CN107236765A - One kind fermentation Jujun grasses bio-based and preparation method thereof - Google Patents
One kind fermentation Jujun grasses bio-based and preparation method thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Abstract
The invention discloses a kind of preparation method for Jujun grasses bio-based of fermenting.Mixed bacteria liquid is added on Jujun grasses forage, inoculum concentration is per the 100g mixed bacteria liquids of kg forages 30, sealing and fermenting, fermented 10 30 days in the case where temperature is 20 35 DEG C, to fermentation forage pH value reach 3.5 4.5 between when, fermentation ends, thus obtain ferment Jujun grasses bio-based；Every milliliter described of mixed bacteria liquid contains lactobacillus paracasei 5 × 107‑1×109Individual and Lactobacillus plantarum 9 × 107‑1.25×109It is individual.The fermentation Jujun grasses bio-based of the present invention, its effective active lactic acid bacteria bacterium number is 5 × 108‑5×109Cfu/g, the compositions such as organic matter in the protein in Jujun grasses, sugar and pond progress flourish can be utilized in breeding water body, ammonia nitrogen, nitrite, suppression Measures of Algae in Water Body growth effectively in degraded water body, improve water quality, the use of antibiotic is reduced, promotes the sound development of culture fishery.
Description
Technical field
The invention belongs to microorganism field, and in particular to one kind fermentation Jujun grasses bio-based and preparation method thereof.
Background technology：
Jujun grasses (Pennisetum giganteum z.x.lin) are the upright bunch type plant of perennial grass family, thick egg
White and sugar content is high.Jujun grasses are after the strain fermentations such as inoculated plant lactobacillus, lactobacillus paracasei, pH value reduction, breast
The compositions such as acid, acetic acid increase, lactic acid bacteria bacterium number rolls up simultaneously, effectively raises the use value of Jujun grasses.After fermentation
Jujun grasses can be in whole bag input breeding water body, its effect is, Jujun grasses of fermenting contain the probiotics of a large amount of amphimicrobians, in water
In body environment flourish, effectively drop are carried out using compositions such as organic matters in the protein in Jujun grasses, sugar and pond
Solve the ammonia nitrogen in water body, nitrite, suppress Measures of Algae in Water Body growth, while promoting the growth of other microorganisms in water body, change
Kind water quality.
Lactobacillus plantarum and lactobacillus paracasei are that the Ministry of Agriculture of China allows as one of bacterial strain of feed addictive, and it is
Application of Direct-fed Microbials is developed into more and more.
Jujun grasses bio-based of fermenting is using the Jujun grasses of active lactic acid bacteria as main component, by simple, it is easy to work
The production technology of industry metaplasia production, prepares a kind of probiotics preparation used for aquiculture.The product of the invention can effectively degrade foster
Ammonia nitrogen, the nitrite in water body are grown, suppresses Measures of Algae in Water Body growth, reduces the use of antibiotic, promote culture fishery
Develop in a healthy way.
The content of the invention：
The present invention's is to provide high fermentation Jujun grasses bio-based of a kind of effective active lactic acid bacteria bacterium number and preparation method thereof.
The fermentation Jujun grasses bio-based of the present invention is prepared by the following method, and it comprises the following steps：
Mixed bacteria liquid is added on Jujun grasses forage, inoculum concentration is the sealing hair per kg forage 30-100g mixed bacteria liquids
Ferment, is to ferment 10-30 days at 20-35 DEG C in temperature, when being reached to the pH value of the forage that ferments between 3.5-4.5, fermentation ends, by
This obtains Jujun grasses bio-based of fermenting；
Every milliliter described of mixed bacteria liquid contains lactobacillus paracasei 5 × 107-1×109Individual and Lactobacillus plantarum 9 × 107-
1.25×109It is individual.
Described Jujun grasses forage is that the ripe bacterial strain of the Jujun grasses of fresh harvesting is crushed into 3-10cm and obtained.
The fermentation Jujun grasses bio-based of the present invention, its effective active lactic acid bacteria bacterium number is 5 × 108-5×109Cfu/g, can
Flourish is carried out using compositions such as organic matters in the protein in Jujun grasses, sugar and pond in breeding water body, effectively
Ammonia nitrogen, nitrite, suppression Measures of Algae in Water Body growth in degraded water body, improve water quality, reduce the use of antibiotic, promotion water
Produce the sound development of aquaculture.
Embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
The preparation method of the fermentation Jujun grasses bio-based of embodiment 1
(1) preparation of lactobacillus paracasei bacterium solution
The single bacterium colony of picking lactobacillus paracasei strain from inclined-plane, is seeded to one equipped with 100ml improvement MC trainings respectively
In the 250ml triangular flasks for supporting base, 37 DEG C of quiescent culture 24h are activated；The bacterium solution after activation is connect with volume fraction 4% again
Amount is planted, 37 DEG C of quiescent culture 24h in three 500ml triangular flasks equipped with 350ml is inoculated into, prepares shake-flask seed liquid.
