CN118702831B

CN118702831B - A method for extracting high-purity starch from microalgae and microalgae starch

Info

Publication number: CN118702831B
Application number: CN202411178692.9A
Authority: CN
Inventors: 李冠天; 尹丽菁; 李航; 杨斯琪
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2024-08-27
Filing date: 2024-08-27
Publication date: 2025-01-21
Anticipated expiration: 2044-08-27
Also published as: CN118702831A

Abstract

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting high-purity starch from microalgae and the microalgae starch. A method for extracting high-purity starch from microalgae comprises the steps of microalgae culture, collection and pretreatment, cell wall breaking, starch separation and purification and the like. According to the invention, a sectional culture strategy is adopted to promote starch accumulation, liquid nitrogen pretreatment is utilized to improve wall breaking efficiency, efficient cell wall breaking is realized through combination of multiple enzymes, enzymolysis and ultrasonic crushing, and SDS washing and ultrafiltration technology are combined to carry out deep purification. The method can be applied to various microalgae such as spirulina, chlorella, chlamydomonas reinhardtii and the like, and the obtained starch has the purity of more than or equal to 98 percent and the amylose percentage of 40-75 percent. The method disclosed by the invention is simple in process, environment-friendly and wide in applicability, and the obtained high-purity and high-linear-chain-content microalgae starch has a wide application prospect in the fields of foods, medicines, chemical industry and the like.

Description

Method for extracting high-purity starch from microalgae and microalgae starch

Technical Field

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting high-purity starch from microalgae and the microalgae starch.

Background

Starch is an important polysaccharide substance and is widely applied to the fields of food, medicine, chemical industry and the like. Traditional starch extraction typically relies on terrestrial plants such as corn, potato, and the like. However, these methods have not only environmental impact but also are limited by land resources. The ocean has grown a wide variety of algae with starch as an energy storage material. Currently, studies on algal starch have been mainly focused on brown algae starch, and studies on red algae starch have been little studied for nearly 30 years. Starch is an important component of the cytoplasm of large red algae such as asparagus, gracilaria and the like. Microalgae, which are an emerging biological resource, have rapid growth, high-efficiency photosynthesis and high starch content, are a potential starch source.

However, the prior art is difficult in extracting starch from microalgae, mainly arising from several problems:

(1) The purity is not high, the existing extraction method is difficult to obtain the high-purity microalgae starch, the impurity content is high, and the product quality is affected;

(2) The process is complex, and a plurality of extraction methods involve complex process flows, so that the production cost and the difficulty are increased;

(3) The yield is low, the starch extraction efficiency in the prior art is low, and the requirement of large-scale production is difficult to meet;

(4) Environmental impact-the use of large amounts of organic solvents for part of the extraction process may have negative environmental impact;

(5) The temperature influence is that the partial extraction method uses high-temperature treatment to cause the microalgae starch to be gelatinized in the processing and extraction process, thereby obviously influencing the subsequent product quality.

Therefore, the development of an efficient, environment-friendly and high-purity microalgae starch extraction method is of great significance.

Disclosure of Invention

The invention aims to provide a method for extracting high-purity starch from microalgae, which can simplify the process flow, improve the extraction purity and yield of the starch and obtain a starch product with high linear chain content.

In order to achieve the aim, the invention adopts the following technical scheme that the method for extracting high-purity starch from microalgae comprises the following steps:

(1) Culturing microalgae, namely selecting microalgae species suitable for starch accumulation, culturing the microalgae, and performing nitrogen-deficient culture on the microalgae by using a nitrogen-deficient culture medium after the microalgae enter a platform stage, wherein the microalgae species are selected from any one of spirulina, chlamydomonas, chlorella, red-haired algae, schizochytrium limacinum and Dunaliella salina;

(2) Collecting and preprocessing microalgae, namely harvesting the microalgae 2-10 days after nitrogen-deficiency culture of the microalgae, and performing liquid nitrogen freezing treatment on the harvested microalgae;

(3) Breaking microalgae cells, namely performing enzymolysis on microalgae frozen by liquid nitrogen by using compound enzymes consisting of cellulose, pectase and muramidase, and then thoroughly destroying the cell wall to release starch in cells after ultrasonic crushing treatment, wherein the addition amount of the compound enzymes in the freeze-dried microalgae with unit gram weight is respectively cellulase 1U-4U, pectase 0.5U-3U and muramidase 0.3U-1.5U;

(4) Separating and purifying starch, namely separating the microalgae suspension after wall breaking to obtain a crude starch product, dissolving the crude starch product in water, and washing for multiple times by adopting an SDS solution to remove impurities.

