CN101524180B - Composite microbiological antibiotic and preparation method thereof - Google Patents
Composite microbiological antibiotic and preparation method thereof Download PDFInfo
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- CN101524180B CN101524180B CN2009100718548A CN200910071854A CN101524180B CN 101524180 B CN101524180 B CN 101524180B CN 2009100718548 A CN2009100718548 A CN 2009100718548A CN 200910071854 A CN200910071854 A CN 200910071854A CN 101524180 B CN101524180 B CN 101524180B
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Abstract
The invention provides a composite microbiological antibiotic and a preparation method thereof. The preparation method comprises the steps of respectively culturing lactobacillus rhamnose (A) and lactobacillus acidophilus (B) in an MRS culture medium for 48 hours at 37 DEG C, centrifuging the fermentation liquid at a high speed, adding solid ammonium sulphate into the supernatant, dissolving the generated precipitate in distilled water, purifying the precipitate by gel chromatography and performing freeze dehydration to obtain lactobacillus bacteriocin, adding water in Aspergillus niger thallus based on a ratio of 1:6, milling and crushing by colloid to form homogenate, heating to 55 DEG C, adding 1.5% of cellulase, 1.5% of pectinase, 2% of beta-dextranase and 2% of acid prolealytic enzyme, and mixing uniformly, maintaining temperature at 55 DEG C and hydrolyzing for 6 hours, killing enzyme for 10 minutes at 100 DEG C, after cooling, taking the centrifugal supernatant and performing freeze dehydration to obtain enzymolysis frozen dry powder (C) of mycelium content, and finally uniformly mixing bacteriocin A, bacteriocin B and C in a weight ratio of (15-20):(18-25):(20-35) to obtain the finished product.
Description
(1) technical field
The present invention relates to a kind of wide spectrum composite microbiological antibiotic and preparation method thereof, belong to the food microorganisms technical field.
(2) background technology
So-called microbiological antibiotic is that synthetic justacrine has inhibiting sterilization albumen or peptide material to the class in the environment to kind of the same race or that affiliation is nearer in the microbial metabolism.Other microorganisms in same or similar habitat with it can be killed or suppress to these materials, and all microorganisms that produce antibiotic all exist the specific immunity gene, and the antibiotic of generation can not damage producing bacterium itself.Along with progress of research, found that some have the antibiotic of broad-spectrum antibacterial activity to food spoilage microorganism and pathogenic microorganism.
Lactic acid bacteria antibiotic starts from late nineteen eighties in the research of China, but mainly be aimed at the research of Nisin, the screening and the molecular biological research of new bacterial strain and superior strain, and the bacteriocin that other lactic acid bacteria is produced in theory still the research on using is all not mature enough.1988, Institute of Micro-biology of the Chinese Academy of Sciences began Nisin is carried out basic research theoretical and that use, separated and filtered out the lactic streptococcus strains that can produce nisin Z; China classifies it as food additives amendments in nineteen ninety.To the nineties in 20th century, domestic scholars other bacteriocin labs that just begin one's study.Except that nisin, the research of China's bacteriocin lab mainly concentrates on bacterial strain screening, separation and purification and general biological characteristic research, produces the output that bacterium is improved bacteriocin by optimization of fermentation conditions and mutagenesis bacteriocin.Molecular biology research to bacteriocin is less.Lactic acid bacteria is widely distributed at occurring in nature, and be the dominant bacteria in the normal flora in human and animal's enteron aisle, lactic acid bacteria not only has many physiological functions healthy and helpful to the human and animal, and can produce the material that much has bacteriostasis, as antibacterial substances such as organic acid (as lactic acid), hydrogen peroxide and bacteriocins, these antibacterial substances can not only effectively keep flavours in food products and structural state, spoilage organisms that can also suppress to exist in the food and growth of pathogenic bacteria, thereby prevent the corruption of food, prolong the preservation term of food.The bacteriocin that lactic acid bacteria produces is a kind of antiseptics for natural food and feed addictive with broad prospect of application, and it is easy to by the proteasome degradation in the human body self alimentary canal the animal avirulence, does not cause bad reaction so can not accumulate in vivo.
