CN116375719A - Method for oligomerizing chlorophyll and inhibiting chlorophyll from self-polymerization - Google Patents

Abstract

The invention belongs to the technical field of new materials, and discloses an oligomeric chlorophyll which consists of 2-4 chlorophyll molecules. Meanwhile, the invention also discloses a preparation method of the oligomeric chlorophyll. The oligomeric chlorophyll of the invention is advantageously coated by microcapsules.

Description

A method for oligomeric chlorophyll and inhibition of chlorophyll self-aggregation

technical field

The invention belongs to the field of new materials, and in particular relates to a method for oligomerizing chlorophyll and inhibiting the self-aggregation of chlorophyll.

Background technique

Spirulina is used as a food and dietary supplement because of its many nutrients and health benefits, including chlorophyll. Spirulina has a huge stock in nature and has not been fully utilized. It is a kind of abundant resource reserve and has a good potential application prospect. However, the unique bad smell, taste and taste of spirulina itself and substances that may cause allergic reactions in some people limit its further development and utilization. direct and effective method.

Chlorophyll is widely present in the leaves and fruits of higher plants, presenting a bright green color. It is the main pigment for photosynthesis of plants, and it has a non-negligible effect on the growth of plants. In recent years, research on the benefits of chlorophyll to human health has become increasingly popular, and the efficacy of chlorophyll is worthy of attention. It is currently known that chlorophyll has the functions of lowering cholesterol, detoxifying, improving constipation, anti-aging, anti-inflammation, antibacterial, anti-cancer and anti-mutation for the human body. As a common natural pigment, chlorophyll has a huge storage capacity and can be seen everywhere. With the improvement of people's economic level and the pursuit of health, chlorophyll has a very broad market prospect. However, chlorophyll is not a stable structure, and chlorophyll will become an excited state under light conditions. At this time, chlorophyll will lose electrons, which will greatly increase its oxidation ability, and compete for electrons of other substances, such as water, which will make itself extremely unstable. This process is called photolysis of chlorophyll. It is precisely because of the poor stability and extremely poor water solubility of chlorophyll that limit their practical application, how to use chlorophyll more efficiently to make it beneficial to human health has become a difficult problem.

Microcapsule technology is a technical means to use stable and water-soluble compounds as wall materials alone or in combination to achieve the core matrix of sensitive compounds or chemical substances. Proteins (whey protein, soy protein, gelatin, etc.), polysaccharides (gum arabic, maltodextrin, modified starch, and chitosan, etc.) and lipids (phospholipids, etc.) are usually chosen as common wall materials for encapsulating active compounds. According to different core materials, it is the key of this technology to select the appropriate wall material for packaging to achieve the best packaging effect.

Chlorophyll tends to self-aggregate when encapsulated by wall materials in water solvents. The occurrence of this phenomenon is generally not related to the properties of wall materials, but directly related to the structural properties of chlorophyll itself. In polar organic solvents, chlorophyll can interact with the central magnesium atom in the structural composition of chlorophyll due to the OH group contained therein, and then simultaneously form hydrogen bonds and metal coordination bonds with pyrrole ring nitrogen. This kind of intermolecular interaction force will make the chlorophyll monomers aggregate to form high polychlorophyll, and this phenomenon is carried out spontaneously in polar solvents, such as aqueous solvents, but this high polychlorophyll is for micro Encapsulation is disadvantageous. The particle size of high-poly chlorophyll is relatively large, which will lead to larger particle size of the prepared chlorophyll microcapsules and larger holes on the surface. Such defects will significantly reduce the stability and bioavailability of the microcapsules.

In the prior art, no one has paid attention to the degree of polymerization of chlorophyll. During the research of this project, it was found that the polarity of the organic solvent is an important factor affecting the degree of polymerization of chlorophyll, but to achieve a low degree of polymerization, no matter No matter how to adjust the organic solvent for extraction, the goal cannot be achieved;

High-voltage pulsed electric fields (Pulsed electric fields, PEF) technology is an emerging non-thermal processing technology. Its principle is to apply short-pulse high-voltage electricity of sufficient strength to treat food materials, and to kill food by increasing the permeability of cell membranes. Microorganisms in food materials or to improve the efficiency of obtaining beneficial ingredients from food materials.

