KR102101988B1 - A preparation method for for enzyme treated-spirulina hydrolysate with improved heat stability and increased phycocyanin - Google Patents

Abstract

The present invention relates to a production method of spirulina hydrolysate with a high phycocyanin content, which comprises the steps of: hydrolyzing spirulina with proteolytic enzyme; and obtaining the spirulina hydrolysate.

Description

Method for manufacturing spirulina hydrolyzate with improved thermal stability and increased phycocyanin content manufactured using enzymes {A PREPARATION METHOD FOR FOR ENZYME TREATED-SPIRULINA HYDROLYSATE WITH IMPROVED HEAT STABILITY AND INCREASED PHYCOCYANIN}

The present invention relates to a method for producing a spirulina hydrolyzate having an increased phycocyanin content.

Spirulina is one of the oldest birds on the planet. It is a turquoise blue-green algae that first appeared on the Earth's surface about 3 to 4 billion years ago. It is called Spirulina because it is spirally twisted under a microscope (Yang HN, Lee EH, Kim HM) 1997. Spirulina platensis inhibits anaphylactic reaction.Lif Sci 61: 1237-1244). Spirulina is a non-toxic cyanobacteria that contains polysaccharides, vitamins, minerals, unsaturated fatty acids, carotenoids and phycobiliproteins, which are used as food supplements. Spirulina's biomass consists of 55 to 70% protein, 3 to 9% fat, and 15 to 30% carbohydrates in addition to fiber and pigments.

Phycobiliproteins are phycobilisomes attached to the chloroplast thylakoid membrane and are photosynthetic pigments produced in cells. Cyanobacteria's phycobiliproteins can be divided into three main components: Phycoerythrin (bright pink, red), phycocyanin (dark blue) and allopicocyanin (brighter blue). These phycobiliproteins are widely used in pharmaceuticals, food, cosmetics and fluorescent materials.

Phycocyanin is a water-soluble pigment that is widely used in food (chewing gum, dairy, jelly, etc.), cosmetics (lipstick and eyeliner), fluorescent reagents, and probes for clinical diagnostic equipment. Phycocyanin has been reported to have various physiological activities, and is known to have antioxidant, anti-inflammatory, hepatoprotective and radical scavenging properties. However, despite having various physiological activities of phycocyanin, the use of phycocyanin in food and other applications is limited due to the problem of poor thermal stability.

Meanwhile, the spirulina extraction method is known as a simple extraction method using hot water or an organic solvent and a supercritical extraction method using CO2. The method using hot water or an organic solvent is simple, but the yield of the extract is less than 20%, and there is a big disadvantage of loss of components containing spirulina, and extraction with an organic solvent may cause a problem that the organic solvent remains. In addition, the supercritical extraction method has the disadvantage of requiring expensive equipment.

The present inventors provide an extraction process that improves the extraction efficiency of spirulina and increases the stability of phycocyanin.

An object of the present invention is to provide a method for producing a spirulina hydrolyzate having an increased phycocyanin content.

The present invention,

Hydrolyzing spirulina with a proteolytic enzyme; and

Comprising the step of obtaining a spirulina hydrolysate,

Provided is a method for producing a spirulina hydrolyzate comprising phycocyanin.

Spirulina hydrolyzate produced by the production method of the present invention has a high phycocyanin content.

Figure 1 shows the effect of commercial protease enzymes on (A) C-phycocyanin, (B) allopicocyanine, and (C) Spirulina extract (SE) index in Spirulina. Values are expressed as mean ± standard deviation. Other characters show a significant difference of P <0.05 by Duncan's multiple range test.
FIG. 2 shows the effect of commercial hemicellulase enzymes on (A) C-phycocyanin, (B) allopicocyanine, and (C) Spirulina extract (SE) index in Spirulina. Values are expressed as mean ± standard deviation. Other characters show a significant difference of P <0.05 by Duncan's multiple range test.
FIG. 3 shows the effect of protease enzymes mixed in Spirulina on (A) C-phycocyanin, (B) allopicocyanine, and (C) solids recovery. Values are expressed as mean ± standard deviation. Other characters show a significant difference of P <0.05 by Duncan's multiple range test.
FIG. 4 shows the effect of protease concentrations on (A) C-phycocyanin, (B) allopicocyanine, and (C) solids recovery in Spirulina. Values are expressed as mean ± standard deviation. Other characters show a significant difference of P <0.05 by Duncan's multiple range test.

