CN111100904B - Method for quantitatively detecting kappa-carrageenan by enzyme method - Google Patents
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Abstract
The invention relates to the technical field of biotechnology and biochemical detection, in particular to a method for quantitatively detecting kappa-carrageenan by an enzymatic method. Hydrolyzing kappa-carrageenan into reducing sugar by utilizing the specificity of the kappa-carrageenan, inactivating enzyme, adding p-hydroxybenzoyl hydrazine solution for color reaction, centrifuging, measuring the light absorption value of supernatant at 400-420nm, and comparing with a standard curve to obtain the content of the kappa-carrageenan. The detection method has the advantages of good linear range and high accuracy, and can be popularized and used in the market.
Description
Technical Field
The invention relates to the technical field of biotechnology and biochemical detection, in particular to a method for quantitatively detecting kappa-carrageenan by an enzymatic method.
Background
Carrageenans (also known as carrageenans or carrageenans), which are natural anionic linear polysaccharides of sulfuric acid extracted from the cell walls of red algae such as carrageenans, eucheuma, gigartina, and the like, are of various types depending on the number and attachment position of sulfate groups on galactose and the content of 3,6-anhydrogalactose in the structure. Kappa-carrageenan is one of three common carrageenan types (kappa-, iota-and lambda-carrageenan), consists of alternate sulfated-alpha-D-3,6-anhydrogalactose and sulfated-beta-D-galactose residues, is widely available and has various functions, and is applied to various industrial productions of foods, medicines, cosmetics and the like, and plays roles of coagulator, adhesive, stabilizer, emulsifier, suspending agent, thickener and the like.
Quantitative detection of kappa-carrageenan is a basic link in kappa-carrageenan quantity control, functional research and product development. At present, the commonly used kappa-carrageenan quantitative detection methods are a phenol-sulfuric acid method, a liquid chromatography-tandem mass spectrometry method and the like. The phenol-sulfuric acid method has high sensitivity, does not need expensive instruments, is easily interfered by other polysaccharides in a sample, and is time-consuming and consumes more samples and reagents. The liquid chromatography-tandem mass spectrometry obtains the content of the kappa-carrageenan by hydrolyzing, deriving and pretreating a sample and measuring the content of monosaccharide consisting of the kappa-carrageenan.
P-hydroxybenzoyl hydrazine (pHBH) is an aromatic hydrazide compound which can generate a yellow product with a beta-diketone compound under a strong alkaline condition. Research shows that pHBH can react with reducing sugar such as glucose and the like to generate similar yellow substances under high-temperature and alkaline conditions, the color depth after reaction is in direct proportion to the concentration of the reducing sugar, and the sensitivity of the reaction is high. This provides feasibility for quantitative detection of kappa-carrageenan by enzyme method.
In conclusion, the method for quantitatively detecting the kappa-carrageenan, which has the advantages of high sensitivity, good accuracy and simple and convenient operation, is of great significance.
Disclosure of Invention
The invention aims to solve the technical problem that the commonly used kappa-carrageenan quantitative detection method is a phenol-sulfuric acid method and a liquid chromatography-tandem mass spectrometry method, and the two methods respectively have the problems of poor reproducibility, complex operation, time waste and labor waste.
In order to solve the problems, the invention provides a method for quantitatively detecting kappa-carrageenan by an enzyme method, which specifically degrades the kappa-carrageenan into reducing sugar by utilizing specific kappa-carrageenan enzyme, utilizes the color reaction of p-hydroxybenzoyl hydrazine (pHBH), and measures the light absorption value at 400-420nm to measure the content of the kappa-carrageenan.
In order to achieve the purpose, the invention is realized by the following technical scheme: an enzyme method for quantitatively detecting kappa-carrageenan is disclosed, which utilizes the specificity of kappa-carrageenan to degrade the kappa-carrageenan into kappa-carrageenan tetrasaccharide and a small amount of disaccharide. Inactivating enzyme, adding p-hydroxybenzoyl hydrazine solution for color reaction, centrifuging, measuring light absorption value of supernatant at 400-420nm, and comparing with standard curve to obtain kappa-carrageenan content.
Further, the kappa-carrageenase is kappa-carrageenase Cgk1_ Wa, and the amino acid sequence of the kappa-carrageenase is SEQ ID NO.1.