According to the search, high-voltage pulsed electric field technology is mainly used for cell wall breaking to improve the dissolution of cell contents.

CN111329055A discloses a preparation method of bok choy ferment, comprising the following steps: S1. Weigh bougainvillea, centrifuge to remove water after cleaning, add sterile water and pour it into a refiner for refining, the addition of the sterile water The amount is 8～12% of the total weight of bok choy, through 80 to 120 meshes, to get bok choy slurry; S2. add ethanol solution to the bok choy slurry obtained in step S1, and the addition amount of said ethanol solution is the total bok choy slurry. 6 to 8 times the volume, treated with a high-voltage pulsed electric field for 8 to 10 hours, filtered, and the filter residue was set aside, the filtrate was subjected to vacuum rectification, and ethanol was recovered to obtain a bougainvillea chlorophyll extract; S3. the bougainvillea chlorophyll extract obtained in step S2 and The filter residue is transported to the fermenter through a sterile pipeline, and at the same time, mixed strains are inserted, and the mixed strains are composed of Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus salivarius by 1 : 1:1:1:1 ratio mixing composition, the total volume of the mixed strains inserted is 1/1000 of the total volume of bok choy slurry;

S4. Add the prebiotics into the fermenter in step S3, stir evenly, and ferment for 4 to 5 weeks. The fermentation conditions are: the first week after the inoculation of the mixed strains, the temperature is set at 33-35 ° C, and the mixed strains are inoculated. From the second week, the temperature is set to 28-32°C to obtain the fermentation broth;

S5. The fermented liquid obtained in step S4 is subjected to negative high-pressure pulse treatment for 3 to 5 minutes, and then bottled to obtain

Its instructions record: when extracting chlorophyll from Bougainvillea, a specific high-voltage pulsed electric field with an electric field strength of 30-40kV/cm and a frequency of 4-6Hz can be used to greatly increase the extraction rate of chlorophyll in Bougainvillea. Its specific principle is: the specific high-voltage pulsed electric field treatment provided by the invention can accelerate the movement of molecules in the bok choy slurry, increase the dissolution rate of chlorophyll, and at the same time it can also reduce the particle size of the bok choy slurry and increase the solubility of particles and ethanol contact area, thereby increasing the dissolution rate of chlorophyll. Yet adopt the electric field intensity treatment and pulse that are higher than or lower than that this invention provides all can not improve the stripping rate of chlorophyll;

After fermentation, the fermentation broth is subjected to a specific negative high voltage pulse treatment with a negative pulse high voltage of -10 ~ -5kV and a frequency of 2 ~ 4Hz, which can greatly improve the stability of chlorophyll. The specific principle is: the specific negative high-voltage pulse treatment provided by the present invention can rapidly dissociate charged ions from the fermentation broth, and the negative pulse electric field makes the positive charges in the fermentation broth flow toward the inside, while the negative charges will gather in the fermentation broth. The surface of the liquid can not only prevent the chlorophyll molecules in the fermentation liquid from losing the magnesium in the porphyrin ring to become magnesium chlorophyll, but also improve the stability of chlorophyll.

CN103653125A discloses a production method of a natural chrysanthemum brain active drink, the key lies in the combination of blanching, zinc acetate and ascorbic acid to protect the color, inhibiting the formation of enzymatic oxidation and pheophytin, and maintaining the natural green color of fresh chrysanthemum brain; The bio-enzymatic method of breaking the wall promotes the release of active substances and improves the nutritional and active functions of the product; the use of high-voltage pulsed electric field sterilization effectively retains the biologically active components of chrysanthemum brain, reducing the oxidation and activity loss of phenolic substances. The invention adopts bipolar positive and negative pulse OSU-4L laboratory-scale pulse processor (produced by Ohio State University), which can effectively kill Escherichia coli and pathogenic bacteria in the chrysanthemum brain drink, making it commercially sterile, and the product does not contain Any preservatives have the unique fragrance of chrysanthemum brain, and the shelf life is more than 6 months.

Its manual records: The main purpose of using bipolar positive and negative pulse OSU-4L laboratory-scale pulse processor is to sterilize.

