Ukuhlolwa kwegwele nge-methylene blue: ukubala amangqamuzana aphilayo noma ukukala isikhathi sokuphefumula

Amashubhu amabili abekwe ebhentshini. Womabili aluhlaza okwesibhakabhaka. Kwelinye, amangqamuzana aluhlaza okwesibhakabhaka abonwa ngesibonakhulu asho ukuthi igwele lifile. Kwelinye, ish hubhu elishintsha lisuke kokuluhlaza okwesibhakabhaka lingabi nambala lisho ukuthi igwele liyaphila futhi liyaphefumula. Udayi ofanayo, imiphumela ephambene. Uma udidanisa lezi zinhlobo ezimbili zokuhlolwa, zonke iziphetho ziyaguquka.

Lo mehluko yiwo umongo wale ndatshana. I-methylene blue ingudayi we-redox: esimweni se-oxidation iluhlaza okwesibhakabhaka, kanti esimweni esincishisiwe iba yi-leucomethylene blue engenambala. Ukubala amangqamuzana aphilayo kusebenzisa le nqubo yamakhemikhali ngokuwabuka isikhashana ngesibonakhulu. Ukuhlolwa kokuphefumula kuyisebenzisa ekusabeleni kwengxube yonke okukalwa ngokwesikhathi. Ngakunye kudinga indlela yakho yokuhlola, izilawuli zakho, nokuhlolwa ngokugxeka okufanele.

Ukubala amangqamuzana aphilayo: daya isikhashana, ufunde ngokushesha

Indlela esetshenziswa ezindaweni zokwenza ubhiya yiyona esezingeni elifanayo kakhulu. Lungisa i-methylene blue engu-0.01% w/v ku-sodium citrate engu-2%, uyixube ngesilinganiso esingu-1:1 nengxube yegwele elilengiswe oketshezini, bese uhlola ingxube ngemva kwesikhathi esifushane esinqunyiwe. Izikhathi ezishicilelwe zisukela ku-1 kuya ku-2 imizuzu encwadini ye-Brewing Science Institute kuya cishe ku-5 imizuzu ezindleleni zocwaningo, kanti indlela ye-OIV yokuhlola amagciwane ewayinini idinga ukuthi ukuhlola kwenziwe kungakapheli 10 imizuzu. Udayi engxubeni yokubala ugcina usucishe ube ngu-0.005%. Faka ingxube egunjini lokubala le-Neubauer elithuthukisiwe, bese ekukhulisweni okucishe kube ngu-400x uhlukanisa amangqamuzana aluhlaza okwesibhakabhaka ngokumnyama njengangaphili nalawo angadaywanga njengaphilayo. Endleleni ye-OIV, amangqamuzana aluhlaza okwesibhakabhaka ngokukhanyayo abalwa njengaphilayo, okuyinto esevele ikhomba inkinga yokuncika ekwahluleleni komuntu okuxoxwa ngayo ngezansi.

Ukuminyana kwamangqamuzana kutholakala ngobukhulu nokuma kwegumbi lokubala. Lapho kubalwa isikwele esimaphakathi esiphelele esingu-1 mm, amangqamuzana nge-mL alingana no-N ophindwe ngesici sokuhlanjululwa ophindwe ngo-10^4, lapho u-N eyinani elibalwe kuleyo ndawo, njengoba kuchazwe ezindleleni ze-ASBC zokuhlola igwele. Iphesenti lokuphila lilula kakhulu: amangqamuzana angadaywanga ahlukaniswa ngenani lawo wonke amangqamuzana, bese kuphindwa ngo-100. Bala cishe 400 amangqamuzana ukuze ulinganisele ukuphila, ngokulandela ucwaningo lwe-FEMS lokuqhathanisa ezindaweni zokwenza ubhiya kanye nenqubo ye-OIV. Sebenzisa umthetho owodwa wemingcele ngokungaguquki, ngokwesibonelo ufake amangqamuzana asemigqeni yegridi engezansi nangakwesobunxele, kodwa ungawafaki lawo asemigqeni ephezulu nangakwesokudla.

Amahlumela adinga umthetho ngaphambi kokuba kuqalwe ukubala. Umkhuba ovamile ubala ihlumela njengengqamuzana elizimele lapho selifinyelele cishe engxenyeni yobukhulu bengqamuzana elingumzali, kodwa uma lingaphansi kwalokho libalwa kanye nengqamuzana elingumzali. Noma yimuphi umthetho okhethiwe, uwusebenzise kuzo zonke izikwele futhi uwubhale eshidini lokubala ukuze umuntu olandelayo enze okufanayo.

