CN105087723A - Method for efficiently synthesizing natamycin by coupling mixing precursor with fungal elicitor - Google Patents
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Abstract
Disclosed is a method for efficiently synthesizing natamycin by coupling mixing precursor with fungal elicitor. The method includes fermentation of streptomyces natalensis. The method is characterized in that natamycin mixing precursor is added into a fermentation culture medium by means of fed-batch in the process of fermentation, the fungal elicitor is added in the process of fermentation, the natamycin mixing precursor is composed of sodium propionate, sodium acetate and isoleucine, and the fungal elicitor is fermentation liquid producing . The natamycin mixing precursor and the fungal elicitor are added into the fermentation culture medium, so that yield and purity of natamycin are improved greatly, yield of natamycin reaches more than 5.97g/L and is increased by 573.1% when compared with that acquired when the mixing precursor and the fungal elicitor are not added.
Description
Technical field
The present invention relates to technical field of bioengineering, specifically a kind of method of High-efficient Production tennecetin.
Background technology
Tennecetin (
natamycin) be by Streptomyces natalensis (
streptomycesnatalensis), brown yellow spore streptomycete (
streptomycesgilvosporeus), streptomyces chatanoogensis (
streptomyceschatanoogensis) and streptomyces lydicus (
streptomyceslydicus) etc. the polyene macrolide antibiotics of a kind of efficient, wide spectrum that produces through fermentation.Its molecular formula is C
33h
47nO
13, as shown in Equation 1, this molecule contains two sixteen-ring polyene macrolides to chemical structure, and ring is outer is connected to a marine alga aminosugar by glycosidic link chain.Tennecetin and sterol compound have higher affinity, and by reacting with the sterol compound on cytolemma, trigger cell membrane structure changes and breaks thus, causes the seepage of entocyte, makes necrocytosis.Tennecetin is not only antimycotic, and can also the generation of Antifungi toxin, can be used for treating fungi infestation, comprises candiyeast, mould, aspergillus tubigensis, reaping hook mould etc.Owing to there is not this sterol compound, so tennecetin does not have restraining effect to bacterium in the cell walls of bacterium and cytolemma.Because of its not anti-bacteria fermentation, safety non-toxic, so be also widely used in the foodstuffs industry such as milk-product, meat product and fruits and vegetables, be food preservatives by more than 30 state approval in the world.
Formula 1 tennecetin chemical structural formula
Nineteen fifty-five, the people such as Struykd isolate Streptomyces natalensis from the pedotheque in Natal state, South Africa, and therefrom separation first obtains tennecetin, and China the earliest Zeng Yiming is pimaricin, natamycin etc.External research in tennecetin fermentative production is more deep, and relevant patent report is also many.And the domestic research to tennecetin relatively lags behind, mostly lay particular emphasis on the aspect such as strain improvement and fermentation technology optimization, Yang Dongjing etc. utilize streptomycete resistance to combine with ultraviolet mutagenesis, and obtain tennecetin superior strain, output reaches 2.80g/L; Zhu Hui etc., by gene rearrangement technology, obtain the bacterial strain that tennecetin output can reach 3.52g/L; Luo Jianmei etc., by the optimization to zymotechnique, make tennecetin output reach 5.67g/L; Simultaneously relevant research is also being done by other scientific research institution more domestic.
At present, tennecetin is widely used in the foodstuffs industry such as milk-product, beverage, fruit, meat, and the tennecetin of merchandized handling has had the Myprozine of Cyanamid company of the U.S., the Delvocid of GB Kyowa Hakko of the U.S..In addition, the trade names such as Natafucin, Pimafucin, Natamaxvx are also had.Wherein, Natamycin (
natamaxTx)be that tennecetin and lactose mix in the ratio of 1:1 (mass ratio), can be used for food surfaces rotproofing or directly add in food.But the fermentation level of current domestic tennecetin is also lower, cause cost too high, limit tennecetin and realize large-scale industrial production as a kind of novel natural antiseptic agent in China.