CN110799655A provides a method for processing raw sugarcane juice, comprising: reducing the pH of the sugarcane juice to a pH that substantially eliminates microbial activity; isolating chlorophyll from the sugarcane juice; isolating particles larger than 0.5 microns in diameter from the sugarcane juice; Bacteria denatured polyphenol oxidase (PPO) in sugarcane juice; the denatured polyphenol oxidase was isolated from sugarcane juice.

Its instructions record: wherein said pasteurization includes one or more of the following:

a) heat pasteurization;

b) pulsed electric field (PEF) pasteurization;

c) High pressure pasteurization.

It can be seen that the scheme uses pulsed electric field for sterilization.

CN114364688A provides a process for preparing and purifying proteins from plant materials, as well as compositions and uses comprising the proteins, and its specification records:

1. A process for preparing a purified protein product from plant material, comprising:

a) providing said plant material in a buffered solution comprising a reducing agent;

b) cracking said plant material;

c) separating said lysed plant material into a solid phase and a liquid phase, wherein said liquid phase contains soluble proteins and chlorophyll;

d) condensing the chlorophyll in said liquid phase by heating it to a first set temperature in no more than about 30 minutes and then cooling it to a second set temperature in no more than about 30 minutes, wherein said cooling starts when said liquid phase reaches said first set temperature;

e) contacting the liquid phase in d) with a flocculant and/or adsorbent and mixing for a time sufficient to flocculate and/or adsorb chlorophyll in the liquid phase to the adsorbent to form a flocculated mixture ;

f) separating said flocculated mixture in e) into a solid phase and a liquid phase; and

g) filtering said liquid phase in f) to produce a filtrate containing purified protein.

Wherein the plant material is mechanically lysed using a press, sonicator, pulverizer, using a pulsed electric field, agitating using a nitrogen explosion, using ultrasonic energy or by freezing.

Therefore, it can be seen that this scheme is the effect of using pulsed electric field for cell lysis.

Therefore, the core of this project is how to control chlorophyll to keep it in a stable low poly state, so as to facilitate subsequent utilization.

Contents of the invention

Aiming at the deficiencies in the prior art, the object of the present invention is to provide a method for inhibiting chlorophyll self-aggregation;

In the present invention, the electric field and magnetic field effects of the PEF technology are used to prevent the self-aggregation effect of chlorophyll in the mixed solvent containing alcohol solvent and non-polar organic solvent to obtain the expected oligomerization purpose.

At the same time, the invention also provides an oligomeric chlorophyll.

In order to achieve the purpose of the invention, the present invention adopts the following technical scheme: a kind of oligomeric chlorophyll, and the oligomeric chlorophyll is composed of 2 to 4 chlorophyll molecules.

Simultaneously, the present invention also discloses a method for inhibiting chlorophyll self-aggregation, comprising the following steps:

Step 1: using an alcohol solvent to extract chlorophyll from plant cells to obtain a slurry, adding the slurry to a non-polar organic solvent to obtain an organic solution containing chlorophyll;

Step 2: Treat the organic solution with a bipolar high-voltage pulsed electric field to form oligomeric chlorophyll with a degree of polymerization less than or equal to 4; the electric field strength of the bipolar high-voltage pulsed electric field is 1-10 kV/cm, and the pulse width is 20-40 μs , the frequency is 10-100Hz; the number of pulses is 10-300.

The alcohol solvent described in the present invention may be methanol or ethanol, and from the perspective of safety, ethanol is the most preferred.

Generally speaking, steps 1 and 2 are carried out continuously, that is, step 2 needs to be carried out as soon as possible after the extraction of the organic solution containing chlorophyll, otherwise, the organic solution containing chlorophyll obtained in step 1 is prone to self-polymerization.

In some embodiments of the present invention, the electric field strength of the bipolar high-voltage pulsed electric field is 1kV/cm, 2kV/cm, 3kV/cm, 4kV/cm, 5kV/cm, 6kV/cm, 7kV/cm, 8kV/cm cm, 9kV/cm or 10kV/cm;

In some implementation cases of the present invention, the pulse width of the bipolar high-voltage pulsed electric field is 20 μs, 30 μs or 40 μs;

In some embodiments of the invention, the frequency is 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz or 100 Hz.

