CN103898028A - High-density liquid-state cultivation method of feeding lactobacillus plantarum - Google Patents
High-density liquid-state cultivation method of feeding lactobacillus plantarum Download PDFInfo
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- CN103898028A CN103898028A CN201410151222.3A CN201410151222A CN103898028A CN 103898028 A CN103898028 A CN 103898028A CN 201410151222 A CN201410151222 A CN 201410151222A CN 103898028 A CN103898028 A CN 103898028A
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Abstract
According to the invention, bean pulp or bean cake is used as raw material and is subjected to liquid-state fermentation cultivation to produce high-density actobacillus plantarum, and the invention belongs to the field of preparation of the feeding microbial ecological agents by biotechnology. The number of viable probiotics contained in the microbial ecological agent is very important for evaluating whether product quality is good or not, and high-density preparation of probiotics is the basis for preparing high-quality microbial ecological agent. Proliferation of the number of the lactobacillus plantarum of the probiotics is promoted by adding a carbon source and various trace elements. A good pH environment for growing lactobacillus plantarum well is kept by supplementing calcium carbonate at different times. Density of the lactobacillus plantarum exceeds 1014cfu/ml after fermenting for 48 hours. The number of the viable bacteria per unit volume of the lactobacillus plantarum cultivated by the method disclosed by the invention is high, process formula cost is low, the cultivation method is simple and easy to implement, and suitable for large-scale production. The lactobacillus plantarum microbial ecological agent cultivated by adopting the process can be used as a special-purpose feed leavening agent and feed additive for various types of livestock aquatic animals.
Description
1 technical field
The present invention is the method for the liquid culturing plants Bacterium lacticum of a kind of high-density agent, relates to soybean liquefaction by-product utilized and the feeding probiotics leaven of liquid high-density culture field.
2 background technologies
Fodder industry is domestic important mainstay industry, and development in recent years is rapid, and China has become second-biggest-in-the-world fodder production state.Microbiotic is as fodder additives, can assist animal body opposing pathogenic bacteria, promote the effect of growth with it, since last century, the fifties was brought into use, become consumption maximum in feed, one of additive the most widely.But the use of the research discovery sixties in last century microbiotic in livestock and poultry animal causes resistance, and antibiotic remains appears in animal products.So from 20th century the mid-80, countries in the world start to take measures to limit antibiotic use gradually, start to find the Substitutes For Antibiotic that neither develops immunity to drugs, can promote again livestock birds health growth, improves herding production efficiency simultaneously, so feeding probiotic agent becomes optimal selection.
1989, sharp U.S. feed control official association of Food and Drug Admistraton of the United States Federal (FDA) (AAFCO) has announced a collection of by the probiotic bacterium catalogue of GRAS (being commonly referred to be safe) certification, and bag living plant Bacterium lacticum can Direct-fed animal 44 kinds of interior probiotic bacteriums.2008, the Ministry of Agriculture of China also announced 16 kinds of plant lactobacilluss etc. and can be used for feed additive strain register.Numerous test-results show, above-mentioned probiotic bacterium microbial inoculum has and maintains animal intestinal health, improper release and stress, eliminate animal house peculiar smell, regulate animal body metabolism of fat, improve the function of animal meat product quality.
The condition that should possess as probiotics leaven is: 1. the meta-bolites of bacterial strain is nontoxic to host; 2. couple host has prebiotic effect, as suppressed pathogenic bacterium, increase host immune power etc.; 3. bacterial strain Reproduction Conditions is rough, activity stabilized; 4. can tolerate hydrochloric acid in gastric juice and cholate and survive; 5. contained unit biomass viable bacteria number is abundant.As can be seen here, cultivating high-density agent of lactic acid bacteria is the key of Top Ouality starter.
