CN115678785B - Food-grade chlorella culture medium and culture method - Google Patents
Food-grade chlorella culture medium and culture method Download PDFInfo
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Abstract
The invention belongs to the technical field of microalgae biological culture, and discloses a food-grade chlorella culture medium and a culture method. The chlorella culture medium is based on a food-grade photoautotrophic culture medium, waste yeast liquid and waste liquid recovered in the beer brewing process are fed into the photoautotrophic culture medium and are respectively used as an organic nitrogen source and a carbon source for chlorella culture, and the photoautotrophic culture medium and the heterotrophic culture medium are used for combined mixotrophic culture of photosynthetic autotrophic and heterotrophic culture, so that the food-grade chlorella with high biomass, high protein and safety and no pollution can be obtained. Can also realize the recycling of waste yeast and saccharification waste water of brewery, and reduce environmental pollution.
Description
Technical Field
The invention belongs to the technical field of microalgae biological culture, and particularly relates to a food-grade chlorella culture medium and a culture method.
Background
The chlorella pyrenoidosa is a spherical single-cell microalgae of chlorella of Chlorophyceae, is widely distributed in fresh water, has high growth and propagation speed, is rich in protein and various nutrient substances, is approved by the Ministry of health as a new resource food in 2012, and is widely used in the field of food processing. The application of the chlorella pyrenoidosa in foods is mainly to produce and process in the form of algae powder and extract, and the chlorella pyrenoidosa particles, chlorella tablets or chlorella capsules are directly prepared through the processes of heat treatment, wall breaking and the like. The algae powder can be used as raw material for various foods such as noodles and bread to prepare foods with unique flavor, and can also be used as food additive or nutrition enhancer for increasing color, smell and taste of foods and promoting health.
In the existing culture technology of chlorella pyrenoidosa, the safety of a culture medium is not fully paid attention to and considered. In the conventional chlorella cultivation process, the chlorella is usually cultivated as a raw material of biological energy, feed, fertilizer or pollutant treatment substance, nitrogen sources in a culture medium are usually nitrate, urea and the like, and common phosphorus sources are usually chemical fertilizers. These non-food grade materials contain a large amount of harmful impurities and heavy metals, and during the cultivation process, elements such as boron, etc., which are forbidden to use by food additives, are usually added, and the chlorella pyrenoidosa meeting the food safety requirements cannot be obtained by cultivation under such a culture medium condition. The commercial food-grade chlorella pyrenoidosa powder requires centrifugation of a culture medium to achieve food requirements, and repeated washing treatment is carried out on chlorella pyrenoidosa, so that energy waste and secondary pollution are caused.
The chlorella pyrenoidosa can be subjected to photoautotrophic culture, heterotrophic culture under light or no light condition, and mixotrophic culture under illumination condition. The comparison of different culture modes of the chlorella pyrenoidosa is researched by Guilin and the like, the biomass of the photoautotrophic culture is low, the pollution of an open culture mode is large, and the energy consumption of a closed culture mode is high; the pure heterotrophic culture mode has high yield, low chlorophyll content, low protein content, high fat content and low comprehensive nutritive value of the chlorella pyrenoidosa.
Disclosure of Invention
In view of the above, the present invention provides a food-grade chlorella culture medium and a culture method, which can obtain high biomass, high protein, safe and pollution-free food-grade chlorella by using food-grade raw materials and adopting a photoautotrophic and heterotrophic combined mixotrophic mode.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a chlorella culture medium, which is a photoautotrophic culture medium, and comprises the following components: 1-2g/L of ammonium bicarbonate, 0.02g/L of sodium carbonate, 0.018-0.024g/L of citric acid, 0.006g/L of ferric ammonium citrate, 0.02-0.05g/L of calcium chloride, 0.01-0.05g/L of magnesium sulfate, 0.01-0.03g/L of magnesium chloride, 0.02-0.08g/L, EDTA g/L of dipotassium hydrogen phosphate, 2-3 mu g/L of zinc sulfate, 0.07-0.09 mu g/L of copper sulfate, 1.18-3.15 mu g/L of manganese sulfate and 0.04-0.05 mu g/L of cyanocobalamine.