Summary of the invention
Technical purpose of the present invention is: for the present situation that current tennecetin output is lower, there is provided a kind of simple to operate, and can promote that Streptomyces natalensis efficiently synthesizes the method for tennecetin, the method can significantly improve the output of tennecetin, reduces production cycle and the production cost of tennecetin.
For realizing above-mentioned technical purpose, the technical solution adopted in the present invention is: a kind of method of efficient synthesis tennecetin, comprise Streptomyces natalensis (
streptomycesnatalensis) fermentation, wherein, the mode added with stream during the fermentation adds tennecetin mixing precursor in fermention medium, add fungal elicitor during the fermentation, described tennecetin mixing precursor is selected from Sodium Propionate, sodium acetate and Isoleucine, described fungal elicitor be Penicllium chrysogenum (
penicilliumchrysogenum) fermented liquid.
Wherein, described tennecetin mixing precursor fermenting process the interpolation time preferably ferment 24h-36h time, every two hours add once.Described fungal elicitor fermenting process the interpolation time preferably ferment 60h time.
In the present invention, described tennecetin mixing precursor by mol ratio be the Sodium Propionate of 1:1 ~ 15:1 ~ 15, sodium acetate and Isoleucine form; Its addition is 0.1 ~ 1.0% (V/V) preferably, most preferably 0.6% (V/V).
In the present invention, described fungal elicitor be Penicllium chrysogenum (
penicilliumchrysogenum) fermented liquid, preferred Penicllium chrysogenum in the fermentation medium 28 ~ 34 DEG C of shaking tables cultivates the fermented liquid of 2 ~ 7d, and described fermention medium is containing glucose 20 ~ 30g, yeast extract paste 1 ~ 5g, extractum carnis 5 ~ 10g in every 1000mL.Preferred pH6.8 ~ 7.3 of the fermention medium of this Penicllium chrysogenum.The supernatant liquor of centrifugal gained again after described fungal elicitor preferred fermentation liquor filtered through gauze.Described supernatant liquor is degerming preferably through filtering with microporous membrane.The aperture of described millipore filtration is 0.2 ~ 1.25 μm, preferably 0.45 μm.Described Penicllium chrysogenum is the bacterial strain of any Penicllium chrysogenum Pseudomonas.Addition preferably 1 ~ 10% (V/V) of fungal elicitor, more preferably 6% (V/V).
In the present invention, a better example of preparing of described fungal elicitor is prepared by following methods and obtains:
(1) cultivation of fungi: by slant medium Penicllium chrysogenum (
penicilliumchrysogenum) be inoculated in liquid seed culture medium, 28 ~ 34 DEG C, 150 ~ 220r/min cultivates 1 ~ 7d;
Described slant culture based formulas is contain in every 1000mL: W-Gum 50 ~ 100g, glucose 15 ~ 30g, agar powder 15 ~ 20g;
Described liquid seed culture medium formula is contain in every 1000mL: glucose 20 ~ 30g, yeast extract paste 1 ~ 5g, extractum carnis 5 ~ 10g, pH7.0 ~ 7.5 after sterilizing;
(2) by cultured for step (1) fermented liquid first six layers of filtered through gauze, then 8000 ~ 12000r/min is centrifugal, retains supernatant liquor, finally removes bacterial classification with filtering with microporous membrane, obtains fungal elicitor.
In the present invention, in fermentation adopt Streptomyces natalensis seed to be that this area is conventional, preferred method is by comprising the following steps preparation: be inoculated in liquid seed culture medium by the Streptomyces natalensis on slant medium, cultivates 2 ~ 7d for 26 ~ 32 DEG C; Described slant medium is contain in every 1000mL: glucose 10 ~ 20g, malt extract 1 ~ 5g, yeast extract paste 1 ~ 5g, Tryptones 5 ~ 15g, agar powder 15 ~ 20g; Described liquid seed culture medium is contain in every 1000mL: glucose 15 ~ 30g, peptone 5 ~ 15g, yeast extract paste 5 ~ 10g, sodium-chlor 5 ~ 15g.Wherein, described slant medium and the pH of liquid seed culture medium are 6.5 ~ 8.0, preferred pH7.0 ~ 7.5, most preferably pH7.2.
