CN108823132B - Method for prolonging preservation time of liquid photosynthetic bacteria product - Google Patents

Method for prolonging preservation time of liquid photosynthetic bacteria product Download PDF

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CN108823132B
CN108823132B CN201810746186.3A CN201810746186A CN108823132B CN 108823132 B CN108823132 B CN 108823132B CN 201810746186 A CN201810746186 A CN 201810746186A CN 108823132 B CN108823132 B CN 108823132B
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黎建斌
李大列
黄黎明
杨学明
陈福艳
杨琼
吕敏
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Guangxi Academy of Fishery Sciences
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Abstract

The invention discloses a method for prolonging the preservation time of a liquid photosynthetic bacterium product, and relates to the technical field of microorganism preservation. Adding L-ascorbic acid and iodine-free refined salt into a photosynthetic bacteria liquid to be preserved to obtain a bacterial liquid containing 0.15-0.75 g/L of L-ascorbic acid and 5-12.5 g/L of iodine-free refined salt; and adjusting the pH value of the obtained bacterial liquid to 7.0-8.0 by using a phosphate buffer solution. The adjusted photosynthetic bacteria 5L light green transparent polyethylene plastic is hermetically packaged, the number of effective viable bacteria reaches 95.58% after the photosynthetic bacteria are stored for 6 months under the conditions of room temperature of 15-28 ℃ and natural weak light of 400-600 Lx, and the number of effective viable bacteria reaches 83.49% after the photosynthetic bacteria are stored for 12 months.

Description

Method for prolonging preservation time of liquid photosynthetic bacteria product
Technical Field
The invention belongs to the technical field of microorganism preservation, and particularly relates to a method for prolonging the preservation time of a liquid photosynthetic bacterium product.
Background
Photosynthetic Bacteria (PSB) are the most early prokaryotes that occur on earth and are ubiquitous in nature and have an original light energy synthesis system, and are a general term for Bacteria that perform anaerobic photosynthesis under anaerobic conditions. Photosynthetic bacteria are widely distributed in soil, paddy fields, swamps, lakes, rivers, seas and the like in the nature, and are mainly distributed in anoxic zones to which light can be transmitted in aquatic environments. The photosynthetic bacteria known to date are classified into 7 major groups of about 50 genera, such as chromobacteriaceae, exothiospiraceae, purple non-sulfur bacteria, green sulfur bacteria, multicellular filamentous green bacteria, spirobacteriaceae, and obligate aerobic bacteria containing bacteriochlorophyll, and new species are continuously discovered.
In recent 30 years, photosynthetic bacteria show greater superiority and value in the aspects of organic sewage treatment, aquaculture water quality regulation and thallus utilization, and increasingly arouse great interest in research and exploration of scientific and technological work. Japan is one of the earliest countries for the research and utilization of photosynthetic bacteria, and the related research and application in this respect have been started in the last 40 th century; the research of the application technology of photosynthetic bacteria in China starts in the 80 th early years of the last century, and some experiences and achievements have been accumulated at present.
Most of the rhodospirillum photosynthetic bacteria sold in the current market are liquid products (including concentrated and non-concentrated products), but no matter which liquid product has the fact that the quality guarantee period of the strains is short, the strains die gradually in the storage process, and the quality guarantee period and the application effect of the photosynthetic bacteria products are seriously influenced. The research on the seed preservation method of the photosynthetic bacteria is only limited to the preservation of the strains at present, and the methods comprise solid preservation, soil tube preservation, vacuum freeze drying, liquid nitrogen ultralow temperature preservation and the like, so that the method is not suitable for the preservation of large-scale liquid photosynthetic bacteria commodities. Experts and scholars research on the immobilized storage technology of photosynthetic bacteria, such as an "immobilization method of photosynthetic bacteria" (publication No. CN1810966A), "a preparation method of solid photosynthetic bacteria" publication No. CN104293765A), "fixed photosynthetic bacteria technology" publication No. CN1124293) and the like, although the immobilization technology overcomes the defects of large transportation inconvenience and the like of a packaging container, the photosynthetic bacteria are mostly immobilized by adopting an agar and sodium alginate embedding method in the related application and research at present, the price of a fixing material is higher, the fixing method is complicated, the requirements on instruments and equipment are high, the cell viability is weak, the photosynthetic bacteria are easy to be polluted and the like; the effect of the immobilized photosynthetic bacteria in purifying aquaculture water needs to be further proved, which is also an important reason that the liquid photosynthetic bacteria are still used as main components in the market at present.
