WO2013032308A2 - Novel enzymatic-type time-temperature integrator using a laccase - Google Patents

Novel enzymatic-type time-temperature integrator using a laccase Download PDF

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WO2013032308A2
WO2013032308A2 PCT/KR2012/007041 KR2012007041W WO2013032308A2 WO 2013032308 A2 WO2013032308 A2 WO 2013032308A2 KR 2012007041 W KR2012007041 W KR 2012007041W WO 2013032308 A2 WO2013032308 A2 WO 2013032308A2
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food
tti
pigment
enzymatic
time
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WO2013032308A3 (en
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김기혁
이승주
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동국대학교 산학협력단
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G01N31/229—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators for investigating time/temperature history

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  • the present invention relates to an enzyme type time-temperature integrator (TTI) using laccase and a freshness detection method of food using the same.
  • TTI time-temperature integrator
  • Time temperature intergrator is an indicator of the safety and quality of food according to the time-temperature history that occurs during food handling and distribution (Taoukis and Labuza, 1989; Giannakourou et al., 2005). .
  • TTI is an intelligent packaging which is mainly used for measuring the shelf life of food, distribution control and sterilization. These TTIs are label-type indicators that must be inexpensive, safe, visible and non-toxic before they can be consumed and distributed on most foods.
  • the TTI as well as the above elements should be capable of appropriately reflecting miniaturization, irreversible response, easy measurement of color change, and change in food quality.
  • TTIs known to date are classified into five types: enzymatic type, microbial type, diffusion type, polymer based type, and photochemical type.
  • enzymatic TTIs include lipases (US patents 4,043,871 and 4,284,719), alpha-amylases (Van Loey, 1997; Guiavarc'h, 2004; Yan et al, 2010), beta-glucosidase (Adams and Langley, 1998), etc. This was used.
  • Vitsab's product a commercially available representative enzymatic TTI, is used for intelligent packaging at room temperature. The lipase hydrolyzes lipids and changes the pH by the protons that are released.
  • TTIs using such lipases are vulnerable to the physical state of the substrate.
  • fatty acid hydrolysis of fatty acids bound to sn-1,2,3 positions of triglyceride which is a substrate of lipase
  • the speed changes As the lipase reaction rate depends not only on the pH change but also on the structure of the substrate, a complex reaction rate should be studied when estimating the shelf life of the lipase TTI.
  • the existing enzymatic TTI has used a synthetic pigment as a substrate.
  • enzymatic TTIs using natural pigments as the base substrate are available, not only can they improve visibility, which is one of the requirements for meeting TTI performance, but they can also be used at a lower cost, with a wider range of colors. will be.
  • Lacase (EC1.10.3.2), on the other hand, is an enzyme that contains copper ions and has been used to produce chemicals from the paper industry, wine discoloration, wastewater treatment, oxidation of pigments, and lignin (Viswanath). et al., 2008). Lacase is known as an enzyme that can be easily obtained from fungi, higher plants, bacteria, etc., and has been applied to color modification and enhancement in food and beverage processing in the food industry (Minussi et al., 2002).
  • the present inventors have determined that there is a sufficient possibility of the enzyme as a TTI, and thus, the present inventors have completed the present invention by preparing an enzyme-type TTI using the laccase and establishing a reaction rate according to a time-temperature change.
  • It is still another object of the present invention to provide a method of preparing a foodstuff comprising: (a) loading an enzymatic time-temperature hysteresis indicator with a laccase on the packaging surface of a food product; And (b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
  • the present invention provides an enzymatic time-temperature history indicator using a laccase.
  • the present invention comprises the steps of (a) attaching the enzymatic time-temperature history indicator using the laccase on the packaging surface of the food; And (b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
  • Enzymatic time-temperature hysteresis clock using the laccase of the present invention has various activation energies according to substrates, and is an environmentally friendly TTI having a simple reaction mechanism, which is useful in various food fields such as food distribution, distribution management, and sterilization process. Can be.
  • 1 is a view showing the color change according to the enzymatic oxidation process of laccase and guayacol.
  • Figure 2 is a diagram showing the correlation coefficient between each color system value and absorbance according to the concentration of oxidized guayacol solution.
  • FIG. 4 is a diagram showing the Hue value over time of enzymatic TTI Lacase is 0.260 units (TTI # 2).
  • FIG. 5 shows lnk values for 1 / T of enzymatic TTI with 0.260 units of laccase (TTI # 2).
  • FIG. 6 is a diagram showing Hue values according to time-temperature of enzymatic TTI having a laccase of 0.260 units (TTI # 2).
  • FIG. 7 is a diagram showing the color change with time-temperature of enzymatic TTI having a laccase of 0.260 units (TTI # 2).
  • FIG. 8 is a view showing the color change according to the enzyme reaction with laccase when various pigments are used as a substrate.
  • FIG. 9 is a diagram showing the change in color value during the enzymatic reaction of enzymatic TTI using P1 (lac color) as a substrate and using a laccase.
  • FIG. 10 is a diagram showing F (X) values at various temperatures of enzymatic TTI using P1 (lac dye) as a substrate and using a laccase.
  • FIG. 10 is a diagram showing F (X) values at various temperatures of enzymatic TTI using P1 (lac dye) as a substrate and using a laccase.
  • the present invention provides an enzymatic time-temperature integrator (TTI) using laccases.
  • the enzymatic time-temperature history indicator of the present invention may comprise a substrate of laccase.
  • the substrate may be a synthetic dye or a natural dye, but is not limited thereto.
  • the synthetic pigments include bromothymol blue, methyl orange, Remazol Brilliant Blue R, Drimaren Blue, Reactive Black 5, Acid Blue 25, Methyl green, Acid green 27, Acid viloet 7, Orange II, Tropaeolin O, Anthraquinone pigments such as Congo red, Poly R, Azur B, azo pigments, indigo pigments, industrial pigments, polymeric pigments and heterocylics ( heterocylic) pigments and the like, but is not limited thereto.
  • Examples of the natural pigments include guayacol, rock pigment, bokbunja pigment, gardenia blue pigment, blueberry pigment, berry pigment, paprika pigment, red cabbage pigment, beet red pigment, purple sweet potato pigment, corn pigment and safflower pigment.
  • Carotenoids (carotenoids), quinodis (quinodis), flavonoids (flavonoids), anthocyanins (anthocyanines), beta cyanines (betacyanines) and the like, but are not limited thereto.
  • the enzymatic time-temperature history indicator of the present invention may further include an input unit for inputting information of the TTI, information of the food, or distribution history information of the food until the TTI is attached to the food.
  • the information of the TTI may be product information of the TTI.
  • the food information may be a type of food or a distribution method of the food.
  • the distribution history information of the food may be a time history or a temperature history.
  • the food may be selected from the group consisting of seafood, meat, fruits, vegetables and dairy products, but is not limited thereto.
  • the food may be selected from the group consisting of room temperature distribution food, refrigerated distribution food and frozen distribution food, but is not limited thereto.
  • the enzymatic time-temperature history indicator using the laccase of the present invention has the following characteristics.
  • laccases can react with various substrates, such as o- or p-diphenols, aminophenols, polyamines, liglines, aryl diamines, compounds such as phenol groups, azo dyes and the like compared to other enzymes with substrate specificities. have.
  • substrates of various pigments can be applied to the TTI, and can be developed into TTIs having various activation energies depending on the respective reactants.
  • the enzyme type TTI using lipase is very sensitive to pH change, so the enzyme reaction rate is changed to non-linear of sigmoid, whereas the enzyme type TTI using laccase of the present invention has a gradual enzyme reaction rate. It can be handled by simple kinetics.
  • the enzymatic time-temperature hysteresis clock using the laccase of the present invention has various activation energies according to substrates, and is an environmentally friendly TTI having a simple reaction mechanism. It can be usefully used in the field.
  • the present invention comprises the steps of (a) attaching the enzymatic time-temperature history indicator using the laccase on the packaging surface of the food; And (b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
  • laccase EC 1.10.3.2, DeniLite II, Novozyme Co., Bagsv ⁇ rd, Denmark
  • 50 mM sodium acetate buffer pH 5.0
  • the solution was filtered twice with 0.45 ⁇ m syringe filter (Whatman filter paper, USA).
  • bovine serum albumin an enzyme stabilizer, was added to a concentration of 0.1 mg / mL, and then refrigerated at 4 ° C.
  • guayacol which is one of the substrates of laccase.
  • guayacol was mixed in 3 mL of 50 mM sodium acetate buffer (pH 5.0) to a final concentration of 10 mM, and then left at 30 ° C. in advance.
  • the reaction was initiated by dispensing 20 ⁇ L of the laccase solution prepared in Example 1 into the substrate solution.
  • the active unit of the enzyme was set to 1 unit of the amount of enzyme to oxidize 1 mol mol guayacol per minute.
  • the absorbance (OPTIZEN 3220V, Mecasys Co., Dajeon, Korea) was equipped with a water jacket to maintain a constant temperature.
  • Enzyme-type TTI (Lacasease-Guayacol TTI) prototype using Lacase was prepared by the following procedure. First, dispense 6 mL aliquots of a mixture of 20 mM guayacol (Sigmaldrch Co. Ltd.), 50 mM sodium acetate buffer (pH 5.0) and 0.1 mg / mg bovine serum albumin, and incubate the plate. (HST-103 PID type temperature controlled incubator, Korea, respectively, maintained at 5, 10, 15, 25, 37 °C) to reach a constant temperature.
  • the laccase solution prepared in Example 1 Dilute to contain 0.650 units (TTI # 1), 0.260 units (TTI # 2), 0.162 units (TTI # 3), or 0.104 units (TTI # 4), respectively, and then add 50 to the substrate solution of the 6-well plate. The reaction was initiated by aliquoting in ⁇ l The film was covered in the incubator to prevent evaporation of the solution.
  • the fully oxidized guayacol was diluted to a concentration of 0.047 to 0.281 mM, and then Therefore, the color system CIE-LAB, CIE-LCH, x, y, z values were measured (CR-300, Minolta, Tokyo, Japan), and color values and absorbance (470 nm) values were compared. At this time, the color value was measured on a white background in a wooden box to block light.
  • Abs is absorbance at 470 nm
  • h represents hue value (CIE-Lch).
  • reaction rate of the color reaction values of the four kinds of enzymatic TTIs having different unit amounts of laccase is expressed by the following equation.
  • Y is the color response value
  • k is the reaction rate constant
  • t is the reaction time.
  • the activation energy of the enzymatic TTI of the present invention was calculated by taking the natural log on both sides of the Arrhenius functional formula, as shown in Equation 3 below.
  • Ea is the activation energy
  • R is the gas constant
  • T is the absolute temperature
  • A is the pre-exponential factor
  • Ea / R was obtained by the slope through linear regression analysis between ln k and 1 / T, and lnA was obtained by the y-intercept.
  • the reaction rate constant (k) and the coefficient of crystallization (R 2 ) are obtained by applying the color reaction values of four kinds of enzymatic TTIs having different unit amounts of laccase to Equation 2, and applying the activation energy ( Ea) was obtained.
  • the results are shown in Table 1.
  • the reaction rate determination coefficient value of the enzyme-type TTI using the laccase of the present invention was greater than 0.9093, and it was confirmed that the hue value (Y) was linear with time. This indicates a zero-order reaction, and it can be seen that the enzymatic TTI using the laccase of the present invention can accurately reflect the time-temperature history of the food.
  • the Ea values of the four kinds of TTI having different unit amounts of laccases represented 43.9-45.4 kJ / mol, and the Ea range of predictable food quality degradation was 23.9-65.4 kJ / mol. Therefore, in comparison with the Ea values of the conventionally known food deterioration, the enzymatic TTI using the laccase of the present invention is characterized by enzymatic change (41.84-62.76 kJ / mol), hydrolysis (62.76 kJ / mol), fatty acidization It was confirmed that (41.84-104.6 kJ / mol) can be handled.
  • Hue values over time of TTI with 0.260 units of laccase (TTI # 2) and lnK values for 1 / T are shown in FIGS. 4 and 5, respectively.
  • the time required for four kinds of enzymatic TTIs with different unit amounts of laccase to reach the hue value 50 can be determined using the parameters shown in Equation 4 and Table 2 above. It was. The results are shown in Table 3 below.
  • laccase is used for enzymatic TTI, it is important to use a safe and acceptable substrate.
  • synthetic pigments and natural pigments are harmless and safe to the human body, so when used as a substrate of the TTI has the advantage of easy handling.
  • various pigments were prepared.
  • Synthetic pigments bromothiol blue (D1) and methyl orange (D2) were purchased from Samjeon Chemical (Seoul, Korea).
  • Natural pigment of Rock dye (Lac dye (P1)) is Coccus laccae (Laccifer lacca Kerr) a dendritic material to larvae secretion was purchased in powdered countries (Seoul, Korea)
  • bokbunja pigment (P2) is bokbunja (Rubus coreanus fruit) It was isolated from 20% purity of Bokbunja, purchased from Bread Farm (Cheonan, Korea), and Gardenia blue pigment (P3) was purchased from Napalm (Gyeonggi, Korea).
  • Enzyme stabilizer was used bovine serum albumin (BSA, first grade, Sigma, St. Louis, USA).
  • D1 and D2 were each prepared by dissolving 4 mg in 1 mL ethanol and then diluting 100 times in 0.5 mM acetate buffer (pH 5.0).
  • P1 and P3 were prepared by dissolving 2 mg in 1 mL of 50 mM acetate buffer (pH 5.0), respectively.
  • P2 has a bokbunja content of 20%, 1.25 times more than P1 and P3 were added. Thus, 2.5 mg of P2 was dissolved in 1 mL of 50 mM acetate buffer (pH 5.0).
  • BSA solution was added to a final concentration of 0.1 mg / mL.
  • Substrate solutions of each synthetic or natural pigment prepared above were dispensed into 6-well plates by 6 mL.
  • the TTI reaction was initiated by dispensing 150 ⁇ L of 1 unit of laccase into the solution.
  • the color change after 1 hour, 8 hours, and 54 hours at 25 ° C. is shown in FIG. 8.
  • ⁇ E of the P1 pigment showed the largest change, and ⁇ L was the highest, so that the brightness of the TTI changed the most.
  • DELTA a and DELTA b showed the most distinct change in the D1 dye and the P1 dye.
  • the color value of CIE-Lab was obtained by scanning with a scanner (HP scanjet 2300c, HP Co, California, USA) and analyzing the image using a Photoshop program (7.0, Photoshop Co, San jose, USA).
  • the total color change ⁇ E using color values L, a, and b is expressed by Equation 5 below.
  • * ⁇ L, ⁇ a, and ⁇ b represent brightness, red-green, and yellow, respectively.
  • the color response function of the TTI is a zero-order reaction, which is represented by Equation 6 below.
  • ⁇ E was selected as the color response variable (F) of Equation 6 since the value ⁇ E includes all the information about L, a, and b and is already used as the color response variable.
  • F (X) values at various temperatures are shown in FIG. 10. The rest of the enzymatic TTIs show similar results.
  • reaction rate constant (k) and crystal coefficient (R 2 ) activation energy (Ea) values were determined using the color reaction values of five different enzyme-type TTIs. The results are shown in Table 6.
  • reaction rate determination coefficient value of the enzymatic TTI using the laccase of the present invention was greater than 0.9400, which means that the color reaction function (F) with time was considerably linear.
  • the enzymatic TTI activation energy (Ea) value using the laccase of the present invention was found to have a range of 42.6-66.1 kJ / mol.
  • the enzyme-type TTI using the laccase of the present invention can use a variety of pigments as a substrate, by applying to a variety of food deterioration reaction by selecting a substrate having an activation energy similar to the food deterioration reaction Can be used.
  • Enzymatic time-temperature hysteresis clock using the laccase of the present invention has various activation energies according to substrates, and is an environmentally friendly TTI having a simple reaction mechanism, which is useful in various food fields such as food distribution, distribution management, and sterilization process. Can be.

