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3 ^ DE TAI KHOA HOC TRONG DIEM TIM KIEM CAC CHUNG VI SINH VAT CO HOAT TINH SINH HOC N H A M T A O CHE PHAM LAM SACH NlTdC NUOI TOM MA SO: QG - TD 05-05 Chu tri de tai: GS.TS Pham Van Ty Cdc vien: GS TS Nguyen Lan Dung PGS TS Nguyin Thanh Du^crng TS Bui Viet Ha ThS Dao Thj Lu-ong ThS Tr§n Thj Thanh Huyin ThS Nguyin buy Thjnh CN Pham Dii-c Ngoc KS Nguyen Thj Thuy Hang HA NOI - 2007 DAI HOC OUOC GIA HA NOI rPUNG TAM THONG TIN THU VIEN 0006000666^ CAO TOM TAT a, Tin di tdi: Tim kilm cac chung vi sinh vat c6 hoat tinh sinh hoc nham tao che pham lam sach nude nudi torn b, Chu tri de tdi: GS TS Pham Van Ty c, Cdc can bg tham gia: GS TS Nguyin Lan Dung, PGS TS Nguyen Thanh Duong, TS Bui Viet Ha, ThS Dao Thi Luang, ThS Trkn Thi Thanh Huyin, ThS Nguyen Duy Thinh, CN Pham Due Ngoc, KS Nguyin Thi Thuy Hang d, Muc tiiu vd ngi dung nghien cuu - Muc tieu: • Phan lap va tuyen chon bo vi sinh vat c6 kha nang sinh cac chat cd hoat tinh sinh hoc cao, giup phan giai nhanh cac chat huu co du thua va diet cac vi sinh vat gay benh co hoi ao nuoi • Tu cac chung da lira chon, nghien cuu che tao che pham vi sinh than thien mdi trudng vdi gia re nham lam sach nude nuoi torn, thay the cho viec su dung khang sinh - Noi dung nghien curu • Phan lap va tuyen chon cac chung vi sinh vat co nhung dac diem sau: + Cd kha nang sinh cac chat hoat dong sinh hoc (enzym ngoai bao) phan giai nhanh cac chat hiiu co khong mong muon ao torn + Co kha nang sinh chat khang khuan chdng mot so vi khuan gay benh thudng gap d tdm • Nghien cuu dac diem sinh hoc, phan loai cac chung lira chon • Lira chon dieu kien nuoi cay thich hop cho sinh trudng va sinh tong hop cac hoat chat sinh hoc • Nghien cuu tao che pham vi sinh (probiotic) de xu ly nude ao nuoi tom • Khao nghiem che pham d phong thi nghiem va ao nuoi e, Cdc kit qua chinh Tir 130 chung vi khuan co kha nang sinh cac chat hoat dong sinh hoc phan lap dugc tir cac mau dat, bun thu thap tir nhieu tinh, da chon dugc chung cd hoat tinh cao va CO cac uu diem dung nudi thuy san: - Cac chung V20 va VI1 co kha nang cung luc sinh cac enzym amylaza, chitinaza, proteaza, xenlulaza va lipaza 1 nhu: Proteus sp., E.coli, Pseudomonas spp., Aeromonas spp., dac biet Vibrio spp la vi khuan gay benh phd bien nhSt d tdm Ca chiing tren diu sinh trudng cac mdi trudng thong thudng va cd kha nang chiu mudi nen chiu dugc mdi trudng nude bien Khi nghien curu cac dac dilm phan loai theo phuong phap truyen thdng (Bergey's Manual) kit hop vdi phan tich trinh tir rADN 16S cd the ket luan: - Chung V20 la Bacillus licheniformis - Cac chung V75 va VI1 la subtilis - Chung L40 la Lactobacillus plantarum - Chiing LI I la Enterococcus lactis Da tien hanh nghien ciru cac yeu to anh hudng den sinh trudng va sinh tdng hgp cac enzym ngoai bao, kha nang tdng hgp cac chat diet khuan bao gdm: phan mdi trudng (ngudn cacbon, nito, khoang thich hgp), pH, nhiet do, thdi gian nudi thich hgp Da nghien cuu va che tao cdng che pham probiotic dang bgt va dang dich mang ten EMUNIV-S tu chiing da lira chon Quy trinh san xuat bao gdm: lira chgng