CN112940528A

CN112940528A - Method for extracting leek green pigment, sodium copper chlorophyllin and sodium zinc chlorophyllin prepared by method and stability comparison method

Info

Publication number: CN112940528A
Application number: CN202110218177.9A
Authority: CN
Inventors: 洪军; 王永强; 高建民; 叶延欣; 黄玮玮; 张开放; 李倩; 刘志宏; 蒋梦瑶; 马栋豪; 李美换; 刘丙欣; 马伟皓
Original assignee: Henan Jiuxing Biotechnology Research Institute Co ltd; Henan University of Urban Construction
Current assignee: Henan Jiuxing Biotechnology Research Institute Co ltd; Henan University of Urban Construction
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2021-06-11

Abstract

The invention provides a method for extracting leek green pigment, preparation of chlorophyll copper sodium salt and chlorophyll zinc sodium salt, and stability comparison, and solves the technical problems of less natural green pigment used in food, medicine and cosmetics and poor stability, including: Leek selection, cleaning, pulverization, preparation of fat-soluble green pigment, saponification, acidification, copper formation, static salt formation and application effect, preparation method: washing leek leaves, freeze-drying and pulverizing, compound enzyme-assisted microwave treatment, edible ethanol ultrasonic-assisted extraction Fat-soluble green pigment, heated in a water bath to remove odor, concentrated under reduced pressure, and stored in the dark. In order to facilitate the preservation of leek green pigment and improve the stability, the prepared fat-soluble green pigment was saponified under strong alkaline ultrasonic conditions, magnesium ions were replaced with copper or zinc ions under strong acid conditions, statically formed salts under alkaline conditions, and dried to form Salt samples are easily soluble in water, easy to store, and have better stability than fat-soluble green pigments. They can be used as green pigments in food, medicine, etc., and have both nutritional and health care functions.

Description

Method for extracting leek green pigment, sodium copper chlorophyllin and sodium zinc chlorophyllin prepared by method and stability comparison method

Technical Field

The invention relates to the technical field of natural edible pigment extraction and preparation technology and food additives, in particular to a method for extracting leek green pigment, a method for preparing sodium copper chlorophyllin and sodium zinc chlorophyllin by using the method and a stability comparison method.

Background

Nowadays, the application of pigments in the market is increasing, but the synthetic pigments account for a large portion of the pigments currently used in the market, and natural pigments are not commonly used. A large number of research reports indicate that almost all synthetic pigments cannot provide nutrient substances for human bodies, some synthetic pigments even harm human health, and some synthetic pigments have chemical effects on skin, clothes, fibers and the like, and can cause various adverse reactions after long-term contact; if the food is used excessively or improperly, the food can cause canceration in vivo and harm human health. This has led to a gradual shift in the use of pigments to natural pigments.

Most natural pigments come from animal and plant tissues, minerals, microorganisms and other aspects, the safety is high, and some pigments also have physiological activity, and have the functions of nutrition, health care and the like; some pigments can better simulate the color of natural objects, are more natural in coloring and are more easily accepted by consumers; some pigments also have a special aromatic odor and are added to foods to give a pleasant feeling. With the continuous improvement of the living standard of people in China, safe, additive-free and pure natural pigments are bound to become the mainstream of colorants in the fields of food, daily necessities, medicine and the like.

The main component of the natural green pigment is chlorophyll, most of which is from plant extracts, can be naturally decomposed, generally has no harm such as irritation or corrosion, and has small side effect. The natural chlorophyll can be applied to food and daily chemical products, and also has the functions of improving constipation, reducing cholesterol, resisting aging, expelling toxin, diminishing inflammation, deodorizing, resisting cancer, resisting mutation, resisting anemia, protecting liver and the like in the field of medicine. However, since natural chlorophyll is not stable, it is susceptible to fading and discoloration during its use due to various factors such as light, temperature, oxidation-reduction agent, pH, metal ions, additives, etc., which severely restricts the process of replacing synthetic pigments with natural green pigments. The research shows that the copper sodium salt, the iron zinc sodium salt, the microcapsule and the like are beneficial to the stability of chlorophyll and the improvement of water solubility.

