阅读说明：本技术 一种沙漠蛋白核小球藻异养-稀释-光诱导培养方法 (Heterotrophic-dilution-photoinduction culture method for chlorella pyrenoidosa in desert ) 是由 梁钧 于 2021-08-24 设计创作，主要内容包括：本发明涉及一种沙漠蛋白核小球藻异养-稀释-光诱导培养方法。本发明将异养方式与自养方式的结合方式,大大提高沙漠蛋白核小球藻的生物量和蛋白质含量,达到高效生产大量高蛋白质含量的沙漠蛋白核小球藻的目的,不仅可在短时间内获得较高浓度的藻细胞生物量,还可诱导蛋白核小球藻内蛋白质、叶绿素等物质的积累,改善蛋白核小球藻品质,提高蛋白核小球藻的综合利用价值。此外,该方法具有操作简便、实用性强、易实现连续生产等特点,非常有利于大规模的推广和应用,以快速高效地获得大量的高含量蛋白质的沙漠蛋白核小球藻藻粉,为高品质沙漠蛋白核小球藻藻粉的生产提供技术支撑,满足工业生产的需求。(The invention relates to a heterotrophic-dilution-photoinduction culture method of chlorella pyrenoidosa in desert. The invention greatly improves the biomass and the protein content of the desert chlorella pyrenoidosa by combining the heterotrophic mode and the autotrophic mode, achieves the aim of efficiently producing a large amount of the desert chlorella pyrenoidosa with high protein content, can obtain the biomass of algae cells with higher concentration in a short time, can induce the accumulation of substances such as protein, chlorophyll and the like in the chlorella pyrenoidosa, improves the quality of the chlorella pyrenoidosa and improves the comprehensive utilization value of the chlorella pyrenoidosa. In addition, the method has the characteristics of simple and convenient operation, strong practicability, easy realization of continuous production and the like, is very favorable for large-scale popularization and application, so that a large amount of high-protein content desert chlorella pyrenoidosa algae powder can be quickly and efficiently obtained, technical support is provided for the production of high-quality desert chlorella pyrenoidosa algae powder, and the requirement of industrial production is met.)

1. A heterotrophic-dilution-photoinduction culture method of chlorella pyrenoidosa in desert is characterized in that: the method comprises the following steps:

step one, activation of algal species: taking out plate-streaked chlorella pyrenoidosa from an algae seed resource library, selecting a single algae strain in a sterile workbench, streaking the single algae strain in a solid culture medium, and culturing in an illumination incubator at the culture temperature of 28 ℃ and illumination of 5000lux for 15 days to obtain activated algae seeds;

step two, first-stage seed liquid preparation: selecting 3-5 ring algae from activated algae, inoculating into 100mL triangular flask containing 50mL culture medium, culturing at 28 deg.C under illumination of 5000lux in 50r/min shaking incubator to logarithmic phase 10⁸Obtaining first-grade seed liquid after seed/mL;

step three, preparing a secondary seed solution: inoculating the primary seed solution into a 500mL triangular flask filled with 200mL culture medium in an inoculation amount of 10% (v/v), and culturing for 48-72 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a secondary seed solution;

step four, preparing a third-level seed liquid: inoculating the secondary seed solution into a 1000mL triangular flask filled with 500mL culture medium in an inoculation amount of 10% (v/v), and culturing for 48-72 hours in a shaking culture box at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a tertiary seed solution;

step five, heterotrophic culture: taking the third-stage seed solution as a seed solution for fermentation culture, inoculating the seed solution into a fermentation tank according to the inoculation amount of 15% for culture, wherein the culture temperature of the fermentation tank is 28 ℃, the dissolved oxygen is 25%, the pH value is 7, the rotating speed of the fermentation tank is 50r/min, after the culture is carried out for 72 hours, 2000mL of fresh culture solution is taken every day, and 2000mL of fresh culture solution is supplemented, so that the stability of a semi-continuous culture system is kept;

step six, dilution-light induced culture: in the culture process of the step five, sampling and detecting the cell dry weight of the chlorella pyrenoidosa in the fermentation broth every 24 hours, wherein the initial algae cell concentration is 2.12-4.24g/L in a dry weight state, and when no significant difference exists in biomass detection for two consecutive days, stopping culturing, and the algae cell concentration is 150-200g/L in a dry weight state; diluting the culture solution in the fermentation tank, transferring into a 60L columnar photobioreactor for continuous culture under the culture conditions of 28 ℃, illumination of 10000lux and air ventilation of 1L/min, sampling and detecting every day, and finishing the culture when the protein content has no significant difference in continuous detection for 2 days.

