CN112285237B - Squid freshness evaluation method based on biogenic amine content - Google Patents
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Abstract
The invention discloses a squid freshness evaluation method based on biogenic amine content, belonging to the technical field of food analysis, and the squid freshness evaluation method comprises the following steps: (1) Measuring the content of biogenic amine in the squids during low-temperature storage and normal-temperature storage; (2) Judging the freshness limit range of the biological amine content of the squid at different storage temperatures in the storage period; if the content of the biogenic amine is within a fresh limit range, judging that the squid is a fresh squid; and if the content of the biogenic amine is within the range of the putrefaction limit value, judging the squid to be putrefactive. Compared with the prior art, the method provided by the invention is used for squid freshness evaluation by taking the sum of the contents of histamine, putrescine and tyramine or the sum of the contents of cadaverine and putrescine as an evaluation index at different storage temperatures for the first time, and the evaluation method is scientific, accurate and reliable.
Description
Technical Field
The invention belongs to the technical field of food analysis, and particularly relates to a squid freshness evaluation method based on biogenic amine content.
Background
The squid is an ocean fishery resource with high nutritive value and good flavor, contains a large amount of collagen, taurine, vitamins and a plurality of amino acids necessary for human bodies, has the characteristics of high protein, low fat, low calorie and the like, and is deeply loved by consumers. As an important economic aquatic product, the processing and utilization of the aquatic product are becoming wide. However, the water content of the squid is high, the pH value of the squid is about 5.0-6.8, the squid is an ideal substrate for the growth and the propagation of microorganisms, and in addition, the imperfect cold chain transportation system and the openness in the circulating storage process in the processing, the storage and the transportation processes enable the microorganisms to grow and propagate rapidly, so that the squid can be rotten, and the freshness and the nutritional value of the squid are influenced. Therefore, the method is very important for evaluating the freshness of the squids in all links of production, storage, transportation, sale and consumption.
The freshness evaluation indexes of the aquatic products in the low-temperature storage process mainly comprise physical and chemical indexes such as sensory indexes, texture indexes, volatile basic nitrogen, pH value and the like. Many researches show that the change of the species and the content of the biogenic amine can be used as an index of the change of the freshness of aquatic products, but the biogenic amine of the squid is less researched.
The biogenic amines in the squid comprise common tyramine, cadaverine, putrescine, spermine, spermidine, histamine and the like. During storage, the change of the biogenic amine content is related to the putrefaction degree of the squid and is more closely related to the growth and the propagation of microorganisms in the squid. The change of the content of the biogenic amine is influenced by external environmental conditions and internal factors; when the storage temperature is different, the change of the biogenic amine content in the squids under different storage environments is different. The change of the contents of the biological amines is closely related to the putrefaction characteristics of the squids, and the degree of contents of the biological amines can indicate the putrefaction of the squids, which is one of the problems to be solved by researchers. Therefore, the development of an accurate and reliable squid freshness evaluation method based on the biogenic amine is of great significance.
Disclosure of Invention
Aiming at the problems in the prior art, a squid freshness evaluation method based on biogenic amine content.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a squid freshness evaluation method based on biogenic amine content, which comprises the following specific steps: determining the sum of the contents of histamine, putrescine and tyramine in the squid during the low-temperature storage period and judging the freshness limit range of the sum of the contents; determining the sum of the contents of cadaverine and putrescine in the squid during normal-temperature storage and judging the freshness limit range of the sum of the contents;
if the sum of the contents is within a fresh limit value range, judging the squid to be a fresh squid; and if the sum of the contents is within the range of the corruption limit value, judging the squid to be the corrupted squid.
Further, the fresh limit range under the low-temperature storage condition is less than 45.46mg/100g; the putrefaction limit range is not less than 45.46mg/100g.
Further, the cryopreservation condition means storage at 4 ℃.
Further, the fresh limit range under the normal-temperature storage condition is less than 213.45mg/100g; the putrefaction limit range is not less than 213.45mg/100g.
Further, the normal temperature storage condition refers to storage at 25 ℃.
Further, the manner of measuring the sum of the contents of cadaverine and putrescine and the sum of the contents of histamine, putrescine and tyramine is to use high performance liquid chromatography for measurement.
Further, the specific conditions of the high performance liquid chromatography are as follows: by high performance liquid chromatography C 18 Separating with column, detecting with ultraviolet detector, and performing quantitative analysis with external standard method.