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Abstract

The present invention relates to an enzymatic-type time-temperature integrator (TTI) using a laccase, and to a method for sensing food freshness using the integrator. The enzymatic-type time-temperature integrator using a laccase according to the present invention has various activating energies according to substrates thereof, and can be valuably used as an eco-friendly TTI having a simple reaction mechanism in various food-product fields, e.g. food product delivery, distribution management, and sterilization processes.

Description

라카아제를 이용한 새로운 효소형 시간-온도 이력지시계New Enzymatic Time-Temperature History Clock Using Lacase
본 발명은 라카아제를 이용한 효소형 시간-온도 이력 지시계 (time-temperature integrator, TTI) 및 이를 이용한 식품의 신선도 감지방법에 관한 것이다.The present invention relates to an enzyme type time-temperature integrator (TTI) using laccase and a freshness detection method of food using the same.
시간 온도 이력 지시계(Time temperature intergrator, TTI)는 식품이 취급, 분배되는 동안 발생하는 시간-온도 이력에 따른 식품의 안전성과 품질을 나타내는 지시계이다 (Taoukis and Labuza, 1989; Giannakourou et al., 2005). TTI는 지능형 포장으로써 주로 식품의 유통기한을 측정하거나 식품의 분배 관리, 살균 공정에 적용된다. 이러한 TTI는 레이블 형식의 지시계로, 저가형, 안전성, 가시성, 무독성이어야만 대부분의 식품류에 부착되어 소비되고, 유통될 수 있다. 또한, 상기 요소들뿐 아니라 TTI는 소형화, 비가역적 반응, 색 변화의 쉬운 측정, 식품 품질의 변화를 적절하게 반영할 수 있는 것이어야 한다. Time temperature intergrator (TTI) is an indicator of the safety and quality of food according to the time-temperature history that occurs during food handling and distribution (Taoukis and Labuza, 1989; Giannakourou et al., 2005). . TTI is an intelligent packaging which is mainly used for measuring the shelf life of food, distribution control and sterilization. These TTIs are label-type indicators that must be inexpensive, safe, visible and non-toxic before they can be consumed and distributed on most foods. In addition, the TTI as well as the above elements should be capable of appropriately reflecting miniaturization, irreversible response, easy measurement of color change, and change in food quality.
현재까지 알려진 TTI는 효소형(enzymatic type), 미생물형(microbial type), 확산형(diffusion based type), 고분자형(polymer based type), 광화학형(photochemical type)의 다섯 가지 종류로 분류된다. TTIs known to date are classified into five types: enzymatic type, microbial type, diffusion type, polymer based type, and photochemical type.
그 중 효소형 TTI에는 리파아제 (US patents 4,043,871 및 4,284,719), 알파-아밀라아제 (Van Loey, 1997; Guiavarc'h, 2004; Yan et al, 2010), 베타-글루코시다아제 (Adams and Langley, 1998) 등이 사용되었다. 상용화된 대표적인 효소형 TTI인 Vitsab사의 제품은 상온 유통 지능형 포장을 위하여 사용되며, 리파아제가 지질을 가수분해하며 방출되는 양성자에 의한 pH 변화를 지시약 색의 변화로 나타내게 된다. 그러나, 이러한 리파아제를 이용한 TTI는 기질의 물리적 상태에 취약점이 있다. 즉, 리파아제의 기질이 되는 중성지방(triglyceride)의 sn-1,2,3 위치에 결합된 지방산 (fatty acid)이 트리글리세라이드, 디글리세라이드, 모노글리세라이드로 구조적 변화가 일어나면서, 지방산 가수분해 속도가 변하게되는 문제가 있다. 이렇듯 리파아제의 반응속도가 pH 변화뿐 아니라, 기질의 구조에도 의존적이게 되므로, 리파아제를 이용한 TTI로 유통기한을 예측할 때에는 복잡한 반응속도에 대한 연구가 수행되어야 한다. Among them, enzymatic TTIs include lipases (US patents 4,043,871 and 4,284,719), alpha-amylases (Van Loey, 1997; Guiavarc'h, 2004; Yan et al, 2010), beta-glucosidase (Adams and Langley, 1998), etc. This was used. Vitsab's product, a commercially available representative enzymatic TTI, is used for intelligent packaging at room temperature. The lipase hydrolyzes lipids and changes the pH by the protons that are released. However, TTIs using such lipases are vulnerable to the physical state of the substrate. That is, fatty acid hydrolysis of fatty acids bound to sn-1,2,3 positions of triglyceride, which is a substrate of lipase, changes to triglycerides, diglycerides, and monoglycerides, resulting in fatty acid hydrolysis. There is a problem that the speed changes. As the lipase reaction rate depends not only on the pH change but also on the structure of the substrate, a complex reaction rate should be studied when estimating the shelf life of the lipase TTI.
또한, 기존 효소형 TTI는 합성 색소를 기질로 이용해왔다. 그러나 만약 천연 색소를 기본 기질로 사용하는 효소형 TTI가 가용하다면, TTI 성능의 충족 요건 중 하나인 가시성(visibility)을 향상시킬 수 있을 뿐 아니라, 저가형의 보급, 더 넓은 범위의 색깔을 사용할 수 있을 것이다.In addition, the existing enzymatic TTI has used a synthetic pigment as a substrate. However, if enzymatic TTIs using natural pigments as the base substrate are available, not only can they improve visibility, which is one of the requirements for meeting TTI performance, but they can also be used at a lower cost, with a wider range of colors. will be.
한편, 라카아제(EC1.10.3.2)는 구리이온을 포함하는 효소로서 제지산업, 와인의 변색(discoloration), 폐수 처리, 색소의 산화, 리그닌으로부터 화학물질을 생산해내는 데에 이용되어져 왔다(Viswanath et al., 2008). 라카아제는 곰팡이, 고등식물, 박테리아 등으로부터 쉽게 얻을 수 있는 효소로 알려져 있으며, 식품산업에서 식품이나 음료 가공 시 색의 변형과 향상에 적용된바 있다(Minussi et al., 2002).Lacase (EC1.10.3.2), on the other hand, is an enzyme that contains copper ions and has been used to produce chemicals from the paper industry, wine discoloration, wastewater treatment, oxidation of pigments, and lignin (Viswanath). et al., 2008). Lacase is known as an enzyme that can be easily obtained from fungi, higher plants, bacteria, etc., and has been applied to color modification and enhancement in food and beverage processing in the food industry (Minussi et al., 2002).
그러나 지금까지 라카아제를 TTI에 적용시킨 사례는 없었다. However, there have been no cases of applying laccase to TTI.
이에 본 발명자들은 라카아제가 TTI의 효소로 충분한 가능성이 있다고 판단하고, 라카아제를 이용한 효소형 TTI를 제조하고, 시간-온도 변화에 따른 반응 속도를 정립함으로써 본 발명을 완성하였다. Accordingly, the present inventors have determined that there is a sufficient possibility of the enzyme as a TTI, and thus, the present inventors have completed the present invention by preparing an enzyme-type TTI using the laccase and establishing a reaction rate according to a time-temperature change.
본 발명의 목적은 라카아제를 이용한 효소형 시간-온도 이력 지시계를 제공하는 것이다. It is an object of the present invention to provide an enzymatic time-temperature history indicator using laccases.
본 발명의 또 다른 목적은 (a) 라카아제를 이용한 효소형 시간-온도 이력 지시계를 식품의 포장 표면에 부시키는 단계; 및 (b) 상기 (a) 단계의 시간-온도 이력 지시계의 색의 변화로 식품의 변질 정도를 판별하는 단계;를 포함하는 식품의 신선도 감지 방법을 제공하는 것이다. It is still another object of the present invention to provide a method of preparing a foodstuff comprising: (a) loading an enzymatic time-temperature hysteresis indicator with a laccase on the packaging surface of a food product; And (b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
상기 과제를 해결하기 위해, 본 발명은 라카아제를 이용한 효소형 시간-온도 이력 지시계를 제공한다. In order to solve the above problems, the present invention provides an enzymatic time-temperature history indicator using a laccase.
또한 본 발명은 (a) 라카아제를 이용한 효소형 시간-온도 이력 지시계를 식품의 포장 표면에 부착시키는 단계; 및 (b) 상기 (a) 단계의 시간-온도 이력 지시계의 색의 변화로 식품의 변질 정도를 판별하는 단계;를 포함하는 식품의 신선도 감지 방법을 제공한다. In another aspect, the present invention comprises the steps of (a) attaching the enzymatic time-temperature history indicator using the laccase on the packaging surface of the food; And (b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
본 발명의 라카아제를 이용한 효소형 시간-온도 이력지시계는 기질에 따라 다양한 활성화 에너지를 가지며, 단순한 반응 메커니즘을 갖는 친환경적인 TTI로써 식품의 유통, 분배 관리, 살균 공정 등 다양한 식품 분야에서 유용하게 이용될 수 있다. Enzymatic time-temperature hysteresis clock using the laccase of the present invention has various activation energies according to substrates, and is an environmentally friendly TTI having a simple reaction mechanism, which is useful in various food fields such as food distribution, distribution management, and sterilization process. Can be.
도 1은 라카아제와 구아야콜의 효소적 산화 과정에 따른 색 변화를 나타낸 도이다. 1 is a view showing the color change according to the enzymatic oxidation process of laccase and guayacol.
도 2는 산화된 구아야콜 용액의 농도에 따른 각 칼라 시스템 값 및 흡광도와의 상관계수를 나타낸 도이다.Figure 2 is a diagram showing the correlation coefficient between each color system value and absorbance according to the concentration of oxidized guayacol solution.
도 3은 산화된 구아야콜 용액의 농도와 hue 값 간의 표준 곡선을 나타낸 도이다. 3 shows a standard curve between the concentration of oxidized guayacol solution and hue value.
도 4는 라카아제가 0.260 유닛 (TTI #2)인 효소형 TTI의 시간에 따른 Hue 값을 나타낸 도이다. Figure 4 is a diagram showing the Hue value over time of enzymatic TTI Lacase is 0.260 units (TTI # 2).
도 5는 라카아제가 0.260 유닛 (TTI #2)인 효소형 TTI의 1/T에 대한 lnk 값을 나타낸 도이다.FIG. 5 shows lnk values for 1 / T of enzymatic TTI with 0.260 units of laccase (TTI # 2). FIG.
도 6은 라카아제가 0.260 유닛 (TTI #2)인 효소형 TTI의 시간-온도에 따른 Hue 값을 나타낸 도이다. 6 is a diagram showing Hue values according to time-temperature of enzymatic TTI having a laccase of 0.260 units (TTI # 2).
도 7은 라카아제가 0.260 유닛 (TTI #2)인 효소형 TTI의 시간-온도에 따른 색 변화를 나타낸 도이다.FIG. 7 is a diagram showing the color change with time-temperature of enzymatic TTI having a laccase of 0.260 units (TTI # 2).
도 8은 다양한 색소를 기질로 이용하였을 때, 라카아제와의 효소 반응에 따른 색 변화를 나타낸 도이다. 8 is a view showing the color change according to the enzyme reaction with laccase when various pigments are used as a substrate.
도 9는 P1(락 색소)을 기질로 이용하고 라카아제를 이용한 효소형 TTI의 효소 반응 동안 색 값의 변화 양상을 나타낸 도이다. 9 is a diagram showing the change in color value during the enzymatic reaction of enzymatic TTI using P1 (lac color) as a substrate and using a laccase.
