CN117757687A - Culture method of spirulina rich in vitamin C - Google Patents
Culture method of spirulina rich in vitamin C Download PDFInfo
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Abstract
The invention discloses a method for culturing spirulina rich in vitamin C, which comprises the following steps: s1, indoor culture and screening: preparing a culture medium, inoculating, culturing, detecting and judging; s2, expanding culture of algae species: preparing a culture medium, inoculating and culturing; s3, large-scale cultivation: preparing a culture medium, inoculating and culturing in a large scale. The culture of the spirulina rich in the vitamin C can be carried out on a large scale in months with relatively low natural temperature, the production equipment and human resources which are idle in production stopping in months with relatively low natural temperature are fully utilized, the waste of resources is avoided, and the spirulina with more balanced nutrition and rich in the vitamin C can be cultured.
Description
Technical Field
The invention belongs to the technical field of spirulina cultivation, and particularly relates to a method for cultivating spirulina rich in vitamin C.
Background
Spirulina is a type of original lower photosynthetic organism, named because of the spiral shape of the algae. The spirulina has the protein content of more than 60%, low fat and carbohydrate content and rich trace elements and vitamins, and is one of the most abundant and balanced species discovered so far. The researches show that the spirulina has the effects of enhancing immunity, reducing cholesterol, regulating blood sugar, resisting anoxia, resisting radiation, resisting fatigue, resisting aging, regulating blood pressure and the like. Spirulina is widely considered worldwide to belong to the group of best nutrients in the 21 st century. But with a lower vitamin C content.
At present, researches on a culture method of spirulina rich in selenium, zinc, DHA and other trace elements have been carried out, and the culture method of spirulina rich in vitamin C has not been reported. The vitamin C content in the spirulina is improved, so that the spirulina has richer and more balanced nutrients, better food raw materials are provided for human beings, meanwhile, the spirulina products with the partitions are formed, raw materials are provided for the development of terminal products, and better economic benefits are formed.
In addition, in months of lower natural environment temperature, the cultivation of spirulina is in a shutdown state, so that resource waste and economic loss are caused.
Disclosure of Invention
The invention aims to disclose a method for culturing spirulina rich in vitamin C, which can be used for culturing spirulina rich in vitamin C in a large scale in months with relatively low temperature, and can be used for utilizing production equipment and manpower resources which are idle in production stopping in months with relatively low temperature, so that the spirulina rich in vitamin C with more balanced nutrition can be cultured while avoiding resource waste.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
a method for culturing spirulina rich in vitamin C comprises the following steps:
s1, indoor culture and screening: preparing a culture medium, inoculating, culturing, detecting and judging;
s2, expanding culture of algae species: preparing a culture medium, inoculating and culturing;
s3, large-scale cultivation: preparing a culture medium, inoculating and culturing in a large scale.
Further: the steps S1, S2 and S3 are that the culture medium is NaHCO in the preparation of the culture medium 3 4-5g/L，NaNO 3 0.3-0.6g/L，NaCl 0.5-1g/L，KH 2 PO 4 0.2-0.5g/L，K 2 SO 4 0.2-0.5g/L，FeSO 4 ·7H 2 0.003-0.01g/L of O, 0.003-0.01g/L of EDTA.2Na and the balance: water; CO filling 2 The pH is adjusted to 8-9.
Further: the steps S1, S2 and S3 are that the spirulina seeds in logarithmic growth phase are inoculated, and the spectrophotometry value OD of the spirulina liquid is taken 560nm ＝0.18-0.25。
Further: step S1, inoculating 1-5L of the culture medium to a transparent culture bottle, and placing the culture bottle in a constant-temperature oscillator;
8:00-18:00 a day, the culture temperature is 22-25 ℃, the illumination intensity is 4000-6000Lux, 0.02-0.1g/L carbonic anhydrase and 0.01-0.05g/L gibberellin are added in the culture process, and CO is supplemented 2 ，CO 2 Flow rate0.005-0.015L/min, rotation frequency of the cradle: 20-25rpm, shimmy amplitude: 18-22mm;
the daily culture temperature is 18:00-20:00, the culture temperature is 20-22 ℃, the illumination intensity is 2500-4000Lux, and CO is supplemented 2 ，CO 2 The flow is 0.01-0.02L/min, and the rotation frequency of the cradle is: 18-20rpm, shimmy amplitude: 16-18mm;
20:00 a day to 8:00 a day, the culture temperature is 15-18 ℃, the illumination is stopped, 0.02-0.08g/L of ammonium nitrate and 0.08-0.15g/L of sodium carbonate are added, and the rotation frequency of a shaking table is that: 8-12rpm, shimmy amplitude: 10-12mm;
repeating the operation, culturing for 5 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
Further: in the step S1 described above, a step of,
(1) And (3) detection:
spectrophotometry value OD of algae liquid in culture bottle 560nm The spirulina powder is prepared by spray drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃; detecting the content of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder;
(2) Determination criteria:
1) The algae growth well meets standard: the number of spirals is more than or equal to 3, the body length is more than or equal to 100 mu m, the spiral width is more than or equal to 30 mu m, and the filament width is more than or equal to 7.0 mu m;
2) The algae powder detection index meets the standard: vitamin C not less than 30mg/100g, protein not less than 65%, chlorophyll not less than 900mg/100g, total carotenoid not less than 300mg/100g, phycocyanin not less than 4.5g/100g;
3) The spirulina seeds with the growth condition and the detection index of the spirulina powder meeting the standard are regarded as qualified spirulina seeds, and the spirulina seeds can be further cultivated in a large scale.
