Method for extracting spirulina polypeptide powder

Abstract

The invention provides a method for extracting spirulina polypeptide powder, and relates to the technical field of spirulina polypeptide powder preparation. The method for extracting the spirulina polypeptide powder comprises the following steps: cleaning and airing spirulina, stirring into spirulina mud, and dissolving the spirulina mud in distilled water to obtain a mixed solution; carrying out ultrasonic treatment on the mixed solution, repeatedly thawing and freezing, centrifuging, and collecting supernate to obtain clear liquid for later use; bromelain is added into the clear liquid for standby application for hydrolysis and enzyme deactivation, enzymatic hydrolysate is obtained, bacillus licheniformis is inoculated into the enzymatic hydrolysate, aerobic fermentation and sterilization are conducted, and fermentation liquor is obtained. According to the method, the spirulina is subjected to enzymolysis-fermentation-enzymolysis staged treatment, so that the fishy smell of the spirulina polypeptide finished product prepared from the spirulina can be effectively reduced, the antibacterial property of the spirulina polypeptide is also improved to a certain degree, and the application value of directly preparing the spirulina polypeptide from the spirulina is comprehensively improved.

Description

Technical Field

[0001] This invention relates to the field of spirulina polypeptide powder preparation technology, specifically to a method for extracting spirulina polypeptide powder. Background Technology

[0002] Spirulina contains essential macro and micro elements for humans and animals. It not only contains a large amount of iron, but can also chelate with proteins and phycocyanin to form organic iron, thus facilitating absorption and utilization. Furthermore, spirulina has a short growth cycle, reproduces rapidly, has a high protein content, and the selected polypeptides also possess high nutritional value.

[0003] Currently, the application of spirulina faces the following problems: First, spirulina has a strong fishy smell and poor taste. Second, to enhance absorption, spirulina is usually prepared into spirulina polypeptides. Currently, the preparation of spirulina polypeptides typically involves mixing spirulina powder with water to form a suspension, and then using bio-enzyme engineering technology to hydrolyze spirulina protein to prepare spirulina polypeptides. However, a large amount of the active ingredients in the spirulina powder are lost, resulting in insufficient activity in the final product. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting spirulina polypeptide powder, which can effectively improve the fishy smell and insufficient activity of spirulina during the preparation of spirulina polypeptide powder.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for extracting spirulina polypeptide powder includes the following steps: S1. After washing and drying the spirulina, stir it into a mud. Dissolve the mud in distilled water to obtain a mixed liquid. S2. After sonicating the mixture for 10-20 seconds, repeatedly thaw and freeze, centrifuge for 20-30 minutes, collect the supernatant, and obtain the clear liquid for later use. S3. Add bromelain to the prepared clear liquid and hydrolyze for 140-180 min to inactivate the enzyme and obtain the enzymatic hydrolysate. Inoculate Bacillus licheniformis into the enzymatic hydrolysate and ferment aerobically for 1-2 h. Sterilize to obtain the fermentation broth. S4. Add trypsin to the fermentation broth and hydrolyze for 100-120 minutes to inactivate the enzyme and obtain the hydrolysate. S5. Centrifuge the hydrolysate, collect the supernatant, and spray dry to obtain spirulina polypeptide powder.

[0006] Preferably, the ratio of algal mud to distilled water in step S1 is 1:10-15.

[0007] Preferably, the repeated thawing in step S2 specifically involves freezing the mixture with liquid nitrogen, thawing it in a water bath at 30-40°C, repeating this process 2-5 times, with an ultrasonic power of 700-900W and a centrifugation speed of 8000-9000rpm.

[0008] Preferably, in step S3, before adding bromelain, the hydrolysis temperature is adjusted to 50-60℃, and 0.1mol / L dilute hydrochloric acid is added dropwise to adjust the pH of the prepared supernatant to 7.0-8.0. The mass ratio of bromelain to the prepared supernatant is 3-5:100, and the enzyme activity is 1×10⁻⁶. 6 U / g.