The chlorella culture medium is a mixotrophic culture medium, and waste yeast liquid and waste dreg washing liquid recovered in the beer brewing process are added to the mixotrophic culture medium on the basis of the photoautotrophic culture medium.
Preferably, the volume ratio of the photoautotrophic culture medium, the waste yeast solution and the distilled liquid is 1:2-4:8-12.
Preferably, the waste yeast liquid recovered in the beer brewing process is obtained by autolyzing yeast produced in the beer brewing process, and the dry weight of the yeast is 3-4%.
Preferably, the sugar content of the recovered distilled grain washing liquid is 20-30g/L.
The invention also provides a chlorella culture method, which comprises the following steps: inoculating chlorella into the photoautotrophic culture medium for photoautotrophic culture, and then feeding the chlorella mixotrophic culture medium for mixotrophic culture, wherein the whole process of the culture is illuminated.
Preferably, the temperature of the chlorella culture is 20-25 ℃, and the illumination intensity is 5000-10000Lux.
Preferably, in the culture process, filtered sterile air is introduced into the culture medium, the ventilation amount in the autotrophic stage is 30-60L/h, and the ventilation amount in the mixotrophic stage is 60-300L/h.
Preferably, the photosynthetic autotrophic culture is carried out until the cell number of the chlorella is more than or equal to 10 6 The culture was carried out in a culture medium at a concentration of one liter/mL.
Preferably, the period of the cultivation is 7-8 days.
Preferably, the chlorella is cultivated to log phase and inoculated at 3-4% of the total volume.
The invention has the beneficial effects that:
according to the chlorella photoautotrophic culture medium, food-grade additives and nutrition enhancers are selected as raw materials, and components which are forbidden to be used in food processing such as boric acid, cobalt nitrate and sodium molybdate in the existing chlorella pyrenoidosa culture medium are omitted, so that the culture of the food-grade chlorella is realized according to the food additive use standard GB2760-2014 and the food nutrition enhancer use standard GB 14880-2012. The food-grade photoautotrophic culture medium has the same culture effect on chlorella pyrenoidosa as the BG11 culture medium.
The chlorella culture medium is based on a food-grade photoautotrophic culture medium, waste yeast recovered in the beer brewing process is utilized to replace inorganic nitrogen, saccharification waste water recovered in the beer brewing process is used as carbon, and the chlorella culture medium can be applied to large-scale high-quality food-grade chlorella culture. The invention utilizes waste yeast and saccharification waste water of waste liquid in beer brewing process, realizes recycling of resources and reduces environmental pollution.
According to the culture method of the chlorella, the chlorella is subjected to photoautotrophic in the photoautotrophic culture medium, and then the photoautotrophic culture medium of the chlorella is fed to perform the photoautotrophic culture, so that the high-quality chlorella with high biomass, high protein, safety and no pollution can be obtained in a short time, and the produced chlorella meets the food-grade requirements.
Detailed Description
The invention provides a food-grade chlorella culture medium and a culture method, which realize the food-grade safe production of chlorella, and the produced chlorella has high biomass and high protein content. The chlorella culture medium and the culture method can also be applied to the culture of other known conventional chlorella species, and are particularly suitable for the food-grade culture of edible chlorella species, and the food-grade chlorella culture medium and the culture method are described by taking the chlorella pyrenoidosa as an example in the invention.
The chlorella culture medium comprises a photoautotrophic culture medium and a mixotrophic culture medium. The photoautotrophic culture medium selects food-grade additives and nutrient enhancers, and cultures food-grade chlorella according to food additive use standard GB2760-2014 and food nutrient enhancer use standard GB 14880-2012. As one embodiment, the photoautotrophic culture medium of the present invention selects the following food additives: ammonium bicarbonate GB 1888-2014, sodium carbonate GB1886.1-2021, citric acid GB1886.235-2016, ferric ammonium citrate GB1886.296-2016, calcium chloride GB1886.45-2016, zinc sulfate GB25579-2010, copper sulfate GB29210-2012, manganese sulfate GB29208-2012, magnesium sulfate GB29207-2012, magnesium chloride GB 25584-2010, dipotassium hydrogen phosphate GB1886.334-2021,EDTA GB1886.100-2015, and food nutrition enhancer cyanocobalamin GB 1903.43-2020.
