CN107868767A - A kind of purple laver alga border probiotics and its application - Google Patents

A kind of purple laver alga border probiotics and its application Download PDF

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CN107868767A
CN107868767A CN201711342025.XA CN201711342025A CN107868767A CN 107868767 A CN107868767 A CN 107868767A CN 201711342025 A CN201711342025 A CN 201711342025A CN 107868767 A CN107868767 A CN 107868767A
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probiotics
bacillus
culture medium
seaweed
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李孝灵
杨锐
郑宸
何应芸
何佳莲
陈海敏
骆其君
王亚军
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Ningbo University
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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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Abstract

The present invention provides a kind of probiotics of algae border separation, is bacillus (Bacillus sp.) PS1, its deposit number is CGMCC No.14846；The probiotics that the present invention is screened is used to prepare prebiotic bacterial manure.The present invention is by identifying, screening, cultivating, a series of bacteria preparation technique such as drying purple laver alga border probiotics Bacillus sp.PS1 bacterial strains are handled, a kind of prebiotic bacterial manure of acquisition.Any chemical fertilizer composition, such as sylvite, ammonium salt are not added in technique, realizes real green agriculture.The enzyme activity of growth and relevant enzyme of the product that the present invention obtains to seaweed has humidification, and the growth to Chinese little greens trophosome has obvious facilitation.

Description

A kind of purple laver alga border probiotics and its application
Technical field
The invention belongs to probiotics triage techniques field, more particularly to a kind of purple laver alga border probiotics, and its bacterium prepared Fertilizer.
Background technology
At present, the whole world is faced with the Tough questions of increased population and environmental degradation, and inorganic fertilizer is big in agricultural production Amount, which uses, can also make it that environmental degradation is more serious.The weak point of this inorganic fertilizer mainly has：Soil is destroyed, causes soil The content of organic matter declines, and all kinds of nutrients ratio imbalances, soil starts acidifying, hardened, the physicochemical character of soil and edaphon Fauna is caused its water conservation, fertilizer conservation, poor air permeability by heavy damage, it is difficult to meets the actual growth needs of crops；Crops It is stronger to the dependence of fertilizer and pesticide, it is necessary to increase phase usage amount year by year, otherwise, crop growth is slow, and pest and disease damage is serious, makes Into the underproduction；Agroecological environment deteriorates, and after chemical fertilizer applies farmland, practical efficiency averagely only has 30 ﹪, most of to be arranged with farmland Water flows into rivers and lakes or residued in soil and plant and crops body, not only brings environmental pollution but also jeopardizes the mankind and Sheng The safety and health of life, these all turn into the obstacle of Agricul tural Sustain able Development；The above situation can be organic by applying Fertilizer, bio-fertilizer are improved, but the organic fertilizer and bio-fertilizer of domestic many kinds are very different, and some adds in fertilizer Add substantial amounts of chemical fertilizer, some is simply just processed with livestocks faces, and the amount of application also having is few, it is impossible to as main fertilising, and Price is high, and effect is single, it is difficult to meets production requirement.
Moreover, currently as bacterial strain used in microbial-bacterial fertilizer, it is not to be screened from seaweed；Therefore, Find that application effect does not get a desired effect in actual use.
The content of the invention
It is an object of the invention to provide a kind of probiotics of sea water origin, and the bacterial manure of its preparation, it is existing so as to make up The deficiency of technology.