도 10은 P1 (락 색소)을 기질로 이용하고 라카아제를 이용한 효소형 TTI의 다양한 온도에서의 F(X) 값을 나타낸 도이다.FIG. 10 is a diagram showing F (X) values at various temperatures of enzymatic TTI using P1 (lac dye) as a substrate and using a laccase. FIG.
이하 본 발명에 대하여 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명은 라카아제를 이용한 효소형 시간-온도 이력 지시계(time-temperature integrator, TTI)를 제공한다. The present invention provides an enzymatic time-temperature integrator (TTI) using laccases.
본 발명의 효소형 시간-온도 이력 지시계는 라카아제의 기질을 포함할 수 있다. The enzymatic time-temperature history indicator of the present invention may comprise a substrate of laccase.
상기 기질은 합성 색소 또는 천연 색소일 수 있으며, 이에 한정되지 않는다.The substrate may be a synthetic dye or a natural dye, but is not limited thereto.
상기 합성 색소에는 브로모티몰 블루(Bromothymol blue), 메틸 오렌지(Methyl orange), 리마졸 브릴리언트 블루 R(Remazol Brilliant Blue R), 드리마렌 블루(Drimaren Blue), 리액티브 블랙 5(Reactive Black 5), 애시드 블루 25(Acid Blue 25), 메틸 그린(Methyl green), 애시드 그린 27(Acid green 27), 애시드 바이올렛 7(Acid viloet 7), 오렌지 Ⅱ(Orange Ⅱ), 트로페올린 O(Tropaeolin O), 콩고 레드(Congo red), 폴리 R, 아주르 B 등의 안트라퀴논 (anthraquinone) 색소 계열, 아조 (azo) 색소계열, 인디고 (indigo) 색소 계열, 산업용 색소, 폴리메릭 (polymeric) 색소 및 헤테로사일릭 (heterocylic) 색소 등이 포함되며, 이에 한정되지 않는다.The synthetic pigments include bromothymol blue, methyl orange, Remazol Brilliant Blue R, Drimaren Blue, Reactive Black 5, Acid Blue 25, Methyl green, Acid green 27, Acid viloet 7, Orange II, Tropaeolin O, Anthraquinone pigments such as Congo red, Poly R, Azur B, azo pigments, indigo pigments, industrial pigments, polymeric pigments and heterocylics ( heterocylic) pigments and the like, but is not limited thereto.
상기 천연 색소에는 구아야콜 (guaiacol), 락 색소, 복분자 색소, 치자 청색소, 블루베리 색소, 베리류 색소, 파프리카 색소, 적양배추 색소, 비트레드 색소, 자색고구마 색소, 콘 색소, 잇꽃 색소 등의 카로티노이드계(carotenoids), 키논(quinodis), 플라보노이드계(flavonoids), 안토시아닌계(anthocyanines), 베타시아닌계(betacyanines) 등이 포함되며, 이에 한정되지 않는다. Examples of the natural pigments include guayacol, rock pigment, bokbunja pigment, gardenia blue pigment, blueberry pigment, berry pigment, paprika pigment, red cabbage pigment, beet red pigment, purple sweet potato pigment, corn pigment and safflower pigment. Carotenoids (carotenoids), quinodis (quinodis), flavonoids (flavonoids), anthocyanins (anthocyanines), beta cyanines (betacyanines) and the like, but are not limited thereto.
본 발명의 효소형 시간-온도 이력 지시계에는 TTI의 정보, 식품의 정보 또는 TTI가 식품에 부착되기 전까지의 식품의 유통 이력 정보를 입력하는 입력부가 더 포함될 수 있다.The enzymatic time-temperature history indicator of the present invention may further include an input unit for inputting information of the TTI, information of the food, or distribution history information of the food until the TTI is attached to the food.
상기 TTI의 정보는 TTI의 제품 정보일 수 있다.The information of the TTI may be product information of the TTI.
상기 식품의 정보는 식품의 종류 또는 식품의 유통 방법일 수 있다.The food information may be a type of food or a distribution method of the food.
상기 식품의 유통 이력 정보는 시간 이력 또는 온도 이력일 수 있다.The distribution history information of the food may be a time history or a temperature history.
상기 식품은 어패류, 육류, 과일, 채소 및 낙농제품으로 이루어지는 군으로부터 선택될 수 있으며, 이에 한정되지 않는다. 또한 상기 식품은 상온 유통 식품, 냉장 유통 식품 및 냉동 유통 식품으로 이루어지는 군으로부터 선택될 수 있으며, 이에 한정되지 않는다.The food may be selected from the group consisting of seafood, meat, fruits, vegetables and dairy products, but is not limited thereto. In addition, the food may be selected from the group consisting of room temperature distribution food, refrigerated distribution food and frozen distribution food, but is not limited thereto.
본 발명의 라카아제를 이용한 효소형 시간-온도 이력 지시계는 하기와 같은 특성을 가지고 있다. The enzymatic time-temperature history indicator using the laccase of the present invention has the following characteristics.
첫째, 라카아제는 기질 특이성을 갖는 다른 효소들에 비해서 o- 또는 p-디페놀, 아미노페놀, 폴리아민, 리그린, 아릴 디아민, 페놀기 등의 화합물, 아조 색소 계열 등과 같은 다양한 기질과 반응할 수 있다. 따라서, 다양한 색소의 기질을 TTI에 적용할 수 있으며, 각 반응물에 따라 다양한 활성화 에너지를 갖는 TTI로 개발될 수 있다. First, laccases can react with various substrates, such as o- or p-diphenols, aminophenols, polyamines, liglines, aryl diamines, compounds such as phenol groups, azo dyes and the like compared to other enzymes with substrate specificities. have. Thus, substrates of various pigments can be applied to the TTI, and can be developed into TTIs having various activation energies depending on the respective reactants.
둘째, 기존 리파아제를 이용한 효소형 TTI는 pH 변화에 매우 민감하여 효소 반응 속도가 s자 모양(sigmoid)의 비직선형으로 변화하는데 반해, 본 발명의 라카아제를 이용한 효소형 TTI는 효소 반응 속도가 점진적으로 변화하기 때문에 단순한 반응 속도(kinetics)로 다룰 수 있다. Second, the enzyme type TTI using lipase is very sensitive to pH change, so the enzyme reaction rate is changed to non-linear of sigmoid, whereas the enzyme type TTI using laccase of the present invention has a gradual enzyme reaction rate. It can be handled by simple kinetics.
셋째, 기존의 효소형 TTI는 지시약으로 합성 색소들을 사용하는데 반해, 본 발명의 라카아제를 이용한 효소형 TTI는 주로 아조 계열, 안트라퀴논 계열, 인디고 계열 등의 합성 색소를 탈색시킬 수 있을 뿐만 아니라, 자연계에 존재하는 천연색소들의 색을 변화시킬 수 있기 때문에, 식품에 오염되어도 무해한 환경 친화적인 TTI로 이용될 수 있다. Third, conventional enzymatic TTIs use synthetic pigments as indicators, whereas enzymatic TTIs using the laccases of the present invention not only can decolorize synthetic pigments such as azo, anthraquinone, and indigo, Because it can change the color of natural pigments present in nature, it can be used as an environmentally friendly TTI that is harmless even if contaminated with food.
상기한 바와 같이, 본 발명의 라카아제를 이용한 효소형 시간-온도 이력지시계는 기질에 따라 다양한 활성화 에너지를 가지며, 단순한 반응 메커니즘을 갖는 친환경적인 TTI로써 식품의 유통, 분배 관리, 살균 공정 등 다양한 식품 분야에서 유용하게 이용될 수 있다. As described above, the enzymatic time-temperature hysteresis clock using the laccase of the present invention has various activation energies according to substrates, and is an environmentally friendly TTI having a simple reaction mechanism. It can be usefully used in the field.
또한 본 발명은 (a) 라카아제를 이용한 효소형 시간-온도 이력 지시계를 식품의 포장 표면에 부착시키는 단계; 및 (b) 상기 (a) 단계의 시간-온도 이력 지시계의 색의 변화로 식품의 변질 정도를 판별하는 단계;를 포함하는 식품의 신선도 감지 방법을 제공한다. In another aspect, the present invention comprises the steps of (a) attaching the enzymatic time-temperature history indicator using the laccase on the packaging surface of the food; And (b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다. 단, 하기 실시예 및 실험예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following Examples and Experimental Examples are only for illustrating the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
실시예 1. 라카아제의 정제Example 1 Purification of Lacasees
라카아제(EC 1.10.3.2, DeniLite Ⅱ, Novozyme Co., Bagsværd, Denmark) 70 g을 50 mM 소디움 아세테이트 완충액(pH 5.0) 100 mL에 녹인 후, 실온에서 1 시간가량 균일하게 혼합하였다. 상기 혼합액을 2,650 X g에서 20분 동안 원심분리한 후, 침전물은 버리고, 깨끗한 용액 부분을 모았다. 상기 용액을 0.45 μm 실린지 필터 (Whatman filter paper,, USA)로 두 번 여과하였다. 마지막으로, 효소 안정제인 소혈청알부민 (bovine serum albumin, BSA)이 농도 0.1 mg/mL가 되도록 첨가한 뒤, 4 ℃에서 냉장 보관하였다. 70 g of laccase (EC 1.10.3.2, DeniLite II, Novozyme Co., Bagsværd, Denmark) was dissolved in 100 mL of 50 mM sodium acetate buffer (pH 5.0), followed by uniform mixing at room temperature for 1 hour. After centrifugation of the mixture at 2,650 X g for 20 minutes, the precipitate was discarded and the clear solution portion collected. The solution was filtered twice with 0.45 μm syringe filter (Whatman filter paper, USA). Finally, bovine serum albumin (BSA), an enzyme stabilizer, was added to a concentration of 0.1 mg / mL, and then refrigerated at 4 ° C.
실험예 1. 라카아제 활성 측정Experimental Example 1. Measurement of Lacasease Activity
상기 실시예 1에서 제조한 라카아제가 정상적인 효소 활성을 가지는지 확인하기 위하여, 라카아제의 기질 중 하나인 구아야콜(guaiacol)을 이용하여 하기와 같은 실험을 수행하였다. 먼저, 50 mM 소디움 아세테이트 완충액 (pH 5.0) 3 mL에 구아야콜을 최종 농도가 10 mM이 되게 혼합한 후, 미리 30 ℃에서 방치시켰다. 상기 기질 용액에 실시예 1에서 제조한 라카아제 용액 20 μL를 분주하여 반응을 개시하였다. 효소의 활성단위는 1 분 당 1 μmol의 구아야콜을 산화시키는 효소의 양을 1 유닛으로 정하였다. 이 후 시간에 따라 (0~5시간), 30 ℃, 470 nm(ε470 = 6,740 M-1cm-1)에서 흡광도를 측정하였다. 이때, 흡광도계(OPTIZEN 3220V, Mecasys Co., Dajeon, Korea)에는 워터재킷을 장착하여 일정한 온도를 유지하도록 하였다.In order to confirm that the laccase prepared in Example 1 has normal enzymatic activity, the following experiment was performed using guayacol, which is one of the substrates of laccase. First, guayacol was mixed in 3 mL of 50 mM sodium acetate buffer (pH 5.0) to a final concentration of 10 mM, and then left at 30 ° C. in advance. The reaction was initiated by dispensing 20 μL of the laccase solution prepared in Example 1 into the substrate solution. The active unit of the enzyme was set to 1 unit of the amount of enzyme to oxidize 1 mol mol guayacol per minute. After that time (0 ~ 5 hours), the absorbance was measured at 30 ℃, 470 nm (ε 470 = 6,740 M -1 cm -1 ). At this time, the absorbance (OPTIZEN 3220V, Mecasys Co., Dajeon, Korea) was equipped with a water jacket to maintain a constant temperature.
그 결과를 도 1에 나타내었다. The results are shown in FIG.
도 1에 나타낸 바와 같이, 라카아제는 구아야콜과 반응하여 시간에 따라 갈색으로 변화하는 것을 확인하였다. As shown in FIG. 1, it was confirmed that the laccase reacted with guayacol and changed to brown with time.
실시예 2. 라카아제를 이용한 효소형 TTI (라카아제-구아야콜 TTI) 프로토타입의 제조Example 2. Preparation of Enzymatic TTI (Lacase-Guayacol TTI) Prototypes with Lacasease
