CN105647828A - Fermentation preparation process of Streptomyces marinensis and secondary metabolite Streptochlorin thereof - Google Patents
Fermentation preparation process of Streptomyces marinensis and secondary metabolite Streptochlorin thereof Download PDFInfo
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Abstract
The invention discloses a fermentation preparation process of Streptomyces marinensis and a secondary metabolite thereof. The strain is characterized by having a preservation number of CGMCC No. 11468. The fermentation process specifically includes the steps of: inoculating Streptomyces marinensis to PDA, culturing in an incubator at 27.5 DEG C for 2 d; picking a single colony, inoculating to a seed culture medium, and culturing at 27.5 DEG C for 2 d; inoculating 5% by volume of a seed liquid to a fermentation medium, culturing at 27.5 DEG C for 11 d under the condition of 150 r/min; extracting a metabolite; and finally, separating and purifying a sampling liquid by a high-speed countercurrent chromatography to obtain a product. The process has the advantages of high preparation speed, and high product purity and yield.
Description
Technical field
The present invention relates to fermentable and natural product extraction separation technology field, especially relate to the fermentation preparation technology of a kind of marine streptomyces and secondary metabolite Streptochlorin thereof.
Background technology
Streptochlorin is a kind of indoles microbiotic, and the people such as Jae were separated from the streptomycete 04DH110 of marine source in 2007 and obtain, and found that human body cell is had anti-value-added activity (Jae, SH. by this compound; Jeong, HS.; Lee, HS.; Park, SK.; Kim, HM.; Kwon, HJ.J.Microbiol.Biotechnol.2007,17,1403). In research in recent years, this compound is proved has good anti-tumor activity (Lee, SH.; Shin, HJ.; Kim, DY.; Shim, DW.; Kim, TJ.; Ye, SK.; Won, HS.; Koppula, S.; Kang, TB.; Lee, KH.PLoSOne.2013,8 (9), e74194), anti-inflammatory activity (Shim, DW.; Shin, HJ.; Han, JW.; Shin, WY.; Sun, X.; Shim, EJ.; Kim, TJ.; Kang, TB.; Lee, KH.Int.J.Mol.Sci.2015.16,6902), anti-microbial activity (Zhang, MZ.; Chen, Q.; Xie, CH.; Mulholland, N.; Turner, S.; Irwin, D.; Gu, YC.; Yang, GF.; Clough, J.EuropeanJournalofMedicinalChemistry.2015,92,776) etc.
The fermentation of Streptochlorin is rarely had report by domestic and foreign literature, and in experiment, we utilize general fermentation condition and preparation condition, find that the output of Streptochlorin is few, yield is low, and this becomes the limiting factor that this compound is studied further, is unfavorable for the commercial exploitation in later stage.
Summary of the invention
Technical problem to be solved by this invention is to provide the fermentation preparation technology of a kind of marine streptomyces and secondary metabolite Streptochlorin thereof, the method can significantly improve secondary metabolite Streptochlorin output, the Streptochlorin of preparation high purity quick, efficient.
The present invention solves the problems of the technologies described above the technical scheme adopted: a kind of marine streptomyces, this bacterium is SYYLWHS-1-4 bacterial strain, classification called after streptomycete (Streptomycessp.), being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 30th, 2015, deposit number is CGMCCNo.11468.
This bacterial strain presents faint yellow translucent at the slat chain conveyor initial stage, and bacterium colony is rounded, and smooth surface is moistening, and along with incubation time extends, aerial hyphae grows, and covers whole bacterium colony surface, presents white.