2. The heterotrophic-dilution-photoinduction culture method of Chlorella pyrenoidosa in desert as claimed in claim 1, wherein: the culture medium used in the first step to the fourth step is Basal solid culture medium containing 10g/L glucose.

3. The heterotrophic-dilution-photoinduction culture method of Chlorella pyrenoidosa in desert as claimed in claim 1, wherein: and the culture medium and the additive culture medium used in the fifth step both use a Basal culture medium with the glucose concentration of 20g/L as a Basal culture medium and use glucose as a carbon source.

4. The heterotrophic-dilution-photoinduction culture method of Chlorella pyrenoidosa in desert as claimed in claim 1, wherein: and step five, adjusting the pH of the culture solution in the fermentation tank by using 50% acetic acid and 2mol/L sodium hydroxide.

5. The heterotrophic-dilution-photoinduction culture method of Chlorella pyrenoidosa in desert as claimed in claim 1, wherein: and in the sixth step, the culture medium used for the culture solution in the dilution fermentation tank is a Basal culture medium.

6. The heterotrophic-dilution-photoinduction culture method of Chlorella pyrenoidosa in desert as claimed in claim 1, wherein: and step six, the protein content of the chlorella pyrenoidosa obtained after finishing the culture is 40-55%.

Technical Field

The invention relates to the technical field of microalgae cultivation, in particular to a heterotrophic-dilution-photoinduction culture method for chlorella pyrenoidosa in desert.

Background

Chlorella (Chlorella sp.) is a unicellular, ubiquitous green algae belonging to the phylum Chlorophyta, class Chlorophyceae, family Oomycetaceae, genus Chlorella. Common chlorella species in China include chlorella pyrenoidosa, chlorella ellipsoidea, chlorella vulgaris and the like. Chlorella is rich in protein, unsaturated fatty acid (linoleic acid, linolenic acid, DHA, EPA, etc.), carotenoid, astaxanthin and multiple vitamins, and can be widely applied to the fields of functional food, feed, cosmetics, medicine, etc.

The existing production method of chlorella pyrenoidosa algae powder mainly comprises open autotrophic culture, closed heterotrophic culture and mixed culture. Open autotrophic culture is easy to pollute, culture conditions such as illumination, temperature and the like are easy to be influenced by weather, the concentration of algae cells is low, the harvesting cost is higher, closed heterotrophic culture generally utilizes glucose as a carbon source and adopts high-energy-consumption nitrate nitrogen such as sodium nitrate as a nitrogen source, although the ideal concentration of the algae cells can be obtained, the culture process is always in a dark condition, the contents of algae powder protein, chlorophyll and the like harvested in the later period are lower, the quality and various biological activity functions of the algae cells are obviously insufficient, and hydrochloric acid is used for adjusting the pH value in the culture process, so that fermentation equipment is easily damaged to a certain degree.

The tacrolimus dryland desert green algae as extreme environment microalgae have certain differences in biological characteristics, physiological and biochemical components and the like from green algae in the evolution of tens of thousands of years. The invention provides a heterotrophic-dilution-photoinduction culture method suitable for desert chlorella pyrenoidosa, and aims to solve the problems that the production period of the autotrophic culture of the desert chlorella pyrenoidosa is long, the biomass is low, the protein content of heterotrophic culture algae powder is low and the like, so that the high-quality desert chlorella pyrenoidosa can be obtained in a short time.

Disclosure of Invention

In view of the problems in the prior art, the invention discloses a heterotrophic-dilution-photoinduction culture method of chlorella pyrenoidosa in desert, which comprises the following steps:

step one, activation of algal species: taking out plate-streaked chlorella pyrenoidosa from an algae seed resource library, selecting a single algae strain in a sterile workbench, streaking the single algae strain in a solid culture medium, and culturing in an illumination incubator at the culture temperature of 28 ℃ and illumination of 5000lux for 15 days to obtain activated algae seeds;

step two, first-stage seed liquid preparation: selecting 3-5 ring algae from activated algae, inoculating into 100mL triangular flask containing 50mL culture medium, culturing at 28 deg.C under illumination of 5000lux in 50r/min shaking incubator to logarithmic phase 10⁸Obtaining first-grade seed liquid after seed/mL;