nhan gidng cap I, cap 2, len men tren mdi trudng xdp va djch the; lira chgn chat mang, ty le phdi hgp gidng, thdi gian bao quan Da tien hanh khao nghiem hieu qua cua che pham phdng thi nghiem va tren hien trudng: - Xu ly nude nudi tdm bi d nhiem be kinh - Xii ly biin loang (slime) cua ao nudi d nhiem be kinh Ket qua cho thay che pham cd tac dung cai thien chat lugng nude Cac chi so BOD, COD, NH4, NO ", H2S deu giam so vdi ddi chumg Bun cd mau xam d be ddi chung bun cd mau den va mui thdi Thi nghiem nudi tdm be kinh cd xu ly bang che pham cung cho ket qua truong tir Khao nghiem tai ao gdm loai: ao 20.000 m2 tai Dinh Vu - Hai Phdng cho thay cac chi so NH4, NO2, H2S, BOD, COD d cac ao thi nghiem ludn thap hon so vdi ddi chung Cac dong vat thiiy sinh nhdm Copepoda, Cladocera, Rotatoria cao hon cac loai vi tao lam thirc an cho tdm nhu Chlorella, Skeletonema, Chaetoceros cung tang len tao doc Ceratium vi khuan lam gay benh ban vd tdm giam di ig vdu uoi uiiuiig lit Hiyo, iiaiig simi tang gap doi (2,6-3 tan/ha) so vdi ddi chung la 1,5 - Tai Ddng Nam Dien, Nam Dinh da tien hanh khao nghiem tren 20.000 m^ ao nuoi lim so sanh che pham EMUNIV-S vdi mot so loai che pham dang luu hanh tren thi idng (Biochie cua My, BRF-2 cua Viet Nam) theo phuong phap so sanh tuong d6i Kit la budc dau cho thay: - Dung EMUNIV-S de xu ly ao nuoi tom su cho mau nude dep hon (mau non m6i), ben mau hon va cd tot hon so vdi ddi chung va vdi che pham khac tinh ' 45 den 90 nuoi tha - Khi su dung EMUNIV-S lugng nito tdng so va photpho tdng so bun la lap nhat - Su dung EMUNIV-S cho toe tang trudng tuong ddi cua tom su tot hon so vdi ohe pham lai Ket qua khao nghiem tai phong thi nghiem tai phdng thi nghiem ciing nhu ilu cd thay EMUNIV-S la che pham co hieu qua lam sach nude ao nudi torn va do jm tang nang suat CAC CONG TRINH CONG BO VA SAN PHAM DAO TAO I Cong bo cong trinh Tran Thi Thanh Huyen, Pham Van Ty, Dao Thi Luang, 2007 Tuyen chon vi chuan co kha nang ddi khang vdi mot so tac nhan gay benh thudng gap d torn Gui dang Fap chi Khoa hoc , Dai hoc Quoc gia Ha Noi Nguyen Duy Thinh, Dao Thi Luang, Bui Viet Ha, Pham Due Ngoc, Tran Thi Thanh Huyen, Pham Van Ty, 2007 Thu nghiem che pham vi sinh vat cai thien nude nuoi torn Gui dang Tap chi Khoa hoc , Dai hoc Quoc gia Ha Ngi II Dao tao: thac sT bao ve nam 2006 Tran Thi Thanh Huyen vdi de tai: 'Than lap, tuyen chgn cac chiing vi sinh vat CO hoat tinh sinh hoc cao de tao che pham dung xu ly nhiem moi trudng nuoi torn" Nguyen Duy Thinh vdi de tai: "Su dung che pham vi sinh vat de xii ly nhiem moi trudng nude nudi tdm" Hai khoa luan cu nhan sinh hoc, 2006 / itnn nmn su aung kmn phi cua de tdi Kinh phi dugc c^'p: 300 trieu dong Kinh phi da sii dung: 300 tri6u dong KHOA QUAN L t (Ky va ghi rd ho ten) CHU TRI DE TAI (Ky va ghi rd ho ten) ^ PHO CHU NHIEM KHOA OSTS I>'>4/» Sa^ TS VAsUY^ C''^^ ^j Jf^Ma TRl/C)NG DAI HOC KHOA HOC TlTNHIEN HO HiEu THUONC P^S.I^.Udui^MrLAj^ ^ '%^nh PROJECT SCREENING OF BIO ACTIVE COMPOUNDPRODUCING MICROORGANISMS FOR TREATMENT OF SHRIMP CULTURE WATER CODE NUMBER: QG - TD 05-05 COORDINATOR: PROF.DR PHAM VAN TY HANOI - 