In the present invention, the fermention medium that described Streptomyces natalensis natamycin fermentation preparation adopts is conventional Streptomyces natalensis fermention medium, containing glucose 10 ~ 20g, soybean cake powder 20 ~ 40g in the preferred every 1000mL of the present invention, Zulkovsky starch 20 ~ 40g, yeast extract paste 5 ~ 10g.This fermention medium pH6.5 ~ 8.0, preferred pH7.0 ~ 7.5, most preferably pH7.2.
In the present invention, the fermentation of Streptomyces natalensis is normal condition, preferred leavening temperature 26 ~ 32 DEG C, fermentation time 5 ~ 9d.Preferred 160 ~ 220r/min shaking culture during fermentation.
In the present invention, in fermention medium, the inoculum size of Streptomyces natalensis is conventional, preferably 1 ~ 10% (V/V).
In the present invention, described Streptomyces natalensis is any bacterial strain during conventional Streptomyces natalensis belongs to.
In the present invention, the preparation of described various substratum is all ordinary method, uses distilled water constant volume by after various raw material mixing.Described substratum all will through sterilizing before using.Need adjust pH, after constant volume, carry out adjust ph.The control method of pH value is conventional, regulates with acid or alkali.
In the present invention, test materials used unless otherwise indicated, market all can fetch.
Beneficial effect:
Compared with prior art, the present invention by adding tennecetin mixing precursor and fungal elicitor in fermention medium, substantially increase output and the purity of tennecetin, make the output of tennecetin reach more than 5.97g/L, to compare with the output of fungal elicitor improve 573.1% than not adding mixing precursor.Meanwhile, the technique of synthetic method own is simple, utilization of materials is high, and equipment requirements is low, reduces production cost, facilitates the process of industrialization that it is produced.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described in detail, unreceipted specific experiment condition in following examples, the condition of conditioned disjunction manufacturer suggestion routinely usually.
The invention provides a kind of precursor coupling fungal elicitor that mixes and promote the tennecetin method of efficiently synthesizing, improve the output of tennecetin by adding tennecetin mixing precursor and fungal elicitor to stream in fermented liquid.
In the present invention, described Streptomyces natalensis (
streptomycesnatalensis) be purchased from DSMZ of Institute of Microorganism, Academia Sinica, Penicllium chrysogenum (
penicilliumchrysogenum) be this Laboratories Accession bacterial classification.
embodiment 1
1, the preparation of seed: picking 2 ring spore is in sterile distilled water from Streptomyces natalensis slant medium, make the spore suspension that mass concentration is 5%, then be inoculated in the 250mL triangular flask containing 40mL liquid seed culture medium by the inoculum size of 8% (V/V), 30 DEG C, 180r/min shaking table cultivation 48h.
Described slant culture based formulas is: glucose 12g, malt extract 4g, yeast extract paste 4g, Tryptones 7g, agar powder 20g, and adding distil water is settled to 1000mL, pH7.2 after sterilizing.
Described liquid seed culture medium formula is: glucose 20g, peptone 6g, yeast extract paste 8g, and sodium-chlor 8g, adds water and be settled to 1000mL, pH7.3 after sterilizing.
2, the preparation of fungal elicitor: the Penicllium chrysogenum sterile distilled water on slant medium is made the spore suspension that mass concentration is 5%, and be inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium with the inoculum size of 5% (V/V), 30 DEG C, 180r/min shaking culture 48h.By cultured fermented liquid six layers of filtered through gauze, the then centrifugal 15min of 10000r/min, retain supernatant liquor, be finally that the filtering with microporous membrane of 0.22 μm is degerming with aperture, obtain fungal elicitor.
Described slant culture based formulas is: containing W-Gum 80g, glucose 25g, agar powder 20g in every 1000mL.
Described liquid culture based formulas is: containing glucose 30g, yeast extract paste 4g, extractum carnis 8g, pH7.2 after sterilizing in every 1000mL.