Further, the squid is an equatorial mendelia.
The invention discloses the following technical effects:
the method for evaluating the freshness of the squid takes the content sum of the histamine, the putrescine and the tyramine or the content sum of the putrescine and the cadaverine as an evaluation index for the first time, and the evaluation method is scientific, accurate and reliable.
Drawings
FIG. 1 is a graph showing the change of the contents of 6 kinds of biogenic amines in cold fresh squid during storage at 4 ℃.
FIG. 2 is a graph showing the change of the contents of 6 kinds of biogenic amines in cold fresh squid during storage at 25 ℃.
Detailed Description
The following further illustrates embodiments of the invention, taken in conjunction with the accompanying drawings, which are not to be considered limiting of the invention, but are to be understood as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
The sample pretreatment method of the squid in the following embodiment is based on the determination of biogenic amine in national standard GB 5009.208-2016 food safety national standard food, omits the steps of purifying and extracting a food matrix by organic solvents such as trichloromethane, and the like, adds perchloric acid solution (0.4 mol/L,10 mL) into squid meat (2 g), homogenizes for 2min, and then centrifuges for 10min at 4 ℃ (5000 r/min,1984 RCF) to obtain supernatant. The above extraction steps are repeated once. The two supernatants were mixed and the volume was adjusted to 25mL with perchloric acid (0.4 mol/L) to obtain an extract of biogenic amine. The extract was stored in a centrifuge tube (50 mL) at 4 ℃ and awaited for derivatization. Studies have shown that pH, temperature of derivatization, and time of derivatization all affect the amount of biogenic amine derivatized. Therefore, the optimal steps for derivatization of the biogenic amine are: to the biogenic amine sample extract, 100. Mu.L of 2M sodium hydroxide and 300. Mu.L of saturated sodium bicarbonate were added and mixed well. Then, 2mL of dansyl chloride-acetone solution (10 mg/mL) was added to the mixed solution, and derivatization was performed at 40 ℃ for 45min with exclusion of light. Subsequently, the reaction was mixed with 100 μ L of 25% ammonium hydroxide to remove residual dansyl chloride derivatizing agent. After standing at room temperature for 30min, the solution was made up to 5mL with acetonitrile and passed through a 0.22 μm organic phase needle filter. The sample pretreatment step is simpler overall, the time consumption is shorter, and a better detection effect can be achieved.
In the following examples, the sum of the contents of cadaverine and putrescine and the sum of the contents of histamine, putrescine and tyramine were determined by high performance liquid chromatography, and qualitative and quantitative analysis of biogenic amine was performed by using Agilent 1260 high performance liquid chromatography (Agilent technologies, USA) in combination with external standard method. The biogenic amine is separated by an Agilent Zorbax extended-C18 column (the inner diameter is 4.6MM multiplied by 150 MM), and the trailing phenomenon of chromatographic peaks can be obviously reduced; elution was performed with a binary solution of 0.1M ammonium acetate (solvent A) and acetonitrile (solvent B), the best gradient elution protocol is shown in Table 1, and sufficiently rapid elution of biogenic amine was achieved by varying the ratio of organic phases. The flow rate is set to be 1.0mL/min, the column temperature is set to be 35 ℃, the sample injection amount is 10 mu L, and the detection wavelength of the ultraviolet detector is 254nm.
TABLE 1 HPLC GRADIENT ELIMINATION PROGRAM FOR BIOAMINE ANALYSIS
Example 1
Freshness evaluation method of low-temperature stored squid
(1) Determining the sum of the contents of putrescine, histamine and tyramine in the squid during the low-temperature storage period;
(2) Judging the freshness limit range of the sum of the contents of putrescine, histamine and tyramine; if the sum of the contents of putrescine, histamine and tyramine is less than 45.46mg/100g, judging the cold fresh squid to be fresh; and if the sum of the contents of putrescine, histamine and tyramine is more than or equal to 45.46mg/100g, judging the cold fresh squid to be the putrefactive cold fresh squid.
