JP4968772B2 - Method for producing antifreeze activator, antifreeze activator and frozen food containing the antifreeze activator - Google Patents

Description

  The present invention relates to a method for producing an antifreeze activator, an antifreeze activator, and a frozen food containing the antifreeze activator.

  Although frozen storage is performed for the purpose of storing meat, vegetables, blood, organs and the like for a long period of time, when cooled to a freezing temperature, ice nuclei generated in the cells grow and freeze while being bound. In this case, freeze-concentration in which the ice crystals grown at this time press the tissue in the cell to separate and concentrate occurs, and the cell tissue is damaged or destroyed. For this reason, the meat and vegetables stored frozen have a problem that the taste is impaired. As a method for preventing freeze concentration, it was examined that it is effective to use antifreeze active substances present in the body of fish inhabiting the Arctic and Antarctic. In recent years, in addition to fish that live in the Arctic and Antarctica, it has been found that anti-freezing active substances are contained in smelt and vegetables, and various anti-freezing active substances have been reported (Patent Documents 1 to 3). etc).

JP 2003-250572 A JP 2004-83546 A JP 2001-245659 A

  However, conventionally known antifreeze active substances derived from fish and vegetables are weak against heat and their antifreeze activity is significantly reduced by heating, so that they can be used for foods that require heat treatment. could not. The present invention can solve the above conventional problems, and can provide an antifreeze activator capable of obtaining an excellent antifreeze activator whose antifreeze activity does not decrease even after the heat treatment step, the antifreeze activator, and the antifreeze thereof. An object is to provide a frozen food containing an active agent.

That is, the present invention
(1) Production of an antifreeze activator characterized in that a buffer solution is added to the viscera of a marine mollusc and an extraction treatment is performed, and then the extract is subjected to molecular weight fractionation to collect a fraction having a molecular weight of 10,000 or more. Method,
(2) Extraction obtained by adding a buffer solution to the viscera of marine mollusks and then treating the extract with acetone or ethanol and adding a buffer solution to the treated precipitate for extraction. A method for producing an antifreeze activator comprising molecular weight fractionation of a liquid and recovering a fraction having a molecular weight of 10,000 or more (3) Degreasing the viscera of a marine mollusk, and buffering the precipitate after the treatment A method for producing an antifreeze activator, characterized in that a fraction having a molecular weight of 10,000 or more is recovered by molecular weight fractionation of an extract obtained by subjecting the extract to extraction,
(4) The method for producing an antifreeze active agent according to the above (1) to (3), wherein the internal organs of the marine mollusc are scallop midgut glands,
(5) An antifreeze activator obtained by the production method of any one of (1) to (4) above,
(6) Frozen food containing the antifreeze active agent of (5) above,
Is a summary.

  According to the method of the present invention, an excellent antifreeze active agent can be produced at low cost from the viscera of marine mollusks, which are food wastes. Moreover, after processing using solvents, such as acetone and ethanol, by extracting and fractionating, an antifreeze activator can be obtained efficiently and industrialization becomes easy. The antifreeze activator of the present invention has an excellent antifreeze activity and can be widely used in the fields of food, cosmetics, pharmaceuticals, research reagents and the like. In addition, the antifreeze activator of the present invention is excellent in thermal stability, and the antifreeze activity is not reduced by heating, so that the antifreeze activity is impaired even when used by adding to foods that require heat treatment. There is no. The frozen food containing the antifreeze activator of the present invention can prevent the ice crystals from becoming coarse during frozen storage, and has the effect of not deteriorating the quality of the food.

  The antifreeze active agent of the present invention can be obtained from the viscera of marine mollusks. The internal organs of marine mollusks include craniopods such as squid and octopus, and internal organs of shellfish such as scallops, abalone, shrimp, turban shell, oysters and clams. Part (goro) is preferred, and scallop midgut gland is particularly preferred. These marine mollusks are preferably those that inhabit in a low temperature range. It is also possible to use a marine mollusk acclimatized at a low temperature. In the present invention, the antifreeze activity is an action of suppressing the growth of ice crystals, inhibiting the recrystallization of ice, and lowering the freezing temperature of the solution. The ice crystal whose growth is suppressed by the antifreeze activator of the present invention is confirmed to have an antifreeze activity effect due to its rock shape. Furthermore, the antifreeze activator of the present invention can keep the average diameter of ice crystals at 6 μm or less when held at −9 ° C. for 30 minutes for the effect of inhibiting recrystallization of ice during cryopreservation. Further, the antifreeze activator of the present invention exhibits a thermal hysteresis activity of 0.2 ° C. or higher, retains an activity of 85% or higher even after treatment at 80 ° C. for 1 hour, and has an excellent effect of lowering the freezing temperature of the solution. Have.