[0009] Preferably, in step S3, the temperature of the enzymatic hydrolysate is adjusted to 35-40°C before inoculating it with Bacillus licheniformis, and the mass ratio of Bacillus licheniformis to the enzymatic hydrolysate is 7-9:100.

[0010] Preferably, the content of Bacillus licheniformis in step S3 is 1×10⁻⁶. 8 CFU / g.

[0011] Preferably, in steps S3 and S4, enzyme inactivation is performed by treating in a water bath at 80-90℃ for 15-25 minutes, and sterilization is performed by passing through a microporous membrane with a pore size of 0.22μm.

[0012] Preferably, in step S4, the fermentation broth temperature is adjusted to 45-50℃ before inoculation, the mass ratio of trypsin to fermentation broth is 5-9:100, and the enzyme activity is 2×10⁻⁶. 5 U / g.

[0013] Preferably, the centrifugation in step S5 specifically involves processing at 8000-12000 rpm for 25-35 minutes at 3-6℃.

[0014] This invention provides a method for extracting spirulina polypeptide powder, which has the following advantages compared with the prior art: This invention directly uses Spirulina as a base material for extraction, which avoids the loss of activity after it is made into dry powder, and saves the step of making it into dry powder. It can be used directly after post-processing, thus saving preparation costs.

[0015] This invention employs a staged process of enzymatic hydrolysis-fermentation-enzymatic hydrolysis for spirulina, which can effectively reduce the fishy smell of the finished spirulina polypeptide and also enhance the antibacterial properties of the spirulina polypeptide, thus comprehensively improving the application value of directly preparing spirulina polypeptide from spirulina.

[0016] This invention pre-treats spirulina with bromelain, followed by fermentation with Bacillus licheniformis, and further treats the fermentation broth with trypsin. The scavenging rates of hydroxyl radicals and superoxide anion radicals are maximized when the degree of hydrolysis is 29%. This aims to enhance the antioxidant capacity of spirulina polypeptide powder while increasing the degree of hydrolysis for maximum scavenging, thereby improving the utilization rate of spirulina. Attached Figure Description

[0017] Figure 1 The results are from the determination of the degree of hydrolysis of spirulina protein; Figure 2 The results show the determination of the scavenging ability of spirulina polypeptides against hydroxyl radicals. Figure 3 The results show the determination of the scavenging ability of spirulina polypeptides against superoxide anion free radicals. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Spirulina was purchased from Xindaze Spirulina Co., Ltd. in Fuqing City; bromelain was purchased from Dongheng Huadao, with an enzyme activity of 1×10⁻⁶. 6 U / g food-grade bromelain; Bacillus licheniformis was purchased from Liaoning Huaxing Biotechnology, with a bacterial count of 1×10⁻⁶. 8 CFU / g; trypsin was purchased from Ningxia Xiasheng Industrial Group, with an enzyme activity of 2×10⁻⁶. 5 U / g; Spirulina powder was purchased from Huamo (Shandong) Biotechnology Co., Ltd.

[0020] Example 1: S1. Wash and dry 5 kg of spirulina, then stir it into algae mud. After passing the algae mud through a 60-mesh sieve, dissolve it in distilled water. The ratio of algae mud to distilled water is 1:12 to obtain a mixed liquid. S2. The mixture was ultrasonically treated with a power of 800W and a speed of 8000rpm for 15s, then frozen with liquid nitrogen, and then thawed in a water bath at 35℃. This process was repeated 5 times. The mixture was then centrifuged at 8500rpm for 25min, and the supernatant was collected to obtain the clear liquid for use. S3. Adjust the temperature of the prepared supernatant to 55℃, add 0.1mol / L dilute hydrochloric acid to adjust the pH of the prepared supernatant to 7.5, and add enzyme with an activity of 1×10⁻⁶. 6Hydrate with bromelain (U / g) for 160 min, using a bromelain-to-prepared supernatant ratio of 5:100. Inactivate the enzyme in an 85℃ water bath for 20 min to obtain the hydrolysate. Adjust the hydrolysate temperature to 38℃ before inoculation with a concentration of 1×10⁻⁶. 8 Bacillus licheniformis CFU / g, with a mass ratio of Bacillus licheniformis to enzymatic hydrolysate of 7:100, was aerobic fermented for 1.5 h and then sterilized by passing it through a microporous membrane with a pore size of 0.22 μm to obtain the fermentation broth. S4. Adjust the fermentation broth temperature to 48℃, and add enzyme with an activity of 2×10⁻⁶ to the fermentation broth. 5 Hydrolyze with trypsin at U / g for 110 min, with a trypsin to fermentation broth mass ratio of 5:100. Inactivate enzymes in an 85℃ water bath for 20 min to obtain hydrolysate. S5. Centrifuge the hydrolysate at 10,000 rpm for 30 min at 4℃, collect the supernatant, and spray dry to obtain spirulina polypeptide powder.

