CN104082525B - The application of strange land Klebsiella in barbadosnut pulp reduce toxicity of dwelling - Google Patents
The application of strange land Klebsiella in barbadosnut pulp reduce toxicity of dwelling Download PDFInfo
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- CN104082525B CN104082525B CN201410276120.4A CN201410276120A CN104082525B CN 104082525 B CN104082525 B CN 104082525B CN 201410276120 A CN201410276120 A CN 201410276120A CN 104082525 B CN104082525 B CN 104082525B
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Abstract
The invention discloses a kind of application of strange land Klebsiella in barbadosnut pulp reduce toxicity of dwelling, and the fermented detoxification method of barbadosnut pulp, comprise the steps: that (1) get strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling, activation, obtains seed liquor; (2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein, 20%～100% (v/w) that inoculum concentration is barbadosnut pulp, 50%～150% (v/w) that initial amount of water is barbadosnut pulp; (3) in temperature be the condition bottom fermentation 24～72 hours of 27 DEG C～47 DEG C. The inventive method has improved the degradation rate of Buddhist ripple ester in barbadosnut pulp, shortens fermentation time, does not destroy the nutritional labeling of grouts simultaneously, and method is easy, with low cost, has good market application foreground.
Description
Technical field
The present invention relates to the one strange land Klebsiella (Klebsiellavariicola) of dwelling sends out at barbadosnut pulpApplication in ferment detoxification, belongs to fermentation field.
Background technology
Jatropha curcas (JatrophacurcasL.) is that universally acknowledged most possibly becoming substitute fossil futureThe seeds with huge potentiality to be exploited of the energy, utilize the biodiesel of jatropha curcas oil processing everyIn performance indications, approach and even exceed country " 0 " number diesel oil and national Biodiesel Standards, therefore with fiber cropsCrazy tree fruit production biodiesel has become to be researched and produced now of biodiesel and enlivens direction. LeprosyTree grouts (Jatrophacurcasseedcake, JCSC) are the by-products in production of biodiesel processThing, has very high protein content and higher nutritive value, but because its toxicity is large, is mainly used asFertilizer with or be directly thrown in natural environment, not only seriously wasted a large amount of high-quality protein resources,But also caused serious environmental pollution, if can reduce its toxicity, can be used as feed protein and use.
The main toxicant of barbadosnut pulp be Buddhist ripple ester and derivative thereof, trypsin inhibitor andCurcin. Wherein, trypsin inhibitor and Curcin are thermal instability compositions, simple heating placeReason just can make their molecular configuration change, and can make it large thereby lose activity by heat treatmentAmount reduces, to almost disappearing completely. But Buddhist ripple ester (Phorbol-ester, PE) is heat endurance one-tenthPoint, strong toxicity can tolerate 30min and unaffected at 160 DEG C, and its structure belongs to tetracyclic diterpeneType, crotons alkane is the basic structure part of phorbol ester. What therefore, remove Buddhist ripple ester is barbadosnut pulpThe key factor of utilizing.
At present, remove about barbadosnut pulp in the research of Buddhist ripple ester, be generally physics and chemistry method connectionWith, or the biological method fermenting by microorganism carries out. Because Buddhist ripple ester has heat endurance, so logicalIt is unsatisfactory that overheat treatment method carries out Buddhist ripple ester removal effect. The higher physics of existing removal efficiency andChemistry method for combined use comprises: Buddhist ripple ester is melted in methyl alcohol and irradiates by plasma the Buddhist ripple ester of can degrading,Ethanol cyclic washing also can be removed Buddhist ripple ester, with containing NaHCO3Ethanolic solution can remove the Buddhist in seedRipple ester etc. While utilizing physico-chemical process to carry out detoxification, complicated operation process is loaded down with trivial details, easily cause chemicalsResidual, cost is high, and the nutritional labeling in grouts is caused to certain destruction. Microorganism have breeding fast,The features such as quantity is large, product enzyme system is complicated, utilizing the detoxification of microbial fermentation engineering technology is that one is comparatively effectiveMethod. Bibliographical information adopts pseudomonas aeruginosa (P.aeruginosaPseA) the fermentation fiber crops of can degradingBuddhist ripple ester in the crazy seeds of trees dregs of rice. But pseudomonas aeruginosa is one of the main pathogenic fungi of inside-hospital infection,Often cause postoperative wound infection, also can cause bedsore, abscess, otitis media suppurative etc. or urinary tractThe encountered pathogenic bacteria infecting. Thereby use as feed with the barbadosnut pulp of pseudomonas aeruginosa processing,There is potential potential safety hazard.
