CN104082525A - Application of Klebsiella variicola in fermentation detoxification of Jatropha curcas seed cake - Google Patents
Application of Klebsiella variicola in fermentation detoxification of Jatropha curcas seed cake Download PDFInfo
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- 241001014264 Klebsiella variicola Species 0.000 title claims abstract description 18
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses application of Klebsiella variicola in fermentation detoxification of a Jatropha curcas seed cake and a fermentation detoxification method of the Jatropha curcas seed cake. The method comprises the following steps: (1) taking Klebsiella variicola LY-1 and activating Klebsiella variicola LY-1 so as to obtain a seed liquid; (2) taking the Jatropha curcas seed cake, adding water, carrying out uniform mixing and inoculating the seed liquid prepared in the step (1), wherein the inoculation amount of the seed liquid is 20 to 100% (v/w) of the Jatropha curcas seed cake, and the initial addition amount of water is 50 to 150% (v/w) of the Jatropha curcas seed cake; and (3) carrying out fermentation at 27 to 47 DEG C for 24 to 72 h. The method improves the degradation rate of phorbol ester in the Jatropha curcas seed cake, shortens fermentation time, does not destroy nutritional components in the seed cake, is simple and convenient and has low cost and good market application prospects.
Description
Technical field
The present invention relates to a kind of application of strange land Klebsiella (Klebsiellavariicola) in barbadosnut pulp reduce toxicity of dwelling, belong to fermentation field.
Background technology
Jatropha curcas (Jatrophacurcas L.) is the universally acknowledged seeds with huge potentiality to be exploited that most possibly become following alternative fossil energy, the biodiesel of utilizing jatropha curcas oil processing approaches and even surpasses country's " 0 " number diesel oil and national Biodiesel Standards on property indices, therefore with Jatropha curcas fruit production biodiesel, has become and has researched and produced now of biodiesel and enliven direction.Barbadosnut pulp (Jatrophacurcas seed cake, JCSC) be the accessory substance in production of biodiesel process, there is very high protein content and higher nutritive value, but because of its toxicity large, mainly be used as fertilizer 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 is Buddhist ripple ester and derivative, trypsin inhibitor and Curcin.Wherein, trypsin inhibitor and Curcin are thermal instability compositions, and simple heat treated just can make their molecular configuration change, thereby lose activity, by heat treatment, can make it reduce in a large number, to almost disappearing completely.But Buddhist ripple ester (Phorbol-ester, PE) is heat endurance composition, strong toxicity can tolerate 30min and unaffected at 160 ℃, and its structure belongs to tetracyclic diterpene type, and crotons alkane is the basic structure part of phorbol ester.What therefore, remove Buddhist ripple ester is the key factor that barbadosnut pulp utilizes.
At present, about barbadosnut pulp, remove in the research of Buddhist ripple ester, be generally the coupling of physics and chemistry method, or the biological method fermenting by microorganism carries out.Because Buddhist ripple ester has heat endurance, so it is unsatisfactory to carry out Buddhist ripple ester removal effect by heat treatment method.The physics and chemistry method for combined use that existing removal efficiency is higher comprises: Buddhist ripple ester is melted in methyl alcohol and irradiates by plasma the Buddhist ripple ester of can degrading, and ethanol cyclic washing also can be removed Buddhist ripple ester, with containing NaHCO
3ethanolic solution can remove Buddhist ripple ester in seed etc.While utilizing physico-chemical process to carry out detoxification, complicated operation process is loaded down with trivial details, easily cause that chemicals are residual, cost is high, and the nutritional labeling in grouts is caused to certain destruction.Microorganism has the features such as breeding is fast, quantity is large, product enzyme system is complicated, and utilizing the detoxification of microbial fermentation engineering technology is a kind of comparatively effective method.The Buddhist ripple ester that bibliographical information adopts pseudomonas aeruginosa (P.aeruginosaPseA) fermentation can degrade in the barbadosnut seed 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 the encountered pathogenic bacteria of urinary tract infections.Thereby use as feed with the barbadosnut pulp that pseudomonas aeruginosa is processed, there is potential potential safety hazard.