5 shows the effect of enzyme reaction temperatures in Spirulina on concentrations (A) C-phycocyanin, (B) allopicocyanin, and (C) solids recovery. Values are expressed as mean ± standard deviation. Other characters show a significant difference of P <0.05 by Duncan's multiple range test.
6 shows the effect of enzyme reaction times in Spirulina on concentrations (A) C-phycocyanin, (B) allopicocyanin, and (C) solids recovery. Values are expressed as mean ± standard deviation. Other characters show a significant difference of P <0.05 by Duncan's multiple range test.
Figure 7 shows the change in CR values after various temperatures and pHdptj duf treatment for 30 minutes.
8 shows changes in CR values after heat treatment at 95 ° C. for 10 minutes by addition of cyclodextrin (CD) and / or tannic acid.
9 shows changes in CR values after heat treatment at 95 ° C. for 10 minutes by addition of cysteine.
10 shows changes in CR values after heat treatment at 95 ° C. for 10 minutes by adding cysteine during extraction of copulaline.
11 is scanning results before and after heat treatment by adding cysteine when extracting copulaline.
12 shows changes in CR values after heat treatment at various temperatures and pH for 30 minutes.

The present invention,

Hydrolyzing spirulina with a proteolytic enzyme; and

Comprising the step of obtaining a spirulina hydrolysate,

It relates to a method for producing a spirulina hydrolyzate containing phycocyanin.

Hereinafter, the present invention will be described in detail.

Spirulina hydrolysis steps

The manufacturing method of the present invention includes the step of hydrolyzing spirulina with a proteolytic enzyme. The proteolytic enzyme is at least one selected from the group consisting of copulaline and protamax, and may preferably be copulaline. The hydrolysis step may be performed by treating 4 to 10 parts by weight of protease with respect to 100 parts by weight of spirulina, and may be performed by hydrolysis for 1.5 to 6 hours. The hydrolysis step may be performed under the conditions of pH 4.5 to pH 8, preferably under the conditions of pH 4.5 to pH 6.5.

In this case, the hydrolysis step may be performed by treating spirulina with proteolytic enzyme and cysteine. When spirulina is hydrolyzed, the stability of phycocyanin in spirulina hydrolyzate is increased by treating cysteine together. That is, the stability of phycocyanin in the hydrolyzate hydrolyzed spirulina using an enzyme and cysteine is higher than that of a hydrolyzate hydrolyzed spirulina only with an enzyme.

At this time, the hydrolysis step may be performed by treating cysteine with 0.5 to 3 parts by weight based on 100 parts by weight of spirulina. Preferably, the hydrolysis step may be performed by treating 4 to 10 parts by weight of protease and 0.5 to 3 parts by weight of cysteine with respect to 100 parts by weight of spirulina.

Spirulina hydrolyzate obtaining step

The production method of the present invention includes the step of obtaining a spirulina hydrolyzate. At this time, the method of obtaining the spirulina hydrolyzate may be a method commonly used in the art or known to be usable, and is not particularly limited.

Spirulina hydrolyzate of the present invention

Spirulina hydrolyzate produced by the production method of the present invention is characterized by high phycocyanin content. Preferably, the spirulina hydrolyzate produced by the production method of the present invention has a higher phycocyanin content than the hydrolyzate hydrolyzed spirulina or spirulina with other enzymes. Preferably, the spirulina hydrolyzate produced by the production method of the present invention has a higher phycocyanin content than the hydrolyzate hydrolyzed spirulina with a flavozyme, particularly a high C-phycocyanin content, an allopicocyanin content and a SE index (index) is also high.