Chinese patent CN102329739B discloses a strain and has made bio-bacterial manure for the little seeds of a tung oil tree grouts that fermentAspergillus versicolor (AspergillusversicolorA003); Chinese patent CN102358887BOne strain is disclosed for removing the streptomyces fimicarius (Streptomyces of little seeds of a tung oil tree grouts toxicityFimicarius3). Chinese patent CN102316998A discloses the Buddhist ripple ester removal side in organic matterThe manufacturer of method, the organic manufacture method of high protein content, high protein content organic matter, feedMethod and feed, will contain organic matter and the Bacillus natto (Bacillus of Buddhist ripple ester compositionSubtilisvar.natto) mix, by making it ferment to decompose Buddhist ripple ester, fermentation time is 2～4Week. Chinese patent CN103509738A discloses a kind of day ditch dimension enterobacteria and has sent out at barbadosnut pulpApplication in ferment detoxification, day ditch dimension enterobacteria Enterobactergergoviaezxy-15, can be effectiveReduce barbadosnut pulp toxic component, fermentation time 8 days, the clearance of Buddhist ripple ester is up to 55.6%.Chinese patent CN103509737A discloses a kind of morganella morganii and at barbadosnut pulp reduce toxicityIn application, morganella morganii (Morganellamorganii) zxy-12-4, can effectively reduce leprosyTree grouts toxic component, fermentation time 3 days, the rate of descent of Buddhist ripple ester total amount is up to 73.0%. ChinaPatent CN103734559A discloses a kind of fermented detoxification method of barbadosnut pulp, utilizes day ditch dimension intestinesBacillus and morganella morganii mix, and reduce barbadosnut pulp toxicity, fermentation time 2 days, the going of Buddhist ripple esterExcept rate is up to 51.54%. In the disclosed method of prior art, fermentation time is longer mostly, if fermentation timeShort, the degradation rate of Buddhist ripple ester is undesirable again, is unfavorable for industrial production.
The strange land Klebsiella of dwelling is the mutation of Klebsiella, the disclosed application of prior art, as: inState patent CN102344898B discloses a strain and has become the Klebsiella bacterial strain of dwelling(Klebsiellavariicola) HUB-IV-005, has inhibiting trees for termite gut nitrogen-fixing bacteriaEndophyte tunning is cure of termite effectively. Chinese patent CN103484399A discloses a strainEndophytic bacterium SH-1 and application thereof, produce bacterial strain and become the klebsiella of dwelling(Klebsiellavariicola) SH-1, the Alternaria alternate that alternaric bacteria is caused, black spot of cabbage,Early blight of tomato has significant prevention effect. Have no the report that it is applied to barbadosnut pulp detoxification.
Summary of the invention
The invention provides one strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling existsApplication in barbadosnut pulp reduce toxicity.
The present invention's purposes of strange land Klebsiella in barbadosnut pulp reduce toxicity of dwelling.
The bacterial strain deposit number of described strange land Klebsiella (Klebsiellavariicola) LY-1 of dwellingFor CICIMB1743.
The fermented detoxification method of barbadosnut pulp of the present invention, comprises the steps:
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 20%～100% (v/w, i.e. volume mass ratio) of barbadosnut pulp, and initial amount of water is fiber crops50%～150% (v/w) of crazy tree grouts;
(3) in temperature be the condition bottom fermentation 24～72 hours of 27 DEG C～47 DEG C.
In step (1), described in the dwell bacterial strain deposit number of strange land Klebsiella LY-1 be CICIMB1743。
In step (1), described in the dwell seed liquor of strange land Klebsiella LY-1 be prepared as follows:Adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated into LB liquid respectivelyIn culture medium, shaken cultivation 14h under 37 DEG C, 180r/min, makes bacterial classification concentration reach 108cfu/mL。
In step (2), described barbadosnut pulp is prepared as follows: get jatropha curcas seed, squeezeGet de-oiling, pulverize, cross 40 mesh sieves.