SEQ ID NO.1：
TSQNLRPLNAKPGENWTIKWNRSDDFNQSRPNNAVDYGKWQRNPGQVQTWTWDND NNAKEVNGVLNLTARFDDAGADRNIFQNCGGATNDLFYTSAMLKSYSKGVYGYYEAKIK GANLFPGVAPAFWMYSDIDDSLTQEGAIRYSEVDVVEMTQRGNRVSGNEMIMDHNLHAIV TTRNKVTSNGYTFQLDTNGNKIPLTNNEITTNKGRRWFRPGNPEVSHDQENVTGQASDPN RFDPRAAFHTYGCRIDQNWITWYVDNREIGRKANTKWHRPMNVALSLGIRAPYTTFCNNA FALPTRQFALNNQNKFPQTMQVDYIRVWELDGNNTNNNPPTEPIDPPSNNNLPSSGSTINLK ASNNNKFITVVSNNSNTLKTTANSGTGNNQKFTITNTSDGFVSLKSSANNKFVTATSITNSP LRVGASKAFNRQKFRIETSNGKIVLKAKINNKYIVANNNVLEANGNNKNAALKFDITNFSS TANLIKKEETLEISNLK
Furthermore, the optimum reaction temperature of the enzyme is 25 ℃, the maximum activity of the enzyme can be achieved at normal temperature, the optimum reaction pH value is 8.0, the enzyme is basically kept stable in the pH value range of 6.0-10.0, the storage stability is good, and the enzyme can be stably stored for at least 3 months at 4 ℃. The enzyme has good reaction specificity, has high activity on kappa-carrageenan, but has no degradation effect on other marine polysaccharides such as agar, algin, iota-carrageenan and the like. In summary, the kappa-carrageenase Cgk _ Wa has good application potential, is particularly applied to detection of kappa-carrageenase content, has the characteristics of simple and convenient operation, high accuracy and good stability, and can react at normal temperature, so that the situation that substrates are changed at high temperature is avoided, (iota-and lambda-carrageenans, algin, agar and the like can be degraded at high temperature to generate reducing sugar, and degradation products can also undergo color reaction with pHBH to cause higher actual detection results), and further the quantitative accuracy is influenced.
The enzyme has a sequence similarity of up to 40% with other known enzymes (the closest sequence is kappa _ P.LL1 produced by Pseudomonas sp.LL1). Phylogenetic trees were constructed using MEGA6 with Cgk _ Wa and kappa-carrageenase sequences that have been annotated as the GH16 family, with the results shown in fig. 2: it can be seen that kappa-carrageenase Cgk1_ Wa is in the phylogenetic tree of GH16 family kappa-carrageenase. Therefore, cgk _ Wa is a new member of the kappa-carrageenase GH16 family. Blast analysis of kappa-carrageenase Cgk _ Wa amino acid sequences and multiple sequence alignment of Cgk _ Wa with 7 kappa-carrageenase sequences known from the GH16 family using ClustalX2 resulted in the following table in figure 3: the 7 kappa-carrageenases were CgkA _ P.c (Genbank CAA 50624.1) derived from Pseudomonas carrageeneova, cgkX _ P.QY203 (Genbank AFV 39914.1) derived from Pseudomonas sp.QY203, cgk-K142a _ P.t (Genbank BAJ 3579) derived from Pseudomonas tetraodonis, respectively kappa _ P.LL1 from Pseudomonas sp.LL1 (Genbank ADD 92366.1), cgkS _ S.Kz7 from Shewanella sp.Kz 7 (Genbank AHN 05534.1), cgkA _ Z.g from Zobellia galactavorans (Genbank CAZ 94309.1) and CgkZ _ Z.M-2 from Zobellia sp.M-2 (Genbank AGS43006.1). As can be seen from fig. 3, cgk _ Wa shows different degrees of specificity at other sites besides strictly conserved at key catalytic sites, and Blast confirmed that Cgk _ Wa has only the highest similarity of 40% compared to the kappa _ p.ll1 sequence produced by Pseudoalteromonas sp.ll1, indicating that Cgk _ Wa is a novel kappa-carrageenase in the GH16 family.