Above-mentioned shake-flask seed liquid is connected to the 30L seeds equipped with molasses fluid nutrient medium with the inoculum concentration of volume fraction 5%
Fermented in tank, liquid amount is volume fraction 75%, 37 DEG C of fermentation temperature, quiescent culture, culture to zymotic fluid pH is left for 4.0
The right side, this zymotic fluid is second level seed liquor.
Above-mentioned 30L second level seed liquor is connected to the 300L seeds equipped with molasses fluid nutrient medium with 8% inoculum concentration
Fermented in tank, liquid amount be volume fraction 80%, 37 DEG C of fermentation temperature, quiescent culture, culture to zymotic fluid pH be 4.0,
Lactobacillus paracasei bacterium solution is obtained, zymotic fluid viable count is detected.
(2) Lactobacillus plantarum bacterium solution is prepared according to the method same with above-mentioned lactobacillus paracasei bacterium solution
1st, Modified MC agar：
1000ml is added water to, pH to 6.0 ± 0.2 is adjusted.Solid medium adds the agar of mass fraction 2%.Sterilizing is standby
2nd, molasses fluid nutrient medium：
1000ml is added water to, pH to 6.8 ± 0.2 is adjusted, sterilized standby.
(3) preparation for Jujun grasses of fermenting
The ripe bacterial strain of Jujun grasses of fresh harvesting is taken, Jujun grasses are crushed to 3cm or so, then will by fresh weight about 2000kg
Lactobacillus paracasei bacterium solution and Lactobacillus plantarum bacterium solution by volume 1：The mixed bacteria liquid of 9 ratios mixing is inoculated into the grass after crushing
On material, every milliliter described of mixed bacteria liquid contains lactobacillus paracasei 5 × 107It is individual, Lactobacillus plantarum 9 × 107Individual, inoculum concentration is
Per kg forages inoculation 30g mixed bacteria liquids, stir, special fermentation bag is loaded immediately, per packed 30kg, be placed in 20 degrees Celsius
Lower fermentation 30 days.Period detects material pH value and viable count.Fermentation ends when pH 4.5, thus obtain fermentation Jujun grasses life
Thing base, the storage of end product shady place.Measure Lactobacillus plantarum and lactobacillus paracasei total viable count reaches 5 × 108Cfu/g,
(4) measure of fermentation Jujun grasses bio-based viable count
1 the method for plate culture count
1.1 weigh the biological based specimen 10g of fermentation Jujun grasses, are put into and fill 90ml sterilized waters and the triangle burning with bead
In bottle, shaking table 200rpm vibration 20min stand 20~30S, sample suspension are made.
1.2 draw 1mL sample suspensions with 1ml aseptic straws, are put into the test tube for filling 9mL aqua sterilisas, shake well makes
It is well mixed, that is, it is made 1：100 uniform dilutions.
1.3 draw 1 with 1mL aseptic straws：10 dilution 1mL, blow slowly to inject along tube wall and contain in 9mL aqua sterilisa test tubes,
Test tube is shaken, is well mixed, makes 1：1000 dilution.1mL sterilized straws separately are taken, by aforesaid operations order, 10 times is done and passs
Increase dilution, be respectively prepared 10-4、10-5、10-6……10-9Gradient dilution liquid, so often incremental dilution once, that is, uses 1 instead
1mL sterilized straws.
1.4 2-3 acceptable diluent degree of selection (typically take 10-6、10-7、10-8), with 100 μ L micropipettors of sterilizing, respectively take
0.1mL is injected on the blank plate of sterilizing, and whole surface is uniformly applied to sterile push rod, is subsequently poured into and is had cooled down to 45 DEG C
The Modified MC agar of left and right, each dilution factor makees 3 flat boards.
1.5 are inverted in plate in 37 DEG C of ± 1 DEG C of constant incubators after culture 48h ± 2h, choose clump count in 30-300
Between plate count.
2 Lactobacillus plantarums, lactobacillus paracasei colonial morphology
Morphological feature：Lactobacillus plantarum falls projection, rounded, and surface is smooth, fine and closely woven, in rice white or light yellow；Pair is dry
Lactobacillus paracasei bacteria colony white or canescence, circular, the smooth moistening in surface, neat in edge, projection.
3 colony countings
The selection of 3.1 dilution factors
3.1.1 dilution factor of the average colony between 30-300 should be selected, extension rate is multiplied by and reports it
If 3.1.2 there is two dilution factors.Its grow clump count between 30-300, then regarding how to come between the two determine
If determining it than less than or equal to 2, its average should be reported；If more than 2, reporting wherein less numeral.