A fermentation preparation technology of marine streptomyces secondary metabolite Streptochlorin, comprises the following steps:
(1) solid culture
The marine streptomyces that the deposit number being deposited in-80 DEG C of refrigerators is CGMCCNo.11468 is coated on solid plate substratum, 27.5 DEG C of incubators is cultivated 48h and obtains dull and stereotyped bacterial classification;
(2) seed culture
Get the dull and stereotyped bacterial classification of well-grown, it is inoculated in the liquid nutrient medium that liquid amount is 50%, at 27.5 DEG C, cultivate 48h when 150r/min and obtain seed liquor;
(3) fermentation culture
Getting the seed liquor of well-grown, by volume the inoculum size of mark 5% is inoculated in the liquid nutrient medium that liquid amount is 50%, in 27.5 DEG C, cultivates 11d and obtain fermented liquid when 150r/min;
(4) fermented liq process
Get fermented liquid to add isopyknic ethyl acetate and fully extract once, collect extraction liquid, get raffinate again and add isopyknic ethyl acetate re-extract once, get raffinate again and add isopyknic extraction into ethyl acetate once, merge three extraction gained extraction liquids, rotary evaporated to dryness, again redissolves with methyl alcohol, obtains into sample liquid;
(5) preparation of Streptochlorin
Sample liquid carries out separation and purification to entering to adopt high speed adverse current chromatogram (HSCCC) method, obtains marine streptomyces secondary metabolite Streptochlorin monomer.
The formula of the solid plate substratum described in step (1) is as follows: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g, agar 15g.
Liquid culture based formulas described in step (2) and step (3) is as follows: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g.
Step (5) is specially:
A. get sherwood oil, ethyl acetate, methyl alcohol and pure water mix in the ratio of 9:0.8:5:5 after as the two-phase system of high-speed counter-current chromatograph;
B. by upper as stationary phase using sherwood oil and ethyl acetate in two-phase system, lower to moving phase using methyl alcohol and pure water, stationary phase is entered high-speed counter-current chromatograph by the speed pump of 50mL/min until instrument pipeline is full of stationary phase, regulate high-speed counter-current chromatograph rotating speed, after rotating speed reaches 800r/min, moving phase is entered high-speed counter-current chromatograph with the speed pump of 5mL/min until moving phase and stationary phase reach the laggard sample of balance;
C. start-up detector starts monitoring, collects the elutriant that the appearance time containing Streptochlorin is 42min to 52min part, obtains purity and reach 90% and above marine streptomyces secondary metabolite Streptochlorin monomer after concentrating under reduced pressure.
Compared with prior art, it is an advantage of the current invention that: the present invention makes public for the first time the fermentation preparation technology of a kind of marine streptomyces and secondary metabolite Streptochlorin thereof, this fermentation process makes the output of Streptochlorin increase substantially, and reaches 3.37mg/L. In addition, the HSCCC method that preparation Streptochlorin uses contrasts other preparation methods and has the features such as sample size is big, flow velocity fast, purity height, especially can solve the problem that when utilizing other chromatographic processes to prepare, sample recovery rate is low. The commercial exploitation of the scientific research of Streptochlorin and later stage is all significant by the present invention.
Above-mentioned marine streptomyces, this bacterium is SYYLWHS-1-4 bacterial strain, classification called after streptomycete (Streptomycessp.), deposit number is CGMCCNo.11468, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 30th, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Accompanying drawing explanation
Fig. 1 is ultra-high efficiency liquid chromatography (UPLC) figure that Streptochlorin enters sample liquid;
Fig. 2 is ultra-high efficiency liquid chromatography (UPLC) figure of the Streptochlorin monomer obtained after HSCCC purifying;
Fig. 3 is the HSCCC spectrogram that Streptochlorin enters sample liquid;
Fig. 4 is the core magnetic qualification H spectrum of the Streptochlorin monomer obtained after HSCCC purifying;
Fig. 5 is the core magnetic qualification C spectrum of the Streptochlorin monomer obtained after HSCCC purifying;
Fig. 6 is the MS spectrogram of the Streptochlorin monomer obtained after HSCCC purifying.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
