CN104894028A - Fishery ocean microbial ecological preparation and preparation method thereof - Google Patents
Fishery ocean microbial ecological preparation and preparation method thereof Download PDFInfo
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- CN104894028A CN104894028A CN201510339159.0A CN201510339159A CN104894028A CN 104894028 A CN104894028 A CN 104894028A CN 201510339159 A CN201510339159 A CN 201510339159A CN 104894028 A CN104894028 A CN 104894028A
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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 invention discloses a fishery ocean microbial ecological preparation. The preparation comprises lactobacillus pantarum and pediococcus pentosaceus, wherein the microbial content ratio of lactobacillus pantarum to pediococcus pentosaceus is 2:1. The invention further discloses a preparation method of the fishery ocean microbial ecological preparation. The preparation method comprises a first step of activation and culture expansion of strains; a second step of preparation of a fermentation medium; a third step of fermentation cultivation; a fourth step of filtering and material discharging. A culture medium formula facilitating large-scale production is screened out for lactobacillus pantarum and pediococcus pentosaceus, the feed efficiency is greatly improved, metabolites of lactobacillus pantarum and pediococcus pentosaceus are synergistic, the efficiency of the metabolites of lactobacillus pantarum and pediococcus pentosaceus is much higher than that of a metabolite of lactobacillus pantarum or pediococcus pentosaceus in production application, and popularization and application of lactobacillus pantarum and pediococcus pentosaceus microbial ecological preparation for aquaculture are effectively promoted.
Description
Technical field
The present invention relates to the technical field of probiotics, particularly relate to a kind of fishing ocean probiotics and preparation method thereof.
Background technology
It is more and more serious that current culture fishery also exists disease, traditional microbiotic and chemical industry sterilizing agent hard to carry on, consequent aquaculture water discharges and has carried out severe contamination to cultivation zone, photosynthetic bacterium utilisation technology constantly progress in aquaculture from the nineties, in recent years, the application of probiotics in aquaculture was in the ascendant, the technology that genus bacillus is promoted in aquaculture reaches its maturity, referential foundation is provided to the application of microorganism in culture fishery, TaiWan, China the nineties is just applied to aquaculture milk-acid bacteria, so far certain application level is possessed, China just just starts the investigation and application of milk-acid bacteria, milk-acid bacteria has field planting cultivated animals enteron aisle and suppresses pathogenic bacteria and aid digestion absorption, eliminate the organism of water body, the characteristics such as objectionable impurities, provide sound assurance to aquaculture Sustainable development.
Plant lactobacillus (Lactobacillus plantarum) belongs to lactobacillaceae, lactobacillus, Gram-positive.Plant lactobacillus is homofermentative lactic bacteria, only produces lactic acid during the fermentation, is typical facultative anaerobe, has the ability of very strong fermentable carbohydrates, comparatively salt tolerant, has synergy with other milk-acid bacteria.Pediococcus pentosaceus (Pediococcus pentosaceus) belongs to coccus, genus lactubacillus, Gram-positive, facultative anaerobic, have and produce that high, the stable performance of acid, degradation effect are good, the more high advantage of salt tolerance, be usually used in stalk fermentation and fodder additives.Plant lactobacillus and Pediococcus pentosaceus are that in the Ministry of Agriculture's No. 2045 bulletin " fodder additives kind catalogue (2013) " in 2013, regulation can the feed level microbe additive bacterial classification of Direct-fed cultivated animals as probiotics.Plant lactobacillus and Pediococcus pentosaceus are two different sortses of milk-acid bacteria.
In the prior art, plant lactobacillus and Pediococcus pentosaceus are normally respectively used to the microbe additive as aquatic living things, for both integrated uses, also rarely have report.In view of this, the present inventor studies and devises a kind of fishing ocean probiotics and preparation method thereof, and this case produces thus.
Summary of the invention
The object of the present invention is to provide a kind of fishing ocean probiotics and preparation method thereof; the present invention is to plant lactobacillus and Pediococcus pentosaceus; filter out the culture medium prescription of large-scale production; to improve feed efficiency, and then promote plant lactobacillus and Pediococcus pentosaceus probiotics applying in aquaculture.
For achieving the above object, the present invention solves the technical scheme of its technical problem and is:
A kind of fishing ocean probiotics, comprises plant lactobacillus and Pediococcus pentosaceus, and the bacterium number of described plant lactobacillus and described Pediococcus pentosaceus is than being 2:1.