If 3.1.3 the average colony number of all dilution factors is all higher than 300.It should then multiply by dilution factor highest average colony number
It is reported with extension rate.
If 3.1.4 all dilution factor average colony numbers are respectively less than 30, it should be multiplied by by the minimum average colony number of dilution factor dilute
Release multiple and report it.
3.2 calculate：
Lactobacillus plantarum and lactobacillus paracasei total viable count (cfu/g)=flat-plate bacterial colony average of same dilution factor three
× extension rate × 10
(5) application effect of the fermentation Jujun grasses bio-based in aquaculture
The fermentation Jujun grasses bio-based of above-mentioned gained is put into Pelteobagrus fulvidraco culture pond by 10kg/ mus of meter Shui Shen consumption
In, every month, cultivating pool depth of water 1.5m, 10 mu of area measured water sample pH 8.2, ammonia nitrogen before input Jujun grasses using once
Initial concentration is 0.55mg/L, and nitrite concentration is 0.65mg/L, water sampling analysis in the 90th day, and ammonia nitrogen concentration is 0.2mg/L,
Nitrite concentration is 0.1mg/L, and ammonia nitrogen degradation rate is 63.63%, and nitrite degradation rate is 84.61%.
The preparation method of the fermentation Jujun grasses bio-based of embodiment 2
(1) preparation of lactobacillus paracasei bacterium solution
The lactobacillus paracasei single bacterium colony of picking strain from inclined-plane, is seeded to one equipped with 100ml improvement MC trainings respectively
In the 250ml triangular flasks for supporting base (be the same as Example 1), 37 DEG C of quiescent culture 24h are activated；Again by the bacterium solution after activation with body
The inoculum concentration of fraction 4%, is inoculated into 37 DEG C of quiescent culture 24h in three 500ml triangular flasks equipped with 350ml, prepares shaking flask kind
Sub- liquid.
Above-mentioned shake-flask seed liquid is connected to the 30L seeds equipped with molasses fluid nutrient medium with the inoculum concentration of volume fraction 5%
Fermented in tank, liquid amount is volume fraction 75%, 37 DEG C of fermentation temperature, quiescent culture, culture to zymotic fluid pH is left for 4.0
The right side, obtains second level seed liquor.
Above-mentioned 30L second level seed liquor is connected to equipped with molasses fluid nutrient medium with the inoculum concentration of volume fraction 8%
Fermented in 300L seeding tanks, liquid amount is volume fraction 80%, 37 DEG C of fermentation temperature, quiescent culture, culture to zymotic fluid
PH is 4.0, obtains lactobacillus paracasei bacterium solution, detects zymotic fluid viable count.
(2) Lactobacillus plantarum bacterium solution is prepared according to the method same with lactobacillus paracasei bacterium solution
(3) preparation for Jujun grasses of fermenting
The ripe bacterial strain of Jujun grasses of fresh harvesting is taken, Jujun grasses are crushed to 6cm or so, then will by fresh weight about 2000kg
Lactobacillus paracasei bacterium solution and Lactobacillus plantarum bacterium solution by volume 3：7 mixed bacteria liquid is inoculated on the forage after crushing, institute
Every milliliter of the mixed bacteria liquid stated contains lactobacillus paracasei 3 × 108It is individual, Lactobacillus plantarum 7 × 108Individual, inoculum concentration connects per kg forages
Kind of 60g mixed bacteria liquids, stir, special fermentation bag are loaded immediately, per packed 40kg, are placed under 28 degrees Celsius and ferment 20
My god.Period detects material pH value and viable count.Fermentation ends when pH 4.0, thus obtain Jujun grasses bio-based of fermenting, most
Finished product shady place is stored afterwards.Lactobacillus plantarum and lactobacillus paracasei total viable count in fermentation Jujun grasses bio-based is measured to reach
1.0×109cfu/g。
(4) application effect of the fermentation Jujun grasses bio-based in aquaculture
In consumption input grass carp culture pond by the fermentation Jujun grasses bio-based of above-mentioned gained by 10kg/ mus of meter Shui Shen,
Every month, cultivating pool depth of water 1m, 10 mu of area measured water sample pH 8.0, ammonia nitrogen is initial before input Jujun grasses using once
Concentration is 0.52mg/L, and nitrite concentration is 1.65mg/L, water sampling analysis in the 90th day, and ammonia nitrogen concentration is 0.1mg/L, nitrous
Hydrochlorate concentration is 0.3mg/L, and ammonia nitrogen degradation rate is 80.76%, and nitrite degradation rate is 81.82%.
