CN1259849C - Special lactobacillus inoculum for silage and its making method - Google Patents
Special lactobacillus inoculum for silage and its making method Download PDFInfo
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Abstract
The present invention relates to a special lactobacillus inoculant for ensilage and a preparing method thereof. The lactobacillus inoculant for ensilage, of the present invention, contains two kinds of activated xerotus afer fr initial powder, cellulase, hemicellulase, carbohydrate, manganese sulphate, diatomite, etc. The special lactobacillus inoculant for ensilage, of the present invention, can be prepared with a method of drying under intermediate temperature and reduced pressure and coating with gelatin-starch or a viable bacteria freezing and drying method. Experiments prove: in the final finished products of the present invention, the number of viable bacteria is greater than 90 billion CFU/g, is 8 times higher than that of similar products in foreign countries, and achieves an international leading level; the present invention solves the problem of product shelf life, and when the products of the present invention is preserved at the temperature of minus 20 DEG C for 110 days, the survival rate of the products of the present invention is 100%; experiments of feeding cow herds in a large scale prove that by using the products of the present invention, milk yield is increased by 283kg per cow every year.
Description
Technical field:
The invention belongs to field of feed processing, specifically a kind of special lactobacillus inoculum for silage and manufacture method thereof.
Technical background:
China's dairy development is very fast, past 5 annual production has increased by 5 times, reach 4,890,000 by 2000 and (comprise adult good breed of cattle, the improvement ox, the young ox and the meat dual-purpose ox that gives milk), 8,270,000 tons of milk total outputs, actual nearly 7 kilograms of people's annual drink milk, if with day only 0.25 kilogram of calculating of drink per capita, nearly 81 kilograms of people's annual drink milk amount, on present foundation level, also has the development space more than 10 times, China's milk cow development can be predicted and in quite over a long time, vigorous pace can be kept, but incompatible with it is that China's milk cow production technology level and advanced international standard have a long way to go, the U.S. is up to the 7462 kilograms/first year, China is 2500-3000 kilogram/first year only, one of them very important reasons lack quality silage exactly.Especially lack additive such as ensilage fermentation agent (Silage Inoculant Bacteria efficiently again in the existing silage making technology, be called for short SIB), present Chinese ensilage output is about 5,000 ten thousand tons (fresh weights), and the maximum potential market demand of green storage inoculating agent is 500 tons (adding product ensilage fermentation agent 10 grams of the present invention with ensilage per ton calculates).The green storage inoculating agent has become last 15-20 of American-European 20th century of developed country and has come ruminant feed and feed addictive to develop one of the most active field.Europe and North America market green storage inoculating agent product surpass kind more than 200, and Chinese up to now like product is blank.To the ensiling Study on Technology, mainly concentrate on two aspects abroad: the one, the research of Silaging method itself is as hay silage, additive ensiling, ensiling special equipment, compact technique etc.The additive ensiling is to the outer adding preservative agent (as formaldehyde, sulfurous acid etc.) of greenfeed, organic acid (formic acid) or mineral acid (sulfuric acid, hydrochloric acid etc.), stopping the activity of microorganism in the greenfeed, thereby reaches the preservation purpose.Its advantage is to make the pH value of ensiling drop to the degree that needs, thus some aerobic and losses anaerobic fermentation in eliminating or the minimizing ensilage; Shortcoming is that mineral metabolism, the especially calcium that mineral acid may influence in the animal body can produce negative balance; It is more that formaldehyde can make protein digestibility reduce, and the organic substance digestibility also may decrease; Formic acid can make the digestibility of storage crude fibre and nitrogen reduce, and is because the general quantity of additional substance is less, higher to the specification requirement that greenfeed mixes in addition.The 2nd, in ensilage, artificially add microorganism formulation, enter the lactic fermentation stage fast to quicken ensilage.Its technical essential is: 1. artificially add some bacterial strain and auxiliary additive, the Artificial Control ensilage starts lactic fermentation so that the pH value is reduced to optimum range in the shortest time; 2. reduce the consumption of nutriment in the ensilage as much as possible, reach the purpose that improves nutrient composition content.3. too do not pursue crude fibre external degradation rate, and emphasize to improve and keep the ensiling quality and help ensilage digestion and metabolism in animal body.