Further: step S2, culturing, namely culturing qualified spirulina seeds screened in step S1 in a 100-300L flat-plate photobioreactor;
8:00 a day after inoculation-18:00, culturing at 22-25deg.C under illumination of 4000-6000Lux, adding carbonic anhydrase 0.02-0.1g/L and gibberellin 0.01-0.05g/L during culturing, and supplementing CO 2 ，CO 2 The flow is 0.005-0.015L/min;
the daily culture temperature is 18:00-20:00, the culture temperature is 20-22 ℃, the illumination is 2500-4000Lux, and CO is supplemented 2 ，CO 2 The flow is 0.01-0.02L/min;
20:00 a day to 8:00 a day, the culture temperature is 15-18 ℃, the illumination is stopped, and 0.02-0.08g/L of ammonium nitrate and 0.08-0.15g/L of sodium carbonate are added;
further: step S3, large-scale cultivation is to cultivate the spirulina obtained by the enlarged cultivation in the step S2 in a cultivation pool;
8:00-18:00 a day, controlling the temperature to be 20-25 ℃, controlling the illumination intensity to be 4000-6000Lux, adding 0.02-0.1g/L of carbonic anhydrase and 0.01-0.05g/L of gibberellin, continuously stirring, and supplementing CO at the stirring speed of 25-35r/min 2 ，CO 2 The flow is 0.005-0.015L/min;
the temperature is controlled at 18:00-20:00 a day, the temperature is controlled at 18-22 ℃, the illumination intensity is 2500-4000Lux, stirring is continuously carried out, the stirring speed is 20-25r/min, and CO is supplemented 2 ，CO 2 The flow is 0.01-0.02L/min;
20:00 a day to 8:00 a day, controlling the temperature to 12-18 ℃ and no light, adding 0.02-0.08g/L of ammonium nitrate and 0.08-0.15g/L of sodium carbonate, continuously stirring, and keeping the stirring speed at 8-12r/min;
repeating the operation, culturing for 7 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected respectively, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope;
spectrophotometry value OD of algae liquid in culture pond 560nm The spirulina powder is prepared by collecting the spirulina mud, spraying and drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃.
Further: the pH of the algae liquid is always kept between 8 and 9 in the culture in the steps S1 and S2 and the culture in the step S3, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2 。
Remarks: OD (optical density) 560nm The spectrophotometric value obtained by measuring the concentration of spirulina seed liquid at 560nm by an ultraviolet spectrophotometer is shown.
Compared with the prior art, the invention has the beneficial effects that:
(1) The conventional culture temperature of the spirulina is about 20-25 ℃, and the growth and propagation of the spirulina can be influenced by the excessively low temperature (the temperature is lower than 10 ℃). The cultivation method disclosed by the invention utilizes relatively low-temperature stimulation and oxidative stress, timely supplements nutrition ingredients, screens spirulina seeds with higher vitamin C content accumulation, carries out scale cultivation, improves the vitamin C content in the cultivated spirulina powder by more than or equal to 30mg/100g compared with that in the spirulina powder cultivated by the common cultivation method by more than 200%, has unaffected protein, chlorophyll, total carotenoid and phycocyanin nutrient contents, has lower bacterial count in the algae liquid, and can obtain spirulina powder with more balanced nutrition ingredients and better quality.
(2) The cultivation method provided by the invention has the advantages of low cultivation cost and simplicity and convenience in operation, and can be applied to large-scale production.
(3) The natural temperature is lower in 11 months per year to 3 months per year, the spirulina cultivated by the conventional method has low yield, and the spirulina is in a production stopping state, so that production equipment and workers are idle, and resource waste and enterprise economic loss are caused. The cultivation method disclosed by the invention can be used for carrying out large-scale cultivation of the spirulina rich in vitamin C in months with relatively low temperature, fully utilizing production equipment and human resources which are idle in the production stopping period, avoiding resource waste, increasing income, solving the problem of labor consumption in the production stopping period of spirulina cultivation enterprises, and being applicable to winter in the south part of China, completely avoiding yield reduction or production stopping, greatly reducing economic loss and improving economic benefit.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1:
a method for culturing spirulina rich in vitamin C comprises selecting spirulina seeds of the type of GFJ (1), TFJ (1), FJ II, TFJ (2), FJD2, JX II, 6310XY, CH-5, and CH, numbered 1-10 in sequence, respectively.