2007 PROJECT SCREENING OF BIOACTIVE COMPOUNDPRODUCING MICROORGANISMS FOR TREATMENT OF SHRIMP CULTURE WATER CODE NUMBER: QG - TD 05-05 Coordinator: Prof Dr Pham Van Ty Members: Prof Dr Nguyen Lan Dung Assoc Prof Dr Nguyen Thanh Duong Dr Bui Viet Ha MSc Dao Thi Luong MSc Tran Thi Thanh Huyen MSc Nguyen Duy Thinh BSc Pham Due Ngoc Eng Nguyen Thi Thuy Hang HANOI - 2007 SUMMARY a Project title: Screening of bioactive compound-producing microorganisms for treatment of shrimp culture water b, Coordinator: Prof Dr Pham Van Ty c Members: Prof Dr Nguyen Lan Dung, Assoc Prof Dr Nguyen Thanh Duong, Dr Bui Viet Ha, MSc Tran Thi Thanh Huyen, MSc Dao Thi Luong, MSc Nguyen Duy Thinh, BSc Pham Due Ngoc, Eng Nguyen Thi Thuy Hang d, Objectives and main contents - Objectives: • Isolation and screening of microorganisms, which are capable of rapidly degrading undesirable organic substances in shrimp culture pond and possess antibacterial properties against common diseases in shrimp • Production of cheap and friendly environmental-friendly microbial preparation using the selected strains to improve shrimp culture water This preparation can be used as a replacement of the traditional method (antibiotics) - Main contents Isolated and screened characteristics: microbial strains processing the following • Isolated and screened microbial strains processing the following characteristics: (i) capable of secreting bioactive substances (enzymes) to rapidly decompose undesirable organic substances in shrimp culture pond; (ii) capable of producing anti-bacterial substances against common pathogenic bacteria in shrimps • Investigated biological and classification characteristics of the selected strains • Optimized the culture conditions to obtain maximal growth and bioactive compound production • Studied on manufacture of microbial preparation (probiotic) by using selected strains for treatment of shrimp culture pond • Tested the effectiveness of this preparation using shrimp culture tanks and ponds e Key results - 130 bioactive compound-producing bacterial strains were isolated from soil and slime samples from different provinces Among these, five strains were selected basing on their high activity in producing bioactive compound and their potential application to shrimp culture - Two strains V20 and VI1 were capable of secreting multiple enzymes, including amylase, protease, cellulose, chitinase and lipase - Three strains V75, L40, and VI1 exhibited antibacterial activity against shrimp pathogens, including Proteus sp., E.coli, Pseudomonas spp., Aeromonas spp., especially Vibrio spp (the most common pathogen) - All five above strains could grow in regular medium and in high salt medium, indicating that these strains can grow in seawater - Results of classification study using traditional method (Bergey's Manual) and I6S ribosomal DNA sequence analysis revealed that: + V20 is Bacillus licheniformis + V75 and VI1 are B subtilis + L40 is Lactobacillus plantarum + LI I is Enterococcus lactis - Affects of culture condition on growth, exogenous enzyme synthesis and anti-bacterial