CN102787087A - Lactobacillus casei and application of lactobacillus casei to feed - Google Patents
Lactobacillus casei and application of lactobacillus casei to feed Download PDFInfo
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- CN102787087A CN102787087A CN2012102774544A CN201210277454A CN102787087A CN 102787087 A CN102787087 A CN 102787087A CN 2012102774544 A CN2012102774544 A CN 2012102774544A CN 201210277454 A CN201210277454 A CN 201210277454A CN 102787087 A CN102787087 A CN 102787087A
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Abstract
The invention discloses a strain of lactobacillus casei and application of the lactobacillus casei to feed. The Llactobacillus casei L-1 is obtained through screening and separation from soil, the preservation number is CCTCC (China Center for Type Culture Collection) M2012150. The strain can be fermented to produce phospholipase A2, the enzyme yield is high, the strain is applied to the feed field, oil crops containing plant phospholipid or processing byproducts of oil crops are used as fermentation substrates, ammonium sulfate, sodium chloride and corn starch are used as energy supply bodies, the strain is used as a fermentation strain, and feed additives are prepared through solid fermentation. The plant phospholipid conversion rate of the feed additives is high, good antioxidation and antibacterial capability is realized, and no toxic or side effect is caused on animals. Feeding experiments show that when the feed additives are added into a pig feed premix compound, the feed intake and the daily weight increase of pigs can be improved, the feed-weight ratio is reduced, and a growth promotion effect is realized.
Description
Technical field
The invention belongs to biofermentation technique and field of fodder, be specifically related to a strain and produce the lactobacterium casei of Phosphoric acid esterase A2 and the application in feed thereof.
Background technology
In recent years; Probiotics is at home and abroad used in the fodder industry and is on the increase, and utilizes the green feed additive of nontoxic, the nonreactive noresidue of probiotic microorganism fermentative prodn, has the digestibility that can improve feed; Improve animal immunizing power, prolong functions such as shelf-lives.As one type of main probiotic bacterium; Probiotic lactobacillus possesses a series of good probiotic properties because of it, such as: acid resistance, tolerance bile acide, in digestive tube, decide reproductive growth and alleviate not anti-disease, the different sudden change of resistance, reduction serum cholesterol, antitumor, suppress enteric pathogenic bacteria, improve intestinal microflora and enhance immunity function etc. and be widely used in the fields such as food fermentation, animal-feed and health care.
Plant phosphatide is one type of fatty cpd that contains the phosphatide element, and mainly being present in soybean, Semen Brassicae campestris, peanut, sunflower seeds etc. has in the oil crops.Phospholipase A2 (Phospholipase A2; PLA2), promptly phosphatide-2-Acyl-hydrolase (EC 3.1.1.4) is the enzyme that plays an important role in the plant phosphatide catabolic process; The hydrolysis reaction of its catalyzing glycerol phosphatide C-2 position ester bond generates lysophospholipid and lipid acid.Lysophospholipid is a kind of good emulsifying agent, emulsifying dispersant, foodstuff additive and antiseptic-germicide, and its effect is superior to common phosphatide, has been natural additive for foodstuff by multinational approvals such as China, Japan.In fodder industry, through with the plant in the feedstuff raw material phospholipid modified be lysophospholipid, can make it have good oxidation-resistance and broad-spectrum antimicrobial ability, also can improve the carrotenoid particularly specific absorption and the bioavailability of zeaxanthin.Patent CN96190502 has reported through in feed, adding exogenous Phospholipase A2; Chicken feed transformation efficiency and non-digestibility of ruminating calf have significantly been improved to fat; In addition, in the foodstuff of fish and Crustacean, add Phospholipase A2 and also can improve feed conversion rate.This patent is further pointed out, in animal-feed, adds Phospholipase A2 and can reduce the incorporation of expensive batching such as VITAMINs or tinting material in feed.
At present, prior art is in feed, to add streptomyces species, produces Phospholipase A2 through fermentation, thereby reaches the purpose of lysophospholipid in the supplement feed.But; Because strain properties; There are some shortcomings in streptomycete in the feed fermentation process, slow like strain growth, the fermentation viable count is few, yield of enzyme is not high, plant in different strains toxicity proterties differ greatly etc., directly influence the transformation efficiency of plant phosphatide in the fermenting process; Cause feed conversion rate not high, the promotes growth DeGrain.