Preferably, in step (1), the nitrogen-deficient medium of schizochytrium limacinum is free of a phosphorus source.

Preferably, in step (1), the nitrogen-deficient medium of chlamydomonas is free of sulfur sources.

Preferably, in step (1), the concentration of sodium chloride in the nitrogen-deficient medium of Dunaliella salina is 3M.

Preferably, in the step (3), the complex enzyme of the rambutan further comprises beta-glucanase, and the addition amount of the beta-glucanase is 0.3-0.6U.

Preferably, in the step (3), the complex enzyme of the schizochytrium limacinum also comprises chitinase, and the addition amount of the chitinase is 0.4-0.8U.

Preferably, in the step (3), the complex enzyme of Dunaliella salina salt further comprises alkaline protease, and the addition amount of the alkaline protease is 0.2-0.5U.

Preferably, in the step (4), the volume ratio of the SDS solution to the crude starch water solution is 10:1, and the mass volume concentration of the SDS solution is 1% -16%.

The invention further provides the microalgae starch prepared by the method, wherein the total starch content in the microalgae starch is more than or equal to 98%, and the amylose percentage is 40-75%.

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

(1) The microalgae starch with the purity of more than 98 percent is obtained through the purification process of multiple steps, which is obviously superior to the prior art;

(2) The high amylose content is that the proportion of the obtained amylose is 40-75% by adopting a sectional culture strategy and precise process control, which is higher than that of the starch of the traditional crops;

(3) The efficiency is high, the cell wall breaking rate is up to 95% through liquid nitrogen freezing pretreatment and multiple enzyme combined wall breaking, and the extraction efficiency of the starch is greatly improved;

(4) The whole extraction process is relatively simple, and the industrial production is easy to realize;

(5) The environment-friendly type water-ethanol composite material mainly uses water and ethanol as solvents, reduces the use of organic solvents, and is more environment-friendly;

(7) The raw materials are various, and the raw materials are suitable for various microalgae, such as spirulina, chlamydomonas and the like, and have wide sources;

(8) The products are diversified, namely starch products with different linear chain contents can be obtained by adjusting the technological parameters, and different application requirements are met.

Drawings

FIG. 1 shows the change of microalgae density and microalgae dry weight in the cultivation process, wherein (a) the change of the microalgae density and (b) the change of the microalgae dry weight are shown;

FIG. 2 shows the variation of starch content during microalgae cultivation in accordance with an embodiment of the present invention;

FIG. 3 is a graph showing the particle size distribution of microalgae starch according to an embodiment of the present invention;

FIG. 4 is a transmission electron microscope image of microalgae starch provided by the embodiment of the invention;

FIG. 5 is a scanning electron microscope image of microalgae starch provided by an embodiment of the invention;

FIG. 6 is a graph showing the effect of different breaking methods on the wall breaking rate of microalgae according to the embodiment of the invention.

Detailed Description

The technical scheme of the invention is described in detail below with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

Example 1 this example provides a method for extracting high purity starch from microalgae, comprising the following steps:

(1) Microalgae cultivation, namely selecting spirulina (Spirulina platensis) as an experimental object. The spirulina is inoculated in a modified Zarouk culture medium, and the culture conditions are that the temperature is 28+/-1 ℃, the illumination intensity is 6000 lux, and the light-dark ratio is 14:10h. After the logarithmic growth phase (about 8 days), as shown in fig. 1 (a), the algal liquid was collected by centrifugation and transferred to a medium containing no nitrogen source for continuous culture for 7 days to promote starch accumulation. The dry weight and starch content of microalgae reached a maximum on day 7 of cultivation, at which time the dry weight was 4.8 g/L, as shown in FIG. 1 (b), and the starch content was 56%, as shown in FIG. 2.

