CN1807592A - Method for producing lactic acid bacteria formulation and lactic acid bacteria bacteriocin using ethanol draff liquid - Google Patents
Method for producing lactic acid bacteria formulation and lactic acid bacteria bacteriocin using ethanol draff liquid Download PDFInfo
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Abstract
This invention discloses a method for preparing lactobacillus preparation and lactobacillus bacteriocino with waste alcohol distiller liquid, and it belongs to bio-preparation prosessing sphere, comprising steps of: get corn alcohol distiller liquid after alcoholic fermentation; solid-liquid separate the alcohol distiller liquid to get clear licquid; obtain expand-culture medium from the clear licquid; expand culture bacterial gradually; obtain fermention medium from the clear licquid; ferment the expand-culture bacterial in fermention medium; lastly separate fermention liquid to get lactobacillus preparation and lactobacillus bacteriocino. which has high added value and is produced by using alcohol distiller liquid, especially corn alcohol distiller liquid. It can prolongs industrial chain of corn processing; can improves corn's value in use; can recycle resource and can depress the manufacturing cost of lactobacillus preparation and lactobacillus bacteriocino.
Description
Technical field
The present invention relates to the working method of lactic acid bacteria formulation and bacteriocin lab in a kind of biotechnological formulation processing and preparing field, more particularly, relate to a kind of particularly corn alcohol waste dregs liquid method of producing lactic acid bacteria biological preparation and bacteriocin lab of alcohol waste lees solution of utilizing.
Background technology
Alcohol waste lees solution (Vinasse) is the by product of fermentative Production alcohol, is a kind of organic waste water of high density.The quantity discharged in China every year is up to more than 3,000 ten thousand tons, and this is not only the resource significant wastage, simultaneously also serious environment pollution.
Bacteriocin lab is a kind of active substance that some milk-acid bacteria (galactococcus and Bacterium lacticum) is produced, and promptly a class that produces by the rrna synthesis mechanism in metabolism has the polypeptide or the precursor polypeptide of bacteriostatic activity.
The bacteriocin NISIN that streptococcus acidi lactici produced, promptly lacticin is the antiseptics for natural food of world health organisation recommendations, since its security and high efficiency, the extremely favor of consumers in general and foodstuff production producer.
Milk-acid bacteria is in close relations with the mankind's, especially in recent years, the application of milk-acid bacteria is the attention of clinical medicine circle extremely, aspect clinical medicine, the milk-acid bacteria effect is as follows: 1, can treat the intestinal function disorder with milk-acid bacteria as whole intestines level ecological agent, keep enteron aisle thalline balance; 2, lactic acid bacteria formulation has therapeutic action to oral disease (carious tooth etc.); 3, antitumor action; 4, immunity effect effect; 5, delay body aging; 6, treat bacillary colpitis mycotica.
In addition, milk-acid bacteria also can be applicable in the feed, can be used as probiotics and plays and promote to grow, disease preventing and treating and improve the quality of poultry product.
Simultaneously, milk-acid bacteria also can be used as foodstuff additive, improves acidity and improves local flavor, increases nutrient and improves nutritive value, plays the prolongation effective period of food quality, prevents putrid effect.
But well-known, lactic-acid-bacterium is galactococcus particularly, and its nutritional needs is the comparison harshness, is therefore producing milk-acid bacteria preparation alive, particularly produces the meta-bolites of bacteriocin lab and so on, and its raw materials cost is very expensive.Production cost of products is high, so the price of product is also very high, as, about 1300 yuan of every kilogram of market price of nisin (Nisin).
Alcohol waste lees solution is rich in growth, the metabolism desired nutritional material that can supply multiple lactic-acid-bacterium, as soluble protein, and 18 seed amino acids, inorganic salt and multivitamin or the like are shown in table 1 in the accompanying drawing.In addition, contain abundant carbon source, nitrogenous source, vitamin B group and growth hormone in the corn alcohol waste dregs liquid.Wherein depleted filtrate contains total reducing sugar 0.6-0.7%, total nitrogen 0.36-0.45%, and VITAMIN machine inorganic salt content is shown in table 2 in the accompanying drawing.With it is the culture medium culturing lactic-acid-bacterium; Fermenting lactic acid bacterium ecological agent and bacteriocin lab etc. are the very selections of science.