Summary of the invention
Above-mentioned defective to the prior art existence; The purpose of this invention is to provide a strain lactobacterium casei; Said lactobacterium casei can ferment and produce Phospholipase A2, and yield of enzyme is high, and another object of the present invention provides the application of a kind of said lactobacterium casei in feed; As fermentation strain, prepare fodder additives with this bacterial strain through solid state fermentation.
Technical scheme of the present invention is following:
One strain lactobacterium casei (
Lactobacillus casei) L-1, deposit number is: CCTCC No.M2012150.
The present invention also provides the application of a kind of said lactobacterium casei in feed; Be to utilize said lactobacterium casei on solid-state fermentation culture medium, to carry out solid state fermentation; Fermentation finishes, and carries out air-dryly in 30～40 ℃, makes fodder additives; Said solid-state fermentation culture medium is to be matrix with the oil crops that contain phosphatide or its processed side product, and adds ammonium sulfate, sodium-chlor, W-Gum and water.
Its further technical scheme is:
Said oil crops comprise one or more in soybean, Semen Brassicae campestris, peanut, the sunflower seeds.
Said processed side product comprises one or more in soybean cake powder, rapeseed meal, peanut meal, the sunflower seed dregs.
The grinding particle size of said matrix is 20～80 orders.
Said solid-state fermentation culture medium added ingredients is counted with the weight percentage that accounts for said matrix: W-Gum 1～8%, ammonium sulfate 0.1～1.0%, sodium-chlor 0.5～0.8%.
The add-on of said water accounts for 55～70% of said solid-state fermentation culture medium gross weight.
Said solid state fermentation conditions is: said lactobacterium casei inoculum size accounts for 1～5% of said matrix gross weight, and the fermentation original ph is 6.0～7.0, and fermenting process article temperature is 25～40 ℃, and fermentation time is 24～36h.
The method of use of above-mentioned made fodder additives is: this additive is no more than 10% ratio in its weight percent directly adds in the animal feed premix material.
Said lactobacterium casei (Lactobacillus casei) L-1 obtains through the following steps screening and separating:
From gathering 28 parts of different soils samples near the pork pig plant of Yancheng, Jiangsu, Nantong, Suzhou, Wuxi four ground different scales, respectively take by weighing 2g, add respectively in the 98mL SPSS; In 37 ℃ of 20min that in shaking table, vibrate, shaking speed 120rpm draws the 1mL supernatant and adds in the LB liquid nutrient medium; In 37 ℃ of shaking culture 6h, shaking speed 120rpm is with 10000 times of gained pregnant solution dilutions; With the spreading rod separate application to LB solid medium (contain weight percent be 20% yolk liquid); In thermostat container, behind the cultivation 48h, obtain the bacterial strain that 13 strains are grown better and can be produced transparent circle in 37 ℃, the size of logical more transparent loop diameter and colony diameter ratio is analyzed the product Phospholipase A2 ability of this 13 strain bacterial strain; Obtain the bacterial strain of 3 strain enzymatic productivities higher (being that transparent circle diameter and colony diameter ratio are bigger); Numbering 01,02,03, picking list bacterium colony carries out enlarged culturing with LB liquid nutrient medium (containing the glucose that mass concentration is 20g/L) respectively, and compares according to the product Phospholipase A2 ability of above-mentioned analytical procedure to this three strains bacterial strain; Final definite No. 02 bacterial strain is the optimum bacterial strain of yield of enzyme, the called after lactobacterium casei (
Lactobacillus casei) L-1.
Lactobacterium casei provided by the present invention (
Lactobacillus casei) L-1, be preserved in Chinese typical culture collection center (CCTCC) on April 27th, 2012, deposit number: CCTCC M 2012150, address: China, Wuhan, Wuhan University.This bacterial strain is hereinafter to be referred as lactobacterium casei L-1 CCTCC M 2012150.
Said lactobacterium casei L-1 CCTCC M 2012150 has following microbial characteristic:
1. morphological specificity
Lactobacterium casei L-1 CCTCC M 2012150 anaerobism on MRS solid differential medium is cultivated 48h, and the bacterium colony of formation is white in color, and colony diameter is 1～3mm, rounded protuberance, surperficial little rough; Use observation by light microscope, gramstaining is positive, and rod-short does not produce gemma, and atrichia does not move.