Chinese patent CN 102329739 B disclose a strain and for the little seeds of a tung oil tree grouts that ferment, have made the aspergillus versicolor (Aspergillusversicolor A003) of bio-bacterial manure; Chinese patent CN 102358887 B disclose a strain for removing the streptomyces fimicarius (Streptomyces fimicarius 3) of little seeds of a tung oil tree grouts toxicity.Chinese patent CN 102316998 A disclose manufacture method and the feed of Buddhist ripple ester removal method in organic matter, the organic manufacture method of high protein content, high protein content organic matter, feed, the organic matter that contains Buddhist ripple ester composition is mixed with Bacillus natto (Bacillus subtilisvar.natto), by making it ferment to decompose Buddhist ripple ester, fermentation time is 2～4 weeks.Chinese patent CN 103509738 A disclose a kind of day ditch and have tieed up enterobacteria and the application in barbadosnut pulp reduce toxicity thereof, day ditch dimension enterobacteria Enterobactergergoviae zxy-15, can effectively reduce barbadosnut pulp toxic component, fermentation time 8 days, the clearance of Buddhist ripple ester is up to 55.6%.Chinese patent CN 103509737 A disclose a kind of morganella morganii and the application in barbadosnut pulp reduce toxicity thereof, morganella morganii (Morganellamorganii) zxy-12-4, can effectively reduce barbadosnut pulp toxic component, fermentation time 3 days, the rate of descent of Buddhist ripple ester total amount is up to 73.0%.Chinese patent CN 103734559 A disclose a kind of fermented detoxification method of barbadosnut pulp, utilize day ditch dimension enterobacteria and morganella morganii to mix, and reduce barbadosnut pulp toxicity, fermentation time 2 days, and the clearance of Buddhist ripple ester is up to 51.54%.In the disclosed method of prior art, fermentation time is longer mostly, if fermentation time is short, 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: Chinese patent CN 102344898 B disclose a strain and have become Klebsiella bacterial strain (Klebsiellavariicola) HUB-IV-005 of dwelling, and for termite gut nitrogen-fixing bacteria, have effectively cure of termite of inhibiting trees endophyte tunning.Chinese patent CN 103484399 A disclose plants endogenetic bacterium SH-1 and an application thereof, produce bacterial strain and become klebsiella (Klebsiellavariicola) SH-1 of dwelling, the Alternaria alternate that alternaric bacteria is caused, black spot of cabbage, early blight of tomato have significant prevention effect.Have no the report that it is applied to barbadosnut pulp detoxification.
Summary of the invention
The invention provides a kind of application of strange land Klebsiella (Klebsiellavariicola) LY-1 in barbadosnut pulp reduce toxicity of dwelling.
The present invention's purposes of strange land Klebsiella in barbadosnut pulp reduce toxicity of dwelling.
Described bacterial strain deposit number of dwelling strange land Klebsiella (Klebsiellavariicola) LY-1 is CICIM B1743.
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, 20%～100% (v/w that inoculum concentration is barbadosnut pulp, be volume mass ratio), 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 ℃～47 ℃.
In step (1), described in the dwell bacterial strain deposit number of strange land Klebsiella LY-1 be CICIM B1743.
In step (1), described seed liquor of dwelling strange land Klebsiella LY-1 is prepared as follows: adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated in LB fluid nutrient medium respectively, shaken cultivation 14h under 37 ℃, 180r/min, makes bacterial classification concentration reach 10
8cfu/mL.
In step (2), described barbadosnut pulp is prepared as follows: get jatropha curcas seed, squeeze de-oiling, pulverize, cross 40 mesh sieves.
In step (2), 33%～40% (v/w) that described inoculum concentration is barbadosnut pulp, 100%～105% (v/w) that initial amount of water is barbadosnut pulp.
In step (3), described temperature is 37 ℃; 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 improves 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.
Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
The specific embodiment of form, is described in further detail foregoing of the present invention again by the following examples.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 information centre and (is subordinate to organization: Southern Yangtze University), bacterial strain deposit number is CICIM B1743, generic name: Klebsiella, kind of name adds word: variicola, feature: bacterium colony is little and smooth, white, edge and smooth surface, Gram-negative.
The specific embodiment
Material
Bacterial classification: the present invention strange land Klebsiella (Klebsiellavariicola) LY-1 of dwelling, is called for short bacterial strain LY-1.
Barbadosnut pulp: get jatropha curcas seed, squeeze de-oiling, pulverize, cross 40 mesh sieves.
Huo Pianjiao township, Ren Li township, Yongsheng County, jatropha curcas seed Cai Yu Yunnan Province of the present invention, gets ripe, natural drying seed then and tests.