라카아제를 이용한 효소형 TTI (라카아제-구아야콜 TTI) 프로토타입을 하기와 같은 과정을 거쳐 제조하였다. 먼저, 6-웰 플레이트에 20 mM 구아야콜 (Sigmaldrch Co. Ltd.), 50 mM 소디움 아세테이트 완충액 (pH 5.0) 및 0.1 mg/mg 소혈청알부민의 혼합액을 6 mL씩 분주하고, 상기 플레이트를 항온 배양기 ((HST-103 PID type temperature controlled incubator, Korea, 각각 5, 10, 15, 25, 37 ℃로 유지됨)에 놓아 일정한 온도에 도달하도록 하였다. 이 후, 실시예 1에서 제조한 라카아제 용액을 각각 0.650 유닛 (TTI #1), 0.260 유닛 (TTI #2), 0.162 유닛 (TTI #3) 또는 0.104 유닛 (TTI #4)씩 포함되도록 희석한 후, 상기 6-웰 플레이트의 기질 용액에 각각 50 μl씩 분주하여 반응이 개시되도록 하였다. 항온 배양기 안에서는 용액의 증발을 막기 위해 필름을 덮어주었다. Enzyme-type TTI (Lacasease-Guayacol TTI) prototype using Lacase was prepared by the following procedure. First, dispense 6 mL aliquots of a mixture of 20 mM guayacol (Sigmaldrch Co. Ltd.), 50 mM sodium acetate buffer (pH 5.0) and 0.1 mg / mg bovine serum albumin, and incubate the plate. (HST-103 PID type temperature controlled incubator, Korea, respectively, maintained at 5, 10, 15, 25, 37 ℃) to reach a constant temperature. After this, the laccase solution prepared in Example 1 Dilute to contain 0.650 units (TTI # 1), 0.260 units (TTI # 2), 0.162 units (TTI # 3), or 0.104 units (TTI # 4), respectively, and then add 50 to the substrate solution of the 6-well plate. The reaction was initiated by aliquoting in μl The film was covered in the incubator to prevent evaporation of the solution.
실험예 2. 라카아제를 이용한 효소형 TTI (라카아제-Experimental Example 2. Enzyme-type TTI (Lacase) using Lacase 구아야콜 TTI) 프로토타입의 비색 분석 (colorimetry)Colorimetry of Guayacol TTI) Prototype
상기 실시예 2에서 제조한 라카아제를 이용한 효소형 TTI 프로토타입이 실제 TTI로 이용될 수 있는지 확인하기 위하여, 완전히 산화된 구아야콜을 최종 농도 0.047~0.281 mM의 농도로 희석한 후, 농도에 따라 일반적인 칼라 시스템인 CIE-LAB, CIE-LCH, x, y, z 값을 측정(CR-300, Minolta, Tokyo, Japan)하고, 색 값과 흡광도 (470 nm)값을 비교하였다. 이때, 색 값은 빛을 차단하기 위하여 나무 상자 안의 하얀색 바탕 위에서 측정하였다.In order to confirm that the enzymatic TTI prototype using the laccase prepared in Example 2 can be used as the actual TTI, the fully oxidized guayacol was diluted to a concentration of 0.047 to 0.281 mM, and then Therefore, the color system CIE-LAB, CIE-LCH, x, y, z values were measured (CR-300, Minolta, Tokyo, Japan), and color values and absorbance (470 nm) values were compared. At this time, the color value was measured on a white background in a wooden box to block light.
그 결과를 도 2에 나타내었다. The results are shown in FIG.
도 2에 나타낸 바와 같이, 각각의 칼라 시스템 값 및 흡광도와 다양한 농도의 산화된 구아야콜 용액 간의 상관계수 중, 흡광도값 (r=1)과 hue 값 (r= -0.998)이 가장 높은 상관관계를 보임을 확인하였다. As shown in FIG. 2, the absorbance value (r = 1) and the hue value (r = −0.998) have the highest correlation among the respective color system values and the absorbance values and the correlation coefficients between the oxidized guayacol solutions of various concentrations. Confirmed to show.
상기 결과를 바탕으로, Hue 값과 흡광도 간의 선형 회귀 분석식을 구하였으며 (R2 = 0.995), 이를 하기 수학식 1에 나타내었다. Based on the above results, a linear regression equation between Hue value and absorbance was obtained (R 2 = 0.995), which is shown in Equation 1 below.
수학식 1
Figure PCTKR2012007041-appb-M000001
 Equation 1
Figure PCTKR2012007041-appb-M000001
* Abs는 470 nm에서의 흡광도, h는 hue 값 (CIE-Lch)을 나타냄.Abs is absorbance at 470 nm, h represents hue value (CIE-Lch).
상기 수학식 1에서 Y 변수로 hue 값을 이용한 표준곡선을 도 3에 나타내었다. A standard curve using hue values as Y variables in Equation 1 is shown in FIG. 3.
도 3에 나타낸 바와 같이, 산화된 구아야콜의 농도가 증가할수록 hue 값이 감소함을 확인하였다. As shown in Figure 3, it was confirmed that the hue value decreases as the concentration of oxidized guayacol increases.
실험예 3. 라카아제를 이용한 효소형 TTI (라카아제-구아야콜 TTI) 프로토타입의 반응 속도 및 아레니우스 활성화 에너지 분석Experimental Example 3. Analysis of reaction rate and Areneus activation energy of enzymatic TTI (Lacasease-Guayacol TTI) prototype using Lacase
상기 실시예 2에서 제조한 라카아제를 이용한 효소형 TTI (라카아제-구아야콜 TTI) 프로토타입의 반응 속도 및 아레니우스 활성화 에너지를 구하기 위하여 하기와 같은 실험을 수행하였다.In order to determine the reaction rate and the Areneus activation energy of the enzyme-type TTI (Lacase-Guayacol TTI) prototype using the laccase prepared in Example 2, the following experiment was performed.
먼저, 라카아제의 유닛 량이 다른 네 가지 종류의 효소형 TTI의 색 반응 값의 반응 속도는 하기 수학식 2와 같다.First, the reaction rate of the color reaction values of the four kinds of enzymatic TTIs having different unit amounts of laccase is expressed by the following equation.
수학식 2
Figure PCTKR2012007041-appb-M000002
 Equation 2
Figure PCTKR2012007041-appb-M000002
* Y는 색 반응 값, k는 반응 속도 상수, t는 반응 시간을 나타냄. * Y is the color response value, k is the reaction rate constant, and t is the reaction time.
또한, 본 발명의 효소형 TTI의 활성화 에너지는 하기 수학식 3에 나타낸 바와 같이, 아레니우스 함수식 양 변에 자연로그를 취함으로써 계산되었다. In addition, the activation energy of the enzymatic TTI of the present invention was calculated by taking the natural log on both sides of the Arrhenius functional formula, as shown in Equation 3 below.
수학식 3
Figure PCTKR2012007041-appb-M000003
 Equation 3
Figure PCTKR2012007041-appb-M000003
* Ea는 활성화에너지, R은 기체상수, T는 절대온도, A는 pre-exponential factor를 나타냄. * Ea is the activation energy, R is the gas constant, T is the absolute temperature, and A is the pre-exponential factor.
이때, lnk 와 1/T 간의 선형 회귀 분석을 통하여 기울기로 Ea/R를 구하고, y 절편으로 lnA를 구하였다. At this time, Ea / R was obtained by the slope through linear regression analysis between ln k and 1 / T, and lnA was obtained by the y-intercept.
라카아제의 유닛 량이 다른 네 가지 종류의 효소형 TTI의 색 반응 값을 수학식 2에 적용하여 반응속도 상수(k) 및 결정계수(R2)를 구하고, 상기 수학식 3에 적용하여 활성화에너지 (Ea) 값을 구하였다. 그 결과를 표 1에 나타내었다. The reaction rate constant (k) and the coefficient of crystallization (R 2 ) are obtained by applying the color reaction values of four kinds of enzymatic TTIs having different unit amounts of laccase to Equation 2, and applying the activation energy ( Ea) was obtained. The results are shown in Table 1.
표 1
TTI 온도 (℃) k(△hue/hour) R2* Ea(kJ/mole) R2
#1(0.650 Unit) 5 16.418 0.9939 45.44 0.9674
10 11.136 0.9859
15 5.2859 0.9914
25 2.9482 0.9738
37 2.4741 0.9787
# 2(0.260 Unit) 5 12.061 0.9865 46.92 0.9704
10 6.946 0.9816
15 4.1352 0.9485
25 2.0679 0.9513
37 1.5397 0.9975
# 3(0.162 Unit) 5 7.0412 0.9564 44.36 0.9554
10 2.987 0.944
15 2.4777 0.9093
25 1.2265 0.9765
37 0.897 0.9687
# 4(0.104 Unit) 5 5.5005 0.9768 43.90 0.9897
10 2.6804 0.9574
15 1.7336 0.9717
25 1.0438 0.9801
37 0.8591 0.9765
Table 1
TTI Temperature (℃) k (△ hue / hour) R 2 * Ea (kJ / mole) R 2
# 1 (0.650 Unit) 5 16.418 0.9939 45.44 0.9674
10 11.136 0.9859
15 5.2859 0.9914
25 2.9482 0.9738
37 2.4741 0.9787
# 2 (0.260 Unit) 5 12.061 0.9865 46.92 0.9704
10 6.946 0.9816
15 4.1352 0.9485
25 2.0679 0.9513
37 1.5397 0.9975
# 3 (0.162 Unit) 5 7.0412 0.9564 44.36 0.9554
10 2.987 0.944
15 2.4777 0.9093
25 1.2265 0.9765
37 0.897 0.9687
# 4 (0.104 Unit) 5 5.5005 0.9768 43.90 0.9897
10 2.6804 0.9574
15 1.7336 0.9717
25 1.0438 0.9801
37 0.8591 0.9765
(* 결정계수)(* Coefficient of determination)
표 1에 나타낸 바와 같이, 본 발명의 라카아제를 이용한 효소형 TTI의 반응속도 결정계수 값은 0.9093보다 컸으며, 이를 통해 hue 값(Y)이 시간에 따라 직선형을 보임을 확인하였다. 이는 0차 반응을 나타내는 것으로, 본 발명의 라카아제를 이용한 효소형 TTI가 식품의 시간-온도 이력을 정확하게 반영할 수 있음을 알 수 있다. As shown in Table 1, the reaction rate determination coefficient value of the enzyme-type TTI using the laccase of the present invention was greater than 0.9093, and it was confirmed that the hue value (Y) was linear with time. This indicates a zero-order reaction, and it can be seen that the enzymatic TTI using the laccase of the present invention can accurately reflect the time-temperature history of the food.
또한, 라카아제의 유닛 량이 다른 네 가지 종류의 TTI의 Ea 값은 43.9~45.4 kJ/mol을 나타냄을 확인하였으며, 예측 가능한 식품 품질 저하의 Ea 범위는 23.9-65.4 kJ/mol임을 확인하였다. 따라서 기존에 알려진 전형적인 식품 품질 저하의 Ea 값과 비교해볼 때, 본 발명의 라카아제를 이용한 효소형 TTI는 효소적 변화 (41.84-62.76 kJ/mol), 가수분해 (62.76 kJ/mol), 지방산화 (41.84-104.6 kJ/mol) 를 다룰 수 있음을 확인하였다. 특히, 본 발명의 라카아제를 이용한 효소형 TTI는 현재 상업적으로 이용되고 있는 효소형 TTI (Vitsab AB, Malmo, Sweden) C2-15d (Ea = 50.2 kJ/mole) 및 확산형 TTI (Ea = 33-50 kJ/mole) (Pocas et al., 2008)와도 견줄만한 활성화 에너지 값을 나타내었다. In addition, it was confirmed that the Ea values of the four kinds of TTI having different unit amounts of laccases represented 43.9-45.4 kJ / mol, and the Ea range of predictable food quality degradation was 23.9-65.4 kJ / mol. Therefore, in comparison with the Ea values of the conventionally known food deterioration, the enzymatic TTI using the laccase of the present invention is characterized by enzymatic change (41.84-62.76 kJ / mol), hydrolysis (62.76 kJ / mol), fatty acidization It was confirmed that (41.84-104.6 kJ / mol) can be handled. In particular, enzymatic TTIs using the laccases of the present invention are enzymatic TTIs (Vitsab AB, Malmo, Sweden) C2-15d (Ea = 50.2 kJ / mole) and diffusion TTIs (Ea = 33-) which are currently commercially available. 50 kJ / mole) (Pocas et al., 2008) showed an activation energy value comparable.
대표적으로, 라카아제가 0.260 유닛 (TTI #2)인 TTI의 시간에 따른 Hue 값 및 1/T에 대한 lnK 값을 각각 도 4 및 도 5에 나타내었다.Representatively, Hue values over time of TTI with 0.260 units of laccase (TTI # 2) and lnK values for 1 / T are shown in FIGS. 4 and 5, respectively.
