Method for improving heat stability of Laba garlic pigment extract
Technical Field
The invention relates to a method for improving heat stability of a Laba garlic pigment extract, belonging to the field of agricultural product processing.
Background
China is the main producing country of garlic, and the yield of the garlic accounts for three quarters of the total world yield. In addition to fresh food, the garlic is also processed into products such as mashed garlic, garlic juice, dehydrated garlic slices, garlic powder and the like, and the Laba garlic is a traditional garlic product in China, and the green color is a color-developing substance of the Laba garlic, wherein the green pigment is a color formed by combining yellow pigment and blue pigment. The blue pigment and yellow pigment are natural pigments, and researches show that the Laba garlic pigment alcohol extract has the effects of resisting oxidation, removing free radicals, resisting tumors and the like. The stability of the pigment is the precondition of color presentation and function representation of the Laba garlic product. The research shows that the Laba garlic pigment is sensitive to heat and light, and the pH value also has an influence on the Laba garlic pigment. The Chinese patent ZL200910077868.0 discloses that natural active substance propenyl cysteine sulfoxide extracted from onion juice is added into Laba garlic, so that green pigment is continuously formed, the Laba garlic which originally has only 1-month shelf life is prolonged to 3-6 months, and the stability of the pigment is improved.
The heat stability of the Laba garlic is of great importance to the influence of the pigment of the Laba garlic in the fresh food storage process and the cooking and heating process. Research shows that after the Laba garlic is subjected to alcohol extraction, the Laba garlic is heated in a water bath at 40 ℃ for 30min, and the loss rates of the blue pigment and the yellow pigment are only 1.1 percent and 0.7 percent. The loss rate increases with the increase of the heating temperature, and the blue pigment is lost by 14.7 percent and 27.9 percent at 80 ℃ and 100 ℃; yellow pigment loss 15.4% and 27.1%. Heating in a 60 deg.C water bath, the two pigments lost 4.7% and 5.9%, respectively. Therefore, the method for improving the thermal stability of the pigment is provided, and the guarantee of the efficacy and the color stability can be provided for further application of the pigment.
Disclosure of Invention
The invention aims to provide a method for improving the thermal stability of a Laba garlic pigment extract, which utilizes DNA and protein to improve the thermal stability of the Laba garlic pigment extract.
The method for improving the heat stability of the Laba garlic pigment extract comprises the step of adding DNA or protein into the Laba garlic pigment extract.
In specific implementation, the solution of the Laba garlic pigment extract can be mixed with the solution of the DNA or the solution of the protein, so that the thermal stability of the blue pigment and the yellow pigment extract in the Laba garlic pigment extract can be obviously improved.
Specifically, the Laba garlic pigment extract reacts with the DNA or the protein under the following conditions to realize the combination of the Laba garlic pigment extract and the DNA or the protein;
the temperature can be 15-25 ℃ and the time can be 10-30 minutes.
The DNA may be calf thymus DNA and the protein may be bovine serum albumin.
The mass ratio of the Laba garlic pigment extract to the DNA can be 500-800: 1, such as 750: 1;
the mass ratio of the Laba garlic pigment extract to the protein can be 700-1000: 1, as 857: 1.
the Laba garlic pigment extract is prepared according to the following method:
(1) crushing fresh garlic into mashed garlic, and adding an acetic acid aqueous solution into the mashed garlic until the mashed garlic becomes green;
the fresh garlic is stored at the temperature of 0-4 ℃ for 1-2 months to break dormancy;
the volume percentage of acetic acid in the acetic acid aqueous solution is 4.5-5.5%;
(2) extracting the mashed garlic by adopting ethanol water solution I, and concentrating the obtained filtrate I to obtain a crude pigment extract I;
the pH value of the ethanol water solution I is 2.5-4.0;
adjusting the pH value of the ethanol water solution I by adopting formic acid, acetic acid, trifluoroacetic acid, phosphoric acid or a mixture thereof;
the ethanol in the ethanol water solution I is 75-85% in percentage by volume;
the ratio of the mashed garlic to the ethanol aqueous solution I is as follows: 1 g: 1-8 mL;
the extraction times are 2-4 times;
(3) extracting the ethanol solution of the pigment crude extract I by using ethyl acetate, discarding an ethyl acetate phase, retaining an ethanol phase, and concentrating the ethanol phase to obtain a pigment crude extract II;
the volume ratio of the pigment crude extract I to the ethanol to the ethyl acetate is 1: 1-3: 1-3;
(4) separating the pigment crude extract II by adopting macroporous resin, eluting by adopting an ethanol water solution II, collecting eluent with ultraviolet absorption at the wavelength of 440nm and 590nm, concentrating and drying to obtain the pigment crude extract II;
the pH value of the ethanol water solution II is 2.5-4.0;
the conditions for the macroporous resin separation are as follows:
the pigment crude extract II is prepared from the following components in a proportion of 1 g: dissolving 2-4 mL of the solution in water and then sampling;
firstly, washing with an aqueous solution I with the pH value of 2.5-4.0, and then eluting with an ethanol aqueous solution III with the pH value of 2.5-4.0;
the macroporous resin is XAD-7 macroporous resin, AB-8 macroporous resin or YWD-01 macroporous resin;
after the step (4), the preparation method also comprises the step of performing ultrafiltration treatment on the Laba garlic pigment extract by adopting an ultrafiltration membrane;
the cutoff molecular weight of the ultrafiltration membrane is 3000-5000 daltons;
before ultrafiltration, dissolving the garlic pigment extract in an aqueous solution II with the pH value of 2.5-4.0;
the ratio of the Laba garlic pigment extract to the aqueous solution II is 1 g: 6-15 mL;
the conditions of the ultrafiltration were as follows:
the transmembrane pressure is 3-5 bar, and the flow rate is 20-35 mL/min.
