CN101086006B - Composite type biological surfactant and its production method - Google Patents
Composite type biological surfactant and its production method Download PDFInfo
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Abstract
The invention relates to a kind of complex biological surface active agent and the preparation method. It is prepared with bacillus subtilis, and the comprised component and their weight proportion are as follows: fat 98.1+- 0.2%, protein 19.1+- 0.2%, sugar 0.7+-0.1%, the critical micelle concentration of said complex biological surface active agent are 4.2- 5.0 g/l and 1.25- 1.39 g/l respectively, and they reduce interfacial force of distilled water from 78 m/m to 38.4- 39.8 mN/m, and 40.3- 40.8 mN/m. The invention is characterized in that it enriches biological surface active agent species generated by bacillus subtilis, and make it possible for bacillus subtilis wide application in environmental pollution treatment.
Description
Technical field
The present invention relates to a kind of new composite type biological surfactant that producing bacillus subtilis gives birth to and to the suitable extracting method of this bio-surfactant.
Background technology
Bio-surfactant is paid attention to by people gradually because of it has toxicological harmless or low toxic and biodegradable, and just progressively replaces " conventional surfactant " in industrial application.Yet, bio-surfactant cost costliness and in industrial application the problems such as finiteness of kind become its extensive adopted bottleneck in industry.In solid refuse compost field, bio-surfactant can improve the compost microenvironment, thereby promotes the decomposition of microorganism to organic waste, improves the processing efficiency of rubbish in the compost.Garbage compost is the reaction system of a complexity, and multiple bio-surfactant acting in conjunction will more help improving the compost microenvironment in compositing system.But if the multiple microorganism that can produce bio-surfactant is inoculated in the compost environment simultaneously, may can not grow well simultaneously because of mutual antagonistic action between the microorganism also produces multiple bio-surfactant simultaneously.Therefore, make the least possible microbe species in same compost environment system, produce multiple bio-surfactant effectively and become an outlet that addresses this problem.Ideal state is exactly that a kind of microorganism can produce bio-surfactant number of different types, that function is different.
The bio-surfactant that the producing bacillus subtilis of mentioning in the documents and materials is in the past given birth to is mainly 3 kinds of lipopeptid class materials, i.e. surfactins, iturins and fengycins.These 3 kinds of lipopeptid type biological surfactants can obtain by the method for acid deposition, and this has illustrated that also these 3 kinds of lipopeptid type biological surfactants will lose some character of their bio-surfactant under the extreme acidic conditions.Therefore, subtilis can being produced a kind ofly has surface-active bio-surfactant too at the extreme acidic conditions will make that this microorganism obtains using more widely in industry.
Summary of the invention
The objective of the invention is to overcome above-mentioned the deficiencies in the prior art, a kind of also have under extremely sour condition active composite type biological surfactant and its production method are provided.
For addressing the above problem, the present invention by the following technical solutions:
A kind of composite type biological surfactant, give birth to by producing bacillus subtilis, the composition that comprises following quality percentage composition: the lipid (binding substances that comprises pure lipid and lipid and other materials, as lipopeptid, lipoprotein or glycolipid etc.) 98.1 ± 0.2%, protein 19.1 ± 0.2%, carbohydrate 0.7 ± 0.1%; This composite type biological surfactant micelle-forming concentration is respectively 5.0～5.4g/L and 1.33～1.39g/L, and this moment, they dropped to 38.4～38.9mN/m and 40.3～40.7mN/m with the surface tension value of distilled water from 78mN/m respectively.
A kind of method of producing above-mentioned composite type biological surfactant may further comprise the steps:
With subtilis in 30～37 ℃ down behind the activation 24h, be inoculated in the enrichment culture liquid, 30～37 ℃ of temperature, cultivate 24h under rotating speed 200～250rpm condition, with the bacterial classification after the enrichment by volume mark 2～5% be inoculated in and carry out aerobic fermentation in the aerobic fermentation nutrient solution and cultivate, culture condition is: 30～37 ℃ of temperature, rotating speed 150～200rpm, Ventilation Rate 1.0～1.2vvm, the air that feeds is crossed 0.45 μ m filter membrane and is carried out filtration sterilization, determines the generation and the aerobic fermentation incubation time of composite type biological surfactant in the invention by the variation of measuring aerobic fermentation nutrient solution surface tension value.