  The antifreeze active agent of the present invention can be obtained by molecular weight fractionation of a visceral extract of a marine mollusc such as a scallop midgut gland. The internal organs may be raw or used after being heat-treated such as boiling or steaming. When performing the extraction process from the internal organs, the internal organs are pasted and homogenized by a food processor or the like as a pretreatment. As the extract, a buffer solution such as a potassium phosphate buffer solution, a sodium phosphate buffer solution, an ammonium hydrogen carbonate buffer solution, or a Tris-HCl buffer solution can be used. The buffer solution preferably has a pH of 6-9 and a salt concentration of 5-100 mmol / L. The buffer solution is preferably added in an amount of 1 to 5 times the amount of paste and stirred to extract the antifreeze active agent in the buffer solution.

  After the extraction treatment, the supernatant liquid and the precipitate are separated by centrifugation and the supernatant liquid is recovered. After separating the supernatant liquid, the buffer is further added to the precipitate for extraction, and the process of collecting the supernatant liquid is repeated a plurality of times to increase the recovery rate of the antifreeze active agent. This process is preferably repeated about 2 or 3 times. Further, the antifreeze active ingredient can be concentrated by adding acetone or ethanol to the buffer extract. Concentration also reduces the amount of buffer solution and simplifies the subsequent operations. By performing such treatment with acetone or ethanol, an enzyme such as protease contained in the viscera is inactivated, and an activator with high antifreeze activity can be obtained. Acetone or ethanol is preferably added in an amount of 1 to 5 times the amount of the extract. After addition and stirring, the supernatant is removed by centrifugation and the precipitate containing the antifreeze active agent is recovered. The buffer is added again, extraction is performed, and the supernatant is recovered. Next, the recovered supernatant is subjected to molecular weight fractionation, components having a molecular weight of less than 10,000 are removed, and fractions having a molecular weight of 10,000 or more are collected, whereby the antifreeze activator of the present invention can be obtained. In order to fractionate a component having a molecular weight of less than 10,000 and a component having a molecular weight of 10,000 or more in the extract, dialysis, ultrafiltration, or the like can be employed. Ultrafiltration is suitable for molecular weight fractionation processing with high accuracy, and dialysis is suitable for molecular weight fractionation treatment of a large-volume extract. When the volume of the extract is small, components having a molecular weight of 10,000 or more can be fractionated only by ultrafiltration. However, when the volume of the extract is large, ultrafiltration can be performed after dialysis. preferable. In addition, it is preferable to perform microfiltration prior to dialysis or ultrafiltration because clogging of the dialysis membrane or ultrafiltration membrane can be prevented. As the membrane used for the fractionation, a cellulose-based membrane such as regenerated cellulose or cellulose acetate, or a synthetic polymer-based membrane such as polysulfone is used, and ultrafiltration is suitable for separating components having a molecular weight of 10,000 or more. It is. Moreover, as a membrane for separating components having a molecular weight of 10,000 or more, a cellulose membrane or a PVDF membrane is suitable.

  In the method of the present invention, delipidation of lipids contained in the viscera before extraction by adding a buffer solution to the viscera of marine mollusks increases the degree of purification of the resulting antifreeze active agent, resulting in high antifreeze. An antifreeze activator having activity can be obtained. The fat contained in the internal organs can be degreased by heat treatment of the internal organs, solvent treatment, or a combination of heat treatment and solvent treatment. As the solvent used for degreasing, chloroform, hexane, ether, acetone, ethanol, methanol, butanol, etc. can be used, but acetone, hexane, hexane and ethanol are preferably used, and when hexane and ethanol are used in combination, treatment with hexane And the treatment with ethanol may be performed separately or simultaneously using a hexane-ethanol mixed solution. Prior to the degreasing treatment, it is preferable to carry out a dehydration treatment by freeze drying, heat drying or the like, if necessary, but it is not necessary to do so.