In step (2), 33%～40% (v/w) that described inoculum concentration is barbadosnut pulp, initially adds waterAmount is 100%～105% (v/w) of barbadosnut pulp.
In step (3), described temperature is 37 DEG C; The time of described fermentation is 48 hours.
The inventive method utilization strange land Klebsiella LY-1 of dwelling carries out reduce toxicity to barbadosnut pulp,Greatly improve the degradation rate of Buddhist ripple ester in barbadosnut pulp, shorten fermentation time, do not destroy grouts simultaneouslyNutritional labeling, method is easy, with low cost.
Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area,Do not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make other various ways amendment,Replace or change.
The detailed description of the invention of form by the following examples, remakes further foregoing of the present inventionDetailed description. But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
The present invention strange land Klebsiella LY-1 of dwelling derives from Chinese Universities ' industrial microorganism resource and informationCenter (be subordinate to organization: Southern Yangtze University), bacterial strain deposit number is CICIMB1743, generic name:Klebsiella, plants name and adds word: variicola, feature: bacterium colony is little and smooth, white, edgeAnd smooth surface, Gram-negative.
Detailed description of the invention
Material
Bacterial classification: the present invention strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling, is called for short bacteriumStrain LY-1.
Barbadosnut pulp: get jatropha curcas seed, squeeze de-oiling, pulverize, cross 40 mesh sieves.
Jatropha curcas seed of the present invention is adopted in Huo Pianjiao township of Ren Li township, Yongsheng County, Yunnan Province, get then ripe, fromSo dry seed is tested.
Key instrument
Waters515 high performance liquid chromatograph (U.S.), UT-1900 ultraviolet-visible spectrophotometer (northJing Puxi all purpose instrument factory), AE-240 electronic analytical balance (0.0001g), SB-300DTR ultrasonic waveFrequency sweep cleaning machine, RE-52AA Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant), the omnipotent powder of JL-100Broken machine (Shanghai Guang Sha Trade Co., Ltd.), SpectraMaxM2/M2e type multifunctional enzyme linked immune analysisInstrument (MolercularDevices company of the U.S.), spiral cold pressing expeller, biochemical cultivation case, shaking table,Aseptic operating platform, high-pressure sterilizing pot, gavage pin.
Reagent:
Enzyme labeling two is anti-: goat anti-rabbit igg-HRP (Beijing company of Zhong Shan Golden Bridge), N, N, N ', N '-TMB (tetramethyl benzidine) nitrite ion (U.S. Amresco).
All reagent is to be analyzed purely, and chromatogram agents useful for same is chromatographically pure, all purchased from Chengdu section dragon reagent public affairsDepartment. Chromatographic silica gel (160-200 order).
Extraction and the detection method of Buddhist ripple ester in solid state fermentation grouts
(1) extraction of Buddhist ripple ester
Adopt carrene extraction method: to containing in grouts or grouts fermentate triangular flask, add 40mL at every turnCarrene to carry out 20min ultrasonic, so repeats 6 times. Ultrasonic liquid collection is carried out to suction filtration and revolves steaming,Obtain Buddhist ripple ester concentrate and dissolve with methyl alcohol. After the centrifugal 10min of 12000r/min, get againThe organic membrane filtration of 0.22 μ m for clear liquid, obtains the methanol solution that contains Buddhist ripple ester.
(2) HPLC detection method
Detect by high performance liquid chromatography, high-efficient liquid phase chromatogram condition is: (contain 0.175% with 80% acetonitrilePhosphoric acid) as mobile phase, using 8% methyl alcohol as cleaning mobile phase, using pure acetonitrile as protection liquid mobile phase;Detection wavelength is that 280nm, flow velocity are that 1.0mL/min, column temperature are that 25 DEG C, sample size are 20 μ L, in additionMark method, as basis, is determined the content of Jatropha curcas Buddhist ripple ester in sample by calculated by peak area, formula isY=3025300x-33.38, wherein y be peak area and, x is Buddhist ripple ester content (mg/g).