External production practices prove, add microorganism ensiling agent and can effectively improve the ensiling quality in ensilage, obtain good input-output ratio.Ensiling is microbiologic population's growth and decline evolution process of a complexity, the experience three phases: the firstth, and aerobic stage, the aerobic microorganism breeds in a large number, and this stage is long more, and the nutriment consumption of ensilage is big more; The secondth, anaerobic stages, along with oxygen depletion, various lactic acid bacterias begin a large amount of breedings, utilize sugar fermentation to produce lactic acid and a large amount of acetate, formic acid and propionic acid, and when lactic acid reaches a certain amount of, when pH dropped to 4.0 left and right sides, other various bacterium all were suppressed.Guarantee the sugar sufficient supplies, shorten the time of this section and can improve the ensiling quality.Phase III is the stabilization sub stage.Lactic acid continues breeding, and pH reduced to 3.8 o'clock, and the growth of lactic acid bacteria itself also is suppressed, and Biochemical processes all in the ensilage all stop, if oxygen free condition does not change long preservation to go down.On external ensiling technical research emphasis concentrates on the SIB manufacturing and uses over nearly 20 years.Simulating nature ensilage microbiologic population growth and decline characteristics, development can promote the live lactobacillus preparation of ensilage rapid lactic acid fermentation, join in the ensilage when silage making, to improve the quality of ensilage.Cardinal principle is that the use of these active bacteria formulations can reach following effect: (1) is reduced the ensilage heat and is distributed, (2) reduce the dry loss amount, (3) improve the dry uptake of animal for ensilage, (4) improve breeding performonce fo animals, (5) prolong ensilage and deposit the cellar for storing things time, (6) safety non-toxic.External lactic acid bacteria the most widely of adopting comprises Lactobacillus plantarum Lactobacillus plantarum, lactobacillus acidophilus Lactobacillus acidophilus, lactobacillus xylosus Lactobacillus xylosus, Pediococcus pentosaceus Pediococcus pentosaceus, have a liking for sour sheet coccus Pediococcus acidilactici, produce propionic acid bacteria Propionibacterium shermanii etc., fermentation medium main component carbon source is generally glucose, lactose, soluble starch and sucrose, nitrogenous source is generally whey powder, yeast culture and hydrolyzed animal protein, the batch fermentation tunning separates general the employing and filters, ultrafiltration or centrifugal, highly concentrated; Separating obtained high-purity thalline or employing spray-drying (loss late height) or employing freeze drying (cost height), the latter requires to have corresponding freezing drying protective agent, except freeze-drier mainly comes from the one-time investment, protective agent is the key that decision reduces viable bacteria freeze-drying loss and prolongs the product shelf life, and protective agent mainly is made up of skimmed milk power or maltodextrin, glycerine, calcium carbonate, sucrose, maltose, sodium glutamate, inositol, manganese sulfate etc.; External SIB product such as Britain produce SIL-ALL and the U.S. produces total viable count of lactic acid bacteria＞10,000,000,000 CFU/ tons (ensilage) that the Sil-Early assurance provides, Canada AgMaster produces the total viable count of lactic acid bacteria 10-60 ten thousand CFU/ tons (ensiling clover, corn etc.) that the LAB assurance provides, so commodity total viable count of lactic acid bacteria amount is generally 1-10 hundred million CFU/g on the market, shelf life 1 year, the freezing preservation of general employing, merchandise cost is higher.To sum up analyze, the restrictive factor that influences this area product promotion is viable bacteria resist technology and dry technology.
Summary of the invention:
The present invention is exactly at the problems referred to above, has proposed a kind of special lactobacillus inoculum for silage and manufacture method thereof based on Lactobacillus plantarum and Pediococcus pentosaceus.
Contained bacterium comprises in ensilage fermentation agent of the present invention (SIB): Lactobacillus plantarum and Pediococcus pentosaceus.Lactobacillus plantarum (LP) characteristic: nose circle straight-bar bacterium, the 0.9-1.2 micron is wide usually, and the 3-8 micron is long, and is single, and paired or one-tenth short chain can move the Gram stained positive; Chemoheterotrophic bacteria, the nutritional requirement complexity, anaerobism, acidproof, optimal pH 5.5-6.2.Optimum temperature 30-35 degree; Homofermentation produces DL-lactic acid; The fructose that can turn sour, galactolipin, glucose, lactose, maltose, mannose, melibiose, raffinose, rhamnose, ribose, sucrose etc.; Growth course individuality is before this grown up, after split into chain, be split into single individuality or short chain again.Pediococcus pentosaceus (PP) characteristic, cell sphere, diameter 0.8-1.0 micron, paired or tetrad shape; Do not move; The Gram stained positive; Chemoheterotrophic bacteria, the nutritional requirement complexity; Microaerobion, the catalase feminine gender; Optimum temperature 35 degree; Acidproof, grow at PH4.5-7.0.Homofermentation produces DL-lactic acid, and general L (+) mapping structure is preponderated; The arabinose that can ferment, ribose, maltose, trehalose, glucose etc.; Growth course by two that in succession along vertical plane alternately fission be the tetrad shape, being split by the tetrad shape is two or is that eight shapes are further divided into two tetrad shapes by the fission of tetrad shape that the growth course of Pediococcus pentosaceus is exactly the process of division again.Existing Lactobacillus plantarum and Pediococcus pentosaceus all can be used for the present invention.
Ensilage special lactobacillus inoculum of the present invention agent contains two kinds of former powder of the dried bacterium of work, cellulase, hemicellulase, carbohydrate, manganese sulfate, diatomite etc., consisting of of its each composition: the former powder of Lactobacillus plantarum: 50-500 hundred million CFU/ gram, preferred 10,000,000,000 CFU/ gram; The former powder of Pediococcus pentosaceus: 500-5000 hundred million CFU/ gram, preferred 80,000,000,000 CFU/ gram; Cellulase: 1000-1500U/ gram, preferred 1000U/ gram; Hemicellulase: 1500-3000U/ gram, preferred 1500U/ gram; Carbohydrate: DEXTROSE ANHYDROUS 1-2 gram, preferred 1.5 grams; Manganese sulfate: 0.2 gram-0.5 gram, preferred 0.3 gram; Diatomite: 0.05-0.15 gram, preferred 0.10 gram.
Below be described in detail ensilage special lactobacillus inoculum agent manufacture method of the present invention
1, the method for gelatin-starch double-contracting quilt and middle temperature vacuum drying manufacturing storage feed special lactobacillus inoculum agent
The vacuum middle temperature is dried protective agent of this method is by physiological saline, gelatin, and compositions such as starch, the content of each composition are (g/L); According to bacterium mud: gelatin (W/W), 1: 0.3-0.5, preferred 1: 0.4; Bacterium mud: starch (W/W), 1: 2-3, preferred 1: 2.5 ratio; Gelatin and starch are respectively with mixing well in bacterium mud behind the physiological saline solution.