This example a study of 4 ℃ cryopreservation of squid in equatorial brachypodium harvested from the northwest pacific (see figure 1) shows: the range of 6 biogenic amines is 0.67-6.59 mg/kg (putrescine), 1.51-3.36 mg/kg (cadaverine), 21.03-32.26 mg/kg (histamine), 1.05-6.61 mg/kg (tyramine), 3.67-11.20 mg/kg (spermidine) and 10.84-17.94 mg/kg (spermine) during a storage period of up to 12 days. In the early stage of the study, the content of biogenic amine of other species except histamine (21.03 mg/kg) in the sample is relatively low (0.67-10.84 mg/kg). During the cold storage period, the content of 5 kinds of biogenic amines was continuously increased, except that the content of cadaverine was not changed much (1.51-3.36 mg/kg). After being refrigerated for 12 days, the content of the histamine is the highest, namely 32.26mg/kg, and the content is increased remarkably, which indicates that the histamine is the most main amine substance and is a biogenic amine which is highly related to the time change during the storage period of the squid. After being refrigerated for 12 days, the content of the spermine (17.94 mg/kg) and the spermidine (11.20 mg/kg) is higher than that of the putrescine and the tyramine, but the content of the putrescine and the tyramine is obviously increased after being stored for 10 to 12 days, and the increase is far larger than that of the spermine and the spermidine. This shows that the content of histamine, putrescine and tyramine can be used for the freshness evaluation of the squid during low-temperature storage.
Therefore, if the sum of the contents of putrescine, histamine and tyramine in the squid during the low-temperature storage is measured to be within the range of the freshness limit value, the squid is judged to be fresh. Adopt simultaneously that freshness evaluation index (TVB-N) commonly used to appraise this cold fresh squid, the evaluation result is fresh cold fresh squid, and the evaluation result is unanimous with this embodiment evaluation result.
Example 2
Method for evaluating freshness of squid stored at normal temperature
(1) Determining the sum of the contents of putrescine and cadaverine in the squid during normal-temperature storage;
(2) Judging the freshness limit value range of the sum of the contents of putrescine and cadaverine; if the sum of the contents of putrescine and cadaverine is less than 213.45mg/100g, judging the squid to be fresh; and if the sum of the contents of putrescine and cadaverine is more than or equal to 213.45mg/100g, judging the squid to be the putrefactive squid.
This example study of squid stored at ambient temperature at 25 ℃ on equatorial brachypodium harvested from the northwest pacific (see fig. 2) shows that: the change of the biogenic amine at 25 ℃ before 20 hours of storage is different from the change of the biogenic amine at 4 hours after storage, and the content of the biogenic amine is between 0.67 and 23.32 mg/kg. The tyramine content is remarkably increased from 1.38mg/kg to 9.01mg/kg within 12-16 hours, and the growth rate is higher than that of tyramine under the storage condition of 4 ℃. In the initial phase of the study, histamine content was highest in all biogenic amines, consistent with the 4 ℃ condition, but the change in histamine during storage was relatively small (21.93-22.78 mg/kg), and after 16 hours, histamine content increased to 24.76mg/kg. Histamine content is generally related to histamine decarboxylase in seafood, which catalyzes the formation of more histamine from histidine during storage. Unlike at 4 ℃, after 12 hours of storage of squid at 25 ℃, the spermidine content decreases and then tends to stabilize (1.53-2.71 mg/kg), which may be caused by the conversion of spermidine to putrescine under the action of spermidine synthase. The contents of putrescine and cadaverine are greatly increased after 20 hours of storage (cadaverine: 7.04-105.93 mg/kg; putrescine: 14.57-107.52 mg/kg) and become two main potential spoilage indicators. Meanwhile, the content of putrescine and cadaverine is much higher than that of other biogenic amines. This shows that the content of putrescine and cadaverine can be used for evaluating the freshness of the squid during normal-temperature storage.
And thus, when the sum of the contents of putrescine and cadaverine in the squid during the normal-temperature storage is measured to be within the putrefaction limit value range, the squid is judged to be putrefactive. Meanwhile, the squid is evaluated by adopting a common freshness evaluation index (TVB-N), the evaluation result is a rotten squid, and the evaluation result is consistent with the evaluation result of the embodiment.
