CN115678785A - 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 washing liquid recovered in the beer brewing process are added into the photoautotrophic culture medium in a flowing mode, and are respectively used as organic nitrogen sources and carbon sources for chlorella culture to carry out the mixotrophic culture mode of combining photoautotrophy and heterotrophy, so that the food-grade chlorella with high biomass, high protein, safety and no pollution can be obtained. Can also realize the recycling of waste yeast and waste water of saccharifying and washing the lees in beer brewing 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 Chlorococcales of Chlorophyceae, is widely distributed in freshwater, has high growth and reproduction speed, is rich in protein and various nutrient substances, is approved as new resource food by Ministry of health in 2012, and is widely used in the field of food processing at present. The application of the chlorella pyrenoidosa in food is mainly to produce and process the chlorella pyrenoidosa in the form of algae powder and extract, directly prepare chlorella pyrenoidosa granules, chlorella tablets or chlorella capsules by the processes of heat treatment, wall breaking and the like. The algae powder can be used as raw material to be added into various foods such as noodles and bread to prepare foods with unique flavor, and can also be used as food additive or nutrition enhancer to increase color, aroma and health promotion effects of foods.
In the existing culture technology of the chlorella pyrenoidosa, the safety of a culture medium is not fully paid attention and considered. In the traditional culture process of chlorella, the chlorella is usually cultured as raw materials of biological energy, feed, fertilizers or pollutant treatment substances, nitrogen sources in a culture medium of the chlorella are usually nitrates, urea and the like, and common phosphorus sources are mostly fertilizers. The non-food grade raw materials contain a large amount of harmful impurities and heavy metals, elements such as boron which are forbidden to be used by food additives are usually added in the culture process, and the chlorella pyrenoidosa which meets the food safety requirement cannot be obtained by culturing under the condition of the culture medium. In order to meet food requirements, a commercially available food-grade chlorella pyrenoidosa algae powder needs to centrifuge a culture medium and repeatedly wash chlorella, so that energy waste and secondary pollution are caused.
The Chlorella pyrenoidosa can be subjected to photoautotrophic culture, heterotrophic culture in the presence or absence of light, and mixotrophic culture in the presence of light. Guilin and the like research the comparison of different culture modes of chlorella pyrenoidosa, the biomass of 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 this, the invention provides a food-grade chlorella culture medium and a culture method thereof, which utilize food-grade raw materials to obtain food-grade chlorella with high biomass, high protein, safety and no pollution through a mixotrophic mode of photoautotrophy and heterotrophy combination.
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, 0.001g/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 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 to the waste yeast liquid to the spent wash liquid is 1: 2-4: 8-12.
Preferably, the waste yeast liquid recovered in the beer brewing process is obtained by autolysis liquefaction of yeast produced in the brewing process of the beer, and the dry weight of the yeast is 3-4%.
Preferably, the sugar content of the recovered spent 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 adding the chlorella mixotrophic culture medium for mixotrophic culture, wherein the culture process is illuminated in the whole process.
Preferably, the temperature for culturing the chlorella is 20-25 ℃, and the illumination intensity is 5000-10000Lux.
Preferably, filtered sterile air is introduced into the culture medium in the culture process, the ventilation of the autotrophic stage is 30-60L/h, and the ventilation of the mixotrophic stage is 60-300L/h.
Preferably, the photoautotrophic culture is carried out until the cell number of chlorella is more than or equal to 10 6 The cells/mL were cultured mixedly.
Preferably, the period of the culture is 7 to 8 days.
Preferably, the chlorella is cultured 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, 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 eliminated, and 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 culture effect of the food-grade photoautotrophic culture medium on the chlorella pyrenoidosa is equal to that of a BG11 culture medium.