A preparation method for fishing ocean probiotics, comprises the following steps:
The activation of step one, bacterial classification and enlarged culturing:
One-level is cultivated: in Bechtop, get inclined-plane preserve single bacterium colony in 100ml sterilizing MRS substratum, be placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h; Plant lactobacillus and Pediococcus pentosaceus respectively inoculate 4 bottles, and object is activated spawn; Get bacterium liquid coating MRS flat board after 24h multiple, be placed in quiescent culture 24h in 35 DEG C of thermostat containers, treat that bacterium colony grows, be placed in 4 DEG C of refrigerator conservations, stand-by;
Secondary is cultivated: in Bechtop, get inclined-plane preserve single colony inoculation in 200ml sterilizing MRS, and two kinds of each inoculations 6 bottles altogether of bacterium, are placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h;
Third stage culture: get in cultured bacterium liquid 10ml to 2000ml sterilizing MRS substratum, be placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h, obtain seed liquor, adopt boulton process to produce plant lactobacillus 2kg, Pediococcus pentosaceus 30kg, bacterium number all reaches hundred million grades;
The preparation of step 2, fermention medium:
Peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, dipotassium hydrogen phosphate 2g, sodium acetate 2g, disodium citrate 2g, bitter salt 0.5g, Manganese sulfate pentahydrate 0.2g, tween-80 1ml, is settled to 1L;
Step 3, fermentation culture:
Described plant lactobacillus and described Pediococcus pentosaceus are inoculated in described fermention medium by bacterium number than the mixed bacterium for 2:1, inoculum size is 2%, be that 12:1 adds glucose and soy peptone by C:N in described fermention medium, leavening temperature 35 DEG C, initial p H are 6.5, stir as 12h stirs 1 time, each churning time is 5min, reaches less than 4.0 for fermentation termination with PH;
Step 4, filtration discharging:
After fermentation to terminal, filtration discharging is carried out to fermented liquid, then carries out packing, obtained this fishing ocean probiotics.
As the optimal way of embodiment, the bacterium number of described mixed bacterium is 1,000,000,000/g.
The present invention is to plant lactobacillus and Pediococcus pentosaceus; filter out the culture medium prescription of large-scale production; substantially increase feed efficiency; the meta-bolites of plant lactobacillus and Pediococcus pentosaceus has synergistic ability; usefulness far away higher than monomer in production application, effectively facilitates plant lactobacillus and Pediococcus pentosaceus probiotics applying in aquaculture.
Accompanying drawing explanation
The ratio of Fig. 1 two kinds of milk-acid bacterias is on the impact of its meta-bolites;
Fig. 2 carbon source is on the impact of two kinds of lactobacter growths;
Fig. 3 nitrogenous source is on the impact of two kinds of lactobacter growths;
Fig. 4 buffering salt is on the impact of two kinds of lactobacter growths;
Fig. 5 somatomedin is on the impact of two kinds of lactobacter growths;
The impact that Fig. 6 different vaccination amount is fermented on plant lactobacillus and Pediococcus pentosaceus;
The impact that Fig. 7 different fermentations temperature is fermented on plant lactobacillus and Pediococcus pentosaceus;
The impact that the different initial pH of Fig. 8 ferments on plant lactobacillus and Pediococcus pentosaceus.
Embodiment
Embodiment 1
A kind of fishing ocean probiotics, comprises plant lactobacillus and Pediococcus pentosaceus, and the bacterium number of described plant lactobacillus and described Pediococcus pentosaceus is than being 2:1.
The fermented liquid getting this proportioning test carries out cultivation pilot scale, throws something and feeds through 10 days, and the shrimp individuality of experimental group is obviously greater than other groups, and enteron aisle is comparatively thick, and wriggling ability is strong, ingests fast.Therefore, the best ratio of fermentation of plant lactobacillus and Pediococcus pentosaceus is 2:1.
We carry out milk-acid bacteria to the juvenile prawn of 0.7cm and throw something and feed, contrast each group that does not does not throw something and feed, effect display is particularly evident, the juvenile prawn of throwing something and feeding according to 2:1 proportioning, and when showing energetic, frightened, spring reaction comparatively obviously, enteron aisle is comparatively thick, wriggling ability is strong, tail fan launches the features such as amplitude is larger.