substance synthesis, including media (carbon, nitrate, mineral), pH, temperature, and culture duration were investigated - Liquid and power forms of Probiotic preparation were successfully developed and called EMUNIV-S EMUNIV-S is a mixture of selected strains - The effectiveness of the preparation was tested in tanks and in culture pond: + Treated the contaminated shrimp culture water in tanks with the preparation + Treated the contaminated slime in tanks with the preparation - The results revealed that the preparation improves the water quality There was a decrease in indicators such as BOD, COD, NH4, NO2 H2S in treated tanks when compared to untreated control The slime was gray in treated tanks while in untreated control, it was dark and stink - Application of the preparation to ponds (three 20,000-m^ ponds at Dinh Vu-Hai Phong) gave similar results Moreover, there was an increase in favorable microflora including Copepoda, Cladocera, Rotatoria, Chlorella, Skeletonema, Chaetoceros In contrast, there was a decrease in bad microflora including Ceratium and cyanobacteria - Population of Vibrio parahaemolyticus was reduced 10 times Survival of shrimp in treated and untreated pond was 64-70% and 41%, respectively Shrimp production in treated pond was 2.6-3 ton/ha, double the production in untreated pond (1,5 ton/ha) In Dong Nam Dien, Hai Phong province, an experiment was designed to compare the effectiveness of EMUNIV-S with two conunercial preparations (Biochie-USA and BRF-2-Vietnam) (Biochie cua My, BRF-2 ciia Viet Nam) The preliminary results were as follows (in comparison to the two commercial preparations): - Water in shrimp culture pond treated with EMUNIV-S was clearer and greener - Nitrate and Phosphate amount in slime from EMUNIV-S treated pond was lower - Growth rate of shrimp in EMUNIV-S treated pond was higher In conclusion, all above results suggest that treatment with EMUNIV-S leads to improved culture water and enhanced shrimp production PUBLICATIONS AND TRAINING ACTIVITY I Two publications Tran Thi Thanh Huyen, Pham Van Ty, Dao Thi Luong Screening of bacteria producing bioactive compound against common pathogens in shrimps Science magazine 5/2007 Nguyen Duy Thinh, Dao Thi Luong, Bui Viet Ha, Pham Due Ngoc, Tran Thi Thanh Huyen and Pham Van Ty An experiment on using microbial preparation for water quality improvement of shrimpculture ponds Vietnam National University Hanoi Journal of Science 2007 II Training activity: MSc completed in 2006 1 Tran Thi Thanh Huyen, project: "Isolation and screening of bioactive compound-producing microorganisms for treatment of contaminated shrimp culture ponds." Nguyen Duy Thinh, project: "Application of microbial preparation for treatment of contaminated shrimp culture ponds" PROJECT COORDINATOR FACULTY OF BIOLOGY /i-SSoC ^rio^ -^^ f%.a^ TucfH A^ht'^ w\ vdi^ •i^Akam ^^' PROJECT IMPLEMENTING ORGANIZATION HANOI UNIVERSITY OF SCIENCE, VNU Lcn NOI D A U La mot nhung nuoc co dieu kien thien nhien im dai, nganh nuoi thuy san cua Viet Nam nhung nam qua da phat trien vugt bac, dat dien tich tren 700 nghin