CN110157621A - A kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells - Google Patents

A kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells Download PDF

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CN110157621A
CN110157621A CN201910383261.9A CN201910383261A CN110157621A CN 110157621 A CN110157621 A CN 110157621A CN 201910383261 A CN201910383261 A CN 201910383261A CN 110157621 A CN110157621 A CN 110157621A
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photosynthetic bacteria
gram
preservative agent
algae
living cells
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CN110157621B (en
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吕和鑫
齐兵兵
刘翠华
贾士儒
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Tianjin University of Science and Technology
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

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Abstract

The invention discloses a kind of preparation methods of highly enriched long-acting preservative agent of microalgae living cells, by the way that by photosynthetic bacteria capsula Rhodopseudomonas Rhodopseudomonas capsulate and the thermophilic Halomonas Halomonas campisalis of Halophiles, fermented and cultured obtains bacterium mud in glutamate culture medium, then bacterium mud is embedded in alginate carrier particle, continue that inorganic salts will be contained, vitamin, the nutrient matrixes powder adsorption such as organic carbon source is in carrier granular, obtain photosynthetic bacteria carrier granular nutritive salt hybrid particles, after spray-dried and powder homogeneous, the highly enriched long-acting preservative agent of microalgae living cells is made.This preservative agent uses upper simple, it is only necessary to a certain amount of preservative agent powder be added in concentration algae solution, bacterium is rised in value in embedded particles, and the metabolin of generation can maintain the algae concentrate ecological balance.This method is simple and easy, and spring and autumn three quarters can maintain concentration 10-30 days vigor of algae living cells and occur without corruption.