Disclosure of Invention
In view of the above, the present invention provides a method for prolonging the storage time of a liquid photosynthetic bacteria product.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for prolonging the storage time of a liquid photosynthetic bacterium product, which uses a phosphate buffer solution to adjust the pH value of a photosynthetic bacterium liquid to be stored to 7.0-8.0, and stores the photosynthetic bacterium liquid at 15-28 ℃.
Preferably, the phosphate buffer is a potassium dihydrogen phosphate buffer with a mass concentration of 1-2%.
Preferably, before the pH value of the photosynthetic bacteria liquid to be preserved is adjusted, L-ascorbic acid is added into the photosynthetic bacteria liquid to be preserved, and the final concentration of the L-ascorbic acid is 0.15-0.75 g/L.
Preferably, before the pH value of the photosynthetic bacteria liquid to be preserved is adjusted, iodine-free salt is further added into the photosynthetic bacteria liquid to be preserved, and the final adding concentration of the iodine-free salt is 5-12.5 g/L.
Preferably, after the pH value of the photosynthetic bacteria liquid to be preserved is adjusted, the method further comprises the step of hermetically packaging.
Preferably, the container adopted by the sealed package is a light green transparent polyethylene plastic bottle.
Preferably, the volume of the light green transparent polyethylene plastic bottle is 3-10L, and the containing coefficient is 80-96%.
Preferably, the photosynthetic bacteria bacterial liquid is obtained by inoculating photosynthetic bacteria into an optimized culture medium and culturing for 6-8 days; the optimized culture medium comprises 1000 parts of water and 2-3.5 parts of CH by mass3COONa, 0.5-1 part of NH4Cl, 0.6-1.2 parts of KH2PO40.3-0.6 parts of MgSO4·7H2O, 0.8-1.5 of yeast extract and 0.03-0.05 part of CaCl20.015 to 0.025 parts of MnSO4And 0.002-0.003 part of FeSO4。
Preferably, the photosynthetic bacteria comprise salt-tolerant photosynthetic bacteria capable of growing at a salinity of 50% o.
Preferably, the number of effective viable bacteria in the photosynthetic bacteria liquid is more than or equal to 4.0 multiplied by 109CFU/ml。
Has the advantages that:
the invention provides a method for prolonging the storage time of a liquid photosynthetic bacterium product, which uses a phosphate buffer solution to adjust the pH value of a photosynthetic bacterium liquid to be stored to 7.0-8.0, and can obviously prolong the storage time of the photosynthetic bacterium product when the photosynthetic bacterium liquid is stored at 15-28 ℃. And (3) experimental verification: the pH value of the photosynthetic bacteria liquid to be preserved is adjusted to 7.0-8.0 by using a phosphate buffer solution, after the photosynthetic bacteria liquid is preserved for 12 months under the room-temperature natural low-light condition, the survival rate of effective viable bacteria can reach more than 50 percent, and is improved by at least 8 percent compared with the preservation method under other pH conditions.
Furthermore, the method provided by the invention further limits that L-ascorbic acid is added into the photosynthetic bacteria liquid, so that the final concentration of the L-ascorbic acid in the preservation liquid is 0.15-0.75 g/L. And (3) experimental verification: after the photosynthetic bacteria liquid treated by the method is stored for 12 months under the room-temperature natural low-light condition, the survival rate of effective viable bacteria can reach more than 60 percent, and is improved by at least 7 percent compared with the storage method without adding L-ascorbic acid.
Furthermore, the method provided by the invention further adds iodine-free salt on the basis of adding L-ascorbic acid into the photosynthetic bacteria liquid to be preserved, so that the final concentration of the iodine-free salt is 5-12.5 g/L. And (3) experimental verification: after the photosynthetic bacteria liquid treated by the method is stored for 12 months under the room-temperature natural low-light condition, the survival rate of effective live bacteria can reach more than 70 percent, and is improved by at least 15 percent compared with the storage method without adding iodine-free salt.
Furthermore, the method provided by the invention further defines a packaging container, and the light green transparent polyethylene plastic bottle is adopted for sealing and packaging. And (3) experimental verification: after the photosynthetic bacteria liquid treated by the method is stored for 12 months under the room-temperature natural low-light condition, the survival rate of effective viable bacteria can reach more than 80 percent, and is improved by at least 5 percent compared with the storage method using other packaging materials.