step three, preparing a secondary seed solution: inoculating the primary seed solution into a 500mL triangular flask filled with 200mL culture medium in an inoculation amount of 10% (v/v), and culturing for 48-72 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a secondary seed solution;

step four, preparing a third-level seed liquid: inoculating the secondary seed solution into a 1000mL triangular flask filled with 500mL culture medium in an inoculation amount of 10% (v/v), and culturing for 48-72 hours in a shaking culture box at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a tertiary seed solution;

step five, heterotrophic culture: taking the third-stage seed solution as a seed solution for fermentation culture, inoculating the seed solution into a fermentation tank according to the inoculation amount of 15% for culture, wherein the culture temperature of the fermentation tank is 28 ℃, the dissolved oxygen is 25%, the pH value is 7, the rotating speed of the fermentation tank is 50r/min, after the culture is carried out for 72 hours, 2000mL of fresh culture solution is taken every day, and 2000mL of fresh culture solution is supplemented, so that the stability of a semi-continuous culture system is kept;

step six, dilution-light induced culture: in the culture process of the step five, sampling and detecting the cell dry weight of the chlorella pyrenoidosa in the fermentation broth every 24 hours, wherein the initial algae cell concentration is 2.12-4.24g/L in a dry weight state, and when no significant difference exists in biomass detection for two consecutive days, stopping culturing, and the algae cell concentration is 150-200g/L in a dry weight state; diluting the culture solution in the fermentation tank, transferring into a 60L columnar photobioreactor for continuous culture under the culture conditions of 28 ℃, illumination of 10000lux and air ventilation of 1L/min, sampling and detecting every day, and finishing the culture when the protein content has no significant difference in continuous detection for 2 days.

As a preferred embodiment of the present invention, the culture medium used in the first to fourth steps is Basal solid culture medium containing 10g/L glucose.

In a preferred embodiment of the present invention, the medium used in step five and the additional medium are both Basal medium with a glucose concentration of 20g/L and glucose as a carbon source.

As a preferable mode of the present invention, in the fifth step, the pH of the culture solution in the fermentation tank is adjusted by using 50% acetic acid and 2mol/L sodium hydroxide.

In a preferred embodiment of the present invention, the culture medium used in the culture solution in the dilution fermenter in the sixth step is Basal medium.

As a preferable scheme of the invention, the protein content of the chlorella pyrenoidosa obtained after the culture is finished in the step six is 40-55%.

The invention has the beneficial effects that: the heterotrophic-dilution-photoinduction culture method for the chlorella pyrenoidosa in the desert provided by the invention has the advantages that the combination of the heterotrophic mode and the autotrophic mode greatly improves the biomass and the protein content of the chlorella pyrenoidosa in the desert, achieves the aim of efficiently producing a large amount of chlorella pyrenoidosa with high protein content, can obtain the biomass of algae cells with higher concentration in a short time, can induce the accumulation of substances such as protein, chlorophyll and the like in the chlorella pyrenoidosa, improves the quality of the chlorella pyrenoidosa and improves the comprehensive utilization value of the chlorella pyrenoidosa. In addition, the method has the characteristics of simple and convenient operation, strong practicability, easy realization of continuous production and the like, is very favorable for large-scale popularization and application, so that a large amount of high-protein content desert chlorella pyrenoidosa algae powder can be quickly and efficiently obtained, technical support is provided for the production of high-quality desert chlorella pyrenoidosa algae powder, and the requirement of industrial production is met.

Drawings

FIG. 1 is a photograph of a separated and purified Chlorella pyrenoidosa employed in the present invention;

FIG. 2 is a graph of the protein content of heterotrophic and heterotrophic-photoinduced desert Chlorella pyrenoidosa;

FIG. 3 is a chlorophyll content diagram of light-induced desert Chlorella pyrenoidosa at different dilution times;

FIG. 4 is a graph showing the content of protein in desert Chlorella pyrenoidosa induced by light with different dilution times.