A kind of marine streptomyces, this bacterium is SYYLWHS-1-4 bacterial strain, classification called after streptomycete (Streptomycessp.), deposit number is CGMCCNo.11468, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 30th, 2015. This bacterium colony presents faint yellow translucent at the slat chain conveyor initial stage, and bacterium colony is rounded, and smooth surface is moistening, and along with incubation time extends, aerial hyphae grows, and covers whole bacterium colony surface, presents white. The concrete steps that this fermenting marine streptomyces produces secondary metabolite Streptochlorin are as follows:
(1) solid culture
The marine streptomyces that the deposit number being deposited in-80 DEG C of refrigerators is CGMCCNo.11468 is coated on solid plate substratum, 27.5 DEG C of incubators is cultivated 48h and obtains dull and stereotyped bacterial classification; Wherein the formula of solid plate substratum is as follows: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g, agar 15g;
(2) seed culture
Get the dull and stereotyped bacterial classification of well-grown, it is inoculated in the liquid nutrient medium that liquid amount is 50%, at 27.5 DEG C, cultivate 48h when 150r/min and obtain seed liquor;
(3) fermentation culture
Getting the seed liquor of well-grown, by volume the inoculum size of mark 5% is inoculated in the liquid nutrient medium that liquid amount is 50%, in 27.5 DEG C, cultivates 11d and obtain fermented liquid when 150r/min; Wherein liquid culture based formulas is as follows: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g;
(4) fermented liq process
Get fermented liquid to add isopyknic ethyl acetate and fully extract once, collect extraction liquid, get raffinate again and add isopyknic ethyl acetate re-extract once, get raffinate again and add isopyknic extraction into ethyl acetate once, merge three extraction gained extraction liquids, rotary evaporated to dryness, again redissolves with methyl alcohol, obtains into sample liquid; HPLC detected result is as shown in Figure 1; Illustrate that Streptochlorin is the main component in process secondary fermentation liquid, also have some impurity in addition;
(5) preparation of Streptochlorin
Sample liquid carries out separation and purification to entering to adopt high speed adverse current chromatogram (HSCCC) method, obtains marine streptomyces secondary metabolite Streptochlorin monomer, and detailed process is:
A. get sherwood oil, ethyl acetate, methyl alcohol and pure water mix in the ratio of 9:0.8:5:5 after as the two-phase system of high-speed counter-current chromatograph;
B. by upper as stationary phase using sherwood oil and ethyl acetate in two-phase system, lower to moving phase using methyl alcohol and pure water, stationary phase is entered high-speed counter-current chromatograph by the speed pump of 50mL/min until instrument pipeline is full of stationary phase, regulate high-speed counter-current chromatograph rotating speed, after rotating speed reaches 800r/min, moving phase is entered high-speed counter-current chromatograph with the speed pump of 5mL/min until moving phase and stationary phase reach the laggard sample of balance;
C. start-up detector starts monitoring, collects the elutriant that the appearance time containing Streptochlorin is 42min to 52min part, obtains purity and reach 90% and above marine streptomyces secondary metabolite Streptochlorin monomer after concentrating under reduced pressure.
The marine streptomyces secondary metabolite Streptochlorin monomer obtained after purifying is carried out HPLC detection, and as shown in Figure 2, as shown in Figure 2, obtain after HSCCC purifying is single Streptochlorin chromatographic peak to result.
Streptochlorin qualitative analysis: this streptomycete is carried out mass propgation, extraction into ethyl acetate, uses HSCCC separation and purification, obtains monomeric compound, carries out core magnetic and mass spectrum qualification, and result is as shown in Fig. 4, Fig. 5 and Fig. 6.
Streptochlorin compound structure is identified:
(1) molecular weight (MS) measures: compound molecular weight adopts direct injection to measure at HPLC-MS chromatographic instrument, Mass Spectrometry Conditions: adopt electron spray ionisation source ion source temperature 100 DEG C, desolventizing temperature 250 DEG C, desolventizing nitrogen flow rate 400L/h, taper hole blowback nitrogen. Quadrupole sweep limit m/z50-500. TOF ion flight mode adopts V model. Use leu-enkaphalin, as external standard, object ion is carried out accurate mass locking. Adopt positive ion mode, kapillary ionization voltage 2.8kV, sampling taper hole voltage 80V, collision cell energy 5-80V. The MS spectrogram obtained is such as Fig. 6;
(2) NMR measures: sample dissolution in deuterated methanol, NMR [1H,13C, homonuclearcorrelationspectroscopy (COSY), heteronuclearsinglequantumcoherencespectroscopy (HSQC), with heteronuclearmultiplebondcorrelationspectroscopy (HMBC)] measure with VarianINOVA400 chromatographic instrument, measure1H operates when 400MHz, measures13C operates when 100MHz, and tetramethylsilane (TMS) is as interior mark. The NMR obtained1H and13C spectrogram is such as Fig. 4, Fig. 5.