Present invention firstly provides a kind of algae border separation probiotics, be bacillus (Bacillus sp.) PS1, in On October 31st, 2017 Institute of Microorganism, Academia Sinica being deposited in positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.14846；
The probiotics that the present invention is screened is used to prepare prebiotic bacterial manure；
Another aspect of the present invention provides a kind of prebiotic bacterial manure, is to use prepared by screened probiotics fermention；
As the specific restriction of embodiment, wherein the culture medium for probiotics of fermenting, is in 2216E seawater liquid Except dusty yeast, the preparation of Sargassum horneri extract is added, the pH value of culture medium is adjusted to 5.4-5.6 or so；
Described Sargassum horneri extract is the supernatant abandoned using Sargassum horneri after production Sargassum horneri carbohydrate gum or fucoxanthin crystallization It is concentrated to form；
Above-mentioned Sargassum horneri extract can use kelp or seaweed, the fragrant plant mentioned in ancient texts and asparagus such as sea-tangle, sargassum fusifome etc. large-scale Red algae processes fucoidin, sodium alginate, and the discarded object of the algaeization product such as fucoxanthin or agar, red seaweed polysaccharide substitutes.
The present invention is by identifying, screening, cultivating, a series of bacteria preparation technique such as drying to purple laver alga border probiotics Bacillus sp.PS1 bacterial strains are handled, and obtain a kind of prebiotic bacterial manure.Any chemical fertilizer composition is not added in technique, such as Sylvite, ammonium salt etc., realize real green agriculture.The enzyme activity of growth and relevant enzyme of the product that the present invention obtains to seaweed has increasing Use is pretended, and the growth to Chinese little greens trophosome has obvious facilitation.
Brief description of the drawings
Fig. 1：Superoxide dismutase (SOD) vitality test figure；
Fig. 2：Catalase (CAT) vitality test figure；
Fig. 3：Peroxidase (POD) vitality test figure.
Embodiment
Bacterial strain uses therefor Bacillus sp.PS1 of the present invention are located away from cultivation of porphyra sea area, are one plant of seawater bacterias.The bacterial strain There is wide spectrum and stronger bacteriostatic activity to staphylococcus aureus, aquatic pathogenic bacteria etc., and there are more polyketide synthase (PKS I) approach, it may be possible to which it shows a kind of mode of bacteriostatic activity.Research finds that PS1 co-cultures with porphyra haitanensis, at 20 DEG C or so Under the conditions of thermophilic, the growth of seaweed can be promoted,；PS1 and its extract can also promote the life of terrestrial plant hair Chinese cabbage trophosome It is long.Therefore, it is significant as bacterial manure to develop it.
The present invention is described in detail with reference to embodiment.
Embodiment 1：The screening of bacterial strain
From Nantong, Qingdao, the thallus of porphyra on the ground of Wenzhou District of Zhejiang Province three and Dongtai seaweed shell conchocelis And its seawater of surrounding isolates seaweed Epiphytic bacteria, and bacteriostatic activity scalping is carried out, and combined with Protocols in Molecular Biology 16S rDNA sequential system developmental analysis, PKS I gene screenings are carried out to the bacterium of tool broad-spectrum antibacterial activity, determine PKS I bases Because of producing strains.Final screening obtains purpose bacterial strain PS1
With bacterial 16 S rRNA universal primers, PCR amplification bacterial strains PS1 16S rRNA conserved region sequences obtain 1426bp's Sequence.Bacterial strain sequencing result submission EzBioCloud is compared, the higher bacterial strain of similitude is downloaded from retrieval result 16S rRNA sequences select homology to carry out Phylogenetic Analysis higher, part retrieval result is as follows as comparison other: Bacillus stratosphericus 41KF2a (T) (NR_042336), Bacillus aerophilus28K (T) (AJ831844), Bacillus altitudinis 41KF2b (T) (AJ831842), Bacillus pumilus ATCC 7061 (T) (ABRX01000007.1), Bacillus safensis FO-036b (T) (AF234854), Bacillus Atrophaeus JCM 9070 (T) (AB021181), Bacillus siamensis KCTC 13613 (T) (AJVF01000043), Bacillus amyloliquefaciens subsp.plantarum FZB42 (T) (CP000560), Bacillus subtilis subsp.subtilis NCIB 3610 (T) (ABQL01000001), Bacillus methylotrophicus CBMB205(T)(EU194897).Will with the ClustalW in sequence synthesis analysis software MEGA 4 The 16S rRNA sequences of the aimed strain sequence high with the homology retrieved and bacillus type strain are compared, Carry out phylogenetic analysis, structure bacterial strain PS1 systematic evolution tree.Bacterial strain PS1 16S rRNA sequencing and sequence analysis As a result show, bacterial strain and bacillus similitude highest, according to bacterial strain PS1 form, physiological characteristic, 16S rDNA homologys with And Phylogenetic analysis, the results showed that, bacterial strain PS1 should belong to bacillus.By the bacillus (Bacillus of screening Sp. the Chinese Academy of Sciences that) PS1 strains are deposited on October 31st, 2017 positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 is micro- Biological study China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.14846。