(2) Microalgae collection and pretreatment, namely, collecting algae by using a centrifugal machine (5000 rpm,10 min). The collected algae are washed for 3 times by deionized water, and then are subjected to quick freezing treatment by liquid nitrogen. And (5) storing the quick-frozen algae in a refrigerator at the temperature of-80 ℃ for later use.

(3) Cell wall breaking by taking 10 g (dry weight) quick frozen algae, adding 100 mL of acetic acid buffer (50 mM) with pH of 5.0. Cellulase (2U/g algal body), pectase (1U/g algal body) and muramidase (0.5U/g algal body) were added. Enzymatic hydrolysis is carried out for 2 hours at 37 ℃. Subsequently, the enzymatic hydrolysate was placed in an ultrasonic breaker (power 400W, work 5s, batch 5 s) for a total treatment time of 10 min.

(4) Starch separation and purification, namely centrifuging the crushed algae liquid (8000 rpm,15 min), and collecting precipitate. The pellet was resuspended in 100mL deionized water, 10mL strength 1% (w/v) SDS solution was added and treated in a 30℃water bath for 30 min. The SDS washing step was repeated 3 times. Finally, the washed sample was subjected to ultrafiltration purification by an ultrafiltration membrane of 100 kDa molecular weight cut-off and was replaced 5 times with deionized water.

(5) Starch drying and preservation the purified starch suspension was freeze-dried (-50 ℃) for 48 hours. The dried starch samples were stored in a desiccator in a sealed manner. The starch dry weight was 3.87g, the amylose percentage was 40% and the starch purity was 98%. As shown in FIG. 3, the microalgae have particle diameter of 0.5-3 μm, and their transmission electron microscope and scanning electron microscope are shown in FIG. 4 and FIG. 5.

Example 2 this example provides a method for extracting high purity starch from chlorella, comprising the following steps:

(1) Microalgae cultivation, namely selecting chlorella (Chlorella vulgaris) as an experimental object. Inoculating Chlorella into BG11 culture medium under the culture conditions of 25+ -1deg.C, light intensity 5000 lux, and light-dark ratio 12:12h. After the logarithmic growth phase (about 5 days), the algae liquid was collected by centrifugation and transferred to a medium without nitrogen source for continuous cultivation for 7 days to promote starch accumulation and maximize the starch content in the microalgae.

(2) Microalgae collection and pretreatment the algae were collected using a centrifuge (4500 rpm,15 min). The collected algae are washed for 3 times by deionized water, and then are subjected to quick freezing treatment by liquid nitrogen. And (5) storing the quick-frozen algae in a refrigerator at the temperature of-80 ℃ for later use.

(3) Cell wall breaking by taking 8g (dry weight) quick frozen algae, adding 80 mL of citric acid buffer (50 mM) with pH of 5.5. Cellulase (2.5U/g algal), pectinase (1.5U/g algal) and muramidase (0.8U/g algal) were added. Enzymatic hydrolysis is carried out for 2.5 hours at 40 ℃. Subsequently, the enzymatic hydrolysate was placed in an ultrasonic breaker (power 450W, work 8 s, batch 5 s) and the total treatment time was 12 min.

(4) Starch separation and purification, namely centrifuging the crushed algae liquid (9000 rpm,20 min), and collecting precipitate. The pellet was resuspended in 80 mL deionized water, 8 mL strength 12% (w/v) SDS solution was added and treated in a 35℃water bath for 35 min. The SDS washing step was repeated 4 times. Finally, the washed sample was subjected to ultrafiltration purification by an ultrafiltration membrane of 50 kDa molecular weight cut-off and was replaced 6 times with deionized water.

(5) Starch drying and preservation the purified starch suspension was spray dried (inlet temperature 180 ℃ and outlet temperature 85 ℃). The dried starch samples were stored in a sealed brown glass bottle. The starch dry weight is 3.87 g, the amylose percentage is 36% and the starch purity is 97%.