Key instrument
Waters515 high performance liquid chromatograph (U.S.), UT-1900 ultraviolet-visible spectrophotometer (Beijing Pu Xi all purpose instrument factory), AE-240 electronic analytical balance (0.0001g), SB-300DTR ultrasonic sweep-frequency cleaning machine, RE-52AA Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant), JL-100 Universalpulverizer (Shanghai Guang Sha Trade Co., Ltd.), SpectraMax M2/M2e type multifunctional enzyme linked immune analysis instrument (U.S. Molercular Devices company), 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).
It is pure that all reagent is analysis, and chromatogram agents useful for same is chromatographically pure, all purchased from Chengdu Ke Long reagent company.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 carrene at every turn and carry out 20min ultrasonic, so repeat 6 times.Ultrasonic liquid collection is carried out to suction filtration and revolve steaming, obtain Buddhist ripple ester concentrate and dissolve with methyl alcohol.After the centrifugal 10min of 12000r/min, get the organic membrane filtration of 0.22 μ m for supernatant again, obtain the methanol solution that contains Buddhist ripple ester.
(2) HPLC detection method
By high performance liquid chromatography, detect, high-efficient liquid phase chromatogram condition is: using 80% acetonitrile (containing 0.175% phosphoric 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 ℃, sample size are 20 μ L, using external standard method as basis, by calculated by peak area, determine the content of Jatropha curcas Buddhist ripple ester in sample, formula is y=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 ℃, standby.
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 in LB fluid nutrient medium respectively, is placed in 37 ℃, the constant temperature oscillator of 180r/min and carries out the cultivation of bacteria suspension, and incubation time is 14h, makes bacterial classification concentration reach 10
8cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Take Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content is 2.32mg/g).
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly adds after sterilized water, bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, with the barbadosnut pulp fermenting, do blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt high performance liquid chromatography to detect the content of Buddhist ripple ester, and calculate 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, 20% (v/w) that inoculum concentration is 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 ℃.
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 ℃, standby.
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 in LB fluid nutrient medium respectively, is placed in 37 ℃, the constant temperature oscillator of 180r/min and carries out the cultivation of bacteria suspension, and incubation time is 14h, makes bacterial classification concentration reach 10
8cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Take Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content is 2.32mg/g).
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly adds after sterilized water, bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, with the barbadosnut pulp fermenting, do blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt high performance liquid chromatography to detect the content of Buddhist ripple ester, and calculate 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, 100% (v/w) that inoculum concentration is 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 ℃.
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 ℃, standby.
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 in LB fluid nutrient medium respectively, is placed in 37 ℃, the constant temperature oscillator of 180r/min and carries out the cultivation of bacteria suspension, and incubation time is 14h, makes bacterial classification concentration reach 10
8cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Take Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content is 2.32mg/g).
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly adds after sterilized water, bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, with the barbadosnut pulp fermenting, do blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt high performance liquid chromatography to detect the content of Buddhist ripple ester, and calculate 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, 40% (v/w) that inoculum concentration is 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 ℃.
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 ℃, standby.
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 in LB fluid nutrient medium respectively, is placed in 37 ℃, the constant temperature oscillator of 180r/min and carries out the cultivation of bacteria suspension, and incubation time is 14h, makes bacterial classification concentration reach 10
8cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Take Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content is 2.32mg/g).
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly adds after sterilized water, bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, with the barbadosnut pulp fermenting, do blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt high performance liquid chromatography to detect the content of Buddhist ripple ester, and calculate 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, 40% (v/w) that inoculum concentration is 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 ℃.
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 ℃, standby.
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 in LB fluid nutrient medium respectively, is placed in 37 ℃, the constant temperature oscillator of 180r/min and carries out the cultivation of bacteria suspension, and incubation time is 14h, makes bacterial classification concentration reach 10
8cfu/mL, obtains bacterial strain LY-1 seed liquor.
1.3 solid state fermentation tests
Take Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content is 2.32mg/g).
Unpasteurized solid-state fermentation culture medium is placed in respectively to 100mL triangular flask, evenly adds after sterilized water, bacterium liquid, with aseptic breathable sealing film sealing, select different condition to carry out solid state fermentation test, with the barbadosnut pulp fermenting, do blank test.
After fermentation, grouts, without sterilizing, directly adopt carrene method to carry out Buddhist ripple ester and extract, and adopt high performance liquid chromatography to detect the content of Buddhist ripple ester, and calculate 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, 40% (v/w) that inoculum concentration is 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 ℃.
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 Design Expert8.0 software to set up a kind of Mathematical Modeling of optimization of fermentation conditions.