The chlorella mixotrophic culture medium is based on a food-grade photoautotrophic culture medium, utilizes waste yeast recovered in the beer brewing process to replace inorganic nitrogen, utilizes saccharified spent grain washing wastewater recovered in the beer brewing process as a carbon source, and can be applied to large-scale high-quality food-grade chlorella culture. The invention utilizes the waste yeast and the waste water of saccharifying and washing the lees in the beer brewing process, realizes the recycling of resources and reduces the environmental pollution.
The culture method of the chlorella of the invention comprises the steps of carrying out photoautotrophy on the chlorella in a photoautotrophy culture medium, then feeding the chlorella to the culture medium for mixotrophic culture, thus obtaining high-quality chlorella with high biological quantity, high protein, safety and no pollution in a short time, and the produced chlorella meets the requirement of food grade.
Detailed Description
The invention provides a food-grade chlorella culture medium and a culture method thereof, which realize food-grade safe production of chlorella and produce chlorella with high biological content and high protein content. The chlorella culture medium and the culture method of the invention 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.
The chlorella culture medium comprises a photoautotrophic culture medium and a mixotrophic culture medium. The photoautotrophic culture medium selects food-grade additives and nutrition enhancers, and cultures the food-grade chlorella according to the food additive use standard GB2760-2014 and the food nutrition enhancers use standard GB 14880-2012. As an embodiment, the photoautotrophic 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 GB1881881886.100-2015, and food nutrition enhancer cyanocobalamine GB 1903.43-2020.
As an embodiment, the photoautotrophic 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, 0.001g/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 skilled person can arbitrarily select the concentration within a suitable range depending on the kind of the above-mentioned components.
The method for preparing the photoautotrophic culture of the present invention is not particularly limited, and the preparation may be performed by a conventional method in the art. As an implementation mode, zinc sulfate, copper sulfate, cyanocobalamin and manganese sulfate are respectively added into 1L of water to prepare a microelement mother solution, and microelements are required to be completely dissolved. Preparing a culture medium: adding dipotassium hydrogen phosphate, EDTA, citric acid, magnesium sulfate, magnesium chloride, sodium carbonate, calcium chloride, ferric ammonium citrate and ammonium bicarbonate into purified water in sequence, and adding trace elements. The preparation solution is added one by one in sequence, and the next operation is carried out after complete dissolution is ensured. The prepared culture medium is sterilized for use.
The chlorella mixotrophic culture medium of the invention is prepared by mixing the waste yeast liquid in the beer brewing process and the waste washing liquid in the beer brewing process in the photoautotrophic culture medium.
The waste yeast liquid for brewing beer is obtained by autolysis and liquefaction of redundant yeast generated in the process of brewing beer. Preferably, the dry weight of the waste brewery yeast is 3-4%. The waste yeast can be autolyzed and liquefied into amino acid and polypeptide at 45-55 ℃ and used as a chlorella culture nitrogen source to promote the growth of chlorella.
The beer brewing spent grain washing liquid is waste spent grain washing water collected after the brewing beer saccharification is finished, the optimized sugar content is 20-30g/L, and the spent grain washing liquid can be used as a food-grade carbon source to promote the growth and the propagation of chlorella.
Preferably, the yeast solution and the spent wash are mixed and sterilized, and are added into the photoautotrophic culture medium in the culture tank in a flowing mode.
Preferably, the photoautotrophic medium, the beer brewing waste yeast liquid and the beer brewing spent wash liquid are mixed at a volume ratio of 1: 2 to 4: 8 to 12, and more preferably at a volume ratio of 1: 3: 10.
The invention also provides a culture method of the food-grade chlorella, which comprises the steps of inoculating the chlorella into the photoautotrophic culture medium for photoautotrophic culture, then feeding the chlorella into the mixotrophic culture medium for mixotrophic culture, and illuminating in the whole culture process.
The chlorella strain used in the invention is propagated and cultured in the photoautotrophic culture medium, and inoculated into the culture medium for amplification culture after growing to logarithmic phase. The chlorella strain is preferably cultured in a shake flask at the temperature of 20-25 ℃ and under the continuous illumination of 4000-6000Lux, and the shake flask is shaken 2-3 times per day.