Detailed Description
The invention provides a method for prolonging the storage time of a liquid photosynthetic bacterium product, which is characterized in that the pH value of a photosynthetic bacterium liquid to be stored is adjusted to 7.0-8.0 by using a phosphate buffer solution, and the photosynthetic bacterium liquid is stored at the temperature of 15-28 ℃, so that the storage time of the photosynthetic bacterium product can be obviously prolonged.
In the invention, the photosynthetic bacteria bacterial liquid is preferably obtained by inoculating photosynthetic bacteria into an optimized culture medium for culture. The photosynthetic bacteria are preferably salt-tolerant photosynthetic bacteria capable of growing under the condition of 50 per mill of salinity. The salt-tolerant photosynthetic bacteria preferably comprise rhodopseudomonas palustris, rhodopseudomonas capsulata or rhodopseudomonas sphaeroides, and the optimum salinity of the photosynthetic bacteria is about 15 per mill, and the photosynthetic bacteria can still grow under the condition of 50 per mill of salinity. In the invention, the inoculation amount of the inoculation is preferably 30-50%, more preferably 35-40%, and the inoculation amount refers to the volume ratio of the strain to the optimized culture medium. In the present invention, the temperature of the culture is preferably 25 to 35 ℃, more preferably 28 to 32 ℃, and most preferably 30 ℃. The illumination intensity of the culture is preferably 6000-8000 Lx, and more preferably 7000 Lx. The culture time is preferably 6-8 days, and more preferably 7 days.
In the present invention, the optimized medium comprises water, CH3COONa，NH4Cl，KH2PO4，MgSO4·7H2O, yeast extract, CaCl2，MnSO4And FeSO4. Based on 1000 parts of water, the CH3The preferred mass part of COONa is 2-3.5 parts, and the more preferred mass part is 3 parts; the NH4The mass part of Cl is preferably 0.5-1 part, and more preferably 0.8 part; the KH2PO4Is preferably 0.6 to 1.2 parts by mass, more preferably 1.0 part by mass, of the MgSO4·7H2The mass part of O is preferably 0.3-0.6 part, and more preferably 0.5 part; the yeast extract is preferably 0.8-1.5 parts by mass, and more preferably 1.2 parts by mass; the CaCl is2The mass part of (b) is preferably 0.03 to 0.05 part, and more preferably 0.04 part; the MnSO4The mass part of (b) is preferably 0.015 to 0.025 part, and more preferably 0.02 part; the FeSO4The amount of (b) is preferably 0.002 to 0.003 part by mass, more preferably 0.0025 part by mass. In the invention, the pH value of the optimized culture medium is 6.5-7.0. Inoculating the photosynthetic bacteria into an optimized culture medium for culturing for 7-8 days to obtain photosynthetic bacteria liquid with effective viable count of not less than 4.0 × 109CFU/ml。
The pH value of the photosynthetic bacteria liquid to be preserved is adjusted to 7.0-8.0 by using a phosphate buffer solution. In the present invention, the phosphate buffer is preferably a potassium dihydrogen phosphate buffer; the mass concentration of the potassium dihydrogen phosphate buffer solution is preferably 1% to 2%, and more preferably 1.5%. The pH value of the photosynthetic bacteria liquid to be preserved is preferably adjusted to 7.2-7.8, and more preferably to 7.5.
According to the invention, before the pH value of the photosynthetic bacteria liquid to be preserved is adjusted by using a phosphate buffer solution, L-ascorbic acid is preferably added into the photosynthetic bacteria liquid to be preserved, and the final concentration of the L-ascorbic acid is preferably 0.15-0.75 g/L, and more preferably 0.5 g/L. The addition of the L-ascorbic acid can quickly deoxidize the photosynthetic bacteria, slow down the cell decay of the bacteria and increase the stability and the activity of the bacteria in the preservation process.
According to the invention, the L-ascorbic acid is preferably added with iodine-free salt, and the final concentration of the iodine-free salt in the bacterial liquid is preferably 5-12.5 g/L, and more preferably 10 g/L. The addition of iodine-free salt can prevent contamination of photosynthetic bacteria such as blue (green) algae during storage.