Ishidi lokubala (liphrinte)

Leli shidi lokubala liyinsiza yokuqala eyenzelwe le ndatshana. Likopishele encwadini yamanothi noma uphrinte elilodwa ngesampula ngayinye.

Ukuphila (%) = 100 x angadaywanga / (angadaywanga + aluhlaza okwesibhakabhaka ngokumnyama). Ingqikithi yamangqamuzana nge-mL = N x isici sokuhlanjululwa x 10^4, lapho u-N etholakala endaweni echaziwe yegumbi lokubala. Bhala isikhathi sokudaya eduze komphumela, ngoba inani lokuphila elingenaso isikhathi salo alikwazi ukuphinde litholakale ngokwethembeka.

Kungani amanani okuphila eguquguquka

Isikhathi yisiphazamisi sokuqala, futhi sithinta imiphumela ngezindlela zombili. Amangqamuzana anomsebenzi we-metabolism anciphisa udayi nge-redox, ngakho ukufunda kusenesikhathi kungawathola amangqamuzana aphilayo engakaqedi ukuwunciphisa bese kuwahlukanisa ngokungeyikho njengafile. Kodwa ukuchayeka isikhathi eside ngokwakho kunobuthi futhi kwakha amangqamuzana aluhlaza okwesibhakabhaka alimele ngempela, kukhiqize ukudaya okukhomba ukufa ngokungamanga. Ngakho indlela yokufundisa yaselabhorethri icacisa ukuthi ukubala kufanele kwenziwe kungakapheli 5 imizuzu, njengoba kubhalwe endleleni yase-Wisconsin yokufundisa ngokuhlola igwele. Indlela ephephile yokukuchaza ukuthi ukubala nge-methylene blue kuncike esikhathini, ngakho khetha isikhathi esisodwa esifushane futhi usigcine singaguquki kuzo zonke izisampula ochungechungeni.

Izisampula ezinamaphesenti aphansi okuphila zinokwenzelela kwesibili. Indlela esezingeni elifanayo ixwayisa ngokuthi i-methylene blue ingalinganisela ukuphila kwangempela kube ngaphezulu kwalokho okuyikho uma kungaphansi cishe kuka-80%, ngoba amangqamuzana asakwazi ukunciphisa udayi nge-redox awagcini ngempela amandla okuzalana. Izincwadi eziphathelene nokwenza ubhiya zichaza ukungathembeki okufanayo lapho ukuphila kwehle kakhulu. Thatha i-methylene blue njengendlela esheshayo yokulinganisela kuphela, bese uqinisekisa izinqumo ezibalulekile lapho ukuphila kuphansi, njengokuphinde usebenzise igwele ukuqalisa ukuvutshelwa, ngokubala amakoloni emapuletini noma ngodayi wokuhlola ohambisanayo.

Imiphumela yezinye izakhi zesampula iza ngokwesithathu. Indlela ye-OIV isebenza eduze kwe-pH 4.6 futhi ixwayisa ngokuthi izisampula ezine-pH ephansi egcinwa iqinile yi-buffer zivimbela ukuziphatha okufanele kukadayi; iphinde yeluleke ukuba ukuhlolwa kungasetshenziswa lapho ushukela ungaphezu kuka-100 g/L, ngoba amangqamuzana abonakala eluhlaza okwesibhakabhaka ngokukhanyayo ngendlela edukisayo. Ukuqhathanisa kokuhlola ama-pH okudaya kwathola ukuthi ukuzwela kwashintsha kakhulu phakathi kwebanga elahlolwa, okuhambisana nocwaningo lwendlela ye-methylene blue encike ku-pH. Imfucumfucu iyisicupho esihlukile: izinhlayiya ezidayiwe ezingxubeni zokusanhlamvu ezingcolile zifana namangqamuzana afile futhi zandisa ukwehluka kwemiphumela phakathi kwabahloli.