The enlarged culture of the chlorella is preferably carried out in a culture tank, and the existing fermentation equipment for brewing beer can be selected directly for production, so that the comprehensive utilization of the equipment is realized. Before cultivation, equipment is required to be sterilized, and the equipment comprises a cultivation tank, a valve, an air filter and a pipeline.
As an embodiment, the method for sterilizing the culture tank comprises: washing the material tank, heating purified water to boil, boiling for 20min, CIP circulating pump for 20min, and introducing into the culture tank, and keeping the temperature for 30min. The valve of the tank body is repeatedly washed by boiling water for 3 to 6 times, and each time lasts for 10 to 20min. The air filter is sterilized by steam flow for 40-60s preferably, and the air is ventilated for 3 times. And after the equipment is sterilized, switching on a ventilation system.
According to the invention, the light supplement lamp is arranged in the inner wall of the culture tank so as to meet the requirement of chlorella on illumination. The installation of light filling lamp all goes on under sterile condition, seals after the installation and prevents that miscellaneous fungus from getting into.
The invention inoculates the chlorella growing to logarithmic phase into the culture tank, the inoculation amount is preferably culture3-4% of the nutrient volume. Adding photoautotrophic culture medium via batch feeding port under photoautotrophic condition according to growth requirement of Chlorella, and sealing (under alcohol mist) when Chlorella cell number is greater than or equal to 10 6 The mixotrophic culture medium of the invention is fed at one/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 ventilation rate in the mixotrophic stage is preferably 60-300L/h, more preferably 100-250L/h.
The temperature of the chlorella of the present invention is preferably 20 to 25 ℃ and the illumination intensity is preferably 5000 to 10000Lux, more preferably 7000 to 9000Lux. The culture period of the chlorella is 7-8 days, and the obtained chlorella has high biological content, high protein content, safety and no pollution, and can be used as food-grade chlorella for production and use.
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 is derived from the fresh water algae seed bank of the culture Collection of the national academy of sciences typically, and may be purchased commercially, and the source thereof is not limited by the present invention.
Example 1
Before the chlorella pyrenoidosa is cultured, the mixing tank is washed clean, 60L of purified water is used for heating to boil, boiling is carried out for 20min, the mixture is led into the culture tank, heat preservation is carried out for 30min, sterilization is carried out, and the culture tank is emptied. The air filtration device is sterilized at high temperature using steam. Preparing 50L food grade photoautotrophic culture medium in a sterilized material preparation tank, heating to boil, keeping the temperature for 20min, boiling for 10min (circulation), introducing into a culture tank, cooling with ice water to 25 deg.C (technological parameters: circulation 10s stops for 10min, keeping at 25 deg.C, and keeping at constant temperature in production cycle).
The food-grade photoautotrophic culture medium comprises the following components: 1.5g/L of ammonium bicarbonate; 0.02g/L of sodium carbonate; 0.02g/L of citric acid; 0.006g/L of ammonium ferric citrate; 0.03g/L of calcium chloride; magnesium sulfate 0.03g/L; 0.02g/L of magnesium chloride; dipotassium phosphate 0.05g/L; EDTA0.001g/L; trace elements: zinc sulfate 2.5 mug/L; copper sulfate 0.08 μ g/L; 2.5 mu g/L of manganese sulfate; cyanocobalamin 0.05. Mu.g/L.
And placing a full-spectrum light supplement lamp, and illuminating in the whole process in the culture process, wherein the illumination intensity is 8000Lux.
Air is introduced into the culture medium of the culture tank, and an aeration autotrophic stage of 45L/h and a mixotrophic stage of 200L/h 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 sterilized mixotrophic culture medium is fed-batch at one/mL, and the mixotrophic culture medium consists of waste beer brewing yeast liquid (dry weight is 3-4%), waste spent washing liquid (optimized sugar content is 25 g/L) recycled from beer brewing and photoautotrophic culture medium which are mixed according to the volume ratio of 3: 10: 1, and the culture period is 8 days.