Detailed Description

Example 1

The invention relates to a heterotrophic-dilution-photoinduction culture method of chlorella pyrenoidosa in desert, which adopts the technical scheme that the method comprises the following steps:

step one, activation of algal species: taking out plate-streaked chlorella pyrenoidosa from an algae seed resource library, selecting a single algae strain in a sterile workbench, streaking the single algae strain in a solid culture medium, culturing in an illumination incubator, wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing at 28 ℃ under illumination of 5000lux for 15 days to obtain activated algae seeds;

step two, first-stage seed liquid preparation: selecting 3-ring algae from activated algae, inoculating into 100mL triangular flask containing 50mL culture medium (10 g/L glucose-containing Basal solid culture medium), culturing at 28 deg.C under illumination of 5000lux and 50r/min in shaking incubator until logarithmic phase 10⁸Obtaining first-grade seed liquid after seed/mL;

step three, preparing a secondary seed solution: inoculating the primary seed solution into a 500mL triangular flask filled with 200mL culture medium in an inoculation amount of 10% (v/v), wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing for 48 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a secondary seed solution;

step four, preparing a third-level seed liquid: inoculating the secondary seed solution into a 1000mL triangular flask filled with 500mL culture medium in an inoculation amount of 10% (v/v), wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing for 48 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a tertiary seed solution;

step five, heterotrophic culture: inoculating the three-stage seed solution serving as a seed solution for fermentation culture into a fermentation tank according to the inoculation amount of 15% for culture, wherein a culture medium and an addition culture medium in the fermentation tank both use a Basal culture medium with the glucose concentration of 20g/L as a Basal culture medium, use glucose as a carbon source, the culture temperature of the fermentation tank is 28 ℃, the dissolved oxygen is 25%, the pH is 7, the rotation speed of the fermentation tank is 50r/min, after 72 hours of culture, 2000mL of a material is taken every day, 2000mL of fresh culture solution is supplemented, the pH of the culture solution in the fermentation tank is adjusted by using 50% acetic acid and 2mol/L sodium hydroxide, and the stability of a semi-continuous culture system is kept;

step six, dilution-light induced culture: in the culture process of the fifth step, sampling and detecting the cell dry weight of the chlorella pyrenoidosa in the fermentation broth every 24 hours, wherein the initial algae cell concentration is 2.12g/L in a dry weight state, and when no significant difference exists in biomass detected for two consecutive days, stopping culturing, and the algae cell concentration is 150g/L in a dry weight state; adding a Basal culture medium into a fermentation tank to dilute the culture solution, transferring the culture solution into a 60L columnar photobioreactor for continuous culture under the conditions of 28 ℃, illumination of 10000lux and air ventilation of 1L/min, sampling and detecting every day, and finishing the culture when the protein content has no significant difference in detection for 2 consecutive days; the protein content of the chlorella pyrenoidosa obtained after the culture is finished is 40-55%.

Example 2

The invention relates to a heterotrophic-dilution-photoinduction culture method of chlorella pyrenoidosa in desert, which adopts the technical scheme that the method comprises the following steps:

step one, activation of algal species: taking out plate-streaked chlorella pyrenoidosa from an algae seed resource library, selecting a single algae strain in a sterile workbench, streaking the single algae strain in a solid culture medium, culturing in an illumination incubator, wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing at 28 ℃ under illumination of 5000lux for 15 days to obtain activated algae seeds;

step two, first-stage seed liquid preparation: selecting 5-ring algae from activated algae, inoculating into 100mL triangular flask containing 50mL culture medium (10 g/L glucose-containing Basal solid culture medium), culturing at 28 deg.C under illumination of 5000lux and 50r/min in shaking incubator until logarithmic phase 10⁸Obtaining first-grade seed liquid after seed/mL;

step three, preparing a secondary seed solution: inoculating the primary seed solution into a 500mL triangular flask filled with 200mL culture medium in an inoculation amount of 10% (v/v), wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing for 60 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a secondary seed solution;

step four, preparing a third-level seed liquid: inoculating the secondary seed solution into a 1000mL triangular flask filled with 500mL culture medium in an inoculation amount of 10% (v/v), wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing for 60 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a tertiary seed solution;

step five, heterotrophic culture: inoculating the three-stage seed solution serving as a seed solution for fermentation culture into a fermentation tank according to the inoculation amount of 15% for culture, wherein a culture medium and an addition culture medium in the fermentation tank both use a Basal culture medium with the glucose concentration of 20g/L as a Basal culture medium, use glucose as a carbon source, the culture temperature of the fermentation tank is 28 ℃, the dissolved oxygen is 25%, the pH is 7, the rotation speed of the fermentation tank is 50r/min, after 72 hours of culture, 2000mL of a material is taken every day, 2000mL of fresh culture solution is supplemented, the pH of the culture solution in the fermentation tank is adjusted by using 50% acetic acid and 2mol/L sodium hydroxide, and the stability of a semi-continuous culture system is kept;