The method also preferably comprises the step of hermetically packaging after the pH value of the photosynthetic bacteria liquid to be preserved is adjusted by using the phosphate buffer solution. The container adopted by the sealed package is preferably a light green transparent polyethylene plastic bottle. The color of the bottle body is light green, so that a light source required by the growth of a plurality of mixed bacteria and algae can be filtered, and the phenomenon that cells are aged and adhered to walls is avoided. The volume of the light green transparent polyethylene plastic bottle is preferably 3-10L, and more preferably 5L. The filling coefficient of the light green transparent polyethylene plastic bottle is preferably 80-96%, and more preferably 90-95%.
The prepared bacterium liquid is preferably stored under the condition of normal temperature natural weak light, wherein the normal temperature is preferably 15-28 ℃, and more preferably 25 ℃; the intensity of the natural weak light is preferably 400-600 Lx, and more preferably 500 Lx.
The method for prolonging the preservation time of the liquid photosynthetic bacteria product provided by the invention is described in detail with reference to the following examples, but the method is not to be construed as limiting the scope of the invention.
Example 1
Preparation of liquid photosynthetic bacteria liquid
The strain source is as follows: the strain is rhodopseudomonas palustris, rhodopseudomonas capsulata and rhodopseudomonas spheroids, is obtained by enrichment, separation and culture from the bottom mud of a high-yield shrimp pond in coastal areas of Guangxi by Guangxi aquatic science research institute, and can still grow when the salinity is 15 per mill and is 50 per mill.
Preparing an amplification culture solution: weighing the following components in parts by weight per liter of water: CH (CH)3COONa3.0g，NH4Cl 0.8g，KH2PO41.0g，MgSO4·7H20.50g of O, 1.2g of yeast extract and CaCl240mg，MnSO420mg，FeSO42.5mg, and the pH value is adjusted to 7.0 +/-0.2 for later use.
And (3) amplification culture: culturing with 5L colorless transparent polyethylene plastic bottle, inoculating the stored three-stage strain liquid into sterilized optimized culture medium with 35% volume ratio, with a loading coefficient of 95% (v/v), stirring, controlling temperature at 30 deg.C and illumination intensity of 7000Lx, standing and culturing outdoors for 7d, and stopping culturing when the cell culture solution is deep red to obtain deep red high-activity liquid photosynthetic bacteria.
According to the test requirements, 5 batches of scale-up culture were carried out in succession according to the above process, totaling 2.5 tons for use. The results of the liquid photosynthetic bacteria culture are shown in Table 1.
TABLE 1 liquid photosynthetic bacteria culture results
Figure BDA0001724424170000061
The data results in table 1 show that: the culture results of 5 batches of liquid photosynthetic bacteria are similar, and the average thallus concentration, the mixed bacteria rate and the pH value are respectively 4.46 multiplied by 109CFU/ml, less than or equal to 4.8 percent and 9.0, deep red and sticky, which indicates that the culture process of the photosynthetic bacteria is stable and can be used for large-scale production.
Example 2
The liquid photosynthetic bacteria liquid of batch No. 1 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 1.5% potassium dihydrogen phosphate buffer solution was used to adjust the initial pH of the bacteria liquid sample to 7.0, and after being stored for 6 months and 12 months at room temperature under natural low light conditions, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: the liquid photosynthetic bacteria are preserved at pH of 7.0, and the number of effective viable bacteria after 6 months is 3.09 × 109CFU/ml, survival rate of 68.67%; the effective viable bacteria amount after 12 months is 2.48 multiplied by 109CFU/ml, survival rate of 55.11%.
Example 3
The liquid photosynthetic bacteria liquid of batch No. 1 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 1.5% potassium dihydrogen phosphate buffer solution was used to adjust the initial pH of the bacteria liquid sample to 7.5, and after being stored for 6 months and 12 months at room temperature under natural low light conditions, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: the liquid photosynthetic bacteria are preserved at pH 7.5, and the number of effective viable bacteria after 6 months is 3.12 × 109CFU/ml, survival rate 69.33%; the effective viable bacteria amount after 12 months is 2.59 multiplied by 109CFU/ml, survival rate of 57.56%.
Example 4
The liquid photosynthetic bacteria liquid of batch No. 1 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 1.5% potassium dihydrogen phosphate buffer solution was used to adjust the initial pH of the bacteria liquid sample to 8.0, and after being stored for 6 months and 12 months at room temperature under natural low light conditions, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: the liquid photosynthetic bacteria are preserved at pH of 8.0, and the number of effective viable bacteria after 6 months is 2.98 × 109CFU/ml, survival rate of 66.22%; the effective viable bacteria amount after 12 months is 2.37 multiplied by 109CFU/ml, survival rate of 52.67%.