Sodium copper chlorophyllin or sodium zinc chlorophyllin is one of chlorophyll derivatives, and is also a natural green edible pigment. The sodium copper chlorophyllin or its derivative can be easily absorbed by human body, has effect in promoting metabolism, and can be used as copper preparation or zinc preparation for supplementing trace element copper. Researches prove that the sodium copper chlorophyllin has good curative effects on infectious liver diseases, duodenal ulcer, gastric ulcer, trauma healing and other pathological changes, and is a safe and nontoxic natural pigment with good water solubility, good colorability and high stability. In addition, researches show that sodium copper chlorophyllin can be widely used as a basic pigment of cosmetics and a colorant of toothpaste, is used as a multifunctional daily chemical raw material, is widely applied to various green medicinal toothpastes at home and abroad, and has obvious effects of treating periodontitis, canker sore, removing halitosis and the like. The sodium zinc chlorophyllin can be widely applied to medicines, cosmetics and beverages, and has a certain effect on treating cancers.

Leek is also called as "herba polygoni multiflori", which belongs to a perennial herb of liliaceae, and the root, stem, leaf, flower and seed of leek are all useful and are treasure throughout the body. The leek is rich in protein, fat, carbohydrate, cellulose, chlorophyll, a large amount of vitamins, and minerals such as calcium, phosphorus, iron, etc. Leek is not only a vegetable rich in nutrition, but also has a magical effect in preventing and treating diseases.

A cultivation base for high-quality leeks in leaf counties of Pingtian mountain city, Henan province has multiple varieties and large planting amount, is sold as fresh vegetables and leek seeds, and can be used for extracting and preparing natural green pigment mainly containing chlorophyll for the second-crop leeks with low price in 5-6 months or late-selling leeks so as to improve the additional value of leeks products, can be directly applied to foods, medicines and daily necessities as a coloring agent, and can also be used as a natural chlorophyll substance to play a role in preventing and treating diseases.

Therefore, the method has important significance for researching the extraction of the green pigment in the Chinese chives and the stability of the green pigment after salifying.

Disclosure of Invention

Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the method for extracting the leek green pigment, the sodium copper chlorophyllin and the sodium zinc chlorophyllin prepared by the method, and the stability comparison, so that the technical problems and the defects that the natural green pigment used in the existing food, medicine and cosmetics is less, has poor stability and is easily influenced by factors such as pH, light, heat, oxidation reducing agent and the like to fade or brown are effectively solved.

The method for extracting the leek green pigment is characterized by comprising the following steps:

removing yellow leaves on the Chinese chives, cleaning, cutting into sections, then putting the sections into a vacuum freeze dryer for freeze drying, then crushing the sections by using a crusher, and screening the crushed sections by using a 40-60-mesh sieve for dark low-temperature storage for later use;

step two, weighing 10g of leek powder or directly taking cleaned fresh leeks and homogenizing by a homogenizer according to the proportion of 1: 1;

adding a complex enzyme water solution containing cellulase and pectinase with the final mass concentration of 0.3% and 0.1% according to the material-liquid ratio of 1:4, adjusting the pH value of the environment to 4.8 by using salicylic acid, performing water bath at 45 ℃ for 60min, treating for 60S under the condition that the microwave power is 600W, filtering to remove supernatant, and collecting filter residue A for later use;

step four, adding the filter residue A into 95% edible ethanol according to the liquid-material ratio of 20:1 ml/g;

step five, extracting the green pigment under the conditions that the ultrasonic temperature is 60 ℃, the ultrasonic time is 60min and the ultrasonic power is 200W, performing suction filtration by using a vacuum suction filter after extraction is finished, collecting filtrate B and filter residue C, and extracting in a dark place in the extraction process;

step six, adding petroleum ether organic solvent into the filter residue C according to the liquid-material ratio of 10:1ml/g, extracting according to the step five until the filter residue is colorless, and collecting a filtrate D after solid-liquid separation;

step seven, finally, mixing the extracted filtrate B and the extracted filtrate D, heating the mixture in water bath at 50 ℃ for 30-60min to remove odor, and then carrying out reduced pressure distillation and concentration to 1 g/ml;

and step eight, obtaining the fat-soluble green pigment mainly containing chlorophyll.

A method for preparing sodium copper chlorophyllin, which is characterized by comprising the following steps:

s1: saponification; pouring the prepared fat-soluble green pigment mainly containing chlorophyll into a beaker, adjusting the pH to 12-13 by using 10% NaOH, and saponifying for 30-40min under the conditions that the ultrasonic temperature is 60 ℃ and the ultrasonic power is 200W;

s2: completely saponifying; taking out after saponification is finished, cooling to room temperature, filtering to remove unsaponifiable sticky impurities, transferring the saponified solution into a separating funnel, adding petroleum ether with the same volume as the saponified solution, fully vibrating, standing for layering, and obtaining completely saponified product with yellow upper layer;