Above-mentioned MRS solid differential medium composition is counted by grams per liter: glucose 20g/L, peptone 10 g/L, yeast powder 5 g/L, Carnis Bovis seu Bubali cream 10 g/L; Anhydrous sodium acetate 5 g/L, diammonium hydrogen citrate 2 g/L, potassium hydrogenphosphate 2 g/L; MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.58 g/L, manganese sulfate monohydrate 0.25 g/L, tween 80 1 g/L; Agar powder 15 g/L add water to 1L, regulate pH value to 6.4～6.6.
2. physiological and biochemical property
The facultative heterofermentation lactose of lactobacterium casei L-1 CCTCC M 2012150 abilities; Can utilize glucose, lactose, fructose, semi-lactosi, cellobiose, SANMALT-S, seminose, melizitose, ribose, sucrose, trehalose, sorbyl alcohol, N.F,USP MANNITOL preferably; Can slowly utilize starch; Can not utilize melibiose, cottonseed sugar, rhamnosyl, wood sugar, pectinose, H
2S test, indole test, gelatin liquification test, nitrate reduction test are all negative, and the bacterial strain optimum growth temperature is 37 ℃.
In conjunction with above-mentioned morphological specificity and physiological and biochemical property, can identify bacterial strain lactobacterium casei L-1 CCTCC M of the present invention 2012150 for lactobacterium casei (
Lactobacillus casei).
Useful technique effect of the present invention is:
The present invention relates to a strain lactobacterium casei L-1 CCTCC M 2012150, said lactobacterium casei can ferment and produce Phospholipase A2, and the fermentation viable count is many, and yield of enzyme is high; Said lactobacterium casei is applied to field of fodder, is fermentation substrate with oil crops or its processed side product that contains plant phosphatide, with ammonium sulfate, sodium-chlor, W-Gum as energy donor; With this bacterial strain as fermentation strain; Prepare fodder additives through solid state fermentation, plant phosphatide transformation efficiency high (reaching as high as 63.6%) makes it possess good oxidation-resistance and antibacterial ability in the said fodder additives; Again because originally as probiotic bacterium, there is not toxic side effect in fermentation strain to animal; Prove through feeding experiment, said fodder additives is added in the pig feed Preblend, can improve pig food consumption and day weight gain only, reduce the material anharmonic ratio, have growth promoting function.
Embodiment
Do further description below in conjunction with the embodiment specific embodiments of the invention, following examples are convenient to understand better the present invention, but do not limit the present invention.
Used lactobacterium casei seed liquor adopts following method to cultivate in following examples: the lactobacterium casei L-1 CCTCC M 2012150 that the inclined-plane is preserved or glycerine is preserved is inoculated in the liquid seed culture medium, leaves standstill in 37 ℃ and cultivate 12h.The liquid seed culture medium composition is counted by grams per liter: glucose 20 g, yeast powder 10 g, peptone 10 g, potassium hydrogenphosphate 2 g, diammonium hydrogen citrate 2 g, tween 80 1 g, sodium acetate 5 g, MgSO
40.25 g, MnSO
4H
2O 0.05 g, all the other compositions are water, regulate pH value to 7.0, in 121 ℃ of autoclaving 20min.
Embodiment 1
Take by weighing soybean cake powder 5kg, rapeseed meal 1kg, sunflower seeds 0.1kg is crushed to 20 orders with kibbler with it; Be mixed with matrix, add water 10.9kg, when adding water, add W-Gum 0.061kg, ammonium sulfate 0.0061kg; Sodium-chlor 0.037kg stirs, and is mixed with solid-state fermentation culture medium; The solid-state fermenter of packing into is in 100 ℃ of boiling 30min, with subsequent use after the aseptic chilled air cools; Get above-mentioned lactobacterium casei seed liquor to account for the inoculum size of said matrix gross weight 1%; Be inoculated in the above-mentioned solid-state fermentation culture medium, solid state fermentation conditions is following: the fermentation original ph is 6.0, and fermenting process article temperature is 25 ℃; Fermentation time is 24h; Fermentation ends is carried out air-dryly in air dryer in 30 ℃, make fodder additives.With this additive pack sealing, be kept at ventilation, cool place, dry place.Adopt MRS solid medium pour plate counting process counting lactobacterium casei quantity during the fermentation, lactobacterium casei quantity reaches 9.1 * 10
9Cfu/g; Measure the transformation efficiency of phosphatide in the made fodder additives according to the said detection method of CNCA 2009B550 " phosphatase assay colourimetry in the import and export food ", the phosphatide transformation efficiency is 50.5%.