Take Li Ren township, Yongsheng County, Yunnan Province barbadosnut pulp as raw material (Buddhist ripple ester initial content is 2.32mg/g).
At fermentation time, be under the condition of 48 hours, choose fermentation temperature and inoculum concentration, initial 3 factors of amount of water, utilize Design Expert8.0 software to carry out Box-Behnken design to the impact of the strange land Klebsiella LY-1 solid state fermentation degraded barbadosnut pulp Buddhist ripple ester of dwelling, the degradation rate of Buddhist ripple ester is analog value, each factor is got 3 levels, utilize Design Expert8.0 to carry out analytical test data, further optimize the parameter value (in Table 1) of each factor.
Table 1 experimental design factor level and coding
Through Design Expert8.0, carry out response surface analysis result of the test, the results are shown in Table 2.
Table 2 Box-Behnken response surface experimental design and result
According to the regression analysis of response surface coefficient, the fit equation (binary regression equation) that obtains this model is: Y=82.95+1.16 * X
1-2.73 * X
2-0.36 * X
3+ 3.54 * X
1* X
2+ 2.73 * X
1* X
3-4.05 * X
2* X
3-8.24 * X
1 2-4.76 * X
2 2-3.06 * X
3 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, X1
2, X2
2, X3
2on the impact of Buddhist ripple ester degradation rate extremely significantly (P<0.001), illustrate inoculum concentration, fermentation temperature and initially amount of water be the key factor in sweat, having the greatest impact with inoculum concentration wherein.Significant interaction (P<0.01) between fermentation temperature and inoculum concentration, inoculum concentration and initial amount of water.Lose and intend item not remarkable (P>0.05), 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 that regression equation degree of fitting is good; The multiple correlation coefficient of regression equation is 0.9951, shows available this model explanation of variation of 99.51% Buddhist ripple ester degradation rate, better with actual conditions matching.Proofreading and correct coefficient correlation is 0.9887, and the Buddhist ripple 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 provides a suitable model for strange land Klebsiella (Klebsiellavariicola) the solid state fermentation degraded barbadosnut seed grouts Buddhist ripple ester of dwelling.There is the corresponding actual value of point of safes (0.1775,0.3196,0.2318) (37.12,32.81,104.73) in regression model, most degradation rate estimated value is 83.42%.
Set up Mathematical Modeling is verified:
According to the optimal value of each factor, in conjunction with actual test conditions, fermentation temperature is 37 ℃, inoculum concentration 33% (v/w), and 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 situation of strange land Klebsiella (Klebsiellavariicola) the solid state fermentation degraded barbadosnut seed grouts Buddhist ripple ester of dwelling.
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, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein, 40% (v/w) that inoculum concentration is barbadosnut pulp, 100% (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature, be respectively the constant temperature bottom fermentation 48 hours of 27 ℃, 32 ℃, 37 ℃, 42 ℃, 47 ℃.
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 degradation rate of Buddhist ripple ester is along with the rising of fermentation temperature first raises and reduces afterwards, and when fermentation temperature is 37 ℃, the degradation rate of Buddhist ripple ester is maximum, reaches 78.51%.
Test explanation, while using bacterial strain LY-1 of the present invention to barbadosnut pulp reduce toxicity, fermentation temperature is preferably 37 ℃.
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, is barbadosnut pulp in inoculum concentration respectively 20%, 40%, 60%, 80%, under the condition of 100% (v/w), 100% (v/w) that initial amount of water is barbadosnut pulp;
(3) in temperature, be the constant temperature bottom fermentation 48 hours of 37 ℃.
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 degradation rate of Buddhist ripple ester, along with the increase of inoculum concentration, first raises and reduces afterwards, and when inoculum concentration is 40%, the degradation rate of Buddhist ripple ester is maximum, reaches 81.35%.
Test explanation, during for barbadosnut pulp reduce toxicity, the inoculum concentration of bacterial strain LY-1 of the present invention is preferably 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, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein, 40% (v/w) that inoculum concentration is barbadosnut pulp, under the condition of 50%, 75%, 100%, 125%, 150% (v/w) that is barbadosnut pulp at initial amount of water respectively;
(3) in temperature, be the constant temperature bottom fermentation 48 hours of 37 ℃.
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 degradation rate of Buddhist ripple ester, along with the increase of initial amount of water, first raises and reduces afterwards, and when initial amount of water is 100%, the degradation rate of Buddhist ripple ester is maximum, reaches 82.87%.
Test explanation, while using bacterial strain LY-1 of the present invention to barbadosnut pulp reduce toxicity, initial amount of water is preferably 100%.