In addition, the spirulina hydrolyzate of the present invention not only has a high phycocyanin content, but also has the feature of increasing the stability of phycocyanin in the spirulina hydrolyzate.

The spirulina hydrolyzate of the present invention has a c-phycocyanin content of 1 mg / g to 10 mg / g, preferably 1 mg / g to 5 mg / g. Spirulina hydrolyzate of the present invention has an allopicocyanine content of 1 mg / g to 10 mg / g, preferably 1 mg / g to 5 mg / g.

Spirulina hydrolyzate of the present invention can be used in the manufacture of food, cosmetics, reagents, clinical equipment, medicine, etc., and can also be used for other purposes. The use of the spirulina hydrolyzate of the present invention is not particularly limited.

Advantages and features of the present invention, and methods for achieving them will be clarified with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains. It is provided to completely inform the person having the scope of the invention, and the present invention is only defined by the scope of the claims.

<Materials and methods>

material

Spirulina used in the experiment was 100% Earthrise® Natural Spirulina (Arthrospira platensis) powder and was a product of EARTHRISE NUTRITIONALS, LLC (USA). Examples of cell wall degrading enzymes include Cytolase PLC5, Rapidase TF, Novozymes®, Ultraflo® L, Ultraflo® Max, and Viscozyme® L from Vision Biochem, and Flavozyme®, Protamex, DSM Food specialtie, Collupulin MG from Novozymes® as proteolytic enzymes. Used.

Spirulina Enzyme Treatment

Spirulina powder was suspended in 30 ml citric acid buffer (0.05M citrate buffer, pH 5.0) at a concentration of 5% (w / v). The enzyme was decomposed for 12 hours under the reaction conditions of Table 1 by adding an amount corresponding to 3% of the substrate. The reaction solution after the enzyme treatment was boiled for 15 minutes to stop the enzyme reaction, and then centrifuged at 5000 rpm for 20 minutes to obtain a supernatant, which was lyophilized.

Reaction conditions of enzymes for spirulina hydrolysis

Method of analysis

The extraction yield was expressed as solid recovery, and was calculated based on the weight ratio of the used raw spirulana powder and the freeze-dried extract. The obtained phycocyanin (allophycocyanin) and allophycocyanin (allophycocyanin) was calculated by the following equations 1 and 2 by measuring the absorbance of the supernatant obtained after enzymatic hydrolysis at 620 nm and 652 nm, respectively.

<Equation 1>

<Equation 2>

A620: absorbance at 620 nm

A652: absorbance at 652 nm

Stability of phycocyanin pigment

The stability evaluation according to the change of pH and temperature of the phycocyanin aqueous solution pigment was performed. Specifically, a solution of 1% (v / v) of phycocyanin extract at pH 5.0, 6.0, 7.0 using citrate phosphate buffer was heat treated at 50, 60, and 70 ° C for 30 minutes, respectively, before and after heat treatment at 620 nm. The absorbance was measured to evaluate the stability of the pigment. At this time, the stability of the pigment was expressed by the relative residual amount of phycocyanin (CR,%) before and after heat treatment according to Equation 3.

<Equation 3>

Evaluation of stability improvement ability of phycocyanin

A solution was prepared by adding phycocyanin extract to 1% (v / v) in Citrate buffer (pH 5.0). Then, the effects of phycocyanin pigment stability were measured by adding guagum, cysteine, and cystine to the prepared solution. At this time, dye stability was evaluated by measuring absorbance at 620 nm before and after the heat treatment at a temperature of 90 ° C.

<Result>

<Experiment 1> Content of phycocyanin extract according to proteolytic enzyme

The phycocyanin content of spirulina according to the addition of protease, flavozyme, and protease, which are powdery hydrolytic enzymes, was analyzed.