As one embodiment, the photoautotrophic culture medium of the present invention comprises the following components: 1-2g/L of ammonium bicarbonate, 0.02g/L of sodium carbonate, 0.018-0.024g/L of citric acid, 0.006g/L of ferric ammonium citrate, 0.02-0.05g/L of calcium chloride, 0.01-0.05g/L of magnesium sulfate, 0.01-0.03g/L of magnesium chloride, 0.02-0.08g/L, EDTA g/L of dipotassium hydrogen phosphate, 2-3 mu g/L of zinc sulfate, 0.07-0.09 mu g/L of copper sulfate, 1.18-3.15 mu g/L of manganese sulfate and 0.04-0.05 mu g/L of cyanocobalamine. Those skilled in the art can arbitrarily select a suitable concentration range according to the kind of the above-mentioned components.
The method for preparing the photoautotrophic culture is not particularly limited, and the photoautotrophic culture can be prepared by a conventional method in the art. As an implementation mode, zinc sulfate, copper sulfate, cyanocobalamine and manganese sulfate are respectively taken and added into 1L of water to prepare a trace element mother solution, and trace elements are required to be completely dissolved. Preparing culture medium: sequentially adding dipotassium hydrogen phosphate, EDTA, citric acid, magnesium sulfate, magnesium chloride, sodium carbonate, calcium chloride, ferric ammonium citrate and ammonium bicarbonate into purified water, and adding microelements. The prepared solutions are added one by one according to the sequence, so that the next operation is carried out after complete dissolution. The prepared culture medium is sterilized for use.
The chlorella culture medium is prepared by mixing waste yeast liquid in the beer brewing process and waste distillers' grains washing liquid in the beer brewing process in an photoautotrophic culture medium.
The waste yeast liquid for brewing is obtained by autolyzing redundant yeast generated in the brewing process. Preferably, the dry weight of the spent brewery yeast is 3-4%. The waste yeast can be autolyzed into amino acid and polypeptide at 45-55 ℃ and is used as an algae-cultivating nitrogen source to promote the growth of chlorella.
The beer brewing distilled grain washing liquid is waste distilled grain washing water collected after beer saccharification is finished, the sugar content is optimized to be 20-30g/L, and the beer brewing distilled grain washing liquid can be used as a food-grade carbon source to promote the growth and propagation of chlorella.
Preferably, in the present invention, the yeast solution and the spent wash are both sterilized by mixing and fed to the photoautotrophic medium in the culture tank.
Preferably, the volume ratio of the photoautotrophic culture medium, the waste beer fermentation broth and the brewery spent wash in the present invention is 1:2-4:8-12, more preferably 1:3:10.
The invention also provides a method for culturing food-grade chlorella, which comprises the steps of inoculating the chlorella into the photoautotrophic culture medium for photoautotrophic culture, and then feeding the chlorella with the photoautotrophic culture medium for photoautotrophic culture, wherein the whole process of the culture is illuminated.
The chlorella species used in the invention are propagated and cultivated in the photoautotrophic culture medium, and are inoculated into the culture medium for expansion cultivation after growing to the logarithmic phase. The chlorella species are preferably cultured in shake flasks, the temperature of the propagation culture is 20-25 ℃, continuous illumination is carried out at 4000-6000Lux, and shake flasks are carried out 2-3 times per day.
The enlarged culture of the chlorella is preferably carried out in a culture tank, and the chlorella is produced by directly using the existing fermentation equipment for brewing beer, so that the equipment is comprehensively utilized. Before culturing, the equipment needs to be sterilized, including a culture tank, a valve, an air filter and a pipeline.