  In the frozen food containing the antifreeze activator of the present invention, the growth of ice crystals is inhibited, so that the drip at the time of thawing is reduced and the food texture of the food can be improved. Moreover, the quality of frozen food during storage can be improved by suppressing recrystallization of ice during freezing. The amount of the antifreeze activator of the present invention added to the frozen food is preferably about 10 to 100 ppm.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
100 g of scallop midgut gland was made into a paste using a food processor, 100 ml of Tris-HCl buffer (100 mmol, pH = 8.0) was added thereto, and the mixture was stirred while cooling with ice water. Then 10000 r. p. m. The supernatant was separated and recovered from the precipitate. To the separated precipitate, 100 ml of the same buffer was added, and the mixture was stirred in the same manner while cooling with ice water. p. m. The supernatant was recovered by centrifugation at 15 minutes and mixed with the previous supernatant. This supernatant is purified by microfiltration and then desalted by dialysis with a cellophane tube with a molecular weight cut-off of about 10,000, and further, components with a molecular weight of less than 10,000 are removed with an ultrafiltration membrane with a molecular weight fraction of 10,000. An antifreeze activator comprising a fraction having a molecular weight of 10,000 or more was obtained.

  Table 1 shows the results of testing the ice crystal growth inhibitory effect when the obtained antifreeze activator is used without heating and the ice crystal growth inhibitory effect when heated at 80 ° C. for 1 hour. Show. Table 1 also shows the effect of the antifreeze activator on the inhibition of recrystallization of ice crystals and the results of the thermal hysteresis measurement test.

Test of growth inhibition effect of ice crystals After heating a sample solution of 1 mg / ml concentration to 80 ° C. and then cooling to 20 ° C., the same solution was heated to 20 ° C. without heating. 1 μL of each sample was set on the stage of a microscope equipped with a temperature controller, cooled to −50 ° C. at a rate of 100 ° C./min, then heated to −10 ° C. at a rate of 100 ° C./min, then 5 The temperature was raised to -2 ° C at a rate of ° C / min to form an ice single crystal. The single crystal was cooled at a rate of 1 ° C./min, and changes in the morphology of ice crystals were observed. On the other hand, a 1 mg / ml concentration sample of the antifreeze activator was heated at 80 ° C. for 1 hour, and then the same test was performed.

Test of recrystallization inhibition effect Prepare a sample solution of antifreeze activator so that the sucrose concentration is 30%, heat this to 80 ° C, then heat the same solution as the heat-treated sample cooled to 20 ° C. 1 μL of each non-heated sample adjusted to 20 ° C. without being heated is set on the stage of a microscope equipped with a temperature control device, cooled to −80 ° C. at a rate of 100 ° C./minute, and then rapidly frozen. The temperature was raised to −9 ° C. at a rate of ° C./minute, the temperature was kept constant, the number of ice crystals after 30 minutes was measured, and the number of ice crystals when the antifreeze activator-free product was set to 1 Shown as a ratio. In addition, the minimum and maximum values of the crystal are shown.

Thermal hysteresis measurement test After heating a sample solution of antifreeze activator at a concentration of 1 mg / ml to 80 ° C., the sample was heated to 20 ° C. and then heated to 20 ° C. without heating the same solution. Each sample was cooled to −50 ° C. at a rate of 100 ° C./min, then heated to −10 ° C. at a rate of 100 ° C./min, and then raised to −2 ° C. at a rate of 5 ° C./min. Ice single crystals were formed. The ice single crystal was cooled at a rate of 1 ° C./min, the time until the ice crystal began to grow was measured, and the thermal hysteresis was calculated from the following equation (1). From the thermal hysteresis of the heat-treated sample heated to 80 ° C. and the thermal hysteresis of the non-heated sample, the residual rate of hysteresis was determined from the following equation (2). The results are shown in Table 1.

(Equation 1)
Thermal hysteresis: TH (° C) = 60 ^-1 (° C / sec) x measurement time (sec) (1)

(Equation 2)
Residual rate = heating heat hysteresis value / non-heating heat hysteresis value × 100 (2)

Example 2
100 g of scallop midgut gland was made into a paste using a food processor, 100 ml of Tris-HCl buffer (100 mmol, pH = 8.0) was added thereto, and the mixture was stirred with cooling with ice water, followed by 10,000 r.p. p. m. The supernatant was separated and recovered from the precipitate. 100 ml of the same buffer was added to the separated precipitate, and the mixture was stirred in the same manner while cooling with ice water. p. m. The supernatant was recovered by centrifugation at 15 minutes and mixed with the previous supernatant. Three times the amount of acetone was added to the collected supernatant and stirred under ice-water cooling, and then allowed to stand at 4 ° C. overnight. After standing overnight, 12000 r. p. m. Was centrifuged for 30 minutes, and the supernatant was separated and removed to recover the precipitate. After air-drying the precipitate, 100 ml of Tris-HCl buffer (100 mmol, pH = 8.0) was added and stirred, and then 12000 r. p. m. And centrifuged for 60 minutes to recover the supernatant. The supernatant was purified by microfiltration to 0.2 μm, dialyzed with a cellophane tube with a molecular weight cut-off of about 10,000, and desalted, and further with a molecular weight fraction of 10000 and a molecular weight of less than 10,000. The antifreeze activator comprising a fraction having a molecular weight of 10,000 or more was obtained. The obtained antifreeze activator was subjected to the same single crystal growth inhibitory effect, recrystallization inhibitory effect, and thermal hysteresis measurement test as in Example 1. The results are shown in Table 1.