Bacteriocin lab is not a broad-spectrum antiseptic as foodstuff antibiotic, and is apparent in view to the gram-positive bacteria fungistatic effect, but fungies such as Gram-negative bacteria, mould, yeast are pressed down DeGrain extremely.Patent of the present invention selects for use two strains of lactic acid bacteria composite fermentations to produce bacteriocin, composite in proportion with contents such as fungal cell's polysaccharide, prepare a kind of microorganism composite antibiosis element with broad-spectrum antibacterial action, it is fresh-keeping to be mainly used in varieties of food items, this antibiotic is not subjected to the restriction of temperature, pH value, and it is obvious that mould, yeast and varieties of food items spoilage organisms are suppressed effect.
(3) summary of the invention
The object of the invention is to provide a kind of composite microbiological antibiotic and preparation method thereof.
The object of the present invention is achieved like this: related ratio is mass ratio except that other has indicating among the present invention, and product of the present invention adopts such method to prepare:
1. preparation process
1.1 lactic acid bacteria mixed culture fermentation prepares bacteriocin
1.1.1 bacterial classification source
Fermentation strain is bought in Chinese industrial microorganism fungus kind preservation center, title and being numbered: Lactobacillus rhamnosus CICC6141 (A), lactobacillus acidophilus CICC 6075 (B).
1.1.2 sweat
1.2.2.1MRS the preparation of culture medium
Peptone 1%, beef extract 1%, yeast extract 0.5%, K
2HPO
40.2%, citric acid diamines 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1%, MgSO
47H
2O0.058%, MnSO
44H
2O 0.025%, and all the other are deionized water, and pH6.2-6.4 sterilized 20 minutes for 121 ℃.
1.2.2.2 the activation of fermentation strain
Measure 0.9% physiological saline 10ml respectively in vitro in 5, be cooled to 30 ℃ in 20 minutes through 121 ℃ of sterilizations, respectively the freeze-dried vaccine powder in Lactobacillus rhamnosus CICC 6141 and the lactobacillus acidophilus CICC 6,075 two strains of lactic acid bacteria peace bottle is all poured under germ-free condition in 0.9% the physiological saline, concussion makes its dissolving, static activation is 30 minutes in 30 ℃ of insulating boxs, and is standby.
1.2.2.3 lactic acid bacteria composite fermentation
Lactic acid bacteria after the activation is inoculated in respectively in the 1.2.2.1 culture medium by 10% inoculum concentration, and 37 ℃ of anaerobism are cultivated 48h, finish fermentation.
1.1.3 the leaching process of bacteriocin
1.1.3.1 the preparation of bacteriocin crude extract
In 10000 rev/mins, high speed centrifugation 15min gets supernatant with cultured bacterial strain fermentation liquor, places 4 ℃ of preservations standby.
1.1.3.2 ammonium sulfate precipitation bacteriocin
The centrifuged supernatant for preparing is contained in the conical flask after handling with 0.45 μ m bacteriological filtration film, every bottled 100mL that has, and adding solid ammonium sulfate then respectively, to make its saturation degree be 70%, shaken overnight.8000 rev/mins, 4 ℃ centrifugal 30min, collecting precipitation is used a small amount of dissolved in distilled water.
1.1.3.3 gel chromatography bacteriocin
With preswollen Sephacryl S-100 gel column adopt sodium acetate buffer (0.05mol/L, pH5.1), balance SephacrylS-100 gel column.Adopting applied sample amount is the bacteriocin (sample behind the ammonium sulfate precipitation) of 1mL, uses the sodium acetate buffer elute protein, and elution speed is 0.3mL/min, and collecting eluent with fraction collector, every 10min collects 1 pipe, every pipe 3mL, collect the component of No. 15 test tubes, this component is the bacteriocin behind the purifying.