The kappa-carrageenase Cgk _ Wa has high activity on kappa-carrageenin, but has no degradation effect on other marine polysaccharides such as agar, algin, iota-carrageenin and the like. The optimum reaction temperature is 25 ℃, the storage stability is at least 30 days at 4 ℃, the optimum reaction pH value is 8.0, the stability is basically kept in the pH value range of pH 6.0-10.0, the enzyme kinetic constant Km is 0.07mg/mL, and the Kcat is 27.54s -1 Km/Kcat of 165.63. Mu.M -1 s -1 . As can be seen from the above, compared with other kappa-carrageenases, the kappa-carrageenase Cgk1_ Wa of the invention has the advantages of excellent enzymatic properties, good stability, easy storage, strong specificity for substrate combination and high enzymolysis rate, and is used for detecting the quantitative kappa-carrageenaseThe detected ideal enzyme can react at normal temperature, so that the change of a substrate at high temperature is avoided, and the quantitative accuracy is influenced.
Furthermore, the nucleotide sequence of the gene for coding the kappa-carrageenase is SEQ ID NO.2 or all genes capable of being translated into SEQ ID NO.1.
SEQ ID NO.2：
ACTTCTCAAAATTTAAGACCGCTTAATGCCAAACCTGGAGAAAATTGGACTATCAA ATGGAATCGTTCCGATGATTTTAATCAATCCAGACCAAACAATGCTGTTGATTATGGTAA ATGGCAAAGAAACCCAGGTCAAGTACAAACATGGACTTGGGACAACGACAATAATGCC AAAGAAGTGAATGGTGTTTTAAATTTAACAGCTCGATTTGATGATGCTGGAGCAGATAG AAATATTTTTCAGAATTGTGGTGGTGCTACCAATGATTTATTCTACACATCTGCAATGCTT AAATCTTATTCCAAAGGAGTTTACGGCTATTATGAAGCCAAAATTAAAGGTGCCAATTTG TTTCCTGGTGTAGCTCCTGCATTTTGGATGTATAGTGATATTGATGATAGTTTAACCCAAG AGGGAGCTATTAGATATAGCGAAGTAGATGTGGTAGAAATGACTCAAAGAGGTAACAGA GTAAGTGGTAATGAAATGATTATGGATCATAATTTACACGCTATTGTTACTACCCGTAACA AAGTAACTTCTAACGGTTATACATTTCAATTAGATACTAATGGAAATAAAATTCCTTTGAC CAATAATGAGATAACCACCAACAAGGGACGTAGATGGTTTAGACCTGGAAATCCAGAA GTAAGTCACGACCAAGAAAATGTAACAGGTCAAGCTAGTGATCCTAACAGATTTGATCC TAGAGCTGCATTTCACACATATGGATGTAGAATAGATCAAAATTGGATTACATGGTATGTT GATAACAGAGAAATTGGAAGAAAGGCCAATACAAAATGGCATAGACCTATGAATGTGGC ATTATCTTTAGGGATTAGAGCTCCTTATACTACATTTTGTAACAATGCCTTTGCATTGCCTA CCAGACAGTTTGCCTTAAACAATCAAAATAAGTTTCCACAAACAATGCAAGTTGATTAC ATAAGAGTATGGGAACTAGATGGAAATAATACAAACAACAATCCGCCAACTGAACCTAT AGATCCTCCAAGCAACAACAATTTACCTAGTTCTGGTAGTACTATCAATTTAAAAGCTTC CAATAACAACAAATTTATTACTGTAGTAAGCAACAACAGCAATACATTAAAAACAACGG CTAATAGTGGAACTGGAAACAATCAAAAATTTACAATAACAAACACATCAGATGGCTTT GTTAGCTTAAAATCTTCTGCAAATAATAAATTTGTAACAGCAACTTCTATTACAAATTCCC CCTTAAGAGTAGGAGCTTCTAAAGCTTTTAATAGACAAAAATTTAGAATTGAAACTTCTA ATGGTAAAATTGTATTAAAAGCTAAAATTAACAACAAGTATATTGTAGCAAACAACAATG TATTAGAAGCTAATGGAAATAATAAAAATGCAGCCTTAAAGTTTGACATTACTAATTTTAG TAGCACAGCTAACCTAATTAAAAAAGAAGAAACGTTAGAAATATCTAATCTTAAGAACA ATGTTCTTGTTAAATTAGAGAATACTAATAGTATTAAAAACATTAGTTTGTTTACTCTTTTA GGAAGTTTAATCTATCACAATTCTACCAGTGAAAACAATTTCAACTTACATAAAAATCAA TTTCAAAAAGGTATTTACGTATTAGAAATAAGTTCTAACACAGGAATTAAAACCCAAAA AATAATTATAGATTAA
Further, the method for quantitatively detecting the kappa-carrageenan by the enzyme method specifically comprises the following steps:
(1) Preparing a kappa-carrageenan solution: weighing chemical-grade or above-purity kappa-carrageenan, dissolving in a buffer solution, and preparing a kappa-carrageenan standard solution with a concentration gradient;
(2) preparation of a 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; this is because a concentrated pHBH reagent is easy to store in an acidic environment, but develops color in an alkaline environment, which is necessary for color development.