s3: acidifying and placing copper; putting the extract liquor at the lower layer of the liquid separating funnel in the S2 into a beaker, adjusting the pH value to 1.5-2.0 by using 10% HCl, adding acid to fully react, and changing the solution into yellow brown to generate pheophorbide;

detecting the content of magnesium ions;

adding 10% copper sulfate solution, wherein the addition amount of copper ions in the copper sulfate solution is 1.5-2.0 times of the theoretical amount of magnesium ions contained in chlorophyll molecules;

slowly adding the mixture while stirring, performing ultrasonic water bath at 60 ℃ for 1h to gradually replace magnesium ions in chlorophyll by copper ions, recovering the solution to green and generating dark green copper chlorophyllin acid precipitate, performing suction filtration, and repeatedly washing the obtained copper chlorophyllin acid with distilled water to remove water-soluble impurities; then washing the mixture for three times by using 40% ethanol solution;

s4: salifying; after copper is placed, dissolving the chlorophyllin cuprate in S3 in 95% edible ethanol, adding 10% NaOH solution while stirring, adjusting the pH value of the solution to 12-13, and standing for salification;

s5: removing impurities and drying; then repeatedly washing with petroleum ether to obtain a wet product of sodium copper chlorophyllin, and removing ethanol and petroleum ether;

and drying to obtain the finished product of sodium copper chlorophyllin.

A method for preparing sodium zinc chlorophyllin, which is characterized by comprising the following steps:

a, placing 10ml of extracted fat-soluble green pigment with the concentration of 1g/ml and mainly containing chlorophyll in a round-bottom flask;

b: saponification; adding 10% NaOH to adjust pH to 12-13, saponification temperature is 60 deg.C, saponification time is 30min, and ultrasonic power is 200W;

c: extracting; adding petroleum ether into the cooled saponified solution according to the ratio of 1:1, standing for several hours for layering, wherein the lower layer is dark green saponified solution;

d: acidifying; after collecting the dark green subnatant, adjusting the pH to 2-3 by using 10% HCl;

e: substituting zinc; adding the acidized solution into a zinc sulfate solution according to the liquid-material ratio of 1:1, carrying out ultrasonic treatment at the temperature of 70 ℃ for 1h at the ultrasonic power of 200W, and cooling to obtain a chlorophyllin zinc acid precipitate;

f: washing with distilled water for 3 times after filtering to obtain zinc chlorophyllin acid;

g: salifying; adding 10ml of 95% edible ethanol, adding 10% NaOH to adjust the pH value to 12-13, and standing for 1h to form salt;

h: filtering, placing in a 45 ℃ oven, and drying to obtain the sodium zinc chlorophyllin.

The invention provides an extraction method of leek chlorophyll, preparation application and stability comparison thereof, the raw material of the green pigment is leek, the source is convenient to obtain and has a large acquisition amount, the extraction and preparation process is simple, the green pigment can be directly used as a food additive to be applied to food and medicines, no special smell exists, the coloring effect is good and stable, and the green pigment is suitable for industrial application.

Drawings

FIG. 1 is a detection map of leek green pigment of the present invention.

FIG. 2 is a detection spectrum of sodium copper chlorophyllin according to the present invention.

FIG. 3 is a detection spectrum of the sodium zinc chlorophyllin of the present invention.

FIG. 4 shows sodium copper chlorophyllin powder according to example two of the present invention.

FIG. 5 shows the powder of sodium zinc chlorophyllin in example III of the present invention.

FIG. 6 is a graph showing the experimental results of the effect of light irradiation on the fat-soluble chlorophyll of leek.

FIG. 7 is a graph showing a control experiment of the effect of light irradiation on sodium copper chlorophyllin and sodium zinc chlorophyllin according to the present invention.

FIG. 8 is a graph of a control experiment showing the effect of temperature on sodium copper chlorophyllin and sodium zinc chlorophyllin according to the present invention.

FIG. 9 is a graph of a control experiment showing the effect of pH values of different pH values on sodium copper chlorophyllin and sodium zinc chlorophyllin according to the present invention.

FIG. 10 is a graph of a control experiment showing the effect of the oxidizing agent and the reducing agent of the present invention on sodium copper chlorophyllin and sodium zinc chlorophyllin.

FIG. 11 is a control experiment chart showing the effect of the food additives of the present invention, such as salt, sucrose, starch, etc., on sodium copper chlorophyllin and sodium zinc chlorophyllin.

Detailed Description

The foregoing and other aspects, features and advantages of the invention will be apparent from the following more particular description of embodiments of the invention, as illustrated in the accompanying drawings in which reference is made to figures 1 to 11. The contents of the following examples are all referred to in the attached drawings of the specification.