The preparation method of the fermentation Jujun grasses bio-based of embodiment 3
(1) preparation of lactobacillus paracasei bacterium solution
The lactobacillus paracasei single bacterium colony of picking strain from inclined-plane, is seeded to one equipped with 100ml improvement MC trainings respectively
In the 250ml triangular flasks for supporting base, 37 DEG C of quiescent culture 24h are activated；The bacterium solution after activation is connect with volume fraction 4% again
Amount is planted, 37 DEG C of quiescent culture 24h in three 500ml triangular flasks equipped with 350ml is inoculated into, prepares shake-flask seed liquid.
Above-mentioned shake-flask seed liquid is connected to the 30L seeds equipped with molasses fluid nutrient medium with the inoculum concentration of volume fraction 5%
Fermented in tank, liquid amount is volume fraction 75%, 37 DEG C of fermentation temperature, quiescent culture, culture to zymotic fluid pH is left for 4.0
The right side, thus obtains second level seed liquor.
Above-mentioned 30L seed liquor is connected to the 300L kinds equipped with molasses fluid nutrient medium with the inoculum concentration of volume fraction 8%
Fermented in sub- tank, liquid amount is volume fraction 80%, 37 DEG C of fermentation temperature, quiescent culture, culture is to zymotic fluid pH
4.0, lactobacillus paracasei bacterium solution is thus obtained, zymotic fluid viable count is detected.
(2) Lactobacillus plantarum bacterium solution is prepared according to the method same with lactobacillus paracasei bacterium solution
(3) preparation for Jujun grasses of fermenting
The ripe bacterial strain of Jujun grasses of fresh harvesting is taken, Jujun grasses are crushed to 10cm or so, then will by fresh weight about 2000kg
Lactobacillus paracasei bacterium solution and Lactobacillus plantarum bacterium solution by volume 5：5 mixed bacteria liquid is inoculated on the forage after crushing, institute
Every milliliter of the mixed bacteria liquid stated contains lactobacillus paracasei 1 × 109It is individual, Lactobacillus plantarum 1.25 × 109It is individual, inoculum concentration grass per kg
Material inoculation 100g mixed bacteria liquids, stir, special fermentation bag are loaded immediately, per packed 50kg, be placed in 35 degrees Celsius and issue
Ferment 10 days.Period detects material pH value and viable count.The fermentation ends of pH 3.5 are treated, Jujun grasses bio-based of fermenting thus is obtained,
Measure this fermentation Jujun grasses bio-based Lactobacillus plantarum and lactobacillus paracasei total viable count reaches 5 × 109cfu/g。
(4) application effect of the fermentation Jujun grasses bio-based in aquaculture
In the consumption input shrimp aquaculture pool by the fermentation Jujun grasses bio-based of above-mentioned gained by 10kg/ mus of meter Shui Shen, often
Using once, cultivating pool depth of water 1m, 10 mu of area measures water sample pH 7.5, ammonia nitrogen is initially dense before input Jujun grasses within individual month
Spend for 0.42mg/L, nitrite concentration is 0.75mg/L, water sampling analysis in the 90th day, ammonia nitrogen concentration is 0.1mg/L, nitrous acid
Salinity is 0.2mg/L, and ammonia nitrogen degradation rate is 76.19%, and nitrite degradation rate is 73.33%.
Claims (3)
1. a kind of preparation method for Jujun grasses bio-based of fermenting, it is characterised in that comprise the following steps：
Mixed bacteria liquid is added on Jujun grasses forage, inoculum concentration is the sealing and fermenting per kg forage 30-100g mixed bacteria liquids,
Temperature is to ferment 10-30 days at 20-35 DEG C, and when being reached to the pH value of fermentation forage between 3.5-4.5, thus fermentation ends obtain
To fermentation Jujun grasses bio-based；
Every milliliter described of mixed bacteria liquid contains lactobacillus paracasei 5 × 107-1×109Individual and Lactobacillus plantarum 9 × 107-1.25
×109It is individual.
2. preparation method according to claim 1, it is characterised in that described Jujun grasses forage is by the huge of fresh harvesting
Juncao maturation bacterial strain is crushed to 3-10cm and obtained.
3. the fermentation Jujun grasses bio-based that a kind of preparation method according to described in claim 1 or 2 is prepared.
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| CN114806951A (en) * | 2022-05-06 | 2022-07-29 | 福建省农业科学院农业生物资源研究所 | Preparation method for producing lactobacillus microcapsule microbial inoculum by fermenting grass juice |
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| CN114806951A (en) * | 2022-05-06 | 2022-07-29 | 福建省农业科学院农业生物资源研究所 | Preparation method for producing lactobacillus microcapsule microbial inoculum by fermenting grass juice |
| CN115247134A (en) * | 2022-07-25 | 2022-10-28 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as fermentation medium of paecilomyces lilacinus for nematode biocontrol bacteria |
| CN115404181A (en) * | 2022-07-25 | 2022-11-29 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as lactobacillus plantarum fermentation medium |
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