2. get the fermented liquid high speed centrifugation degerming after above-mentioned aerobic fermentation is cultivated, centrifugal force is 11000～12000 * g, time 15～20min; Regulate supernatant liquor pH1.5～2.0 with HCl solution, then 0～4 ℃ of following standing over night, leave standstill liquid high speed centrifugation again, centrifugal force 10000～12000 * g, time 15～20min; The supernatant liquor of getting after centrifugal adds ethyl acetate in Erlenmeyer flask, be placed on then in the incubator with maximum speed of revolution rotational oscillation 5～6min, fall standing demix in separating funnel, subnatant adds the ethyl acetate with same volume last time once more, continue extraction as stated above, repeat 3 times, collect upper strata liquid.
3. the upper strata liquid with above-mentioned collection rotates evaporates to dryness in 40～45 ℃ of following vacuum, and lyophilize again can obtain composite type biological surfactant of the present invention.
Enrichment culture liquid composition is in the described step 1: NaCl 4.0～5.0g, peptone 4.0～5.0g, beef powder 2.5～3.0g, distilled water 1000mL, pH7.0～7.2; Described fermentation culture consists of: glucose 40～45g, NH
4NO
36.8～7.3g, K
2HPO
43H
2O 13.8～14.3g, NaH
2PO
4H
2O 1.95～2.05g, MgSO
47H
2O0.5～0.6g, MnSO
4H
2O 0.036～0.04g, yeast extract paste 0.5～0.6g, distilled water 1000mL, pH7.0～7.2; The aerobic fermentation incubation time is preferably 24h; V in the described step 2
Supernatant liquor: V
Ethyl acetate=9: 4～10: 3.
Advantage of the present invention:
The present invention makes producing bacillus subtilis give birth to a kind of new composite type biological surfactant, enriched the kind of the bio-surfactant that producing bacillus subtilis gives birth to, make subtilis in industry, particularly obtain using more widely aspect the control of environmental pollution to become a kind of may.Aspect the compost of the solid refuse in environmental pollution improvement, the adding of bio-surfactant will help improving the compost microenvironment, promote microorganism and organic density and the intensity of contacting, and improve organic speed of microbiological deterioration and thoroughness.The effect that different bio-surfactants play in compositing system is each has something to recommend him.Discover, when adding multiple bio-surfactant in the compositing system simultaneously, will significantly improve microorganism organic degradation rate.But various microorganisms survive in same system and may be because of each other antagonistic actions when breeding and the industrial treatment effect is had a negative impact.Therefore, if can allow the bio-surfactant of the abundant kind of microorganisms of the least possible kind, the negative impact that will reduce and avoid this antagonistic action to bring.The bio-surfactant that the producing bacillus subtilis of mentioning in the documents and materials is in the past given birth to is mainly 3 kinds of lipopeptid class materials, i.e. surfactins, iturins and fengycins.The present invention finds that subtilis suitably can produce a kind of new biological emulsifier under the situation, increased the kind of the bio-surfactant that it produces, make subtilis in industry, particularly obtain using more widely aspect the compost of environmental pollution improvement to become a kind of may.
In addition, the leaching process of this bio-surfactant is to adopt ethyl acetate in the water solution system of pH1.5～2.0 it to be extracted.The issuable lipopeptid type biological surfactant of subtilis this moment is all by sedimentation.This has embodied this composite type biological surfactant to a certain extent and has been better than those by the characteristics of settled bio-surfactant at antiacid aspect of performance.
Description of drawings
Fermented liquid surface tension value situation over time among Fig. 1: the embodiment 1;
Fermented liquid pH among Fig. 2: the embodiment 1, thalline absorbancy and receive liquid thalline absorbancy situation over time;
The micelle-forming concentration of gained bio-surfactant among Fig. 3: the embodiment 1;
The micelle-forming concentration of gained bio-surfactant among Fig. 4: the embodiment 2.
Embodiment
Embodiment 1: produce composite type biological surfactant
The bacterial classification of using among the present invention is subtilis (numbering CCTCC Bacillus subtilis AB93108), and this bacterial classification is available from China typical culture collection center, and inoculates once bacterial classification preservation under 4 ℃ of conditions per 1～February on slant medium.The bacterial classification of getting refrigeration is in 30 ℃ of activation 24h down, then in the aseptic technique platform with transfering loop from slant medium picking one ring bacterial classification inoculation in seed culture fluid, seed culture fluid in advance in the high pressure steam pot at 115 ℃ of sterilization 30min down.Postvaccinal seed culture fluid is placed in the constant incubator in 30 ℃, cultivates under the 200rpm condition.Consisting of of seed culture fluid: NaCl 5.0g, peptone 5.0g, beef powder 3.0g, distilled water 1000mL, and regulate pH7.0 with 1M NaOH.