(3) the degradation rate computing formula of Buddhist ripple ester
Buddhist ripple ester degradation amount represents with the degradation rate of Buddhist ripple ester:
Embodiment 1 adopts the inventive method to barbadosnut pulp reduce toxicity
1.1 culture medium
1) LB fluid nutrient medium: moist heat sterilization 20min at 121 DEG C, for subsequent use.
2) solid-state fermentation culture medium: barbadosnut pulp powder 5g, water, pH nature, not sterilizing.
The preparation of 1.2 bacteria suspensions
Adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated into LB respectivelyIn fluid nutrient medium, be placed in 37 DEG C, the constant temperature oscillator of 180r/min and carry out the cultivation of bacteria suspension,Incubation time is 14h, makes bacterial classification concentration reach 108Cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Taking Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content as2.32mg/g)。
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly add sterilized water,After bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, not enterThe barbadosnut pulp of row fermentation does blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt highEffect liquid phase chromatogram method detects the content of Buddhist ripple ester, and calculates the degradation rate of Buddhist ripple ester.
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 20% (v/w) of barbadosnut pulp, 100 (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be the condition bottom fermentation 48 hours of 37 DEG C.
After testing, under above-mentioned fermentation condition, the degradation rate of Buddhist ripple ester is 78.27%.
Embodiment 2 adopts the inventive method to barbadosnut pulp reduce toxicity
1.1 culture medium
1) LB fluid nutrient medium: moist heat sterilization 20min at 121 DEG C, for subsequent use.
2) solid-state fermentation culture medium: barbadosnut pulp powder 5g, water, pH nature, not sterilizing.
The preparation of 1.2 bacteria suspensions
Adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated into LB respectivelyIn fluid nutrient medium, be placed in 37 DEG C, the constant temperature oscillator of 180r/min and carry out the cultivation of bacteria suspension,Incubation time is 14h, makes bacterial classification concentration reach 108Cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Taking Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content as2.32mg/g)。
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly add sterilized water,After bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, not enterThe barbadosnut pulp of row fermentation does blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt highEffect liquid phase chromatogram method detects the content of Buddhist ripple ester, and calculates the degradation rate of Buddhist ripple ester.
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 100% (v/w) of barbadosnut pulp, 100 (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be the condition bottom fermentation 48 hours of 37 DEG C.
After testing, under above-mentioned fermentation condition, the degradation rate of Buddhist ripple ester is 72.09%.
Embodiment 3 adopts the inventive method to barbadosnut pulp reduce toxicity
1.1 culture medium
1) LB fluid nutrient medium: moist heat sterilization 20min at 121 DEG C, for subsequent use.
2) solid-state fermentation culture medium: barbadosnut pulp powder 5g, water, pH nature, not sterilizing.
The preparation of 1.2 bacteria suspensions
Adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated into LB respectivelyIn fluid nutrient medium, be placed in 37 DEG C, the constant temperature oscillator of 180r/min and carry out the cultivation of bacteria suspension,Incubation time is 14h, makes bacterial classification concentration reach 108Cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Taking Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content as2.32mg/g)。
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly add sterilized water,After bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, not enterThe barbadosnut pulp of row fermentation does blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt highEffect liquid phase chromatogram method detects the content of Buddhist ripple ester, and calculates the degradation rate of Buddhist ripple ester.
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 40% (v/w) of barbadosnut pulp, 100 (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be the condition bottom fermentation 72 hours of 37 DEG C.
After testing, under above-mentioned fermentation condition, the degradation rate of Buddhist ripple ester is 82.47%.
Embodiment 4 adopts the inventive method to barbadosnut pulp reduce toxicity
1.1 culture medium
1) LB fluid nutrient medium: moist heat sterilization 20min at 121 DEG C, for subsequent use.
2) solid-state fermentation culture medium: barbadosnut pulp powder 5g, water, pH nature, not sterilizing.
The preparation of 1.2 bacteria suspensions
Adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated into LB respectivelyIn fluid nutrient medium, be placed in 37 DEG C, the constant temperature oscillator of 180r/min and carry out the cultivation of bacteria suspension,Incubation time is 14h, makes bacterial classification concentration reach 108Cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Taking Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content as2.32mg/g)。
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly add sterilized water,After bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, not enterThe barbadosnut pulp of row fermentation does blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt highEffect liquid phase chromatogram method detects the content of Buddhist ripple ester, and calculates the degradation rate of Buddhist ripple ester.