At present, the cultural method of milk-acid bacteria has two classes: solid medium cultivation and liquid culture method.Due to genus lactubacillus facultative anaerobe, therefore liquid culture method is preferably for this type of probiotic's culture.Liquid culture method need provide the composition such as carbon source, nitrogenous source, trace element that can meet lactobacillus growth, and carbon source is as lactose, maltose, sucrose, glucose etc., and nitrogenous source is as peptone, polypeptide, milk-protein, yeast extract paste, corn steep liquor etc.In order to keep the growing environment that lactobacillus is good, someone adopts stream to add the acidity of the alkaline matter neutralise broths such as sodium carbonate, sodium hydroxide, potassium hydroxide, ammoniacal liquor, improves the biomass of lactobacillus.The selects of fourth etc., taking soy peptone, yeast lixiviate powder and vitamin complex and trace element etc. as substratum, adopt alkaline stream to add and keep fermented liquid pH6.0-6.8, three kinds of mixing lactic acid bacterium viable counts 9 × 10 in nutrient solution
9～1 × 10
10cfu/ml.Ma Ming cultivates and has a liking for yogurt bar, lactobacillus fermentum, three kinds of mixing lactic acid bacteriums of enterococcus faecalis with compositions such as soy peptone, yeast lixiviate powder, glucose, inorganic salt, halfcystines, and stream adds alkali lye during this time, and fermented liquid is viable count 3.0～5.0 × 10 after centrifugal drying
12cfu/g.Wei Lihua etc. are with the composition fermentation culture milk-acid bacterias such as soybean peptides, compound fruit and vegetable juice, raw dairy, viable count 8.9 × 10 in fermented liquid
10～2.7 × 10
11cfu/ml.
Dregs of beans is that organic solvent is carried the by product after oil, soya-bean cake is that mechanical expression is carried the by product after oil, soybean cake protein accounts for 33%-38%, the about 44%-48% of dregs of beans protein, and soya-bean cake or dregs of beans are all except the abundant high-quality nitrogenous source of high protein, amino acid after degrease.In dregs of beans or soya-bean cake, contain the syrup compound that exceedes 30%, comprise sucrose, raffinose, stachyose and polysaccharide, wherein first three kind soybean oligosaccharide can be utilized by probiotic bacterium metabolism, for probiotic bacterium growth provides good carbon source.Be the conventional edible oil of common people resident in China's soybean oil, liquefaction enterprise at home number is numerous, liquefaction by product soya-bean cake or dregs of beans sufficient raw, and soybean peptides, soy peptone is with low cost relatively, is the natural medium of preparation probiotic bacterium microbial inoculum high-quality.
Milk-acid bacteria is the main probiotic bacterium in animal intestinal, is familiar with and accepts to detoxification milk-acid bacteria probiotic agent or fermented feed at home and abroad by people.Quality product and cost are the life of product, can probiotic bacterium microbial inoculum be received in and depend on to a great extent and obtain the size of probiotic bacterium biomass and the number of cost, utilize liquefaction by product dregs of beans or soya-bean cake for basic material, it is the effective way of producing Cheap highly effective probiotic agent that high density fermentation is cultivated milk-acid bacteria microbial inoculum.
3 summary of the invention
The present invention relates to soybean liquefaction by product dregs of beans or soya-bean cake comprehensive utilization and the feeding probiotics leaven of liquid high-density culture field.The probiotic bacterium quantity of feed fermentation agent is one of leading indicator of weighing feed fermentation agent quality good or not, is also the key factor that reduces fermented feed preparation cost.The present invention is intended to utilize the raw material of lower cost, and by fermentation technology optimization, feeding fermented dose of preparation high-density plant lactobacillus, solves that current feeding probiotic agent viable bacteria number is low, the problem of fermented feed high expensive.