[0021] Example 2: This embodiment follows the preparation method of Example 1, except for the amount of bromelain, Bacillus licheniformis, and trypsin added, specifically: The mass ratio of bromelain to the prepared supernatant was 3:100, the mass ratio of Bacillus licheniformis to the enzymatic hydrolysate was 9:100, and the mass ratio of trypsin to the fermentation broth was 9:100.

[0022] Comparative Example 1: This comparative example follows the preparation method of Example 1, except that steps S3 and S4 are different, while all other steps are the same, specifically: S3. Adjust the temperature of the prepared supernatant to 55℃, add 0.1mol / L dilute hydrochloric acid to adjust the pH of the prepared supernatant to 7.5, and add enzyme with an activity of 1×10⁻⁶. 6 The bromelain was hydrolyzed for 160 min at a concentration of U / g. The mass ratio of bromelain to the prepared supernatant was 5:100. The enzyme was inactivated in an 85℃ water bath for 20 min to obtain the enzymatic hydrolysate. S4. Adjust the temperature of the enzyme hydrolysate to 48℃, and add enzyme with an activity of 2×10 to the fermentation broth. 5 Hydrolyze with trypsin at U / g for 110 min, with a trypsin to fermentation broth mass ratio of 5:100. Inactivate enzymes in an 85℃ water bath for 20 min to obtain hydrolysate. Comparative Example 2: This comparative example follows the preparation method of Example 1, except that steps S3 and S4 are different, while all other steps are the same, specifically: S3. Adjust the temperature of the prepared solution to 48℃, and add enzyme with an activity of 2×10 to the fermentation broth. 5Hydrolyze the fermentation broth with trypsin at a concentration of U / g for 110 min, maintaining a trypsin-to-fermentation broth mass ratio of 5:100. Inactivate the enzyme in an 85℃ water bath for 20 min to obtain the hydrolysate. Adjust the hydrolysate temperature to 38℃ before inoculating with a concentration of 1×10⁻⁶. 8 Bacillus licheniformis CFU / g, with a mass ratio of Bacillus licheniformis to enzymatic hydrolysate of 7:100, was aerobic fermented for 1.5 h and then sterilized by passing it through a microporous membrane with a pore size of 0.22 μm to obtain the fermentation broth. S4. Adjust the fermentation broth temperature to 55℃, add 0.1mol / L dilute hydrochloric acid to adjust the pH of the prepared supernatant to 7.5, and add enzyme with an activity of 1×10⁻⁶. 6 The bromelain was hydrolyzed for 160 min at a concentration of U / g. The mass ratio of bromelain to the prepared supernatant was 5:100. The enzyme was inactivated in an 85℃ water bath for 20 min to obtain the hydrolysate. Comparative Example 3: This comparative example follows the preparation method of Example 1, except that steps S3 and S4 are different, while all other steps are the same, specifically: S3. After adjusting the temperature of the prepared solution to 38℃, the inoculation concentration is 1×10⁻⁶. 8 Bacillus licheniformis (CFU / g) was inoculated at a ratio of 7:100 to the prepared supernatant. Aerobic fermentation was carried out for 1.5 h, followed by sterilization through a 0.22 μm microporous membrane to obtain the fermentation broth. The fermentation broth temperature was adjusted to 55℃, and the pH of the prepared supernatant was adjusted to 7.5 by adding 0.1 mol / L dilute hydrochloric acid. An enzyme with an activity of 1 × 10⁻⁶ was then added. 6 The bromelain was hydrolyzed for 160 min at a concentration of U / g. The mass ratio of bromelain to the prepared supernatant was 5:100. The enzyme was inactivated in an 85℃ water bath for 20 min to obtain the enzymatic hydrolysate. S4. Adjust the temperature of the enzyme hydrolysate to 48℃, and add enzyme with an activity of 2×10 to the fermentation broth. 5 Hydrolyze with trypsin at U / g for 110 min, with a trypsin to fermentation broth mass ratio of 5:100. Inactivate enzymes in an 85℃ water bath for 20 min to obtain hydrolysate. Comparative Example 4: This comparative example follows the preparation method of Example 1, except that steps S3, S4, and S5 are different, while all other steps are the same, specifically: S3. Adjust the temperature of the prepared supernatant to 55℃, add 0.1mol / L dilute hydrochloric acid to adjust the pH of the prepared supernatant to 7.5, and add enzyme with an activity of 1×10⁻⁶. 6 Hydrate with bromelain (U / g) for 160 min, using a bromelain-to-prepared supernatant ratio of 5:100. Inactivate the enzyme in an 85℃ water bath for 20 min to obtain the hydrolysate. Adjust the hydrolysate temperature to 48℃ and add enzyme with an activity of 2×10⁻⁶ U / g to the fermentation broth. 5Hydrolyze with trypsin at U / g for 110 min, with a trypsin to fermentation broth mass ratio of 5:100. Inactivate enzymes in an 85℃ water bath for 20 min to obtain hydrolysate. S4. After adjusting the hydrolysate temperature to 38℃, the inoculation concentration is 1×10⁻⁶. 8 Bacillus licheniformis CFU / g, with a mass ratio of Bacillus licheniformis to enzymatic hydrolysate of 7:100, was aerobic fermented for 1.5 h and then sterilized by passing it through a microporous membrane with a pore size of 0.22 μm to obtain the fermentation broth. S5. Centrifuge the fermentation broth at 10,000 rpm for 30 min at 4℃, collect the supernatant, and spray dry to obtain spirulina polypeptide powder.