Embodiment 2
Take by weighing soybean cake powder 5kg, rapeseed meal 2kg is crushed to 30 orders with kibbler with it, is mixed with matrix; Add water 9kg, when adding water, add W-Gum 0.21kg, ammonium sulfate 0.035kg; Sodium-chlor 0.049kg stirs, and is mixed with solid-state fermentation culture medium; The solid-state fermenter of packing into is in 100 ℃ of boiling 30min, with subsequent use after the aseptic chilled air cools; Get above-mentioned lactobacterium casei seed liquor to account for the inoculum size of said matrix gross weight 2%; Be inoculated in the solid-state fermentation culture medium, solid state fermentation conditions is following: the fermentation original ph is 6.0, and fermenting process article temperature is 30 ℃; Fermentation time is 36h; Fermentation ends is carried out air-dryly in air dryer in 40 ℃, make fodder additives.With this additive pack sealing, be kept at ventilation, cool place, dry place.Adopt MRS solid medium pour plate counting process counting lactobacterium casei quantity during the fermentation, lactobacterium casei quantity reaches 1.2 * 10
10Cfu/g; Measure the transformation efficiency of phosphatide in the made fodder additives according to the said detection method of CNCA 2009B550 " phosphatase assay colourimetry in the import and export food ", the phosphatide transformation efficiency is 63.6%.
Embodiment 3
Take by weighing soybean cake powder 1000kg, rapeseed meal 200kg, sunflower seed dregs 30kg; With kibbler it is crushed to 40 orders, is mixed with matrix, add water 1797kg; When adding water, add W-Gum 61.5kg, ammonium sulfate 3.69kg, sodium-chlor 6.15kg; Stir, be mixed with solid-state fermentation culture medium, insert the cement fermentation vat; Get above-mentioned lactobacterium casei seed liquor to account for the inoculum size of said matrix gross weight 3%; Be inoculated in the solid-state fermentation culture medium, solid state fermentation conditions is following: the fermentation original ph is 6.5, and fermenting process article temperature is 35 ℃; Fermentation time is 24h; Fermentation ends is carried out air-dryly in air dryer in 30 ℃, make fodder additives.With this additive pack sealing, be kept at ventilation, cool place, dry place.Adopt MRS solid medium pour plate counting process counting lactobacterium casei quantity during the fermentation, lactobacterium casei quantity reaches 1.3 * 10
10Cfu/g; Measure the transformation efficiency of phosphatide in the made fodder additives according to the said detection method of CNCA 2009B550 " phosphatase assay colourimetry in the import and export food ", the phosphatide transformation efficiency is 61.8%.
Embodiment 4
Take by weighing soybean cake powder 1000kg, rapeseed meal 200kg, sunflower seed dregs 30kg; With kibbler it is crushed to 70 orders, is mixed with matrix, add water 3037kg; When adding water, add W-Gum 73.8kg, ammonium sulfate 9.84kg, sodium-chlor 6.15kg; Stir, be mixed with solid-state fermentation culture medium, insert the cement fermentation vat; Get above-mentioned lactobacterium casei seed liquor to account for the inoculum size of said matrix gross weight 4%; Be inoculated in the solid-state fermentation culture medium, solid state fermentation conditions is following: the fermentation original ph is 7.0, and fermenting process article temperature is 30 ℃; Fermentation time is 36h; Fermentation ends is carried out air-dryly in air dryer in 40 ℃, make fodder additives.With this additive pack sealing, be kept at ventilation, cool place, dry place.Adopt MRS solid medium pour plate counting process counting lactobacterium casei quantity during the fermentation, lactobacterium casei quantity reaches 7.6 * 10
9Cfu/g; Measure the transformation efficiency of phosphatide in the made fodder additives according to the said detection method of CNCA 2009B550 " phosphatase assay colourimetry in the import and export food ", the phosphatide transformation efficiency is 56%.