실험예 4. 시간-온도에 따른 반응 모식화Experimental Example 4. Schematic representation of time-temperature
기존에 알려진 Yan 등의 (2008) 시간-온도에 따른 색 반응 모식화에 따라 본 발명의 본 발명의 라카아제를 이용한 효소형 TTI의 시간-온도에 따른 색 반응을 시뮬레이션하였다. 모식화 식을 수학식 4에 나타내었다.(2008) Time-Temperature Color Reaction Scheme of Yan et al. (2008) simulates the time-temperature color reaction of enzymatic TTI using the laccase of the present invention. The schematic expression is shown in equation (4).
수학식 4
Figure PCTKR2012007041-appb-M000004
 Equation 4
Figure PCTKR2012007041-appb-M000004
* Y는 색 반응, T는 절대온도, t는 시간, a-d는 상수 파라미터 (constant parameters)를 나타냄.* Y stands for color response, T stands for absolute temperature, t stands for time, and a-d stands for constant parameters.
상기 수학식 4를 최적화 하기 위해 SAS 9.1 (SAS Institute Inc., USA) 프로그램을 통해 비선형 회귀분석을 실시하였으며, 시간-온도에 따른 색 값을 삼차원의 표면 플롯으로 나타내기 위하여, Minitab ver. 14.0 (Minitab Inc., USA)을 사용하였다. In order to optimize the equation 4, nonlinear regression analysis was performed through the SAS 9.1 (SAS Institute Inc., USA) program, and in order to represent color values according to time-temperature as three-dimensional surface plots, Minitab ver. 14.0 (Minitab Inc., USA) was used.
라카아제의 유닛 량이 다른 네 가지 종류의 효소형 TTI에서 비선형 회기분석을 통해 도출한 a, b, c, d의 파라미터를 하기 표 2에 나타내었다. The parameters of a, b, c, d derived from nonlinear regression analysis in four kinds of enzymatic TTIs with different unit amounts of laccase are shown in Table 2 below.
표 2
TTI 파라미터
a b c d R2* p**
#1(0.650 Unit) -0.1691 74.4042 -0.1481 1.7844 0.995 <0.0001
#2(0.260 Unit) -0.0310 71.7793 -0.1704 0.7975 0.996 <0.0001
#3(0.162 Unit) -0.0168 72.7277 -0.1359 0.7015 0.996 <0.0001
#4(0.104 Unit) -0.0161 73.3902 -0.1313 0.9274 0.996 <0.0001
TABLE 2
TTI parameter
a b c d R 2 * p **
# 1 (0.650 Unit) -0.1691 74.4042 -0.1481 1.7844 0.995 <0.0001
# 2 (0.260 Unit) -0.0310 71.7793 -0.1704 0.7975 0.996 <0.0001
# 3 (0.162 Unit) -0.0168 72.7277 -0.1359 0.7015 0.996 <0.0001
# 4 (0.104 Unit) -0.0161 73.3902 -0.1313 0.9274 0.996 <0.0001
(*결정계수, ** 비선형 회귀분석의 유의성을 위한 에러 확률)(* Decision coefficient, ** error probability for significance of nonlinear regression)
대표적으로 라카아제가 0.260 유닛 (TTI #2)인 효소형 TTI의 시간-온도에 따른 hue 값 반응을 도 6에 나타내었으며, 같은 방식으로 나머지 종류의 효소형 TTI의 파라미터 값을 구하였다. Representative time-temperature hue value response of the enzyme-type TTI with 0.260 units of laccase (TTI # 2) is shown in FIG. 6, and the parameter values of the other types of enzyme-type TTIs were obtained in the same manner.
또한, 대표적으로 라카아제가 0.260 유닛 (TTI #2)인 효소형 TTI가 일정한 온도에서 특정 hue 값에 도달하기 위해 걸리는 시간에 대한 정보를 제공하기 위하여, 시간-온도에 따른 색 변화를 도 7에 나타내었다. 상기 결과는 식품의 취급 및 수송 시에 시간에 대한 이력을 평가하기 위한 방법으로 이용될 수 있다. In addition, in order to provide information on the time it takes for the enzymatic TTI, which is typically 0.260 units of laccase (TTI # 2) to reach a specific hue value at a constant temperature, the color change with time-temperature is shown in FIG. 7. Indicated. The results can be used as a method for evaluating the history of time in the handling and transportation of food.
라카아제의 유닛 량이 다른 네 가지 종류의 효소형 TTI가 hue 값 50 (더 이상 색 변화가 일어나지 않는 가장 낮은 값)에 도달하기 위해 필요한 시간을 상기 수학식 4와 표 2에 나타낸 파라미터를 이용하여 구하였다. 그 결과를 하기 표 3에 나타내었다.The time required for four kinds of enzymatic TTIs with different unit amounts of laccase to reach the hue value 50 (the lowest value at which no color change occurs) can be determined using the parameters shown in Equation 4 and Table 2 above. It was. The results are shown in Table 3 below.
표 3
TTI hue 값 50에 도달하기 위해 필요한 시간
5 ℃ 10 ℃ 15 ℃ 25 ℃ 37 ℃
# 1(0.650 Unit) 28.9 14.4 9.6 5.8 3.9
# 2(0.260 Unit) 141.5 70.3 46.8 28.1 19
# 3(0.162 Unit) 273 135.3 90.2 54.1 36.6
# 4(0.104 Unit) 291.4 145.3 96.9 58.1 29.3
TABLE 3
TTI time required to reach hue value 50
5 ℃ 10 ℃ 15 ℃ 25 ℃ 37 ℃
# 1 (0.650 Unit) 28.9 14.4 9.6 5.8 3.9
# 2 (0.260 Unit) 141.5 70.3 46.8 28.1 19
# 3 (0.162 Unit) 273 135.3 90.2 54.1 36.6
# 4 (0.104 Unit) 291.4 145.3 96.9 58.1 29.3
표 3에 나타낸 바와 같이, 라카아제의 유닛 량이 다른 네 가지 종류의 효소형 TTI에서 효소 활성이 감소할수록 반응 속도가 감소하고, hue 값 50에 도달하는 시간이 증가함을 확인하였다. 상기 결과를 통하여, 효소형 TTI에 포함되는 라카아제의 양을 조절함으로써 색 변화 시간을 조절할 수 있고, 이에 서로 다른 유통기간을 갖는 다양한 식품 품질 평가에 적용될 수 있음을 확인하였다. As shown in Table 3, it was confirmed that as the enzyme activity decreases in four kinds of enzymatic TTIs having different unit amounts of laccase, the reaction rate decreases and the time to reach hue value 50 increases. Through the above results, it was confirmed that the color change time can be adjusted by adjusting the amount of laccase contained in the enzymatic TTI, and thus it can be applied to various food quality evaluations having different shelf life.
실시예 3. 라카아제를 이용한 효소형 TTI에 이용하기 위한 색소 준비Example 3 Pigment Preparation for Use in Enzymatic TTI with Lacasease
라카아제를 효소형 TTI에 사용한다면, 안전하고 받아들일 수 있는 기질을 사용하는 것이 중요하다. 특히, 합성 색소와 천연 색소는 인체에 무해하고 안전하므로 TTI의 기질로 사용될 경우 취급이 용이한 장점이 있다. 이에, 라카아제를 이용한 효소형 TTI에 합성 색소 또는 천연 색소 기질을 이용할 수 있는지 확인하기 위하여, 다양한 색소를 준비하였다. If laccase is used for enzymatic TTI, it is important to use a safe and acceptable substrate. In particular, synthetic pigments and natural pigments are harmless and safe to the human body, so when used as a substrate of the TTI has the advantage of easy handling. Thus, in order to confirm whether a synthetic dye or a natural pigment substrate can be used for the enzymatic TTI using laccase, various pigments were prepared.
합성 색소인 브로모티올 블루 (D1)와 메틸 오렌지 (D2)는 삼전 케미컬(Seoul, Korea)로부터 구매하였다. 천연 색소인 락 색소(Lac dye (P1))는 Coccus laccae (Laccifer lacca Kerr) 유충이 분비하는 수지상 물질로 가루나라(Seoul, Korea)에서 구매하였으며, 복분자 색소(P2)는 복분자(Rubus coreanus fruit)로부터 분리한 것으로 복분자 20% 순도를 가지고 있으며, 브레드팜(Cheonan, Korea)에서 구매하였고, 치자 청색소(Gardenia blue pigment (P3))는 내팜(Gyeonggi, Korea)에서 구매하였다. 효소 안정제는 소혈청알부민(BSA, first grade, Sigma, St. Louis, USA))을 사용하였다.Synthetic pigments bromothiol blue (D1) and methyl orange (D2) were purchased from Samjeon Chemical (Seoul, Korea). Natural pigment of Rock dye (Lac dye (P1)) is Coccus laccae (Laccifer lacca Kerr) a dendritic material to larvae secretion was purchased in powdered Countries (Seoul, Korea), bokbunja pigment (P2) is bokbunja (Rubus coreanus fruit) It was isolated from 20% purity of Bokbunja, purchased from Bread Farm (Cheonan, Korea), and Gardenia blue pigment (P3) was purchased from Napalm (Gyeonggi, Korea). Enzyme stabilizer was used bovine serum albumin (BSA, first grade, Sigma, St. Louis, USA).
실시예 4. 라카아제를 이용한 효소형 TTI (라카아제-색소 TTI) 프로토타입의 제조Example 4 Preparation of Enzymatic TTI (Lacase-Color TTI) Prototype Using Lacase
상기 실시예 3에서 제조한 합성 색소 (D1, D2) 또는 천연 색소 (P1, P2, P3) 용액을 이용하여 라카아제를 이용한 효소형 TTI (라카아제-색소 TTI) 프로토타입을 제조하기 위해, 합성 색소 또는 천연 색소의 기질 용액을 하기와 같이 준비하였다. In order to prepare an enzyme-type TTI (lacase-pigment TTI) prototype using laccase using the synthetic dye (D1, D2) or natural pigment (P1, P2, P3) solution prepared in Example 3, Substrate solutions of pigments or natural pigments were prepared as follows.
D1과 D2는 각각 4 mg을 1 mL 에탄올에 용해시킨 뒤, 0.5 mM 아세테이트 완충액 (pH 5.0)에 100배 희석하여 준비하였다.D1 and D2 were each prepared by dissolving 4 mg in 1 mL ethanol and then diluting 100 times in 0.5 mM acetate buffer (pH 5.0).
P1과 P3은 각각 2 mg을 1 mL의 50 mM 아세테이트 완충액 (pH 5.0)에 용해시켜 준비하였다. P1 and P3 were prepared by dissolving 2 mg in 1 mL of 50 mM acetate buffer (pH 5.0), respectively.
P2는 20%의 복분자 함량을 가지므로, P1, P3보다 1.25 배 가량을 더 넣어주었으며, 이에 P2 2.5 mg을 1 mL의 50 mM 아세테이트 완충액 (pH 5.0)에 용해시켜 준비하였다.Since P2 has a bokbunja content of 20%, 1.25 times more than P1 and P3 were added. Thus, 2.5 mg of P2 was dissolved in 1 mL of 50 mM acetate buffer (pH 5.0).
BSA 용액은 최종농도 0.1mg/mL가 되도록 첨가하였다. BSA solution was added to a final concentration of 0.1 mg / mL.
pH 5.0에서 각 색소의 색을 하기 표 4에 나타내었으며, 그 구조식을 화학식 1 내지 5에 나타내었다. The color of each pigment at pH 5.0 is shown in Table 4 below, and the structural formulas are shown in Chemical Formulas 1 to 5.
표 4
종류 색소의 색깔
합성색소브로모티올 블루 (D1)메틸 오렌지 (D2) 노랑오렌지
천연색소락 색소(Lac dye (P1))복분자 색소(P2) 청색소(Gardenia blue pigment (P3)) 빨강보라 핑크파랑
Table 4
Kinds Color of pigment
Synthetic Pigment Bromothiol Blue (D1) Methyl Orange (D2) Yellow and orange
Natural pigments lock dye (Lac dye (P1)) bokbunja pigment (P2) blue cattle (Gardenia blue pigment (P3)) Red Purple Pink Blue
화학식 1
Figure PCTKR2012007041-appb-C000001
 Formula 1
Figure PCTKR2012007041-appb-C000001
화학식 2
Figure PCTKR2012007041-appb-C000002
 Formula 2
Figure PCTKR2012007041-appb-C000002
화학식 3
Figure PCTKR2012007041-appb-C000003
 Formula 3
Figure PCTKR2012007041-appb-C000003
화학식 4
Figure PCTKR2012007041-appb-C000004
 Formula 4
Figure PCTKR2012007041-appb-C000004
화학식 5
Figure PCTKR2012007041-appb-C000005
 Formula 5
Figure PCTKR2012007041-appb-C000005
상기에서 제조한 각각의 합성 색소 또는 천연 색소의 기질 용액을 6 mL씩 6-웰 플레이트에 분주하였다. 상기 용액에 1 유닛의 라카아제 150 μL을 분주하여 TTI 반응을 개시하였다. 25 ℃에서 1 시간, 8 시간, 54 시간이 지난 후 색 변화를 도 8에 나타내었다. Substrate solutions of each synthetic or natural pigment prepared above were dispensed into 6-well plates by 6 mL. The TTI reaction was initiated by dispensing 150 μL of 1 unit of laccase into the solution. The color change after 1 hour, 8 hours, and 54 hours at 25 ° C. is shown in FIG. 8.