100 mg of enzyme was added to a solution containing 5% (v / v) of substrate spirulina and reacted at 45 ° C for 9 hours. When the enzyme collupulin was added, the contents of C-phycocyanin and allopicocyanin were significantly higher than before and after the reaction compared to flavozyme and protamax. In the spirulina extraction (SE) index, which is a method of indicating the content of nucleic acids in the spirulina extract according to the following formula 4, the enzyme corupulin and protamax were added for 9 hours, which was significantly higher. Through this, it was determined that protease, copulaline and protamax, are hydrolytic enzymes suitable for phycocyanin extraction rather than aminopeptidase, flavozyme (FIG. 1).

<Equation 4>

D: dilution multiple,

W1: dry weight of Spirulina extract (mg / mL),

W2: Spirulina raw weight before extraction (mg / mL)

<Experimental Example 2> Content of phycocyanin extract according to water-soluble fibrinolytic enzyme

In the solution containing 5% (v / v) of the substrate spirulina, liquid forms of the hydrolase enzymes cytolase (pectinase), rapidase (pectinase), ultraflo L (beta-glucanase, arabinase, hemicellulase, cellulase, xylnase) and viscozyme L ( 1 mL of beta-glucanase, hemicellulase, cellulase, xylnase) was added and reacted at 45 ° C. for 9 hours, and the content and SE index of C-phycocyanin and allopicocyanine were determined by absorbance analysis.

As a result, the contents of C-phycocyanin and allopicocyanin after the reaction using the enzyme ultraflo L were significantly higher compared to before the reaction, and cytolase, rapidase and viscozyme L were significant pico after 9 hours reaction There was no increase in cyanine content. In addition, the SE index also showed a significantly higher value when reacted with the enzyme ultraflo L (FIG. 2).

<Experiment 3> Content of phycocyanin extract according to proteolytic enzyme mixing

The content of phycocyanin extract according to proteolytic enzyme mixing was evaluated using a solution containing 5% (v / v) of substrate spirulina. In the following experiments, unless otherwise indicated, enzymatic hydrolysis was performed using a solution containing spirulina 5% (v / v) (100% by weight solution). In this experiment, the effect according to the mixing ratio of the proteomic enzyme protamex and copululin, which showed a high content of phycocyanin extraction when compared to the water-soluble fibrinolytic enzyme, was analyzed. At this time, 1 mL of the hydrolase in the liquid form was added to the solution containing 5% (v / v) of the substrate spirulina, and the solid recovery rate (yield) was calculated according to Equation 5 below.

<Equation 5>

DW: dry weight of Spirulina extract (mg / mL),

V: volume of solvent,

SW: dry weight of Spirulina extract (mg / mL)

As a result, the content of C-phycocyanin was significantly higher when the enzyme was used alone, and the content of allopicocyanin was significant when the protamex and colupulin were mixed in a weight ratio of 3: 2. Was highly extracted. In addition, there was no significant difference in the solids recovery rate, and it was judged that the use of copululin alone was more convenient for phycocyanin extraction than the mixture of protamax and copululin, which are proteolytic enzymes (FIG. 3).

<Experimental Example 4> Content of phycocyanin extract according to concentration when extracting spirulina enzyme

In order to select the amount of proteolytic enzyme cholupulin that was determined to be suitable for spirulina extraction, it was evaluated as follows. Spirulina suspension (containing Spirulina 5% (v / v)) is added to 100% (v / v) of enzymes up to 0.5, 1.0, 2.0 and 3.0% (v / v), reacted at 45 ° C for 12 hours, and then no enzyme is added. The control group and C-phycocyanin and allopicocyanin content and solid recovery were compared and analyzed.