3, during fermentation culture, seed liquor is inoculated in the 250mL triangular flask containing 40mL tennecetin fermention medium by the inoculum size of 5% (V/V), 30 DEG C, 180r/min shaking culture, when fermenting 24h, according to the method for adding every two hours once, in fermented liquid, additional proportion is the mixing precursor (Sodium Propionate: sodium acetate: Isoleucine=1:7:1) of 0.6% (V/V), and the ratio when fermenting 60h in 5% (V/V) adds fungal elicitor.Whole fermenting process totally 7 days.
Described tennecetin fermentative medium formula is: glucose 20g, soybean cake powder 30g, Zulkovsky starch 30g, yeast extract paste 8g, pH7.2 after sterilizing.
4, the mensuration of tennecetin output: get 1mL natamycin fermentation liquor, adds 9mL methyl alcohol, and fully after vibration, the centrifugal 10min of 12000r/min, retains supernatant liquor, and the output detecting tennecetin by HPLC method reaches 5.99g/L.
embodiment 2
1, the preparation of seed: picking 2 ring spore is in sterile distilled water from Streptomyces natalensis slant medium, make the spore suspension that mass concentration is 5%, then be inoculated in the 250mL triangular flask containing 40mL seed culture medium by the inoculum size of 8% (V/V), 32 DEG C, 200r/min shaking table cultivation 48h.
Described slant culture based formulas is: glucose 12g, malt extract 5g, yeast extract paste 5g, Tryptones 12g, agar powder 20g, and adding distil water is settled to 1000mL, pH7.2 after sterilizing.
Described seed culture based formulas is: glucose 25g, peptone 8g, yeast extract paste 6g, and sodium-chlor 10g, adds water and be settled to 1000mL, pH7.0 after sterilizing.
2, the preparation of fungal elicitor: the Penicllium chrysogenum sterile distilled water on slant medium is made the spore suspension that mass concentration is 5%, and be inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium with the inoculum size of 5% (V/V), 28 DEG C, 200r/min shaking culture 72h.By cultured fermented liquid six layers of filtered through gauze, the then centrifugal 10min of 12000r/min, remove thalline, be that the filtering with microporous membrane of 0.22 μm is degerming with aperture, obtain fungal elicitor.
Described slant culture based formulas is: containing W-Gum 80g, glucose 30g, agar powder 18g in every 1000mL.
Described liquid culture based formulas is: containing glucose 20g, yeast extract paste 5g, extractum carnis 6g, pH7.3 after sterilizing in every 1000mL.
3, during liquid fermenting, seed liquor is inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium by the inoculum size of 8% (V/V), 28 DEG C, 200r/min shaking culture, when fermenting 24h, according to the method for adding every two hours once, in fermented liquid, additional proportion is the mixing precursor (Sodium Propionate: sodium acetate: Isoleucine=1:7:1) of 0.6% (V/V), and the ratio when fermenting 60h in 6% (V/V) adds fungal elicitor.Whole fermenting process totally 7 days.
Described tennecetin fermentative medium formula is: glucose 10g, soybean cake powder 40g, Zulkovsky starch 35g, yeast extract paste 6g, pH7.2 after sterilizing.
4, the mensuration of tennecetin output: get 1mL natamycin fermentation liquor, adds 9mL methyl alcohol, and fully after vibration, the centrifugal 10min of 12000r/min, retains supernatant liquor, and the output detecting tennecetin by HPLC method reaches 5.97g/L.
embodiment 3
1, the preparation of seed: picking 2 ring spore is in sterile distilled water from Streptomyces natalensis slant medium, make the spore suspension that mass concentration is 5%, then be inoculated in the 250mL triangular flask containing 40mL seed culture medium by the inoculum size of 8% (V/V), 26 DEG C, 200r/min shaking table cultivation 168h.
Described slant culture based formulas is: glucose 10g, malt extract 5g, yeast extract paste 5g, Tryptones 5g, agar powder 15g, and adding distil water is settled to 1000mL, pH7.2 after sterilizing.
Described seed culture based formulas is: glucose 15g, peptone 5g, yeast extract paste 10g, and sodium-chlor 2g, adds water and be settled to 1000mL, pH7.0 after sterilizing.
