CN101020895B - Plant lactobacillus fermenting process for preparing ensilage with rich conjugated linoleic acid - Google Patents
Plant lactobacillus fermenting process for preparing ensilage with rich conjugated linoleic acid Download PDFInfo
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- CN101020895B CN101020895B CN2007100196553A CN200710019655A CN101020895B CN 101020895 B CN101020895 B CN 101020895B CN 2007100196553 A CN2007100196553 A CN 2007100196553A CN 200710019655 A CN200710019655 A CN 200710019655A CN 101020895 B CN101020895 B CN 101020895B
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Abstract
The invention relates to a method for preparing a silage riching in conjugated linoleic acid by fermenting plant lactobacillus, and belongs to the technical field of microbe and animal nutrition and feed. The strain Lactobacillus plantarum ANCLA01 of the invention was preserved in China Gerenal Microbiological Culture Collector Center on January 8th 2007 with the preservation number of CGMCC No. 1906. The plant lactobacillus ANCLA01 is prepared through separating from corn silage, screening, purifying and culturing, named the Lactobacillus plantarum ANCLA01. The plant lactobacillus ANCLA01 is applied to the silage, which can improve the conjugated linoleic acid content in the silage and produce functional silage with rich conjugated linoleic acid.
Description
Technical field
The invention belongs to microorganism and animal nutrition and feed technical field, be specifically related to the new bacterial strain of a kind of high yield conjugated linolic acid (CLA, Conjugated linoleic acid) and utilize its fermentative production to be rich in the functional silage of CLA.
Background technology
Anticancer, functions such as promotion is grown, inhibition accumulation of fat, enhancing body immunological competence that conjugated linolic acid (CLA) has can improve pig, the fowl speed of growth and lean ratio, are the research focuses of subjects such as current medicine, protective foods and Animal nutrition.U.S. NRC classifies CLA as unique animal source lipid acid with antitumous effect.There are 4 kinds of isomer at least in CLA, and " Cis-9, trans-11-linolic acid " is main among the CLA, the active a kind of isomer of tool.CLA in ruminating animal such as milk and beef, the mutton food more than 90% is " Cis-9, a trans-11-linolic acid ", is human topmost CLA natural origin.Therefore, the milk that CLA content is high is called as functional milk, and beef, mutton that CLA content is high are called as functional foodstuff.At present, people improve CLA content in milk from ruminants, the meat by two kinds of diet nutrient regulatory pathways: the one, and daily ration adds grease or changes daily ration adipose-derived, with the linoleic content of CLA metabolism substrate in the increase cud, and then improve CLA content in the livestock product; The 2nd, directly add CLA or ruminally-protected CLA (promptly crossing cud CLA).But because CLA costs an arm and a leg, it is too high to add cost in the feed, uses few in the production practice.Therefore, the animal nutrition and feed scholar is seeking other suitable, cost effective method that improves CLA content in the livestock product.
Summary of the invention
Technical problem to be solved by this invention is: the deficiency that is intended to overcome above-mentioned direct interpolation conjugated linolic acid and changes the daily ration lipid conformation, provide a kind of can the high yield conjugated linolic acid the new bacterial strain of plant lactobacillus and utilize its fermentative preparation to be rich in the method for conjugated linolic acid silage, to improve the content of conjugated linolic acid in the ruminant.
The new bacterial strain of plant lactobacillus of the present invention system is from separation from corn silage, screening, purifying and cultivate called after plant lactobacillus ANCLA01 (Lactobacillus plantarum ANCLA01).
The method that this plant lactobacillus fermentative preparation is rich in the conjugated linolic acid silage is: after earlier the strains A NCLA01 of this high yield conjugated linolic acid being spread cultivation in batches, again tieback makes the linolic acid fermentation in the Vegetable oil lipoprotein be converted into conjugated linolic acid in ensilings such as corn, Radix Dauci Sativae, clover and beet again.
Description of drawings
Fig. 1 is the photo that plant lactobacillus ANCLA01 of the present invention takes at microscopically;
Fig. 2 is a plant lactobacillus ANCLA01 bacterial strain pcr amplification band photo of the present invention;
Fig. 3 is the ability of different strains bio-transformation conjugated linolic acid;
Fig. 4 is the utilization of plant lactobacillus ANCLA01 to carbohydrate in the fermentation substrate.