This method comprises the strain inclined plane cultivation, is inoculated in fermentation tank, regulates the known microorganisms expansion of those skilled in that art such as PH, fermentation incubation step, and the major technique of the method for the invention is characterised in that the post-processing technology of culture, that is:
(1) put jar after, direct continuous centrifugal;
(2) according to bacterium mud: gelatin (W/W), 1: 0.3-0.5, preferred 1: 0.4; Bacterium mud: starch (W/W), 1: 2-3, ratio was mixed well in preferred 1: 2.5, added 2-3 again and doubly measured starch, put into the mixer mixing, and 40 mesh sieves are crossed in the back;
(3) sieving is laid on pallet, 1.0-2.0cm behind the bed of material goes into vacuum drier and dried 3-5 hour down in 20-60 ℃, makes its water content＜5%;
(4) the effective viable bacteria colony counting of former bacterium powder;
(5) by the prescription of ensilage special lactobacillus inoculum provided by the invention agent, take by weighing the amount of each composition, composite, promptly get the present invention and get product.
The method of 2, freeze drying manufactured ensilage special lactobacillus inoculum agent
The cryoprotector of this method is by physiological saline, skimmed milk power, and Vc, monosodium glutamate, inositol, soft white sugar is formed, and the content of each component is (g/L): skimmed milk power 180-260, preferred 230, soft white sugar 30-50, preferred 40; Vc60-90, preferred 75; Monosodium glutamate 50-70, preferred 60; Inositol 40-60, preferred 50, mend physiological saline to 1 liter.
This method comprises the strain inclined plane cultivation, is inoculated in fermentation tank, regulates the known microorganisms expansion of those skilled in that art such as pH, fermentation incubation step, and the major technique of the method for the invention is characterised in that the post-processing technology of culture, that is:
(1) put jar after, press fermentating liquid volume 1-3% and stir to add Sodium Meta Hexa Phosphate 62 and 67 P2O5 contents, leave standstill continuous centrifugal after 10-30 minute;
(2) add cryoprotector: bacterium mud and protective agent physiological saline solution, bacterium mud and cryoprotector by weight (1: 2.0--3.0) mixing and stirring;
(3) be laid on pallet, the thick 1.5-2.0cm bed of material was gone into refrigerator dry 30-36 hour, made its water content＜5%;
(4) grind freeze-dried material, cross 40 mesh sieves, get former bacterium powder;
(5) the effective viable bacteria colony counting of former bacterium powder;
(6) by the prescription of ensilage special lactobacillus inoculum provided by the invention agent, take by weighing the amount of each component, composite, promptly get product of the present invention.
The present invention compares than prior art has following advantage:
1, the present invention can make PP and LP bacterial strain reach about 3,000,000,000 CFU/ml and 1,500,000,000 CFU/ml respectively at the zymotic fluid biomass, through separating-concentrating--the dry former bacterium powder that obtains reaches 4,200 hundred million CFU/g respectively and more than 38,400,000,000 CFU/g, prepare the finished product number of viable at last greater than 90,000,000,000 CFU/g, higher 8 times than external like product, reach the international leading level;
2, the invention solves the problem of product shelf life, realized the industrialization of ensiling special lactobacillus preparation, product of the present invention was kept at the survival rate 100% under-20 ℃ in 110 days;
3, the present invention has studied the mechanism pattern of microbial inoculant for the influence of silage corn sweat first, has disclosed the theoretical mechanism that microorganism ensiling agent could guarantee and improve the silage corn quality;
4, extensive cows feeding experiment proves, uses product year milk-increasing quantity 283kg/ head of the present invention.
Brief description of drawings:
Fig. 1 is that middle temperature drying under reduced pressure and gelatin-starch double-contracting are store feed special lactobacillus inoculum agent process chart by manufactured
Fig. 2 is a viable bacteria freeze drying manufactured ensilage special lactobacillus inoculum agent process chart
Experimental example 1: narrate experimental example of the present invention below in conjunction with accompanying drawing
1, microorganism is cultivated:
(1) bacterial classification: any bacterial strain of Lactobacillus plantarum (LP) and Pediococcus pentosaceus (PP);
(2) seed culture medium (the MRS culture medium, %): see " lactic acid bacteria classification identify and experimental technique " (China Light Industry Press, Ling Daiwen chief editor, 1999, Beijing).Prepare after 115-116 ℃, the 30min sterilization waits to reduce to the room temperature inoculation, leaves standstill under 36 ± 2 ℃ and cultivates 20-30h.The obvious retrogradation of seed culture medium after having cultivated, color shoal (back of sterilizing is because of temperature drift, and the culture medium color is darker), and the microscopy thalli growth is good, do not have to make a variation, and carry out jar cultivation greatly behind the assorted bacterium of nothing.
(3) fermentation medium and condition of culture (g/L): peptone 17.1, yeast extract 6.5, glucose 39.2, sodium acetate 4.6, KH
2PO
42, MgSO
4.7H
2O 0.093, MnSO
4.H
2O 0.047, Tween-80 3.4, distilled water 1L.500 liters of canned 400L culture mediums are after 115 ℃ of sterilization 20min, with culture medium (general every 2h transfers once) pH=6.0 ± 0.2 in ammoniacal liquor initial pH of allotment culture medium and the maintenance sweat, be cooled to 37 ℃ of inoculations (1.2%), at the incubation medium speed is 60r/min, throughput is zero, fermentation temperature is controlled at 36 ± 2 ℃, fermentation period 36h.Viable count LP＞1.5 * 10 in zymotic fluid during fermentation ends
9CFU/ml, PP＞3 * 10
9CFU/ml.
2, gelatin-starch double-contracting is handled fermentation afterproduct and middle temperature vacuum drying technique by method:
Gelatin of the present invention-starch double-contracting is handled the fermentation afterproduct by method and middle temperature vacuum drying method is:
(1) put jar after, direct continuous centrifugal, 16,000r/mim, centrifugal.