Example 3
After boiling the scallops together with the shellfish, extraction and molecular weight fractionation were performed in the same manner as in Example 2 except that the midgut gland extracted by opening the shellfish was used, and an antifreeze activator containing a fraction having a molecular weight of 10,000 or more Obtained. The obtained antifreeze activator was subjected to the same single crystal growth inhibitory effect, recrystallization inhibitory effect, and thermal hysteresis measurement test as in Example 1. The results are shown in Table 1.

Example 4
After making 100 g of scallop midgut gland into a paste using a food processor, it was washed twice with 10 times the amount of midgut gland and then centrifuged to collect the precipitate. It was degreased by washing twice with a double amount of hexane. To the midgut gland after the degreasing treatment, 100 ml of Tris-hydrochloric acid buffer (100 mmol, pH = 8.0) was added and stirred under cooling with ice water. Then 12000 r. p. m. And centrifuged for 60 minutes to recover the supernatant. This supernatant was purified by microfiltration to 0.2 μm, then desalted by dialysis using a cellophane tube with a molecular weight cut-off of about 10,000, and then further with a molecular weight fraction of 10,000 on a cellulose ultrafiltration membrane. Less than components were removed to obtain an antifreeze activator comprising a fraction having a molecular weight of 10,000 or more. The obtained antifreeze activator was subjected to the same single crystal growth inhibitory effect, recrystallization inhibitory effect, and thermal hysteresis measurement test as in Example 1. The results are shown in Table 1.

Comparative Example 1
The same test as in Example 1 was performed except that the antifreeze active protein obtained from Wakasagi was used. Table 1 shows the results of the single crystal growth suppression effect, the recrystallization inhibition effect, and the thermal hysteresis measurement test.

Freeze concentration test One drop of red ink was dropped into 50 ml of water, and 100 ppm of the antifreeze activator of Example 2 heat-treated at 80 ° C. was added thereto and stirred to prepare a test solution. Similarly, 100 ppm of the antifreeze activator of Comparative Example 1 was added and stirred to prepare a test solution. When these test solutions were stored frozen at −20 ° C. for one week, in the case of the test solution to which the antifreeze activator of Comparative Example 1 was added, the red ink was freeze-concentrated, whereas that of Example 2 In the case of the test solution to which antifreeze was added, the red ink remained uniformly dispersed.

Example 5
When 3 g of agar was added to 100 g of water and the agar had melted by heating, a sample prepared by adding 10 ppm of the antifreeze activator of Example 2 and stirring was poured into a petri dish and solidified. After solidifying, it was stored frozen at −20 ° C. for one week, then thawed, and the influence of the antifreeze active agent at the time of freezing and thawing was examined. A similar test was performed on a sample with no antifreeze activator added and a sample prepared using the antifreeze activator of Comparative Example 1 instead of the antifreeze activator of Example 2. In the agar using the antifreeze activator of Example 2, the drip was reduced after thawing as compared with the case of using the antifreeze activator-free sample and the antifreeze activator of Comparative Example 1. Moreover, when the agar was dried, the shrinkage of the agar was suppressed and the tissue destruction was small.

Claims (6)

A method for producing an antifreeze active agent, comprising: adding a buffer solution to the viscera of a marine mollusk and performing an extraction treatment; Extraction was performed by adding a buffer solution to the viscera of marine mollusks, then treating the extract with acetone or ethanol, and adding the buffer solution to the precipitate after the treatment to extract the molecular weight of the extract. A method for producing an antifreeze activator comprising fractionating and collecting a fraction having a molecular weight of 10,000 or more. The internal organs of marine mollusks are degreased, and the extract obtained by adding a buffer solution to the precipitate after the treatment and extracting it is subjected to molecular weight fractionation to collect a fraction having a molecular weight of 10,000 or more. Method for producing antifreeze activator. The method for producing an antifreeze active agent according to any one of claims 1 to 3, wherein the viscera of the marine mollusk is a scallop midgut gland. The antifreeze activator obtained by the manufacturing method in any one of Claims 1-4. A frozen food comprising the antifreeze activator according to claim 5.