2, the preparation of fungal elicitor: the Penicllium chrysogenum sterile distilled water on slant medium is made the spore suspension that mass concentration is 5%, and be inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium with the inoculum size of 5% (V/V), 34 DEG C, 150r/min shaking culture 24h.By cultured fermented liquid six layers of filtered through gauze, the then centrifugal 10min of 8000r/min, remove thalline, be that the filtering with microporous membrane of 0.22 μm is degerming with aperture, obtain fungal elicitor.
Described slant culture based formulas is: containing W-Gum 50g, glucose 15g, agar powder 15g in every 1000mL.
Described liquid culture based formulas is: containing glucose 30g, yeast extract paste 1g, extractum carnis 5g, pH7.5 after sterilizing in every 1000mL.
3, during liquid fermenting, seed liquor is inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium by the inoculum size of 10% (V/V), 26 DEG C, 200r/min shaking culture, when fermenting 36h, according to the method for adding every two hours once, in fermented liquid, additional proportion is the mixing precursor (Sodium Propionate: sodium acetate: Isoleucine=1:4:15) of 1.0% (V/V), and the ratio when fermenting 60h in 10% (V/V) adds fungal elicitor.Whole fermenting process totally 5 days.
Described tennecetin fermentative medium formula is: glucose 20g, soybean cake powder 20g, Zulkovsky starch 40g, yeast extract paste 5g, pH6.5 after sterilizing.
4, the mensuration of tennecetin output: get 1mL natamycin fermentation liquor, adds 9mL methyl alcohol, and fully after vibration, the centrifugal 10min of 12000r/min, retains supernatant liquor, and the output detecting tennecetin by HPLC method reaches 6.01g/L.
embodiment 4
1, the preparation of seed: picking 2 ring spore is in sterile distilled water from Streptomyces natalensis slant medium, make the spore suspension that mass concentration is 5%, then be inoculated in the 250mL triangular flask containing 40mL seed culture medium by the inoculum size of 8% (V/V), 30 DEG C, 200r/min shaking table cultivation 72h.
Described slant culture based formulas is: glucose 20g, malt extract 1g, yeast extract paste 1g, Tryptones 5g, agar powder 18g, and adding distil water is settled to 1000mL, pH7.4 after sterilizing.
Described seed culture based formulas is: glucose 30g, peptone 15g, yeast extract paste 5g, and sodium-chlor 12g, adds water and be settled to 1000mL, pH7.8 after sterilizing.
2, the preparation of fungal elicitor: the Penicllium chrysogenum sterile distilled water on slant medium is made the spore suspension that mass concentration is 5%, and be inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium with the inoculum size of 5% (V/V), 30 DEG C, 180r/min shaking culture 168h.By cultured fermented liquid six layers of filtered through gauze, the then centrifugal 10min of 10000r/min, remove thalline, be that the filtering with microporous membrane of 0.22 μm is degerming with aperture, obtain fungal elicitor.
Described slant culture based formulas is: containing W-Gum 100g, glucose 20g, agar powder 20g in every 1000mL.
Described liquid culture based formulas is: containing glucose 25g, yeast extract paste 4g, extractum carnis 10g, pH7.0 after sterilizing in every 1000mL.
3, during liquid fermenting, seed liquor is inoculated in the 250mL triangular flask containing 40mL liquid nutrient medium by the inoculum size of 1% (V/V), 32 DEG C, 200r/min shaking culture, when fermenting 30h, according to the method for adding every two hours once, in fermented liquid, additional proportion is the mixing precursor (Sodium Propionate: sodium acetate: Isoleucine=1:1:1) of 0.1% (V/V), and the ratio when fermenting 60h in 1% (V/V) adds fungal elicitor.Whole fermenting process totally 9 days.
Described tennecetin fermentative medium formula is: glucose 15g, soybean cake powder 30g, Zulkovsky starch 20g, yeast extract paste 10g, and after sterilizing, pH is 8.0.
