCCAAGCGTATTCTGCATCAAATGTATTATTTA AAAATCTATATTGTAAAGGTGGAGTAACTCTTAGACTAGAAACTGATGACAAAACCATG AAAGATGCTGTTAAAAATGGTGGTAAGTTATTTGGATTGAGAAATATTTACGCAGATATG ATAAAATGTACTAGTGGACTTTGCCCTATCATGTTTTCTCCTCACTTTACTGAAAACGGA AAAATTACAGCAAGAAATATTACCGCAACTGGATGTGCTTTTGCTGTAAGAGTTGAACA CGGGTTTATTGAAGTTTTTGACACTAATAAAACTTATGCTTTAACCTCATCTGGAGGAAA CCAATTTAAAAACTTTATTGCTGGTAAAATA TCAGGAACTGGAAACTCTTCTAAATTCAT AGGCAACCAATACAAGAGAGCTAATGGCACACAGTGGGCTATTAGATTATCTGACGCTT CTATAAACGGCTCTCTAGATCCATACATCACTAATCAAATTGGATATTTAAAAAATGGTAG TTTTGAAAGCACAACCATAGAAAATGTAACAACAATTTACAAACCTACAAACGCCAAAT TAAAACAATCCTTTTTACCATTTATCCCTTGTAATGATTGGACTAGCAAGATTAAAAATCC TACTGACACAGGAATGGGGAATGGTTTTGAATACTATGGACCTTCTTTAGGAGAAAGAT TTGACAACACAAATGGTACCAACTCTAATGGAAACTACATCATCAATGTAAATGGAACA ACTACTAGGTTTTCAACTGTGAGAAACATTCTTTACAACACCCCAACCGCCTGTACAAG TAATGCATATGGTACAATTCCTACCACTTCTAATAGTCCTGGGTTATAA

Further, the method for the quantitative detection of iota-carrageenan by enzymatic method specifically comprises the following steps:

(1) Preparation of iota-carrageenan solution: take chemical-grade or above-purity iota-carrageenan and dissolve it in a buffer to prepare a iota-carrageenan standard solution with a concentration gradient;

(2) Preparation of pHBH solution: Weigh pHBH and dissolve it in HCl, then add NaOH to adjust the pH of the solution to alkaline, and prepare a 10-100 mg/mL pHBH solution. This is because the concentrated pHBH reagent in an acidic environment is easier to store, but the color development must be in an alkaline environment.

(3) Drawing of quantitative standard curve: get the different concentration ι-carrageenan solutions prepared in step (1) and mix with appropriate ι-carrageenan enzyme solution respectively to react; place 5-10min in 100 ℃ of metal baths after reaction to make enzyme Inactivation; then add pHBH solution, develop color in a metal bath at 100°C for 5-10min, rapidly cool to room temperature and centrifuge to take the supernatant, measure the absorbance of the supernatant, the detection wavelength is 400-420nm (pHBH and reducing sugar The reaction product has a good linear relationship in this wavelength range, and the accuracy is high); the iota-carrageenan solution of the same concentration gradient is mixed with the inactivated enzyme solution to repeat the above reaction, and the absorbance value is measured as a control, and then the different concentrations of ι - the corresponding absorbance value increment of carrageenan solution; With the concentration of ι-carrageenan standard solution as the abscissa, the absorbance value increment of each concentration 1-carrageenan is the ordinate, draws under specific reaction conditions by linear fitting standard curve;

(4) Sample determination: add a certain amount of iota-carrageenase to the sample and repeat the reaction of step (3); Substitute the absorbance value increment into the standard curve under conditions such as corresponding enzyme amount, reaction time, reaction temperature, and reaction pH , calculate the iota-carrageenan concentration in the reaction system, and then can obtain the iota-carrageenan content in the sample.

Further, the pH of the buffer solution in step (1) is 7.0-9.0, and the enzyme activity is relatively high in this pH range.