Description

A kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells
Technical field
The present invention relates to a kind of a large amount of microalgae concentrate long-term preservations of hot season to maintain and mention with cell viability in transport High method.
Background technique
Aquatic microalgae such as chlorella, Dunaliella salina, haematococcus pluvialis etc., since microalgae itself contains a large amount of nutrients Matter and pigment content are present optimal aquatic feeds.Present bait algae mainly has microalgae living cells and microalgae cell dry powder.It is micro- Algae living cells is full of nutrition, but is easy dead, corruption during transportation.Existing microalgae concentrate transport generallys use low The means of transportation of temperature refrigeration, leads at high cost, the problems such as frustule vigor is low.
The transport of present bait algae is mainly the following method.
(1) microalgae low-temperature transport, low temperature are often referred to temperature at 0-4 DEG C, at these temperatures, such to transport every milliliter 2 ×1010Cell concentration, there is a problem of at high cost, and algae vigor reduces significant, is not suitable for algae living and adjusts breeding water body water quality Situation.
(2) microalgae is transported at room temperature, and microalgae is divided into illumination and dark two ways in being transported at room temperature, but in light conditions Under, frustule concentration is low, every milliliter 10 general6A cell is hereinafter, be more than every milliliter 107Frustule concentrate 24 hours in i.e. Corruption, conevying efficiency is low, at high cost.
(3) algae dry powder transports, and the feed algae of the method is dead cell, and a part of nutriment has been lost in algae It loses, loses original biological value, for certain aquatic livestocks, algae living is its type of uniquely ingesting.It is not suitable for algae tune living The case where saving breeding water body water quality.
According to above-mentioned analysis, bait algae there are shipping concentrations in transportational process under spring, summer, autumn low, transport at present Apart from the problems such as short, at high cost.Therefore high concentration microalgae cell vital activity can be kept under tropical temperatures by finding one kind Store method has a good application prospect.
According to retrieval, it is found that following several patents document is related to the present invention, is respectively:
1, patent (201610330167.3) " a kind of store method of chlorella ", this method is by by chlorella concentrate It is mixed with sterile water, then adds glycerol, tea extract, potassium sorbate and saved, facilitate the storage of a small amount of chlorella algae Hiding and transport.And this method mainly emphasizes the long-term preservation and transport of highly enriched frustule, keeps the high vigor of frustule, saves Contain photosynthetic bacteria in agent, maintains the ecological circulation of sealing algae concentrate, it is at low cost, it is pollution-free to water body.
2, patent (00107546.2) " a kind of fresh living microbial phycophyte food and preparation method thereof ", this method passes through algae solution is dense Contracting is added in protection liquid, is injected into special container, and obtained algae safety can be fabricated to food.But keep microalgae Activity time is short, and small scale, at high cost.This method is mainly the protection to extensive Mirco algal food during transportation, and Directly preservative agent is added in concentrate, is not necessarily to speciality container, it is easy to operate；Preservation liquid is living body photosynthetic bacteria, at low cost, right Water body does not pollute.
3, patent (200410036386.8) " preparation method and its product of unit cell dried chlamydomonas product and application ", this method are logical It crosses algae concentration, dry, vacuum or nitrogen-filled packaging, convenient transportation, but frustule is dead in the patent, in frustule Part nutriment loss.This method is that the vigor of extensive fresh living microbial phycophyte is maintained and improved, and is suitable for marine industry pair The demand of active somatic cell.
Summary of the invention
It is simple and convenient the object of the present invention is to provide a kind of applied widely, it can largely be saved under tropical conditions and fortune The preparation method of defeated microalgae cell concentrate preservative agent.Preservative agent ingredient be the original salinity of natural water body, vitamin, amino acid with The photosynthetic bacteria contained in natural water and thermophilic Halomonas, do not pollute water body and at low cost.
The present invention is to be achieved through the following technical solutions:
A kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells, it is characterised in that include the following steps:
(1) production of bacterium mud is mixed
By photosynthetic bacteria capsula Rhodopseudomonas Rhodopseudomonas capsulate, the thermophilic Halomonas of Halophiles Halomonas campisalis with fermentor respectively to be trained in 10% inoculum concentration access glutamate culture medium respectively It supports, 22-32 DEG C of temperature, incubation time is 36-48 hours.Total bacteria count reaches 2 × 10 in fermentation liquid10Cfu/ml, by two kinds of bacterium Fermentation liquid 1: 1 mixes, and then high speed centrifugation obtains photosynthetic bacteria bacterium mud.
(2) production of photosynthetic bacteria water holding carrier granular
Bacterium mud is embedded to form photosynthetic bacteria carrier granular: the sodium alginate soln and photosynthetic bacteria bacterium mud for being 20g/L by concentration Volume ratio 1: 1 is uniformly mixed, and the calcium chloride solution of 10g/L is then added into mixed liquor, is stirred simultaneously, until solution Muddiness is no longer deepened, and solidification 15-40 minutes is then allowed to stand.It is filtered using 1500 mesh filter clothes, obtains photosynthetic bacteria water holding carrier Particle.
(3) production of photosynthetic bacteria carrier granular nutritive salt hybrid particles