As a result, the content of C-phycocyanin and allopicocyanin and the recovery of solid content were increased in the experimental group containing proteolytic enzyme colupulin compared to the control group, and the enzyme was 3.0% (v / v). It was judged that the use was suitable for Spirulina extraction (Fig. 4).

<Experimental Example 5> Evaluation of the content of phycocyanin extract according to temperature when extracting spirulina enzyme

Enzymes 3.0% (v / v) and 4.0% (v / v) to 100% (v / v) spirulina turbidity (containing 5% (v / v) spirulina) spirulina to screen the reaction temperature of the enzyme cholupulin suitable for spirulina extraction After adding v), the mixture was reacted at 30 ° C, 40 ° C and 50 ° C for 12 hours.

As a result, the use of 4% (v / v)% of cholupoline at the enzyme reaction temperature of 30 ° C and 50 ° C compared to 3% (v / v) enzyme significantly increased the content of C-phycocyanin and aloe. It was confirmed that phycocyanin was extracted. At 40 ° C, 3% (v / v) enzyme was found to be more efficient than 4% (v / v) enzyme, but 4% (v / v / v / v), considering total C-phycocyanin and allopicocyanin content. v) The reaction was performed at 50 ° C. for 12 hours by adding an enzyme, and a significantly higher extraction rate could be expected (FIG. 5).

<Experiment 6> Content of phycocyanin extract over time when extracting spirulina enzyme

C-phycocyanin and allopicos extracted hourly at 40 ° C after adding 3% (v / v)% or 4% (v / v) enzyme to select the optimal reaction time in spirulina extraction using enzyme Non-content and solids recovery were analyzed.

As a result, C-phycocyanin and allopicocyanin content with high use of 4% (v / v) enzyme were shown in all reaction times, and in the case of C-phycocyanin, 2 and 4 hours after enzyme addition Significantly high content in time was measured. In addition, allopicocyanin in Spirulina was extracted with the highest content in a 4 hour enzyme reaction. The solid content recovery did not show a significant difference after using the enzyme at two concentrations and the 4-hour reaction (FIG. 6).

<Experimental Example 7> Stability according to the pH and temperature of the phycocyanin pigment

Pigment stability according to pH and temperature changes was evaluated.

As a result, as the enzyme treatment temperature increased, the CR value, which is a relative residual amount of phycocyanin before and after the enzyme treatment, decreased, that is, the stability decreased rapidly. In addition, when looking at the effect of the change in pH, pH 6.0 was slightly higher than pH 5.0 or pH 7.0. When pH 5.0 and pH 7.0 were compared, stability at pH 5.0 was high (Fig. 7).

<Experiment 8> phycocyanin pigment stability according to Cyclodextin and tannic acid additives

To increase the pigment stability of phycocyanin, CD (cyclodextrin) and tannic acid were added, respectively, and the change in absorbance at 620 nm after heat treatment at pH 5.0 and 95 ° C. was measured. At this time, in order to measure the protective effect of phycocyanin by the capture effect, stability of phycocyanin was measured by adding alpha-CD, beta-CD, gamma-CD and tannic acid 0.2% (v / v), respectively.

As a result, in the case of the control group without the addition of CD, before the heat treatment, after the heat treatment, and with the addition of tannic acid, the CR value was 100% before the heat treatment, but decreased to about 12% after the heat treatment. Looked. In addition, it was confirmed that CD addition and tannic acid addition were independent of phycocyanin stability as the CR value tended to decrease when CD was expected to have a trapping effect (FIG. 8).

<Experimental Example 9> Evaluation of pigment stability of phycocyanin according to the addition of cysteine

The stability of phycocyanin according to the addition of cysteine known to possess SH-group was evaluated.

To the solution prepared by adding phycocyanin extract to 1% (v / v) in citrate buffer (pH 5.0), cysteine was added to 0.1, 0.2, and 0.5% (v / v), respectively. Then, after heat treatment at 95 ° C for 10 minutes, the CR value was measured to measure the stability of phycocyanin according to the addition of cysteine.