| Component | The corn alcohol waste dregs liquid | The molasses-spirit waste dregs liquid |
| BOD(mg/l) | 15000-25000 | 50000-60000 |
| Solid substance (%) | 7.5 | 8.5 |
| Carbohydrate (%) | 0.5 | 0.9 |
| Protein (%) | 2.3 | 0.8 |
| Ash content (%) | 1.5 | 2.5 |
| VITAMIN | More | More |
The composition of table 1 alcohol waste lees solution
| The VITAMIN inorganic salt | Content (%) | Metabolic function |
| V B1 | 1.2×10 -5 | Constitute decarboxylase, transaldolase, the prothetic group of transketolase, with the oxidative decarboxylation of a-ketone acid, ketone group shifts relevant |
| V B2 | 2.28×10 -5 | The precursor of flavine nucleosides FMN and FAD, they constitute the prothetic group of flavoprotein, shift hydrogen |
| Pantothenic acid | 4.36×10 -5 | The precursor of coenzyme A, the prothetic group of acetyl carrier shifts acyl group, involved in sugar and lipid acid synthetic |
| Nicotinic acid | 2.4×10 -4 | The precursor of NAD and NADP is the coenzyme of desaturase, participates in passing hydrogen process and redox reaction |
| V B6 | 1.75×10 -5 | Pyridoxal phosphate is an amino acid, racemase, the prothetic group of transaminase and decarboxylase |
| Folic acid | 1.1×10 -6 | The commentaries on classics that participates in carbon back claims that with synthetic fast cry of certain animals, pyrimidine, Nucleotide, amino acid is relevant |
| V E | 1.12×10 -4 | Participate in redox reaction |
| Choline | 9.6×10 -3 | Participate in metabolism of fat |
| Vitamin H | 1.1×10 -6 | The prothetic group of various carboxylases at amino acid, works in lipid acid and the carbohydrate metabolism |
| P | 0.026 | Nucleic acid, the composition of phosphoric acid and coenzyme |
| S | 0.0074 | The composition of sulfur-containing amino acid and sulfur-bearing VITAMIN |
| K | 0.035 | The cofactor of fructokinase etc. is kept potential difference and osmotic pressure |
| Mg | 0.013 | The cofactor of many enzymes |
VITAMIN and inorganic salt content and metabolic function in the table 2 corn alcohol waste dregs liquid filtrate
At present, fully utilize alcohol waste lees solution both at home and abroad and mainly contain following several method:
(1) produces (SCP) feed;
(2) production full price dry feed (DDGS);
(3) biogas fermentation;
(4) utilize its fermentative production fungi bacterium powder and fungus polysaccharide.
From existing data, do not find to have the report that utilizes alcohol waste lees solution to produce lactic acid bacteria formulation and bacteriocin lab as yet both at home and abroad.
Summary of the invention
The present invention is directed to the problems referred to above, the method that provides a kind of utilization to produce lactic acid bacteria formulation and bacteriocin lab as the alcohol waste lees solution of by product has solved behind the corn processed alcohol usually waste dregs liquid as waste discharge, causes the problem of the wasting of resources.
In order to address the above problem, the invention provides a kind of method of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation, its scheme comprises the step that obtains alcohol waste lees solution after the zymamsis, alcohol waste lees solution is the corn alcohol waste dregs liquid, and method also comprises the steps: S11, obtains clear liquid: described alcohol waste lees solution solid-liquid separation is obtained clear liquid; S12, substratum obtains to spread cultivation: get the clear liquid that step S11 obtains, add 0-0.5% yeast extract paste, 0-4% sucrose, transfer pH value to 5.5-6.5; S13, get bacterial classification and carry out spreading cultivation step by step of triangular flask, seeding tank with the inoculum size of 3-10%; S14, acquisition fermention medium: get the clear liquid that step S11 obtains, add the 0-0.5% yeast extract paste; 0-4% sucrose; 0-4% glucose; 0-0.2% potassium primary phosphate (KH
2PO
4); The accent pH value is 5.5-6.5; S15, fermentation: after the conventional sterilization, the inoculum size of pressing 3-10% to fermentor tank, and remains on 0.15-0.5kg/cm with the method that charges into nitrogen with tank pressure with the bacterial classification inoculation that spreads cultivation among the S13
2, 25-35 ℃ of discontinuity stirs 200-500 rev/min, stirs 4-8 minute in every 1.5-3 hour, cultivates 18-36 hour, transfers pH value to 1.75-2.25, leaves standstill 8-16 hour; S16, acquisition lactic acid bacteria formulation: will leave standstill the filtering fermentation liquor of back gained among the step S15, and collect filter cake, vacuum-drying becomes the solid lactic acid bacteria preparation.
The above-mentioned method of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation, it further improves or characteristics are, obtains the clear liquid method to be in step S11: utilize whizzer that described alcohol waste lees solution centrifugation is obtained clear liquid; Perhaps utilize filter type to obtain clear liquid; After concentrating again or with the clear liquid that centrifugal or filter type obtains once more thin up obtain clear liquid.