Embodiment 2
A preparation method for fishing ocean probiotics, comprises the following steps:
The activation of step one, bacterial classification and enlarged culturing:
One-level is cultivated: in Bechtop, get inclined-plane preserve single bacterium colony in 100ml sterilizing MRS substratum, be placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h; Plant lactobacillus and Pediococcus pentosaceus respectively inoculate 4 bottles, and object is activated spawn; Get bacterium liquid coating MRS flat board after 24h multiple, be placed in quiescent culture 24h in 35 DEG C of thermostat containers, treat that bacterium colony grows, be placed in 4 DEG C of refrigerator conservations, stand-by;
Secondary is cultivated: in Bechtop, get inclined-plane preserve single colony inoculation in 200ml sterilizing MRS, and two kinds of each inoculations 6 bottles altogether of bacterium, are placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h;
Third stage culture: get in cultured bacterium liquid 10ml to 2000ml sterilizing MRS substratum, be placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h, obtain seed liquor, adopt boulton process to produce plant lactobacillus 2kg, Pediococcus pentosaceus 30kg, bacterium number all reaches hundred million grades;
Described MRS substratum is: peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, dipotassium hydrogen phosphate 2g, sodium acetate 2g, disodium citrate 2g, bitter salt 0.5g, Manganese sulfate pentahydrate 0.2g, and tween-80 1ml, is settled to 1L.
The preparation of step 2, fermention medium:
Peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, dipotassium hydrogen phosphate 2g, sodium acetate 2g, disodium citrate 2g, bitter salt 0.5g, Manganese sulfate pentahydrate 0.2g, tween-80 1ml, is settled to 1L;
Step 3, fermentation culture: described plant lactobacillus and described Pediococcus pentosaceus are inoculated in described fermention medium by bacterium number than the mixed bacterium for 2:1, inoculum size is 2%, be that 12:1 adds glucose and soy peptone by C:N in described fermention medium, leavening temperature 35 DEG C, initial p H are 6.5, stir as 12h stirs 1 time, each churning time is 5min, reaches less than 4.0 for fermentation termination with PH;
Step 4, filtration discharging: after fermentation to terminal, filtration discharging is carried out to fermented liquid, then carries out packing, obtained this fishing ocean probiotics.
As the optimal way of embodiment, the bacterium number of described mixed bacterium is 1,000,000,000/g.
Embodiment 3 plant lactobacillus and the research of Pediococcus pentosaceus concentration proportioning
The Proportionality design of plant lactobacillus and Pediococcus pentosaceus is: 1:1,1:2,1:3,1:4,2:1,3:1 six ratios.Be seeded in MRS substratum (121 DEG C, 30min sterilizing) in proportion, be placed in 35 DEG C of water bath with thermostatic control incubators slowly concussion cultivation 12h, get bacterium liquid and detect bacterium number and PH, each ratio three is parallel, test-results represents with mean value, and result is as shown in table 1 and Fig. 1.
The ratio of table 1 two kinds of milk-acid bacterias is on the impact of its meta-bolites
| Plant lactobacillus/Pediococcus pentosaceus | 1:1 | 1:2 | 1:3 | 2:1 | 3:1 |
| Test 1 group | 5.2 | 4.7 | 3.8 | 3.9 | 3.7 |
| Test 2 groups | 5.1 | 4.3 | 4.4 | 3.4 | 3.8 |
| Test 3 groups | 4.7 | 4.5 | 3.8 | 3.2 | 3.5 |
| PH (mean value) | 5 | 4.5 | 4 | 3.5 | 3.7 |
| Test 1 group | 13 | 19 | 17 | 23 | 19 |
| Test 2 groups | 13 | 20 | 21 | 19 | 15 |
| Test 3 groups | 15 | 15 | 22 | 18 | 14 |
| Breeding bacteria (10 8Cuf/ml) (mean value) | 14 | 18 | 20 | 20 | 16 |
Because the meta-bolites of plant lactobacillus and Pediococcus pentosaceus exists synergy, therefore in order to better economic benefit, the proportioning of these two kinds of bacterial strains is studied, determination methods judges with PH height and bacterium number height, as shown in Figure 1, when the ratio of plant lactobacillus and Pediococcus pentosaceus is 2:1, PH is lower, bacterium number is not low yet simultaneously, and therefore plant lactobacillus and the best proportioning of fermenting of Pediococcus pentosaceus are 2:1.In addition, the fermented liquid getting this proportioning test carries out cultivation pilot scale, throws something and feeds through 10 days, and the shrimp of 2:1 proportioning of throwing something and feeding fermented liquid is obviously greater than other groups, and enteron aisle is energetic, ingests fast.