Further, the amount of enzyme added in step (2) is 1-1000U, the reaction time is 10-30min, and the reaction temperature is 20-50°C. Within the above parameter range, ι-carrageenase has high enzymolysis activity, which can ensure the rapid progress of the enzymolysis reaction; the amount of enzyme added and the reaction time should correspond to each other, and if the amount of enzyme added is small, the reaction time should be increased to ensure the stability of the sample. Complete enzymatic digestion.

Further, the reducing sugar in the sample is removed according to the national standard GB5009.88-2014 method before the measurement in step (4).

The beneficial effects of the present invention are:

(1) The invention provides a method for enzymatic quantitative detection of ι-carrageenan, using ι-carrageenan enzyme to specifically degrade ι-carrageenan in a lower temperature sample to form reducing sugars, which can be A color reaction occurs with pHBH, and the color depth of the solution is proportional to the reducing sugar concentration. By detecting the increase in absorbance before and after the reaction of the reaction solution, the content of iota-carrageenan in the test sample can be known. The detection method has good linear range and high accuracy, and can be popularized and used in the market.

(2) Provide a kind of iota-carrageenan enzyme, its enzymatic property is good, good stability, easy storage, simultaneously for the specificity of substrate binding, enzymolysis rate is fast, is used for detecting iota-carrageenan quantitatively Ideal enzyme for detection.

Description of drawings

Fig. 1: iota-carrageenase Cgi1_Wf target gene agarose gel electrophoresis pattern of the present invention;

Fig. 2: the electrophoresis figure after the purification of iota-carrageenase Cgi1_Wf of the present invention;

Figure 3: Cgi1_Wf and all the phylogenetic trees that have been annotated for GH82 family ι-carrageenase construction; wherein, the black box is ι-carrageenase Cgi1_Wf;

Figure 4: Cgi1_Wf multiple sequence alignment results; wherein, the black boxes and white characters are the conserved residues of ι-carrageenase.

detailed description

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Embodiment 1: Cloning expression and acquisition of iota-carrageenase Cgi1_Wf in Escherichia coli

Culture Wenyingzhuangia fucanilytica CZ1127 ^T in 2216E medium until the end of the logarithm and extract the whole genome DNA, design upstream and downstream primers (5'- GACACGGATCCAAAGACAAAAGTAGCAGTAAATGATACTACA; 5'- GACACCTCGAGCTATTGATATACTCTTACATAATCTATTTC) according to the target gene, and use the whole genome as a template for PCR, PCR reaction conditions The procedure was: 95°C for 3 minutes, 94°C for 30s, 58°C for 30s, 72°C for 150s, 22 cycles, and finally 72°C for 5 minutes to obtain the ι-carrageenase Cgi1_Wf gene fragment, which was connected to the DH5α vector to form a recombinant plasmid. The recombinant plasmids were introduced into BL21(DE3) competent cells to form recombinant strains. The expression was induced by isopropylthiogalactoside in LB medium containing kanamycin, the induction temperature was 17°C, and the induction time was 12h. Collect the bacteria by centrifugation, add a certain amount of 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate (Na ₂ HPO ₄ -NaH ₂ PO ₄ ) buffer to suspend, and then perform ultrasonic disruption in an ice-water bath (power 400W, working 2s, interval 6s, cycle 99 times), centrifuge to collect the supernatant, which is the crude enzyme solution of iota-carrageenase Cgi1_Wf.

The intracellular supernatant enzyme liquid after sonication was purified by affinity chromatography on the HisTrapTM HP chromatographic column. The SDS-PAGE analysis results are shown in Figure 2. The purified enzyme protein is a single band band, indicating that its purity is good. The activity of the purified enzyme is 88.25U/mg (1U activity is defined as the activity of generating 1μmol reducing sugar within 1min).