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 40% (v/w) of barbadosnut pulp, 150 (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be the condition bottom fermentation 48 hours of 37 DEG C.
After testing, under above-mentioned fermentation condition, the degradation rate of Buddhist ripple ester is 71.70%.
Embodiment 5 adopts the inventive method to barbadosnut pulp reduce toxicity
1.1 culture medium
1) LB fluid nutrient medium: moist heat sterilization 20min at 121 DEG C, for subsequent use.
2) solid-state fermentation culture medium: barbadosnut pulp powder 5g, water, pH nature, not sterilizing.
The preparation of 1.2 bacteria suspensions
Adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated into LB respectivelyIn fluid nutrient medium, be placed in 37 DEG C, the constant temperature oscillator of 180r/min and carry out the cultivation of bacteria suspension,Incubation time is 14h, makes bacterial classification concentration reach 108Cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Taking Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content as2.32mg/g)。
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly add sterilized water,After bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, not enterThe barbadosnut pulp of row fermentation does blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt highEffect liquid phase chromatogram method detects the content of Buddhist ripple ester, and calculates the degradation rate of Buddhist ripple ester.
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 40% (v/w) of barbadosnut pulp, 100 (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be the condition bottom fermentation 48 hours of 37 DEG C.
After testing, under above-mentioned fermentation condition, the degradation rate of Buddhist ripple ester is 82.87%.
Embodiment 6 response surface method optimization of fermentation conditions are set up model
In order more easily the fermentation condition in large production in the future to be selected, the present invention utilizes DesignExpert8.0 software is set up a kind of Mathematical Modeling of optimization of fermentation conditions.
Taking Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content as2.32mg/g)。
Be under the condition of 48 hours at fermentation time, choose fermentation temperature and inoculum concentration, initial amount of water 3Individual factor, utilizes DesignExpert8.0 software to fall the strange land Klebsiella LY-1 solid state fermentation of dwellingSeparate the impact of barbadosnut pulp Buddhist ripple ester and carry out Box-Behnken design, the degradation rate of Buddhist ripple ester is correspondingValue, each factor is got 3 levels, utilizes DesignExpert8.0 to carry out analytical test data, enters oneStep is optimized the parameter value (in table 1) of each factor.
Table 1 experimental design factor level and coding
Carry out response surface analysis result of the test through DesignExpert8.0, the results are shown in Table 2.
Table 2Box-Behnken response surface experimental design and result
According to the regression analysis of response surface coefficient, obtain the fit equation (binary regression equation) of this modelFor: Y=82.95+1.16 × X1-2.73×X2-0.36×X3+3.54×X1×X2+2.73×X1×X3-4.05×X2×X3-8.24×X1 2-4.76×X2 2-3.06×X3 2。
Binary regression equation is carried out to variance analysis and the variance analysis of response surface result, the results are shown in Table 3 tables 4.
The variance analysis of table 3 binary regression equation
The variance analysis of table 4 response surface result
As shown in Table 3:
X2、X1X2、X2X3、X12、X22、X32On extremely significantly (P < 0.001) of the impact of Buddhist ripple ester degradation rate,Illustrate that inoculum concentration, fermentation temperature and initial amount of water are the key factors in sweat, wherein with inoculationHaving the greatest impact of amount. Significant interaction between fermentation temperature and inoculum concentration, inoculum concentration and initial amount of water(P<0.01). Lose and intend not significantly (P>0.05) of item, illustrate in data and there is no abnormity point, model is suitable.
From table 3 and table 4:
Regression model is (P < 0.001) extremely significantly, loses and intends not significantly (P=0.2721), shows regression equation planRight good; The multiple correlation coefficient of regression equation is 0.9951, shows 99.51% Buddhist ripple ester degradation rateChange available this model explanation, better with actual conditions matching. Proofreading and correct coefficient correlation is 0.9887, Buddhist rippleThe ester degraded flow process coefficient of variation is 0.92%, and signal to noise ratio is 38.438, illustrates that model credibility is higher.