(2) according to bacterium mud weight, add and mix well with physiological saline solution good gelatin and bacterium mud, add starch again, put into the mixer mixing, 40 mesh sieves are crossed in the back.Bacterium mud: gelatin (W/W) 1: 0.4; With bacterium mud: 1: 2.5 ratio of starch (W/W) is formed and is allocated.
(3) sieving is laid on pallet, the thick 1.0-2.0CM of the bed of material goes into 40 ℃ of oven dry of vacuum drier 3-5 hour, makes its water content＜5%.
(4) the effective viable bacteria colony counting of former bacterium powder;
(5) according to the preferred 10,000,000,000 CFU/ gram of the former powder of Lactobacillus plantarum; The former powder 80,000,000,000 CFU/ gram of Pediococcus pentosaceus; Cellulase 1000U/ gram; Hemicellulase 1500U/ gram; DEXTROSE ANHYDROUS 1.5 grams; Manganese sulfate 0.3 gram; Diatomite 0.10 gram. take by weighing the each component amount successively, composite product of the present invention.
The middle temperature vacuum drying protective agent of this method is allocated by physiological saline, gelatin, and compositions such as starch, each components contents is according to bacterium mud: gelatin (W/W) 1: 0.4; With bacterium mud: 1: 2.5 ratio of starch (W/W) is formed.
3, the viable bacteria freeze drying process is handled the fermentation afterproduct:
The cryodesiccated method of viable bacteria of the present invention is:
(1) put jar after, press fermentating liquid volume 1.5% and stir and add Sodium Meta Hexa Phosphate 62 and 67 P2O5 contents, leave standstill continuous centrifugal after 20 minutes, 16,000r/min.
(2) add cryoprotector: with physiological saline solution and allotment cryoprotector, bacterium mud and cryoprotector by weight 1: 2.0--3.0, mixing in preferred 1: 2.5 stirs.
(3) be laid on pallet, the thick 1.5-2.0CM bed of material is gone into refrigerator (NL-5 type vacuum freeze drier, Nanjing medical instruments factory) drying, and 30-36h makes its water content＜5%.
(4) grind freeze-dried material, cross 40 mesh sieves, get former bacterium powder, water content＜5%.
(5) the effective viable bacteria colony counting of former bacterium powder;
(6) according to the preferred 10,000,000,000 CFU/ gram of the former powder of Lactobacillus plantarum; The former powder 80,000,000,000 CFU/ gram of Pediococcus pentosaceus; Cellulase 1000U/ gram; Hemicellulase 1500U/ gram; DEXTROSE ANHYDROUS 1.5 grams; Manganese sulfate 0.3 gram; Diatomite 0.10 gram. take by weighing the each component amount successively, composite product of the present invention.
The cryoprotector of this method is allocated by physiological saline, skimmed milk power, and Vc, monosodium glutamate, inositol, soft white sugar are formed, and the content of each component be (g/L): skimmed milk power 230, soft white sugar 40, Vc 75, monosodium glutamate 60, inositol 50, benefit physiological saline to 1 liter.
4, product shelf life viable count is observed
Low temperature is preserved and is beneficial to the prolongation shelf life.Especially-20 ℃ following preservation effect is best, takes second place under 4 ℃ and the room temperature; The PP bacterium is under-20 ℃ and 4 ℃, and the interior number of viable of storage life did not reduce fully in 90 days; Viable bacteria inactivation inactivation 16% only under the room temperature.The preservation effect of LP bacterium is obviously far short of what is expected under similarity condition, 0%, 4 ℃ of following inactivation rate＜5% of normal temperature 78% ,-20 ℃ of following inactivations of following 110 days inactivations.Therefore, low temperature is preserved and is vacuum-packed for prolonging this product shelf life and guaranteeing that product quality is necessary.
Embodiment 2: count with Pediococcus pentosaceus in the product Lactobacillus plantarum of the present invention
1, culture medium
(1) 1% aseptic peptone buffer agent: accurately take by weighing the 5g peptone and be dissolved in the 500ml deionized water, be sub-packed in conical flask, place autoclave, sterilized 30 minutes down at 121.5 ℃, standby.
(2) MRS (Mgnn, Rogosa, Sharpe) medium component and preparation (g/l): beef peptone 10g, yeast soaks powder 5g, beef extract 10g, glucose 20g, Tween 80 1.0g, ammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.1g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2.0g, deionized water 1000ML, pH=6.1-6.3,25 ℃ of temperature. medium pH is regulated by ammoniacal liquor.Take by weighing agent dissolves in the 1000ml deionized water according to above prescription, be sub-packed in conical flask after the stirring.Then, sterilized 30～40 minutes under 121.5 ℃ of conditions, when equitemperature dropped to 45 ℃, dull and stereotyped under aseptic condition, every ware culture medium 15～20ml places cooling, and was standby.
2, in the product Lactobacillus plantarum of the present invention and the total clump count of Pediococcus pentosaceus viable bacteria detect
(1) takes by weighing sample 1g in the MRS culture medium that 100ml sterilized, in 37 ℃ of shaking tables, activate 2h.
(2) draw the aseptic peptone buffer agent of 9ml respectively in being numbered 1～8 sterile test tube.
(3) dilution: shake up sample, taking-up 1mL is put in No. 1 test tube and makes 10
-1Dilution, filled in tampon, in the eddy current mixed instrument, mixed 1～2 minute.Take out 1mL from No. 1 test tube again and be put in No. 2 test tubes, make 10
-2Dilution mixes.Make 10 successively
-1～10
-8The dilution of eight gradients.