Embodiment 2: Cloning expression and acquisition of iota-carrageenase Cgi1_Wf in Bacillus subtilis

Wenyingzhuangia fucanilytica CZ1127 ^T was cultured in 2216E medium until the logarithmic phase and the whole genome DNA was extracted, and the upstream and downstream primers (5'-GGCTAATGGGCAGCAGCCATCATCA;5'-AATGAATTATGTAAGAGTATATCAATAG were designed according to the target gene, and the whole genome was used as a template for PCR as in Example 1, Obtain iota-carrageenase Cgi1_Wf gene fragment, be connected to pHT01 carrier, constitute recombinant plasmid.Recombinant plasmid is transformed in subtilis competent cell, screens positive clone and utilizes isopropyl thiogalactoside in LB nutrient solution Induction expression was carried out, the induction temperature was 38°C, and the induction time was 14h. The cells were collected by centrifugation, suspended by adding a certain amount of 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution, and then ultrasonically disrupted in an ice-water bath (power 400W, Work 2s, gap 6s, cycle 99 times), centrifuge to collect supernatant, this is the crude enzyme liquid of iota-carrageenase Cgi1_Wf.Repeat the purification operation in Example 1, obtain pure enzyme liquid, detect the activity of recombinant enzyme fermented liquid It is 140.71U/mg.

Embodiment 3: Cloning expression and acquisition of iota-carrageenase Cgi1_Wf in Pichia pastoris

Cultivate Wenyingzhuangia fucanilytica CZ1127 ^T in 2216E medium until the end of the logarithm and extract the whole genome DNA, design upstream and downstream primers (5'-TTATAAATGGGCAGCAGCCATCATCA;5'-GCATGCAGATTATGTAAGAGTATATCAATAG) according to the target gene, and use the whole genome as a template to perform PCR as in Example 1 , obtain the iota-carrageenase Cgi1_Wf gene fragment, connect it to the pPIC9k vector to form a recombinant plasmid, add it to Pichia pastoris GS115 competent cells to form a recombinant cell; screen positive clones and inoculate them in YPD medium, and cultivate them at 30°C for 20h. Then inoculated in BMGY medium, cultured on a shaker at 30°C and 200rpm until OD600=2.0, collected the bacteria by centrifugation, discarded the supernatant, resuspended the pellet with BMMY medium, and induced culture with methanol at 200rpm at 29°C for 72h. After the induction, the supernatant was collected by centrifugation, which was the crude enzyme solution. The activity of the recombinant enzyme fermentation broth was detected to be 59.44U/mg.

Embodiment 4: Carry out accuracy verification to the method of the present invention

Utilize the inventive method and phenol-sulfuric acid method to measure the iota-carrageenan content in the sample:

(1) Preparation of iota-carrageenan solution: Weigh chemical grade iota-carrageenan and dissolve it in 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer at pH 8.0, and prepare concentrations of 1.00mg/mL and 3.00mg respectively /mL, 5.00mg/mL, 7.00mg/mL, 9.00mg/mL iota-carrageenan standard solution;

(2) Preparation of pHBH solution: Weigh pHBH and dissolve it in 2mol/L HCl to prepare a 200mg/mL pHBH mother liquor, mix the mother liquor with 2mol/L NaOH solution at a ratio of 1:9, and prepare a 20mg/mL pHBH solution.

(3) Drawing of quantitative standard curve: Take 375 μL of the above-prepared iota-carrageenan solutions with different concentrations and react with 10UCgi1_Wf (buffer solution to 375 μL) at 25°C for 25 min. After the reaction, place in a metal bath at 100°C for 10 minutes to inactivate the enzyme, add 250 μL of pHBH solution to develop color in a metal bath at 100°C for 8 minutes, rapidly cool to room temperature, and centrifuge to take the supernatant, and measure the absorbance of the supernatant at 415 nm; , the inactivated enzyme solution of the same concentration gradient is mixed with the ι-carrageenan solution to repeat the operation, and its absorbance value is measured as a contrast, thereby calculating the corresponding absorbance value increment of different concentration solutions; Concentration is the abscissa, and the corresponding absorbance increment is the ordinate, and the standard curve under specific reaction conditions is obtained by linear fitting as y=1.3425x ⁺ 0.0158, and the R2 value is 0.9991.