Pearson product difference correlation analysis was performed on the changes of 5 kinds of biogenic amine content and volatile basic nitrogen (TVB-N) value of freshness index during storage at 4 ℃ and 25 ℃ of the squids in example 1 and example 2, and the correlation coefficient of biogenic amine with TVB-N and storage time at different storage temperatures is shown in Table 2. Cadaverine (R) was removed under storage conditions at 4 ℃ 2 = 0.868) and the content of the other 5 biogenic amines is well correlated with the storage time (R) 2 = 0.962-0.994); the storage time at 25 ℃ is also clearly linear with the biogenic amine level (R) 2 =0.646 to 0.969). On the other hand, the biogenic amine level of the squid meat is positively correlated with TVB-N (R) in the processes of cold storage and normal temperature storage 2 ＝0.556～0.966，p<0.05). Especially the content of putrescine (R) 2 = 0.894-0.957) is linearly related to the TVB-N values of the squid sample at the two temperatures. There is also a correlation between putrescine and cadaverine during squid storage (R) 2 = 0.937-0.995). This result is related to the interaction between the precursor amino acids of the corresponding biogenic amines, the precursor of putrescine being ornithine and the precursor of cadaverine being lysine. Due to the good correlation between the biogenic amine and the TVB-N, the biogenic amine can effectively evaluate the quality of the squid, so that the aquatic product evaluation is accurate and reliable.
TABLE 2 correlation of biogenic amine, volatile base Total Nitrogen (TVB-N) with storage time at 4 ℃ and 25 ℃
Note: ** ：P<0.01, the difference is very significant; * ：P<0.05, significant variability.
From the above analysis, 3 kinds of biogenic amines, such as histamine, putrescine and tyramine, can be used as biogenic amines with putrefaction characteristics when the squid is stored at low temperature of 4 ℃, so that the sum of the contents of the 3 kinds of biogenic amines can be used as a freshness index for evaluating the freshness of the squid during low-temperature storage. According to the freshness grading condition of the squid meat under the storage condition of 4 ℃, the biogenic amine index (sum of the contents of histamine, putrescine and tyramine) for squid freshness evaluation can be determined as follows: fresh (< 45.46mg/100 g) and putrefaction (. Gtoreq. 45.46mg/100 g).
From the above analysis, 2 biogenic amines, i.e. putrescine and cadaverine, can be used as biogenic amines with putrefaction characteristics when the squid is stored at the normal temperature of 25 ℃, so that the sum of the contents of the 2 biogenic amines can be used as a freshness index for evaluating the freshness of the squid during the normal temperature storage. According to the freshness grading condition of the squid meat under the storage condition of 25 ℃, the biogenic amine index (sum of putrescine and cadaverine contents) for squid freshness evaluation can be determined as follows: fresh (< 213.45mg/100 g) and putrefaction (. Gtoreq. 213.45mg/100 g).
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (3)
1. A squid freshness evaluation method based on biogenic amine content is characterized in that the squid freshness evaluation is carried out by taking the content sum of histamine, putrescine and tyramine or the content sum of putrescine and cadaverine as an evaluation index,
the method comprises the following specific steps: when the storage condition is low temperature, determining the sum of the contents of histamine, putrescine and tyramine in the squid and judging the limit value range of the sum of the contents of the histamine, putrescine and tyramine;
if the sum of the contents of histamine, putrescine and tyramine is within the freshness limit range, judging the squid to be a fresh squid; if the sum of the contents of histamine, putrescine and tyramine is within the range of the putrefaction limit value, the squid is judged to be putrefactive;
under the condition of low-temperature storage, the range of the freshness limit value is less than 45.46mg/100g, and the range of the putrefaction limit value is more than or equal to 45.46mg/100g;
the low-temperature storage condition refers to storage at 4 ℃;
when the storage condition is normal temperature, determining the sum of the contents of cadaverine and putrescine in the squid and judging the limit range of the sum of the contents of the cadaverine and the putrescine;
if the sum of the contents of the cadaverine and the putrescine is within the freshness limit range, judging that the squid is a fresh squid; if the sum of the contents of the cadaverine and the putrescine is within the putrefaction limit value range, judging the squid to be a putrefactive squid;
under the condition of normal temperature storage, the limit range of freshness is less than 213.45mg/100g, and the limit range of putrefaction is more than or equal to 213.45mg/100g;
the normal-temperature storage condition refers to storage at 25 ℃;
the squid is an equatorial tender fish.
2. The squid freshness evaluation method based on the biogenic amine content according to claim 1, wherein the manner of measuring the sum of the contents of cadaverine and putrescine and the sum of the contents of histamine, putrescine and tyramine is to use high performance liquid chromatography for measurement.
3. The squid freshness evaluation method based on the biogenic amine content according to claim 2, wherein the specific conditions of the high performance liquid chromatography are as follows: separating with high performance liquid chromatography C18 column, detecting with ultraviolet detector, and performing quantitative analysis with external standard method.
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