(4) selected dilution factor is got each 100 μ L of three samples of dilution bacterium liquid and is applied to respectively on three flat boards, and numbering is placed sealing after 30 minutes, is inverted in 37 ℃ of biochemical incubators, cultivates 48～72 hours.Make a blank simultaneously.
(5) cultivate the back numeration, every flat-plate bacterial colony be effective flat board between 30～300.
(6) with a denumerable dilution factor (10
-8) three effective bacterium colonies of flat board (CFU/g), calculating mean value is as total lactic acid bacteria number.
(7) count results: what of extension rate and sampling can calculate three result of the test lactic acid bacterias according to and to add up to: 2.7 * 10
11, 7.2 * 10
10, 3.5 * 10
10CFU/g.
3, Lactobacillus plantarum, Pediococcus pentosaceus clump count detect
(1) selected dilution factor.Get 10
-8Each 100ul of dilution bacterium liquid evenly is applied on three flat boards.Numbering is placed sealing after 30 minutes, is inverted in 37 ℃ of biochemical incubators, cultivates 24～48 hours.Make a blank simultaneously.(2) behind the cultivation 48h, on the MRS flat board, choose 100 bacterium colonies at random, examine under a microscope the morphological feature of bacterium, bacillus is a Lactobacillus plantarum, and spherical bacteria is a Pediococcus pentosaceus.Calculate the ratio of Lactobacillus plantarum and Pediococcus pentosaceus then.According to the lactic acid bacteria sum of numeration and the ratio of Lactobacillus plantarum and Pediococcus pentosaceus, calculate Lactobacillus plantarum and Pediococcus pentosaceus quantity (CFU/g), three different batch products count results of producing of product of the present invention see Table 1.
Table 1, Lactobacillus plantarum, Pediococcus pentosaceus quantity and sum (CFU/g)
| Batch | The lactic acid bacteria sum | LP∶PP | LP | PP |
| 1 | 3.8×10 11 | 1∶9 | 3.8×1010 | 3.42×10 11 |
| 2 | 7.9×10 12 | 1∶9 | 7.9×10 11 | 7.11×10 12 |
| 3 | 8.2×10 10 | 2∶8 | 1.64×10 9 | 6.56×10 10 |
Embodiment 3, lactobacillus inoculum agent of the present invention are to the influence of corn silage quality and milk cow production performance
1. green storage inoculating agent of the present invention
Ensilage use amount per ton is 10 grams, ratio according to 5 grams per liters is dissolved in the water drying powdery Inoculant fully, with the mechanically-operated knapsack spraying machine SIB-HA inoculation liquid is sprayed to then and has shredded in the horizental silo on the ensiling bed of material of completing, whenever complete Inoculant solution of spraying behind one deck ensilage, with caterpillar tractor compacting back and forth, other links are handled according to conventional ensiling measure, are used for milk cow feeding experiment use in the coming year.
2. ensilage quality evaluation
In the cattle farm, the north suburb in Beijing with fresh whole-plant corn (in the spring maize kind beautiful No. 4), with cutting the chopping of hand hay cutter machine, length is less than 50mm, inoculate SIB-HA according to the method described above, insert the Plastic Drum (10 liter capacity) with Double layer lid then by hand, every barrel of ensilage loading amount is 11.5 ± 0.5 kilograms, tamp compress after, the bucket inner plastic bag is sealed, and plastic drum lid lid is tight, and with the adhesive tape reality of carefully mouth being obturaged.Use for indoor mechanism research sampling.Adorn 100 barrels altogether.
(adding or do not add SIB-HA) handled in each ensiling 3 repetitions, after ensiling is made the 1st day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 25 days, 40 days, 55 days, 70 days, 85 days, be used for destructiveness (disposable) sampling in 100 days, every barrel is only used or takes a sample once.Sampling back sample is kept under the 20-22 ℃ of room temperature.
After fully mixing, the sample that will take from 3 repetitions carries out the mensuration of following items: dry (DM) content (Dewar and McDonald, 1961), and pH (Woolford, 1984), total nitrogen (
N) and crude protein (CP), ammoniacal nitrogen (NH
3-N), water soluble carbohydrates (soluble sugar), volatile fatty acid (VFA) and lactic acid (Wilson and Wilkins, 1978).
3. milk cow production performance test
3 semi-underground type horizental silos (three is cement wall surface) are adopted in this test, and the volume of each horizental silo is 1200 tons.Using method 2.2 same corn material and methods are made silage corn.2 horizental silos are made inoculation SIB-HA and are handled, and another one is not for inoculating contrast.Test is carried out in the cattle farm, the north suburb in Beijing.Use 62 Chinese Fresians to carry out feeding experiment, their average calving 2.7 parity, be 145 days average lactation period, according to their age, body weight, the calving number, the calving date and the output of milk are selected pairing.62 cow heads are divided into two groups, 31 every group.
Two groups of test oxen feed basal diet, fine fodder, and sheep's hay and straw, and the ensilage that adds or do not add SIB-HA, the basal diet prescription sees Table 2, and experimental period is 70 days, and preceding 10 days is preliminary trial period.
The each output of milk (3 times/day) of record every test ox every day was done a milk-quality analysis every 7 days, produce Combi Foss 300 serial dairy products analysis-e/or determining milk sample butterfat percnetage, lactoprotein, lactose and total solids contents with Denmark.
4, statistical analysis adopts SAS Stat (Anon, 1985) that test data is carried out statistical analysis.