The method of middle-high density culturing plants Bacterium lacticum of the present invention comprises the following steps: one, utilize dregs of beans or soya-bean cake for raw material, pulverized after 20 order～40 mesh sieves, getting the lower fine powder 30g of sieve packs in 500ml triangle cake, be made into 6% liquid nutrient medium, in liquid nutrient medium, add glucose to concentration 4%～8%, add micro-final concentration to sodium acetate 1.5%, ammonium citrate 0.3%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.6%, manganous sulfate 0.075%, cooling after sterilizing.Two, access plant lactobacillus seed liquor, inoculum size 0.1%～0.5%, 39 DEG C of standing for fermentation 45h～48h.Three, between yeast phase, divide and add calcium carbonate 3 times: in the time fermenting to 6h, in fermented liquid, add calcium carbonate 0.5%; In the time of fermentation 21h, in fermented liquid, add calcium carbonate 0.5%; In the time fermenting to 36h, in fermented liquid, add calcium carbonate 0.5%; Four, fermentation ends, measures plant lactobacillus cell density 1.13 × 10 in fermented liquid
13cfu/ml zymocyte liquid～3.30 × 10
14cfu/ml zymocyte liquid; Meanwhile, fermented liquid 4000rpm is centrifugal, gets precipitation, and asepsis vacuum packing is feeding liquid probiotic agent; Or precipitate through lyophilize, asepsis vacuum packing is feeding solid-state probiotic agent.
Compare existing similar forage plant Bacterium lacticum probiotics leaven, the present invention takes full advantage of the feature of liquefaction by product dregs of beans and the contained material lower fat of soya-bean cake, high protein, adopt microorganism pure culture technigne to increase the biomass of probiotics leaven, reduce production cost, improved the added value of by product.Technological operation of the present invention is easy, culture condition Wen Li, probiotic bacterium biomass is high, raw material availability good, feeding fermented dose that makes has cost and probiotic bacterium quantity double dominant concurrently, and the plant lactobacillus that adopts this kind of technique to cultivate can be used as special feed starter and the fodder additives of multiple beasts, birds and aquatic products animal.
4 brief description of the drawings
Fig. 1 is production technological process of the present invention
5 embodiments
Embodiment one: one, to utilize dregs of beans or soya-bean cake be raw material, pulverized after 20 order～40 mesh sieves, getting the lower fine powder 30g of sieve packs in 500ml triangle cake, be made into 6% liquid nutrient medium, in liquid nutrient medium, add glucose to concentration 8%, add micro-final concentration to cooling room temperature after sodium acetate 1.5%, ammonium citrate 0.3%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.6%, 0.075%, 121 DEG C of sterilizing 15min of manganous sulfate.Two, the plant lactobacillus seed liquor of access, inoculum size 0.1%～0.5%, 39 DEG C of standing for fermentation 45h～48h.Three, between yeast phase, divide and add calcium carbonate 3 times: in the time fermenting to 6h, in fermented liquid, add calcium carbonate 0.5%; In the time of fermentation 21h, in fermented liquid, add calcium carbonate 0.5%; In the time fermenting to 36h, in fermented liquid, add calcium carbonate 0.5%; Four, fermentation ends, measures plant lactobacillus cell density 1.13 × 10 in fermented liquid
13cfu/ml zymocyte liquid～3.30 × 10
14cfu/ml zymocyte liquid; Meanwhile, fermented liquid 4000rpm is centrifugal, gets precipitation, and asepsis vacuum packing is feeding liquid probiotic agent; Or precipitate through lyophilize, asepsis vacuum packing is feeding solid-state probiotic agent.
Embodiment two: what present embodiment was different from embodiment one is the preparation process of plant lactobacillus seed liquor: preparation MRS liquid nutrient medium is sub-packed in test tube, 10ml/ test tube, cooling room temperature after 121 DEG C of sterilizing 15min, encircle/the test tube of plant lactobacillus inclined-plane thalline 1 that access has activated, 37 DEG C leave standstill cultivation 24h.Other step is identical with embodiment one.
Embodiment three: what present embodiment was different from embodiment one is that fermentation 45h finishes, and measures plant lactobacillus viable count 1.13 × 10 in fermented liquid
13cfu/ml zymocyte liquid.Other step is identical with embodiment one.
Embodiment four: what present embodiment was different from embodiment one is that fermentation 48h finishes, and measures plant lactobacillus viable count 3.30 × 10 in fermented liquid
14cfu/ml zymocyte liquid.Other step is identical with embodiment one.