도 8에 나타낸 바와 같이, 54 시간이 지난 후에 색소의 색은 점차 연해지고, 사라지는 경향을 보였다. As shown in FIG. 8, after 54 hours, the color of the pigment gradually became softer and disappeared.
이러한 색의 변화를 CIE-Lab으로 나타내었으며, 그 결과를 표 5에 나타내었다. This color change is represented by CIE-Lab, and the results are shown in Table 5.
표 5
색소의 종류 효소 처리 전 효소 처리 54 시간 후 색 변화
L a b L a b DL Da Db DE
D1 68 -6 44 70 -4 8 2 2 -36 36.1
D2 64 5 58 65 -5 27 1 -10 -31 32.6
P1 44 38 41 62 2 21 18 -36 -20 44.9
P2 55 11 0 62 -2 16 7 -13 16 21.8
P3 29 5 -23 41 7 -17 12 2 6 13.6
Table 5
Type of pigment Before enzyme treatment After 54 hours of enzyme treatment Color change
L a b L a b DL Da Db DE
D1 68 -6 44 70 -4 8 2 2 -36 36.1
D2 64 5 58 65 -5 27 One -10 -31 32.6
P1 44 38 41 62 2 21 18 -36 -20 44.9
P2 55 11 0 62 -2 16 7 -13 16 21.8
P3 29 5 -23 41 7 -17 12 2 6 13.6
표 5에 나타낸 바와 같이, P1 색소의 △E가 가장 큰 변화를 나타내었으며, △L이 가장 높아 TTI의 밝기가 가장 많이 변화되었다. 또한, △a 와 △b는 D1 색소와 P1 색소가 가장 뚜렷한 변화를 보였다. As shown in Table 5, ΔE of the P1 pigment showed the largest change, and ΔL was the highest, so that the brightness of the TTI changed the most. In addition, DELTA a and DELTA b showed the most distinct change in the D1 dye and the P1 dye.
이러한 색 변화의 차이는 단지 CIE-Lab값의 변화로 설명되어, 특정 기질을 선택하는 데에 불충분한 면이 존재한다. 특히, 주어진 색과 비슷한 색이 상호 작용할 때, 사람의 눈은 색의 차이를 더 크게 인지하는 경향이 있기 때문에, TTI를 적용한 제품에 적절한 배경 색의 선택을 고려할 필요가 있다. These differences in color change are explained only by changes in the CIE-Lab values, and there are insufficient aspects to select a specific substrate. In particular, when colors similar to a given color interact with each other, the human eye tends to perceive the color difference more, and therefore, it is necessary to consider the selection of a suitable background color for the TTI applied product.
실험예 5. 라카아제를 이용한 효소형 TTI (라카아제-색소 TTI) 프로토타입의 반응 속도 및 아레니우스 활성화 에너지 분석Experimental Example 5. Analysis of reaction rate and Areneus activation energy of enzymatic TTI (lacase-pigment TTI) prototype using laccase
상기 실시예 4에서 제조한 라카아제를 이용한 효소형 TTI (라카아제-색소 TTI) 프로토타입과 동일한 방법으로, 각각의 색소 기질 용액에 1 유닛의 라카아제 150 μL을 분주하여 TTI 반응을 개시하였다. 각각의 플레이트를 10 ℃, 15 ℃, 25 ℃ 또는 37 ℃ 항온 배양기(HST-103, Korea)에서 배양하였으며, 용액의 증발을 막기 위해 필름을 덮어주었다.In the same manner as the enzymatic TTI (lacase-pigment TTI) prototype using the laccase prepared in Example 4, 150 μL of 1 unit of laccase was dispensed into each pigment substrate solution to initiate a TTI reaction. Each plate was incubated in a 10 ° C., 15 ° C., 25 ° C. or 37 ° C. incubator (HST-103, Korea) and covered with a film to prevent evaporation of the solution.
주어진 시간에 따라 CIE-Lab의 색 값은 스캐너(HP scanjet 2300c, HP Co, California, USA)로 스캔한 뒤 포토샵 프로그램(7.0, Photoshop Co, San jose, USA)을 이용한 이미지 분석을 통해 구하였고, L, a, b의 색 값을 이용한 총 색 변화(△E)는 하기 수학식 5와 같다. According to the given time, the color value of CIE-Lab was obtained by scanning with a scanner (HP scanjet 2300c, HP Co, California, USA) and analyzing the image using a Photoshop program (7.0, Photoshop Co, San jose, USA). The total color change ΔE using color values L, a, and b is expressed by Equation 5 below.
수학식 5
Figure PCTKR2012007041-appb-M000005
 Equation 5
Figure PCTKR2012007041-appb-M000005
* △L, △a, △b 은 각각 밝기, 빨강-초록, 노랑을 나타냄. * ΔL, Δa, and Δb represent brightness, red-green, and yellow, respectively.
TTI의 색 반응 함수는 0차 반응으로, 하기 수학식 6과 같다.The color response function of the TTI is a zero-order reaction, which is represented by Equation 6 below.
수학식 6
Figure PCTKR2012007041-appb-M000006
 Equation 6
Figure PCTKR2012007041-appb-M000006
* F는 △E, k는 반응속도상수, t는 반응 시간을 나타냄. * F is ΔE, k is reaction rate constant, and t is reaction time.
대표적으로, P1을 기질로 이용한 라카아제를 이용한 효소형 TTI에서 효소 반응 동안 색 값의 변화 양상을 확인하였다. 그 결과를 도 9에 나타내었다. Representatively, in the enzymatic type TTI using laccase using P1 as a substrate, the change of color value was confirmed during the enzymatic reaction. The results are shown in FIG.
도 9에 나타낸 바와 같이, 색 반응 변수 중 △E, L, a, b 모두 시간에 따라 그 변화 값이 선형을 이루는 것을 확인하였다. As shown in FIG. 9, it was confirmed that ΔE, L, a, and b among the color response variables were linearly changed in time.
그 중 △E 값이 L, a, b에 대한 정보를 모두 포함하고 색 반응 변수로 이미 사용되고 있는 값이므로 수학식 6의 색 반응 변수 (F)로 △E를 선정하였다. 대표적으로, P1을 기질로 이용한 라카아제를 이용한 효소형 TTI에서, 다양한 온도에서의 F(X) 값을 도 10에 나타내었다. 나머지 종류의 효소형 TTI 역시 유사한 결과를 나타낸다. Among them, ΔE was selected as the color response variable (F) of Equation 6 since the value ΔE includes all the information about L, a, and b and is already used as the color response variable. Representatively, in the enzymatic TTI using laccase using P1 as a substrate, F (X) values at various temperatures are shown in FIG. 10. The rest of the enzymatic TTIs show similar results.
색소의 종류가 다른 다섯 가지 종류의 효소형 TTI의 색 반응 값을 이용하여 반응속도 상수(k), 결정계수(R2) 활성화에너지 (Ea) 값을 구하였다. 그 결과를 표 6에 나타내었다. The reaction rate constant (k) and crystal coefficient (R 2 ) activation energy (Ea) values were determined using the color reaction values of five different enzyme-type TTIs. The results are shown in Table 6.
표 6
TTI 온도 (℃) k (1/min) R2 * Ea (kJ/mol) R2 **
D1 10 0.0705 0.9933 57.4 0.9562
15 0.1566 0.9899
25 0.2235 0.9869
37 0.7089 0.9784
D2 10 0.0183 0.9469 66.1 0.9454
15 0.0544 0.9804
25 0.0851 0.9648
37 0.2679 0.9672
P1 10 0.1108 0.9892 42.6 0.9733
15 0.1566 0.9899
25 0.227 0.9858
37 0.57 0.9499
P2 10 0.0409 0.9728 59.2 0.9870
15 0.0826 0.9862
25 0.1594 0.9744
37 0.4019 0.9616
P3 10 0.0207 0.9708 53.2 0.9991
15 0.0325 0.9813
25 0.0635 0.9659
37 0.1526 0.9400
Table 6
TTI Temperature (℃) k (1 / min) R 2 * Ea (kJ / mol) R 2 **
D1 10 0.0705 0.9933 57.4 0.9562
15 0.1566 0.9899
25 0.2235 0.9869
37 0.7089 0.9784
D2 10 0.0183 0.9469 66.1 0.9454
15 0.0544 0.9804
25 0.0851 0.9648
37 0.2679 0.9672
P1 10 0.1108 0.9892 42.6 0.9733
15 0.1566 0.9899
25 0.227 0.9858
37 0.57 0.9499
P2 10 0.0409 0.9728 59.2 0.9870
15 0.0826 0.9862
25 0.1594 0.9744
37 0.4019 0.9616
P3 10 0.0207 0.9708 53.2 0.9991
15 0.0325 0.9813
25 0.0635 0.9659
37 0.1526 0.9400
(* k의 결정계수, ** Ea의 결정계수)(* k coefficient, ** Ea coefficient)
표 6에 나타낸 바와 같이, 본 발명의 라카아제를 이용한 효소형 TTI의 반응속도 결정계수 값은 0.9400보다 컸으며, 이는 시간에 따른 색 반응 함수(F)가 상당히 선형을 나타냄을 의미한다.As shown in Table 6, the reaction rate determination coefficient value of the enzymatic TTI using the laccase of the present invention was greater than 0.9400, which means that the color reaction function (F) with time was considerably linear.
또한, 본 발명의 라카아제를 이용한 효소형 TTI 활성화 에너지(Ea) 값은 42.6-66.1 kJ/mol의 범위를 갖는 것으로 확인되었다.In addition, the enzymatic TTI activation energy (Ea) value using the laccase of the present invention was found to have a range of 42.6-66.1 kJ / mol.
전형적인 식품 품질 저하의 활성화 에너지를 표 7에 나타내었다. The activation energy of typical food deterioration is shown in Table 7.
표 7
식품 반응의 종류 Ea (kJ/mol) 이용가능한 본 발명의 라카아제를 이용한 TTI의 종류(Ea)
확산 제어 (Diffusion controlled) 0 - 63 D1, D2, P2, P3
가수분해 (Hydrolysis) 63
효소 반응 (Enzymic) 42 - 63 D1, D2, P1, P2, P3
지질 산화 (Lipid oxidation) 42 - 105
영양 손실 (Nutrient loss) 84 - 126 D2
미생물 성장 (Microbial growth) 84 - 251
TABLE 7
Type of food reaction Ea (kJ / mol) Available Types of TTIs Using the Lacasees of the Invention (Ea)
Diffusion controlled 0-63 D1, D2, P2, P3
Hydrolysis 63
Enzymatic reaction 42-63 D1, D2, P1, P2, P3
Lipid oxidation 42-105
Nutrient loss 84-126 D2
Microbial growth 84-251
표 7에 나타낸 바와 같이, 본 발명의 라카아제를 이용한 효소형 TTI는 다양한 색소를 기질로 이용할 수 있어, 식품의 품질 저하 반응과 비슷한 활성화 에너지를 갖는 기질을 선택함으로써 다양한 식품 품질 저하 반응에 적용하여 사용할 수 있다. As shown in Table 7, the enzyme-type TTI using the laccase of the present invention can use a variety of pigments as a substrate, by applying to a variety of food deterioration reaction by selecting a substrate having an activation energy similar to the food deterioration reaction Can be used.
보다 구체적으로는, D2를 이용한 라카아제 효소형 TTI의 활성화 에너지(Ea =66.1 kJ/mol)는 가수분해의 활성화 에너지(Ea = 63 kJ/mol)와 비슷하므로, 이는 가수분해로 인한 식품의 품질 저하를 예측하는 데에 사용될 수 있다. 또한, P2를 이용한 라카아제 효소형 TTI의 활성화 에너지 (Ea = 59.2 kJ/mol)는 효소적 분해, 지방 산화에 의한 식품 품질 저하에 사용될 수 있다. More specifically, the activation energy (Ea = 66.1 kJ / mol) of the laccase enzyme type TTI using D2 is similar to the activation energy of hydrolysis (Ea = 63 kJ / mol), which means that the quality of the food due to hydrolysis is high. Can be used to predict degradation. In addition, the activation energy (Ea = 59.2 kJ / mol) of the laccase enzyme type TTI using P2 can be used for degrading food quality by enzymatic degradation and fat oxidation.
본 발명의 라카아제를 이용한 효소형 시간-온도 이력지시계는 기질에 따라 다양한 활성화 에너지를 가지며, 단순한 반응 메커니즘을 갖는 친환경적인 TTI로써 식품의 유통, 분배 관리, 살균 공정 등 다양한 식품 분야에서 유용하게 이용될 수 있다. Enzymatic time-temperature hysteresis clock using the laccase of the present invention has various activation energies according to substrates, and is an environmentally friendly TTI having a simple reaction mechanism, which is useful in various food fields such as food distribution, distribution management, and sterilization process. Can be.