Exemplary embodiments of the present invention will be described below with reference to the accompanying drawings.

The first embodiment is as follows:

The extraction process is carried out in dark condition, can reduce decomposition of chlorophyll to obtain liposoluble green pigment mainly containing chlorophyll, is easily soluble in organic solvent, can be directly used as colorant in food, medicine and daily necessities, and can be stored at 4 deg.C in dark condition for more than 1 month.

For convenience of storage, prolonging shelf life and increasing chlorophyll usage, the prepared concentrated fat-soluble green pigment is substituted by copper or zinc ions in the following examples to form salt substances.

The attached figure 1 of the specification is a detection spectrum of chlorophyll in leek green pigment.

Example two:

a method of preparing sodium copper chlorophyllin comprising the steps of:

detecting the content of magnesium ions;

and drying to obtain the finished product of sodium copper chlorophyllin.

The sodium copper chlorophyllin obtained by the method is dark green and has metallic luster, is a powdery substance, is easy to dissolve in water, has no precipitate, is slightly soluble in ethanol, methanol and chloroform, and is insoluble in petroleum ether, acetone and diethyl ether, and all indexes of the sodium copper chlorophyllin meet the requirements of GB 26406-2011.

Description accompanying figure 2 is the detection map of sodium copper chlorophyllin of the present invention.

And figure 4 in the specification is chlorophyll copper sodium salt powder in example two.

Example three:

And FIG. 5 shows the powder of sodium zinc chlorophyllin in example III.

More specifically, the method comprises the following steps:

the other specific detailed preparation process comprises the following steps:

cleaning and cutting fresh leek leaves, then putting the fresh leek leaves into a vacuum freeze dryer for freeze drying, and then crushing the fresh leek leaves by a crusher, wherein the material-liquid ratio of the fresh leek leaves to the material-liquid ratio of 1: treating with microwave at a ratio of 4: 60s, adding 95% edible ethanol at a liquid-to-material ratio of 20: 1(ml/g), extracting chlorophyll at 60 deg.C for 60min under 200W, collecting filtrate, repeatedly extracting the residue with the same method (generally 2 times), mixing the filtrates, heating in 50 deg.C water bath to remove odor, vacuum filtering, concentrating with rotary evaporator to 1g/ml to obtain liposoluble green pigment mainly containing chlorophyll, and storing in brown sealed bottle. Preparing a leek chlorophyll solution to be tested, adding 1ml of leek chlorophyll extracting solution (1 g/ml) into 95% edible ethanol for diluting by 10 times, and performing spectral scanning by using an ultraviolet spectrophotometer in a 600-700nm interval. The green pigment is not stable under illumination, and needs to be stored at low temperature of 4 deg.C in dark to avoid contact with oxidant and reductant.

Firstly, the influence of illumination on chlorophyll extracted from Chinese chives is verified:

taking 100 mu L of 1g/ml chive chlorophyll concentrated solution, adding absolute ethyl alcohol to fix the volume to 10ml, placing the solution in an indoor illumination place, taking 1ml of solution from the 100-time diluted solution on the 2 nd day, adding absolute ethyl alcohol to fix the volume to 10ml (10000 times diluted before and after), measuring the light absorption value of the solution, taking 1ml of solution from the 100-time diluted solution on the 3 rd day, adding absolute ethyl alcohol to fix the volume to 10ml, measuring the light absorption value of the solution, and determining the result until the 7 th day as the attached figure 6 of the specification:

as shown in the attached figure 6 of the specification, from a line graph, the light absorption value of the chive chlorophyll is gradually reduced along with the prolonging of the illumination time, the OD value on the 3 rd day is reduced by about 36 percent compared with that on the 1 st day, and the yellow brown color is kept after the 7 th day, which indicates that the chive green pigment is unstable under illumination and should be stored in a dark place.

In order to facilitate the preservation and stability of the leek green pigment, the prepared water-soluble green pigment is saponified for 30min under the conditions that the pH value is 12-13, the ultrasonic temperature is 60 ℃ and the ultrasonic power is 200W, the saponified solution is added with isovolumetric petroleum ether, the lower layer extract is collected after layering, then the pH value is adjusted to 1.5-2.0, 10% copper sulfate solution is added, ultrasonic treatment is carried out for 1-2h at 60 ℃ to generate chlorophyll copper acid precipitate, suction filtration is carried out, after impurity removal, the chlorophyll copper acid precipitate is dissolved in 95% edible ethanol, the pH value of the solution is adjusted to 12-13, standing is carried out to form salt, and after ethanol removal, sodium copper chlorophyllin is obtained, namely solid powder. The sodium copper chlorophyllin is easily soluble in water, convenient for storage, and has better stability than liposoluble green pigment, and can be used in food, medicine and daily necessities.