Embodiment five: raw material dregs of beans or soya-bean cake concentration single factor experiment: dregs of beans or soya-bean cake are raw material, pulverized and get the lower fine powder of sieve after 20 order～40 mesh sieves and pack in right amount in 500ml triangle cake, be made into respectively 4%, 5%, 6%, 7%, 8% liquid nutrient medium, in liquid nutrient medium, add glucose to concentration 8% respectively again, between yeast phase, do not add calcium carbonate, fermentation 48h finishes, and measures respectively plant lactobacillus cell density 2.58 × 10 in fermented liquid
13cfu/ml zymocyte liquid, 3.35 × 10
13cfu/ml zymocyte liquid, 4.87 × 10
13cfu/ml zymocyte liquid, 1.51 × 10
13cfu/ml zymocyte liquid, 0.77 × 10
13cfu/ml fermentation mattress liquid, preferred feedstock concentration 6% is advisable.Other step is identical with embodiment one.
Embodiment six: glucose concn single factor experiment: dregs of beans or soya-bean cake are raw material, pulverized after 20 order～40 mesh sieves, getting the lower fine powder 30g of sieve packs in 500ml triangle cake, in liquid nutrient medium, add respectively glucose to concentration 4%, 5%, 6%, 7%, 8%, between yeast phase, do not add calcium carbonate, fermentation 48h finishes, and measures respectively plant lactobacillus cell density 1.62 × 10 in fermented liquid
13cfu/ml zymocyte liquid, 1.61 × 10
13cfu/ml zymocyte liquid, 1.96 × 10
13cfu/ml zymocyte liquid, 1.93 × 10
13cfu/ml zymocyte liquid, 5.32 × 10
13cfu/ml zymocyte liquid, preferably glucose concn 4%～8% is advisable.Other step is identical with embodiment one.
Embodiment seven: trace element combination addition test: dregs of beans or soya-bean cake are raw material, pulverized after 20 order～40 mesh sieves, getting the lower fine powder 30g of sieve packs in 500ml triangle cake, be made into 6% liquid nutrient medium, in liquid nutrient medium, add glucose to concentration 8%, add trace element by following five groups respectively, cooling room temperature after 121 DEG C of sterilizing 15min, the plant lactobacillus seed liquor of access, inoculum size 0.1%～0.5%, 39 DEG C of standing for fermentation, between yeast phase, do not add calcium carbonate, fermentation 48h measures plant lactobacillus cell density in first group to the 5th group fermented liquid and is respectively 4.2 × 10
12cfu/ml zymocyte liquid, 8.6 × 10
12cfu/ml zymocyte liquid, 1.02 × 10
13cfu/ml zymocyte liquid, 2.07 × 10
13cfu/ml zymocyte liquid, 2.13 × 10
13cfu/ml zymocyte liquid, preferably the 4th group of trace element addition is advisable.Other step is identical with embodiment one.
| Concentration (W/V) | Sodium acetate | Ammonium citrate | Dipotassium hydrogen phosphate | Magnesium sulfate | Manganous sulfate |
| First group | 0.75％ | 0.15％ | 0.3％ | 0.3％ | 0.0375％ |
| Second group | 1％ | 0.2％ | 0.4％ | 0.4％ | 0.05％ |
| The 3rd group | 1.25％ | 0.25％ | 0.5％ | 0.5％ | 0.0625％ |
| The 4th group | 1.5％ | 0.3％ | 0.6％ | 0.6％ | 0.075％ |
| The 5th group | 1.75％ | 0.35％ | 0.7％ | 0.7％ | 0.0875％ |
Claims (9)
1. a processing method for the liquid fermentation culture plant lactobacillus of high-density, is characterized in that comprising the following steps: (1) utilizes dregs of beans or soya-bean cake is raw material, is made into liquid nutrient medium after pulverizing, adds carbon source and various trace elements, cooling after sterilizing; (2) access logarithmic phase plant lactobacillus seed liquor, 39 DEG C of standing for fermentation 45h～48h; (3), between yeast phase, divide and add calcium carbonate 3 times; (4) fermentation ends, centrifugal, get precipitation, asepsis vacuum packing is feeding liquid probiotic agent; Or precipitate through lyophilize, asepsis vacuum packing is feeding solid-state probiotic agent.