The optimal screening of embodiment 4 plant lactobacillus and Pediococcus pentosaceus substratum
The impact that 4.1 different carbon source are cultivated plant lactobacillus and Pediococcus pentosaceus
On MRS liquid nutrient medium basis, select compounding sugar 5 kinds of carbon sources of sucrose, glucose, maltose, lactose and glucose and lactose mass ratio 2:1, massfraction is all 5%, by 2% inoculum size inoculation seed liquor in fermention medium, and initial pH6.0, culture temperature 35 DEG C, be placed in quiescent culture 18h in thermostat container, measure the colony number of nutrient solution, often organize test three parallel, result represents with mean value, and result is as shown in table 2 and Fig. 2.
Table 2 carbon source is on the impact of two kinds of lactobacter growths
| Carbon source kind | Sucrose | Glucose | Maltose | Lactose | Compounding sugar | Blank |
| Test 1 group | 8.5 | 23.0 | 9.0 | 10.8 | 8.6 | 7.6 |
| Test 2 groups | 8.9 | 16.0 | 10.3 | 11.3 | 9.9 | 8.4 |
| Test 3 groups | 9.6 | 21.0 | 9.5 | 6.4 | 11.5 | 8.0 |
| Average fermentation bacterium number (10 8cuf/ml) | 9.0 | 20.0 | 9.6 | 9.5 | 10.0 | 8.0 |
As can be seen from Fig. 2 and table 2, promote that the optimum carbon source of two kinds of lactobacter growths is glucose.During using glucose as carbon source, the zymophyte concentration of gained is apparently higher than other carbon sources, and concentration can reach 20.0 × 108cuf/ml, and sucrose, maltose, lactose and compounding sugar are close as the impact effect of carbon source to two kinds of lactobacter growths.
The impact that 4.2 different nitrogen sources are cultivated plant lactobacillus and Pediococcus pentosaceus
On MRS liquid nutrient medium basis, select the leaching of soy peptone, Tryptones, yeast powder, SODIUMNITRATE, ammonium sulfate 5 kinds of nitrogenous sources, massfraction is all 1%.By 2% inoculum size inoculation seed liquor in fermention medium, initial pH6.0, culture temperature 35 DEG C, is placed in quiescent culture 18h in thermostat container, measures the colony number of nutrient solution, and often organize test three parallel, result represents with mean value, and result is as shown in table 3 and Fig. 3.
Table 3 nitrogenous source is on the impact of two kinds of lactobacter growths
| Nitrogenous source kind | Soy peptone | Trypsin | Yeast leaching powder | SODIUMNITRATE | Ammonium sulfate | Blank |
| Test 1 group | 18.8 | 17.5 | 17.0 | 11.3 | 11.0 | 7.6 |
| Test 2 groups | 19.6 | 17.9 | 17.0 | 13.8 | 12.5 | 8.4 |
| Test 3 groups | 18.6 | 18.6 | 16.9 | 12.7 | 12.5 | 8.0 |
| Average fermentation bacterium number (10 8cuf/ml) | 19.0 | 18.0 | 17.0 | 12.6 | 12.0 | 8.0 |
As can be seen from Fig. 3 and table 3, promote that the optimum nitrogen source of two kinds of lactobacter growths is soy peptones.During using soy peptone as nitrogenous source, the zymophyte concentration of acquisition is the highest, is 19.0 × 108cuf/ml, Trypsin and yeast leaching powder take second place as nitrogenous source effect, SODIUMNITRATE and ammonium sulfate effect the poorest.
The impact that 4.3 different buffering salts are cultivated plant lactobacillus and Pediococcus pentosaceus
On MRS liquid nutrient medium basis, select buffering salt: light calcium carbonate, dipotassium hydrogen phosphate, sodium-acetate, Calcium hydrogen carbonate.Massfraction is 5 ‰.By 2% inoculum size inoculation seed liquor in fermention medium, initial medium PH=6.5, culture temperature 35 DEG C, is placed in constant incubator quiescent culture 18h, measures colony number, and often organize test three parallel, result represents with mean value, and result is as shown in table 4 and Fig. 4.