From the description, the peak spectrum of sodium copper chlorophyllin shown in figure 2 has two absorption peaks at 408nm and 634nm, wherein the absorption peak at 408nm is maximum.

From the description, the peak map of the sodium zinc chlorophyllin shown in figure 3 has two absorption peaks at the wavelengths of 412nm and 632nm, wherein the absorption peak at 412nm is the maximum.

A method for comparing the stability effects of common factors on sodium copper chlorophyllin and sodium zinc chlorophyllin comprises the following steps:

1) extracting fat-soluble green pigment from the Chinese chives;

2) respectively preparing sodium copper chlorophyllin and sodium zinc chlorophyllin according to the fat-soluble green pigment extracted in the step 1);

3) and preparing a test solution:

respectively weighing 0.1g of sodium copper chlorophyllin and sodium zinc chlorophyllin prepared from the same batch of fat-soluble green pigment, dissolving with distilled water, and diluting to a constant volume of 1L, wherein the solution is used as a test solution for detecting stability and effect;

4) and absorbance detection:

absorbance detection is carried out by using an enzyme-labeling instrument, wherein the detection wavelength of sodium copper chlorophyllin is 408nm, and the detection wavelength of sodium zinc chlorophyllin is 412 nm;

control of the effect of common factors on the stability of sodium copper chlorophyllin and sodium zinc chlorophyllin:

verification of the effect of illumination on the stability of sodium copper chlorophyllin and sodium zinc chlorophyllin:

taking 10ml of the above prepared test solution of sodium copper chlorophyllin and sodium zinc chlorophyllin, setting 3 times for each sample, respectively putting the samples into 3 20ml glass test tubes with plugs, placing the samples in indoor illumination places, respectively measuring the light absorption value of the solution by a microplate reader at certain time points of 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days, and taking 100 plus 200 microliter of the solution, wherein the result is shown in the attached figure 7 of the specification.

As can be seen from the attached fig. 7 in the specification, the change of sodium zinc chlorophyllin is large and the change of sodium copper chlorophyllin is small in the first 2 days, and the influence of light on the sodium zinc chlorophyllin is larger than that on the sodium copper chlorophyllin in the whole 7 days.

Therefore, sodium copper chlorophyllin is relatively stable to sodium zinc chlorophyllin under light conditions.

Secondly, verifying the influence of temperature on the stability of sodium copper chlorophyllin and sodium zinc chlorophyllin:

and respectively putting 10ml of the prepared test solution of sodium copper chlorophyllin and 10ml of sodium zinc chlorophyllin into a 20ml test tube, sealing, setting 3 times of each sample, putting the samples into a water bath kettle at 40 ℃, 60 ℃, 80 ℃ and 100 ℃ for water bath for 20min, cooling to room temperature, taking unheated sample liquid as a reference and taking a corresponding solvent as a blank, and taking 100-fold 200 mu L of solution to measure absorbance by using an enzyme-linked immunosorbent assay instrument, wherein the result is shown in the attached figure 8 of the specification.

As can be seen from the description in fig. 8:

the change of the sodium copper chlorophyllin at the temperature of 40-60 ℃ is small, the influence on the sodium copper chlorophyllin is small, the stability of the sodium copper chlorophyllin is reduced when the temperature exceeds 80 ℃, and the influence of the temperature on the stability of the sodium copper chlorophyllin is small from the change of OD value.

The stability of the sodium zinc chlorophyllin is reduced in the temperature range of 40-80 ℃, although the change is also small, the change is large compared with that of the sodium copper chlorophyllin, and the OD value tends to be stable after the temperature exceeds 80 ℃.

Thus, sodium copper chlorophyllin is more stable than sodium zinc chlorophyllin under the influence of temperature.

And thirdly, verifying the comparison of the influence of acid and alkali with different pH values on the stability of sodium copper chlorophyllin and sodium zinc chlorophyllin:

respectively taking 10ml of the prepared test solution sodium copper chlorophyllin and 10ml of the prepared test solution sodium zinc chlorophyllin, putting the test solution sodium copper chlorophyllin and the test solution sodium zinc chlorophyllin into a 20ml test tube, setting 3 times of repetition for each sample, adjusting the pH value of the sodium copper chlorophyllin and the pH value of the sodium zinc chlorophyllin with 1% NaOH and 10% HCl solution to prepare solutions with the pH values close to 2,4,6,8,10 and 12, standing the solutions at room temperature for 1 hour after uniformly mixing, and measuring the absorbance of the sodium copper chlorophyllin and the sodium zinc chlorophyllin under the corresponding pH conditions, wherein the result is shown in the attached figure.