With the seed culture fluid of cultivating 24h by volume the inoculum size of mark 2% be inoculated in the fermentation culture, fermented liquid consists of: glucose 40g, NH
4NO
36.8g, K
2HPO
43H
2O 13.8g, NaH
2PO
4H
2O 1.95g, MgSO
47H
2O 0.5g, MnSO
4H
2O 0.036g, yeast extract paste 0.5g, distilled water 1000mL, and regulate pH7.0 with 1M NaOH.Fermentation culture also descends sterilization 30min at 115 ℃ in advance in the high pressure steam pot, the postvaccinal fermentation culture of the bottled 250mL of the taper of each 500mL, be placed in the constant incubator, in 30 ℃ of temperature, rotating speed 200rpm, cultivate under the condition of Ventilation Rate 1.0vvm, the air of feeding is crossed the filter membrane of 0.45 μ m and is sterilized.The foam that produces in the fermenting process flows in the 500mL receiving bottle by outlet pipe, and 10mL regulates pH2.0 with 1N HCl distilled water is housed in the receiving bottle in advance.
Every 12h gets fermented liquid 20mL, centrifugal 15min under 5600 * g, measure the surface tension value (as Fig. 1) of supernatant liquor with automatic interfacial tensimeter, surface tension dropped to 39.2mN/m by initial 61.3mN/m when fermented liquid was cultivated 24h, the surface tension value of fermented liquid reaches minimum, means the output maximum of bio-surfactant in fermented liquid this moment.The thalline that residues in after centrifugal in the centrifuge tube dilutes with sterilized water, under the 600nm wavelength, measure thalline absorbancy (as Fig. 2) with ultraviolet spectrophotometer, linear according to certain condition hypothallus concentration and thalline absorbancy, subtilis reached the logarithmic phase later stage when as seen fermented liquid was cultivated 24h, at this moment the cell concentration maximum.The generation that we can obtain this kind bio-surfactant in conjunction with Fig. 1 and Fig. 2 is the growth relationship type.Thalli growth situation in the receiving bottle can obtain (as Fig. 2) with the same method of said determination fermented liquid thalline absorbancy by getting 5.0mL bacterium liquid, and cell concentration is along with the foamy amount that flows in the receiving bottle increases and increases.Directly get the 30mL fermented liquid and measure its pH (as Fig. 2), can find that the optimum growh environment of subtilis in this kind fermentation culture is neutral little slant acidity, the pH of correspondence was 6.83 when fermented liquid cultivation 24h cell concentration reached maximum.
Get the fermented liquid centrifugal 15min under 12000 * g that cultivates 24h and remove thalline, with being put in 4 ℃ of following standing over night behind the 6N HCl adjusting supernatant liquor pH2.0.Leave standstill liquid centrifugal 15min under 12000 * g, the issuable lipopeptid type biological surfactant of subtilis (comprising surfactins, iturins and fengycins etc.) is removed.Get supernatant liquor 450mL after centrifugal in the 1000mL Erlenmeyer flask, add the 200mL ethyl acetate, be placed on then in the incubator with maximum speed of revolution rotational oscillation 5min, fall standing demix in the 1000mL separating funnel then, subnatant adds and last the long-pending ethyl acetate of same volume once more, continue extraction as stated above, repeat 3 times, upper strata liquid is collected sealing and is deposited.The organic extraction of collecting is rotated evaporate to dryness, lyophilizes again in 40 ℃ of following vacuum.The composite type biological surfactant that obtains after the drying is the thick material of reddish-brown.
Embodiment 2: produce composite type biological surfactant
As embodiment 1, get the bacterial classification that refrigerates under 4 ℃ of conditions and activate 24h down in 37 ℃, picking one ring bacterial classification inoculation is in seed culture fluid from slant medium with transfering loop in the aseptic technique platform then, and seed culture fluid descends sterilization 30min at 115 ℃ in advance in the high pressure steam pot.Postvaccinal seed culture fluid is placed in the constant incubator in 37 ℃, cultivates under the 250rpm condition.Consisting of of seed culture fluid: NaCl 4.0g, peptone 4.0g, beef powder 2.5g, distilled water 1000mL, and regulate pH7.2 with 1MNaOH.