Embodiment 5
Take by weighing soybean cake powder 100kg, rapeseed meal 20kg, peanut meal 2kg; With kibbler it is crushed to 80 orders, is mixed with matrix, add water 211kg; When adding water, add W-Gum 9.76kg, ammonium sulfate 1.22kg, sodium-chlor 0.976kg; Stir, be mixed with solid-state fermentation culture medium, insert the cement fermentation vat; Get above-mentioned lactobacterium casei seed liquor to account for the inoculum size of said matrix gross weight 5%, be inoculated in the solid-state fermentation culture medium, solid state fermentation conditions is following: the fermentation initial water is divided into 70%; The fermentation original ph is 7.0; Fermenting process article temperature is 40 ℃, and fermentation time is 36h, fermentation ends; In air dryer, carry out air-dryly in 40 ℃, make fodder additives.With this additive pack sealing, be kept at ventilation, cool place, dry place.Adopt MRS solid medium pour plate counting process counting lactobacterium casei quantity during the fermentation, lactobacterium casei quantity reaches 5.1 * 10
9Cfu/g; Measure the transformation efficiency of phosphatide in the made fodder additives according to the said detection method of CNCA 2009B550 " phosphatase assay colourimetry in the import and export food ", the phosphatide transformation efficiency is 52.9%.
Through the contrast feeding experiment effect of the present invention is described below:
The one-factor experiment design is adopted in this test, with 120 body weight be 25kg (± 0.5kg) pork pig is divided into two groups at random, a test group, a control group, each 60 pig is divided into 6 repeating groups respectively, 10 pigs of each repeating groups.With embodiment 5 made additives by weight percentage 8% ratio add (wherein the contained component of 92% pig premix is that commercially available Z824 type pig is used Preblend) in the pig premix to; Again with this pig premix by weight percentage 4% ratio add (wherein the weight percent of 96% the contained component of pig complete feed is: corn 55%, dregs of beans 23%, wheat bran 17.5%, fish meal 0.5%) in the pig complete feed to, be used for feeding experiment group pork pig; With commercially available Z824 type pig with Preblend by weight percentage 4% ratio directly add (wherein the weight percent of 96% contained each component of pig complete feed as stated) in the pig complete feed to, the control group pork pig is used to feed.Fed continuously 42 days.Feed intake, add feedings and managements such as water, dosing, epidemic prevention by the normal breeding way in pig farm, measure average daily food consumption, day weight gain and material anharmonic ratio, test-results is seen table 1.
Table 1
Can be known that by table 1 data the average daily food consumption of test group is higher than control group with the weight average that increases day by day, test group material anharmonic ratio reduces; This presentation of results; With bacterial strain lactobacterium casei L-1 CCTCC M 2012150 of the present invention as fermentation strain; Preparing fodder additives according to process for solid state fermentation according to the invention also will be applied in the pig breeding production; Can improve pig average daily food consumption and day weight gain only, reduce the material anharmonic ratio, have growth promoting function.
Embodiment 1～5 relates to each raw material and is the commercial goods, relates to each equipment and is this area conventional equipment.
Claims (9)
- One strain lactobacterium casei ( Lactobacillus casei) L-1, deposit number is: CCTCC No.M2012150.
- 2. the application of the said lactobacterium casei of claim 1 in feed; It is characterized in that: on solid-state fermentation culture medium, carry out solid state fermentation with said lactobacterium casei; Fermentation finishes, and carries out air-dryly in 30～40 ℃, makes fodder additives; Said solid-state fermentation culture medium is to be matrix with the oil crops that contain phosphatide or its processed side product, and adds ammonium sulfate, sodium-chlor, W-Gum and water.
- 3. according to the application of the said lactobacterium casei of claim 2 in feed, it is characterized in that: said oil crops comprise one or more in soybean, Semen Brassicae campestris, peanut, the sunflower seeds.
- 4. according to the application of the said lactobacterium casei of claim 2 in feed, it is characterized in that: said processed side product comprises one or more in soybean cake powder, rapeseed meal, peanut meal, the sunflower seed dregs.