[0023] Comparative Example 5: This comparative example follows the preparation method of Example 1, except that step S3 is different, while all other steps are the same, specifically: S3. After adjusting the temperature of the prepared solution to 38℃, the inoculation concentration is 1×10⁻⁶. 8 Bacillus licheniformis CFU / g, with a mass ratio of Bacillus licheniformis to enzymatic hydrolysate of 7:100, was aerobic fermented for 1.5 h and then sterilized by passing it through a microporous membrane with a pore size of 0.22 μm to obtain the fermentation broth. Comparative Example 6: This comparative example follows the preparation method of Example 1, except that step S4 is omitted and step S5 is adjusted, while all other steps remain the same. Specifically: S4. Centrifuge the fermentation broth at 10,000 rpm for 30 min at 4℃, collect the supernatant, and spray dry to obtain spirulina polypeptide powder.

[0024] Comparative Example 7: This comparative example follows the preparation method of Example 1, except that spirulina is directly replaced with spirulina powder; all other steps are the same, specifically: S1. Dissolve 5 kg of spirulina powder in distilled water, with a spirulina powder to distilled water ratio of 1:12, to obtain a mixed solution. Detection: 1. Determination of the degree of hydrolysis of spirulina protein Based on the preparation methods in Examples 1-2 and Comparative Examples 1-7, the degree of hydrolysis of spirulina protein was determined by pH-stat method. The specific calculation method is shown in formula (1), and the specific results are shown in Table 1. Figure 1 As shown: DH = h / h tot ×100% (1) DH - Degree of hydrolysis of spirulina protein; h - Number of peptide bonds broken by hydrolysis; h tot - Total number of bonds.

[0025] Table 1 As shown in the table above, Examples 1, 2, 4 and 7 show better degrees of hydrolysis.