With the seed culture fluid of cultivating 24h by volume the inoculum size of mark 5% be inoculated in the fermentation culture, fermented liquid consists of: glucose 45g, NH
4NO
37.3g, K
2HPO
43H
2O 14.3g, NaH
2PO
4H
2O 2.05g, MgSO
47H
2O 0.6g, MnSO
4H
2O 0.04g, yeast extract paste 0.6g, distilled water 1000mL, and regulate pH7.2 with 1M NaOH.Fermentation culture also descends sterilization 30min at 115 ℃ in advance in the high pressure steam pot, the postvaccinal fermentation culture of the bottled 250mL of the taper of each 500mL, be placed in the constant incubator, in 37 ℃ of temperature, rotating speed 150rpm, cultivate under the condition of Ventilation Rate 1.2vvm, the air of feeding is crossed the filter membrane of 0.45 μ m and is sterilized.The foam that produces in the fermenting process flows in the 500mL receiving bottle by outlet pipe, and 10mL regulates pH1.5 with 1N HCl distilled water is housed in the receiving bottle in advance.
Every 12h gets fermented liquid 20mL, centrifugal 15min under 5600 * g, measure the surface tension value of supernatant liquor with automatic interfacial tensimeter, similar to Example 1, surface tension reached minimum with sample value when fermented liquid was cultivated 24h, drop to 39.5mN/m by initial 61.3mN/m, mean the output maximum of bio-surfactant in fermented liquid this moment.The thalline that residues in after centrifugal in the centrifuge tube dilutes with sterilized water, measures the thalline absorbancy with ultraviolet spectrophotometer under the 600nm wavelength, and subtilis reaches the logarithmic phase later stage when similarly finding fermented liquid cultivation 24h, at this moment the cell concentration maximum.Here the generation that proves this kind bio-surfactant once more is the growth relationship type.Directly get the 30mL fermented liquid and measure its pH, can find that the optimum growh environment of subtilis in this kind fermentation culture is similar to Example 1, be the little slant acidity of neutrality, the pH of correspondence was 6.81 when fermented liquid cultivation 24h cell concentration reached maximum.
Get the fermented liquid centrifugal 20min under 11000 * g that cultivates 24h and remove thalline, with being put in 0 ℃ of following standing over night behind the 6N HCl adjusting supernatant liquor pH1.5.Leave standstill liquid centrifugal 20min under 10000 * g, the issuable lipopeptid type biological surfactant of subtilis (comprising surfactins, iturins and fengycins etc.) is removed.Get supernatant liquor 500mL after centrifugal in the 1000mL Erlenmeyer flask, add the 150mL ethyl acetate, be placed on then in the incubator with maximum speed of revolution rotational oscillation 6min, fall standing demix in the 1000mL separating funnel then, isolating subnatant adds and last the long-pending ethyl acetate of same volume once more, continue extraction as stated above, repeat 3 times, upper strata liquid is collected sealing and is deposited.The organic extraction of collecting is rotated evaporates to dryness in 45 ℃ of following vacuum, and lyophilize again obtains the thick composite type biological surfactant material of reddish-brown.
Embodiment 3
The composition and the character of embodiment 1 and 2 prepared bio-surfactants:
1. lipid (lipids) content
Get the 0.15g bio-surfactant, (V: V=2: 1) lipid material is extracted in the mixing solutions dissolving, and centrifugal 10min under 7332 * g collects supernatant liquor then, and the residue in the centrifuge tube continues to extract as stated above, repeats 3 times with the 6.0mL chloroform/methanol.The organic extraction of collecting in 46 ℃ of following rotary evaporation in vacuo, is weighed, and the lipid quality percentage composition that records in the bio-surfactant is 98.1 ± 0.2%.Because present method utilization is the solubility of lipid material in organism, the lipid content that therefore records is the total mass percentage composition of pure lipid and it and the formed binding substances of other materials (as lipopeptid, lipoprotein or glycolipid class material etc.).
2. protein content
Protein content adopts the Xylene Brilliant Cyanine G method to measure in the bio-surfactant, adopts bovine serum albumin as standard, and the absorbing wavelength of light is chosen to be 595nm.Measure in the experiment that proteinic quality percentage composition is 19.1 ± 0.2% in the bio-surfactant.
3. carbohydrate content
Carbohydrate content records by the phenolsulfuric acid method in the bio-surfactant, adopts glucose as standard, and the absorbing wavelength of light is chosen to be 490nm.Measure in the experiment that carbohydrate quality percentage composition is 0.7 ± 0.1% in the bio-surfactant.
4. micelle-forming concentration and surface tension value
As Fig. 3, shown in 4, this composite type biological surfactant micelle-forming concentration is respectively 4.2～5.0g/L and 1.25～1.39g/L, and this moment, they dropped to 38.4～39.8mN/m and 40.3～40.8mN/m with the surface tension value of distilled water from 78mN/m respectively.