The above-mentioned method of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation, it further improves or characteristics are, utilizes NaOH and HCl to transfer pH value among step S12, the S14; Transfer pH value with phosphoric acid among the step S15; In addition, the described solid lactic acid bacteria preparation that step S16 is obtained is made powdery lactic acid bacteria biological preparation; And in step S13, comprise: S131, female plant activation: mother is planted receive in the container that the 10-50ml liquid nutrient medium is housed, 25-35 ℃ of static cultivation 16-36 hour, and prove conclusively the pure of bacterial classification with the method that microscopy and inclined-plane are coated with; It is that ratio in 3-10% is linked into test tube strains in the 150ml triangular flask that the 100ml liquid nutrient medium is housed that S132, triangular flask spread cultivation, cultivated 16-36 hour for 25-35 ℃, press the inoculum size of 3-10% again, bacterial classification in the 150ml triangular flask is linked in the 5000ml triangular flask that the 3000ml liquid nutrient medium is housed 25-35 ℃ of static cultivation 16-36 hour; S133, seeding tank spread cultivation be by the inoculum size of 3-10% with the bacterial classification inoculation of triangular flask in seeding tank, and guarantee that with the method for inflated with nitrogen tank pressure is 0.15-0.5kg/cm
2Discontinuity stirs 200-500 rev/min; Stirred 25-35 ℃ of static cultivation 8-36 hour in every 1.5-3 hour 4-8 minute.
The present invention also provides a kind of method of utilizing alcohol waste lees solution to produce bacteriocin lab, its scheme comprises that zymamsis obtains the step of alcohol waste lees solution, alcohol waste lees solution is the corn alcohol waste dregs liquid, and method also comprises the steps: S21, described alcohol waste lees solution solid-liquid separation is obtained clear liquid; S22, substratum obtains to spread cultivation: get the clear liquid that step S21 obtains, add 0-0.5% yeast extract paste, 0-4% sucrose, the accent pH value is 5.5-6.5; S23, substratum obtains to spread cultivation: get bacterial classification and carry out spreading cultivation step by step of triangular flask, seeding tank with the inoculum size of 3-10%; S24, acquisition fermention medium: get the clear liquid that step S21 obtains, add the 0-0.5% yeast extract paste; 0-4% sucrose; 0-4% glucose; 0-0.2% potassium primary phosphate (KH
2PO
4); The accent pH value is 5.5-6.5; S25, fermentation: after the conventional sterilization, the inoculum size of pressing 3-10% to fermentor tank, and remains on 0.15-0.5kg/cm with the method that charges into nitrogen with tank pressure with the bacterial classification inoculation that spreads cultivation among the S13
2, 25-35 ℃ of discontinuity stirs 200-500 rev/min, stirs 4-8 minute in every 1.5-3 hour, cultivates 18-36 hour, transfers pH value to 1.75-2.25, leaves standstill 8-16 hour; S26, obtain bacteriocin lab, comprising: S261, collect filtrate after filtration, filter the material of holding back 20,000 molecular weight through ultrafilter again, collect the ultrafiltration filtered solution; Described ultrafiltration filtered solution ultrafiltration is once more held back material between the 2000-20000 molecular weight; S262, hold back in the concentrated solution to step S261 and to add skim-milk or salt NaCl or sex change dextrin and make the concentrated solution water content be transferred to 50-60%, concentrated solution; The described concentrated solution that S263, S262 obtain is through the centrifugal spray dryer spraying drying, actual conditions is that inlet temperature transfers to 200-230 ℃, and air outlet temperature transfers to 90 ℃, and spouting liquid is 500-1000kg/h (kilogram/per hour), must consolidate the shape bacteriocin lab, product volume is 250-500kg/h.
The above-mentioned method of utilizing alcohol waste lees solution to produce bacteriocin lab, it further improves or characteristics are, obtains described clear liquid method among the step S21 to be: utilize whizzer that described alcohol waste lees solution centrifugation is obtained clear liquid; Perhaps utilize filter type to obtain clear liquid; After concentrating again or with the clear liquid that centrifugal or filter type obtains once more thin up obtain clear liquid.
The above-mentioned method of utilizing alcohol waste lees solution to produce bacteriocin lab, it further improves or characteristics also are, utilizes NaOH and HCl to transfer pH value among step S22, the S24; Transfer pH value with phosphoric acid among the step S25, the described solid shape bacteriocin lab crushing packing that in addition step S26 is obtained gets the bacteriocin product.
The above-mentioned method of utilizing alcohol waste lees solution to produce bacteriocin lab, it further improves or characteristics also are, comprise among the step S23: S231, the female kind activate: mother is planted receive in the container that the 10-50ml liquid nutrient medium is housed, 25-35 ℃ of static cultivation 16-36 hour, and pure with the method conclusive evidence bacterial classification of microscopy and inclined-plane coating; It is that ratio in 3-10% is linked into test tube strains in the 150ml triangular flask that the 100ml liquid nutrient medium is housed that S232, triangular flask spread cultivation, cultivated 16-36 hour for 25-35 ℃, press the inoculum size of 3-10% again, bacterial classification in the 150ml triangular flask is linked in the 5000ml triangular flask that the 3000ml liquid nutrient medium is housed 25-35 ℃ of static cultivation 16-36 hour; S233, seeding tank spread cultivation be by the inoculum size of 3-10% with the bacterial classification inoculation of triangular flask in seeding tank, and guarantee that with the method for inflated with nitrogen tank pressure is 0.15-0.5kg/cm
2Discontinuity stirs 200-500 rev/min; Stirred 25-35 ℃ of static cultivation 8-36 hour in every 1.5-3 hour 4-8 minute.