This compound is light yellow crystal, HR-ESI-MSm/z:219.0303 [M+H]+, calculated value is 218.0303. This compound1H and13CNMR data:1HNMR(500MHz,CD3OD, TMS) ppm:7.28 (2H, dt, J=24.0), 7.45 (1H, d, J=8.1), 7.81 (1H, d, J=2.7), 7.88 (1H, s), 8.09 (1H, d, J=7.9), 8.65 (1H, s);13CNMR(126MHz,CD3OD, TMS) ppm:103.93,111.61,120.93,121.32,121.83,123.43,123.46,124 .56,135.80,143.34,147.72.
Interpretation: measuring through HPLC-MS and NMR, the molecule of compound S treptochlorin is C11H7ClN2O, the molecular weight of compound S treptochlorin is, chemical structural drawing is as follows,��
Streptochlorin quantitative analysis: watersC18 reverse-phase chromatographic column (1.7 ��m, 2.1 �� 100mm), sampling volume 1 �� L, flow velocity 5mL/min, gradient elution, in 10min, by 10% methyl alcohol and 90% water to 90% methyl alcohol and 10% water, about Streptochlorin5.7min goes out peak, under 220nm wavelength, color atlas is carried out integration, typical curve according to its chromatographic peak area and the Streptochlorin of mensuration carries out quantitative analysis, calculate marine streptomyces secondary metabolite Streptochlorin monomer purity and can reach 90%, often liter of fermented liquid can produce Streptochlorin3.37mg.
Specific embodiment two
1, the determination of culture condition remarkably influenced factor
To the yeast powder (X of substratum1), Zulkovsky starch (X2), MgSO4(X3), K2HPO4(X4), CaCl2(X5), initial pH value (X6), mediumvolume(X7), temperature(X8), marinumsalt(X9) nine factors carry out variable analysis. Each variable all sets a height (+1) low (-1) 2 levels (concrete numerical value is in table 1). (wherein X is tested with the Plackett-Burman of Minitab Software Create 12 factor 20 times10��X11And X12For pseudo-variable), responsively it is worth (Y) with Streptochlorin output (mg/L), by the effect value of each factor of Mintab software analysis, the factor selecting influential effect higher does further investigation. Experimental design is in table 1 and the results are shown in Table 2, and analytical results is in table 3.
The level of each variable in table 1 culture medium prescription
The Plackeet-Burman experiment that table 212 factor is 20 times
Table 3 analytical results
As shown in Table 3, it is yeast powder (X to Streptochlorin yield effect relatively big (P��0.05)1), Zulkovsky starch (X2) and K2HPO4(X4).
2, the determination of best medium
Center combination design principle according to Box-Behnken, 3 levels are respectively got to by above-mentioned determined 3 factors of specific embodiment two experimental result, carry out the response surface analysis of 20 test points, remarkably influenced factor is carried out further optimization, for other non-principal factors, all it is averaged level value (concrete numerical value is in table 4). Responsively it is worth (Z) with the output (mg/L) of Streptochlorin, by Mintab, experimental data is analyzed. Experimental design is in table 4 and the results are shown in Table 5, and analytical results is in table 6.