(4) Sample determination: Weigh an appropriate amount of sample, wash it with 85% ethanol solution after grinding to remove reducing sugar, discard the ethanol solution, and continue for 3 times. After desugaring, the sample was dried overnight in an oven at 40°C, and after drying, the sample was dissolved in buffer. Take 375 μL of the sample solution, repeat the operation described in step 3, substitute the absorbance value increment into the standard curve y=1.3425x+0.0158, calculate the concentration of iota-carrageenan in the reaction system, and then convert the content of iota-carrageenan in the sample .

(5) Determination of the iota-carrageenan content in the sample according to the phenol-sulfuric acid method in the national standard SN/T 4260-2015.

Three parallel determinations were carried out for each method, and the results are shown in the table below.

The method of the invention Phenol-sulfuric acid method Determination of parallel 1 (mg/mL) 10.24 9.62 Determination of parallel 2 (mg/mL) 9.88 9.43 Determination of parallel 3 (mg/mL) 9.50 10.20 Determination of average value (mg/mL) 9.87 9.75

From the above results, it can be seen that the method measurement result of the present invention has no deviation substantially from the phenol-sulfuric acid method measurement result, indicating that the method of the present invention has good accuracy.

Embodiment 5: Carry out quantitative specificity verification to the method of the present invention

The iota-carrageenan in three kinds of mixed solutions is quantified respectively with the inventive method.

Step 1, preparation of iota-carrageenan solution: Weigh chemical-grade iota-carrageenan and dissolve it in 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer at pH 7.0 to prepare iota-carrageenan with a concentration of 4.00 mg/mL glue solution; prepare agar, κ-carrageenan, and alginate solutions with a concentration of 2.00 mg/mL at the same time; mix the agar, κ-carrageenan, and alginate solutions with ι-carrageenan solutions in equal volumes to prepare Mixed solution, the iota-carrageenan concentration of mixed solution is 2.00mg/mL.

Step 2, preparation of pHBH solution: Weigh pHBH and dissolve it in 2mol/L HCl to prepare 200mg/mL pHBH mother liquor, and mix the mother liquor with 2mol/L NaOH solution at a ratio of 1:9 to prepare a 20mg/mL pHBH solution.

Step 3, drawing of the quantitative standard curve: take 375 μL of the above-prepared iota-carrageenan solutions of different concentrations and react with 50 U Cgi1_Wf (buffer solution to 375 μL) at 25° C. for 25 minutes. After the reaction, place in a metal bath at 100°C for 8 minutes to inactivate the enzyme, add 250 μL of pHBH solution to develop color in a metal bath at 100°C for 5 minutes, rapidly cool to room temperature, and centrifuge to take the supernatant, and measure the absorbance of the supernatant at 400 nm; , the inactivated enzyme solution of the same concentration gradient is mixed with the ι-carrageenan solution to repeat the operation, and its absorbance value is measured as a contrast, thereby calculating the corresponding absorbance value increment of different concentration solutions; Concentration is the abscissa, and the corresponding absorbance increment is the ordinate. Through linear fitting, the standard curve under specific reaction conditions is y=1.3844x ⁺ 0.0226, and the R2 value is 0.9990.

Step 4, sample determination: take 375 μL of the mixed solution, repeat the operation described in step 3, substitute the absorbance value increment into the standard curve y=1.3844x+0.0226, calculate the ι-carrageenan concentration in the reaction system, and then convert the mixed iota-carrageenan content in solution.

The parallel determination was performed three times, and the determination results are as follows.