The Laba garlic pigment extract comprises a Laba garlic blue pigment extract and a Laba garlic yellow pigment extract, and in order to research the influence of DNA or protein on the thermal stability of the Laba garlic pigment extract, the Laba garlic blue pigment extract and the Laba garlic yellow pigment extract need to be separated, and the method can be carried out according to the following steps: separating with gel resin column (Sephadex G-25, Sephadex LH-20, Sephadex G-20 or Sephadex G-15); according to the weight volume ratio g/mL of 1: dissolving the extract in an acid aqueous solution with the pH value of 2.5-3.5 (adjusting the pH value by formic acid, acetic acid, trifluoroacetic acid or phosphoric acid) at a ratio of 2-4, taking a mixed solution of the acid aqueous solution with the pH value of 2.5-3.5 (adjusting the pH value by formic acid, acetic acid, trifluoroacetic acid or phosphoric acid) and an organic solvent (ethanol, acetonitrile or n-butyl alcohol) as an eluent for elution, wherein the volume percentage of the organic solvent in the eluent is 50-70%, controlling the flow rate at 1-4 mL/min, collecting eluents with characteristic absorption peaks at 440nm and 590nm respectively, mixing the eluents with the same absorption peak, concentrating, and freeze-drying under the pressure of 0.22MPa to obtain yellow powder and blue powder to obtain the yellow and blue pigment extract of the Laba garlic.
The invention provides a method for improving the thermal stability of a Laba garlic pigment extract based on the influence of DNA or protein on the thermal stability of the Laba garlic pigment extract (Laba garlic blue pigment extract and Laba garlic yellow pigment extract). The embodiment of the invention shows that the degradation rate can be reduced by 20-25% by heating the Laba garlic blue pigment extract for 1 hour at 60 ℃ after combining with DNA, and the degradation rate can be reduced by 15-18% after combining with protein; the Laba garlic yellow pigment extract can reduce the degradation rate by 45-50% by heating at 60 ℃ for 1 hour after being combined with DNA, and can reduce the degradation rate by 30-35% after being combined with protein.
Drawings
FIG. 1 is the UV-visible scanning spectrum of Laba garlic blue pigment extract and yellow pigment extract.
FIG. 2 shows the effect of calf thymus DNA and bovine serum albumin on the heat stability of the extract of Laba garlic blue pigment.
FIG. 3 shows the effect of calf thymus DNA and bovine serum albumin on the heat stability of the extract of Garlic flavins.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of Laba Garlic blue pigment and yellow pigment extract
Storing 1kg fresh Bulbus Allii at 4 deg.C for 1 month, breaking dormancy, peeling, and cleaning. Pulverizing, placing in a beaker, adding 5% acetic acid water solution (v: v), and making the mashed Bulbus Allii green at room temperature for 4 days.
Extracting with 80% ethanol water solution with pH 3 (adjusted with acetic acid) at room temperature for 3 times (each time for 12 hr). The material-liquid ratio of the mass of the raw materials to the volume of the extracting solution is 1: and 3, filtering the materials extracted each time to obtain filtrate, mixing the filtrate, and concentrating by rotary evaporation at the temperature of 35 ℃ to obtain a crude pigment extract.
Dissolving the crude extract in ethanol to obtain an ethanol solution, and extracting with ethyl acetate, wherein the volume ratio of the crude extract to the ethyl acetate to the ethanol is 1: 2: 2; extracting for 3 times, discarding ethyl acetate phase (liposoluble impurities such as allicin remained in pigment), mixing ethanol extractive solutions, and rotary evaporating at 35 deg.C for concentration.
And (3) mixing the pigment crude extract according to a weight-volume ratio g/mL of 1: 2, dissolving in double distilled water, and purifying by macroporous resin XAD-7. Washing 4 column volumes with water (pH 3 adjusted with formic acid) at flow rate of 4mL/min, eluting the pigment component with ethanol (pH 3 adjusted with formic acid) at flow rate of 4mL/min, collecting eluates at 440nm and 590nm, rotary evaporating at 35 deg.C for concentration, and freeze drying to obtain green powder.
According to the following steps of 1: dissolving the crude extract of laba garlic pigment in double distilled water with pH of 3 (pH adjusted by acetic acid) according to a weight-to-volume ratio (g/mL) of 10, performing ultrafiltration by using an ultrafiltration membrane (polyether sulfone) with a molecular weight cutoff of 5000 daltons, removing other impurities with a molecular weight of more than 5000 daltons, controlling the transmembrane pressure to be 4bar and the flow rate to be 25mL/min, and freeze-drying the filtrate to obtain the laba garlic green pigment extract.