(3) Drawing a quantitative standard curve: mixing the kappa-carrageenan solutions with different concentrations prepared in the step (1) with a proper amount of kappa-carrageenan enzyme solution respectively to react; after the reaction, the mixture is placed in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme; adding pHBH solution, developing in 100 deg.C metal bath for 5-10min, rapidly cooling to room temperature, centrifuging to obtain supernatant, measuring light absorption value of the supernatant, and detecting wavelength of 400-420nm; mixing the kappa-carrageenan solution with the same concentration gradient with the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the mixture as a control, and then calculating the light absorption value increment corresponding to the kappa-carrageenan solution with different concentrations; taking the concentration of the kappa-carrageenan standard solution as an abscissa and the light absorption value increment of each concentration of kappa-carrageenan 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 kappa-carrageenase into the sample solution to repeat the reaction in the step (3); substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme adding amount, reaction time, reaction temperature, reaction pH and the like, and calculating the concentration of the kappa-carrageenan in the reaction system so as to obtain the content of the kappa-carrageenan in the sample.
Further, the pH value of the buffer solution in the step (1) is 7.0-10.0, and the enzyme activity is higher in the pH range, thereby being beneficial to full enzymolysis.
Further, the addition amount of the enzyme in the step (2) is 1-1000U, the reaction time is 5-40min, the reaction temperature is 20-35 ℃, and Cgk _ Wa has higher enzymolysis activity and can keep stable within the range of 20-35 ℃; in addition, within the parameter range, the kappa-carrageenase, particularly Cgk1_ Wa, has higher enzymolysis activity, and ensures the rapid proceeding of the enzymolysis reaction; the addition amount of the enzyme and the reaction time need to correspond to each other, and if the addition amount of the enzyme is small, the reaction time is increased, so that the complete enzymolysis of the sample is ensured.
Furthermore, before the determination in the step (4), reducing sugar in the sample is removed according to the method of national standard GB 5009.88-2014.
The invention has the beneficial effects that:
(1) The invention provides a method for quantitatively detecting kappa-carrageenan by an enzyme method, which utilizes that kappa-carrageenan can specifically degrade kappa-carrageenan in a sample at a lower temperature to form reducing sugar, the reducing sugar can perform a color reaction with pHBH, the color depth of a solution is in direct proportion to the concentration of the reducing sugar, and the content of the kappa-carrageenan in the detected sample can be obtained by detecting the light absorption value increment before and after the reaction of a reaction solution. The detection method has the advantages of good linear range and high accuracy, and can be popularized and used in the market.
(2) A kappa-carrageenase is provided, which has a sequence similarity of only 40% at the maximum with other known enzymes. The enzyme has the advantages of excellent enzymological property, good stability, easy storage, strong specificity for substrate combination and high enzymolysis rate, and is an ideal enzyme for detecting quantitative detection of kappa-carrageenan.