Research finds that PS1 bacterial strains have wide spectrum and stronger bacteriostatic activity to staphylococcus aureus, aquatic pathogenic bacteria etc., And there is more polyketide synthases (PKS I) approach, it may be possible to which it shows a kind of mode of bacteriostatic activity.Research finds, PS1 and altar Seaweed co-cultures, and under the conditions of 20 DEG C or so of thermophilic, when the two is in reciprocal dynamic equilibrium, microbes promote seaweed Growth, while can also alleviate the attack of opportunistic fungus；PS1 can equally promote the life of terrestrial plant hair Chinese cabbage trophosome It is long.
Embodiment 2：PS1 fermentation condition
Bacterial strain Bacillus sp.PS1 growth is divided into lag phase, exponential phase and stationary phase.Can from growth curve Go out, within 0~4h this periods, for the growth rate of bacterial strain close to 0, bacterial strain is in lag phase；In 4~19h, bacterial strain is quick Growth, viable count substantial increase, bacterial strain are in exponential phase of growth；After 20h, bacterial strain quantity is stable around a definite value, bacterium Strain is plateau in stationary phase.
Using medium component, cultivation temperature, initial pH as factor, three horizontal, design orthogonal experiments are set respectively.Mapping is every PS1 growth curve in individual treatment group.Tested with reference to more than, contrast growth curve, select optimal fermentation condition.
As a result show：2216E culture mediums remove yeast powder component, and add Sargassum horneri extract, 30 DEG C, under the conditions of pH 5.5 The upgrowth situation of bacterial strain is best.It can be found that the primary and secondary order for influenceing growth factor is by the size of R values：Culture medium> Originate pH>Temperature.The culture medium be the Sargassum horneri processing waste by the use of 10% as source of nutrition, substitute the yeast in culture medium Powder, the leftover bits and pieces that algae can be made full use of to process not only improves the fermentation efficiency of bacterial strain, and can save cost, has Important utilization and promotional value.
Table 1：Fermentation condition envirment factor and level
Table 2：PS1 strain fermentation condition optimizing Orthogonal experiment results
Table 3：PS1 strain fermentation condition optimizings orthogonal experiment is analyzed
Research finds that the growth of bacterial strain that Sargassum horneri extract screens to the present invention has a facilitation, in Sargassum horneri extract not Only abundant glucide, algae gelatin substance, also there is regulatory function containing abundant active material, some of materials, Such as mannitol is advantageous to removal of the bacterial strain of the present invention to nitrate nitrogen.
Embodiment 3：The physicochemical property of bacterial strain
1st, the influence that PS1 grows to seaweed
Porphyra haitanensis after recovery is cleaned 3 times in seawater is sterilized, then is sterilized seawater and repeatedly washed, and is examined under low-powered microscope Look into, it is ensured that without miscellaneous algae and foreign matter on blade, it is good to select growth conditions, the complete blade of eucaryotic cell structure, is inhaled as far as possible with blotting paper Seaweed leaf water is received, porphyra haitanensis is processed into diameter about 6mm seaweed disk using the method for punching, and use up with blotting paper Amount absorbs seaweed leaf water, then the blade with 0.1 ± 0.01g is made.After the sterilized seawer washing of above blade, with 0.7% KI soaks 10min, adds ampicillin (final concentration of 300 μ g/mL), kanamycins (final concentration of 100 μ g/mL), celebrating greatly Mycin (final concentration of 100 μ g/mL), sterileization processing is carried out to seaweed, is taken out after 18-20h, cleaned seaweed with antiseptic sea water and justify Piece 3 times, seaweed nutrient solution overnight incubation is transferred to, it is standby thoroughly to remove residual antibiotic.The sample that will be handled well after 1~2 day Random equal portions are tested.Seaweed optimum culturing temperature is 18 ± 1 DEG C, 15-18 μm of ol/m of intensity of illumination2S, Light To Dark Ratio L：D =12h：12h.