Comparative example 1
The liquid photosynthetic bacteria liquid of batch No. 1 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 1.5% potassium dihydrogen phosphate buffer solution was used to adjust the initial pH of the bacteria liquid sample to 6.5, and after being stored for 6 months and 12 months at room temperature under natural low light conditions, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: the liquid photosynthetic bacteria are preserved at pH of 6.5, and the number of effective viable bacteria after 6 months is 2.46 × 109CFU/ml, survival rate 54.67%; the effective viable bacteria amount after 12 months is 1.64 multiplied by 109CFU/ml, survival rate of 36.44%.
Comparative example 2
The liquid photosynthetic bacteria liquid of batch No. 1 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 1.5% potassium dihydrogen phosphate buffer solution was used to adjust the initial pH of the bacteria liquid sample to 9.0, and after being stored for 6 months and 12 months at room temperature under natural low light conditions, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: the liquid photosynthetic bacteria are preserved at pH of 9.0, and the number of effective viable bacteria after 6 months is 2.40 × 109CFU/ml, survival rate 53.33%; the effective viable bacteria amount after 12 months is 1.51 multiplied by 109CFU/ml, survival rate of 33.56%.
From the results of examples 2 to 4 and comparative examples 1 to 2, it can be seen that: the survival rate of the liquid photosynthetic bacteria is better in the range of pH value 7.0-8.0, wherein the preservation effect is the best when the pH value is 7.5, and the preservation effect is the worst when the pH value is 6.5 and the pH value is 9.0, which indicates that the environment with partial acid and partial alkali is not beneficial to the preservation of the liquid photosynthetic bacteria product, and the absorption of the photosynthetic bacteria to nutrient substances is directly influenced.
Example 5
The liquid photosynthetic bacteria of batch 3 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 0.15 g/L-ascorbic acid was added to the bacteria liquid, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, and after storing for 6 months and 12 months at room temperature under natural low light, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: adding 0.15 g/L-ascorbic acid into the bacterial liquid, and keeping the effective viable bacteria number of 3.31 × 10 after 6 months9CFU/ml, survival rate of 75.23%; the number of effective viable bacteria is 2.65 multiplied by 10 after 12 months of storage9CFU/ml, survival rate of 60.23%.
Example 6
The liquid photosynthetic bacteria of batch 3 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 0.50 g/L-ascorbic acid was added to the bacteria liquid, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, and after storing for 6 months and 12 months at room temperature under natural low light, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: adding 0.50 g/L-ascorbic acid into the bacterial liquid, and keeping the effective viable bacteria number of 3.54 × 10 after 6 months9CFU/ml, survival rate of 80.45%; the number of effective viable bacteria is 3.13 multiplied by 10 after 12 months of storage9CFU/ml, survival rate 71.14%.
Example 7
The liquid photosynthetic bacteria of batch 3 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 0.75 g/L-ascorbic acid was added to the bacteria liquid, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, and after storing for 6 months and 12 months at room temperature under natural low light, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: adding 0.75 g/L-ascorbic acid into the bacterial liquid, and keeping the effective viable bacteria number of 3.47 × 10 after 6 months9CFU/ml, survival rate of 78.86%; the number of effective viable bacteria is 2.98 multiplied by 10 after 12 months of storage9CFU/ml, survival rate of 67.73%.
Comparative example 3
The liquid photosynthetic bacteria of batch 3 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken out without adding L-ascorbic acid, the initial pH of the bacteria was adjusted to 7.5 with 1.5% potassium dihydrogen phosphate buffer, and after storing for 6 months and 12 months at room temperature under natural low light, samples were taken to determine the number of effective viable bacteria of the bacteria and the survival rate was calculated.
The results show that: the number of effective viable bacteria after 6 months of preservation of the bacterial liquid without addition of L-ascorbic acid was 2.93X 109CFU/ml, survival rate of 66.59%; the number of effective viable bacteria is 2.34 multiplied by 10 after 12 months of storage9CFU/ml, storageThe activity rate was 53.18%.