The experimental results are as follows: after the end of the culture period, the cell density was 47.5g/L and the protein content was 59.4%.
Example 2
Before the chlorella pyrenoidosa is cultured, the mixing tank is washed clean, 60L of purified water is used for heating to boil, boiling is carried out for 20min, the mixture is led into the culture tank, heat preservation is carried out for 30min, sterilization is carried out, 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 preparation tank, heating to boil, keeping the temperature for 20min, boiling for 10min (circulation), introducing into a culture tank, cooling with ice water to 25 deg.C (process parameters: circulation for 10s and stop for 10min, and keeping at 25 deg.C and constant temperature in production cycle).
The food-grade photoautotrophic culture medium comprises the following components: 1.2g/L of ammonium bicarbonate; 0.02g/L of sodium carbonate; 0.018g/L of citric acid; 0.006g/L of ammonium ferric citrate; 0.02g/L of calcium chloride; magnesium sulfate 0.01g/L; 0.01g/L of magnesium chloride; dipotassium hydrogen phosphate 0.02g/L; EDTA0.001g/L; trace elements: zinc sulfate 2 mug/L; copper sulfate 0.07 μ g/L; manganese sulfate is 1.32 mu g/L; cyanocobalamin 0.04. Mu.g/L.
And placing a full-spectrum light supplement lamp, and illuminating in the whole process in the culture process, wherein the illumination intensity is 10000Lux.
Air is introduced into the culture medium of the culture tank, the aeration rate is set to 35L/h in the autotrophic stage and 120L/h in the mixotrophic stage, 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 Is fed-batch at one/mLThe sterilized mixotrophic culture medium consists of waste beer brewing yeast liquid (dry weight is 3%), waste beer brewing washing liquid (optimized sugar content is 20 g/L) and photoautotrophic culture medium which are mixed according to the volume ratio of 4: 11: 1, and the culture period is 8 days.
The experimental results are as follows: after the culture period, the cell density was 45.2g/L and the protein content was 56.23%.
Example 3
Before culturing Chlorella pyrenoidosa, washing the mixing tank, boiling with 60L purified water for 20min, introducing into the culture tank, maintaining the temperature for 30min, sterilizing, and emptying the culture tank. The air filtration device is sterilized at high temperature using steam. Preparing 50L food grade photoautotrophic culture medium in a sterilized material preparation tank, heating to boil, keeping the temperature for 20min, boiling for 10min (circulation), introducing into a culture tank, cooling with ice water to 25 deg.C (technological parameters: circulation 10s stops for 10min, keeping at 25 deg.C, and keeping at constant temperature in production cycle).
The food-grade photoautotrophic culture medium comprises the following components: 2g/L of ammonium bicarbonate; 0.02g/L of sodium carbonate; 0.024g/L of citric acid; 0.006g/L ferric ammonium citrate; 0.05g/L of calcium chloride; magnesium sulfate 0.05g/L; 0.03g/L of magnesium chloride; dipotassium hydrogen phosphate 0.08g/L; EDTA0.001g/L; trace elements: zinc sulfate 3 mug/L; copper sulfate 0.09 μ g/L; 3.05 mu g/L of manganese sulfate; cyanocobalamin 0.05. Mu.g/L.
And placing a full-spectrum light supplement lamp, and illuminating in the whole process in the culture process, wherein the illumination intensity is 6000Lux.
Air is introduced into the culture medium of the culture tank, and the aeration rate autotrophic stage is set at 60L/h, the mixotrophic stage 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 sterilized mixotrophic culture medium is fed-batch at one/mL, and the mixotrophic culture medium consists of waste beer brewing yeast liquid (dry weight 4%), waste beer brewing spent washing liquid (sugar content is 30g/L after optimization) and photoautotrophic culture medium which are mixed according to the volume ratio of 2: 8: 1, and the culture period is 7 days.