As one embodiment, the method for sterilizing the culture tank includes: washing the batching tank, heating purified water to boil, boiling for 20min, CIP circulating pump for 20min, and introducing into culture tank for heat preservation for 30min. The boiling water of the tank valve is repeatedly washed for 3-6 times, and each time is 10-20min. The air filter according to the invention is preferably sterilized by steam flow, with aeration for 3 times, 40-60s each. And after the sterilization of the equipment is finished, the ventilation system is connected.
The invention installs the light supplementing lamp in the inner wall of the culture tank to meet the requirement of the chlorella on illumination. The installation of light filling lamp is all gone on under aseptic condition, seals after the installation and prevents miscellaneous fungus entering.
The invention inoculates the chlorella species grown to log phase into the culture tank, and the inoculation amount is preferably 3-4% of the culture volume. According to the growth requirement of chlorella, under the condition of photoautotrophic, adding photoautotrophic culture medium in batches through a feed inlet and sealing (under alcohol fog), when the cell number of chlorella is more than or equal to 10 6 The culture medium of the invention was fed at a volume of each mL. Introducing filtered sterile air into the culture medium in the culture process, wherein the ventilation amount in the autotrophic stage is 30-60L/h, and more preferably 40-50L/h; the aeration rate in the polyculture stage is preferably 60 to 300L/h, more preferably 100 to 250L/h.
The temperature of the chlorella controlled culture of the invention is preferably 20-25 ℃, the illumination intensity is preferably 5000-10000Lux, and more preferably 7000-9000Lux. The culture period of the chlorella in the method is 7-8 days, and the obtained chlorella has high biomass, high protein content, safety and no pollution and can be used for producing food-grade chlorella.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Chlorella pyrenoidosa (Chlorella pyrenoidosa) used in the following examples of the present invention was obtained from the fresh water algae seed stock of the national academy of sciences typical culture preservation committee, and was also commercially available, and the present invention is not limited to the source thereof.
Example 1
Before the chlorella pyrenoidosa is cultured, the batching tank is washed clean, 60L of purified water is used for heating to boiling, boiling is carried out for 20min, the mixture is introduced into a culture tank for heat preservation for 30min for sterilization, and the culture tank is emptied. The air filtration device is sterilized using steam at high temperature. Preparing 50L of food-grade photoautotrophic culture medium in a sterilized material mixing tank, heating to boiling, maintaining the temperature for 20min, continuously boiling for 10min (circulation), introducing into a culture tank, cooling with ice water to 25deg.C (technological parameters: circulation for 10s, stopping for 10min, maintaining at 25deg.C, and keeping constant temperature in production period).
The food-grade photoautotrophic culture medium comprises the following components: ammonium bicarbonate 1.5g/L; sodium carbonate 0.02g/L; citric acid 0.02g/L; ferric ammonium citrate 0.006g/L; 0.03g/L of calcium chloride; 0.03g/L of magnesium sulfate; 0.02g/L of magnesium chloride; dipotassium hydrogen phosphate 0.05g/L; EDTA0.001g/L; trace elements: zinc sulfate 2.5 μg/L; copper sulfate 0.08. Mu.g/L; 2.5 mug/L of manganese sulfate; cyanocobalamin 0.05 μg/L.
And a full spectrum light supplementing lamp is arranged, and the whole process of illumination is carried out in the culture process, wherein the illumination intensity is 8000Lux.
Air is introduced into the culture medium of the culture tank, 45L/h of the autotrophic stage with ventilation and 200L/h of the mixotrophic stage are set, and the temperature is maintained at 25 ℃. Observing the growth condition of the chlorella pyrenoidosa, wherein the cell number of the chlorella pyrenoidosa is more than or equal to 10 6 The culture medium after the sterilization is fed in each mL, the culture medium is formed by mixing waste yeast liquid (dry weight is 3-4 percent) for beer brewing, waste distillers' grains washing liquid (sugar content is 25g/L after optimization) for beer brewing and photoautotrophic culture medium according to the volume ratio of 3:10:1, and the culture period is 8 days.
Experimental results: cell density at the end of the culture period was 47.5g/L and protein content was 59.4%.