Drawings
FIG. 1: an electrophoretogram of purified kappa-carrageenase Cgk _ Wa of the invention;
FIG. 2 is a schematic diagram: cgk1_ Wa and all phylogenetic trees annotated as GH16 family kappa-carrageenase constructs; wherein the black frame is kappa-carrageenase Cgk1_ Wa;
FIG. 3: cgk1_ Wa multiple sequence alignment; wherein, the black box is the conserved residue of the kappa-carrageenase.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1: cloning, expressing and obtaining kappa-carrageenase Cgk _ Wa in escherichia coli
Culturing Wenyingzhuangia aestuarii OF219 in 2216E culture medium until the end OF logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-GACACGGATCCACTTCTCAAAATTTAAGACCGCTTAATGC; 5' -GACACCTCGAGTTAATCTATAATTATTTTTTGGGTTTTAATTCC) according to a target gene, and carrying out PCR by taking the whole genome as a template, wherein the PCR reaction conditions are as follows: 95 ℃ for 3min,94 ℃ for 30s,58 ℃ for 30s,72 ℃ for 150s,22 cycles, and finally 72 ℃ for 5min to obtain a kappa-carrageenase Cgk _ Wa gene fragment, which is connected to a DH5 alpha vector to form a recombinant plasmid. The recombinant plasmid is introduced into BL21 (DE 3) competent cells to construct a recombinant strain. In an LB culture medium containing kanamycin, isopropyl thiogalactoside is used for induction expression, the induction temperature is 17 ℃, and the induction time is 12h. Centrifuging to collect thallus, adding 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate (Na) 2 HPO 4 -NaH 2 PO 4 ) Suspending the buffer solution, then carrying out ultrasonic disruption in an ice-water bath (power 400W, work for 2s, gap 6s, circulation for 99 times), centrifuging and collecting supernatant, namely the crude enzyme solution of the kappa-carrageenase Cgk _ Wa.
And (3) performing affinity chromatography purification on the target protein in the supernatant by using a HisTrpTM HP chromatographic column on the intracellular supernatant enzyme liquid after the ultrasonication, wherein the SDS-PAGE analysis result is shown in figure 2, and the purified enzyme protein is a single band, which shows that the purity is good. The activity of the purified enzyme was 40.28U/mg (1U activity was defined as the activity to produce 1. Mu. Mol of reducing sugar in 1 min).
Example 2: kappa-carrageenase Cgk _ Wa clone expression in bacillus subtilis and its obtaining method
Culturing Wenyingzhuangia aestuarii OF219 in 2216E medium until late logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-GTACACGGATCCACTTCTCAAAATTTAAGACCGCTT; 5' -TTAGCTCGAGTTAATCTATAA) according to target geneTTATTTTTTGGGTTTACCG) using the whole genome as a template, and performing PCR as in example 1 to obtain a kappa-carrageenase Cgk _ Wa gene fragment, which was ligated to a pHT01 vector to construct a recombinant plasmid. Transforming the recombinant plasmid into bacillus subtilis competent cells, screening positive clones, and carrying out induced expression in LB culture solution by using isopropyl thiogalactoside, wherein the induction temperature is 38 ℃ and the induction time is 14h. Centrifuging to collect thallus, adding a certain amount of 20mM Na 2 HPO 4 -NaH 2 PO 4 Suspending the buffer solution, then carrying out ultrasonic disruption in an ice-water bath (power 400W, work for 2s, gap 6s, circulation for 99 times), centrifuging and collecting supernatant, namely the crude enzyme solution of the kappa-carrageenase Cgk _ Wa. The purification procedure of example 1 was repeated to obtain a pure enzyme solution, and the activity of the recombinase fermentation broth was determined to be 81.61U/mg.
Example 3: clone expression and acquisition of kappa-carrageenase Cgk _ Wa in pichia pastoris
Culturing Wenyingzhuangia aestuarii OF219 in a 2216E culture medium until the end OF logarithm, extracting whole genome DNA, designing an upstream primer and a downstream primer (5 '-GGAATGGATCCACTTCTCAAAATTTAAGACCGCTTAA; 5' -CCGTATCGAGTTAATCTATAATTATTTTTTGGGTTTTAGGCC) according to a target gene, carrying out PCR (polymerase chain reaction) by taking the whole genome as a template according to an embodiment 1 to obtain a kappa-carrageenase Cgk _ Wa gene fragment, connecting the fragment to a pPIC9k vector to form a recombinant plasmid, and adding the recombinant plasmid into a Pichia pastoris GS115 competent cell to form a recombinant cell; screening positive clones, inoculating to YPD medium, culturing at 30 deg.C for 20h, inoculating to BMGY medium, shake culturing at 30 deg.C and 200rpm until OD600=2.0, centrifuging to collect thallus, discarding supernatant, resuspending the precipitate in BMMY medium, and inducing with methanol at 29 deg.C and 200rpm for 72h. After the induction is finished, centrifuging and collecting supernatant fluid to obtain crude enzyme liquid. The activity of the recombinase fermentation liquor is detected to be 72.24U/mg.