As can be seen in the description accompanying fig. 9:

the absorbance of sodium copper chlorophyllin and sodium zinc chlorophyllin gradually increases with the increase of the pH value, which shows that the sodium copper chlorophyllin and the sodium zinc chlorophyllin can increase the stability under stronger alkaline conditions. However, sodium copper chlorophyllin has a higher OD value than sodium zinc chlorophyllin under acidic, neutral or alkaline conditions, wherein the OD values of the two are partially overlapped at a pH of 8 to 10.

Therefore, sodium copper chlorophyllin is more stable than sodium zinc chlorophyllin under different acid-base conditions.

And fourthly, verifying the influence of the oxidizing agent and the reducing agent on the stability of the sodium copper chlorophyllin and the sodium zinc chlorophyllin:

and (3) respectively putting 10ml of the prepared test solution of sodium copper chlorophyllin and 10ml of sodium zinc chlorophyllin into a 20ml test tube, setting each sample for 3 times, respectively adding the H202 and Na2SO3 solutions with the final concentrations of 0%, 0.30%, 0.60% and 0.90%, uniformly mixing, standing at room temperature for 1H, and measuring the absorbance of the solution, wherein the result is shown in the attached figure 10 of the specification.

As can be seen from the description accompanying FIG. 10:

the oxidizing agent has a greater influence on sodium copper chlorophyllin than the reducing agent, and the influence is greater at high concentrations. High concentrations of redox agents should be avoided for preservation and application of sodium copper chlorophyllin.

The OD value of the oxidizing agent to the sodium zinc chlorophyllin is larger than that of the reducing agent to the sodium zinc chlorophyllin at the concentration of 0-0.40%, the OD value of the oxidizing agent to the sodium zinc chlorophyllin is smaller than that of the reducing agent to the sodium zinc chlorophyllin at the concentration of 0.40% -0.80%, and the OD value of the oxidizing agent to the sodium zinc chlorophyllin is larger than that of the reducing agent to the sodium zinc chlorophyllin at the concentration of 0.80% -0.90%.

However, the OD value of sodium zinc chlorophyllin is greater than the OD value of sodium copper chlorophyllin at various concentrations, both reducing and oxidizing agents.

Therefore, zinc sodium chlorophyllin is more stable than copper sodium chlorophyllin for reducing and oxidizing agents.

Verifying the influence of different food additives on the stability of sodium copper chlorophyllin and sodium zinc chlorophyllin by comparison:

and (3) respectively putting 10ml of the prepared test solution of sodium copper chlorophyllin and 10ml of sodium zinc chlorophyllin into a 20ml test tube, setting 3 times for each sample, respectively adding salt and sucrose with final concentrations of 0%, 2%, 4% and 6%, uniformly mixing, standing at room temperature for 1h, and measuring the absorbance of the solution, wherein the result is shown in the attached figure 11 of the specification.

As can be seen from the description of FIG. 11:

the food additives of sucrose and salt with the mass concentration of 2-6% can reduce the stability of sodium copper chlorophyllin, particularly, the sucrose with the mass concentration of 2% can reduce the stability of sodium copper chlorophyllin more, and the salt with the mass concentration of 2% has no obvious influence on the stability.

Similarly, the food additive sucrose with the mass concentration of 2% -6% can improve the stability of the sodium zinc chlorophyllin, and especially has the best stability when the concentration is 4%; the table salt has no obvious influence on the sodium zinc chlorophyllin, but the table salt with the mass concentration of 4% has better stability.

In the case of the same food additive, the overall OD value of the sodium zinc chlorophyllin is higher than that of the sodium copper chlorophyllin.

Therefore, under the condition of the food additive, the stability of the sodium zinc chlorophyllin is better than that of the sodium copper chlorophyllin.

Compared with the prior art, the invention has the beneficial effects that:

1. the leeks contain rich chlorophyll, and the leeks have the characteristics of rapid growth, easy culture, no limitation of places and seasons, and adjustable production scale, can be directly used as a food additive, are favorable for saving cost, can ensure resource supply, improve economic benefit, and have the nutrition and health care functions of the leeks.