B+BE- groups：To be put into 25 porphyra haitanensis disks in each conical flask, add bacterial strain PS1 (1.8 × 108Cells), 10 it is parallel, be put into illumination box and cultivate, rock 4-5 times daily, culture uses vernier caliper measurement in 12 days Its diameter.B-BE- groups：25 porphyra haitanensis disks will be put into each conical flask, 10 parallel, is put into illumination box and trains Support, rock 4-5 times daily, the time of the measurement of diameter and method are consistent with B+BE- groups.B-BE+ groups：By in each conical flask 25 porphyra haitanensis disks are put into, add bacterial strain PS1 extracts 5ml, 10 parallel, is put into illumination box and cultivates, shakes daily Shake 4-5 times, the time of the measurement of diameter and method are consistent with B+BE- groups.As a result show PS1 or its metabolin to porphyra haitanensis all It is obviously promoted growth (P<0.05).
Moreover, because the bacterial strain that is screened of the present invention is the fungal component with seaweed, screened compared to other from seawater The bacterial strain of bacillus, the production facilitation effect of bacterial strain of the invention to porphyra haitanensis are more preferable.
2nd, PS1 SOD disproportionation performances
SOD is disproportionated O2- it is H2O2And O2, be intracellular reactive oxygen metabolism key enzyme.It is at the beginning, right in incubation According to group and add the SOD enzyme activity of bacterium group all to show ascendant trend, be at 1 hour, the ascendant trend of control group, which is significantly greater than, to be added Bacterium group, reach top within 4 hours, hence it is evident that higher than adding bacterium group.Although and add bacterium group also rising, the unobvious of rising, Substantially also reached top when 4 hours.Last two groups of experiments tend towards stability substantially, finally add bacterium group after 8 hours It is constantly in slightly above control group (Fig. 1).But by analysis after software SPSS16.0 analyses, two groups do not have significant difference change Change (P>0.05).
CAT and POD is H in plant2O2Scavenger enzyme, with SOD act synergistically, maintain activity in vivo Oxygen Balance.It is real Test result and show the activity of since second hour CAT enzymes more than control group, until just declining after 8 hours, behind two The CAT enzymatic activitys of group experiment reached a peak (Fig. 2) again simultaneously at 12 hours.Control group and add bacterium group present at the beginning The trend of liter, before two hours plus bacterium treatment group POD activity is apparently higher than control group, and control group, which exceedes, after two hours adds bacterium group and one All apparently higher than bacterium treatment group is added before 10 hours, two groups of experiment POD enzymatic activitys are begun to decline after 8 hours, are finally tended to It is stable, but control group is also slightly higher than plus bacterium treatment group (Fig. 3).
Result above shows that seaweed POD and SOD enzyme activity are obvious high between 2-8 hours are cultivated under sterile culture state In adding bacterium treatment group, 8 as a child two groups of experiments all tend towards stability and are close.But software SPSS16.0 analyzes it by analysis Afterwards, two groups do not have significant difference to change (P>0.05).