In the above-mentioned method for inhibiting the self-aggregation of chlorophyll, a bipolar high-voltage pulsed electric field is used for a treatment time of 10 to 60 minutes.

In some embodiments of the present invention, the treatment time of the bipolar high-voltage pulsed electric field is 10 min, 20 min, 30 min, 40 min, 50 min or 60 min.

In some embodiments of the present invention, the number of bipolar high-voltage pulsed electric fields is 10, 20, 30, 40, 50, 100, 200 or 300.

In the above method for inhibiting chlorophyll self-aggregation, the treatment temperature in step 2 is 20-40°C.

Generally speaking, it is enough to control it at room temperature.

In the present invention, the non-polar organic solvent can be selected as: n-hexane or benzene.

In the above-mentioned method for inhibiting chlorophyll self-aggregation, the step 1 is specifically:

Step 11: Extracting chlorophyll from plants by using an alcoholic organic solvent, supplemented by ultrasonic vibration extraction, to obtain a crude extract;

Step 12: adding the crude extract into a non-polar organic solvent to obtain an organic solution containing chlorophyll.

More specifically, in the step 11, chlorophyll is extracted from plants by using absolute ethanol in combination with ultrasonic vibration extraction and crushing, homogeneous stirring and extraction;

The ultrasonic sound energy density is 1.0-1.6W/g, the ultrasonic action time is 0.5-2.5h, and the weight ratio of solid to liquid is 1:10-20; after extraction, the crude chlorophyll extract is obtained by centrifugation;

The step 12 specifically includes: adding n-hexane to the crude chlorophyll extract, and collecting the supernatant to obtain the spirulina chlorophyll extract.

Preferably, the plants are green algae plants or seed plants.

As an example, the algae can be selected from Spirulina maxima, Spirulina platensis, Chlorella, Pyramid marine algae or Ulva pore;

Seed plants of choice are kale, spinach, lettuce, or alfalfa.

Preferably, the concentration of chlorophyll in the organic solution is 100 μg/mL˜300 μg/mL, and the purity of chlorophyll is 70˜90%.

It should be noted that, in addition to chlorophyll, there are some oil-soluble substances extracted from cells in the organic solvent, so 70-90% chlorophyll purity means: the content of chlorophyll in the solid phase in the organic solvent is 70% ~90wt%.

Compared with the prior art, the present invention has the following beneficial effects:

The invention reduces the degree of self-aggregation of chlorophyll in the mixed solvent containing alcohol solvent and non-polar organic solvent by treating the organic solvent containing chlorophyll through the high-voltage pulse electric field.

At the same time, we also found that the chlorophyll treated by the high-voltage pulsed electric field promoted the interaction between phytol and ester bonds in the structure of chlorophyll oligomers and the hydrophobic residues of tyrosine and tryptophan in the structure of whey protein isolate, making the preparation The resulting chlorophyll microcapsules have smaller particle size, better encapsulation effect and better stability.

In terms of its principle, the electric field and magnetic field effects of the high-voltage pulsed electric field can cause changes in the ionization degree and charge number of the polar groups of the compound in the solution, thereby changing the weak non-polar covalent bonds and intermolecular forces in the compound molecules. Furthermore, it prevents the OH group contained in the polar solvent from interacting with the central magnesium atom in the chlorophyll structure, and simultaneously forms hydrogen bonds and metal coordination bonds with the pyrrole ring nitrogen, so as to realize the synthesis of chlorophyll with a low degree of polymerization.

It should be noted that the use of ethanol to extract chlorophyll is low in extraction cost and easy to operate. On the other hand, by adding it to a non-polar organic solvent and controlling the ethanol content, a self-polymerizing environment can be created. Under the support of the electric field, relatively restrained self-polymerization is maintained; therefore, from the perspective of necessity, the use of ethanol for extraction and ethanol-n-hexane mixed solvent for post-treatment is an effective guarantee for the realization of oligomerization.