As a result, the CR value showed a low value around 10% after heat treatment, and the addition of cysteine did not significantly affect the CR value, and was not effective in increasing the pigment stability of phycocyanin (FIG. 9).

<Experimental Example 10> Evaluation of pigment stability according to the addition of cysteine and cystine when extracting spirulina enzyme

As a result of evaluating in Experimental Example 9, it was found that there is no effect of an additive for increasing pigment stability of phycocyanin contained in spirulina. This seems to be due to the structural changes of phycocyanin-containing phycobiliproteins by copululin, a protease during the extraction of spirulina, and cysteine and cystine having an SS bond containing a SH group during enzyme decomposition by copululin. Pigment stability of phycocyanin prepared by adding (cystine) was evaluated.

As a result, the CR value of the extract (Enzyme) by copulaline (Enzyme) was higher than that of the extract (control, Con) of Spirulina extracted using Citrate buffer (pH 5.0). On the other hand, when extracting cysteine by an enzyme (collupulin), CR values of phycocyanin pigment obtained by adding 0.1, 0.2, and 0.5% (v / v) respectively are 33.8, 43.4, and 58.4%, which are extracted using only citrate buffer or enzyme. It showed higher CR value than phycocyanin pigment. Therefore, it was confirmed that it was effective to increase the pigment stability by adding cysteine when extracting the enzyme using copulaline (FIG. 10).

11 is a photograph of a change in absorbance and a change in pigment according to a change in wavelength after heat treatment. It can be confirmed that the pigment stability of phycocyanin obtained by adding cysteine during extraction with copululin is higher than that of phycocyanin extracted with citrate buffer or phycocyanin extracted with copulaline (x in the graph of FIG. 11). Axis: wavelength (nm), y axis: optical density (OD)).

<Experimental Example 11> Evaluation of the stability of the phycocyanin pigment contained in the cholupullin extract by adding cysteine

From the results of Experimental Example 10, it was confirmed that the addition of cysteine when extracting phycocyanin pigment from spirulina by copulaline has an effect of increasing pigment stability. Therefore, the color stability of phycocyanin obtained by the addition of cysteine during extraction of copululin was evaluated according to the change in temperature.

As a result of measuring the CR values after 30 minutes of heat treatment of phycocyanin solutions of pH 5.0, 6.0, and 7.0 at 50, 60, and 70 ° C, the change in CR value according to the temperature change gradually decreased. The effect on the pH at was less than 30 ° C and 40 ° C on the dye stability by pH, but at 60 ° C and 70 ° C, the CR value of pH 7.0 was slightly decreased compared to pH 5.0 or 6.0. At high temperature, pH 5.0 showed a slightly higher CR value, but there was no significant difference from pH 6.0 (FIG. 12).

Therefore, when extracting phycocyanin from spirulina from the above experimental results, copululin is effective, and when extracting phycocyanin from spirulina using copululin, cysteine is 0.2% (v / v) or 0.5% (v / v). When added, it was confirmed that the phycocyanin pigment stability increased.

Claims (9)

Hydrolyzing spirulina with copulaline and cysteine; and
Obtaining a spirulina hydrolyzate,
At this time, 4 to 10 parts by weight of copulaline and 0.5 to 3 parts by weight of cysteine are treated with respect to 100 parts by weight of spirulina.
Method for producing a spirulina hydrolyzate having a c-phycocyanin content of 1 mg / g to 10 mg / g.
delete delete delete According to claim 1,
The hydrolysis step is a manufacturing method characterized in that it is carried out by hydrolysis for 1.5 hours to 6 hours.
According to claim 1,
The hydrolysis step is a production method characterized in that it is carried out under the conditions of pH 4.5 to pH 8.
delete delete According to claim 1,
The spirulina hydrolyzate is a production method characterized in that the allopicocyanin content is 1 mg / g to 10 mg / g.

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