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, obtains seed liquor;
(2) get barbadosnut pulp, add water, mix, seed liquor prepared by inoculation step (1), wherein, 40% (v/w) that inoculum concentration is barbadosnut pulp, 100% (v/w) that initial amount of water is barbadosnut pulp;
(3) under being the constant temperature of 37 ℃, temperature ferments 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 degradation rate of Buddhist ripple ester raises along with the prolongation of fermentation time, and when fermentation time is 48 hours, the degradation rate of Buddhist ripple ester reaches 82.16%, then extends fermentation time, and 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 preferably 48 hours.
Above-mentioned result of the test explanation: in fermentation process of the present invention, inoculum concentration is in the scope of 20%～100% (v/w), initial amount of water is in the scope of 50%～150% (v/w), fermentation temperature is in the scope of 27 ℃～47 ℃, 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 toxicity test
The solid state fermentation detoxification of 5.1 pairs of different places of production grouts
Pian Jiao township, Yongsheng County, the Yunnan Province barbadosnut pulp (Buddhist ripple ester initial content is 0.840mg/g) of take is raw material, in inoculum concentration, be 33% (v/w), initial amount of water is under the condition of 105% (v/w), in 37 ℃ of constant incubators, ferments 48 hours, repeats 3 times.
Adopt HPLC detection method, after being fermented, barbadosnut pulp Buddhist ripple ester content is 0.109mg/g, and 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/T 6432-1994 to measure crude protein content, adopt GB/T 6433-2006 to measure crude fat content, adopt GB/T 6434-2006 to measure crude fibre, adopt GB/T50094-2010 to measure ash content, adopt GB/T 18246-2000 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, and strange land Klebsiella (Klebsiellavariicola) the solid state fermentation barbadosnut pulp of dwelling does not destroy the nutritional labeling of barbadosnut pulp.
5.3 animal acute toxicity test
Test determines that detoxification meal maxima solubility in 0.5% sodium carboxymethylcellulose (CMC) solution is 0.3g/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, male and female 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 is filled with and is fed the grouts of detoxification treatment (after test 2.3.5 reduce toxicity, 121 ℃ of sterilizing 20min, carry out vacuum drying after sterilizing) suspension; Test III group is filled with and is fed untreated grouts (Pian Jiao township, Yongsheng County, Yunnan Province barbadosnut pulp (Buddhist ripple ester initial content is 0.840mg/g, and 121 ℃ of sterilizing 20min carry out vacuum drying after sterilizing)) suspension.After gavage is processed, Continuous Observation is 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 its application is very safe, and method of the present invention can successfully be sloughed the Buddhist ripple ester in grouts, to the further utilization of grouts, provides safety guarantee.
In sum, fermentation process of the present invention, can slough the Buddhist ripple ester in barbadosnut pulp easily, and 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 detoxification does not have large change, good without acute toxicity, security, can be directly as animal feed, and the further utilization for barbadosnut pulp, provides reliable approach.
Claims (8)
1. the purposes of strange land Klebsiella (Klebsiellavariicola) in barbadosnut pulp reduce toxicity of dwelling.
2. purposes according to claim 1, is characterized in that: described in the dwell bacterial strain deposit number of strange land Klebsiella (Klebsiellavariicola) LY-1 be CICIM B1743.
3. 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;
(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 ℃～47 ℃.
4. method according to claim 3, is characterized in that: in step (1), described in the dwell bacterial strain deposit number of strange land Klebsiella LY-1 be CICIM B1743.
5. method according to claim 3, it is characterized in that: in step (1), described seed liquor of dwelling strange land Klebsiella LY-1 is prepared as follows: adopt the activation of LB solid medium to dwell after the Klebsiella LY-1 of strange land, picking is inoculated in LB fluid nutrient medium respectively, shaken cultivation 14h under 37 ℃, 180r/min, makes bacterial classification concentration reach 10
8cfu/mL.
6. method according to claim 3, is characterized in that: in step (2), described barbadosnut pulp is prepared as follows: get jatropha curcas seed, squeeze de-oiling, pulverizing, crosses 40 mesh sieves.
7. method according to claim 3, is characterized in that: in step (2), and 33%～40% (v/w) that described inoculum concentration is barbadosnut pulp, 100%～105% (v/w) that initial amount of water is barbadosnut pulp.
8. method according to claim 3, is characterized in that: in step (3), described temperature is 37 ℃; The time of described fermentation is 48 hours.
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