Plant lactobacillus ANCLA01 of the present invention (Lactobacillus plantarum ANCLA01) has been carried out in accordance with regulations preservation: depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center abbreviates CGMCC as; Address: China. Beijing. Zhong Guan-cun, Institute of Microorganism, Academia Sinica; Preservation date: on January 8th, 2007; The numbering CGMCCNo.1906 that preservation is registered on the books.
Embodiment:
1. materials and methods
1.1 material: corn silage is collected in Hefei City white Supreme Being's dairy industry Development Co., Ltd; Substratum: pH6.5-6.8, the MRS broth culture, the PY substratum, MRS solid medium, MRS semisolid medium, MRS liquid nutrient medium and benefit are added 0.1g (100ml)
-1The MRS liquid nutrient medium of LA.Substratum is with the preceding 20min that all sterilizes under 121 ℃ of high pressure steam.
1.2 main agents: LA and CLA: purity 〉=99%, available from Sigma company; Bacterial genomes DNA extraction test kit, PCR test kit and glue reclaim test kit, available from precious biotechnology (Dalian) company limited; Other reagent is analytical pure.
1.3 bacterial screening:
1.3.1 primary dcreening operation: gather choose immediately after the ensiling Oranoleptic indicator preferably the ensiling branches and leaves immerse in 37 ℃ of distilled waters, remove branches and leaves after ten minutes, draw fresh even soak solution 10mL and be added to 500ml and be equipped with in the Erlenmeyer flask of 300mL MRS broth culture.37 ℃ static increase bacterium 96h after, dilution spread plate method is toppled over the MRS agar plate, cultivates 48h in 37 ℃ of anaerobism behind four rides, picking list bacterium colony percutaneous puncture-inoculation is cultivated rear defences for 37 ℃ and is preserved in refrigerator in the MRS semisolid medium.
1.3.2 multiple sieve: primary dcreening operation is preserved bacterial classification after 37 ℃ of activation, and the inoculum size by 5% is inoculated in the band nut test tube (d18mmx160mm) that 10mL MRS liquid nutrient medium is housed, and wherein linoleic acid content is 0.1g (100mL) in the MRS liquid nutrient medium
-1N
2Drive away air, measure CLA behind the anaerobically fermenting 24h, produce the CLA bacterium with screening.
1.4CLA measure: with the normal hexane is solvent, and the CLA standard specimen is made into the solution of different concns, is reference with the hexane, in 233nm place its absorption value of mensuration, is X-coordinate with CLA concentration (ug/mL), and light absorption value is an ordinate zou, the drawing standard curve.(1998) method operations such as Jiang are pressed in the extraction of CLA in the fermented liquid.Afterwards, institute is obtained fatty acid extract 5mL n-hexane dissolution, with the fermentation broth extract of not inoculating is reference, with ultraviolet spectrophotometer 00-350nm scope interscan again, read the absorption value at charateristic avsorption band 233nm place, calculate the content (Pariza etc., 1999) of CLA in the fermented liquid according to typical curve.Reference: substratum adds and the linolic acid of sample with amount, does not connect bacterial classification, the same sample of other step.
1.5 strain identification
1.5.1 bacterium colony and morphologic observation: after the superior strain activation that obtains, be inoculated on the MRS plate culture medium by four district's collimation methods, anaerobism is observed colony characteristics after cultivating 24h.Observe strain morphology feature, length range and width range etc. down in opticmicroscope behind the gramstaining.
1.5.2 Physiology and biochemistry is identified: with reference to " uncle's outstanding Bacteria Identification handbook the 9th edition (1994) is carried out experiments such as catalase reaction, gelatin liquification test, hydrogen sulfide reaction, indole reaction, temperature growth to the conjugated linolic acid superior strain that obtains and carried out preliminary evaluation.Produce evaluations of classifying of physiology such as acid, aerogenesis experiment and seminose, raffinose and biochemical aspect from glucose, experiment is with reference to " the 9th edition (1994) milk-acid bacteria classification evaluation of the outstanding Bacteria Identification handbook of uncle and experimental technique carry out concrete operations.
1.5.3 molecular biology identification: segmental recovery of purpose and purifying are all operated by the test kit description of step behind the pcr amplification of the preparation of template, bacterial strain 16SrDNA and the agarose gel electrophoresis.The purpose fragment that is recovered to is delivered to by precious biotechnology (Dalian) company limited then and checked order.With sequencing result use landed bacterium among BLAST software and the GenebanK the 16SrRNA sequence relatively, find and land of poor qualityly with the highest the landing the bacterial strain sequence number and access it of its similarity, carry out diversity ratio.