ι-carrageenan, agar gum ι-carrageenan, κ-carrageenan ι-carrageenan, alginate Determination of parallel 1 (mg/mL) 2.05 2.08 1.98 Determination of parallel 2 (mg/mL) 2.02 1.99 2.04 Determination of parallel 3 (mg/mL) 2.04 1.95 2.04 Determination of average value (mg/mL) 2.04 2.01 2.02 Relative error(%) 2.0% 0.5% 1.0%

It can be seen from the above results that the method of the present invention has good quantitative specificity.

Embodiment 6: measure the iota-carrageenan content in a meat product

(1) Preparation of iota-carrageenan solution: Weigh chemical grade iota-carrageenan and dissolve in 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution with pH 8.0, and prepare concentrations of 0.20mg/mL and 0.40mg respectively /mL, 0.60mg/mL, 0.80mg/mL, 1.00mg/mL iota-carrageenan standard solution;

(2) Preparation of pHBH solution: Weigh pHBH and dissolve it in 2mol/L HCl to prepare a 200mg/mL pHBH mother liquor, mix the mother liquor with 2mol/L NaOH solution at a ratio of 1:9, and prepare a 20mg/mL pHBH solution.

(3) Drawing of quantitative standard curve: take 375 μL of iota-carrageenan solutions with different concentrations prepared above and react with 100 UCgi1_Wf (buffer solution supplemented to 375 μL) at 35° C. for 15 min. After the reaction, place in a metal bath at 100°C for 5 minutes to inactivate the enzyme, add 250 μL of pHBH solution to develop color in a metal bath at 100°C for 5 minutes, rapidly cool to room temperature, and centrifuge to take the supernatant, and measure the absorbance of the supernatant at 410 nm; , the inactivated enzyme solution of the same concentration gradient is mixed with the ι-carrageenan solution to repeat the operation, and its absorbance value is measured as a contrast, thereby calculating the corresponding absorbance value increment of different concentration solutions; Concentration is the abscissa, and the corresponding absorbance increment is the ordinate. Through linear fitting, the standard curve under specific reaction conditions is y=1.4031x ⁺ 0.0419, and the R2 value is 0.998.

(4) Sample determination: Weigh an appropriate amount of sample, wash it with 85% ethanol solution after grinding to remove reducing sugar, discard the ethanol solution, and continue for 3 times. After desugaring, the sample was dried overnight in an oven at 40°C, and after drying, the sample was dissolved in buffer. Take 375 μL of sample solution, repeat the operation described in step (3), substitute the absorbance value increment into the standard curve y=1.4031x+0.0419, calculate the iota-carrageenan concentration in the reaction system, and then convert the ι-carrageenan concentration in the meat product - Carrageenan content is 10.4 mg/mL.

Embodiment 7: measure the iota-carrageenan content in a kind of jelly

(1) Preparation of iota-carrageenan solution: Weigh chemical grade iota-carrageenan and dissolve in 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution with pH 8.0, and prepare concentrations of 0.30mg/mL and 0.60mg respectively /mL, 0.90mg/mL, 1.20mg/mL, 1.50mg/mL iota-carrageenan standard solution;

(2) Preparation of pHBH solution: Weigh pHBH and dissolve it in 2mol/L HCl to prepare a 200mg/mL pHBH mother liquor, mix the mother liquor with 2mol/L NaOH solution at a ratio of 1:9, and prepare a 20mg/mL pHBH solution.

(3) Drawing of quantitative standard curve: Take 375 μL of iota-carrageenan solutions with different concentrations prepared above and react with 200 UCgi1_Wf (buffer solution supplemented to 375 μL) at 30° C. for 20 min. After the reaction, place in a metal bath at 100°C for 5 minutes to inactivate the enzyme, add 250 μL of pHBH solution to develop color in a metal bath at 100°C for 5 minutes, rapidly cool to room temperature, and centrifuge to take the supernatant, and measure the absorbance of the supernatant at 415 nm; , the inactivated enzyme solution of the same concentration gradient is mixed with the ι-carrageenan solution to repeat the operation, and its absorbance value is measured as a contrast, thereby calculating the corresponding absorbance value increment of different concentration solutions; Concentration is the abscissa, and the corresponding absorbance increment is the ordinate, and the standard curve under specific reaction conditions is obtained by linear fitting as y=1.3726x ⁺ 0.0429, and the R2 value is 0.9993.