Pass through technique scheme, the present invention utilizes alcohol waste lees solution to produce the method for lactic acid bacteria formulation and bacteriocin lab, especially by utilizing the corn alcohol waste dregs liquid to produce lactic acid bacteria formulation and such as the meta-bolites of bacteriocin lab, reduce production costs significantly, turn waste into wealth and realize the utilization once more of resource.Simultaneously also make the corn deep processing industrial chain obtain extension, greatly improved the utility value of corn.Its advantage is as follows:
(1) provides a kind of new way of utilizing alcohol waste lees solution to produce high value added product.
(2) industrial chain of corn deep processing has been prolonged, the utility value of corn has been improved.
(3) because in process of production, adopted membrane technique, held back filtered solution, can use again by backwater fully less than 2000 molecular weight.Promptly can be used for producing, also can be used in life simultaneously.
Description of drawings
Fig. 1 is that the present invention utilizes alcohol waste lees solution to produce the process flow diagram of lactic acid bacteria formulation and bacteriocin lab method.
Embodiment
Utilize alcohol waste lees solution to produce the technology of lactic acid bacteria formulation and bacteriocin lab method as shown in Figure 1.Comprising, obtain alcohol waste lees solution after the zymamsis; Utilize alcohol waste lees solution centrifugation solid sediment and supernatant liquor.
Also comprise, utilize throw out to make the full price dry feed.
Also comprise, once filter acquisition filtered liquid I after utilizing the supernatant liquor fermentation, filtered liquid I filters once more and obtains filtered liquid II, and the liquid that filtered liquid II holds back 2,000 to 20,000 molecular weight concentrates back acquisition concentrated solution with salt, to concentrated solution spraying, the dry bacteriocin lab that obtains.And utilize filtered liquid worker's concentrated solution I drying to make the full price dry feed.
Comprise also, utilize supernatant liquor to filter and to obtain wet thallus that wet thallus is with protective material, vacuum-drying, and acquisition lactic acid bacteria formulation after pulverizing.
The bacterial classification that the present invention uses is: streptococcus acidi lactici, enterococcus faecalis, lactobacillus gasseri, lactobacillus crispatus, short lactobacillus, and the conventional bacterial classification of other kinds.Above-mentioned bacterial classification all derives from industrial microorganism institute of Liaoning Normal University, and the method for culture presevation is a freeze-drying.
The method of strain expanded culture adopts the conventional method that spreads cultivation step by step, comprises the steps:
(1) obtaining liq substratum: get the corn alcohol waste dregs liquid, after filtration or 3000 rev/mins, and centrifugal 20 minutes, get filtrate or supernatant liquor, add the 0-0.5% yeast extract paste; 0-4% sucrose utilizes NaOH and HCl to transfer PH to 5.5-6.5.
(2) femalely plant activation: mother is planted receive in the container that 10-50ml (milliliter) liquid nutrient medium is housed, 25-35 ℃ of static cultivation 16-36 hour, and prove conclusively the pure of bacterial classification with the method that microscopy and inclined-plane are coated with.
The female kind of preferred test tube activates, and original seed is received in the test tube that the 10ml liquid nutrient medium is housed, and cultivates 24 hours for 30 ℃, and be single by microscopy and inclined-plane coating conclusive evidence bacterial classification.
(3) to spread cultivation be that ratio in 3-10% is linked into test tube strains in the 150ml triangular flask that the 100ml liquid nutrient medium is housed to triangular flask, cultivated 16-36 hour for 25-35 ℃, press the inoculum size of 3-10% again, bacterial classification in the 150ml triangular flask is linked in the 5000ml triangular flask that the 3000ml liquid nutrient medium is housed 25-35 ℃ of static cultivation 16-36 hour.
Preferably the test tube kind is linked in the 150ml triangular flask that the 100ml liquid nutrient medium is housed, in 5% ratio the seed 90ml in the 150ml triangular flask is linked in the 5000ml volumetrical triangular flask that the 3000ml liquid nutrient medium is housed again in 5% ratio.
(4) seeding tank spread cultivation be by the inoculum size of 3-10% with the bacterial classification inoculation of triangular flask in seeding tank, and guarantee that with the method for inflated with nitrogen tank pressure is 0.15-0.5kg/cm
2(kilogram/square centimeter); Discontinuity stirs 200-500 rev/min; Stirred 25-35 ℃ of static cultivation 8-24 hour in every 1.5-3 hour 4-8 minute.
Preferably the triangular flask kind is inoculated in the seeding tank, protects tank pressure 0.3kg/cm with inflated with nitrogen in 5% ratio
2Discontinuity stirs 320 rev/mins; Stirred 5 minutes in per 2 hours.