Example 2
Before the chlorella pyrenoidosa is cultured, the batching tank is washed clean, 60L of purified water is used for heating to boiling, boiling is carried out for 20min, the mixture is introduced into a culture tank for heat preservation for 30min for sterilization, and the culture tank is emptied. The air filtration device is sterilized using steam at high temperature. Preparing 50L of food-grade photoautotrophic culture medium in a sterilized material mixing tank, heating to boiling, maintaining the temperature for 20min, continuously boiling for 10min (circulation), introducing into a culture tank, cooling with ice water to 25deg.C (technological parameters: circulation for 10s, stopping for 10min, maintaining at 25deg.C, and keeping constant temperature in production period).
The food-grade photoautotrophic culture medium comprises the following components: ammonium bicarbonate 1.2g/L; sodium carbonate 0.02g/L; citric acid 0.018g/L; ferric ammonium citrate 0.006g/L; 0.02g/L of calcium chloride; 0.01g/L magnesium sulfate; 0.01g/L of magnesium chloride; dipotassium hydrogen phosphate 0.02g/L; EDTA0.001g/L; trace elements: zinc sulfate 2 μg/L; copper sulfate 0.07. Mu.g/L; manganese sulfate 1.32. Mu.g/L; cyanocobalamin 0.04 μg/L.
And a full spectrum light supplementing lamp is arranged, and the whole process of illumination is carried out in the culture process, wherein the illumination intensity is 10000Lux.
Air is introduced into the culture medium of the culture tank, the aeration autotrophic stage is set to 35L/h, the mixotrophic stage is set to 120L/h, and the temperature is maintained at 24 ℃. Observing the growth condition of the chlorella pyrenoidosa, wherein the cell number of the chlorella pyrenoidosa is more than or equal to 10 6 The culture medium after the sterilization is fed at the volume ratio of each mL, the culture medium is formed by mixing waste yeast liquid (dry weight 3 percent) of beer brewing, waste distillers' grains washing liquid (sugar content 20g/L after optimization) of beer brewing and photo-autotrophic culture medium according to the volume ratio of 4:11:1, and the culture period is 8 days.
Experimental results: cell density 45.2g/L after the end of the culture period, protein content 56.23%.
Example 3
Before the chlorella pyrenoidosa is cultured, the batching tank is washed, 60L of purified water is used for heating to boiling, boiling is carried out for 20min, the mixture is introduced into a culture tank for heat preservation for 30min for sterilization, and the culture tank is emptied. The air filtration device is sterilized using steam at high temperature. Preparing 50L of food-grade photoautotrophic culture medium in a sterilized material mixing tank, heating to boiling, maintaining the temperature for 20min, continuously boiling for 10min (circulation), introducing into a culture tank, cooling with ice water to 25deg.C (technological parameters: circulation for 10s, stopping for 10min, maintaining at 25deg.C, and keeping constant temperature in production period).
The food-grade photoautotrophic culture medium comprises the following components: 2g/L ammonium bicarbonate; sodium carbonate 0.02g/L; citric acid 0.024g/L; ferric ammonium citrate 0.006g/L; 0.05g/L of calcium chloride; 0.05g/L of magnesium sulfate; 0.03g/L of magnesium chloride; dipotassium hydrogen phosphate 0.08g/L; EDTA0.001g/L; trace elements: zinc sulfate 3 μg/L; copper sulfate 0.09. Mu.g/L; 3.05 mug/L of manganese sulfate; cyanocobalamin 0.05 μg/L.
And a full spectrum light supplementing lamp is arranged, and the whole process of illumination is carried out in the culture process, wherein the illumination intensity is 6000Lux.
Air is introduced into the culture medium of the culture tank, the aeration autotrophic stage is set at 60L/h, the mixotrophic stage is set at 300L/h, and the temperature is maintained at 23 ℃. Observing the growth condition of the chlorella pyrenoidosa, wherein the cell number of the chlorella pyrenoidosa is more than or equal to 10 6 The culture medium after the sterilization is fed at the volume ratio of 2:8:1, and the culture period is 7 days.