4, the mensuration of tennecetin output: get 1mL natamycin fermentation liquor, adds 9mL methyl alcohol, and fully after vibration, the centrifugal 10min of 12000r/min, retains supernatant liquor, and the output detecting tennecetin by HPLC method reaches 6.12g/L.
Claims (9)
1. the method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin, comprise the fermentation of Streptomyces natalensis, it is characterized in that: the mode added with stream during the fermentation adds tennecetin mixing precursor in fermention medium, add fungal elicitor during the fermentation, described tennecetin mixing precursor by mol ratio be the Sodium Propionate of 1:1 ~ 15:1 ~ 15, sodium acetate and Isoleucine form, described fungal elicitor is the fermented liquid of Penicllium chrysogenum.
2. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 1, it is characterized in that: containing glucose 10 ~ 20g in the every 1000mL of described fermention medium, soybean cake powder 20 ~ 40g, Zulkovsky starch 20 ~ 40g, yeast extract paste 5 ~ 10g.
3. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 1, it is characterized in that: described Streptomyces natalensis inoculum size is in the fermentation medium 1 ~ 10% (V/V), leavening temperature is 26 ~ 32 DEG C, and fermentation time is 5 ~ 9d.
4. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 3, it is characterized in that: the described addition of tennecetin mixing precursor in Streptomyces natalensis fermention medium is 0.1 ~ 1.0% (V/V), the interpolation time is the 24h ~ 36h after fermentation starts, and every 2h adds once.
5. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 3, it is characterized in that: described fungal elicitor is Penicllium chrysogenum fermented liquid, in its fermention medium, under 28 ~ 34 DEG C of conditions, 2 ~ 7d cultivated by shaking table, containing glucose 20 ~ 30g in the every 1000mL of fermention medium wherein, yeast extract paste 1 ~ 5g, extractum carnis 5 ~ 10g; The addition of described fungal elicitor in Streptomyces natalensis fermention medium is 1 ~ 10% (V/V), and the interpolation time is the 60h after fermentation starts.
6. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 1, it is characterized in that, described fungal elicitor is prepared by following steps and obtains:
The cultivation of step one, fungi: the Penicllium chrysogenum on slant medium is inoculated in liquid seed culture medium, 28 ~ 34 DEG C, cultivate 1 ~ 7d under 150 ~ 220r/min condition;
Containing W-Gum 50 ~ 100g, glucose 15 ~ 30g, agar powder 15 ~ 20g in the every 1000mL of described slant medium;
Containing glucose 20 ~ 30g, yeast extract paste 1 ~ 5g in the every 1000mL of described liquid seed culture medium, extractum carnis 5 ~ 10g, pH are 7.0 ~ 7.5;
Step 2, by cultured for step one fermented liquid first six layers of filtered through gauze, get filtrate, then 8000 ~ 12000r/min is centrifugal, retains supernatant liquor, afterwards, with filtering with microporous membrane, obtains fungal elicitor.
7. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 1, it is characterized in that, the Streptomyces natalensis seed adopted that ferments is prepared by following steps: be inoculated in liquid seed culture medium by the Streptomyces natalensis on slant medium, cultivates 2 ~ 7d for 26 ~ 32 DEG C;
Containing glucose 10 ~ 20g, malt extract 1 ~ 5g, yeast extract paste 1 ~ 5g, Tryptones 5 ~ 15g, agar powder 15 ~ 20g in the every 1000mL of described slant medium;
Containing glucose 15 ~ 30g, peptone 5 ~ 15g, yeast extract paste 5 ~ 10g, sodium-chlor 2 ~ 12g in the every 1000mL of described liquid seed culture medium.
8. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 1, is characterized in that: pH6.5 ~ 8.0 of described fermention medium.
9. a kind of method mixing precursor coupling fungal elicitor and efficiently synthesize tennecetin according to claim 1, is characterized in that: described Streptomyces natalensis is any bacterial strain during Streptomyces natalensis belongs to.
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CN113337552A (en) * | 2021-06-03 | 2021-09-03 | 淮北师范大学 | Epsilon-polylysine-natamycin co-production method based on fermentation waste mushroom residue interactive recycling |
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