(4) Sample determination: Weigh an appropriate amount of sample, wash it with 85% ethanol solution after grinding to remove reducing sugar, discard the ethanol solution, and continue for 3 times. After desugaring, the sample was dried overnight in an oven at 40°C, and after drying, the sample was dissolved in buffer. Take 375 μL of sample solution, repeat the operation described in step 3, substitute the absorbance value increment into the standard curve y=1.3726x+0.0429, calculate the concentration of iota-carrageenan in the reaction system, and then convert the iota-carrageenan in the jelly The content is 12.45mg/mL.

Finally, it should be noted that although the above examples have described the specific implementation of the present invention, they are not intended to limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims limited. All modifications or equivalent replacements shall be included in the protection scope of the present invention.

sequence listing

<110> Ocean University of China

<120> A method for the quantitative detection of iota-carrageenan by enzymatic method

<130> Ocean University of China

<140> 1

<141> 2019-12-10

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 492

<212> PRT

<213> Artificial Sequence

<400> 1

Asn Glu Ile Glu Gln Glu Ile Thr Ala Asn Asn Gln Gln Glu Phe Asn

1 5 10 15

Leu Ser Arg Glu Ala Lys Thr Gly Ile Thr Ser Thr Gly Tyr Asn Ser

20 25 30

Thr Asn Tyr Phe Gln Pro Pro Thr Asn Leu Pro Thr Lys Asn Phe Thr

35 40 45

Gly Ser Thr Ser Thr Gln Leu Gln Thr Leu Ile Asn Asn Ser Ser Thr

50 55 60

Gly Ala Ile Ile Lys Ile Pro Lys Lys Thr Tyr Asn Trp Gly Glu Ile

65 70 75 80

Lys Leu Lys Ser Lys Ile Gln Leu Glu Ile Glu Ser Gly Thr Ile Ile

85 90 95

Lys Pro Ser Asn Asn Asn Ile Lys Arg Ile Phe Ser Ile Gly Ser Ser

100 105 110

Gly Asn Gly Thr Arg Val Thr Asp Val Ser Ile Ile Gly Val Gly Gly

115 120 125

Lys Phe Thr Ile Asp Leu Ser Ala Thr Ala Asn Leu Asn Gln Asn Met

130 135 140

Ala Val Ile Lys Met Gly Arg Val Ser Asn Phe Lys Ile Ser Asn Phe

145 150 155 160

Ile Ile Lys Asp Arg Arg Thr Ser Leu Ala Ser Ile Leu Leu Asn Tyr

165 170 175

Ile Pro Ser Asn Ser Asp Asn Glu Pro Tyr Pro Lys Asn Gly Val Ile

180 185 190

Glu Lys Ile Asn Gln Thr Gly Ile Ser His Thr Gly Tyr Gly Leu Ile

195 200 205

Gln Ala Tyr Ser Ala Ser Asn Val Leu Phe Lys Asn Leu Tyr Cys Lys

210 215 220

Gly Gly Val Thr Leu Arg Leu Glu Thr Asp Asp Lys Thr Met Lys Asp

225 230 235 240

Ala Val Lys Asn Gly Gly Lys Leu Phe Gly Leu Arg Asn Ile Tyr Ala

245 250 255

Asp Met Ile Lys Cys Thr Ser Gly Leu Cys Pro Ile Met Phe Ser Pro

260 265 270

His Phe Thr Glu Asn Gly Lys Ile Thr Ala Arg Asn Ile Thr Ala Thr

275 280 285

Gly Cys Ala Phe Ala Val Arg Val Glu His Gly Phe Ile Glu Val Phe

290 295 300