Claims (8)

  1. 라카아제 (laccase)를 이용한 효소형 시간-온도 이력지시계(time-temperature integrator, TTI).Enzymatic time-temperature integrator (TTI) using laccase.
  2. 제 1항에 있어서, 상기 시간-온도 이력지시계는, 기질로 합성 색소 또는 천연 색소를 이용한 것을 특징으로 하는, 효소형 시간-온도 이력지시계.The enzymatic time-temperature hysteresis clock according to claim 1, wherein the time-temperature hysteresis clock uses a synthetic dye or a natural dye as a substrate.
  3. 제 2항에 있어서, 상기 합성 색소는 브로모티몰 블루(Bromothymol blue), 메틸 오렌지(Methyl orange), 리마졸 브릴리언트 블루 R(Remazol Brilliant Blue R), 드리마렌 블루(Drimaren Blue), 리액티브 블랙 5(Reactive Black 5), 애시드 블루 25(Acid Blue 25), 메틸 그린(Methyl green), 애시드 그린 27(Acid green 27), 애시드 바이올렛 7(Acid viloet 7), 오렌지 Ⅱ(Orange Ⅱ), 트로페올린 O(Tropaeolin O), 콩고 레드(Congo red), 폴리 R 및 아주르 B로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는, 효소형 시간-온도 이력지시계.The method of claim 2, wherein the synthetic pigment is bromothymol blue, methyl orange, Remazol Brilliant Blue R, Drimaren Blue, Reactive Black 5 (Reactive Black 5), Acid Blue 25, Methyl green, Acid green 27, Acid viloet 7, Orange II, Tropeolin Enzymatic time-temperature history clock, characterized in that at least one selected from the group consisting of O (Tropaeolin O), Congo red (Congo red), poly R and Azur B.
  4. 제 2항에 있어서, 상기 천연 색소는 구아야콜 (guaiacol), 락 색소, 복분자 색소, 치자 청색소, 블루베리 색소, 베리류 색소, 파프리카 색소, 적양배추 색소, 비트레드 색소, 자색고구마 색소, 콘 색소 및 잇꽃 색소로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는, 효소형 시간-온도 이력지시계.According to claim 2, wherein the natural pigment is guayacol (guaiacol), lac pigment, bokbunja pigment, gardenia blue pigment, blueberry pigment, berry pigment, paprika pigment, red cabbage pigment, beet red pigment, purple sweet potato pigment, corn Enzymatic time-temperature history clock, characterized in that at least one selected from the group consisting of pigments and safflower pigments.
  5. 제 1항에 있어서, 상기 시간-온도 이력 지시계는 TTI의 정보, 식품의 정보 또는 TTI가 식품에 부착되기 전까지의 식품의 유통 이력 정보를 입력하는 입력부가 더 포함되어 있는 것을 특징으로 하는, 효소형 시간-온도 이력지시계.According to claim 1, wherein the time-temperature history indicator further comprises an input unit for inputting information of the TTI, food information or distribution history information of the food until the TTI is attached to the food, enzyme type Time-temperature resume clock.
  6. (a) 라카아제를 이용한 효소형 시간-온도 이력 지시계를 식품의 포장 표면에 부착시키는 단계; 및 (a) attaching an enzymatic time-temperature history indicator with laccase to the packaging surface of the food; And
    (b) 상기 (a) 단계의 시간-온도 이력 지시계의 색의 변화로 식품의 변질 정도를 판별하는 단계;를 포함하는 식품의 신선도 감지 방법.(b) determining the degree of deterioration of the food by changing the color of the time-temperature history indicator of step (a).
  7. 제 6항에 있어서, 상기 식품은 어패류, 육류, 과일, 채소 및 낙농제품으로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는, 식품의 신선도 감지 방법.The method of claim 6, wherein the food is at least one selected from the group consisting of seafood, meat, fruit, vegetables and dairy products.
  8. 제 6항에 있어서, 상기 식품은 상온 유통 식품, 냉장 유통 식품 및 냉동 유통 식품으로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는, 식품의 신선도 감지 방법.The method according to claim 6, wherein the food is at least one selected from the group consisting of a room temperature distribution food, a refrigerated distribution food, and a frozen distribution food.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103645268A (en) * 2013-12-25 2014-03-19 济南出入境检验检疫局检验检疫技术中心 Preparation method of acid violet 6B standard sample
CN103712934A (en) * 2014-01-03 2014-04-09 江西省农业科学院农产品质量安全与标准研究所 Label-free ultraviolet absorption spectrometry method for monitoring laccase conformational change
KR101463097B1 (en) * 2014-06-09 2014-11-20 한윤석 label
KR101485333B1 (en) * 2014-06-09 2015-01-26 한윤석 label
KR101485334B1 (en) * 2014-06-09 2015-01-26 한윤석 label
CN110617899A (en) * 2019-09-04 2019-12-27 河南牧业经济学院 Solid enzyme type time-temperature indicator and preparation method thereof