Indoor culture and screening
1. Preparation of culture medium
NaHCO 3 4.5g/L，NaNO 3 0.4g/L，NaCl 1g/L，KH 2 PO 4 0.2g/L，K 2 SO 4 0.3g/L，FeSO 4 ·7H 2 O0.005 g/L, EDTA.2Na 0.005g/L, balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Inoculation of
Taking No. 1-10 spirulina seeds in logarithmic growth phase, respectively inoculating into 3L transparent culture bottle, and spectrophotometrically measuring OD of the algae liquid 560nm 10-30 algae were randomly selected from each flask, and their length was measured with a microscope with an eyepiece micrometer, and growth of algae was recorded.
3. Culturing
Placing the inoculated culture bottle in a constant temperature oscillator, culturing at 25 deg.C and light intensity of 5000Lux at 8:00-18:00 a day, adding carbonic anhydrase 0.05g/L and gibberellin 0.01g/L during culturing, and supplementing CO 2 ，CO 2 Flow rate 0.005L/min, constant temperature culture shaking table rotational frequency: 25rpm, shimmy amplitude: 22mm;
the daily culture temperature is 18:00-20:00, the culture temperature is 20 ℃, the illumination intensity is 3000Lux, and CO is supplemented 2 ，CO 2 The flow rate is 0.01L/min, and the rotation frequency of the constant temperature culture shaking table is: 20rpm, shimmy amplitude: 18mm;
20:00 a day to 8:00 a day, the culture temperature is 15 ℃, the illumination is stopped, 0.03g/L of ammonium nitrate and 0.08g/L of sodium carbonate are added, and the rotation frequency of a constant temperature culture shaking table is constant: 10rpm, shimmy amplitude: 10mm;
the pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2 ；
Repeating the operation, culturing for 5 daysMonitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
4. Detecting and judging
(1) Detection of
Spectrophotometry value OD of algae liquid in culture bottle 560nm The spirulina powder is prepared by spray drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
(2) Decision criterion
1) The algae growth well meets standard: the number of spirals is more than or equal to 3, the body length is more than or equal to 100 mu m, the spiral width is more than or equal to 30 mu m, and the filament width is more than or equal to 7.0 mu m.
2) The algae powder detection index meets the standard: vitamin C not less than 30mg/100g, protein not less than 65%, chlorophyll not less than 900mg/100g, total carotenoid not less than 300mg/100g, and phycocyanin not less than 4.5g/100g.
3) The spirulina seeds with the growth condition and the detection index of the spirulina powder meeting the standard are regarded as qualified spirulina seeds, and the spirulina seeds can be further cultivated in a large scale.
Example 2:
a method for culturing spirulina rich in vitamin C comprises selecting spirulina seeds of the type of GFJ (1), TFJ (1), FJ II, TFJ (2), FJD2, JX II, 6310XY, CH-5, and CH, numbered 1-10 in sequence, respectively.
Indoor culture and screening
1. Preparation of culture medium
NaHCO 3 4g/L，NaNO 3 0.6g/L，NaCl 0.5g/L，KH 2 PO 4 0.5g/L，K 2 SO 4 0.5g/L，FeSO 4 ·7H 2 O0.003 g/L, EDTA.2Na 0.01g/L, the balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Inoculation of
Taking a pairInoculating 1-10 spirulina seeds in several growth periods into 5L transparent culture bottle, respectively, and spectrophotometrically measuring OD of the algae solution 560nm 10-30 algae were randomly selected from each flask, and their length was measured with a microscope with an eyepiece micrometer, and growth of algae was recorded.
3. Culturing
Placing the inoculated culture bottle in a constant temperature oscillator, culturing at a temperature of 22 ℃ and an illumination intensity of 4000Lux at 8:00-18:00 a day, adding carbonic anhydrase 0.1g/L and gibberellin 0.05g/L during culturing, and supplementing CO 2 ，CO 2 Flow rate is 0.015L/min, and rotation frequency of the constant temperature culture shaking table is high: 25rpm, shimmy amplitude: 22mm;
the daily culture temperature is 18:00-20:00, the culture temperature is 21 ℃, the illumination intensity is 2500Lux, and CO is supplemented 2 ，CO 2 The flow rate is 0.02L/min, and the rotation frequency of the constant temperature culture shaking table is: 18rpm, shimmy amplitude: 16mm;
20:00 a day to 8:00 a day, the culture temperature is 15 ℃, the illumination is stopped, 0.08g/L of ammonium nitrate and 0.1g/L of sodium carbonate are added, and the rotation frequency of a constant temperature culture shaking table is constant: 8rpm, shimmy amplitude: 12mm;
the pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2 ；
Repeating the operation, culturing for 5 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
4. Detecting and judging
(1) Detection of
Spectrophotometry value OD of algae liquid in culture bottle 560nm The spirulina powder is prepared by spray drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
(2) Decision criterion
1) The algae growth well meets standard: the number of spirals is more than or equal to 3, the body length is more than or equal to 100 mu m, the spiral width is more than or equal to 30 mu m, and the filament width is more than or equal to 7.0 mu m.