The Laba garlic green pigment consists of a blue pigment and a yellow pigment, and in order to separate the blue pigment from the yellow pigment, the Laba garlic green pigment extract is further applied to a gel resin column Sephadex LH-20, wherein the weight-volume ratio g/mL is 1: 2 in 3mL of acidic water at pH 3 (pH adjusted with formic acid): ethanol ═ 4: eluting 6 (volume ratio) eluents, controlling the flow rate at 2.5mL/min, respectively collecting eluents with characteristic absorption peaks at 440nm and 590nm, mixing and concentrating, freeze-drying under the pressure of 0.22MPa to obtain yellow powder and blue powder, and obtaining Laba garlic yellow pigment extract and Laba garlic blue pigment extract, wherein the ultraviolet visible scanning spectra of the Laba garlic yellow pigment extract and the Laba garlic blue pigment extract are shown in figure 1, and it can be seen that the eluents respectively have absorption peaks at 440nm and 590nm, which are the absorption peak wavelengths of typical Laba garlic yellow pigment and Laba garlic blue pigment.
Example 2 experiment of increasing thermal stability of pigment after DNA binding with Laba garlic blue pigment and yellow pigment extract
Dissolving calf thymus DNA in double distilled water to obtain 0.8mg/mL solution, and placing in a refrigerator at 4 deg.C overnight. The Laba garlic blue pigment and the Laba garlic yellow pigment extract are respectively prepared into 600mg/mL solution by double distilled water for later use, and are stored at low temperature in the dark.
Adding 0.8mg/mL calf thymus DNA solution 3mL into 10mL test tube, adding 600mg/mL Laba garlic blue pigment extract solution 3mL into double distilled water 3mL, and reacting at 20 deg.C for 10 min. After heating at 60 ℃ for 0 and 1 hour respectively, the sample is scanned by an ultraviolet-visible spectrophotometer. The Laba garlic yellow extract was treated in the same manner as described above.
The content of blue pigment and yellow pigment extract is represented by 590nm and 440nm ultraviolet absorption values respectively. The results are shown in FIGS. 2 and 3. It can be seen that the blue pigment and the yellow pigment extract are both degraded after heating at 60 ℃ for 1 hour, the degradation rate of the blue pigment extract is 30.26%, and the degradation rate after DNA binding is 23.47%, which shows that the degradation rate after DNA binding is reduced by 22.44% ((30.26% -23.47%)/30.26% × 100%), and the thermal stability of the blue pigment extract is obviously improved. After heating at 60 ℃ for 1 hour, the yellow pigment extracts are degraded, the degradation rate of the yellow pigment extracts is 22.58 percent, the degradation rate after DNA combination is 11.49 percent, the degradation rate after DNA combination is reduced by 49.11 percent (22.58 to 11.49 percent)/22.58 percent multiplied by 100 percent), and the thermal stability of the yellow pigment extracts is obviously improved.
Example 3 experiment of improving thermal stability of pigment after protein binding with Laba garlic blue pigment and yellow pigment extract
Bovine serum albumin was dissolved in phosphate buffer pH 6.9 to make a 0.7mg/mL solution. Preparing the Laba garlic blue pigment and the Laba garlic yellow pigment extract into 600mg/mL solutions respectively by using phosphate buffer solutions for later use, and storing the solutions at low temperature in the dark.
Adding 0.7mg/mL bovine serum albumin solution 3mL into a 10mL test tube, adding 3mL Laba garlic blue pigment solution extract with the concentration of 600mg/mL, adding 3mL Laba garlic blue pigment extract solution with the concentration of 600mg/mL into a 3mL reference tube, and reacting at 20 ℃ for 10 min. After heating at 60 ℃ for 0 and 1 hour respectively, the sample is scanned by an ultraviolet-visible spectrophotometer. The Laba yellow extract is treated by the same method.
The content of blue pigment and yellow pigment extract is represented by 590nm and 440nm ultraviolet absorption values respectively. As shown in fig. 2 and 3, it can be seen that the blue pigment and the yellow pigment extract are degraded after heating at 60 ℃ for 1 hour, the degradation rate of the blue pigment extract is 30.26%, and the degradation rate after binding bovine serum albumin is 24.96%, indicating that the degradation rate after binding bovine serum albumin is reduced by 17.51% ((30.26% -24.96%)/30.26% × 100%), and the thermal stability of the blue pigment extract is significantly improved. After the yellow pigment extract is heated for 1 hour at the temperature of 60 ℃, the yellow pigment extract is degraded by 22.58 percent, the degradation rate after the combination of the yellow pigment extract and the bovine serum albumin is 14.87 percent, which shows that the degradation rate of the combined bovine serum albumin is reduced by 34.14 percent (22.58 to 14.87 percent)/22.58 percent multiplied by 100 percent), and the thermal stability of the yellow pigment extract is obviously improved.