nuoi va vuon len thuoc nhom cac quoc gia co san lugng nuoi tr6ng thuy san dung dau the gioi Nam 2006 tong kim ngach xuat khau thuy san dat tren ty USD, mot nua la tu tom, chi dung sau Trung Quoc va Thai Lan Nganh nuoi thiiy san thirc sir da giai quyet van de viec lam cho ngudi lao dong, tang thu nhap, gop phan xoa doi giam ngheo va tang trudng kinh t6 cho dat nuoc Ngay 11/1/2006 Thii tuong Chinh phu da ky Quy^ dinh s6 10/2006/QDTTG phe duyet Quy hoach tong the phat trien nganh thuy san den nam 2010 va djnh huong den nam 2020 Theo nganh thuy san phai phan dau tro nganh san xuat hang hoa Ion, san pham phai co chat luong tot, co sue canh tranh cao tren thi truong the gioi de dua nganh tiep tuc phat trien mot each ben vmig De nghj quyet ciia chinh phii tro hien thirc la mot nhiem vu day kho khan, chiing ta dang phai doi mat voi nhung thach thuc Ion ve nhiem moi truong, lam cho djch benh cang nhieu va cang phuc tap, khien cho riii ro ciia nganh nuoi torn cang Ion, nhieu noi co nguy co mat trang tom chet co den 100%, gay thiet hai nang ne cho nguoi dan Truoc day de phong chong benh cho tom, nguoi dan thuong dung hoa chat va chat khang sinh Ngay cac bien phap khong dugc ap dung nua du lugng hoa chat va chat khang sinh ton du san pham EU da thong bao se kiem tra 100% tom nhap khau vao cac nuoc EU tir Viet Nam Nam 2006 Nhat Ban ban hang Ion ciia Viet Nam, ciing da canh bao ve du lugng khang sinh torn Viet Nam, ma neu khong cai thien thi se co nguy co mat thi truong nay, Moi truong va benh la hai khia canh ciia mot van de, chiing co tac dong qua lai voi Phan Ion dich benh torn xuat phat tir nhiem moi trudng Do vay, viec tao mot che pham sinh hoc de xu ly nhiem moi trudng co y nghla song doi vdi nganh nuoi thuy san noi chung va nganh nuoi tom noi rieng Hien tai chiing ta khong kiem soat het dugc cac che pham sinh hoc dang luu hanh tren thi trudng Cac che pham cua nude ngoai khong nhimg dugc ban vdi gia qua dat ma chua nhieu loai vi sinh vat ma mot tung vao thien nhien chiing ta khong kiem soat dugc Xuat phat tu nhung ly neu tren, muc tieu ciia de tai nghien curu cua chiing toi nham: - Tuyen chgn bo vi sinh vat cd kha nang sinh chat cd hoat tinh sinh hgc cao giiip giiip phan giai nhanh cac chat hiru co du thira va diet cac vi sinh vat gay benh CO hoi ao nuoi - Tu cac chiing da lira chon, tao che pham vi sinh than thien moi trudng vdi gia re dung xu ly nhiem moi trudng thay the cho viec su dung khang sinh nghe nuoi torn Trong ban bao cao nay, ten de tai cung da dugc nit ggn, mot so ngi dung nghien curu da dugc chinh sua (bo phan che pham thiic an, thu ggn dien tich thi nghiem) theo y kien cua "Hoi dong danh gia de cuong de tai trgng diem" 9/4/2005 Nhan sir tham gia de tai cung thay doi thuyen chuyen cong tac Cuoi Cling chiing toi muon nhan co hoi giri Idi cam on chan tdi Dai hoc Quoc gia Ha noi da cap kinh phi, Ban lanh dao trudng Dai hgc Khoa hgc Tu nhien, Ban Khoa hgc cong nghe Ban Ke hoach - Tai chinh, phdng Khoa hgc Cong nghe Ban Chu nhiem Khoa Sinh hgc da tao dieu kien thuan Igi de chiing toi trien khai de tai Trong qua trinh thuc hien de tai chiing