Table 4 buffering salt is on the impact of two kinds of lactobacter growths
| Buffering salt kind | Light calcium carbonate | Dipotassium hydrogen phosphate | Sodium-acetate | Calcium hydrogen carbonate | Blank |
| Test 1 group | 13.5 | 20.2 | 13.4 | 17.4 | 7.6 |
| Test 2 groups | 13.9 | 17.6 | 16.7 | 15.9 | 8.4 |
| Test 3 groups | 14.6 | 18.9 | 17.9 | 17.7 | 8.0 |
| Average fermentation bacterium number (10 8cuf/ml) | 14.0 | 18.9 | 16.0 | 17.0 | 8.0 |
As can be seen from Fig. 4 and table 4, promote that the buffering salt of two kinds of lactobacter growths is dipotassium hydrogen phosphates.When taking dipotassium hydrogen phosphate as buffering salt, the zymophyte concentration of acquisition is the highest, is 18.9 × 108cuf/ml.
The impact that 4.4 different somatomedins are cultivated plant lactobacillus and Pediococcus pentosaceus
On MRS liquid nutrient medium basis, the growth selection factor: tomato juice, corn steep liquor, vitamin complex, yeast extract paste, wort.Massfraction is 5 ‰, by 2% inoculum size inoculation seed liquor in fermention medium, and initial medium PH=6.5, culture temperature 35 DEG C, is placed in constant incubator quiescent culture 18h, measures colony number, often organize test three parallel, result represents with mean value, and result is as shown in table 5 and Fig. 5.
Table 5 somatomedin is on the impact of two kinds of lactobacter growths
| Somatomedin kind | Corn steep liquor | Vitamin complex | Tomato juice | Yeast extract paste | Wort | Blank |
| Test 1 group | 17.6 | 10.3 | 18.9 | 17 | 15.7 | 7.6 |
| Test 2 groups | 17.4 | 13.6 | 18.6 | 16.1 | 15.4 | 8.4 |
| Test 3 groups | 16.6 | 12.1 | 20.4 | 15.8 | 16.6 | 8.0 |
| Average fermentation bacterium number (10 8cuf/ml) | 17.2 | 12.0 | 19.3 | 16.3 | 15.9 | 8.0 |
As can be seen from Fig. 5 and table 5, promote that the optimum growh factor of two kinds of lactobacter growths is tomato juices.
The optimization of the present embodiment to substratum is based upon on the basis of MRS substratum, is optimized Carbon and nitrogen sources.Result shows: optimum carbon source is glucose, and optimum nitrogen source is soy peptone, optimized buffer salt is dipotassium hydrogen phosphate.The present embodiment also further study the growth effect of somatomedin to two kinds of milk-acid bacterias, and result display tomato juice is the most effective.
The optimization of embodiment 5 plant lactobacillus and Pediococcus pentosaceus mixing fermentation culture condition
The selection of 5.1 inoculum sizes
Select different vaccination amount 1%, 2%, 3%, 5%, 9%, inoculation seed liquor is in fermention medium, initial pH6.0, culture temperature 36 DEG C, be placed in quiescent culture 18h in thermostat container, measure the colony number of nutrient solution, often organize test three parallel, result represents with mean value, and result is as shown in table 6 and Fig. 6.
The impact that table 6 different vaccination amount is fermented on plant lactobacillus and Pediococcus pentosaceus
| Inoculum size | 1％ | 2％ | 3％ | 5％ | 7％ |
| Test 1 group | 7.8 | 19.8 | 12.9 | 18.1 | 11.2 |
| Test 2 groups | 8.6 | 18.6 | 14.7 | 16.8 | 16.7 |
| Test 3 groups | 10.6 | 21.6 | 11.4 | 13.1 | 15.6 |
| Average fermentation bacterium number (10 8cuf/ml) | 9 | 20 | 13 | 16 | 14.5 |
As seen from Figure 6, when the fermentation inoculum size of plant lactobacillus and Pediococcus pentosaceus is 2%, zymophyte number reaches 20 × 108cuf/ml, determines that 2% for optimum inoculation amount.Inoculum size is too high, and thalli growth is bad, may be because the nutritive substance in fermentation starting stage substratum can not meet the demand of a large amount of thalli growth.