Asp Thr Asn Lys Thr Tyr Ala Leu Thr Ser Ser Gly Gly Asn Gln Phe

305 310 315 320

Lys Asn Phe Ile Ala Gly Lys Ile Ser Gly Thr Gly Asn Ser Ser Ser Lys

325 330 335

Phe Ile Gly Asn Gln Tyr Lys Arg Ala Asn Gly Thr Gln Trp Ala Ile

340 345 350

Arg Leu Ser Asp Ala Ser Ile Asn Gly Ser Leu Asp Pro Tyr Ile Thr

355 360 365

Asn Gln Ile Gly Tyr Leu Lys Asn Gly Ser Phe Glu Ser Thr Thr Ile

370 375 380

Glu Asn Val Thr Thr Ile Tyr Lys Pro Thr Asn Ala Lys Leu Lys Gln

385 390 395 400

Ser Phe Leu Pro Phe Ile Pro Cys Asn Asp Trp Thr Ser Lys Ile Lys

405 410 415

Asn Pro Thr Asp Thr Gly Met Gly Asn Gly Phe Glu Tyr Tyr Gly Pro

420 425 430

Ser Leu Gly Glu Arg Phe Asp Asn Thr Asn Gly Thr Asn Ser Asn Gly

435 440 445

Asn Tyr Ile Ile Asn Val Asn Gly Thr Thr Thr Arg Phe Ser Thr Val

450 455 460

Arg Asn Ile Leu Tyr Asn Thr Pro Thr Ala Cys Thr Ser Asn Ala Tyr

465 470 475 480

Gly Thr Ile Pro Thr Thr Ser Asn Ser Pro Gly Leu

485 490

<210> 2

<211> 1479

<212>DNA

<213> Artificial Sequence

<400> 2

aacgagattg aacaagaaat caccgcaaac aatcaacaag aatttaacct ctctagagaa 60

gctaaaaccg gaataacttc aacaggatat aattctacaa actattttca acccccaacc 120

aacctaccca caaaaaattt cactggaagt acaagtacac aacttcaaac acttattaat 180

aatagcagca ctggagccat tattaaaatc cctaaaaaaa cgtataactg gggagaaatc 240

aaattaaaat ccaaaataca attagaaata gaaagtggaa ctattattaa accatctaac 300

aataatatta aacgcatttt cagtattggt agttctggta atggtactag agttactgat 360

gttagtatta taggtgtagg cggaaaattt acaatagacc tatctgccac cgctaaccta 420

aaccaaaaca tggcagtgat aaaaatggga agagtatcta attttaaaat ctctaatttt 480

atttattaaag acagaagaac ttcactagca tcaatcttac taaattacat cccatcaaac 540

tctgacaatg aaccttatcc taaaaatggt gtcatcgaaa aaattaacca aacaggaatt 600

tcacacacag gttatgggtt aatccaagcg tattctgcat caaatgtatt atttaaaaat 660

ctatattgta aaggtggagt aactcttaga ctagaaactg atgacaaaac catgaaagat 720

gctgttaaaa atggtggtaa gttatttgga ttgagaaata tttacgcaga tatgataaaa 780

tgtactagtg gactttgccc tatcatgttt tctcctcact ttactgaaaa cggaaaaatt 840

acagcaagaa atttaccgc aactggatgt gcttttgctg taagagttga acacgggttt 900

attgaagttt ttgacactaa taaaacttat gctttaacct catctggagg aaaccaattt 960

aaaaacttta ttgctggtaa aatatcagga actggaaact cttctaaatt cataggcaac 1020

caatacaaga gagctaatgg cacacagtgg gctattagat tatctgacgc ttctataaac 1080

ggctctctag atccatacat cactaatcaa attggatatt taaaaaatgg tagttttgaa 1140

agcacaacca tagaaaatgt aacaacaatt tacaaaccta caaacgccaa attaaaacaa 1200

tcctttttac catttatccc ttgtaatgat tggactagca agattaaaaa tcctactgac 1260

acaggaatgg ggaatggttt tgaatactat ggaccttctt taggagaaag atttgacaac 1320

acaaatggta ccaactctaa tggaaactac atcatcaatg taaatggaac aactactagg 1380

ttttcaactg tgagaaacat tctttacaac accccaaccg cctgtacaag taatgcatat 1440

ggtacaattc ctaccacttc taatagtcct gggttataa 1479

Claims (7)