Lokhu kwehluka kukaliwe, akusekelwe nje ezindabeni zabantu. Ocwaningweni lwezinyanga eziyisithupha endaweni yokwenza ubhiya olwabikwa ekuqhathaniseni igwele kwe-FEMS, izilinganiso ezimbili eziphindiwe ze-methylene blue zaba nesilinganiso sokuchezuka okujwayelekile esingu-6.1%, uma kuqhathaniswa no-0.2% ku-flow cytometry esebenzisa i-oxonol, kanti abahloli ababili bahluka ngamaphuzu ephesenti afinyelela ku-13 kusampula efanayo. Ukuqhathanisa kwango-2023 kwezindlela zokukala ukuphila kwathola ukuthi ukubuka ngesibonakhulu se-fluorescence ne-flow cytometry kwakunemba kakhulu, kuyilapho kubonisa ukuthi ukusebenza kuncike ohlotsheni lwegwele nasekwakhekeni kwesampula. Incazelo yakudala yendlela ka-Painting no-Kirsop (PMID 24430080) isalokhu iyisisekelo sokubhekisela, kanti ukubuyekezwa okubanzi kokuhlolwa kokuphila nobungqabavu begwele (PMID 25154541) kubeka i-methylene blue phakathi kwezindlela ezihambisanayo, hhayi ezikhipha impendulo eqinisekile. I-trypan blue ayiyona ngokuzenzakalelayo indlela engcono lapha: ikhomba ukulahleka kobuqotho bolwelwesi, kanti i-methylene blue ikhomba ukungeniswa kukadayi kanye nomsebenzi wokuwunciphisa nge-redox, ngakho amangqamuzana acindezelekile angahlukaniswa ngokwehlukile ngale dayi emibili.

Ukuhlolwa kokuphefumula: kala isikhathi sokunyamalala kombala oluhlaza okwesibhakabhaka

Ukuhlolwa kwesibili akusebenzisi isibonakhulu nhlobo. Igwele, i-glucose, kanye ne-methylene blue ehlanjululiwe kufakwa eshubhini elivaliwe, bese iwashi likala ukuthi kuthatha isikhathi esingakanani ukuba umbala oluhlaza okwesibhakabhaka unyamalale. Njengoba amangqamuzana enza i-metabolism, akhiqiza amandla okunciphisa nge-redox aguqula udayi oluhlaza okwesibhakabhaka ube sesimweni esingenambala, kuyilapho i-oksijini encibilikile iphinde iwubuyisele esimweni se-oxidation. Lapho amangqamuzana esenciphise i-oksijini, ukuncishiswa nge-redox sekudlula ukubuyiselwa esimweni se-oxidation, bese umbala uyanyamalala eshubhini. Ngakho iphuzu lokuphela libonisa ibhalansi phakathi kokuphefumula, ukuminyana kukadayi, nokutholakala kwe-oksijini, futhi kufanele libikwe njengesikhathi sokuncishiswa kwe-methylene blue nge-redox noma sokunyamalala kombala, hhayi njengezinga eliqondile lokusetshenziswa kwe-oksijini yamangqamuzana.

Sebenzisa udayi ohlanjululwe kakhulu kunalowo osetshenziswa ekubaleni ukuphila ukuze isikhathi sibe sebangeni elilungele ukukalwa. Incwadi ye-AQA A level yomsebenzi waselabhorethri odingekayo ibeka lokhu ngendlela ehlelekile: 1 g we-glucose ku-100 mL wamanzi afudunyezwe acishe abe ngu-30 C, 5 g wegwele elomisiwe elifakwa ngaphambi nje kokusetshenziswa, kanye nodayi wokusebenza ocishe ube ngu-0.001% olungiswa ngokuhlanjulula 0.1 mL wesixazululo sokuqala esingu-1% kuze kufinyelelwe ku-100 mL. Xuba 2 mL wengxube yegwele ne-glucose elilengiswe oketshezini no-2 mL kadayi wokusebenza, uvumele kufinyelele ezingeni lokushisa lokuhlolwa, uyinyakazise kanye imizuzwana engu-10, uqale isibali-sikhathi, bese ungaphinde uyinyakazise. Ukunyakazisa kubuyisela i-oksijini futhi kwenza ish hubhu liphinde libe luhlaza okwesibhakabhaka, okuwukuphazamiseka okukhulu kakhulu okudalwa inqubo. I-AQA iphawula ukuthi le ngxube ngokuvamile kufanele ilahlekelwe umbala kungakapheli cishe 5 imizuzu ku-35 C, ngezilinganiso ezimaphakathi zokuhlola ezingu-275 s ku-20 C, 128 s ku-35 C, kanye no-145 s ku-45 C. Lezi ziyizibonelo zendlela eyodwa yokulungiselela, hhayi izilinganiso ezisebenza yonke indawo. Ukuhlobana okulinganisekayo phakathi kwezinga lokuncishiswa nge-redox negwele elinomsebenzi we-metabolism ngaphansi kwezimo ezilawulwayo kusekelwa izifundo ezikhethekile zokuncishiswa, kuhlanganise nomsebenzi ofakwe ohlwini njenge-PMID 25773544 kanye nocwaningo lwendlela inqubo esebenza ngayo ku-Yeast (2014).