Embodiment 4：The research of influences of the PS1 to hair Chinese cabbage seed germination percentage
By experiment be divided into clear water group, 2216E sea water mediums group, to array, steady group.Each experimental group is made following Processing：The seed of hair Chinese cabbage is placed in clear water respectively, is pulled out after 4h and is air-dried and be placed in the culture dish for being covered with filter paper, will be dry It is placed on filter paper after net sterile continuous wetting, to keep seed to moisten, is then placed in culture dish in 28 DEG C of constant incubator. Every group of experiment in triplicate, every repeatedly 20 seeds.Germination percentage is recorded after 4d.Every group of filter paper is trained with clear water, 2216E seawater respectively Support base, living bacterial liquid, broken zymotic fluid infiltration.Experiment be divided into clear water group, 2216E sea water mediums group, to array, steady group.It is right Each experimental group makees following handle：The approximate hair Chinese cabbage seedling of size is chosen, per potted plant 1 plant of kind, 10 basins are often handled, in sowing After 5d, clear water group clear water pouring root, 2216E sea water mediums group culture medium pouring root, to array 5ml viable bacteria liquid irrigating root, The steady group broken fermentation liquid irrigating roots of 5ml.Unified harvesting plant after pouring root 12d.Determine its change of height.
From table 4, it can be seen that the plant height of sproutings and seedling of the bacterial strain PS1 on Chinese little greens seed is without influence (P> 0.05) blade area and Chinese little greens fresh weight (P can extremely be improved, but significantly<0.001), illustrate that the bacterium is sought to Chinese little greens The growth for supporting body has obvious facilitation.
Table 4：The influence that PS1 bacterial strains germinate and grown to Chinese little greens
Embodiment 5：The preparation of prebiotic bacterial manure
Preparation method, specifically include following steps：
(1) go bail for the refrigeration for being hidden in -80 DEG C of refrigerators purple laver alga border probiotics Bacillus sp.PS1 strains in equipped with In the 250ml of 100ml 2216E seawater fluid nutrient mediums conical flask, 37 DEG C of shaking table, 110r/min culture 12h are put, obtains bacterium Activating solution；
(2) 10% bronze medal is added in equipped with 100ml 2216E seawater liquid except the 250ml of dusty yeast culture medium conical flask Algae extract, pH to 5.5 is adjusted, obtains culture medium；
(3) take bacterium activating solution 1ml to be added in the culture medium that step (2) prepares, put 30 DEG C of shaking table, 110r/min cultures 36h, obtain zymotic fluid；
(4) zymotic fluid is put into centrifuge, 2000r/min centrifugation 15min, a layer solid is removed in separation, and resulting solid is For the bacterial manure of the present invention.
Bacterial manure is used for experiment and the data of the fertilizer efficiency of seaweed：
Porphyra haitanensis after recovery is cleaned 3 times in seawater is sterilized, then is sterilized seawater and repeatedly washed, and is examined under low-powered microscope Look into, it is ensured that without miscellaneous algae and foreign matter on blade, it is good to select growth conditions, the complete blade of eucaryotic cell structure, is inhaled as far as possible with blotting paper Seaweed leaf water is received, porphyra haitanensis is processed into diameter about 6mm seaweed disk using the method for punching, and use up with blotting paper Amount absorbs seaweed leaf water, then the blade with 0.1 ± 0.01g is made.After the sterilized seawer washing of above blade, with 0.7% KI soaks 10min, adds ampicillin (final concentration of 300 μ g/mL), kanamycins (final concentration of 100 μ g/mL), celebrating greatly Mycin (final concentration of 100 μ g/mL), sterileization processing is carried out to seaweed, is taken out after 18-20h, cleaned seaweed with antiseptic sea water and justify Piece 3 times, seaweed nutrient solution overnight incubation is transferred to, it is standby thoroughly to remove residual antibiotic.The sample that will be handled well after 1~2 day Random equal portions are tested.Seaweed optimum culturing temperature is 18 ± 1 DEG C, 15-18 μm of ol/m of intensity of illumination2S, Light To Dark Ratio L：D =12h：12h.