2) The algae powder detection index meets the standard: vitamin C not less than 30mg/100g, protein not less than 65%, chlorophyll not less than 900mg/100g, total carotenoid not less than 300mg/100g, and phycocyanin not less than 4.5g/100g.
3) The spirulina seeds with the growth condition and the detection index of the spirulina powder meeting the standard are regarded as qualified spirulina seeds, and the spirulina seeds can be further cultivated in a large scale.
Example 3:
a method for culturing spirulina rich in vitamin C comprises selecting spirulina seeds of the type of GFJ (1), TFJ (1), FJ II, TFJ (2), FJD2, JX II, 6310XY, CH-5, and CH, numbered 1-10 in sequence, respectively.
Indoor culture and screening
1. Preparation of culture medium
NaHCO 3 5g/L，NaNO 3 0.3g/L，NaCl 0.8g/L，KH 2 PO 4 0.2g/L，K 2 SO 4 0.2g/L，FeSO 4 ·7H 2 O0.01 g/L, EDTA.2Na 0.003g/L, balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Inoculation of
Taking No. 1-10 spirulina seeds in logarithmic growth phase, respectively inoculating into 1L transparent culture bottle, and spectrophotometrically measuring OD of the spirulina liquid 560nm 10-30 algae were randomly selected from each flask, and their length was measured with a microscope with an eyepiece micrometer, and growth of algae was recorded.
3. Culturing
Placing the inoculated culture bottle in a constant temperature oscillator, culturing at a temperature of 23 ℃ and an illumination intensity of 6000Lux at 8:00-18:00 a day, adding carbonic anhydrase 0.02g/L and gibberellin 0.01g/L during culturing, and supplementing CO 2 ，CO 2 Flow rate 0.005L/min, constant temperature culture shaking table rotational frequency: 23rpm, shimmy amplitude: 20mm;
the daily culture temperature is 18:00-20:00, the culture temperature is 22 ℃, the illumination intensity is 4000Lux, and CO is supplemented 2 ，CO 2 The flow rate is 0.01L/min, and the rotation frequency of the constant temperature culture shaking table is:19rpm, shimmy amplitude: 17mm;
20:00 a day to 8:00 a day, the culture temperature is 17 ℃, the illumination is stopped, 0.03g/L of ammonium nitrate and 0.09g/L of sodium carbonate are added, and the rotation frequency of a constant temperature culture shaking table is kept: 10rpm, shimmy amplitude: 10mm;
the pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2 ；
Repeating the operation, culturing for 5 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
4. Detecting and judging
(1) Detection of
Spectrophotometry value OD of algae liquid in culture bottle 560nm The spirulina powder is prepared by spray drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
(2) Decision criterion
1) The algae growth well meets standard: the number of spirals is more than or equal to 3, the body length is more than or equal to 100 mu m, the spiral width is more than or equal to 30 mu m, and the filament width is more than or equal to 7.0 mu m.
2) The algae powder detection index meets the standard: vitamin C not less than 30mg/100g, protein not less than 65%, chlorophyll not less than 900mg/100g, total carotenoid not less than 300mg/100g, and phycocyanin not less than 4.5g/100g.
3) The spirulina seeds with the growth condition and the detection index of the spirulina powder meeting the standard are regarded as qualified spirulina seeds, and the spirulina seeds can be further cultivated in a large scale.
Example 4
10 spirulina seeds stored in Yunnan green A biological industry garden company are selected, namely GFJ (1), TFJ (1), FJ II, TFJ (2), FJD2, JX II, 6310XY, CH-5 and CH, and the spirulina seeds are numbered 1-10 in sequence. The culture was carried out according to the culture procedure of the algae species in the technical specification of culture of the spirulina in the sea of DB 5307/T39-2021, and the test group was controlled.
Monitoring the pH of the algae liquid every 2 hours during cultivation; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
After the algae mud is collected, spray-drying the algae mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃ to prepare the spirulina powder. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
Screening assays
The following are the detection data and determination results of the growth condition of algae, the nutrient content of algae powder in the indoor culture of 10 spirulina species in examples 1-4,
TABLE 1 detection data of the growth conditions of spirulina seeds and algae powder nutrient content and determination results
In Table 1, it can be seen that the algal body conditions of No. 1, 3, 4, 9 and 10 spirulina species in the 10 spirulina species meet the judgment standard, and the contents of vitamin C, protein, total carotenoid and phycocyanin in the corresponding spirulina powder meet the judgment standard, and the contents of vitamin C in the spirulina powder meet the judgment standard but do not affect the contents of protein, total carotenoid and phycocyanin. The spirulina seeds 1, 3, 4, 9 and 10 can be judged to be qualified and rich in vitamin C, and the cultivation can be further enlarged.