Table 2. basal diet composition and trophic level (g/kg dry)
| Feedstuff | Feedstuff | ||
| Corn soya-bean cake Cottonseed Meal wheat bran corn silage sheep's hay straw brewex's grains | 171.4 55.6 66.7 55.6 171.0 90.0 50.0 267.9 | Cornstarch albumen bone meal oyster shell whiting salt crude protein crude fibre Ca P | 45.0 10.6 10.6 5.6 149.0 164.5 10.9 5.5 |
5, ensilage performance rating
The analysis result that leaves breadboard barreled ensilage in sees Table 3 and table 4.The result shows, compare with the control group of handling without SIB-HA: the agent of SIB-HA green storage inoculating is to the ensiling quality, and especially the quality at ensiling initial stage has the influence in obvious front, and ensilage pH can be reduced rapidly, the generation of ammonia reduces, and lactic acid and other sour generations increase.Dry matter content is not subjected to the influence (P＜0.05) that SIB-HA handles in the ensilage.
The corn crop protein content was low originally, so the solubility ammonia level is low, was the result among expecting, so storing 7 days, after 15 days and 100 days, SIB-HA handles crude protein and only improves 1.2%, 2.2% and 4% respectively.SIB-HA processing soluble ammonia level is generally compared according to low 5-10% at lay up period, but also has the observation of indivedual time points reverse situation to occur.This has reflected the difficulty of the sampling and analysing with volatile ammonia.Yet the ensilage protein decomposition that SIB-HA handles is obviously compared according to low.SIB-HA handles ensiling dry and crude protein level do not make significant difference (p＞0.05).Handle through the agent of SIB-HA green storage inoculating and to have improved the sugar of ensiling, exception is also arranged, as 4 days, 7 days, the observation of 25 days and 40 days contrasted sugar exactly and handles higher than SIB-HA.This shows that the amylase composition in the SIB-HA preparation brought into play effect.
The agent of table 3. green storage inoculating is handled dry in the silage corn, and the influence of nitrogen and sugar level (g/kg, DM)
| The ensiling fate | Handle | Dry | Ammoniacal nitrogen (accounting for total N) | Crude protein | Sugar |
| 1 2 3 4 5 7 10 15 25 40 55 70 85 100 | Control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA | 191.1 182.8 177.4 184.1 184.1 183.8 175.7 179.9 189.6 184.7 171.0 176.4 177.5 169.3 180.7 182.2 180.9 180.5 178.0 179.7 176.5 180.8 180.9 179.9 176.9 174.1 176.0 179.0 | 75.35±0.96 60.93±2.22 79.37±6.60 73.96±3.21 118.24±7.94 89.24±9.01 112.44±2.33 102.34±8.67 116.64±0.99 117.46±7.77 147.45±9.00 140.63±11.19 191.67±10.82 192.93±12.01 213.13±9.08 228.51±13.20 243.84±21.12 260.00±32.07 239.60±15.37 236.26±16.66 269.65±19.99 247.77±21.10 336.74±23.38 216.71±10.80 288.51±17.17 255.63±15.20 313.88±10.73 290.77±21.31 | 99.38 94.38 99.38 93.13 92.50 90.63 97.50 95.00 89.38 88.75 100.63 98.75 90.00 93.75 100.00 92.50 91.25 88.75 94.38 91.88 106.88 109.38 91.88 98.75 92.50 94.38 91.88 97.50 | 11.25±0.79 12.04±1.10 10.90±0.73 11.41±1.21 10.16±0.76 13.44±1.20 17.36±0.98 15.40±1.11 13.56±0.10 15.70±0.72 15.61±1.38 14.57±0.76 14.65±1.17 15.54±1.02 15.66±1.20 16.63±0.70 17.03±1.28 16.90±1.50 15.62±1.39 14.47±0.88 14.96±1.32 17.23±1.01 14.29±0.89 15.27±1.22 13.02±0.75 15.08±1.14 14.20±0.21 15.27±1.09 |
Sugar fluctuates and keeps higher level is silage corn material result in accordance with expectation, does not have significant difference between the processing.
In ensiling 4 days, with the pH of comparing the ensiling of SIB-HA processed group descend the rapidest, this be handle material can be because of the main cause that can keep comparing the higher protein content of photograph with lactic acid that higher level is arranged and total acid level.Acetate is occupied an leading position in the ensiling volatile fatty acid.Most SIB-HA handle the yield of acetic acid comparison of silage corn according to low.2 time point observations (5 days, 15 days) exception is arranged, and promptly SIB-HA handles the yield of acetic acid comparison according to slightly high.The propionic acid level is lower, just can detect in the time of the 7th day up to ensiling, and amplitude of variation is bigger, and maximum can reach 0.69 (g/kg dry, the 10th day, control group).Generally, propionic acid is uncommon in corn silage.The butyric acid level (data are unlisted) of processed group silage corn is lower than control group ensiling during 7 to 70 days of ensiling.This is to detect butyric acid first in corn silage or other complete stool cereal ensilings, and butyric acid is a factor that is unfavorable for keeping the high-quality silage corn.Think that this may be that the ensiling sample produces because of ingress of air in the acid extractants process before measure equally after sampling.Do not detect the volatility fat aliphatic acid of more senior (C5 and C6).Lactic acid content is occupied an leading position, and exceeds 7-13% at SIB-HA processed group silage corn lactic acid content than control group, and this species diversity reached 16% at the 15th day.Compare with other acid, lactic acid pKa value is low, and it is to cause handling the comparison of silage corn pH value according to low main cause.The trend of total acid level and lactic acid similar trend, in the time of 25 and 55 days except.This shows that the agent of SIB-HA green storage inoculating makes the homotype lactic fermentation occupy an leading position in the corn silage process.