1.1.3.4 freeze-drying concentrates
Bacteriocin behind the purifying carries out freeze-drying and concentrates, and the moisture in the bacteriocin under the condition of vacuum, low temperature behind the eliminating purifying more than the 95-99% finally obtains pulverous bacteriocin.
1.2 the preparation of black-koji mould filament Dissolve things inside
1.2.1 aspergillus niger thalline source
The black-koji mould filament derives from the waste products of citric acid fermentation producer---the aspergillus niger thalline.
1.2.2 preparation method
The aspergillus niger thalline is added water in 1: 6 ratio, through DSH150 model high-shear colloid mill, after pulverizing homogenate, make the fragmentation of black-koji mould filament, broken tissue is less than 5 μ m, aspergillus niger thalline homogenate is heated to 55 ℃, add 1.5% cellulase (20,000 units/gram), 1.5% pectase (20,000 units/gram), 2% 1,4 beta-glucanase (30,000 units/gram), 2% acid protease (50,000 units/gram), 55 ℃ are incubated hydrolysis 6 hours behind the abundant mixing, be warming up to 100 ℃ then, keep 10 minutes enzymes that go out, cooling is after tube centrifuge high speed centrifugation under 10000 rev/mins rotating speed is got centrifuged supernatant, and it is 50-55% that vacuum is concentrated into solid content, carry out freeze drying with reference to the 1.1.3.4 step then, obtain black-koji mould filament Dissolve things inside enzymolysis freeze-dried powder (C).
1.3 the preparation of composite microbiological antibiotic
Be the A bacteriocin by weight: B bacteriocin: C=(15-20): (18-25): the abundant mixing of ratio (20-35), make the composite microbiological antibiotic finished product.
2. effect
Prove the effect of patent of the present invention below by concrete experiment
2.1 test material and method
2.1.1 indication bacterial classification
Salmonella, staphylococcus aureus are with 0.9% physiological saline activation 2-4 hour, equal-volume mixing for standby use.
2.1.2 experimental technique
2.0% plain agar, be chilled to about 50 ℃, be poured in the aseptic plate by every plate 10mL, drying the Oxford cup of getting in advance sterilization with aseptic nipper in the clean bench is placed in the plate of agar, aseptic scraper coating indicator bacteria overnight incubation, preparation contains 0.7% agar indicator bacteria culture medium, be chilled to about 50 ℃, every 100mL soft agar medium inoculation 1mL indicator bacteria is on the flat board of putting the Oxford cup in advance well, topple over 10mL and contain the soft agar medium of indicator bacteria, dry, adding 100 μ L concentration in the hole of Oxford cup is 10
-6G/ml microorganism composite antibiosis element, the air blast diffusion is 3 hours in clean bench, places 35 ℃ of cultivations to measure antibacterial circle diameter with slide measure after 48 hours.
2.2 result and analysis
Transparent around the Oxford cup in the agar plate, do not have the growth of indicator bacteria, and dull and stereotyped other position overgrows with indicator bacteria, illustrates that patent of the present invention can effectively suppress the growth of pathogenic bacteria such as salmonella, staphylococcus aureus.
(4) description of drawings
Fig. 1 is the inhibition design sketch of microorganism composite antibiosis element to salmonella, staphylococcus aureus.
(5) specific embodiment
For a more detailed description below in conjunction with specific embodiment to the present invention:
Embodiment one:
1. preparation process
1.1 lactic acid bacteria mixed culture fermentation prepares bacteriocin
1.1.1 bacterial classification source
Fermentation strain is bought in Chinese industrial microorganism fungus kind preservation center, title and being numbered: Lactobacillus rhamnosus CICC6141 (A), lactobacillus acidophilus CICC 6075 (B).