After this, fermention medium fermentation and production lactic acid bacteria formulation and bacteriocin lab comprise:
At first, preparation fermention medium: adopt corn alcohol waste dregs liquid filtrate to add the 0-0.5% yeast extract paste; 0-2% sucrose; 0-2% glucose; 0-0.2%KH
2PO
4PH is 5.5-6.0.
Fermentation condition is: after the conventional sterilization, the inoculum size of pressing 3-10% to fermentor tank, and remains on 0.15-0.5kg/cm with the method that charges into nitrogen with tank pressure with the bacterial classification inoculation that spreads cultivation among the S13
2, 25-35 ℃ of discontinuity stirs 200-500 rev/min, stirs 4-8 minute in every 1.5-3 hour, cultivates 18-36 hour, transfers pH value to 1.75-2.25, leaves standstill 8-16 hour.
Preferably, the inoculum size by 5%, the bacterium order is 6 hours, 30 ℃ of discontinuity stir, and 350 rev/mins, stirred 5 minutes in per 2 hours, keep tank pressure 0.3kg/cm in jar
2Cultivated 18-36 hour, PH is transferred to 2, leave standstill overnight.
After this, with Plate Filtration or centrifugally collect thalline and filtrate respectively, thalline can be made the bacterium powder by vacuum lyophilization or low-temperature vacuum drying, i.e. the lactic acid bacteria biological preparation.
Perhaps, collect filtrate, through the membrane filtration of 2000 molecular weight, collect the material between the 2000-20000 molecular weight again the ultrafiltration membrance filter of filtrate by 20000 molecular weight; And add skim-milk or NaCl, making it moisture content is about 50%; Then, spraying drying, preferred centrifugal spray dryer, processing condition, 230 ℃ of inlet temperature, 90 ℃ of air outlet temperatures, spouting liquid are 1000kg/h, 500 kilograms/hour of product volumes (kg/h); Last crushing packing becomes the product bacteriocin.
Embodiment 1 corn alcohol waste dregs liquid liquid submerged fermentation is produced lactic acid bacteria formulation
One, the preparation of corn alcohol waste dregs liquid filtrate
Collect fresh alcohol waste lees solution, centrifugal by horizontal centrifuge (north of the Changjiang River, Sichuan machine works produces WL450), solid-liquid separation is got parting liquid and is filtered through flame filter press, collects filtrate, is alcohol waste lees solution filtrate.
Two, the processing condition of lactobacillus-fermented
1, the lactic acid bacteria culturers technology that spreads cultivation:
(1) produce bacterial strain:
Bacterium lacticum Lactobacillus gasseri 1-7 formats;
Lactobacillus crispatus Lactobacillus crispatus 23-5;
Enterococcus faecalis Entercococcus faecalis 23-7;
Lyophilize, the preservation bacterial classification, industrial microorganism institute of Liaoning Normal University provides.
The spread cultivation proportioning of substratum of liquid spawn: corn alcohol waste dregs liquid filtrate adds 0.5% yeast extract paste; Add 2% sucrose; PH6.0-6.5; 15 pounds of sterilizations in 30 fens.
(2) the female kind of test tube activates: mother's kind is inoculated into respectively in the test tube that the 10ml liquid nutrient medium is housed, cultivated 24 hours for 30 ℃, and single by microscopy and inclined-plane coating conclusive evidence.
(3) triangular flask spreads cultivation: the inoculum size by 5%, the test tube kind is linked in the 150ml triangular flask that the 100ml liquid nutrient medium is housed, and cultivated 24 hours for 30 ℃.By 5% inoculum size, the 90ml bacterial classification in the 150ml triangular flask is linked in the 5000ml volume triangular flask that the 3000ml liquid nutrient medium is housed again.Cultivated 24 hours for 30 ℃.
(4) seeding tank spreads cultivation: by 5% inoculum size, with the triangular flask bacterial classification inoculation in seeding tank; Inflated with nitrogen guarantees tank pressure 0.3kg/cm
2Discontinuity stirs 320 rev/mins; Stirred 5 minutes in per 2 hours.
2, fermention medium and fermentation condition
(1) fermention medium: corn alcohol waste dregs liquid filtrate is added yeast extract paste 0.5%; Sucrose 2%; Glucose 1%; Potassium primary phosphate 0.2%; PH5.5-6.0.
(2) fermentation condition: routinely after the sterilization, the inoculum size with 5% makes bacterium that coccus is 6 hours, and bacillus is that 24 hours bacterial classification is linked in the fermentor tank, 30 ℃ of cultivations; Discontinuity stirs 350 rev/mins, stirs 5 minutes in per 2 hours, fills aseptic nitrogen, keeps tank pressure 0.3kg/cm
2, coccus was cultivated 20 hours, and bacillus was cultivated 36 hours, PH was transferred to 2, standing over night with HCl during the fermentation termination.