Experimental results: cell density 46.7g/L and protein content 58.2% after the end of the culture period.
Comparative example 1
The culture of chlorella is carried out by adopting BG11 culture medium, and the culture medium is not fed in. The rest of the procedure is the same as in example 1.
The BG11 culture medium comprises 1.5g/L of sodium nitrate, 0.02g/L of sodium carbonate, 0.006g/L of citric acid, 0.006g/L of ferric ammonium citrate, 0.036g/L of calcium chloride, 0.075g/L of magnesium sulfate, 0.04g/L of dipotassium hydrogen phosphate and 0.001g/L of EDTA0; trace elements: boric acid 2.86 μg/L, manganese chloride 1.18 μg/L, zinc sulfate 0.22 μg/L, copper sulfate 0.08 μg/L, sodium molybdate 0.39 μg/L, and cobalt nitrate 0.05 μg/L.
Experimental results: after the culture, the cell density was 5.04g/L and the protein content was 50.67%.
Comparative example 2
The culture of Chlorella was performed using the food-grade photoautotrophic medium of example 1, without feeding the facultative medium in between. The rest of the procedure is the same as in example 1.
Experimental results: after the culture, the cell density was 5.24g/L and the protein content was 57%.
In conclusion, the results show that the photoautotrophic result of the food-grade chlorella photoautotrophic culture medium reaches and is superior to that of the BG11 universal culture medium. The invention feeds the mixotrophic culture medium on the basis of the photoautotrophic culture medium, and cultures the chlorella in a photosynthetic autotrophic and heterotrophic combined mixotrophic mode, so that the food-grade chlorella with high biomass, high protein and safety and no pollution can be obtained.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. The food-grade chlorella culture medium is characterized by being a mixotrophic culture medium, wherein waste yeast liquid and waste dreg washing liquid for beer brewing are added to the mixotrophic culture medium on the basis of the photoautotrophic culture medium;
the photoautotrophic culture medium consists of the following components: 1-2g/L of ammonium bicarbonate, 0.02g/L of sodium carbonate, 0.018-0.024g/L of citric acid, 0.006g/L of ferric ammonium citrate, 0.02-0.05g/L of calcium chloride, 0.01-0.05g/L of magnesium sulfate, 0.01-0.03g/L of magnesium chloride, 0.02-0.08g/L, EDTA g/L of dipotassium hydrogen phosphate, 2-3 mu g/L of zinc sulfate, 0.07-0.09 mu g/L of copper sulfate, 1.18-3.15 mu g/L of manganese sulfate and 0.04-0.05 mu g/L of cyanocobalamine; the balance being purified water;
the volume ratio of the photoautotrophic culture medium, the waste yeast solution and the distilled grain washing solution is 1:2-4:8-12;
the waste beer brewing yeast liquid is obtained by autolyzing yeast generated in the beer brewing process, and the dry weight of the yeast is 3-4%;
the sugar content of the beer brewing distilled grain washing liquid is 20-30g/L;
the photoautotrophic culture medium is used for photoautotrophic culture, and the photoautotrophic culture medium is used for achieving photoautotrophic culture by flowing to the photoautotrophic culture medium.
2. A method of culturing food-grade chlorella, comprising: inoculating the chlorella into the photoautotrophic culture medium of claim 1 for photoautotrophic culture, and then carrying out the photoautotrophic culture by using the chlorella mixotrophic culture medium of claim 1, wherein the whole process of the culture is illuminated; the culture temperature of the chlorella is 20-25 ℃, and the illumination intensity is 5000-10000Lux; in the culture process, filtered sterile air is introduced into the culture medium, the ventilation amount in the autotrophic stage is 30-60L/h, and the ventilation amount in the mixotrophic stage is 60-300L/h.
3. The method according to claim 2, wherein the photoautotrophic culture is carried out until the number of chlorella cells is not less than 10 6 The culture was carried out in a culture medium at a concentration of one liter/mL.
4. The method according to claim 2, wherein the period of the photoautotrophic culture and the mixotrophic culture is 7 to 8 days.
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