1. A method for quantitatively detecting iota-carrageenan by an enzymatic method is characterized by comprising the following steps: degrading iota-carrageenan into reducing sugar by utilizing the specificity of iota-carrageenan, inactivating enzyme, adding a p-hydroxybenzoyl hydrazine solution for color reaction, measuring the light absorption value of the supernatant at 400-420nm after centrifugation, and comparing with a standard curve to obtain iota-carrageenan content;

the iota-carrageenase is iota-carrageenase Cgi1_ Wf, and the amino acid sequence of the iota-carrageenase is SEQ ID No.1.

2. The enzymatic quantitative iota-carrageenan detection method according to claim 1, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: the iota-carrageenase has the optimum reaction temperature of 25 ℃, can still keep more than 80% of activity at room temperature, has the optimum reaction pH value of 8.0, basically keeps stable in the pH value range of 5.0-9.0, has good storage stability, can be stored stably for at least 30 days at 4 ℃, and can still keep 80% of activity after being placed for 10 days at 25 ℃.

3. The enzymatic quantitative iota-carrageenan detection method according to claim 1, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: the nucleotide sequence of the gene for coding the iota-carrageenase is SEQ ID NO.2 or all genes which can be translated into SEQ ID NO.1.

4. The enzymatic quantitative iota-carrageenan detection method according to claim 1, characterized by comprising the steps of:

(1) Preparing an iota-carrageenan solution: weighing iota-carrageenan with chemical grade or higher purity, dissolving the iota-carrageenan in a buffer solution, and preparing iota-carrageenan standard solution with concentration gradient;

(2) preparation of pHBH solution: weighing pHBH, dissolving the pHBH in HCl, adding NaOH to adjust the pH of the solution to be alkaline, and preparing into a pHBH solution of 10-100 mg/mL;

(3) Drawing a quantitative standard curve: mixing the iota-carrageenan solutions with different concentrations prepared in the step (1) with a proper amount of iota-carrageenan enzyme respectively to react; inactivating the enzyme after the reaction; adding pHBH solution, developing in 100 deg.C metal bath for 5-10min, cooling to room temperature rapidly, centrifuging to obtain supernatant, measuring light absorption value of the supernatant, and measuring the detection wavelength of 400-420nm; mixing the iota-carrageenan solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the mixture as a comparison, and calculating the light absorption value increment corresponding to the iota-carrageenan solution with different concentrations; taking the concentration of the iota-carrageenan standard solution as an abscissa and the light absorption value increment of the iota-carrageenan with each concentration as an ordinate, and obtaining a standard curve under a specific reaction condition through linear fitting;

(4) And (3) sample determination: adding a certain amount of iota-carrageenase into the sample to repeat the reaction in the step (3); and substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme addition amount, reaction time, reaction temperature, reaction pH and the like to calculate the iota-carrageenan concentration in the reaction system, thereby obtaining the iota-carrageenan content in the sample.

5. The enzymatic quantitative iota-carrageenan detection method according to claim 4, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: the pH value of the buffer solution in the step (1) is 7.0-9.0.

6. The enzymatic quantitative iota-carrageenan detection method according to claim 4, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: in the step (3), the addition amount of the enzyme is 1-1000U, the reaction time is 10-30min, and the reaction temperature is 20-50 ℃.

7. The enzymatic quantitative iota-carrageenan detection method according to claim 4, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: and (4) removing reducing sugar in the sample according to a national standard GB 5009.88-2014 method before measurement.

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