Detailed ways

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the examples are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

Example 1

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 1 g of spirulina powder with 10 mL of absolute ethanol, set the ultrasonic sound energy density to 1.0 W/g, and use ultrasonic The time is 0.5h, and the ratio of solid to liquid is 1:10 (w/w).

After extraction, centrifuge at 13,000g at 4°C to obtain about 10mL of crude chlorophyll extract, then add 50mL of n-hexane for purification, and collect the supernatant as the spirulina chlorophyll extract.

Step 2: Use PEF technology to prevent chlorophyll from self-aggregating to form oligomeric chlorophyll with a degree of polymerization of less than 4: place the chlorophyll extract of Spirulina at a temperature of 20°C in a bipolar high-voltage pulsed electric field with a pulse width of 40 μs for 10 minutes, wherein The electric field strength is 1kV/cm, the frequency is 10Hz, and the number of pulses is 10; in order to prevent the self-aggregation of chlorophyll, and form oligomeric chlorophyll with a polymerization degree of less than 4, and finally obtain the oligomeric chlorophyll extract of Spirulina.

Example 2

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 2 g of spirulina powder with 30 mL of absolute ethanol, set the ultrasonic sound energy density to 1.3 W/g, and ultrasonically The time is 1.5h, and the ratio of solid to liquid is 1:15 (w/w).

After extraction, centrifuge at 13,000g at 4°C to obtain about 30mL of crude chlorophyll extract, then add 150mL of n-hexane for purification, and collect the supernatant as Spirulina chlorophyll extract.

Step 2: Use PEF technology to prevent chlorophyll from self-aggregating to form oligomeric chlorophyll with a degree of polymerization of less than 4: place the chlorophyll extract of Spirulina at a temperature of 30°C in a bipolar high-voltage pulsed electric field pulse with a pulse width of 30 μs for 30 minutes, The electric field strength is 5kV/cm, the frequency is 50Hz, and the number of pulses is 150; in order to prevent the self-aggregation of chlorophyll, and form oligomeric chlorophyll with a degree of polymerization less than or equal to 4, and finally obtain the spirulina oligomeric chlorophyll extract .

Example 3

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 5 g of spirulina powder with 100 mL of absolute ethanol, set the ultrasonic sound energy density to 1.6 W/g, and use ultrasonic The time is 2.5h, the ratio of solid to liquid is 1:20 (w/w).

After extraction, centrifuge at 13,000g at 4°C to obtain about 100mL of crude chlorophyll extract, then add 500mL of n-hexane for purification, and collect the supernatant as the spirulina chlorophyll extract.

Step 2: Use PEF technology to prevent the self-aggregation of chlorophyll to form oligomeric chlorophyll with a degree of polymerization of less than 4: place the chlorophyll extract of Spirulina at a temperature of 40°C in a bipolar high-voltage pulsed electric field pulse with a pulse width of 20 μs for 60 minutes, The electric field strength is 10kV/cm, the frequency is 80Hz, and the number of pulses is 300; in order to prevent the self-aggregation of chlorophyll, and form oligomeric chlorophyll with a degree of polymerization less than or equal to 4, and finally obtain the spirulina oligomeric chlorophyll extract .

Example 4

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 1 g of spirulina powder with 10 mL of absolute ethanol, set the ultrasonic sound energy density to 1.0 W/g, and use ultrasonic The time is 0.5h, and the ratio of solid to liquid is 1:10 (w/w).

After extraction, centrifuge at 13,000g at 4°C to obtain about 10mL of crude chlorophyll extract, then add 50mL of n-hexane for purification, and collect the supernatant as the spirulina chlorophyll extract.

Step 2: Use PEF technology to prevent the self-aggregation of chlorophyll to form oligomeric chlorophyll with a degree of polymerization of less than 4: place the chlorophyll extract of Spirulina at a temperature of 20°C in a bipolar high-voltage pulsed electric field pulse with a pulse width of 40 μs for 10 minutes, The electric field strength is 10kV/cm, the frequency is 100Hz, and the number of pulses is 10; in order to prevent the self-aggregation of chlorophyll, and form oligomeric chlorophyll with a degree of polymerization less than or equal to 4, and finally obtain the spirulina oligomeric chlorophyll extract .