Example 5
Randomly selecting spirulina strain CH-5 rich in vitamin C for further enlarged culture
1. Preparation of culture medium
NaHCO 3 4.5g/L，NaNO 3 0.4g/L，NaCl 1g/L，KH 2 PO 4 0.2g/L，K 2 SO 4 0.3g/L，FeSO 4 ·7H 2 O0.005 g/L, EDTA.2Na 0.005g/L, balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Inoculation of
Taking No. 1-10 spirulina seeds in logarithmic growth phase, respectively inoculating into 200L flat-plate photobioreactor, and measuring the spectrophotometric value OD of the algae liquid 560nm ＝0.2。
3. Culturing
The culture temperature is 25 ℃ and illumination is 5000Lux, carbonic anhydrase 0.05g/L and gibberellin 0.01g/L are added during the culture process, and CO is supplemented 2 ，CO 2 The flow rate is 0.005L/min; the cultivation temperature is 20 ℃ and illumination is 3000Lux, and CO is supplemented at 18:00-20:00 daily 2 ，CO 2 The flow rate is 0.01L/min; 20:00 a day to 8:00 a day, the culture temperature is 15 ℃, the illumination is stopped, and 0.03g/L of ammonium nitrate and 0.08g/L of sodium carbonate are added.
The pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2 。
The spirulina strain CH-5 after the expansion culture is further cultivated in a large scale. Adopting a runway type culture pond for culture, wherein the area of the culture pond is 830 square meters, the water depth of the culture pond is 20cm, and the spectrophotometry value OD of algae liquid in the culture pond 560nm ＝0.2。
1. Preparation of culture medium
NaHCO 3 4.5g/L，NaNO 3 0.4g/L，NaCl 1g/L，KH 2 PO 4 0.2g/L，K 2 SO 4 0.3g/L，FeSO 4 ·7H 2 O0.005 g/L, EDTA.2Na 0.005g/L, balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Large-scale cultivation
8:00-18:00 a day, controlling the temperature to 22 ℃, controlling the illumination intensity to 5000Lux, adding 0.1g/L carbonic anhydrase and 0.03g/L gibberellin, continuously stirring at the stirring speed of 30r/min, and supplementing CO 2 ，CO 2 The flow rate is 0.01L/min.
The temperature is controlled at 18 ℃ and the illumination intensity is 3000Lux, the stirring is continuously carried out at the stirring speed of 20r/min and the CO is supplemented at 18:00-20:00 daily 2 ，CO 2 The flow rate is 0.01L/min.
20:00 a day to 8:00 a day, controlling the temperature to 15 ℃, adding 0.05g/L of ammonium nitrate and 0.1g/L of sodium carbonate without illumination, and continuously stirring at the stirring speed of 10r/min.
The pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2。
Repeating the operation, culturing for 7 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
Spectrophotometry value OD of algae liquid in culture pond 560nm The spirulina powder is prepared by collecting the spirulina mud, spraying and drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
Example 6
Further expanding culture of spirulina strain CH-5 rich in vitamin C
1. Preparation of culture medium
NaHCO 3 4g/L，NaNO 3 0.6g/L，NaCl 0.5g/L，KH 2 PO 4 0.5g/L，K 2 SO 4 0.5g/L，FeSO 4 ·7H 2 O0.003 g/L, EDTA.2Na 0.01g/L, the balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Inoculation of
Taking No. 1-10 spirulina seeds in logarithmic growth phase, respectively inoculating into 300L flat-plate photobioreactor, and measuring the spectrophotometric value OD of the algae liquid 560nm ＝0.25。
3. Culturing
Adding carbonic anhydrase 0.1g/L and gibberellin 0.01g/L during culturing at 24deg.C under illumination of 6000Lux at 8:00-18:00 daily, and supplementing CO 2 ，CO 2 The flow rate is 0.015L/min; the cultivation temperature is 22 ℃ and illumination is 4000Lux, and CO is supplemented at 18:00-20:00 daily 2 ，CO 2 The flow rate is 0.02/min; 20:00 a day to 8:00 a day, and culturing at a temperatureThe illumination is stopped at the temperature of 16 ℃, and 0.08g/L of ammonium nitrate and 0.15g/L of sodium carbonate are added.
The pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2。
3. Large-scale cultivation
The spirulina strain CH-5 after the expansion culture is further cultivated in a large scale. Adopting a runway type culture pond for culture, wherein the area of the culture pond is 830 square meters, the water depth of the culture pond is 20cm, and the spectrophotometry value OD of algae liquid in the culture pond 560nm ＝0.2。
1. Preparation of culture medium
NaHCO 3 4g/L，NaNO 3 0.6g/L，NaCl 0.5g/L，KH 2 PO 4 0.5g/L，K 2 SO 4 0.5g/L，FeSO 4 ·7H 2 O0.003 g/L, EDTA.2Na 0.01g/L, the balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Large-scale cultivation
8:00-18:00 a day, controlling the temperature to be 25 ℃, controlling the illumination intensity to be 6000Lux, adding 0.02g/L carbonic anhydrase and 0.01g/L gibberellin, continuously stirring at the stirring speed of 35r/min, and supplementing CO 2 ，CO 2 The flow rate was 0.015L/min.