2. result
2.1 the screening of bacterial classification: when removing the cultivation of air conditions bottom fermentation, isolate 17 strains altogether and generate the stronger bacterial strain of CLA ability.By table 1 result as can be known, isolated bacterial strain CLA growing amount is between 4.316-33.442ug/mL (wet basis), and wherein No. 12 bacterial strain CLA output are up to 33.442ug/mL.This bacterial strain is named as ANCLA01.
2.2 strain identification:
2.2.1 the bacterium colony of bacterial classification and morphologic observation
Observe after the ANCLA01 bacterial strain MRS solid culture and find that bacterium colony projection, circle, smooth surface also are white in color; As shown in Figure 1, oily mirror is observed down and is found that the ANCLA01 bacterial strain is Gram-positive behind the gramstaining, short straight, the blunt circle in two ends of thalline, and no gemma, thalline is wide in 0.9～1.1um scope, and is long in 2～7um scope, becomes monomer, formation or short chain shape between thalline.Therefore, the ANCLA01 colonial morphology is with " the plant lactobacillus morphological specificity described in the outstanding Bacteria Identification handbook of uncle is more consistent.
2.2.2 Physiology and biochemistry check
Sugar fermentating test shows that ANCLA01 bacterial strain glucose fermentation result does not have gas and produces; Growth temperature detects finds that bacterial strain can be bred growth for 15 ℃, does not grow substantially for 45 ℃; Lactic acid generates and detects the test discovery, has lactic acid to generate in the fermented liquid.Take into account the above-mentioned bacterial strains morphological specificity, show this Pseudomonas homofermentation Type B lactobacillus.
2.2.2 molecular biology identification
The bacterial strain diversity ratio is found, 1482 base sequences identical (sequence has been uploaded GenBank, the number of landing EF185922) after 1482 base sequences that the 5th base of ANCLA01 bacterial strain 16S rRNA sequence is later and the 26th base of Lactobacillus plantarum strain L5.This result confirms that the ANCLA01 bacterial strain is strictly plant lactobacillus.
2.3 different grease additions are to the influence of CLA output in the plant lactobacillus ANCLA01 ensiling
Ensiling raw materials such as corn stalk are cut up with a hay cutter to 1～3cm, on one side the ensiling stockpile is pressed in silage tower, cellar for storing things or the bag, on one side by 8 * 10
7The bright sample of CFU/g sprays under the prerequisite of lactic acid nutrient solution (or the bright sample of 200g/t), the sunflower seed oil addition respectively by 2.0,3.0,4.0,5.0,6.0,7.0, the bright sample of 8.0kg/t (rapeseed oil or peanut oil addition by 5.0,6.0,7.0,8.0,9.0,10.0, the bright sample of 11.0kg/t) adds Vegetable oil lipoprotein, found that: when the bright sample of sunflower seed oil addition 5kg/t or rapeseed oil or peanut oil addition 8.0kg/t, CLA output is the highest, reaches 7.22mg/g (DW basis).The result shows that the sunflower seed oil optimum addition is the bright sample of 5kg/t, and rapeseed oil or peanut oil optimum addition are the bright sample of 8kg/t.
2.4. different lactobacillus inoculum amounts are to the influence of CLA output in the plant lactobacillus ANCLA01 ensiling
With ensiling raw materials such as corn stalk hand hay cutters to 1～3cm, the ensiling stockpile is pressed in silage tower, cellar for storing things or the bag, and meanwhile dosage press the bright sample of 5kg/t and add sunflower seed oil, one side inoculating lactic acid bacterium.Lactobacillus inoculum dosage is by 2 * 10
7CFU/g, 3 * 10
7CFU/g, 4 * 10
7CFU/g, 5 * 10
7CFU/g, 6 * 10
7CFU/g, 7 * 10
7The bright sample of CFU/g (or lactic acid bacteria culture solution 50,75,100,125,150, the bright sample of 175g/t), found that: the lactobacillus inoculum amount surpasses 4 * 10
7During CFU/g aquatic foods sample, CLA output no longer obviously increases after reaching 8.84mg/g (DW basis) in the ensiling, shows that the milk-acid bacteria optimum inoculation amount is 4-6 * 10
7The bright sample of CFU/g (or the bright sample of lactic acid bacteria culture solution 100-150g/t).