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102006690B1 (en) * 2018-06-11 2019-08-02 동국대학교 산학협력단 Time-temperature indicator indicating the optimum cooking time
KR102489373B1 (en) * 2020-10-15 2023-01-18 동국대학교 산학협력단 Time-temperature integrator for indicating ripeness of ripening fruits and method for manufacturing the same
CN113430182B (en) * 2021-08-09 2023-01-13 云南师范大学 Bacterial laccase from Astrospiraceae of elephant intestinal tract and gene thereof
KR102667454B1 (en) 2023-06-23 2024-05-20 한국지능형포장산업(주) Indicator that visually informs the storage temperature duration of food using oil-in-water emulsion

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021120A1 (en) * 1996-11-08 1998-05-22 Valtion Teknillinen Tutkimuskeskus Package for decayable foodstuffs
US20070059837A1 (en) * 2003-04-24 2007-03-15 Valtion Teknillinen Tutkimuskeskus Method for noticing changes in a package, method for locating a package, package and system for locating a package

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR930009512B1 (en) * 1991-05-20 1993-10-06 박관화 Indicator for time temperature using phospholipase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021120A1 (en) * 1996-11-08 1998-05-22 Valtion Teknillinen Tutkimuskeskus Package for decayable foodstuffs
US20070059837A1 (en) * 2003-04-24 2007-03-15 Valtion Teknillinen Tutkimuskeskus Method for noticing changes in a package, method for locating a package, package and system for locating a package

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JUNG YOUNG, LEE ET AL.: 'Kinetic modeling for predicting the qualities of beef and color of enzyme time-temperature integrator during storage.' FOOD ENGINEERING PROGRESS vol. 14, no. 3, August 2010, pages 229 - 234 *
SEBOLELO, M.-P. ET AL.: 'The Use of Laccase Based Time Temperature Integrator During Microwave Heating of an Egg Model' CSIR BIOSCIENCE AGROPROCESSING TECHNOLOGY. 07 May 2007, *

Cited By (7)

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CN103712934A (en) * 2014-01-03 2014-04-09 江西省农业科学院农产品质量安全与标准研究所 Label-free ultraviolet absorption spectrometry method for monitoring laccase conformational change
CN103712934B (en) * 2014-01-03 2015-10-28 江西省农业科学院农产品质量安全与标准研究所 A kind of for monitor laccase conformation change exempt from mark ultraviolet absorption spectroscopy
KR101463097B1 (en) * 2014-06-09 2014-11-20 한윤석 label
KR101485333B1 (en) * 2014-06-09 2015-01-26 한윤석 label
KR101485334B1 (en) * 2014-06-09 2015-01-26 한윤석 label
CN110617899A (en) * 2019-09-04 2019-12-27 河南牧业经济学院 Solid enzyme type time-temperature indicator and preparation method thereof

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