From the results of examples 5 to 7 and comparative example 3, it can be seen that: the addition of 0.15-0.75 g/L-ascorbic acid to the bacterial suspension is effective in improving the preservation of the bacterial suspension, while the addition of 0.5 g/L-ascorbic acid is most effective in preserving the bacterial suspension, and the addition of 0.75 g/L-ascorbic acid is less effective.
Example 8
The liquid photosynthetic bacteria of batch 4 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 0.5 g/L-ascorbic acid was added to the bacteria liquid, 5g/L refined salt without iodine was added, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, the bottles were kept at room temperature under natural low light for 6 months and 12 months, and then samples were taken to determine the effective viable count of the bacteria liquid and calculate the survival rate.
The results show that: adding 5g/L iodine-free refined salt into the bacterial liquid, and storing for 6 months until the effective viable bacteria number is 3.74 × 109CFU/ml, survival rate of 83.11%; the number of effective viable bacteria is 3.16 multiplied by 10 after 12 months of storage9CFU/ml, survival rate 70.22%.
Example 9
The liquid photosynthetic bacteria of batch 4 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 0.5 g/L-ascorbic acid was added to the bacteria liquid, 10g/L refined salt without iodine was added, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, the bottles were kept at room temperature under natural low light for 6 months and 12 months, and then samples were taken to determine the effective viable count of the bacteria liquid and calculate the survival rate.
The results show that: adding 10g/L refined salt without iodine into the bacterial liquid, and keeping effective viable bacteria amount of 3.97 × 10 after 6 months9CFU/ml, survival rate of 88.22%; the number of effective viable bacteria is 3.51 multiplied by 10 after 12 months of storage9CFU/ml, survival rate is 78%.
Example 10
The liquid photosynthetic bacteria of batch 4 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken, 0.5 g/L-ascorbic acid was added to the bacteria liquid, 12.5g/L refined salt without iodine was added, finally, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, and after 6 months and 12 months of storage under room temperature natural low light conditions, samples were taken to determine the effective viable count of the bacteria liquid and calculate the survival rate.
The results show that: adding refined salt without iodine 12.5g/L into the bacterial liquid, and keeping effective viable bacteria amount of 3.90 × 10 after 6 months9CFU/ml, survival rate of 86.67%; the number of effective viable bacteria is 3.47 multiplied by 10 after 12 months of storage9CFU/ml, survival rate of 77.11%.
Comparative example 4
The liquid photosynthetic bacteria of batch 4 prepared in example 1 was dispensed into 5L colorless transparent polyethylene plastic bottles, two of the bottles were taken out, no iodine-free refined salt was added, 0.5 g/L-ascorbic acid was added to the bacteria liquid, 1.5% potassium dihydrogen phosphate buffer was used to adjust the initial pH of the bacteria liquid to 7.5, and after being stored at room temperature under natural weak light for 6 months and 12 months, the effective viable count of the bacteria liquid was measured and the survival rate was calculated.
The results show that: the effective viable bacteria count of the bacterial liquid without adding iodine-free refined salt after 6 months of storage is 3.51 multiplied by 109CFU/ml, survival rate of 78%; the number of effective viable bacteria is 2.48 multiplied by 10 after 12 months of storage9CFU/ml, survival rate of 55.11%.
From the results of examples 8 to 10 and comparative example 4, it can be seen that: the addition of 5-12.5 g/L of iodine-free refined salt to the bacterial liquid has a good effect on improving the preservation of the bacterial liquid, and the addition of 10g/L of iodine-free refined salt has the best preservation effect, and the addition of 12.5g/L of iodine-free refined salt is inferior.
Example 11
After 0.5 g/L-ascorbic acid and 10g/L refined salt without iodine were added to the liquid of liquid photosynthetic bacteria of lot 5 prepared in example 1, the initial pH of the liquid was adjusted to 7.5 with 1.5% potassium dihydrogen phosphate buffer, and the adjusted liquid was dispensed into 5L light green transparent polyethylene plastic bottles, and after the bottles were stored at room temperature under natural low light for 6 months and 12 months, the effective viable count of the liquid was measured and the survival rate was calculated.
The results show that: 5L light green transparent polyethylene plasticThe effective viable bacteria number of the material bottle after 6 months of storage is 4.11 multiplied by 109CFU/ml, survival rate 95.58%; the number of effective viable bacteria is 3.59 multiplied by 10 after 12 months of storage9CFU/ml, survival rate of 83.49%.