The level of each variable in table 4 culture medium prescription
The central combination design of table 5Box-Behnken and result
Table 6 analytical results
Each factorial effect coefficient that experimentally analytical results result obtains, sets up the many units quadratic equation of output on three remarkably influenced factors of marine streptomyces meta-bolites Streptochlorin, as follows:
Z=2.9040+0.7703*X1+0.5307*X2+0.4665*X4-0.4688*X1 2-0.5895*X2 2-0.1994*X4 2+0.1315*X1*X2-0.2668*X1*X4-0.2163*X2*X4
The regression equation of gained is asked respectively the first-order partial derivative of three variablees, and makes it be 0, obtain ternary linear function group, solve to obtain X1��X2And X4, convert Streptochlorin output obtain maximum value time optimal culture condition: yeast powder 1.889g/L, Zulkovsky starch 8.636g/L, K2HPO40.359g/L. Other conditions get intermediate value: MgSO40.625g/L, CaCl22.5g/L, sea-water extract 25g/L, liquid amount 50%, initial pH value 7.0, culture temperature 27.5 �� of C. Now the output of meta-bolites Streptochlorin is 3.4179mg/L. For determining the compatibility of experimental model and the actual result set up, repeating experiment 3 times under the fermentation condition optimized, the mean yield obtaining Streptochlorin is 3.37mg/L, and experimental value only differs 1.4% compared with theoretical value, illustrates that this model is reliable.
3, the determination of optimum incubation time
Select the culture medium prescription optimized, the seed liquor of marine streptomyces is accessed in this substratum (1L Erlenmeyer flask, liquid amount is 40%, 5% inoculum size), at 25 DEG C, when 150rpm, cultivate 15d. Sample 10mL extraction into ethyl acetate every day, taking the chromatographic peak area of Streptochlorin under 220nm as response value, it has been found that the Streptochlorin produced for the 11st day is maximum.
Table 7 cultivated days optimization experiment
In sum, with Plackett-Burman experimental design and Box-Behnken Responds Surface Methodology, the fermentation condition affecting this streptomycete product Streptochlorin is optimized, Streptochlorin is carried out qualitative analysis by syncaryon mr and mass spectrum, utilizing ultra-high efficiency liquid chromatography that Streptochlorin is carried out quantitative analysis, the optimal conditions of fermentation finally obtaining this bacterial strain product Streptochlorin is: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g, incubation time 11d, under optimal conditions is cultivated, the Streptochlorin content of generation reaches 3.37mg/L.The HSCCC method that preparation Streptochlorin uses contrasts other preparation methods and has the features such as sample size is big, flow velocity fast, purity height, especially can solve the problem that when utilizing other chromatographic processes to prepare, sample recovery rate is low. This suboptimization substantially increases the output of Streptochlorin, for the later stage industrialized developing of this bacterium has laid good basis.
Certainly, above-mentioned explanation is limitation of the present invention not, and the present invention is also not limited to above-mentioned citing. Change, remodeling, interpolation or the replacement that those skilled in the art make in the essential scope of the present invention, also should belong to protection scope of the present invention, and protection scope of the present invention is as the criterion with claim book.
Claims (6)
1. a marine streptomyces, it is characterized in that: this bacterium is SYYLWHS-1-4 bacterial strain, classification called after streptomycete (Streptomycessp.), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 30th, 2015, and deposit number is CGMCCNo.11468.
2. a kind of marine streptomyces according to claim 1, it is characterised in that: this bacterial strain presents faint yellow translucent at the slat chain conveyor initial stage, and bacterium colony is rounded, smooth surface is moistening, and along with incubation time extends, aerial hyphae grows, cover whole bacterium colony surface, present white.
3. the fermentation preparation technology of a marine streptomyces secondary metabolite Streptochlorin, it is characterised in that comprise the following steps:
(1) solid culture
The marine streptomyces that the deposit number being deposited in-80 DEG C of refrigerators is CGMCCNo.11468 is coated on solid plate substratum, 27.5 DEG C of incubators is cultivated 48h and obtains dull and stereotyped bacterial classification;
(2) seed culture
Get the dull and stereotyped bacterial classification of well-grown, it is inoculated in the liquid nutrient medium that liquid amount is 50%, at 27.5 DEG C, cultivate 48h when 150r/min and obtain seed liquor;
(3) fermentation culture
Getting the seed liquor of well-grown, by volume the inoculum size of mark 5% is inoculated in the liquid nutrient medium that liquid amount is 50%, in 27.5 DEG C, cultivates 11d and obtain fermented liquid when 150r/min;
(4) fermented liq process
Get fermented liquid to add isopyknic ethyl acetate and fully extract once, collect extraction liquid, get raffinate again and add isopyknic ethyl acetate re-extract once, get raffinate again and add isopyknic extraction into ethyl acetate once, merge three extraction gained extraction liquids, rotary evaporated to dryness, again redissolves with methyl alcohol, obtains into sample liquid;
(5) preparation of Streptochlorin
Sample liquid carries out separation and purification to entering to adopt high speed adverse current chromatogram (HSCCC) method, obtains marine streptomyces secondary metabolite Streptochlorin monomer.