Show the agent of 4.SIB-HA green storage inoculating to corn silage pH, acetate, the influence of propionic acid and lactic acid content
| The ensiling fate | Handle | Acetate (mMol/l) | Propionic acid (mMol/l) | Butyric acid (mMol/l) | Lactic acid ‰ (g/kg, DW) | PH |
| 1 2 3 4 5 7 10 15 25 40 55 70 85 100 | Control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA control group SIB-HA | 614.38±69.00 575.91±63.11 690.00±25.31 497.42±33.98 778.54±79.05 749.34±103.19 1122.91±142.44 1116.09±178.86 838.13±111.17 853.36±159.00 916.88±100.47 660.82±73.21 1133.34±166.39 931.85±176.22 702.71±39.97 709.39±88.94 752.09±67.06 527.81±60.90 867.29±77.41 714.09±67.77 1112.50±169.54 797.59±99.18 986.88±134.01 940.19±120.52 1336.04±79.04 1105.44±97.67 916.46±70.08 773.62±13.99 | ND ND ND ND ND ND ND ND ND ND 24.46±3.77 40.68±5.76 19.05±2.02 51.08±4.22 21.08±3.23 32.702.98 12.03±2.83 9.73±2.01 19.05±1.26 25.95±0.32 39.46±0.45 39.60±2.44 16.49±2.81 46.89±2.22 31.35±2.89 72.57±8.02 29.19±0.90 33.24±2.35 | ND ND ND ND ND ND ND ND ND ND 4.09±1.38 6.31±2.12 7.96±2.11 6.36±0.90 7.84±1.18 3.86±1.05 1.93±0.55 1.93±0.57 3.98±0.27 ND 10.34±1.1 ND 3.75±0.78 3.75±0.27 ND ND ND ND | 53.26±3.90 61.44±3.24 61.80±5.55 70.40±8.21 69.75±4.04 79.91±8.09 84.68±7.77 94.14±10.06 82.60±6.54 93.80±7.77 91.58±4.10 102.85±5.79 83.82±6.09 96.96±7.05 99.67±2.07 107.17±10.00 100.97±7.21 112.02±14.44 101.17±13.22 112.51±4.44 102.03±6.66 109.91±3.99 111.24±2.98 120.06±13.65 111.10±7.90 122.58±8.92 110.33±18.00 120.67±7.01 | 4.18 4.16 4.08 4.06 4.05 4.02 3.97 3.95 3.94 3.96 3.90 3.91 3.90 3.88 3.87 3.85 3.78 3.86 3.78 3.76 3.77 3.78 3.54 3.80 3.53 3.68 3.73 3.80 |
*ND represents not detect.
Along with the prolongation of ensiling time, the level of ensiling volatile fatty acid and lactic acid raises, and increase rate is near 1 times.This explanation was at whole 100 days duration of test, and homotype lactic fermentation and heterolactic fermentation are all strengthened greatly.
Lactate level in the ensiling of SIB-HA processed group is higher than control group.Meaningfully, the difference of processed group and control group lactate level always remains on the level of 7-13% at duration of test.Maximum difference reaches 16% (the 15th day); Clearly, this be because the agent of SIB-HA green storage inoculating mainly by forming with lactic acid bacteria with homotype lactic acid fermentation ability, the lactic fermentation type in its leading corn silage process is based on homotype rather than heterolactic fermentation.
On the other hand, the acetate level in the processed group ensiling is lower than control group (except the 5th day and 15 days), and difference reaches 5-8% to 29.8% (the 25th day).Clearly, this is the result that heterolactic fermentation is suppressed.Along with the prolongation of ensiling time, ensiling pH value descends.And, observe the pH value comparison of the processed group silage corn of time point shine low in the overwhelming majority.PH and lactic acid content in the corn silage have confidential relation, and little with the volatile fatty acid relation with contents.This is consistent with this two classes organic acid souring ability difference.
The test of embodiment 4 milk cow production performances
In preceding 30 days of on-test, ensiling of SIB-HA processed group and control group ensiling do not have influence to milk production of cow.After 30 days, processed group ensiling and control group ensiling begin obviously the influence of milk production of cow.The result of raising dairy cattle test shows that SIB-HA handles and makes the comparison of milk cow daily yielding according to high 0.9kg/ head.Thereby year output of milk increases by 283 kilograms/(average 305 days lactation periods).In addition, calculate with the amount of the 20 kilograms of ensilages of searching for food average day of every cow head, 1 ton of silage corn of handling through SIB-HA is because the raising of quality can improve 46.5 kilograms milk yield.This research is also observed, and the corn silage that the SIB-HA that feeds handles is after 30 days, and the output of milk of milk cow improves.Obviously, although newborn quality do not have be improved significantly have an equal in quality milk yield but be improved significantly (table 5).
Use the quality of SIB-HA green storage inoculating agent raising ensilage.From the result of raising dairy cattle test, milk cow production performance is improved, and the output of milk improves, and economic benefit improves.
Table 5 is handled the influence of corn silage to milk production of cow and milk products quality through the agent of SIB-HA green storage inoculating
| Index | Control group | + SIB-HA processed group |
| Milk yield, kg/ head/day butterfat percnetage, the % lactoprotein, the % lactose, the % total solid, % 3.5% fat corrected milk(FCM), kg | 28.54±2.99 4.17±0.44 31.6±0.28 4.75±0.30 12.80±0.53 31.22 | 29.47±3.06 3.98±0.27 31.5±0.31 4.74±0.26 12.47±0.44 31.45 |
Claims (7)
1, a kind of ensilage special lactobacillus inoculum agent, it is characterized in that, each constituent content is: the former powder of Lactobacillus plantarum: 10,000,000,000 CFU/ gram, the former powder of Pediococcus pentosaceus: 80,000,000,000 CFU/ gram, cellulase: 1000U/ gram, hemicellulase: 1500U/ gram, DEXTROSE ANHYDROUS 1.5 grams, manganese sulfate 0.3 gram, diatomite 0.10 gram.