The selection of 5.2 temperature
By optimum inoculation amount inoculation seed liquor in fermention medium, initial pH6.0, respectively at 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C quiescent culture 18h, measure its colony number, often organize test three parallel, result represents with mean value, and result is as shown in table 7 and Fig. 7.
The impact that table 7 different fermentations temperature is fermented on plant lactobacillus and Pediococcus pentosaceus
| Leavening temperature (DEG C) | 25 | 30 | 35 | 40 | 45 |
| Test 1 group | 9.8 | 15.3 | 24.9 | 11.9 | 6.5 |
| Test 2 groups | 9.6 | 16.8 | 25.3 | 12.5 | 5.7 |
| Test 3 groups | 10.6 | 15.9 | 27.8 | 11.6 | 5.8 |
| Average fermentation bacterium number (10 8cuf/ml) | 10 | 16 | 26 | 12 | 6 |
As can be seen from Fig. 7 and table 7, the leavening temperature of plant lactobacillus and Pediococcus pentosaceus is 35 DEG C, and zymophyte number reaches 26 × 108cuf/ml, determines that 35 DEG C for optimum fermentation temp.Temperature variation directly can affect the activity of thalline internal protein, enzyme, and the too high meeting of temperature makes intracellular protein sex change, affects the macromolecular stability such as rrna, RNA; Temperature is too low can reduce enzyme activity, causes the change of cell membrane fluidity, thus affects the normal growth of thalline.
The selection of 5.3 optimal phs
PH value is selected to be respectively 7.5,7.0,6.5,6.0,5.0,4.0, batch fermentation in 15L fermentor tank, inoculate by optimum inoculation amount, optimum temps is cultivated, and stirring velocity controls at 100r/min, measures colony number every 3h, select its optimal ph, often organize test three parallel, result represents with mean value, and result is as shown in table 8 and Fig. 8.
The impact that the different initial pH of table 8 ferments on plant lactobacillus and Pediococcus pentosaceus
| Fermentation initial p H | 7.5 | 7 | 6.5 | 6 | 5 | 4 |
| Test 1 group | 17.6 | 20.6 | 22.7 | 21.6 | 11.3 | 5.4 |
| Test 2 groups | 16.9 | 21.3 | 23.4 | 22.8 | 14.2 | 5.1 |
| Test 3 groups | 16.5 | 18.1 | 22.9 | 18.6 | 13.5 | 4.5 |
| Average fermentation bacterium number (10 8cuf/ml) | 17 | 20 | 23 | 21 | 13 | 5 |
As seen from Figure 8, the fermentation PH of plant lactobacillus and Pediococcus pentosaceus is 6.5 DEG C, and zymophyte number reaches 23 × 108cuf/ml, determines that 6.5 for best fermentation PH.PH value is lower, and suffered by plant lactobacillus, the impact of acid stress is larger, so optimal ph should control about 6.5.
In sum, the optimal conditions of fermentation of plant lactobacillus and Pediococcus pentosaceus is: temperature 35 DEG C, initial p H are 6.5, and inoculum size is 2%, and stir as 12h stirs 1 time, each churning time is 5min.
The application in culture of Penaeus vannamei of embodiment 6 plant lactobacillus and Pediococcus pentosaceus
6.1 materials and methods
6.1.1 object is cultivated
High-elevation breeding pond Penaeus vannamei
6.1.2 place is applied
80 mu of prawn high-elevation breeding pond fields, flat sea, Huidong, Guangdong
Raoping, Guangdong Province 60 mu of prawn high-elevation breeding pond fields
6.1.3 the fermentative production of plant lactobacillus and Pediococcus pentosaceus mixed bacterium
Substratum: newborn ferment (according to MRS formulated) 80kg, bacterial classification 200g (1,000,000,000/g) is that 12:1 suitably adds glucose and soy peptone by C:N.
Fermentation condition: 35 DEG C, 12h stirs 5min, and amount of fermentation is 2 tons/tank, reaches less than 4.0 for fermentation termination with PH, and to ferment 4 tons, 2 tank with this condition, fermentation termination milk-acid bacteria bacterium number is all higher than 1,000,000,000/ml.If need high density fermentation, then need exponential fed-batch carbon source, nitrogenous source, dipotassium hydrogen phosphate.