2. The green pigment mainly containing chlorophyll can be obtained in two forms of fat-soluble and water-soluble, the stability of the water-soluble sodium copper chlorophyllin or the water-soluble sodium zinc chlorophyllin is better than that of the fat-soluble chlorophyll, and the products in 3 different forms have advantages and disadvantages. Compared with sodium zinc chlorophyllin, sodium copper chlorophyllin has higher light, heat and acid-base stability; compared with sodium copper chlorophyllin, the sodium zinc chlorophyllin has better stability to oxidation reducing agent and food additive, and the two water-soluble sodium salts have the functions of supplementing copper preparation and zinc preparation respectively.

Claims

1. an extraction method of leek green pigment, is characterized in that, comprises the following steps:

Step 1. Remove the yellow leaves on the chives, wash them, cut them into sections, then put them into a vacuum freeze dryer for freeze-drying, then pulverize them with a pulverizer, pass through a 40-60 mesh sieve, and store them at low temperature in the dark for future use;

Step 2, take by weighing 10g chive powder or directly take the fresh chives after washing and homogenize with a homogenizer at a ratio of 1:1;

Step 3: Add an aqueous solution containing a final mass concentration of 0.3% cellulase and 0.1% pectinase compound enzyme in a ratio of 1:4, and use salicylic acid to adjust the pH of the environment to 4.8, and water bath at 45°C for 60min , treated for 60S under the condition that the microwave power is 600W, filtered to remove the supernatant, and collected the filter residue A for use;

Step 4, adding the edible ethanol that the volume fraction is 95% by the ratio of the liquid to material ratio of 20:1ml/g to the filter residue A;

Step 5. Under the condition that the ultrasonic temperature is 60°C, the ultrasonic time is 60min, and the ultrasonic power is 200W, the extraction of green pigment is carried out. After the extraction is completed, suction filtration with a vacuum filter is used to collect the filtrate B and the filter residue C. During this extraction process Avoid light extraction;

Step 6, adding petroleum ether organic solvent to the filter residue C at a ratio of 10:1ml/g of liquid to material ratio, and then extracting according to step 5, until the filter residue is colorless, and after solid-liquid separation, the filtrate D is collected;

Step 7. Finally, combine the above-extracted filtrate B and filtrate D with 50°C water bath heating for 30-60min to deodorize, and then concentrate to 1g/ml by distillation under reduced pressure;

Step 8, obtaining a fat-soluble green pigment mainly composed of chlorophyll.

2. a method for preparing copper chlorophyll sodium salt according to the described leek green pigment of claim 1, is characterized in that, comprises the following steps:

S1: Saponification; pour the prepared chlorophyll-based fat-soluble green pigment into a beaker, adjust the pH to 12-13 with 10% NaOH, and saponify 30- 40min;

S2: complete saponification; take out after the saponification is completed, then cool to room temperature, filter to remove unsaponifiable viscous impurities, then move the saponification liquid into a separatory funnel and add the same volume of petroleum ether as the saponification liquid, then fully shake, stand still After layering, the upper layer turns yellow, which means complete saponification;

S3: acidify copper; put the extract from the lower layer of the separatory funnel in the above S2 into a beaker, adjust the pH value to 1.5-2.0 with 10% HCl, add acid to make it fully react, and the solution turns yellow-brown to generate pheophora green acid;

Detect the content of magnesium ions;

Add 10% copper sulfate solution, wherein, the addition amount of copper ions in the copper sulfate solution is added according to 1.5-2.0 times the theoretical amount of magnesium ions contained in the chlorophyll molecule;

Add slowly while stirring and ultrasonically bath for 1 h at 60°C to gradually replace magnesium ions in chlorophyll with copper ions, so that the solution recovers green and generates dark green chlorophyll cupric acid precipitation, then suction filtration, the obtained chlorophyll cupric acid is repeated with distilled water Wash to remove water-soluble impurities; then wash three times with 40% ethanol solution;

S4: forming a salt; after the copper is placed, dissolve the chlorophyll cupric acid described in the above S3 in 95% edible ethanol, add 10% NaOH solution while stirring, adjust the pH value of the solution to 12-13, and statically form a salt;

S5: removing impurities and drying; then repeatedly washing with petroleum ether to obtain a wet product of copper chlorophyll sodium salt, and removing ethanol and petroleum ether;

drying to obtain the finished product of copper chlorophyll sodium salt.