Example 3 this example provides a method for extracting high purity starch from Chlamydomonas reinhardtii comprising the following steps:

(1) Microalgae cultivation, namely selecting Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) as an experimental object. Chlamydomonas reinhardtii was inoculated into TAP medium at a temperature of 23.+ -. 1 ℃ and an illumination intensity of 4500 lux at a light-dark ratio of 16:8h. After the logarithmic growth phase (about 6 days), the algae liquid was collected by centrifugation and transferred to a medium without nitrogen and sulfur sources for continuous cultivation for 10 days to promote starch accumulation and maximize the starch content in the microalgae.

(2) Microalgae collection and pretreatment the algae were collected using a centrifuge (4000 rpm,20 min). The collected algae are washed for 3 times by deionized water, and then are subjected to quick freezing treatment by liquid nitrogen. And (5) storing the quick-frozen algae in a refrigerator at the temperature of-80 ℃ for later use.

(3) Cell wall breaking by taking 6 g (dry weight) quick frozen algae, adding 60 mL of phosphate buffer (50 mM) with pH of 6.0. Cellulase (3U/g of algae), pectinase (2U/g of algae) and muramidase (1U/g of algae) were added. Enzymatic hydrolysis is carried out for 3 hours at 35 ℃. Subsequently, the enzymatic hydrolysate was placed in an ultrasonic breaker (power 500W, work 10 s, batch 8 s) and the total treatment time was 15 min.

(4) Starch separation and purification, namely centrifuging the crushed algae liquid (10000 rpm,25 min), and collecting precipitate. The pellet was resuspended in 60 mL deionized water, 6 mL strength 5% (w/v) SDS solution was added and stirred at room temperature for 40 min. The SDS washing step was repeated 5 times. Finally, the washed sample was subjected to ultrafiltration purification by an ultrafiltration membrane of 30 kDa molecular weight cut-off and was replaced 7 times with deionized water.

(5) Starch drying and preservation the purified starch suspension was freeze-dried (-60 ℃) for 60 hours. And (5) sealing and preserving the dried starch sample under the nitrogen-filled condition. The starch dry weight is 3.06 g, the amylose percentage is 75% and the starch purity is 98%.

Example 4 this example provides a method for extracting high purity starch from red algae, comprising the following steps:

(1) Microalgae cultivation, wherein the microalgae (Porphyridium cruentum) is selected as an experimental object. The rambutan is inoculated into f/2 culture medium, and the culture condition is that the temperature is 22+/-1 ℃, the illumination intensity is 3500 lux, and the light-dark ratio is 14:10h. After the logarithmic growth phase (about 8 days), the algae liquid was collected by centrifugation and transferred to f/2 medium without nitrogen source for continuous cultivation for 6 days to promote starch accumulation and maximize the starch content in microalgae.

(2) Microalgae collection and pretreatment the algae were collected using a centrifuge (4200 rpm,18 min). The collected algae were washed 3 times with 0.5M NaCl solution, and then subjected to liquid nitrogen flash freezing treatment. And (5) storing the quick-frozen algae in a refrigerator at the temperature of-80 ℃ for later use.

(3) Cell wall breaking by taking 7g (dry weight) quick frozen algae, adding 70 mL of acetic acid buffer (50 mM) with pH of 5.8. Cellulase (2.8U/g algal), pectase (1.8U/g algal), muramidase (0.9U/g algal) and beta-glucanase (0.5U/g algal) were added. Enzymatic hydrolysis is carried out for 2.8 hours at 38 ℃. Subsequently, the enzymatic hydrolysate was placed in an ultrasonic breaker (power 480W, work 9 s, batch 6 s) and the total treatment time was 14 min.

(4) Starch separation and purification, namely centrifuging the crushed algae liquid (9500 rpm,22 min), and collecting precipitate. The pellet was resuspended in 70 mL deionized water, 7 mL strength 8% (w/v) SDS solution was added and 38 min stirred at room temperature. The SDS washing step was repeated 4 times. Finally, the washed sample was subjected to ultrafiltration purification by an ultrafiltration membrane of 40 kDa molecular weight cut-off and was replaced 6 times with deionized water.

(5) Starch drying and preservation the purified starch suspension was lyophilized (-55 ℃ C., 56 hours) in vacuo. And (5) hermetically storing the dried starch sample in vacuum packaging. The starch dry weight 3.27g amylose percentage was 42% and the starch purity was 98%.