B+BE- groups：To be put into 25 porphyra haitanensis disks in each conical flask, add bacterial strain PS1 (1.8 × 108Cells), 10 it is parallel, be put into illumination box and cultivate, rock 4-5 times daily, culture uses vernier caliper measurement in 12 days Its diameter.
B-BE- groups：25 porphyra haitanensis disks will be put into each conical flask, 10 parallel, is put into illumination box Culture, rocks 4-5 times daily, and the time of the measurement of diameter and method are consistent with B+BE- groups.B-BE+ groups：By each conical flask In be put into 25 porphyra haitanensis disks, add bacterial strain PS1 extracts 5ml, 10 are parallel, are put into illumination box and cultivate, daily Rock 4-5 times, the time of the measurement of diameter and method are consistent with B+BE- groups.
Bacterial manure is used for the fertilizer efficiency of hair Chinese cabbage
The research of influences of the PS1 to hair Chinese cabbage seed germination percentage
By experiment be divided into clear water group, 2216E sea water mediums group, to array, steady group.Each experimental group is made following Processing：The seed of hair Chinese cabbage is placed in clear water respectively, is pulled out after 4h and is air-dried and be placed in the culture dish for being covered with filter paper, will be dry It is placed on filter paper after net sterile continuous wetting, to keep seed to moisten, is then placed in culture dish in 28 DEG C of constant incubator. Every group of experiment in triplicate, every repeatedly 20 seeds.Germination percentage is recorded after 4d.Every group of filter paper is trained with clear water, 2216E seawater respectively Support base, bacterial manure, broken zymotic fluid infiltration.
The development test that PS1 influences on hair Chinese cabbage growth of seedling
Experiment be divided into clear water group, 2216E sea water mediums group, to array, steady group.Following locate is made to each experimental group Reason：The approximate hair Chinese cabbage seedling of size is chosen, per potted plant 1 plant of kind, often handles 10 basins, after 5d is sowed, clear water group is filled with clear water Root, 2216E sea water mediums group crush zymotic fluid with 5ml bacterial manure pouring root, steady group with culture medium pouring root, to array with 5ml Pouring root.Unified harvesting plant after pouring root 12d.Determine its change of height.
Fertilizer efficiency of the bacterial manure of table 2 to Chinese little greens
From table 2 it can be seen that the plant height of sprouting and seedling of this bacterial manure on Chinese little greens seed is without influence (P>0.05), But it extremely can significantly improve blade area and Chinese little greens fresh weight (P<0.001), illustrate the bacterium to Chinese little greens trophosome Growth has obvious facilitation.

Claims (8)

1. a kind of probiotics of algae border separation, it is characterised in that the deposit number of described probiotics is CGMCC No.14846.
A kind of 2. culture medium for being used to cultivate probiotics described in claim 1, it is characterised in that described culture medium be In 2216E seawater Liquid Cultures, replace prepared by dusty yeast with Sargassum horneri extract, the pH value of culture medium is 5.4-5.6.
3. culture medium as claimed in claim 2, it is characterised in that described Sargassum horneri extract is Sargassum horneri in production Sargassum horneri carbohydrate gum Or the supernatant concentration thing abandoned after fucoxanthin crystallization.
4. culture medium as claimed in claim 2, it is characterised in that described Sargassum horneri extract kelp or the algae of red algae The discarded object for changing product substitutes.
5. application of the probiotics in prebiotic bacterial manure is prepared described in claim 1.
6. a kind of prebiotic bacterial manure, it is characterised in that described prebiotic bacterial manure is the probiotics fermention system described in usage right requirement 1 Standby.
7. application of the probiotics described in claim 1 in algae culturing.
8. application as claimed in claim 7, it is characterised in that described marine alga is seaweed or Chinese little greens.
CN201711342025.XA 2017-12-14 2017-12-14 A kind of purple laver alga border probiotics and its application Pending CN107868767A (en)

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CN113197225A (en) * 2021-04-30 2021-08-03 上海交通大学 Resource utilization method of polyphenol waste liquid from fucoxanthin extraction from brown algae

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