2. method described in claim 1, is characterized in that: raw materials used in step (1) is dregs of beans or soya-bean cake, pulverizes after 20 order～40 mesh sieves, gets the lower fine powder 30g of sieve and packs in 500ml triangle cake, is made into 6% liquid nutrient medium.
3. according to method described in claim 1, it is characterized in that: in described step (1), be glucose to adding carbon source in liquid nutrient medium, concentration 4%～8%.
4. according to method described in claim 1, it is characterized in that: in described step (1), in liquid nutrient medium, add micro-final concentration to sodium acetate 1.5%, ammonium citrate 0.3%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.6%, manganous sulfate 0.075%.
5. according to the method described in claim 1, it is characterized in that: in described step (2), the plant lactobacillus seed liquor of access, inoculum size 0.1%～0.5%, 39 DEG C of standing for fermentation 45h～48h.
6. according to method described in claim 1, it is characterized in that: in described step (3), in the time fermenting to 6h, in fermented liquid, add calcium carbonate 0.5%; In the time of fermentation 21h, in fermented liquid, add calcium carbonate 0.5%; In the time fermenting to 36h, in fermented liquid, add calcium carbonate 0.5%.
7. according to the method described in claim 1, it is characterized in that: in described step (4), after fermentation 45h～48h, measure plant lactobacillus viable count 1.13 × 10 in fermented liquid
13cfu/ml zymocyte liquid～3.30 × 10
14cfu/ml zymocyte liquid.
8. according to the method described in claim 1, it is characterized in that: in described step (4), after fermentation ends, fermented liquid 4000rpm centrifuging and taking precipitation, asepsis vacuum packing is feeding liquid probiotic agent; Or precipitate through lyophilize, asepsis vacuum packing is feeding solid-state probiotic agent.
9. the forage plant Bacterium lacticum agent that described in claim 1-8 any one, method obtains.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105124137A (en) * | 2015-09-30 | 2015-12-09 | 江南大学 | Method for continuously preparing lactic acid bacterial liquid for fermented feed |
| CN110205276A (en) * | 2019-06-21 | 2019-09-06 | 厦门惠盈动物科技有限公司 | A kind of lactobacillus plantarum liquid fermentation medium and its cultural method and application |
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| CN101074426A (en) * | 2007-01-24 | 2007-11-21 | 合肥市科茂隆生物工程有限公司 | Method for producing bean-dregs feed containing conjugated linolic acid by plant lactobacillin fermentation |
| CN101173241A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing L-lactic acid and isoduicitol lactobacillus special for the same |
| CN102051336A (en) * | 2009-10-27 | 2011-05-11 | 中国石油化工股份有限公司 | Lactobacillus casei and application of lactobacillus casei in ferment production of L-lactic acid |
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- 2014-04-12 CN CN201410151222.3A patent/CN103898028A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101074426A (en) * | 2007-01-24 | 2007-11-21 | 合肥市科茂隆生物工程有限公司 | Method for producing bean-dregs feed containing conjugated linolic acid by plant lactobacillin fermentation |
| CN101173241A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing L-lactic acid and isoduicitol lactobacillus special for the same |
| CN102051336A (en) * | 2009-10-27 | 2011-05-11 | 中国石油化工股份有限公司 | Lactobacillus casei and application of lactobacillus casei in ferment production of L-lactic acid |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105124137A (en) * | 2015-09-30 | 2015-12-09 | 江南大学 | Method for continuously preparing lactic acid bacterial liquid for fermented feed |
| CN105124137B (en) * | 2015-09-30 | 2019-02-12 | 江南大学 | A kind of continuous method for preparing the lactic acid bacterial liquid for fermented feed |
| CN110205276A (en) * | 2019-06-21 | 2019-09-06 | 厦门惠盈动物科技有限公司 | A kind of lactobacillus plantarum liquid fermentation medium and its cultural method and application |
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Application publication date: 20140702 |