The temperature is controlled to be 20 ℃ and the illumination intensity is 4000Lux at 18:00-20:00 a day, stirring is continuously carried out, the stirring speed is 25r/min, and CO is supplemented 2 ，CO 2 The flow rate is 0.02L/min.
20:00 a day to 8:00 a day, controlling the temperature to 18 ℃, adding 0.08g/L of ammonium nitrate and 0.15g/L of sodium carbonate without illumination, and continuously stirring at the stirring speed of 12r/min.
The pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2。
Repeating the operation, culturing for 7 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
Spectrophotometry value OD of algae liquid in culture pond 560nm =0.8, and can be harvested, and the harvested algae mudSpray drying at inlet air temperature of less than 180deg.C and outlet air temperature of less than 80deg.C to obtain spirulina powder. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
Example 7
Further expanding culture of spirulina strain CH-5 rich in vitamin C
1. Preparation of culture medium
NaHCO 3 5g/L，NaNO 3 0.3g/L，NaCl 0.8g/L，KH 2 PO 4 0.2g/L，K 2 SO 4 0.2g/L，FeSO 4 ·7H 2 O0.01 g/L, EDTA.2Na 0.003g/L, balance: and (3) water. CO filling 2 The pH is adjusted to 8-9.
2. Inoculation of
Taking No. 1-10 spirulina seeds in logarithmic growth phase, respectively inoculating into 200L flat-plate photobioreactor, and measuring the spectrophotometric value OD of the algae liquid 560nm ＝0.18。
3. Culturing
The culture temperature is 22 ℃ and illumination is 4000Lux, carbonic anhydrase 0.02g/L and gibberellin 0.03g/L are added in the culture process, and CO is supplemented 2 ，CO 2 The flow rate is 0.01L/min; the daily culture temperature is 18:00-20:00, the culture temperature is 21 ℃, the illumination is 2500Lux, and CO is supplemented 2 ，CO 2 The flow rate is 0.015L/min; 20:00 a day to 8:00 a day, the culture temperature is 15 ℃, the illumination is stopped, and 0.02g/L of ammonium nitrate and 0.1g/L of sodium carbonate are added.
The pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2。
The spirulina strain CH-5 after the expansion culture is further cultivated in a large scale. Adopting a runway type culture pond for culture, wherein the area of the culture pond is 830 square meters, the water depth of the culture pond is 20cm, and the spectrophotometry value OD of algae liquid in the culture pond 560nm ＝0.2。
1. Preparation of culture medium
NaHCO 3 5g/L，NaNO 3 0.3g/L，NaCl 0.8g/L，KH 2 PO 4 0.2g/L，K 2 SO 4 0.2g/L，FeSO 4 ·7H 2 O0.01 g/L, EDTA.2Na 0.003g/L, balance: and (3) water. Filling inCO 2 The pH is adjusted to 8-9.
2. Large-scale cultivation
8:00-18:00 a day, controlling the temperature to 20 ℃, controlling the illumination intensity to 4000Lux, adding 0.08g/L carbonic anhydrase and 0.05g/L gibberellin, continuously stirring at the stirring speed of 25r/min, and supplementing CO 2 ，CO 2 The flow rate is 0.005L/min.
The temperature is controlled at 22 ℃ and the illumination intensity is 2500Lux at 18:00-20:00 per day, stirring is continuously carried out at the stirring speed of 20r/min, and CO is supplemented 2 ，CO 2 The flow rate was 0.01.5L/min.
20:00 a day to 8:00 a day, controlling the temperature to 12 ℃, adding 0.02g/L of ammonium nitrate and 0.08g/L of sodium carbonate without illumination, and continuously stirring at the stirring speed of 8r/min.
The pH of the algae liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the supplementation of CO is stopped 2。
Repeating the operation, culturing for 7 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
Spectrophotometry value OD of algae liquid in culture pond 560nm The spirulina powder is prepared by collecting the spirulina mud, spraying and drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
Example 8
And selecting CH-5 spirulina seeds stored by Yunnan green A biological industry garden company, and carrying out large-scale cultivation according to the technical specification of DB 5307/T39-2021-stroke sea spirulina cultivation, wherein the control test group.
Monitoring the pH of the algae liquid every 2 hours during cultivation; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
After the algae mud is collected, spray-drying the algae mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃ to prepare the spirulina powder. Detecting the contents of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder.
Cultivation result
The following is examples 5-8, in which the growth of algae, the status of algae liquid and the content of each nutrient in the corresponding algae powder were detected during the enlarged cultivation of vitamin C-enriched spirulina species CH-5.