Comparative example 1

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 1 g of spirulina powder with 10 mL of absolute ethanol, set the ultrasonic sound energy density to 1.0 W/g, and use ultrasonic The time is 0.5h, and the ratio of solid to liquid is 1:10 (w/w).

After extraction, centrifuge at 13,000g at 4°C to obtain 10mL of crude chlorophyll extract, then add 50mL of n-hexane for purification, and collect the supernatant as Spirulina chlorophyll extract.

Comparative example 2

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 1 g of spirulina powder with 10 mL of absolute ethanol, set the ultrasonic sound energy density to 1.0 W/g, and use ultrasonic The time is 0.5h, and the ratio of solid to liquid is 1:10 (w/w).

After extraction, centrifuge at 13,000g at 4°C to obtain about 10mL of crude chlorophyll extract, then add 50mL of n-hexane for purification, and collect the supernatant as the spirulina chlorophyll extract.

Step 2: Place the Spirulina chlorophyll extract at 20°C in a bipolar high-voltage pulsed electric field with a pulse width of 10 μs for pulse ionization for 10 minutes, where the electric field strength is 0.5kV/cm, the frequency is 200Hz, and the number of pulses is 10 The collected supernatant is the Spirulina chlorophyll extract.

Comparative example 3

Step 1: Prepare spirulina chlorophyll extract: use ultrasonic vibration extraction and crushing homogeneous stirring extraction to extract chlorophyll from 1 g of spirulina powder with 10 mL of absolute ethanol, set the ultrasonic sound energy density to 1.0 W/g, and use ultrasonic The time is 0.5h, and the ratio of solid to liquid is 1:10 (w/w).

After extraction, centrifuge at 13000g at 4°C to obtain about 10mL of crude chlorophyll extract, then add 50mL of polar solvent-ethanol aqueous solution (absolute ethanol: water = 1:2, v/v) for purification, and collect the supernatant as Spirulina Chlorophyll Extract.

Step 2: Place the chlorophyll extract of Spirulina in a bipolar high-voltage pulsed electric field pulse with a pulse width of 40 μs at a temperature of 20 ° C for 10 minutes, wherein the electric field strength is 1 kV/cm, the frequency is 10 Hz, and the number of pulses is 10; The collected supernatant is the Spirulina chlorophyll extract.

performance test 1

Particle size measurement: The particle size of the chlorophyll aggregates in the chlorophyll solutions of Examples 1-5 and Comparative Examples 1-3 was tested by dynamic light scattering (DLS) using Malvern Nanosizer ZS (Malvern, Worcestershire, UK) equipment. Each sample was tested in triplicate at room temperature. The control group used n-hexane to dissolve chlorophyll (chromatographically pure, sigma company).

The results can be referred to Table 1:

Table 1 particle size test results

^ah Different letters in the same column indicate significant differences between each other (p<0.05)

As can be seen from the results in Table 1, compared to the chlorophyll control group dissolved in non-polar solvents, the particle sizes of Examples 1, 2, 3, and 4 are all relatively small, and the particle size ratio is between 3.62 and 1.67 , while the particle diameters of Comparative Examples 1, 2, and 3 are close to the micron level, and the particle diameter ratio exceeds 92. Wherein, there is ethanol in the mixed solvent in Comparative Example 1, and self-polymerization will occur during the standing process; Comparative Example 2 Due to the low electric field strength, it is not enough to inhibit the interaction between the OH group and the central magnesium atom in the structure of chlorophyll, the simultaneous formation of hydrogen bonds and metal coordination bonds with the pyrrole ring nitrogen, resulting in larger particle size; comparative example In 3, the pure ethanol phase is used and also contains water, even under the action of an electric field, a violent self-polymerization reaction will occur.