The experimental results are as follows: after the end of the culture period, the cell density was 46.7g/L, and the protein content was 58.2%.
Comparative example 1
Culturing chlorella with BG11 culture medium without adding mixotrophic culture medium. The rest of the procedure was the same as in example 1.
BG11 medium composition is sodium nitrate 1.5g/L, sodium carbonate 0.02g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L, calcium chloride 0.036g/L, magnesium sulfate 0.075g/L, dipotassium hydrogen phosphate 0.04g/L, EDTA0.001g/L; trace elements: boric acid 2.86 mug/L, manganese chloride 1.18 mug/L, zinc sulfate 0.22 mug/L, copper sulfate 0.08 mug/L, sodium molybdate 0.39 mug/L and cobalt nitrate 0.05 mug/L.
The experimental results are as follows: after the culture is finished, the cell density is 5.04g/L, and the protein content is 50.67%.
Comparative example 2
The chlorella was cultured in the food grade photoautotrophic medium of example 1 without adding a mixotrophic medium. The rest of the procedure was the same as in example 1.
The experimental results are as follows: after the culture is finished, the cell density is 5.24g/L, and the protein content is 57%.
In conclusion, the results show that the food-grade chlorella photoautotrophic culture medium achieves and is superior to a BG11 universal culture medium in photoautotrophic results. The method is characterized in that a mixotrophic culture medium is fed-batch on the basis of the photoautotrophic culture medium, and the chlorella is cultured in a mixotrophic mode combining photoautotrophy and heterotrophy, so that the food-grade chlorella with high biomass, high protein, safety and no pollution can be obtained.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A food-grade chlorella culture medium is characterized in that the culture medium is a photoautotrophic culture medium and comprises the following components: 1-2g/L ammonium bicarbonate, 0.02g/L sodium carbonate, 0.018-0.024g/L citric acid, 0.006g/L ferric ammonium citrate, 0.02-0.05g/L calcium chloride, 0.01-0.05g/L magnesium sulfate, 0.01-0.03g/L magnesium chloride, 0.02-0.08g/L, EDTA, 0.001g/L zinc sulfate, 2-3 mu g/L copper sulfate, 0.07-0.09 mu g/L copper sulfate, 1.18-3.15 mu g/L manganese sulfate, and 0.04-0.05 mu g/L cyanocobalamine.
2. The food-grade chlorella culture medium according to claim 1, wherein the culture medium is a mixotrophic culture medium, and the mixotrophic culture medium is supplemented with waste yeast solution and spent wash solution for beer brewing.
3. The food-grade chlorella culture medium according to claim 2, wherein the photoautotrophic culture medium, the waste yeast liquid and the spent wash liquid are mixed in a volume ratio of 1: 2-4: 8-12.
4. The culture medium of claim 2, wherein the spent brewer's yeast solution is autolyzed and liquefied from yeast produced during brewing, the yeast having a dry weight of 3-4%.
5. The culture medium of claim 2, wherein the sugar content of the beer brewing spent wash is 20-30g/L.
6. A method for culturing food-grade chlorella is characterized by comprising the following steps: the chlorella is inoculated into the photoautotrophic culture medium of claim 1 for photoautotrophic culture, and then the chlorella mixotrophic culture medium of any one of claims 2 to 5 is fed for mixotrophic culture, and the whole culture process is illuminated.
7. The method of claim 6, wherein the chlorella is cultured at a temperature of 20-25 ℃ and an illumination intensity of 5000-10000Lux.
8. The method according to claim 6, wherein filtered sterile air is introduced into the culture medium during the culturing, and the aeration rate in the autotrophic stage is 30-60L/h and the aeration rate in the mixotrophic stage is 60-300L/h.
9. The method of any of claims 6~8, wherein the photoautotrophic culture is performed until chlorella cell number is greater than or equal to 10 6 The cells/mL were cultured mixedly.
10. The method of claim 6~8 wherein the period of culture is 7-8 days.
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