Table 2CH-5 Spirulina species growth conditions and algae powder nutrient content Table in enlarged cultivation
In Table 2, it can be seen that the growth of the algae is good in the enlarged cultivation of the CH-5 spirulina species, and the contents of vitamin C, protein, total carotenoid and phycocyanin in the algae powder reach the judgment standard. The vitamin C content in the spirulina powder obtained by expanding the culture of CH-5 spirulina seeds is increased by more than two times compared with that in the spirulina powder cultured by the common method, and the content of protein, total carotenoid and phycocyanin in the spirulina powder is not influenced. And the bacterial count in the algae liquid is lower, so that the spirulina powder with higher quality can be obtained.
In the test, natural illumination is used for providing a light source conventionally, and when the natural illumination cannot meet the requirement, the natural illumination can be manually supplemented; the temperature is generally natural temperature, and when the natural temperature can not meet the requirement, the temperature can be manually regulated in an auxiliary way.
Gibberellin is a plant growth hormone, one of the major hormones in vitamin C synthesis, which can promote vitamin C synthesis by increasing oxalic acid synthesis activity in non-oxidative pathways; carbonic anhydrase is a Zn-containing enzyme 2+ The metalloenzyme is photosynthetic carbon metabolism regulating enzyme which is suitable for stress conditions of plants, but carbonic anhydrase belongs to organic matters, and the risk of fungus pollution is possibly increased when the carbonic anhydrase is added into spirulina liquid; ammonium nitrate is an ammonium salt which can provide N source for plant growth and is also an oxidant, pesticide and refrigerant to kill microorganisms in the culture pondAnd enemy organisms, sodium carbonate and ammonium nitrate are combined for use, so that the time of low temperature and oxidization stress can be prolonged, and the risk of fungus pollution after carbonic anhydrase is added can be reduced by adding the ammonium nitrate.
The vitamin C in the spirulina can be promoted to be synthesized by multiple low-temperature and oxidation stimulation and adding substances such as ammonium nitrate, gibberellin, carbonic anhydrase and the like in the spirulina culture process. And in the present application, CO is added 2 The pH is adjusted so that the environment is maintained in alkaline conditions while also being supplemented with a carbon source. And in the large-scale cultivation, the rotation speed is reduced to 8-12r/min after ammonium nitrate is added, so that the generation of ammonia gas, explosion and other conditions are avoided, the ammonium nitrate provides a nitrogen source for the growth of algae, the accumulation of vitamin C in spirulina is facilitated in a low-temperature and oxidized stress environment, the content of microorganisms in bath liquid can be reduced by adding the ammonium nitrate, and the condition of microorganisms is inspected by a mirror. The use of ammonium nitrate and sodium carbonate can prolong the time of oxidative stress. Gibberellin increases oxalic acid synthesis activity of non-oxidative pathways, promoting vitamin C synthesis. Carbonic anhydrase at high concentration of CO 2 Reduced environmental activity, low concentration of CO 2 The environmental activity is increased, and the photosynthetic speed is maintained relatively stable to a certain extent.
The temperature in the sea area is lower in 11 months per year to 3 months per year, the yield of conventional spirulina is low, the conventional spirulina is in a production stopping state, and production equipment and workers are idle, so that resource waste and enterprise economic loss are caused. The application of the cultivation method can be used for cultivating the spirulina rich in the vitamin C in months with relatively low temperature, thereby increasing the income and solving the problem of labor utilization in local low-temperature months.
Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the scope and spirit of the principles of this disclosure. More specifically, various variations and modifications may be made to the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will be apparent to those skilled in the art.
Claims (8)
1. A method for culturing spirulina rich in vitamin C is characterized in that: the method comprises the following steps:
s1, indoor culture and screening: preparing a culture medium, inoculating, culturing, detecting and judging;
s2, expanding culture of algae species: preparing a culture medium, inoculating and culturing;
s3, large-scale cultivation: preparing a culture medium, inoculating and culturing in a large scale.
2. The culture method according to claim 1, wherein: the steps S1, S2 and S3 are that the culture medium is NaHCO in the preparation of the culture medium 3 4-5g/L，NaNO 3 0.3-0.6g/L，NaCl 0.5-1g/L，KH 2 PO 4 0.2-0.5g/L，K 2 SO 4 0.2-0.5g/L，FeSO 4 ·7H 2 0.003-0.01g/L of O, 0.003-0.01g/L of EDTA.2Na0 and the balance: water; CO filling 2 The pH is adjusted to 8-9.