2, the method for a kind of production claim 1 lactobacillus inoculum agent, this method comprise the strain inclined plane cultivation, are inoculated in fermentation tank, regulate Ph, fermentation, post-processing step, it is characterized in that treatment step is:
(1) put jar after, direct continuous centrifugal;
(2) warm vacuum drying protective agent in the interpolation; According to bacterium mud weight, what adding 0.3-0.5 doubly measured mixes well with physiological saline solution good gelatin and bacterium mud, adds the starch that 2-3 doubly measures again, puts into the mixer mixing, and 40 mesh sieves are crossed in the back;
(3) sieving is laid on pallet, the thick 1.0-2.0cm of the bed of material goes into vacuum drier and dried 3-5 hour down in 20-60 ℃, makes its water content＜5%;
(4) effectively viable bacteria colony counting;
(5) by the prescription of claim 1, take by weighing the amount of each component, composite, promptly get product of the present invention.
3, the method for a kind of production claim 1 lactobacillus inoculum agent, this method comprise the strain inclined plane cultivation, are inoculated in fermentation tank, regulate Ph, fermentation, post-processing step, it is characterized in that treatment step is:
(1) put jar after, press fermentating liquid volume 1-3% and stir to add Sodium Meta Hexa Phosphate 62 and 67 P2O5 contents, leave standstill continuous centrifugal after 10-30 minute;
(2) add cryoprotector: bacterium mud and protective agent physiological saline solution, bacterium mud and protective agent by weight 1: 2.0-3.0 mixing and stirring;
(3) be laid on pallet, the thick 1.5-2.0cm bed of material was gone into the refrigerator freeze drying 30-36 hour, made its water content＜5%;
(4) grind and coldly do boringly, cross 40 mesh sieves, former bacterium powder;
(5) effectively viable bacteria colony counting;
(6) by the prescription of claim 1, take by weighing the amount of each component, composite, promptly get product of the present invention.
4, production method according to claim 2 is characterized in that, described in warm each components contents of vacuum drying protective agent be (g/L): according to bacterium mud: gelatin (W/W), 1: 0.3-0.5; Bacterium mud: starch (W/W), 1: 2-3; Gelatin and starch are respectively with mixing well with bacterium mud behind the physiological saline solution.
5, production method according to claim 4 is characterized in that, described in warm each components contents of vacuum drying protective agent be (g/L): according to bacterium mud: gelatin (W/W), 1: 0.4; Bacterium mud: starch (W/W), 1: 2.5; Gelatin and starch are respectively with mixing well with bacterium mud behind the physiological saline solution.
6, production method according to claim 3 is characterized in that, each components contents of described cryoprotector is (g/L): skimmed milk power 180-260, soft white sugar 30-50, V
C60-90, monosodium glutamate 50-70, inositol 40-60 mends physiological saline to 1 liter.
7, production method according to claim 6 is characterized in that, each components contents of described cryoprotector is (g/L): skimmed milk power 230, soft white sugar 40, V
C75, monosodium glutamate 60, inositol 50 is mended physiological saline to 1 liter.
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| JP4101729B2 (en) * | 2003-10-22 | 2008-06-18 | 劉 楊 | Livestock feed additive |
| SI2056681T1 (en) | 2006-07-14 | 2011-09-30 | Legarth Lone | Homofermented products |
| CN101449752B (en) * | 2007-11-30 | 2011-04-27 | 河南农业大学 | Composite microbial inoculant for corn silage and use method thereo |
| CN101427739B (en) * | 2008-12-10 | 2011-02-02 | 中国科学院亚热带农业生态研究所 | Green feed storing method |
| CN101878859A (en) * | 2010-07-01 | 2010-11-10 | 西北农林科技大学 | Microbial additive for silage fodder and preparation method thereof |
| DK2654417T3 (en) * | 2010-12-23 | 2018-10-29 | Dupont Nutrition Biosci Aps | COLD PROTECTIVE COMPOSITIONS AND APPLICATIONS THEREOF |
| CN103060228B (en) * | 2012-12-12 | 2014-06-25 | 广州立达尔生物科技股份有限公司 | Lactobacillus plantarum and micro-ecological preparation for silage |
| CN104068294A (en) * | 2014-06-18 | 2014-10-01 | 兰州大学 | Biochemical composite additive |
| CN104073454A (en) * | 2014-06-18 | 2014-10-01 | 兰州大学 | Low-temperature-resistant lactobacillus planetarium and application thereof |
| CN104531566A (en) * | 2014-12-11 | 2015-04-22 | 中国农业大学 | Silage inoculant and application thereof |
| CN106854626A (en) * | 2015-12-08 | 2017-06-16 | 内蒙古沃德生物质科技有限公司 | A kind of maize straw is blue or green（It is yellow）Store the preparation method of enzyme bacterium composite ferment |
| CN105886427A (en) * | 2016-03-31 | 2016-08-24 | 安徽省农业科学院畜牧兽医研究所 | Activating method of lactic acid bacterial liquid used for alfalfa herb ensiling |
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| CN110777095A (en) * | 2019-11-12 | 2020-02-11 | 黑龙江省科学院微生物研究所 | Lactobacillus agent suitable for straw micro-storage and preparation process thereof |
| CN112890019B (en) * | 2021-02-04 | 2022-08-19 | 华中农业大学 | Forage rape silage and preparation method thereof |
| CN112868914A (en) * | 2021-02-04 | 2021-06-01 | 华中农业大学 | Silage rape feed and preparation method and application thereof |
| CN116326679A (en) * | 2023-03-27 | 2023-06-27 | 武汉新华扬生物股份有限公司 | Composition, method and application for improving stress resistance of pediococcus pentosaceus |
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