1.1.2 sweat
1.2.2.1MRS the preparation of culture medium
Peptone 1%, beef extract 1%, yeast extract 0.5%, K
2HPO
40.2%, citric acid diamines 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1%, MgSO
47H
2O0.058%, MnSO
44H
2O 0.025%, and all the other are deionized water, and pH6.2-6.4 sterilized 20 minutes for 121 ℃.
1.2.2.2 the activation of fermentation strain
Measure 0.9% physiological saline 10ml respectively in vitro in 5, be cooled to 30 ℃ in 20 minutes through 121 ℃ of sterilizations, respectively the freeze-dried vaccine powder in Lactobacillus rhamnosus CICC 6141 and the lactobacillus acidophilus CICC 6,075 two strains of lactic acid bacteria peace bottle is all poured under germ-free condition in 0.9% the physiological saline, concussion makes its dissolving, static activation is 30 minutes in 30 ℃ of insulating boxs, and is standby.
1.2.2.3 lactic acid bacteria composite fermentation
Lactic acid bacteria after the activation is inoculated in respectively in the 1.2.2.1 culture medium by 10% inoculum concentration, and 37 ℃ of anaerobism are cultivated 48h, finish fermentation.
1.1.3 the leaching process of bacteriocin
1.1.3.1 the preparation of bacteriocin crude extract
In 10000 rev/mins, high speed centrifugation 15min gets supernatant with cultured bacterial strain fermentation liquor, places 4 ℃ of preservations standby.
1.1.3.2 ammonium sulfate precipitation bacteriocin
The centrifuged supernatant for preparing is contained in the conical flask after handling with 0.45 μ m bacteriological filtration film, every bottled 100mL that has, and adding solid ammonium sulfate then respectively, to make its saturation degree be 70%, shaken overnight.8000 rev/mins, 4 ℃ centrifugal 30min, collecting precipitation is used a small amount of dissolved in distilled water.
1.1.3.3 gel chromatography bacteriocin
With preswollen Sephacryl S-100 gel column adopt sodium acetate buffer (0.05mol/L, pH5.1), balance SephacrylS-100 gel column.Adopting applied sample amount is the bacteriocin (sample behind the ammonium sulfate precipitation) of 1mL, uses the sodium acetate buffer elute protein, and elution speed is 0.3mL/min, and collecting eluent with fraction collector, every 10min collects 1 pipe, every pipe 3mL, collect the component of No. 15 test tubes, this component is the bacteriocin behind the purifying.
1.1.3.4 freeze-drying concentrates
Bacteriocin behind the purifying carries out freeze-drying and concentrates, and the moisture in the bacteriocin under the condition of vacuum, low temperature behind the eliminating purifying more than the 95-99% finally obtains pulverous bacteriocin.
1.2 the preparation of black-koji mould filament Dissolve things inside
1.2.1 aspergillus niger thalline source
The black-koji mould filament derives from the waste products of citric acid fermentation producer---the aspergillus niger thalline.
1.2.2 preparation method
The aspergillus niger thalline is added water in 1: 6 ratio, through DSH150 model high-shear colloid mill, after pulverizing homogenate, make the fragmentation of black-koji mould filament, broken tissue is less than 5 μ m, aspergillus niger thalline homogenate is heated to 55 ℃, add 1.5% cellulase (20,000 units/gram), 1.5% pectase (20,000 units/gram), 2% 1,4 beta-glucanase (30,000 units/gram), 2% acid protease (50,000 units/gram), 55 ℃ are incubated hydrolysis 6 hours behind the abundant mixing, be warming up to 100 ℃ then, keep 10 minutes enzymes that go out, cooling is after tube centrifuge high speed centrifugation under 10000 rev/mins rotating speed is got centrifuged supernatant, and it is 50-55% that vacuum is concentrated into solid content, carry out freeze drying with reference to the 1.1.3.4 step then, obtain black-koji mould filament Dissolve things inside enzymolysis freeze-dried powder (C).