3. The culture method according to claim 1, wherein: the steps S1, S2 and S3 are that the spirulina seeds in logarithmic growth phase are inoculated, and the spectrophotometry value OD of the spirulina liquid is taken 560nm ＝0.18-0.25。
4. The culture method according to claim 1, wherein: step S1, inoculating 1-5L of the culture medium to a transparent culture bottle, and placing the culture bottle in a constant-temperature oscillator;
8:00-18:00 a day, the culture temperature is 22-25 ℃, the illumination intensity is 4000-6000Lux, 0.02-0.1g/L carbonic anhydrase and 0.01-0.05g/L gibberellin are added in the culture process, and CO is supplemented 2 ，CO 2 The flow is 0.005-0.015L/min, and the rotation frequency of the shaking table is: 20-25rpm, shimmy amplitude: 18-22mm;
the daily culture temperature is 18:00-20:00, the culture temperature is 20-22 ℃, the illumination intensity is 2500-4000Lux, and CO is supplemented 2 ，CO 2 The flow is 0.01-0.02L/min, and the rotation frequency of the shaking table is highThe rate is as follows: 18-20rpm, shimmy amplitude: 16-18mm;
20:00 a day to 8:00 a day, the culture temperature is 15-18 ℃, the illumination is stopped, 0.02-0.08g/L of ammonium nitrate and 0.08-0.15g/L of sodium carbonate are added, and the rotation frequency of a shaking table is that: 8-12rpm, shimmy amplitude: 10-12mm;
repeating the operation, culturing for 5 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm 10-30 algae are randomly selected, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope.
5. The culture method according to claim 1, wherein: in the step S1 described above, a step of,
(1) And (3) detection:
spectrophotometry value OD of algae liquid in culture bottle 560nm The spirulina powder is prepared by spray drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃; detecting the content of vitamin C, protein, chlorophyll, total carotenoid and phycocyanin in the spirulina powder;
(2) Determination criteria:
1) The algae growth well meets standard: the number of spirals is more than or equal to 3, the body length is more than or equal to 100 mu m, the spiral width is more than or equal to 30 mu m, and the filament width is more than or equal to 7.0 mu m;
2) The algae powder detection index meets the standard: vitamin C not less than 30mg/100g, protein not less than 65%, chlorophyll not less than 900mg/100g, total carotenoid not less than 300mg/100g, phycocyanin not less than 4.5g/100g;
3) The spirulina seeds with the growth condition and the detection index of the spirulina powder meeting the standard are regarded as qualified spirulina seeds, and the spirulina seeds can be further cultivated in a large scale.
6. The culture method according to claim 1, wherein: step S2, culturing, namely culturing qualified spirulina seeds screened in step S1 in a 100-300L flat-plate photobioreactor;
adding carbonic anhydride during the culture process at the culture temperature of 22-25deg.C and illumination of 4000-6000Lux at 8:00-18:00 daily after inoculationEnzyme 0.02-0.1g/L, gibberellin 0.01-0.05g/L, and supplementing CO 2 ，CO 2 The flow is 0.005-0.015L/min;
the daily culture temperature is 18:00-20:00, the culture temperature is 20-22 ℃, the illumination is 2500-4000Lux, and CO is supplemented 2 ，CO 2 The flow is 0.01-0.02L/min;
20:00 a day to 8:00 a day, culturing at 15-18deg.C, stopping illumination, and adding ammonium nitrate 0.02-0.08g/L and sodium carbonate 0.08-0.15g/L.
7. The culture method according to claim 1, wherein: step S3, large-scale cultivation is to cultivate the spirulina obtained by the enlarged cultivation in the step S2 in a cultivation pool;
8:00-18:00 a day, controlling the temperature to be 20-25 ℃, controlling the illumination intensity to be 4000-6000Lux, adding 0.02-0.1g/L of carbonic anhydrase and 0.01-0.05g/L of gibberellin, continuously stirring, and supplementing CO at the stirring speed of 25-35r/min 2 ，CO 2 The flow is 0.005-0.015L/min;
the temperature is controlled at 18:00-20:00 a day, the temperature is controlled at 18-22 ℃, the illumination intensity is 2500-4000Lux, stirring is continuously carried out, the stirring speed is 20-25r/min, and CO is supplemented 2 ，CO 2 The flow is 0.01-0.02L/min;
20:00 a day to 8:00 a day, controlling the temperature to 12-18 ℃ and no light, adding 0.02-0.08g/L of ammonium nitrate and 0.08-0.15g/L of sodium carbonate, continuously stirring, and keeping the stirring speed at 8-12r/min;
repeating the operation, culturing for 7 days, and monitoring the pH of the algae liquid every 2 hours; monitoring algae liquid spectrophotometry OD once daily at 20:00 560nm And 10-30 algae are randomly selected respectively, the length of the algae is measured by a microscope with an eyepiece micrometer, the growth condition of the algae is recorded, and the microorganism condition is detected by a microscope;
spectrophotometry value OD of algae liquid in culture pond 560nm The spirulina powder is prepared by collecting the spirulina mud, spraying and drying the collected spirulina mud at the air inlet temperature of less than or equal to 180 ℃ and the air outlet temperature of less than or equal to 80 ℃.
8. The culture method according to claim 4, 5 or 7, wherein: in the cultivation in the steps S1 and S2 and the cultivation in the step S3, algaeThe pH of the liquid is always kept between 8 and 9, if the pH of the algae liquid is lower than 8.5, the CO supplementation is stopped 2 。
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