This equation is strange land Klebsiella (Klebsiellavariicola) the solid state fermentation degraded leprosy of dwellingSeeds of trees grouts Buddhist ripple ester provides a suitable model. Regression model exist point of safes (0.1775,0.3196,0.2318) corresponding actual value (37.12,32.81,104.73), most degradation rate is estimatedValue is 83.42%.
Set up Mathematical Modeling is verified:
According to the optimal value of each factor in conjunction with actual test conditions, i.e. 37 DEG C of fermentation temperatures, inoculum concentration 33%(v/w), initial amount of water is to carry out solid state fermentation 48 hours under 105% (v/w), repeats 3 times.Recording the average degradation rate of Buddhist ripple ester is 84.30%, close with theoretical prediction value, and relative deviation is 1.06%.
Presentation of results, the Mathematical Modeling that the present invention sets up is suitable, can predict preferably the strange land of dwellingThe feelings of Klebsiella (Klebsiellavariicola) solid state fermentation degraded barbadosnut seed grouts Buddhist ripple esterCondition.
For beneficial effect of the present invention is described, the invention provides following test example.
The screening of test example 1 fermentation temperature of the present invention
The fermented detoxification method of barbadosnut pulp, comprises the steps:
(1) get strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling, activation, obtainsSeed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 40% (v/w) of barbadosnut pulp, 100% (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be respectively the constant temperature bottom fermentation of 27 DEG C, 32 DEG C, 37 DEG C, 42 DEG C, 47 DEG C48 hours.
After testing, the detoxification efficiency of different fermentations temperature, the results are shown in Table 5.
The detoxification efficiency of table 5 different fermentations temperature
| Temperature | Degradation rate (%) |
| 27℃ | 64.87±2.43 |
| 32℃ | 70.35±2.34 |
| 37℃ | 78.51±1.70 |
| 42℃ | 71.77±2.07 |
| 47℃ | 59.43±2.10 |
| Blank | 0 |
As shown in Table 5:
Bacterial strain LY-1 carries out after solid state fermentation grouts, and Buddhist ripple ester content significantly declines; The degraded of Buddhist ripple esterRate is along with the rising of fermentation temperature first raises and reduces afterwards, in the time that fermentation temperature is 37 DEG C, and the degraded of Buddhist ripple esterRate maximum, reaches 78.51%.
Test explanation, while using bacterial strain LY-1 of the present invention to barbadosnut pulp reduce toxicity, fermentation temperature is preferredIt is 37 DEG C.
The screening of test example 2 inoculum concentrations of the present invention
The fermented detoxification method of barbadosnut pulp, comprises the steps:
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Under the condition of 20%, 40%, 60%, 80%, 100% (v/w) that is barbadosnut pulp in inoculum concentration respectively,100% (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature be the constant temperature bottom fermentation 48 hours of 37 DEG C.
After testing, the detoxification efficiency of bacterial strain LY-1 different vaccination amount, the results are shown in Table 6.
The detoxification efficiency of table 6 bacterial strain LY-1 different vaccination amount
| Inoculum concentration (v/w) | Degradation rate (%) |
| 20％ | 78.27±2.14 |
| 40％ | 81.35±2.46 |
| 60％ | 75.02±1.88 |
| 80％ | 76.10±1.22 |
| 100％ | 72.09±1.05 |
| Blank | 0 |
As shown in Table 6:
Bacterial strain LY-1 carries out after solid state fermentation grouts, and Buddhist ripple ester content significantly declines; The degraded of Buddhist ripple esterRate, along with the increase of inoculum concentration, first raises and reduces afterwards, in the time that inoculum concentration is 40%, and the degradation rate of Buddhist ripple esterMaximum, reaches 81.35%.
Test explanation, during for barbadosnut pulp reduce toxicity, the inoculum concentration of bacterial strain LY-1 of the present invention is preferredBe 40%.
The screening of the initial amount of water of test example 3 the present invention
The fermented detoxification method of barbadosnut pulp, comprises the steps:
(1) get strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling, activation, obtainsSeed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 40% (v/w) of barbadosnut pulp, be barbadosnut pulp at initial amount of water respectively 50%,75%, under the condition of 100%, 125%, 150% (v/w);
(3) in temperature be the constant temperature bottom fermentation 48 hours of 37 DEG C.