step six, dilution-light induced culture: in the culture process of the fifth step, sampling and detecting the cell dry weight of the chlorella pyrenoidosa in the fermentation broth every 24 hours, wherein the initial algae cell concentration is 3.21g/L in a dry weight state, and when no significant difference exists in biomass detected for two consecutive days, stopping culturing, and the algae cell concentration is 170g/L in a dry weight state; adding a Basal culture medium into a fermentation tank to dilute the culture solution, transferring the culture solution into a 60L columnar photobioreactor for continuous culture under the conditions of 28 ℃, illumination of 10000lux and air ventilation of 1L/min, sampling and detecting every day, and finishing the culture when the protein content has no significant difference in detection for 2 consecutive days; the protein content of the chlorella pyrenoidosa obtained after the culture is finished is 40-55%.

Example 3

The invention relates to a heterotrophic-dilution-photoinduction culture method of chlorella pyrenoidosa in desert, which adopts the technical scheme that the method comprises the following steps:

step one, activation of algal species: taking out plate-streaked chlorella pyrenoidosa from an algae seed resource library, selecting a single algae strain in a sterile workbench, streaking the single algae strain in a solid culture medium, culturing in an illumination incubator, wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing at 28 ℃ under illumination of 5000lux for 15 days to obtain activated algae seeds;

step two, first-stage seed liquid preparation: selecting 5-ring algae from activated algae, inoculating into 100mL triangular flask containing 50mL culture medium (10 g/L glucose-containing Basal solid culture medium), culturing at 28 deg.C under illumination of 5000lux and 50r/min in shaking incubator until logarithmic phase 10⁸Obtaining first-grade seed liquid after seed/mL;

step three, preparing a secondary seed solution: inoculating the primary seed solution into a 500mL triangular flask filled with 200mL culture medium in an inoculation amount of 10% (v/v), wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing for 72 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a secondary seed solution;

step four, preparing a third-level seed liquid: inoculating the secondary seed solution into a 1000mL triangular flask filled with 500mL culture medium in an inoculation amount of 10% (v/v), wherein the culture medium is a Basal solid culture medium containing 10g/L glucose, and culturing for 72 hours in a shaking incubator at 28 ℃, under the illumination of 5000lux and at 50r/min to obtain a tertiary seed solution;

step five, heterotrophic culture: inoculating the three-stage seed solution serving as a seed solution for fermentation culture into a fermentation tank according to the inoculation amount of 15% for culture, wherein a culture medium and an addition culture medium in the fermentation tank both use a Basal culture medium with the glucose concentration of 20g/L as a Basal culture medium, use glucose as a carbon source, the culture temperature of the fermentation tank is 28 ℃, the dissolved oxygen is 25%, the pH is 7, the rotation speed of the fermentation tank is 50r/min, after 72 hours of culture, 2000mL of a material is taken every day, 2000mL of fresh culture solution is supplemented, the pH of the culture solution in the fermentation tank is adjusted by using 50% acetic acid and 2mol/L sodium hydroxide, and the stability of a semi-continuous culture system is kept;

step six, dilution-light induced culture: in the culture process of the fifth step, sampling and detecting the cell dry weight of the chlorella pyrenoidosa in the fermentation broth every 24 hours, wherein the initial algae cell concentration is 4.24g/L in a dry weight state, and when no significant difference exists in biomass detected for two consecutive days, stopping culturing, and the algae cell concentration is 200g/L in a dry weight state; adding a Basal culture medium into a fermentation tank to dilute the culture solution, transferring the culture solution into a 60L columnar photobioreactor for continuous culture under the conditions of 28 ℃, illumination of 10000lux and air ventilation of 1L/min, sampling and detecting every day, and finishing the culture when the protein content has no significant difference in detection for 2 consecutive days; the protein content of the chlorella pyrenoidosa obtained after the culture is finished is 40-55%.

The basic solid culture medium comprises the following main components in percentage by weight:

media composition	Final concentration mg/L	Media composition	Final concentration mg/L
				NaNO3	1250	FeSO4·7H2O	49.8
KH2PO4	1250	ZnSO4·7H2O	88.2
				MgSO4·7H2O	1000	MnCl2·4H2O	14.2
EDTA	500	Na2MoO4·2H2O	11.92
				H3BO4	114.2	CuSO4·5H2O	15.7
CaCl2·H2O	111	Co(NO3)2·6H2O	4.9

Components not described in detail herein are prior art.

Although the present invention has been described in detail with reference to the specific embodiments, the present invention is not limited to the above embodiments, and various changes and modifications without inventive changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.