[0026] 2. Determination of the activity of spirulina polypeptides Spirulina polypeptide powder was extracted according to the preparation methods shown in Examples 1-2 and Comparative Examples 1-7, and dissolved in test tubes containing distilled water to a concentration of 20 mg / ml as a sample. The following determinations were then performed: 2.1 Determination of hydroxyl radical scavenging ability Add 2.0 mL of 6 mmol / L ferrous sulfate solution and 2.0 mL of 6 mmol / L hydrogen peroxide solution to each test tube in sequence. Mix well and let stand for 10 min. Then add 2.0 mL of 6 mmol / L salicylic acid solution, mix well and let stand for 30 min. Measure the absorbance at a wavelength of 510 nm (as shown in formula (2)). Set up a test tube with distilled water as a blank control group. The specific results are shown in Table 2 and Figure 2 As shown: Clearance rate (%) = (A0 - (A1 - A2)) / A0 × 100 (2) A0 - Absorbance without sample solution; A1 - Absorbance with sample solution; A2 - Absorbance without salicylic acid solution.

[0027] Table 2 As shown in the table above, the spirulina polypeptide prepared in Example 1 has good performance in scavenging hydroxyl radicals. Combined with the hydrolysis degree data in Table 1, the hydroxyl radical scavenging ability of the spirulina polypeptide prepared in Example 1 is the largest when the hydrolysis degree is 29%. While improving the hydrolysis degree of spirulina and thus increasing the utilization rate of spirulina, it also obtains a strong hydroxyl radical scavenging ability.

[0028] 2.2 Determination of superoxide anion free radical scavenging ability Add 4.5 mL of Tris-HCl buffer (0.05 mol / L, pH 8.2) and 2.4 mL of distilled water to each test tube, shake well, and incubate in a 25°C water bath for 10 min. Add 0.1 mL of pyrogallol, mix well, let stand for 30 min, and quickly add 0.1 mL of concentrated hydrochloric acid to terminate the reaction. Measure the absorbance at 325 nm (as shown in formula (3)). A separate test tube containing only distilled water was set up as a blank control group. The specific results are shown in Table 3 and... Figure 3 As shown: Clearance rate (%) = (A0 - (A1 - A2)) / A0 × 100 (3) A0 - Absorbance without sample solution; A1 - Absorbance with sample solution; A2 - Absorbance without pyrogallol.

[0029] Table 3 As shown in the table above, the spirulina polypeptide prepared in Example 1 has a strong ability to scavenge superoxide anion free radicals. Combining the hydrolysis degree data in Table 1, although the hydrolysis degree of Comparative Examples 2, 4, and 7 is higher than that of Example 1, according to the superoxide anion free radical scavenging rate data in the table above, the sample of Example 1 has a scavenging rate of 66.88% higher than that of the product prepared from spirulina powder (Comparative Example 7), a 27.72% higher than that of Comparative Example 2, and a 64.47% higher than that of Comparative Example 4. That is, the sample prepared in Example 1 has the best superoxide anion free radical scavenging effect.

[0030] 2.3 Antibacterial activity test 6mm diameter circular filter paper discs were prepared using a punch, placed in dry petri dishes, and sterilized in an autoclave. The filter paper discs were then immersed in each sample for 30 minutes. After immersion, they were placed in E. coli culture dishes (agar medium) and incubated at 37°C to observe colony growth. The discs were removed after 24 hours. Separately, filter paper discs were immersed in physiological saline and placed in petri dishes as a control. The specific results are shown in Table 4. Table 4 "+" indicates the presence of bacterial colonies; "-" indicates the absence of bacterial colonies.

[0031] As shown in the table above, the spirulina polypeptide powder prepared in Example 1 exhibits the best antibacterial properties and good durability. Comparative Examples 2 and 3 also show good antibacterial properties, but their antibacterial durability is not high, and they lose their antibacterial properties after 24 hours. Furthermore, the colony formation time in Comparative Example 2 is later than that in Comparative Examples 3 and 4, indicating that enzymatic hydrolysis of the prepared supernatant before bacterial fermentation can improve the antibacterial properties of the prepared spirulina powder.