Claims (7)
1. a composite type biological surfactant is characterized in that this composite type biological surfactant is living by producing bacillus subtilis, and the quality percentage composition of lipid material is 98.1 ± 0.2% in this composite type biological surfactant; Proteinic quality percentage composition is 19.1 ± 0.2% in this composite type biological surfactant; The quality percentage composition of carbohydrate is 0.7 ± 0.1% in this composite type biological surfactant; Described composite type biological surfactant micelle-forming concentration is respectively 4.2～5.0g/L and 1.25～1.39g/L, and they drop to 38.4～39.8mN/m and 40.3～40.8mN/m with the surface tension value of distilled water from 78mN/m respectively; The content of described lipid material is to utilize the solubility of lipid material in organism to measure; Described Protein content is to adopt the Xylene Brilliant Cyanine G method to measure; The content of described carbohydrate is to measure by the phenolsulfuric acid method.
2. method of producing the described composite type biological surfactant of claim 1, it is characterized in that: subtilis is inoculated in the enrichment culture liquid, bacterial classification inoculation after the enrichment is carried out fermentation culture in the aerobic fermentation nutrient solution, determine the generation and the aerobic fermentation incubation time of composite type biological surfactant by the variation of measuring aerobic fermentation nutrient solution surface tension value, get aerobic fermentation nutrient solution high speed centrifugation and remove thalline, method with acid deposition precipitates the issuable lipopeptid type biological surfactant of subtilis then, and separate by high speed centrifugation and to remove, the method for the supernatant liquor after centrifugal by ethyl acetate extraction obtained composite type biological surfactant.
3. according to the method for the described production composite type biological surfactant of claim 2, it is characterized in that may further comprise the steps:
A, with subtilis in 30～37 ℃ down behind the activation 24h, be inoculated in the enrichment culture liquid, 30～37 ℃ of temperature, cultivate 24h under rotating speed 200～250rpm condition, then with the bacterial classification after the enrichment by volume mark 2～5% be inoculated in and carry out aerobic fermentation in the aerobic fermentation nutrient solution and cultivate, culture condition is: 30～37 ℃ of temperature, rotating speed 150～200rpm, Ventilation Rate 1.0～1.2vvm, the air that feeds is crossed 0.45 μ m filter membrane and is carried out filtration sterilization, determines the generation and the aerobic fermentation incubation time of composite type biological surfactant by the variation of measuring aerobic fermentation nutrient solution surface tension value;
B, get the fermented liquid high speed centrifugation degerming after above-mentioned aerobic fermentation is cultivated, centrifugal force is 11000～12000 * g, time 15～20min, regulate supernatant liquor pH1.5～2.0 with HCl solution, then 0～4 ℃ of following standing over night, leave standstill liquid high speed centrifugation again, centrifugal force 10000～12000 * g, time 15～20min, the supernatant liquor of getting after centrifugal adds ethyl acetate in Erlenmeyer flask, be placed on then in the incubator with maximum speed of revolution rotational oscillation 5～6min, fall standing demix in separating funnel, add the ethyl acetate with same volume last time after subnatant is collected once more, continue extraction as stated above, repeat 3 times, collect upper strata liquid;
C, with the upper strata liquid of above-mentioned collection in 40～45 ℃ of following vacuum rotation evaporates to dryness, lyophilize again can obtain composite type biological surfactant.
4. according to the method for claim 2 or 3 described production composite type biological surfactants, it is characterized in that described enrichment culture based component is: NaCl 4.0～5.0g, peptone 4.0～5.0g, beef powder 2.5～3.0g, distilled water 1000mL, pH7.0～7.2.
5. according to the method for claim 2 or 3 described production composite type biological surfactants, it is characterized in that described fermentation culture consists of: glucose 40～45g, NH
4NO
36.8～7.3g, K
2HPO
43H
2O 13.8～14.3g, NaH
2PO
4H
2O 1.95～2.05g, MgSO
47H
2O 0.5～0.6g, MnSO
4H
2O 0.036～0.04g, yeast extract paste 0.5～0.6g, distilled water 1000mL, pH7.0～7.2.
6. according to the method for claim 2 or 3 described production composite type biological surfactants, it is characterized in that described aerobic fermentation incubation time is preferably 24h.
7. according to the method for claim 2 or 3 described production composite type biological surfactants, it is characterized in that described V
Supernatant liquor: V
Ethyl acetate=9: 4～10: 3.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108271776A (en) * | 2017-12-25 | 2018-07-13 | 武汉科诺生物科技股份有限公司 | Bacillus lipopeptid class metabolite is used as the purposes of microbial bacteria agent aid |
| CN108271776B (en) * | 2017-12-25 | 2021-06-11 | 武汉科诺生物科技股份有限公司 | Use of bacillus lipopeptide metabolite as microbial agent aid |
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