Three, the preparation of lactobacillus powder
The fermented liquid that leaves standstill overnight is passed through Plate Filtration or centrifugal collection thalline; Add skimmed milk or casein hydrolysate as the protective material of milk-acid bacteria,, make the bacterium powder, be the lactic acid bacteria freeze drying powder by vacuum lyophilization or low-temperature vacuum drying.
And filtrate part is transferred PH to 3.5 by adding ammoniacal liquor, and this filtrate can be used as the diluent of bacterium powder, before use the bacterium powder is mixed with diluent in the special spray bottle of packing in the airbag, makes aerosol.Or in separately the bacterium powder being incapsulated, make suppository.
Embodiment 2 corn alcohol waste dregs liquid liquid submerged fermentations are produced bacteriocin lab
One, the preparation of corn alcohol waste dregs liquid filtrate
Collect fresh alcohol waste lees solution, centrifugal by horizontal centrifuge (north of the Changjiang River, Sichuan machine works produces WL450), solid-liquid separation is got parting liquid and is filtered through flame filter press, collects filtrate, is alcohol waste lees solution filtrate.
Two, the processing condition of lactobacillus-fermented
1, the lactic acid bacteria culturers technology that spreads cultivation:
(1) produce bacterial strain:
Streptococcus acidi lactici (Lactococcus lactis.LN992);
Short lactobacillus (Lactobacillus brevis);
Lyophilize, the preservation bacterial classification provides by industrial microorganism institute of Liaoning Normal University.
(2) the liquid spawn substratum that spreads cultivation:
Corn alcohol waste dregs liquid filtrate adds 0.5% yeast extract paste; Add 0.5% sucrose; 15 pounds of sterilizations in 30 fens of PH5.5-6.0.
(3) the female kind of test tube activates: mother's kind is inoculated into respectively in the test tube that the 10ml liquid nutrient medium is housed, cultivated 24 hours for 30 ℃, and single by microscopy and inclined-plane coating conclusive evidence.
(4) triangular flask spreads cultivation: the inoculum size by 5%, the test tube kind is linked in the 150ml triangular flask that the 100ml liquid nutrient medium is housed, and cultivated 24 hours for 30 ℃.By 5% inoculum size, the 90ml bacterial classification in the 150ml triangular flask is linked in the 5000ml volume triangular flask that the 3000ml liquid nutrient medium is housed again.Cultivated 24 hours for 30 ℃.
(5) seeding tank spreads cultivation: the inoculum size by 5% is linked into the short lactobacillus bacterial classification in the triangular flask in the seeding tank; Inoculum size by 3% is linked into the streptococcus acidi lactici bacterial classification in the triangular flask in the seeding tank, spreads cultivation respectively.Inflated with nitrogen is protected tank pressure 0.3kg/cm
2, 30 ℃ of cultivations, discontinuity stirs 320 rev/mins, stirs 5 minutes in per 2 hours, and short lactobacillus was cultivated 24 hours, and streptococcus acidi lactici was cultivated 6 hours.
2, fermention medium and zymotechnique
(1) preparation of fermention medium: corn alcohol waste dregs liquid filtrate is added yeast extract paste 0.5%; Sucrose 0.5%; Potassium primary phosphate 0.2%; PH5.5-6.0
(2) zymotechnique: routinely after the sterilization, the inoculum size with 5% makes bacterium that coccus is that 24 hours short lactobacillus is inserted in the fermentor tank, is 6 hours with the bacterium order in addition, the inoculum size with 3% is inoculated into streptococcus acidi lactici in another fermentor tank 30 ℃ of cultivations; Discontinuity stirs 350 rev/mins, stirs 5 minutes in per 2 hours, and inflated with nitrogen keeps tank pressure 0.3kg/cm
2, coccus was cultivated 20 hours, and bacillus was cultivated 36 hours, during the fermentation termination, transferred PH to 2, standing over night with HCl.
Three, the preparation of bacteriocin lab
(1) Plate Filtration: pH value 2, the short lactobacillus fermented liquid that leaves standstill overnight and streptococcus acidi lactici fermented liquid are passed through Plate Filtration respectively, and collect filtrate respectively.
(2) ultrafiltration membrance filter, selection for the first time can be held back the ultra-filtration membrane of the above material of 20000 molecular weight, carries out ultrafiltration, and collects the filtrate of streptococcus acidi lactici and the filtrate of short lactobacillus fermented liquid respectively.And they are held back the membrane filtration second time of 2000 above Middle Molecular Substances respectively, and collect concentrated solution respectively, this concentrated solution is the material between the 2000-20000 molecular weight.
(3) spraying drying is got above-mentioned (2) concentrated solution and is added skim-milk or sex change dextrin and NaCl to make it water content be about 50%.Utilize the centrifugal spray dryer inlet temperature to transfer to 230 ℃, 90 ℃ of air outlet temperatures, spouting liquid are 1000kg/h, and product volume is 500kg/h.