Comparative example 5
After 0.5 g/L-ascorbic acid and 10g/L refined salt without iodine were added to the liquid of liquid photosynthetic bacteria of lot 5 prepared in example 1, the initial pH of the liquid was adjusted to 7.5 with 1.5% potassium dihydrogen phosphate buffer, and the adjusted liquid was dispensed into 5L colorless transparent polyethylene plastic bottles, and after storing for 6 months and 12 months at room temperature under natural low light, the effective viable count of the liquid was measured and the survival rate was calculated.
The results show that: the number of effective viable bacteria after 6 months of preservation in 5L colorless transparent polyethylene plastic bottle is 3.66 multiplied by 109CFU/ml, survival rate 85.12%; the number of effective viable bacteria is 3.35 multiplied by 10 after 12 months of storage9CFU/ml, survival rate of 77.91%.
Comparative example 6
After 0.5 g/L-ascorbic acid and 10g/L refined salt without iodine were added to the liquid of liquid photosynthetic bacteria of lot 5 prepared in example 1, the initial pH of the liquid was adjusted to 7.5 with 1.5% potassium dihydrogen phosphate buffer, and the adjusted liquid was dispensed into 5L green transparent polyethylene plastic bottles, and after storing for 6 months and 12 months at room temperature under natural low light, the effective viable count of the liquid was measured and the survival rate was calculated.
The results show that: the number of effective viable bacteria after 6 months of preservation in 5L of green transparent polyethylene plastic bottle is 3.46 multiplied by 109CFU/ml, survival rate of 80.47%; the number of effective viable bacteria is 3.09 multiplied by 10 after 12 months of storage9CFU/ml, survival rate 71.86%.
From the results of example 11 and comparative examples 5 to 6, it can be seen that: the color of the packaging container has a remarkable effect on the preservation effect of the liquid photosynthetic bacteria, wherein the preservation effect of 5L light green is the best, and 5L is colorless.
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 (5)

1. A method for prolonging the preservation time of a liquid photosynthetic bacteria product is characterized in that a phosphate buffer solution is used for adjusting the pH value of a photosynthetic bacteria liquid to be preserved to 7.0-8.0, and the photosynthetic bacteria liquid is preserved at the temperature of 15-28 ℃;
the stored condition also comprises natural weak light; the intensity of the natural weak light is 400-600 Lx;
before adjusting the pH value of the photosynthetic bacteria liquid to be preserved, adding L-ascorbic acid into the photosynthetic bacteria liquid to be preserved, wherein the final concentration of the L-ascorbic acid is 0.15-0.75 g/L;
before adjusting the pH value of the photosynthetic bacteria liquid to be preserved, adding iodine-free salt into the photosynthetic bacteria liquid to be preserved, wherein the final addition concentration of the iodine-free salt is 5-12.5 g/L;
after the pH value of the photosynthetic bacteria liquid to be preserved is adjusted, the method also comprises the step of sealing and packaging;
the container adopted for sealing and packaging is a light green transparent polyethylene plastic bottle;
the photosynthetic bacteria are salt-tolerant photosynthetic bacteria which can grow under the condition of 50 per mill of salinity;
the salt-tolerant photosynthetic bacteria are rhodopseudomonas palustris, rhodopseudomonas capsulata or rhodopseudomonas sphaeroides.
2. The method according to claim 1, wherein the phosphate buffer is a potassium dihydrogen phosphate buffer having a mass concentration of 1% to 2%.
3. The method according to claim 1, wherein the volume of the light green transparent polyethylene plastic bottle is 3-10L, and the packing coefficient is 80-96%.
4. The method according to any one of claims 1 to 3, wherein the photosynthetic bacteria bacterial liquid is obtained by inoculating photosynthetic bacteria into an optimized culture medium and culturing the photosynthetic bacteria in the optimized culture medium for 6 to 8 timesd, obtaining; the optimized culture medium comprises 1000 parts of water and 2-3.5 parts of CH by mass3COONa, 0.5-1 part of NH4Cl, 0.6-1.2 parts of KH2PO40.3-0.6 parts of MgSO4·7H2O, 0.8-1.5 of yeast extract and 0.03-0.05 part of CaCl20.015 to 0.025 parts of MnSO4And 0.002-0.003 part of FeSO4。
5. The method as claimed in claim 4, wherein the number of active bacteria in the photosynthetic bacteria liquid is 4.0 x 10 or more9CFU/ml。
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