[0032] 3. Odor determination Using the spirulina polypeptide powder prepared according to Examples 1-2 and Comparative Examples 1-7 as samples, 10 testers (5 males and 5 females) were used for sensory evaluation. The degree of fishy smell was divided into 5 levels: 5 points indicates a very strong fishy smell, 4 points indicates a relatively strong fishy smell, 3 points indicates a moderate fishy smell, 2 points indicates a relatively weak fishy smell, 1 point indicates a very slight fishy smell, and 0 points indicates no fishy smell. The average score was taken after 10 people gave their scores individually. The specific results are shown in Table 5.

[0033] Table 5 As shown in the table above, the spirulina polypeptide powder prepared in Example 1 has the least fishy smell and the best treatment effect. Meanwhile, the spirulina polypeptides prepared in Comparative Examples 2, 3, and 4 have a relatively small fishy smell compared to the other comparative examples. Among them, the effect of Comparative Example 2 is more outstanding. It can be seen that the method of enzymatic hydrolysis of the prepared solution followed by bacterial fermentation and then enzymatic hydrolysis can achieve a good effect in removing the fishy smell.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting spirulina polypeptide powder, characterized in that, The extraction method includes the following steps: S1. After washing and drying the spirulina, stir it into a mud. Dissolve the mud in distilled water to obtain a mixed liquid. S2. After sonicating the mixture for 10-20 seconds, repeatedly thaw and freeze, centrifuge for 20-30 minutes, collect the supernatant, and obtain the clear liquid for later use. S3. Add bromelain to the prepared clear liquid and hydrolyze for 140-180 min to inactivate the enzyme and obtain the enzymatic hydrolysate. Inoculate Bacillus licheniformis into the enzymatic hydrolysate and ferment aerobically for 1-2 h. Sterilize to obtain the fermentation broth. S4. Add trypsin to the fermentation broth and hydrolyze for 100-120 minutes to inactivate the enzyme and obtain the hydrolysate. S5. Centrifuge the hydrolysate, collect the supernatant, and spray dry to obtain spirulina polypeptide powder.

2. The preparation method according to claim 1, characterized in that, The ratio of algal mud to distilled water in step S1 is 1:10-15.

3. The preparation method according to claim 1, characterized in that, The repeated thawing in step S2 specifically involves freezing the mixture with liquid nitrogen, thawing it in a water bath at 30-40℃, repeating this process 2-5 times, using an ultrasonic power of 700-900W, and a centrifugation speed of 8000-9000rpm.

4. The preparation method according to claim 1, characterized in that, In step S3, before adding bromelain, the hydrolysis temperature is adjusted to 50-60℃, and 0.1mol / L dilute hydrochloric acid is added dropwise to adjust the pH of the prepared supernatant to 7.0-8.

0. The mass ratio of bromelain to the prepared supernatant is 3-5:100, and the enzyme activity of bromelain is 1×10⁻⁶. 6 U / g.

5. The preparation method according to claim 1, characterized in that, In step S3, the temperature of the enzymatic hydrolysate is adjusted to 35-40°C before inoculating it with Bacillus licheniformis, and the mass ratio of Bacillus licheniformis to the enzymatic hydrolysate is 7-9:

100.

6. The preparation method according to claim 1, characterized in that, The content of Bacillus licheniformis in step S3 is 1×10⁻⁶. 8 CFU / g.

7. The preparation method according to claim 1, characterized in that, In steps S3 and S4, enzyme inactivation is performed by treating the sample in a water bath at 80-90℃ for 15-25 minutes, and sterilization is performed by passing the sample through a microporous membrane with a pore size of 0.22μm.

8. The preparation method according to claim 1, characterized in that, In step S4, the fermentation broth temperature is adjusted to 45-50℃ before inoculation. The mass ratio of trypsin to fermentation broth is 5-9:100, and the enzyme activity is 2×10⁻⁶. 5 U / g.

9. The preparation method according to claim 1, characterized in that, The centrifugation in step S5 specifically involves processing at 8000-12000 rpm for 25-35 minutes at 3-6℃.

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