1.3 the preparation of composite microbiological antibiotic
Be the A bacteriocin by weight: B bacteriocin: C=15: the abundant mixing of 18: 20 ratio, make the composite microbiological antibiotic finished product.
Embodiment two:
1. preparation process
1.1 lactic acid bacteria mixed culture fermentation prepares bacteriocin
1.1.1 bacterial classification source
Fermentation strain is bought in Chinese industrial microorganism fungus kind preservation center, title and being numbered: Lactobacillus rhamnosus CICC6141 (A), lactobacillus acidophilus CICC 6075 (B).
1.1.2 sweat
1.2.2.1MRS the preparation of culture medium
Peptone 1%, beef extract 1%, yeast extract 0.5%, K
2HPO
40.2%, citric acid diamines 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1%, MgSO
47H
2O0.058%, MnSO
44H
2O 0.025%, and all the other are deionized water, and pH6.2-6.4 sterilized 20 minutes for 121 ℃.
1.2.2.2 the activation of fermentation strain
Measure 0.9% physiological saline 10ml respectively in vitro in 5, be cooled to 30 ℃ in 20 minutes through 121 ℃ of sterilizations, respectively the freeze-dried vaccine powder in Lactobacillus rhamnosus CICC 6141 and the lactobacillus acidophilus CICC 6,075 two strains of lactic acid bacteria peace bottle is all poured under germ-free condition in 0.9% the physiological saline, concussion makes its dissolving, static activation is 30 minutes in 30 ℃ of insulating boxs, and is standby.
1.2.2.3 lactic acid bacteria composite fermentation
Lactic acid bacteria after the activation is inoculated in respectively in the 1.2.2.1 culture medium by 10% inoculum concentration, and 37 ℃ of anaerobism are cultivated 48h, finish fermentation.
1.1.3 the leaching process of bacteriocin
1.1.3.1 the preparation of bacteriocin crude extract
In 10000 rev/mins, high speed centrifugation 15min gets supernatant with cultured bacterial strain fermentation liquor, places 4 ℃ of preservations standby.
1.1.3.2 ammonium sulfate precipitation bacteriocin
The centrifuged supernatant for preparing is contained in the conical flask after handling with 0.45 μ m bacteriological filtration film, every bottled 100mL that has, and adding solid ammonium sulfate then respectively, to make its saturation degree be 70%, shaken overnight.8000 rev/mins, 4 ℃ centrifugal 30min, collecting precipitation is used a small amount of dissolved in distilled water.
1.1.3.3 gel chromatography bacteriocin
With preswollen Sephacryl S-100 gel column adopt sodium acetate buffer (0.05mol/L, pH5.1), balance SephacrylS-100 gel column.Adopting applied sample amount is the bacteriocin (sample behind the ammonium sulfate precipitation) of 1mL, uses the sodium acetate buffer elute protein, and elution speed is 0.3mL/min, and collecting eluent with fraction collector, every 10min collects 1 pipe, every pipe 3mL, collect the component of No. 15 test tubes, this component is the bacteriocin behind the purifying.
1.1.3.4 freeze-drying concentrates
Bacteriocin behind the purifying carries out freeze-drying and concentrates, and the moisture in the bacteriocin under the condition of vacuum, low temperature behind the eliminating purifying more than the 95-99% finally obtains pulverous bacteriocin.
1.2 the preparation of black-koji mould filament Dissolve things inside
1.2.1 aspergillus niger thalline source
The black-koji mould filament derives from the waste products of citric acid fermentation producer---the aspergillus niger thalline.