4. the fermentation preparation technology of a kind of marine streptomyces secondary metabolite Streptochlorin according to claim 3, it is characterised in that the formula of the solid plate substratum described in step (1) is as follows: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g, agar 15g.
5. the fermentation preparation technology of a kind of marine streptomyces secondary metabolite Streptochlorin according to claim 3, it is characterised in that the liquid culture based formulas described in step (2) and step (3) is as follows: pure water 1L, yeast extract 1.889g, Zulkovsky starch 8.636g, K2HPO40.359g��MgSO40.625g��CaCl22.5g, sea crystal 25g.
6. the fermentation preparation technology of a kind of marine streptomyces secondary metabolite Streptochlorin according to claim 3, it is characterised in that step (5) is specially:
A. get sherwood oil, ethyl acetate, methyl alcohol and pure water mix in the ratio of 9:0.8:5:5 after as the two-phase system of high-speed counter-current chromatograph;
B. by upper as stationary phase using sherwood oil and ethyl acetate in two-phase system, lower to moving phase using methyl alcohol and pure water, stationary phase is entered high-speed counter-current chromatograph by the speed pump of 50mL/min until instrument pipeline is full of stationary phase, regulate high-speed counter-current chromatograph rotating speed, after rotating speed reaches 800r/min, moving phase is entered high-speed counter-current chromatograph with the speed pump of 5mL/min until moving phase and stationary phase reach the laggard sample of balance;
C. start-up detector starts monitoring, collects the elutriant that the appearance time containing Streptochlorin is 42min to 52min part, obtains purity and reach 90% and above marine streptomyces secondary metabolite Streptochlorin monomer after concentrating under reduced pressure.
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| CN109652483A (en) * | 2018-12-03 | 2019-04-19 | 曲阜师范大学 | A kind of marine actinomycete liquid fermentation production extracts compound and its preparation method and application |
| CN110310881A (en) * | 2019-06-17 | 2019-10-08 | 宁波大学 | For the collision induced dissociation pond of ion cascade mass spectrometry and its application method |
| CN110452940A (en) * | 2019-09-04 | 2019-11-15 | 台州职业技术学院 | A kind of separating and extracting process of the secondary metabolite of streptomycete |
| CN114686540A (en) * | 2022-01-21 | 2022-07-01 | 中国科学院西北生态环境资源研究院 | Efficient fermentation preparation process of streptomyces thermalii secondary metabolite |
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| CN109652483A (en) * | 2018-12-03 | 2019-04-19 | 曲阜师范大学 | A kind of marine actinomycete liquid fermentation production extracts compound and its preparation method and application |
| CN109652483B (en) * | 2018-12-03 | 2022-07-29 | 曲阜师范大学 | Marine actinomycete liquid fermentation product extraction compound and preparation method and application thereof |
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| CN110452940A (en) * | 2019-09-04 | 2019-11-15 | 台州职业技术学院 | A kind of separating and extracting process of the secondary metabolite of streptomycete |
| CN110452940B (en) * | 2019-09-04 | 2021-03-30 | 台州职业技术学院 | Separation and extraction method of secondary metabolite of streptomycete |
| CN114686540A (en) * | 2022-01-21 | 2022-07-01 | 中国科学院西北生态环境资源研究院 | Efficient fermentation preparation process of streptomyces thermalii secondary metabolite |
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