The above experiments show that the method of this patent can effectively reduce the occurrence of chlorophyll self-aggregation.

performance test 2

Performance test of microcapsule preparation

Microcapsule preparation method is as follows:

Dissolve whey protein isolate powder and gum arabic powder in deionized water at a mass ratio of 1:1 to obtain a composite wall material solution with a concentration of 2%, which is cooled to room temperature and stored overnight at 0°C to ensure Fully hydrated. Then the spirulina oligomeric chlorophyll extract and the composite wall material solution were mixed according to the volume ratio of 1:2, placed in a bipolar high-voltage pulsed electric field at room temperature, and ionized with 10 pulses of 100 μs for 10 minutes, wherein the pulsed electric field intensity was 10kV/cm, frequency 10Hz, to promote the interaction between phytol and ester bonds in the structure of oligochlorophyll and the hydrophobic residues of tyrosine and tryptophan in the structure of whey protein isolate. Finally, spirulina oligomeric chlorophyll microcapsules were obtained by freeze-drying.

In the above method, the spirulina oligomeric chlorophyll extract used is prepared from the final products of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3. The concentrations of chlorophyll in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 are similar.

Test item 1: Determination of visual color of spirulina oligomeric chlorophyll microcapsules

The visual color of each microcapsule was measured by a colorimeter at room temperature. Measurements are expressed in Hunter color values, L*, a*, and b* values, representing lightness (0 to 100), greenness (-) to redness (+) and blueness (-) to yellowness (+ ). In addition, the color difference (ΔE*) calculation equation (1) is as follows, which indicates the color intensity of the sample.

In the formula, ΔL*, Δa* and Δb* are the differences between Examples 1, 2, 3 and the control group, respectively.

The visual color of the spirulina oligomeric chlorophyll microcapsules prepared by different embodiments and comparative examples in table 2

Different letters in the same column of ^ad indicate significant differences between each other (p<0.05)

It can be seen from the results in Table 2 that the L ^* of Example 1 is significantly lower than that of the control group, which may be due to the formation of uniform chlorophyll microcapsules and a decrease in brightness. The a ^* values are all negative, because the chlorophyll itself is green, which causes the microcapsules to appear green as a whole, and the a ^* value in Example 1 is the smallest, indicating that the formed microcapsules are relatively uniform. Compared with Example 1, the ΔE ^* of Comparative Examples 1, 2, and 3 are all greater than 4, indicating that there is a difference visible to the naked eye between the Comparative Example and the Example, and this difference is related to the uniformity of the microcapsules.

Test item 2

Detection of Encapsulation Efficiency (EE) of Spirulina Oligomerized Chlorophyll Microcapsules

50 mg of microcapsules were added into 5 mL of ethanol while stirring for 10 min, and then the mixture was centrifuged at 9000×g for 10 min to obtain supernatant A. Another sample (50 mg) was added to 5 mL of ethanol, and treated with an ultrasonic cell disruptor at 350 W for 10 min. Then, centrifuge at 9000×g for 10 min to obtain supernatant B. The chlorophyll content of the supernatants A and B were measured with a UV-visible spectrometer, which represented the surface chlorophyll (S _CHs ) and total chlorophyll (T _CHs ) content of the microcapsule samples, respectively, and the chlorophyll was calculated using equations (2) and (3) encapsulation rate.

C _Chlorophyll ＝6.10×A ₆₆₅ +20.04×A ₆₄₉ (2)

Among them, chlorophyll represents the concentration of CH (μg/mL), A665 and A649 represent the absorbance at 665 and 649 nm, respectively, and 6.10 and 20.04 are transfer coefficients.

The total chlorophyll (T _CHs ), surface chlorophyll (S _CHs ) and encapsulation efficiency (EE) of the spirulina oligomeric chlorophyll microcapsules prepared by different embodiments and comparative examples in table 3

Different letters in the same column of ^ac indicate significant differences between each other (p<0.05)

As can be seen from Table 3, compared with the control groups 1, 2, and 3, the surface chlorophyll content of the microcapsules prepared in Example 1 is the lowest, and shows a significant difference (p<0.05). The embedding rate of Example 1 exceeds more than 92%, which is 11.19%, 9.16% and 12.07% higher than that of the comparisons 1, 2 and 3 respectively, indicating that the oligomeric chlorophyll microcapsules prepared by the patent method have a better embedding rate , which is directly related to the existence of chlorophyll in an oligomeric form.