Example 5 this example provides a method for extracting high purity starch from schizochytrium limacinum, comprising the following steps:

(1) Microalgae cultivation, namely selecting schizochytrium limacinum (Haematococcus pluvialis) as an experimental object. Inoculating schizochytrium limacinum into an OHM culture medium under the culture conditions that the temperature is 24+/-1 ℃, the illumination intensity is 5500 lux and the light-dark ratio is 16:8h. After the logarithmic growth phase (about 7 days), the algae liquid was collected by centrifugation and transferred to OHM medium without nitrogen and phosphorus sources for continuous cultivation for 12 days to promote starch accumulation and maximize starch content in microalgae.

(2) Microalgae collection and pretreatment the algae were collected using a centrifuge (3800 rpm,25 min). The collected algae were washed 3 times with 0.9% NaCl solution, and then subjected to liquid nitrogen flash freezing treatment. And (5) storing the quick-frozen algae in a refrigerator at the temperature of-80 ℃ for later use.

(3) Cell wall breaking, namely taking 9 g (dry weight) quick-frozen algae, and adding 90 mL of citric acid buffer solution (60 mM) with pH of 5.2. Cellulase (3.2U/g algal), pectase (2.2U/g algal), muramidase (1.2U/g algal) and chitinase (0.6U/g algal) were added. Enzymatic hydrolysis is carried out for 3.2 hours at 36 ℃. Subsequently, the enzymatic hydrolysate was placed in an ultrasonic breaker (power 520W, work 12 s, batch 7 s) and the total treatment time was 18 min.

(4) Starch separation and purification, namely centrifuging the crushed algae liquid (10500 rpm,28 min), and collecting precipitate. The pellet was resuspended in 90 mL deionized water, 9 mL strength 16% (w/v) SDS solution was added and treated in a 42℃water bath for 42 min. The SDS washing step was repeated 5 times. Finally, the washed sample was subjected to ultrafiltration purification by an ultrafiltration membrane of 20 kDa molecular weight cut-off and was replaced 8 times with deionized water.

(5) Starch drying and preservation the purified starch suspension was lyophilized (-55 ℃ C., 56 hours) in vacuo. And (5) hermetically storing the dried starch sample in vacuum packaging. The dried starch samples were stored in nitrogen filled aluminum foil bags in a sealed manner. The starch dry weight was finally 4.13g, the amylose percentage was 45% and the starch purity was 98.5%.

Example 6 this example provides a method for extracting high purity starch from dunaliella salina, comprising the following steps:

(1) Microalgae culture, wherein Dunaliella salina (Dunaliella salina) is selected as the experimental object. Dunaliella salina is inoculated into a modified Johnson's culture medium (2M NaCl) under the culture conditions that the temperature is 26+/-1 ℃, the illumination intensity is 6500 lux and the light-dark ratio is 18:6h. After the logarithmic growth phase (about 9 days), the algae liquid was collected by centrifugation and transferred to a high salt medium (3M NaCl) without nitrogen source for continuous cultivation for 8 days to promote starch accumulation and maximize the starch content in microalgae.

(2) Microalgae collection and pretreatment the algae were collected using a centrifuge (3500 rpm,30 min). The collected algae were washed 3 times with 2M NaCl solution and then subjected to liquid nitrogen flash freezing treatment. And (5) storing the quick-frozen algae in a refrigerator at the temperature of-80 ℃ for later use.

(3) Cell wall breaking by taking 5 g (dry weight) quick frozen algae, adding 50 mL HEPES buffer (70 mM, containing 1M NaCl) with pH of 6.2. Cellulase (2.5U/g algal), pectase (1.5U/g algal), muramidase (0.8U/g algal) and saline-alkali protease (0.4U/g algal) were added. Enzymatic hydrolysis is carried out for 2.5 hours at 40 ℃. Subsequently, the enzymatic hydrolysate was placed in an ultrasonic breaker (power 450W, work 8 s, batch 5 s) and the total treatment time was 12 min.

(4) Starch separation and purification, namely centrifuging the crushed algae liquid (11000 rpm,30 min), and collecting precipitate. The pellet was resuspended in 50 mL deionized water, 5 mL strength 10% (w/v) SDS solution was added and treated in a 35℃water bath for 35 min. The SDS washing step was repeated 4 times. Finally, the washed sample was subjected to ultrafiltration purification by an ultrafiltration membrane of 30 kDa molecular weight cut-off and was replaced 7 times with deionized water.