Example 4: the method of the invention is subjected to accuracy verification
The method and the phenol-sulfuric acid method are utilized to determine the content of the kappa-carrageenan in the sample:
(1) Preparing a kappa-carrageenan solution: weighing chemical-grade kappa-carrageenan, dissolving in 20mM Na with pH of 8.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, preparing kappa-carrageenan standard solution with the concentration of 0.50mg/mL, 1.00mg/mL, 1.50mg/mL, 2.00mg/mL and 2.50mg/mL respectively;
(2) preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/L HCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/L NaOH solution according to the weight ratio of 1:9 to prepare 20mg/mL pHBH solution.
(3) Drawing a quantitative standard curve: 375 μ L of the prepared kappa-carrageenan solution with different concentrations is respectively reacted with 100U Cgk1_Wa (buffer solution is filled to 375 μ L) at 30 ℃ for 20min. Standing at 100 deg.C for 5min after reaction to inactivate enzyme, adding 250 μ L pHBH solution, developing at 100 deg.C in metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 410 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the kappa-carrageenan solution, repeating the operation, and measuring the light absorption value of the mixture to serve as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the kappa-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve y =1.1468x +0.0072 under specific reaction conditions by linear fitting, wherein R is 2 The value was 0.9994.
(4) And (3) sample determination: an appropriate amount of the sample was weighed, ground, washed with 85% ethanol solution to remove reducing sugars, and the ethanol solution was discarded for 3 consecutive times. After desugarization, the sample was dried overnight in an oven at 40 ℃ and the sample was dissolved in buffer after drying. And (3) taking 375 mu L of sample solution, repeating the operation in the third step, substituting the light absorption value increment into the standard curve y =1.1468x +0.0072, calculating the concentration of the kappa-carrageenan in the reaction system, and further converting the content of the kappa-carrageenan in the sample.
(5) The kappa-carrageenan content of the samples was determined according to the phenol-sulfuric acid method in the national standard SN/T4260-2015.
Three replicates of each procedure were performed and the results are shown in the table below.
| The method of the invention | Phenol-sulfuric acid process | |
| Assay parallel 1 (mg/mL) | 10.18 | 9.91 |
| Assay parallel 2 (mg/mL) | 9.73 | 9.70 |
| Assay parallel 3 (mg/mL) | 9.80 | 10.08 |
| Determination of average value (mg/mL) | 9.90 | 9.90 |
From the above results, it can be seen that there is substantially no deviation between the measurement results of the method of the present invention and the measurement results of the phenol-sulfuric acid method, indicating that the method of the present invention has good accuracy.
Example 5: quantitative specificity verification of the method of the invention
The method of the invention is used for quantifying the kappa-carrageenan in the three mixed solutions respectively.
Step one, preparing a kappa-carrageenan solution: chemical-grade kappa-carrageenan is weighed and dissolved in 20mM Na with pH7.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, prepare the kappa-carrageenan solution with concentration of 10.00 mg/mL; simultaneously preparing agar, iota-carrageenan and/or carrageenan with the concentration of 10.00mg/mL,Algin solution; and respectively and uniformly mixing the agar solution, the iota-carrageenan solution and the alginate solution with the kappa-carrageenan solution in the same volume to prepare a mixed solution, wherein the kappa-carrageenan concentration of the mixed solution is 5.00mg/mL.
Step two, preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/L HCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/L NaOH solution according to the weight ratio of 1:9 to prepare 20mg/mL pHBH solution.
Step three, drawing a quantitative standard curve: 375 μ L of the prepared kappa-carrageenan solutions with different concentrations were respectively reacted with 10U Cgk1_Wa (buffer solution to 375 μ L) at 25 ℃ for 10min. Standing in 100 deg.C metal bath for 8min after reaction to inactivate enzyme, adding 250 μ L pHBH solution, developing in 100 deg.C metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 415 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the kappa-carrageenan solution, repeating the operation, and measuring the light absorption value of the mixture to serve as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the kappa-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve of y =1.1040x +0.0018 under specific reaction conditions by linear fitting, wherein R is 2 The value is 0.9992.
Step four, sample determination: and (3) respectively taking 375 mu L of mixed solution, repeating the operation in the third step, substituting the light absorption value increment into the standard curve y =1.1040x +0.0018, calculating the concentration of the kappa-carrageenan in the reaction system, and converting the content of the kappa-carrageenan in the mixed solution.