Utilize this plant lactobacillus fermentative preparation to be rich in the method for conjugated linolic acid silage:
1) plant lactobacillus ANCLA01 bacterial strain being carried out batch spreads cultivation: after will preserving twice of bacterial strain activation with above-mentioned MRS liquid nutrient medium, be inoculated in the 250mL triangular flask, and every bottled liquid measure 100mL, 37 ℃ of constant temperature are pressed 2x10 after leaving standstill and cultivating 48～72h again
7CFU/mL adds concentration and adds in the liquid seed fermentation jar, mixes back cultivation and fermentation 48h under 37 ℃ of conditions.
2) inoculation plant lactobacillus ANCLA01 in silage: ensiling raw materials such as corn stalk are cut up with a hay cutter to 1～3cm, on one side the ensiling stockpile is pressed in silage tower, cellar for storing things or the bag, spray plant lactobacillus ANCLA01 liquid and Vegetable oil lipoprotein after above-mentioned spreading cultivation on one side; Plant lactobacillus ANCLA01 dosage of inoculation is by 4-6 * 10
7The bright sample of CFU/g (or the lactic acid bacteria culture solution addition is the bright sample of 100-150g/t), sunflower seed oil addition are the bright sample of 5kg/t (rapeseed oil or peanut oil addition are the bright sample of 8kg/t); At last with silage tower, cellar for storing things or bag compacting sealing; Can enable behind the ensiling spontaneous fermentation 40-60d, more than actual measurement CLA content is all above 7.52mg/g (DW basis).
Claims (2)
1. a plant lactobacillus ANCLA01 is characterized in that described bacterial strain is Lactobacillusplantarum ANCLA01, and this bacterial strain preservation is registered on the books is numbered CGMCCNo.1906.
2. a plant lactobacillus ANCLA01 fermentative preparation is rich in the method for conjugated linolic acid silage, it is characterized in that:
1) the CGMCCNo.1906 plant lactobacillus ANCLA01 bacterial strain that is numbered that preservation is registered on the books carries out batch and spreads cultivation: after will preserving bacterial strain and activate twice with the MRS liquid nutrient medium, be inoculated in the 250mL triangular flask, every bottled liquid measure 100mL, 37 ℃ of constant temperature are pressed 2x10 after leaving standstill and cultivating 48～72h again
7CFU/mL adds concentration and adds in the liquid seed fermentation jar, mixes back cultivation and fermentation 48h under 37 ℃ of conditions;
2) the inoculation preservation volume of registering on the books is number CGMCCNo.1906 plant lactobacillus ANCLA01 in silage: on one side the ensiling stockpile is pressed in silage tower, cellar for storing things or the bag, spray plant lactobacillus ANCLA01 liquid and Vegetable oil lipoprotein through above-mentioned spread cultivation after on one side; Plant lactobacillus ANCLA01 dosage of inoculation is 4-6 * 10
7The bright sample of CFU/g, the Vegetable oil lipoprotein addition is: the bright sample of sunflower seed oil 5kg/t, or bright sample of rapeseed oil 8kg/t or the bright sample of peanut oil 8kg/t; At last with silage tower, cellar for storing things or bag compacting sealing; Behind spontaneous fermentation 40-60d, make the linolic acid fermentation in the Vegetable oil lipoprotein be converted into conjugated linolic acid, promptly get and be rich in the conjugated linolic acid silage.
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| CN102250807B (en) * | 2011-07-06 | 2013-06-19 | 广西工学院 | Microbial agent for Chinese silvergrass ensilage as well as preparation method and application thereof |
| CN103719551B (en) * | 2013-12-27 | 2015-09-16 | 柳州市青钱柳农业科技开发有限公司 | A kind of preparation method being rich in the ensilage of GABA |
| CN104336416B (en) * | 2014-11-03 | 2016-09-28 | 郑州大学 | One lactobacillus plantarum and the application in alfalfa ensilage thereof |
| CN107232409A (en) * | 2017-08-01 | 2017-10-10 | 合肥合丰牧业有限公司 | A kind of high-quality meat sheep feed |
| CN115960767B (en) * | 2022-11-09 | 2024-04-26 | 厦门元之道生物科技有限公司 | Lactobacillus plantarum and application thereof |
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