Ukubonisa ngokuhamba kwesikhathi (insiza yokuqala)

Ithebula elingezansi lingukubonisa kokuqala okwenzelwe le ndatshana kusetshenziswa indlela efana neye-AQA echazwe ngenhla: 2 mL wengxube yegwele ne-glucose kanye no-2 mL kadayi wokusebenza ocishe ube ngu-0.001%, ukunyakazisa kanye imizuzwana engu-10, bese kugcinwa ezimweni zokuhlolwa kungaphazanyiswa. Okufakiwe kuyizibonelo zemiphumela ngendlela iqembu labafundi okufanele liyibhale ngayo, kanti inkomba yezinga ichazwa njengo-1 ohlukaniswe ngesikhathi sokunyamalala kombala ngemizuzwana. Yenza ukuhlola ngamashubhu afanayo bese ufaka amanani akaliwe esikhundleni salezi zibonelo.

Dweba igrafu yenkomba yezinga uma iqhathaniswa nezinga lokushisa lamashubhu A, B, no-C. Ijika liyakhuphuka bese liyisicaba noma lehle, okuyiphethini elindelekile eqhutshwa ama-enzyme, hhayi umugqa oqondile. Amashubhu D, E, no-F awawona amaphuzu edatha kulelo jika. Ayizilawuli ezenza ijika likwazi ukuchazwa.

Izilawuli ezenza ukuhlolwa ngakunye kube nencazelo

Ukuhlolwa kokuphefumula ngakunye kudinga izisampula ezintathu ezihambisana nakho. Igwele elibilisiwe noma elibulewe kanye ne-glucose nodayi kufanele kuhlale kuluhlaza okwesibhakabhaka, okufakazela ukuthi ukuncishiswa okusheshayo nge-redox kuncike ku-metabolism yamangqamuzana aphilayo. Igwele eliphilayo kanye nodayi ngaphandle kwe-glucose eyengeziwe kuyisilawuli somthombo osetshenziswa ekusabeleni, kodwa lindela ukunyamalala kancane kombala noma kunjalo ngoba igwele lisebenzisa imithombo yalo yamandla yangaphakathi. I-glucose nodayi ngaphandle kwegwele kuyisampula eyisisekelo yamakhemikhali, ebalulekile ngoba i-glucose esezimweni ze-alkaline inganciphisa i-methylene blue nge-redox ngaphandle kokuba khona kwamangqamuzana.

Ukubala amangqamuzana aphilayo kudinga uhlobo olwehlukile lokulawula: ukungaguquki kwenqubo. Isixazululo sokuqala sikadayi esifanayo, isilinganiso sokuxuba esifanayo, isikhathi sokudaya esifanayo, indawo yegumbi lokubala efanayo, nemithetho yamahlumela nemingcele efanayo, kubalwe ngaphandle kokwazi ukuthi iyiphi isampula lapho kungenzeka khona. Lapho abahloli ababili bengavumelani ngamaphuzu ayishumi, ishidi lokubala ngokuvamile liveza imbangela: isikhathi esihlukile, umkhawulo ohlukile wokwahlulela umbala oluhlaza okwesibhakabhaka, noma imithetho yamahlumela ehlukile. Amasu okulungisa nokubuka amangqamuzana adaywe axhumene nezindlela ezilula zokudaya amabhaktheriya, kanti ukuhlola okubanzi kwalokho udayi akuphawulayo ezisampuleni ezahlukene kutholakala ku-atlasi yokudaya ngesibonakhulu. Mayelana nefiziksi yokufunda ngokulinganisekayo noma yikuphi ukuqina kombala oluhlaza okwesibhakabhaka, inkomba yokumuncwa echaza izimo ichaza ukuthi kungani i-coefficient ngayinye kufanele ihambisane nezimo zesincibilikisi sayo, i-pH, nokuminyana.

Khetha ukuhlolwa ngokombuzo. Ukuthi mangaki amangqamuzana aphilayo njengamanje kuwumbuzo wokubala, ophendulwa ngesibonakhulu nang eshidi lokubala. Ukuthi leli gwele elikhulisiwe liphefumula ngomfutho ongakanani kuwumbuzo wesivinini senqubo, ophendulwa ngesitophuwashi nangamashubhu alawulwayo. I-methylene blue isebenza kukho kokubili, kodwa hhayi eshubhini elifanayo ngesikhathi esifanayo.