The measurement was carried out in triplicate, and the measurement results were as follows.
| Kappa-carrageenin and agar | Kappa-carrageenan and iota-carrageenan | Kappa-carrageenan and algin | |
| Assay parallel 1 (mg/mL) | 5.05 | 5.08 | 4.98 |
| Assay parallel 2 (mg/mL) | 5.02 | 4.99 | 5.04 |
| Assay parallel 3 (mg/mL) | 5.04 | 4.95 | 5.04 |
| Determination of average value (mg/mL) | 5.04 | 5.01 | 5.02 |
| Relative error (%) | 0.73％ | 0.13％ | 0.40％ |
From the above results, it can be seen that the method of the present invention has good quantitative specificity.
Example 6: determination of kappa-carrageenan content in a ham sausage
(1) Preparing a kappa-carrageenan solution: weighing chemical-grade kappa-carrageenan, dissolving in p20mM Na in H8.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, preparing kappa-carrageenan standard solution with the concentration of 0.5mg/mL, 1.00mg/mL, 1.50mg/mL, 2.00mg/mL and 2.50mg/mL respectively;
(2) preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/L HCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/L NaOH solution according to the weight ratio of 1:9 to prepare 20mg/mL pHBH solution.
(3) Drawing a quantitative standard curve: 375 μ L of the prepared kappa-carrageenan solution with different concentrations is respectively reacted with 20U Cgk1_Wa (buffer solution is filled to 375 μ L) at 35 ℃ for 15min. Standing in 100 deg.C metal bath for 10min after reaction to inactivate enzyme, adding 250 μ L pHBH solution, developing in 100 deg.C metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 410 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the kappa-carrageenan solution, repeating the operation, and measuring the light absorption value of the mixture to serve as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the kappa-carrageenan standard solution as an abscissa and the corresponding absorbance value increment as an ordinate, obtaining a standard curve y =1.1024x +0.0712 under specific reaction conditions by linear fitting, wherein R is 2 The value is 0.9991.
(4) And (3) sample determination: an appropriate amount of the sample was weighed, ground, washed with 85% ethanol solution to remove reducing sugars, and the ethanol solution was discarded for 3 consecutive times. After desugarization, the sample was dried overnight in an oven at 40 ℃ and the sample was dissolved in buffer after drying. And (3) taking 375 mu L of sample solution, repeating the operation in the third step, substituting the light absorption value increment into a standard curve y =1.1024x +0.0712, calculating the concentration of the kappa-carrageenan in the reaction system, and further calculating the content of the kappa-carrageenan in the ham sausage to be 8.12mg/mL.
Example 7: determination of kappa-carrageenan content in a soft candy
(1) Preparing a kappa-carrageenan solution: weighing chemical-grade kappa-carrageenan, dissolving in 20mM Na with pH of 8.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, preparing kappa-carrageenan standard solution with the concentration of 0.30mg/mL, 0.60mg/mL, 0.90mg/mL, 1.20mg/mL and 1.50mg/mL respectively;
(2) preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/L HCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/L NaOH solution according to the weight ratio of 1:9 to prepare 20mg/mL pHBH solution.
(3) Drawing a quantitative standard curve: 375 μ L of the prepared kappa-carrageenan solution with different concentrations is respectively reacted with 100U Cgk1_Wa (buffer solution is filled to 375 μ L) at 30 ℃ for 30min. Standing at 100 deg.C for 7min after reaction to inactivate enzyme, adding 250 μ L pHBH solution, developing color in 100 deg.C metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 415 nm; meanwhile, mixing the inactivated enzyme solution with the same concentration gradient with the kappa-carrageenan solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a contrast, thereby calculating the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the kappa-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve of y =1.1115x +0.0140 under specific reaction conditions by linear fitting, wherein R is 2 The value is 0.9996.
(4) And (3) sample determination: an appropriate amount of the sample was weighed, ground, washed with 85% ethanol solution to remove reducing sugars, and the ethanol solution was discarded for 3 consecutive times. After desugarization, the sample was dried overnight in an oven at 40 ℃ and the sample was dissolved in buffer after drying. And (4) taking 375 mu L of sample solution, repeating the operation in the third step, substituting the light absorption value increment into the standard curve y =1.1115x +0.0140, calculating the concentration of the kappa-carrageenan in the reaction system, and further calculating the content of the kappa-carrageenan in the soft candy to be 17.42mg/mL.