It is mixed by following mass ratio: 100 parts of photosynthetic bacteria carrier granular (milliliter), 5 parts of sodium nitrate (gram), ammonium chloride 10 parts (gram), vitamin B12 0.01 (gram), 400 parts of sodium acetate (gram), 0.01 part of sodium molybdate (gram), 5 parts of sodium glutamate (gram), 5 parts of sodium bicarbonate (gram), 0.4 part of ironic citrate (gram), 2 parts of boric acid (gram).30-60 minutes are stood after mixing, is obtained Photosynthetic bacteria carrier granular nutritive salt hybrid particles.
(4) highly enriched microalgae living cells preservative agent production
Dry photosynthetic bacteria carrier/nutritive salt hybrid particles refinement: grinder grinds the drying for being broken into 10-20um partial size Grain.Highly enriched microalgae living cells preservative agent is made.
(5) highly enriched microalgae living cells preservative agent application method
In 10g preservative agent: preservative agent is added frustule concentration and is higher than every milliliter 100 by the ratio of 1L frustule concentrate Hundred million or more algae concentrate, slightly mixes, room temperature, and sealing avoids be exposed to the sun transport or preservation.
Glutamate medium component are as follows: yeast extract 10g/L, sodium glutamate 5g/L, sodium molybdate 0.005g/L, carbonic acid Hydrogen sodium 2g/L, glucose 10g/L.
The microalgae include deliver vegetables, Dunaliella salina, haematococcus pluvialis, marine chlorella, limnetic chlorella, micro- quasi- ball Any one of algae, flat algae, Phaeodactylum tricornutum, cylindrotheca clostetium, Chaetoceros, Isochrysis galbana, pavlova viridis, purple ball algae
The present invention has technical effect beneficial below compared with prior art:
Under spring and autumn three quarters room temperature mass storage, transport Mirco algal food can be concentrated, and the vigor of frustule can be kept.
The cell that high density saves is living cells, can be directly used as fish and shrimp diet, can be directly used for adjusting culture pond Water quality.
Figure of description
Fig. 1 --- marine chlorella save 20 days with 30 days after photo
Specific embodiment
Below with reference to specific implementation case, the invention will be described in further detail, it is described be explanation of the invention without It is to limit.
Strain selects capsula Rhodopseudomonas Rhodopseudomonas capsulate and the thermophilic Halomonas of Halophiles Halomonas campisalis。
Embodiment 1
Step 1: by cell concentration 2 × 106The capsula Rhodopseudomonas Rhodopseudomonas capsulate of/ml It is inoculated into glutamate culture medium respectively with the thermophilic Halomonas Halomonas campisalis bacterium of Halophiles, 30 DEG C of cultures In 30L fermentor, nutrient solution volume 15L, ventilation culture 48h, capsula Rhodopseudomonas is 3 × 1010Cfu/ml, Halomonas Campisalis strain density is 2.8 × 1010Cfu/ml respectively samples 10L, mixes to obtain 20L mixed bacteria liquid.5000g is centrifuged 10 minutes, Obtain bacterium mud about 100mL.
Step 2: 100ml concentration is mixed for the 100ml bacterium mud collected in the sodium alginate soln and step 1 of 20g/L, It stirs evenly, the calcium chloride solution of 10g/L is then added, is stirred simultaneously, after about 400ml is added, solution muddiness degree No longer deepen, stand 30 minutes, be subsequently poured into the sack that 1500 mesh filter clothes are made into, stand 1h, liquid no longer exosmoses in bag, obtains Obtain the photosynthetic bacteria water holding carrier granular of about 200ml.
Step 3: following ingredient: sodium nitrate 10 is added into the photosynthetic bacteria water holding carrier granular for the 200ml that previous step obtains Gram, 20 grams of ammonium chloride, 0.02 gram of vitamin B12,800 grams of sodium acetate, 0.02 gram of sodium molybdate, 10 grams of sodium glutamate, sodium bicarbonate 10 grams, 0.8 gram of ironic citrate, 4g grams of boric acid.40 minutes are stood after mixing, obtains the mixing of photosynthetic bacteria carrier granular nutritive salt Grain.
Step 4: photosynthetic bacteria carrier granular nutritive salt hybrid particles are spray-dried: 120 DEG C of inlet air temperature, 60 DEG C Leaving air temp.Obtain the dry photosynthetic bacteria carrier/nutritive salt hybrid particles of about 800g.It is ground through 25000 revs/min of minigrinders of household Mill 5 minutes obtains the highly enriched long-acting preservative agent of microalgae living cells that granular size is 10 microns.
Step 5: 12g preservative agent is added in the marine chlorella liquid that 1 liter of cell concentration is 20,000,000,000 cells/mls, set It is placed on to place on 15-30 DEG C of day and night temperature of workshop without sunlight direct projection table top in plastic barrel, after sealing and save.
2 days after being saved, 8 days, 1 month, 2 months separately sampled, and observation concentration algae solution smell variation is dyed with PI-FDA Method carries out survival rate measurement.
As a result as follows:
Table one, different holding time marine chlorella testing results
Sample time Smell Survival rate
0 day Algae nature bilgy odour 98%
2 days Algae nature bilgy odour 98%
8 days Algae nature bilgy odour 97%
1 month Algae nature bilgy odour 96%
2 months Putrefactive odor 40%
Chlorella is during preservation, though cell survival rate is gradually decreased with the extension of preservation time, in one month Survival rate is 96% or more.
Embodiment 2
Step 1 to four with example 1,
Step 5: mixed algae solution is placed on 15-30 DEG C, is saved under dark condition.
2 days after being saved, 8 days, 1 month, 2 months, smell is observed, carries out survival rate measurement with PI-FDA decoration method.
Table 2, different holding time limnetic chlorella testing results
Sample time Smell Survival rate
0 day Algae nature bilgy odour 98%
After 2 days Algae nature bilgy odour 96%
After 8 days Algae nature bilgy odour 95%
After 1 month The slightly bilgy odour algae of peculiar smell 92%
After 2 months Putrefactive odor 12%
In limnetic chlorella preservation process, though cell survival rate is gradually decreased with the extension of preservation time.Save 1 After month, survival rate still has 92%；It is putrid and deteriorated when in two months, only 12% or so frustule existing state.