- 5. according to the application of the said lactobacterium casei of claim 2 in feed, it is characterized in that: the grinding particle size of said matrix is 20～80 orders.
- 6. according to the application of the said lactobacterium casei of claim 2 in feed, it is characterized in that: said solid-state fermentation culture medium added ingredients is counted with the weight percentage that accounts for said matrix: W-Gum 1～8%, ammonium sulfate 0.1～1.0%, sodium-chlor 0.5～0.8%.
- 7. according to the application of the said lactobacterium casei of claim 2 in feed, it is characterized in that: the add-on of said water accounts for 55～70% of said solid-state fermentation culture medium gross weight.
- 8. according to the application of the said lactobacterium casei of claim 2 in feed; It is characterized in that said solid state fermentation conditions is: said lactobacterium casei inoculum size accounts for 1～5% of said matrix gross weight; The fermentation original ph is 6.0～7.0; Fermenting process article temperature is 25～40 ℃, and fermentation time is 24～36h.
- 9. according to the application of the said lactobacterium casei of claim 2 in feed, it is characterized in that the method for use of made fodder additives is: this additive is no more than 10% ratio in its weight percent directly adds in the animal feed premix material.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103070287A (en) * | 2013-02-26 | 2013-05-01 | 湖南省微生物研究所 | Lactobacillus paracasei fermented feed and preparation method thereof, fermenting equipment and application of lactobacillus paracasei fermented feed |
| CN103305437A (en) * | 2013-05-17 | 2013-09-18 | 南京工业大学 | L-ammonium lactate tolerant bacterium and application thereof |
| CN104651391A (en) * | 2015-02-13 | 2015-05-27 | 江南大学 | Recombinant expression and application of lactobacillus casei phospholipase A2 gene |
| CN105661030A (en) * | 2016-01-25 | 2016-06-15 | 刘景阳 | Method for producing fodder from edible fungus residues by means of mixed fermentation by aid of microorganisms and enzyme preparations |
| CN106497828A (en) * | 2016-10-14 | 2017-03-15 | 北京市农林科学院 | A kind of application of lactobacillus casei and its lyophilized formulations in pig feed |
| CN108018248A (en) * | 2018-01-17 | 2018-05-11 | 江南大学 | It is a kind of that there is the Lactobacillus casei for adjusting Bacterial community disorder caused by antibiotic |
| CN111938036A (en) * | 2020-09-14 | 2020-11-17 | 庞勤 | Biological fermentation feed additive for nursery pigs and preparation and use methods thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103070287A (en) * | 2013-02-26 | 2013-05-01 | 湖南省微生物研究所 | Lactobacillus paracasei fermented feed and preparation method thereof, fermenting equipment and application of lactobacillus paracasei fermented feed |
| CN103305437A (en) * | 2013-05-17 | 2013-09-18 | 南京工业大学 | L-ammonium lactate tolerant bacterium and application thereof |
| CN103305437B (en) * | 2013-05-17 | 2014-10-29 | 南京工业大学 | L-ammonium lactate tolerant bacterium and application thereof |
| CN104651391A (en) * | 2015-02-13 | 2015-05-27 | 江南大学 | Recombinant expression and application of lactobacillus casei phospholipase A2 gene |
| CN105661030A (en) * | 2016-01-25 | 2016-06-15 | 刘景阳 | Method for producing fodder from edible fungus residues by means of mixed fermentation by aid of microorganisms and enzyme preparations |
| CN106497828A (en) * | 2016-10-14 | 2017-03-15 | 北京市农林科学院 | A kind of application of lactobacillus casei and its lyophilized formulations in pig feed |
| CN106497828B (en) * | 2016-10-14 | 2019-06-07 | 北京市农林科学院 | A kind of application of Lactobacillus casei and its lyophilized preparation in pannage |
| CN108018248A (en) * | 2018-01-17 | 2018-05-11 | 江南大学 | It is a kind of that there is the Lactobacillus casei for adjusting Bacterial community disorder caused by antibiotic |
| CN111938036A (en) * | 2020-09-14 | 2020-11-17 | 庞勤 | Biological fermentation feed additive for nursery pigs and preparation and use methods thereof |
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| CN102787087B (en) | 2013-10-16 |
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