Finally, it should be noted that the above embodiments describe specific embodiments of the present invention, but do not limit the present invention; it will be understood by those skilled in the art that these are by way of example only and that the scope of the invention is defined by the appended claims. All changes, modifications and equivalents may be resorted to, falling within the scope of the invention.
Sequence listing
<110> China oceanic university
<120> method for quantitatively detecting kappa-carrageenan by enzyme method
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Arg Asn Lys Val Thr Ser Asn Gly Tyr Thr Phe Gln Leu Asp Thr Asn
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Gly Asn Lys Ile Pro Leu Thr Asn Asn Glu Ile Thr Thr Asn Lys Gly
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Claims (7)
1. A method for quantitatively detecting kappa-carrageenan by an enzyme method is characterized by comprising the following steps: degrading kappa-carrageenan into reducing sugar by utilizing the specificity of the kappa-carrageenan, inactivating enzyme, adding p-hydroxybenzoyl hydrazine solution for color reaction, centrifuging, measuring the light absorption value of supernatant at 400-420nm, and comparing with a standard curve to obtain the content of the kappa-carrageenan; the kappa-carrageenase is kappa-carrageenase Cgk _ Wa, and the amino acid sequence of the kappa-carrageenase is SEQ ID NO.1.
2. The method for quantitatively detecting kappa-carrageenan according to claim 1, which comprises the following steps: the optimum reaction temperature of the kappa-carrageenase is 25 ℃, the optimum reaction pH value is 8.0, the kappa-carrageenase basically keeps stable in the pH value range of 6.0-10.0, has good storage stability, and can be stably stored for at least 3 months at 4 ℃; the enzyme kinetic constants Km and Kcat are respectively 0.07mg/mL and 27.54s -1 Km/Kcat of 165.63. Mu.M -1 s -1 。
3. The method for enzymatic quantitative detection of kappa-carrageenan according to claim 1, wherein: the nucleotide sequence of the gene for coding the kappa-carrageenase is SEQ ID NO.2 or all genes which can be translated into SEQ ID NO.1.
4. The method for the enzymatic quantitative detection of kappa-carrageenan according to claim 1, comprising the steps of:
(1) Preparing a kappa-carrageenan solution: weighing chemical-grade or above-purity kappa-carrageenan, dissolving in a buffer solution, and preparing a kappa-carrageenan standard solution with a concentration gradient;
(2) preparation of a 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 kappa-carrageenan solutions with different concentrations prepared in the step (1) with a proper amount of kappa-carrageenan enzyme solution respectively to react; inactivating the enzyme after the reaction; adding a pHBH solution, carrying out color development, quickly cooling to room temperature, centrifuging to obtain a supernatant, and measuring the light absorption value of the supernatant; mixing the kappa-carrageenan solution with the same concentration gradient with the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the mixture as a control, and then calculating the light absorption value increment corresponding to the kappa-carrageenan solution with different concentrations; taking the concentration of the kappa-carrageenan standard solution as an abscissa and the light absorption value increment of each concentration of kappa-carrageenan 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 kappa-carrageenase into the sample solution to repeat the reaction in the step (3); substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme adding amount, reaction time, reaction temperature, reaction pH and the like, and calculating the concentration of the kappa-carrageenan in the reaction system so as to obtain the content of the kappa-carrageenan in the sample.
5. The method for the quantitative detection of kappa-carrageenan according to claim 4, which comprises the following steps: the pH value of the buffer solution in the step (1) is 7.0-10.0.
6. The method for the quantitative detection of kappa-carrageenan according to claim 4, which comprises the following steps: in the step (3), the addition amount of the enzyme is 1-1000U, the reaction time is 5-40min, and the reaction temperature is 20-35 ℃.
7. The method for the quantitative detection of kappa-carrageenan according to claim 4, which comprises the following steps: and (4) removing reducing sugar in the sample according to a method of national standard GB 5009.88-2014 before the determination of the step (4).
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| Title |
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| kappa-carrageenase [Wenyingzhuangia sp. OF219];Shen,J等;《NCBI GenBank》;20170814;第2-3页 * |
| 基于pHBH法的岩藻聚糖硫酸酯酶酶活测定方法;张翠玉等;《中国食品学报》;20130731;第13卷(第7期);第3节 * |
| 来源于Wenyingzhuangia属海洋细菌的一种β-琼胶酶的克隆表达及性质研究;田雪健等;《食品与发酵工业》;20190531;第45卷(第8期);摘要和第1.2.4节 * |
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