(5) Starch drying and preservation the purified starch suspension was dried (45 ℃ C., 48 hours). And grinding the dried starch sample, and sealing and storing in a self-sealing bag. The starch dry weight was finally 2.08g, the amylose percentage was 38% and the starch purity was 98%.

Example 7 in order to verify the beneficial effects of the method of the invention, the following comparative experiments were performed:

(1) The freezing and crushing method comprises adding 1L distilled water into 10 g dry weight spirulina body to obtain suspension, quickly soaking in liquid nitrogen, and maintaining for several minutes until the sample is completely frozen. After thawing the broken sample, the cell debris and starch are separated by centrifugation (8000-12000 rpm, 10-20 min). The cell disruption rate was 20% and the purity of the extracted starch was 83%.

(2) The ultrasonic method was carried out as in example 1, but omitting the pretreatment with liquid nitrogen and the enzymatic hydrolysis treatment steps. The cell disruption rate was 33% and the purity of the extracted starch was 86%.

(3) Conventional enzymatic extraction (without liquid nitrogen pretreatment and ultrasound assistance) was performed as in example 1, but omitting the liquid nitrogen pretreatment and ultrasound disruption steps. The cell disruption rate was 80% and the purity of the extracted starch was 90%.

(4) The combination of freezing and crushing and enzymolysis ultrasonic method is carried out according to the method of the example 1, the cell crushing rate is 98 percent, and the purity of the extracted starch is 98 percent.

The comparison result is shown in fig. 6, the method of the invention is significantly superior to the conventional method in terms of microalgae cell wall breaking rate and starch purity, and amylose with a percentage of 40-75% is obtained, which is higher than that of conventional crop starch.

Claims

1. A method for extracting high-purity starch from microalgae, which is characterized by comprising the following steps:

(3) Breaking microalgae cells, namely carrying out enzymolysis on microalgae frozen by liquid nitrogen by adopting complex enzyme composed of cellulose, pectase and lywallase, then thoroughly destroying cell walls after ultrasonic crushing treatment to release starch in cells, wherein the addition of the complex enzyme is respectively 1U-4U of cellulase, 0.5-U-3U of pectase and 0.3-U-1.5U of lywallase in unit gram weight of freeze-dried microalgae, the complex enzyme of the rambutanella is also provided with beta-glucanase, the addition of the beta-glucanase is 0.3-0.6U, the complex enzyme of the schizochytrium is also provided with chitinase, the addition of the chitinase is 0.4-0.8U, and the complex enzyme of Dunaliella salina is also provided with alkaline protease, and the addition of the alkaline protease is 0.2-0.5U;

(4) Separating and purifying starch, namely separating the microalgae suspension after wall breaking to obtain a crude starch product, dissolving the crude starch product in water, and adopting SDS solution to wash and ultrafiltrate for multiple times to remove impurities.

2. The method for extracting high purity starch from microalgae according to claim 1, characterized in that in step (1), the nitrogen-deficient medium of schizochytrium limacinum contains no phosphorus source.

3. The method for extracting high purity starch from microalgae according to claim 1, characterized in that in step (1), the nitrogen-deficient medium of chlamydomonas is free of sulfur source.

4. The method for extracting high purity starch from microalgae according to claim 1, characterized in that in step (1), sodium chloride concentration in nitrogen deficiency medium of dunaliella salina is 3M.

5. The method for extracting high purity starch from microalgae according to claim 1, characterized in that in the step (4), the volume ratio of the SDS solution to the crude starch water solution is 10:1, and the mass volume concentration of the SDS solution is 1% -16%.

6. The method for extracting high purity starch from microalgae according to claim 1, characterized in that in step (4), ultrafiltration is carried out with ultrafiltration membrane with molecular weight cut-off of 20-100 kDa, and deionized water is used for 5-8 times.

7. The microalgae starch prepared by the method of any one of claims 1-6, wherein the total starch content in the microalgae starch is more than or equal to 98%, and the amylose percentage is 40-75%.