Production Flow Chart: the filtered water-heating (stirring and evenly mixing) of sterilization tank body-cleaning tank body-add to 35 DEG C-stop heating-add substratum while stirring--add bacterium powder after tune substratum PH-to be mixed is even, continuation stirring 5min-by optimal conditions setup control cabinet, when automatic fermentation-PH shows 4.0, fermentation stops-filters discharging-packing.
6.1.4 DATA REASONING and treatment process
Application, using surviving rate, feed coefficient, yield per unit and unit surface pure profit as production performance index, calculates respectively with following formula:
Surviving rate (%)=experiment terminates to live when rear shrimp sum/experiment alive starts shrimp sum × 100%;
Feed coefficient: feed usage quantity/gain in weight;
Unit surface pure profit=(gross income-feeding cost-seed takes-charges for water and electricity-bio-feritlizer expense-pool rent)/area.
6.2 experimental result
We throw in the long SIS Penaeus vannamei seed of 0.7cm, putting seedling density is 100,000 tails, and along with beginning bait throwing in (bait all uses prawn perfect compound feed) after putting seedling, the mixed bacterium mix fodder using us to ferment is thrown something and fed shrimp seedling, within one day, throw something and feed twice, each feeding volume is 5 ‰, after throwing something and feeding continuously 7 days, compares with the juvenile prawn of test control group, crust bright color, enteron aisle is obviously comparatively thick, and wriggle strong, shrimp vitality of subject is stronger.
Milk-acid bacteria mixed bacterium of throwing something and feeding we according to throwing something and feeding continuously 7 days, suspend the principle of 5 days, whole process prawn ingests normally, does not occur other bad phenomenon.
Extra large feed coefficient white shrimp test group is equalled in table 9 Huidong and control group productive capacity contrasts
Note: it is all 90,000 tails/mu that test group and control group put seedling amount, and each shrimp pool is 2 mu
Table 10 Raoping, Guangdong Province white shrimp test group and control group productive capacity contrast
Note: test group and control group are put seedling amount and be 100,000 tails/mu, each shrimp pool is 3 mu
On September 28th, 2013, receipts are carried out to the prawn of Liang Ge plant and catches, and carried out the calculating of surviving rate, output, feed coefficient mensuration and pure profit, the results are shown in Table 9 and table 10.As known from Table 9, test group and control group contrast, and surviving rate is high by 2.80%, weight in average height 4.32g/ tail, output height 180kg/ mu, and feed coefficient is low by 0.12, many 6000 yuan/mu of pure profit.As known from Table 10, test group and control group contrast, and surviving rate is high by 2.22%, weight in average height 2.38g/ tail, output height 200kg/ mu, feed coefficient is low by 0.08, many 5000 yuan/mu of pure profit.Visible, after Liang Ge plant adds plant lactobacillus and Pediococcus pentosaceus mixed bacterium in feed, the Penaeus vannamei surviving rate of cultivation, prawn weight, per mu yield output and per mu yield pure profit are all high than control group, and feed coefficient test group is all low than control group.
6.3 analyze discussion
6.3.1 milk-acid bacteria is on the impact of culture of Penaeus vannamei surviving rate
The results are shown in Table 9, the surviving rate of control group is 77.78%, and lower than the surviving rate 2.22% of test group, simultaneously because the typhoon second half year is more, around culture efficiency is all bad, and disease is a lot, and row pool rate remains high.Experiment shows, milk-acid bacteria can improve non-specific immune systems function, improves anti-stress ability, disease resistance thus improve the surviving rate of prawn.Test group prawn specification, weight are all high than control group, and per mu yield yield trials group is 1.18 times of control group, and feed coefficient test group is lower than control group by 10%, and per mu yield pure profit test group is 1.5 times of control group.Illustrate that plant lactobacillus and Pediococcus pentosaceus can improve the balance of the microorganism species regulated in gut of shrimp, promote that enteron aisle is to the absorption of nutritive substance, accelerates the prawn speed of growth, improve the utilization ratio of bait, reduce production cost.