Test item 3: Detection of DPPH free radical scavenging activity of Spirulina oligomeric chlorophyll microcapsules

Take microcapsules stored at room temperature for 1 month, mix 100 mg of microcapsules with 10 mL of deionized water by ultrasonic wave for 1 min, and then centrifuge at 9000×g for 10 min. Subsequently, 3 mL of the supernatant was mixed with 2 mL of DPPH solution (0.1 mmol/L, dissolved in absolute ethanol), and then reacted at room temperature for 30 min in the dark. Finally, the absorbance of the DPPH mixed solution of the sample was measured at 517 nm with a UV-Vis spectrophotometer. DPPH radical scavenging activity was calculated by formula (5).

Here A _sample represents the absorbance value of the sample solution, A _blank represents the absorbance value of the distilled water instead of the sample solution, and A _control represents the absorbance value of the ethanol instead of the DPPH solution.

The DPPH free radical scavenging rate test result of the spirulina oligomeric chlorophyll microcapsules prepared by different embodiments and comparative examples of table 4

Different letters in the same column of ^ac indicate significant differences between each other (p<0.05)

As can be seen from Table 4, after one month of storage, the DPPH free radical scavenging rate of embodiment 1 is significantly higher than comparative example 1,2,3, and shows significant difference (p<0.05), illustrates that the present invention prepares Oligomeric chlorophyll microcapsules have a more stable embedding structure, which improves the stability of chlorophyll in the microcapsules during storage, thereby improving the antioxidant activity, which is different from the chlorophyll in the form of oligomeric (n≤4) and microcapsules. And the high embedding rate of chlorophyll is related.

The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. An oligomeric chlorophyll, characterized in that the oligomeric chlorophyll consists of 2-4 chlorophyll molecules.

2. A method of inhibiting chlorophyll self-aggregation comprising the steps of:

step 1: extracting chlorophyll from plant cells by adopting an alcohol solvent to obtain slurry, and adding the slurry into a nonpolar organic solvent to obtain a chlorophyll-containing organic solution;

step 2: treating the organic solution by using a bipolar high-voltage pulse electric field to form oligomeric chlorophyll with a polymerization degree of less than or equal to 4; the electric field strength of the bipolar high-voltage pulse electric field is 1-10 kV/cm, the pulse width is 20-40 mu s, and the frequency is 10-100 Hz; the number of pulses is 10-300.

3. A method for inhibiting chlorophyll self-aggregation according to claim 2, characterized in that the bipolar high voltage pulse electric field treatment time is 10-60 min; the treatment temperature of the step 2 is 20-40 ℃.

4. A method of inhibiting chlorophyll self-polymerization according to claim 2, wherein the volume ratio of said slurry and non-polar organic solvent is 1:4-6.

5. A method of inhibiting chlorophyll self-polymerization according to claim 2, wherein said non-polar organic solution is n-hexane or benzene.

6. The method for inhibiting chlorophyll self-aggregation according to claim 5, wherein said step 1 specifically comprises:

step 11: extracting chlorophyll from plants by adopting an alcohol organic solvent and assisted ultrasonic vibration extraction to obtain a crude extract;

step 12: adding the crude extract into nonpolar organic solvent to obtain organic solution containing chlorophyll.

7. The method for inhibiting self-aggregation of chlorophyll according to claim 6, wherein in said step 11, the method of combining ultrasonic vibration extraction and crushing and homogenizing stirring extraction is used for extracting chlorophyll from plants by using absolute ethanol.

8. The method for inhibiting chlorophyll self-aggregation according to claim 7, wherein said step 11 is specifically: the ultrasonic energy density is 1.0-1.6W/g, the ultrasonic action time is 0.5-2.5 h, and the weight ratio of the feed liquid is 1:10-20; obtaining a chlorophyll crude extract through centrifugal operation after extraction;

the step 12 specifically includes: adding n-hexane into the chlorophyll crude extract, and collecting supernatant to obtain spirulina chlorophyll extract.

9. A method of inhibiting chlorophyll self-aggregation according to claim 2, wherein said plant is a green algae plant or a seed plant.

10. The method for inhibiting chlorophyll self-polymerization according to any one of claims 2 to 9, wherein the concentration of chlorophyll in said organic solution is 100 μg/mL to 300 μg/mL, and the chlorophyll purity is 70 to 90%.