Claims (3)

1. a kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells, which comprises the steps of:
(1) by photosynthetic bacteria capsula Rhodopseudomonas Rhodopseudomonas capsulate and the thermophilic Halomonas of Halophiles Halomonas campisalis with fermentor respectively to be trained in 10% inoculum concentration access glutamate culture medium respectively It supports, incubation time is 36-48 hours, and total bacteria count reaches 2 × 1010cfu/ml or more in fermentation liquid, by the fermentation liquid 1 of two kinds of bacterium: 1 mixing, then high speed centrifugation obtains photosynthetic bacteria bacterium mud.
(2) bacterium mud is embedded to form photosynthetic bacteria carrier granular: the sodium alginate soln and photosynthetic bacteria bacterium mud body for being 20g/L by concentration Product is uniformly mixed than 1: 1, and the calcium chloride solution of 10g/L is then added into mixed liquor, is stirred simultaneously, until solution is muddy No longer deepen, is then allowed to stand solidification 15-40 minutes；It is filtered using 1500 mesh filter clothes, obtains photosynthetic bacteria water holding carrier granular.
(3) it is mixed in the following proportions: 100 parts of photosynthetic bacteria carrier granular (milliliter), 5 parts of sodium nitrate (gram), 10 parts of ammonium chloride (gram), 0.01 part of vitamin B12 (gram), 400 parts of sodium acetate (gram), 0.01 part of sodium molybdate (gram), 5 parts of sodium glutamate (gram), carbon Sour 5 parts of hydrogen sodium (gram), 0.4 part of ironic citrate (gram), 2 parts of boric acid (gram) stand 30-60 minutes after mixing, obtain photosynthetic bacteria and carry Body particle nutritive salt hybrid particles.
(4) be spray-dried photosynthetic bacteria carrier granular nutritive salt hybrid particles: 100-160 DEG C of inlet air temperature, 50-80 DEG C goes out Air temperature obtains dry photosynthetic bacteria carrier/nutritive salt hybrid particles.
(5) dry photosynthetic bacteria carrier/nutritive salt hybrid particles refinement: grinder grinds the drying for being broken into 10-20um partial size Grain.
2. a kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells as described in claim 1, which is characterized in that institute The glutamate medium component stated is yeast extract 10g/L, sodium glutamate 5g/L, sodium molybdate 0.005g/L, sodium bicarbonate 2g/L, glucose 10g/L.
3. a kind of preparation method of the highly enriched long-acting preservative agent of microalgae living cells as described in claim 1, which is characterized in that institute The microalgae stated include deliver vegetables, Dunaliella salina, haematococcus pluvialis, chlorella, Nannochloropsis oculata, flat algae, Phaeodactylum tricornutum, barrel mast Any one of algae, Chaetoceros, Isochrysis galbana, pavlova viridis, purple ball algae.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108753623A (en) * 2018-06-12 2018-11-06 兰溪市沉默生物科技有限公司 A kind of chlorella store method
CN112960768A (en) * 2021-02-23 2021-06-15 广东工业大学 Aerobic granular sludge, long-term storage method thereof and activity recovery method after storage
CN114164128A (en) * 2021-12-15 2022-03-11 海南绿藻世界生物科技有限公司 Algae symbiotic bacteria composition, algae bacteria co-culture system and microalgae culture method

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CN107287125A (en) * 2017-08-24 2017-10-24 黑龙江科技大学 A kind of cultural method of chlorella pyrenoidosa
CN108624582A (en) * 2018-05-18 2018-10-09 江苏世邦生物工程科技有限公司 Microorganism formulation for soil remediation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108753623A (en) * 2018-06-12 2018-11-06 兰溪市沉默生物科技有限公司 A kind of chlorella store method
CN112960768A (en) * 2021-02-23 2021-06-15 广东工业大学 Aerobic granular sludge, long-term storage method thereof and activity recovery method after storage
CN112960768B (en) * 2021-02-23 2022-08-26 广东工业大学 Aerobic granular sludge, long-term storage method thereof and activity recovery method after storage
CN114164128A (en) * 2021-12-15 2022-03-11 海南绿藻世界生物科技有限公司 Algae symbiotic bacteria composition, algae bacteria co-culture system and microalgae culture method

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