(4) two kinds of products of crushing packing can be considered as the fermentating metabolism thing of lactic-acid-bacterium, also have the said function of broad-spectrum bacteriocins.
The above; only be the preferable embodiment of the present invention; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, all should be encompassed within protection scope of the present invention.
Claims (10)
1, a kind of method of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation comprises the step that obtains alcohol waste lees solution after the zymamsis, it is characterized in that described alcohol waste lees solution is the corn alcohol waste dregs liquid, and described method also comprises the steps:
S11, acquisition clear liquid: described alcohol waste lees solution solid-liquid separation is obtained clear liquid;
S12, substratum obtains to spread cultivation: get the clear liquid that step S11 obtains, add 0-0.5% yeast extract paste, 0-4% sucrose, transfer pH value to 5.5-6.5;
S13, get bacterial classification and carry out spreading cultivation step by step of triangular flask, seeding tank with the inoculum size of 3-10%;
S14, acquisition fermention medium: get the clear liquid that step S11 obtains, add the 0-0.5% yeast extract paste; 0-4% sucrose; 0-4% glucose; The 0-0.2% potassium primary phosphate; The accent pH value is 5.5-6.5;
S15, fermentation: after the conventional sterilization, the inoculum size of pressing 3-10% to fermentor tank, and remains on 0.15-0.5kg/cm with the method that charges into nitrogen with tank pressure with the bacterial classification inoculation that spreads cultivation among the S13
2, 25-35 ℃ of discontinuity stirs 200-500 rev/min, stirs 4-8 minute in every 1.5-3 hour, cultivates 18-36 hour, transfers pH value to 1.75-2.25, leaves standstill 8-16 hour;
S16, acquisition lactic acid bacteria formulation: the fermented liquid that leaves standstill the back gained among the step S15 is filtered, collect filter cake, vacuum-drying becomes the solid lactic acid bacteria preparation.
According to the described method of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation of claim 1, it is characterized in that 2, the described clear liquid method of acquisition is among the step S11:
Utilize whizzer that described alcohol waste lees solution centrifugation is obtained described clear liquid; Perhaps,
Utilize filter type to obtain described clear liquid; Again or,
After the clear liquid that described centrifugal or filter type is obtained concentrates once more thin up obtain described clear liquid.
3, according to claim 1 or the 2 described methods of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation, it is characterized in that,
Utilize NaOH and HCl to transfer pH value among step S12, the S14; Transfer pH value with phosphoric acid among the step S15.
4, according to claim 1 or the 2 described methods of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation, it is characterized in that, the described solid lactic acid bacteria preparation that step S16 obtains is made powdery lactic acid bacteria biological preparation.
5, according to the described method of utilizing alcohol waste lees solution to produce lactic acid bacteria formulation of claim 4, it is characterized in that, comprise among the step S13:
S131, female plant activation: mother is planted receive in the container that the 10-50ml liquid nutrient medium is housed, 25-35 ℃ of static cultivation 16-36 hour, and prove conclusively the pure of bacterial classification with the method that microscopy and inclined-plane are coated with;
It is that ratio in 3-10% is linked into test tube strains in the 150ml triangular flask that the 100ml liquid nutrient medium is housed that S132, triangular flask spread cultivation, cultivated 16-36 hour for 25-35 ℃, press the inoculum size of 3-10% again, bacterial classification in the 150ml triangular flask is linked in the 5000ml triangular flask that the 3000ml liquid nutrient medium is housed 25-35 ℃ of static cultivation 16-36 hour;
S133, seeding tank spread cultivation be by the inoculum size of 3-10% with the bacterial classification inoculation of triangular flask in seeding tank, and guarantee that with the method for inflated with nitrogen tank pressure is 0.15-0.5kg/cm
2Discontinuity stirs 200-500 rev/min; Stirred 4-8 minute in every 1.5-3 hour, 25-35 ℃, static cultivation 8-36 hour.