3. a method for preparing chlorophyll zinc sodium salt according to the described chive green pigment of claim 1, is characterized in that, comprises the following steps:

A: get 10ml of leaching and complete the concentration that the described fat-soluble green pigment based on chlorophyll of 1g/ml is placed in the round-bottomed flask;

b: saponification; add 10% NaOH to adjust pH to 12-13, saponification temperature is 60℃, saponification time is 30min, ultrasonic power is 200W;

c: extraction; add petroleum ether to the cooled saponification solution according to the condition of 1:1, leave it to stand for several hours to be layered, and the lower layer is a dark green saponification solution;

d: acidification; after collecting the dark green lower layer liquid, use 10% HCl to adjust the pH to 2-3;

e: replace zinc; add the acidified solution to zinc sulfate solution at a liquid-to-material ratio of 1:1, the ultrasonic temperature is 70 ° C, the ultrasonic time is 1 h, the ultrasonic power is 200 W, and the chlorophyll zinc acid precipitate is obtained after cooling;

f: after filtration, use distilled water to wash 3 times to obtain chlorophyll zinc acid;

g: form salt; add 10ml of edible ethanol with a concentration of 95%, add 10% NaOH to adjust the pH to 12-13, and let stand for 1h to form a salt;

h: After filtration, put it into a 45°C oven, and dry to obtain chlorophyll zinc sodium salt.

4. the comparison method of common factor to chlorophyll copper sodium salt and chlorophyll zinc sodium salt stability effect, is characterized in that, comprises the following steps:

1), extract fat-soluble green pigment from chives;

2), prepare chlorophyll copper sodium salt and chlorophyll zinc sodium salt respectively according to the fat-soluble green pigment extracted in step 1);

3) Prepare the test solution:

Weigh 0.1g of chlorophyll copper sodium salt and chlorophyll zinc sodium salt prepared from the same batch of fat-soluble green pigments, dissolve them in distilled water and dilute to 1L, and use this solution as the test solution for stability effect detection;

4), absorbance detection:

The absorbance was detected by microplate reader, the detection wavelength of chlorophyll copper sodium salt was 408 nm, and the detection wavelength of chlorophyll zinc sodium salt was 412 nm;

5), verify the influence of different factors on the stability of chlorophyll copper sodium salt and chlorophyll zinc sodium salt:

①、Verify the effect of light on the stability of chlorophyll copper sodium salt and chlorophyll zinc sodium salt:

Take 10ml of chlorophyll copper sodium salt and chlorophyll zinc sodium salt of the above-mentioned test solutions respectively, set 3 replicates for each sample, put them into 3 20ml glass test tubes with stoppers respectively, and place them in an indoor light place. Take 100-200 μL of solution at a certain time point of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days, and measure the absorbance value of the solution with a microplate reader;

2. Verify the effect of temperature on the stability of chlorophyll copper sodium salt and chlorophyll zinc sodium salt:

Take 10ml of chlorophyll copper sodium salt and chlorophyll zinc sodium salt of the above-mentioned test solutions and put them in a 20ml test tube and seal them. Each sample is set to 3 replicates and placed in a water bath at 40°C, 60°C, 80°C and 100°C. After 20min in water bath, cool to room temperature, take the unheated sample solution as a control and the corresponding solvent as a blank, take 100-200μL of the solution to measure the absorbance with a microplate reader;

3. To verify the effect of acid and alkalinity of different pH on the stability of chlorophyll copper sodium salt and chlorophyll zinc sodium salt:

Take 10ml of chlorophyll copper sodium salt and chlorophyll zinc sodium salt from the above-mentioned test solutions and put them in a 20ml test tube, set 3 repetitions for each sample, and use 1% NaOH and 10% HCl solution to adjust chlorophyll copper sodium salt and chlorophyll zinc sodium salt. The pH value of the salt is close to 2, 4, 6, 8, 10, 12. After mixing, let stand for 1 h at room temperature and then measure the chlorophyll copper sodium salt and chlorophyll zinc sodium salt under the corresponding pH conditions. the absorbance of the solution;

④. Verify the effects of oxidants and reducing agents on the stability of chlorophyll copper sodium salt and chlorophyll zinc sodium salt:

Take 10ml of chlorophyll copper sodium salt and chlorophyll zinc sodium salt from the above-mentioned test solutions and put them in a 20ml test tube, set 3 replicates for each sample, and add H2O2 with final concentrations of 0%, 0.30%, 0.60% and 0.90% respectively. and Na2SO3 solution, after mixing, let stand for 1 h at room temperature and measure the absorbance of the solution;

⑤、Verify the effect of different food additives on the stability of copper chlorophyll sodium salt and sodium chlorophyll zinc salt:

Take 10ml of chlorophyll copper sodium salt and chlorophyll zinc sodium salt from the above-mentioned test solutions and put them in a 20ml test tube, set 3 repetitions for each sample, and add the final concentrations of 0%, 2%, 4%, and 6% respectively. Concentrations of salt and sucrose were mixed and allowed to stand for 1 h at room temperature to measure the absorbance of the solution.