After testing, the detoxification efficiency of different initial amount of water, the results are shown in Table 7.
The detoxification efficiency of the different initial amount of water of table 7
| Initial amount of water (v/w) | Degradation rate (%) |
| 50％ | 66.08±1.12 |
| 75％ | 77.87±1.48 |
| 100％ | 82.87±1.07 |
| 125％ | 72.09±3.35 |
| 150％ | 71.70±2.68 |
| Blank | 0 |
As shown in Table 7:
Bacterial strain LY-1 carries out after solid state fermentation grouts, and Buddhist ripple ester content significantly declines; The degraded of Buddhist ripple esterRate, along with the increase of initial amount of water, first raises and reduces afterwards, in the time that initial amount of water is 100%, and Buddhist ripple esterDegradation rate maximum, reach 82.87%.
Test explanation, while using bacterial strain LY-1 of the present invention to barbadosnut pulp reduce toxicity, initial amount of water is excellentElect 100% as.
The screening of test example 4 fermentation times of the present invention
The fermented detoxification method of barbadosnut pulp, comprises the steps:
(1) get strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling, activation, obtainsSeed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 40% (v/w) of barbadosnut pulp, 100% (v/w) that initial amount of water is barbadosnut pulp;
(3) under temperature is the constant temperature of 37 DEG C, ferment respectively 24 hours, 36 hours, 48 hours,60 hours, 72 hours.
After testing, the detoxification efficiency of different fermentations time, the results are shown in Table 8.
The detoxification efficiency of table 8 different fermentations time
| Time (hour) | Degradation rate (%) |
| 24 | 54.87±3.11 |
| 36 | 75.48±1.49 |
| 48 | 82.16±3.33 |
| 60 | 81.19±2.02 |
| 72 | 82.47±1.53 |
| Blank | 0 |
As shown in Table 8:
Bacterial strain LY-1 carries out after solid state fermentation grouts, and Buddhist ripple ester content significantly declines; The degraded of Buddhist ripple esterRate raises along with the prolongation of fermentation time, and in the time that fermentation time is 48 hours, the degradation rate of Buddhist ripple ester reachesTo 82.16%, then extend fermentation time, the degradation rate of Buddhist ripple ester does not almost raise.
Test explanation, while using bacterial strain LY-1 of the present invention to barbadosnut pulp reduce toxicity, fermentation time is preferredIt is 48 hours.
Above-mentioned result of the test explanation: in fermentation process of the present invention, inoculum concentration is 20%～100% (v/w's)In scope, initial amount of water is in the scope of 50%～150% (v/w), and fermentation temperature is at 27 DEG C～47 DEG CScope in, fermentation time, within the scope of 24～72 hours, all can reach technique effect of the present invention.
Test example 5 is used the grouts after the inventive method reduce toxicity to carry out nutrient component determining and acute poisonProperty is tested
The solid state fermentation detoxification of 5.1 pairs of different places of production grouts
Taking Pian Jiao township, Yongsheng County, Yunnan Province barbadosnut pulp (Buddhist ripple ester initial content is as 0.840mg/g) as formerMaterial, is 33% (v/w) in inoculum concentration, and initial amount of water is under the condition of 105% (v/w), in 37 DEG CIn constant incubator, ferment 48 hours, repeat 3 times.
Adopt HPLC detection method, after obtaining fermenting, barbadosnut pulp Buddhist ripple ester content is 0.109mg/g,The degradation rate of Buddhist ripple ester reaches 87.03%.
The mensuration of grouts nutritional labeling before and after 5.2 reduce toxicities
Measure the nutritional labeling of above-mentioned barbadosnut pulp reduce toxicity front and back.
Adopt GB/T6432-1994 to measure crude protein content, adopt GB/T6433-2006 to measure thick fatFat content, adopts GB/T6434-2006 to measure crude fibre, adopts GB/T50094-2010 to measure ash content,Adopt GB/T18246-2000 to measure amino acid content.