6.3.2 milk-acid bacteria impact that white shrimp is grown
Through the observation of whole breeding process, the feed mixing carrying out milk-acid bacteria is thrown something and fed, low by 0.08 than control group of the feed coefficient of test group, describe milk-acid bacteria and be colonizated in the digestive function that enteron aisle inner membrance can improve enteron aisle, improve the specific absorption of bait, also can provide nutrition simultaneously, reduce the feeding volume of feed, the anti-stress ability of white shrimp can also be improved simultaneously.
Milk-acid bacteria is along with residual bait discharge and can also purify water with aquaculture water, alleviates the pollution pressure of cultivation later stage water body.
From table display, use milk-acid bacteria feed of throwing something and feeding to substantially increase culture benefit, reject the antibiotic cultural method of tradition use, for aquaculture Sustainable development provides strong experimental evidence.
This project construction 4 tons of plant lactobacilluss and Pediococcus pentosaceus mixing fishing ocean probiotics, and be applied in more than 140 mu of cultured area, one is use in Huidong, Guangdong Ping Hai80Mu plant, another uses in Raoping, Guangdong Province 60 mu of prawn high-elevation breeding pond fields, result is used to show the organic contamination that this product can reduce water body, purifying water body, also can suppress, kill sex pheromone, and can be used as foodstuff additive, supplement the nutrients, improve the GI profitable strain of cultivated animals, reach the object of bionomic control.Improve the food ration of aquatic animal simultaneously, accelerate its growth, Shortening culturing period, thus create larger economic benefit.The use of probiotics, instead of conventional antibiotic, thus reduces the malignant bacteria resistance increase because antibiotic excessive use causes, and the breeding environment eubiosis is destroyed, and enters human body, jeopardize human health etc. through food chain.
All distortion that those of ordinary skill in the art can directly derive from the disclosure of invention or associate, all should think protection scope of the present invention.
Claims (3)
1. a fishing ocean probiotics, is characterized in that: comprise plant lactobacillus and Pediococcus pentosaceus, and the bacterium number of described plant lactobacillus and described Pediococcus pentosaceus is than being 2:1.
2. a preparation method for fishing ocean as claimed in claim 1 probiotics, is characterized in that: comprise the following steps:
The activation of step one, bacterial classification and enlarged culturing:
One-level is cultivated: in Bechtop, get inclined-plane preserve single bacterium colony in 100ml sterilizing MRS substratum, be placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h; Plant lactobacillus and Pediococcus pentosaceus respectively inoculate 4 bottles, and object is activated spawn; Get bacterium liquid coating MRS flat board after 24h multiple, be placed in quiescent culture 24h in 35 DEG C of thermostat containers, treat that bacterium colony grows, be placed in 4 DEG C of refrigerator conservations, stand-by;
Secondary is cultivated: in Bechtop, get inclined-plane preserve single colony inoculation in 200ml sterilizing MRS, and two kinds of each inoculations 6 bottles altogether of bacterium, are placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h;
Third stage culture: get in cultured bacterium liquid 10ml to 2000ml sterilizing MRS substratum, be placed on 35 DEG C of water bath with thermostatic control shaking tables and cultivate 24h, obtain seed liquor, adopt boulton process to produce plant lactobacillus 2kg, Pediococcus pentosaceus 30kg, bacterium number all reaches hundred million grades;
The preparation of step 2, fermention medium:
Peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, dipotassium hydrogen phosphate 2g, sodium acetate 2g, disodium citrate 2g, bitter salt 0.5g, Manganese sulfate pentahydrate 0.2g, tween-80 1ml, is settled to 1L;
Step 3, fermentation culture:
The mixed bacterium of described plant lactobacillus and described Pediococcus pentosaceus 2:1 is by weight ratio inoculated in described fermention medium, inoculum size is 2%, be that 12:1 adds glucose and soy peptone by C:N in described fermention medium, leavening temperature 35 DEG C, initial p H are 6.5, stir as 12h stirs 1 time, each churning time is 5min, reaches less than 4.0 for fermentation termination with PH;
Step 4, filtration discharging:
After fermentation to terminal, filtration discharging is carried out to fermented liquid, then carries out packing, obtained this fishing ocean probiotics.
3. the preparation method of a kind of fishing ocean as claimed in claim 2 probiotics, is characterized in that: the bacterium number of described mixed bacterium is 1,000,000,000/g.
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