6, a kind of method of utilizing alcohol waste lees solution to produce bacteriocin lab comprises that zymamsis obtains the step of alcohol waste lees solution, it is characterized in that described alcohol waste lees solution is the corn alcohol waste dregs liquid, and described method also comprises the steps:
S21, described alcohol waste lees solution solid-liquid separation is obtained clear liquid;
S22, substratum obtains to spread cultivation: get the clear liquid that step S21 obtains, add 0-0.5% yeast extract paste, 0-4% sucrose, the accent pH value is 5.5-6.5;
S23, substratum obtains to spread cultivation: get bacterial classification and carry out spreading cultivation step by step of triangular flask, seeding tank with the inoculum size of 3-10%;
S24, acquisition fermention medium: get the clear liquid that step S21 obtains, add the 0-0.5% yeast extract paste; 0-4% sucrose; 0-4% glucose; The 0-0.2% potassium primary phosphate; The accent pH value is 5.5-6.5;
S25, fermentation: after the conventional sterilization, the inoculum size of pressing 3-10% to fermentor tank, and remains on 0.15-0.5kg/cm with the method that charges into nitrogen with tank pressure with the bacterial classification inoculation that spreads cultivation among the S13
2, 25-35 ℃ of discontinuity stirs 200-500 rev/min, stirs 4-8 minute in every 1.5-3 hour, cultivates 18-36 hour, transfers pH value to 1.75-2.25, leaves standstill 8-16 hour;
S26, acquisition bacteriocin lab comprise:
S261, collect filtrate after filtration, filter the material of holding back 20,000 molecular weight through ultrafilter again, collect the ultrafiltration filtered solution; Described ultrafiltration filtered solution ultrafiltration is once more held back material between the 2000-20000 molecular weight;
S262, in the concentrated solution that step S261 holds back, add skim-milk or NaCl or sex change dextrin and make described concentrated solution water content be transferred to 50-60%;
The described concentrated solution that S263, S262 obtain is through the centrifugal spray dryer spraying drying, actual conditions is that inlet temperature transfers to 200-230 ℃, and air outlet temperature transfers to 90 ℃, and spouting liquid is 500-1000kg/h, must consolidate the shape bacteriocin lab, product volume is 250-500kg/h.
According to the described method of utilizing alcohol waste lees solution to produce bacteriocin lab of claim 6, it is characterized in that 7, the described clear liquid method of acquisition is among the step S21:
Utilize whizzer that described alcohol waste lees solution centrifugation is obtained described clear liquid; Perhaps,
Utilize filter type to obtain described clear liquid; Again or,
After the clear liquid that described centrifugal or filter type is obtained concentrates once more thin up obtain described clear liquid.
8, according to claim 6 or the 7 described methods of utilizing alcohol waste lees solution to produce bacteriocin lab, it is characterized in that, utilize NaOH and HCl to transfer pH value among step S22, the S24; Transfer pH value with phosphoric acid among the step S25.
9, according to claim 6 or the 7 described methods of utilizing alcohol waste lees solution to produce bacteriocin lab, it is characterized in that, the described solid shape bacteriocin lab that step S26 is obtained pulverize, pack the bacteriocin product.
10, according to the described method of utilizing alcohol waste lees solution to produce bacteriocin lab of claim 9, it is characterized in that, comprise among the step S23:
S231, female plant activation: mother is planted receive in the container that the 10-50ml liquid nutrient medium is housed, 25-35 ℃ of static cultivation 16-36 hour, and prove conclusively the pure of bacterial classification with the method that microscopy and inclined-plane are coated with;
It is that ratio in 3-10% is linked into test tube strains in the 150ml triangular flask that the 100ml liquid nutrient medium is housed that S232, triangular flask spread cultivation, cultivated 16-36 hour for 25-35 ℃, press the inoculum size of 3-10% again, bacterial classification in the 150ml triangular flask is linked in the 5000ml triangular flask that the 3000ml liquid nutrient medium is housed 25-35 ℃ of static cultivation 16-36 hour;
S233, seeding tank spread cultivation be by the inoculum size of 3-10% with the bacterial classification inoculation of triangular flask in seeding tank, and guarantee that with the method for inflated with nitrogen tank pressure is 0.15-0.5kg/cm
2Discontinuity stirs 200-500 rev/min; Stirred 25-35 ℃ of static cultivation 8-36 hour in every 1.5-3 hour 4-8 minute.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101475973B (en) * | 2009-01-20 | 2010-08-18 | 海南椰国食品有限公司 | Bacteria cellulose production culture medium containing alcohol wastewater |
| CN101822310A (en) * | 2010-05-06 | 2010-09-08 | 江南大学 | Method for cultivating lactobacillus and producing live lactobacillus feed by utilizing scum |
| CN102132794A (en) * | 2011-04-02 | 2011-07-27 | 河南科技大学 | Production method of complete feed for chicken |
| CN111466573A (en) * | 2020-04-13 | 2020-07-31 | 金华银河生物科技有限公司 | Concentration and extraction process of high-activity lactic acid bacteria metabolites |
-
2006
- 2006-01-04 CN CN 200610005231 patent/CN1807592A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101475973B (en) * | 2009-01-20 | 2010-08-18 | 海南椰国食品有限公司 | Bacteria cellulose production culture medium containing alcohol wastewater |
| CN101822310A (en) * | 2010-05-06 | 2010-09-08 | 江南大学 | Method for cultivating lactobacillus and producing live lactobacillus feed by utilizing scum |
| CN102132794A (en) * | 2011-04-02 | 2011-07-27 | 河南科技大学 | Production method of complete feed for chicken |
| CN102132794B (en) * | 2011-04-02 | 2012-11-14 | 河南科技大学 | Production method of complete feed for chicken |
| CN111466573A (en) * | 2020-04-13 | 2020-07-31 | 金华银河生物科技有限公司 | Concentration and extraction process of high-activity lactic acid bacteria metabolites |
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