Before and after reduce toxicity, the measurement result of barbadosnut pulp nutritional labeling is as shown in table 9.
Barbadosnut pulp Analysis of Nutritive Composition before and after table 9 reduce toxicity
As shown in Table 9:
Before and after fermentation, barbadosnut pulp nutritional labeling changes not quite, the strange land Klebsiella of dwelling(Klebsiellavariicola) solid state fermentation barbadosnut pulp does not destroy the nutrition one-tenth of barbadosnut pulpPoint.
5.3 animal acute toxicity test
Test determines that detoxification meal maxima solubility in 0.5% sodium carboxymethylcellulose (CMC) solution is0.3g/mL; It is each 1mL that test mice can bear maximum gavage dosage, every day 2 times.
Get body weight 30 of the kunming mices of 20g (± 2g), be divided at random 3 groups, 10 every group, femaleMale half and half. First raise 7 days, carry out gavage processing after determining the healthy and adequacy test environment of mouse.
Test I group is control group, fills with and feeds 0.5% the carboxymethylcellulose sodium solution of being prepared by physiological saline;Test II group fill with feed the grouts of detoxification treatment (after test 2.3.5 reduce toxicity, 121 DEG C of sterilizing 20min,After sterilizing, carry out vacuum drying) suspension; Test III group is filled with and is fed untreated grouts (Yongsheng County, Yunnan Province sheet(Buddhist ripple ester initial content is 0.840mg/g to angle township barbadosnut pulp, and 121 DEG C of sterilizing 20min, after sterilizingCarry out vacuum drying)) suspension. After gavage is processed, Continuous Observation 14 days, records mouse animation.
Animal acute toxicity test result, in table 10.
Barbadosnut pulp acute toxicity test in mice before and after table 10 reduce toxicity
Result of the test explanation, the grouts after the inventive method detoxification are to mouse without acute toxicity, and it shouldWith being very safe, method of the present invention can successfully be sloughed the Buddhist ripple ester in grouts, and grouts are entered to oneStep utilization provides safety guarantee.
In sum, fermentation process of the present invention, can slough the Buddhist ripple ester in barbadosnut pulp easily,Fermentation time is short, and the degradation rate of Buddhist ripple ester is high, is convenient to suitability for industrialized production; Grouts nutritional labeling after detoxificationThere is no large change, good without acute toxicity, security, can be directly as animal feed, be Jatropha curcasThe further utilization of grouts, provides reliable approach.
Claims (6)
1. dwell strange land Klebsiella (Klebsiellavariicola) in barbadosnut pulp reduce toxicityPurposes, described in the dwell bacterial strain deposit number of strange land Klebsiella (Klebsiellavariicola) LY-1 beCICIMB1743。
2. a fermented detoxification method for barbadosnut pulp, is characterized in that: it comprises the steps:
(1) get the strange land Klebsiella LY-1 of dwelling, activation, obtains seed liquor;
Described bacterial strain deposit number of dwelling strange land Klebsiella LY-1 is CICIMB1743;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein,Inoculum concentration is 20%～100% (v/w) of barbadosnut pulp, and initial amount of water is barbadosnut pulp50％～150％(v/w)；
(3) in temperature be the condition bottom fermentation 24～72 hours of 27 DEG C～47 DEG C.
3. method according to claim 2, is characterized in that: in step (1), described in dwell differentThe seed liquor of ground Klebsiella LY-1 is prepared as follows: adopt the activation of LB solid medium to dwellAfter the Klebsiella LY-1 of strange land, picking is inoculated in LB fluid nutrient medium respectively, 37 DEG C, 180r/minLower shaken cultivation 14h, makes bacterial classification concentration reach 108cfu/mL。
4. method according to claim 2, is characterized in that: in step (2), and described leprosyTree grouts are prepared as follows: get jatropha curcas seed, squeeze de-oiling, pulverize, cross 40 mesh sieves,.
5. method according to claim 2, is characterized in that: in step (2), and described inoculationAmount is 33%～40% (v/w) of barbadosnut pulp, initial amount of water is barbadosnut pulp 100%～105％(v/w)。
6. method according to claim 2, is characterized in that: in step (3), and described temperatureIt is 37 DEG C; The time of described fermentation is 48 hours.
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