1.2.2 preparation method
The aspergillus niger thalline is added water in 1: 6 ratio, through DSH150 model high-shear colloid mill, after pulverizing homogenate, make the fragmentation of black-koji mould filament, broken tissue is less than 5 μ m, aspergillus niger thalline homogenate is heated to 55 ℃, add 1.5% cellulase (20,000 units/gram), 1.5% pectase (20,000 units/gram), 2% 1,4 beta-glucanase (30,000 units/gram), 2% acid protease (50,000 units/gram), 55 ℃ are incubated hydrolysis 6 hours behind the abundant mixing, be warming up to 100 ℃ then, keep 10 minutes enzymes that go out, cooling is after tube centrifuge high speed centrifugation under 10000 rev/mins rotating speed is got centrifuged supernatant, and it is 50-55% that vacuum is concentrated into solid content, carry out freeze drying with reference to the 1.1.3.4 step then, obtain black-koji mould filament Dissolve things inside enzymolysis freeze-dried powder (C).
1.3 the preparation of composite microbiological antibiotic
Be the A bacteriocin by weight: B bacteriocin: C=20: the abundant mixing of 25: 35 ratio, make the composite microbiological antibiotic finished product.
Claims (1)
1. the preparation method of a composite microbiological antibiotic is characterized in that:
(1) preparation of bacteriocin lab:
With Lactobacillus rhamnosus CICC 6141, lactobacillus acidophilus CICC 6075 respectively in the MRS culture medium 37 ℃ cultivated 48 hours, get supernatant behind the zymotic fluid high speed centrifugation and add solid ammonium sulfate, with the precipitation dissolved in distilled water that produces, behind gel chromatography, carry out freeze drying, obtain Lactobacillus rhamnosus bacteriocin and lactobacillus acidophilus bacteriocin;
(2) preparation of mycelium Dissolve things inside enzymolysis freeze-dried powder:
The aspergillus niger thalline is added water in 1: 6 ratio, after colloid mill is pulverized homogenate, be heated to 55 ℃, add 1.5% cellulase, 1.5% pectase, 2% 1,4 beta-glucanase, 2% acid protease, 55 ℃ are incubated hydrolysis 6 hours behind the mixing, be warming up to 100 ℃ then, keep 10 minutes enzymes that go out, get centrifuged supernatant after the cooling and carry out freeze drying acquisition black-koji mould filament Dissolve things inside enzymolysis freeze-dried powder;
(3) preparation of microorganism composite antibiosis element:
Lactobacillus rhamnosus bacteriocin, lactobacillus acidophilus bacteriocin and step 2 that step 1) is obtained) the black-koji mould filament Dissolve things inside enzymolysis freeze-dried powder that obtains is by weight (15-20): (18-25): the abundant mixing of ratio (20-35), make finished product.
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| CN102578441B (en) * | 2011-12-15 | 2013-09-25 | 宁夏盐池西部畅享农业生物科技有限公司 | Method for producing silage using compound microorganism fermentation straws |
| CN104970086B (en) * | 2015-07-14 | 2018-07-31 | 湖南农业大学 | Bio-preservative and its preparation method and application |
| CN108148789B (en) * | 2018-03-06 | 2021-05-25 | 河南科技学院 | Lactobacillus rhamnosus and application thereof in preparation of bacteriocin |
| CN109266568A (en) * | 2018-08-19 | 2019-01-25 | 东北农业大学 | A kind of prebiotic function Lactobacillus rhamnosus and its application with high bacteriocinogeny |
| CN114732941B (en) * | 2022-03-22 | 2022-12-09 | 安徽徽科生物工程技术有限公司 | Preparation method of cleaning and antibacterial gel |
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| CN1543826A (en) * | 2003-11-11 | 2004-11-10 | 樊兆武 | Lactic acid bacteria element acidifying powder and method for producing the same |
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| CN1280773A (en) * | 2000-08-09 | 2001-01-24 | 郭兴华 | Natural antibacterial composition and its application |
| CN1543826A (en) * | 2003-11-11 | 2004-11-10 | 樊兆武 | Lactic acid bacteria element acidifying powder and method for producing the same |
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