toi cung nhan dugc sir giiip d5 cua nhieu can bg khoa hgc va dong nghiep, nhan dip chiing toi xin chan cam on nhiing su giiip quy bau Hd noi, thdng nam 2007 Tap the can bo tham gia de tai CHrCfNG TONG QUAN VAI TRO CUA CHE PHAM SINH HOC TRONG NUOI TRONG THUY SAN DAT VAN DE Viet Nam la nude cd tiem nang Idn nudi thiiy san, vdi bd bien dai 3260 km, hau am ap quanh nam, Viet Nam co tren 30 tinh nudi thiiy san Tinh den nam 2003, ca nude cd 530.000 nuoi tdm, nam 2005 tong dien tich nudi da len den 750.000 Tuy nhien, sir phat trien at nghe nudi thuy san, dac biet la nghe nudi tom d cac tinh ven bien dan den sir biing no dich benh tren dien rgng, gay thiet hai nang ne cho ngudi dan Sir pha san cua nhimg ngudi dan d Kien Thuy - Thai Binh cung nhu d nhieu dia phuong khac la bai hoc dau xdt chi chay theo quy mo nudi ma khong quan tam tdi yeu to mdi trudng Sir nhiem xay rat nghiem trgng chinh ao nudi va moi trudng sinh thai xung quanh thai mot lugng Idn cac chat hiju co tir thiic an du thua, tir phan va cac chat thai khac cung nhu sir ton dong ciia cac loai hoa chat, chat khang sinh dugc sir dung qua trinh nuoi ma khong dugc xir ly Benh cua thuy san thu&ng bat ngudn tir chinh moi tru&ng ao nudi Viec hinh Idp biin day sir tich tu lau ciia cac chat hiru co, chat can va la noi sinh song cua cac vi sinh vat gay thoi, cac vi sinh vat sinh cac doc nhu amoniac, nitrit, hydrosulflia VI SINH VAT GAY BENH Cac vi sinh vat gay benh cho tom rat da dang, cd the la virut, vi khuAn, n4m sgi va nguyen sinh dong vat Mot so nhiem tir tdm gidng, cdn phAn Idn cac tac nhan cd san ao ho nudi torn, Benh virut co khoang 15-16 loai, thudng lay lan nhanh va lam cho ty le tdm chet cao Cac benh bien la: - Benh BMV (Monodon Baculovirus) thugc ho Baculoviridae - Benh dom trang (WSSV - White spot syndrome virus) virut thuoc ho Nimaviridae gay nen - B?nh dau vang (YHV - Yellow head virus) virut tuong tu Coronaviridae gay nen - Benh hoai tir dudi vd va co quan tao mau (IHHNV - Infectious hypodermal and hematoietic necrosis virus) - Benh teo gan virut HPV - Hepatopancreatic parvolike virus thuoc hg Parvoviridae Bfnh vi khuan rat da dang bao gom cac benh nhu den vd, den mang, phat sang, nau mang, rung dudi, ddm den, phan trang Vi khuan gay benh cd the thugc chi Vibrio, Aeromonas Pseudomonas Tuy nhien phd bien nhat van la cac loai thuoc chi Vibrio nhu: V harveyi V alginolyticus, V parahaemolyticus, V vulnificus, V harveyi la loai chu yeu gay benh phat sang Hau het vi khuan gay benh la bgn co hoi, chiing thudng gay nhiem cac trudng hgp tdm da hi nhiem tac nhan khac, bi stress moi trudng, bj Id loet tir trudc hoac thirc an thieu can doi ve mat dinh dudng Benh nam: Nam Muco bam vao mang, Fusarium lam den mang, mot so nam khac nhu Lagenidium, Haliphtoros phat trien luon dudi Idp vd chitin Nam cdn gay benh dd than tiet aflatoxin Benh nguyen sinh dong vat bao gdm Gregarina ky sinh rugt lam tom cham Idn, benh tom bong Microsporea bam vao co van lam due co Mot so ky sinh phia ngoai nhu Zoothammium spp., Epistylis spp gay can trd chirc nang ho hap Phan lan cdc vi sinh vat gay benh ke tren Id mot phan cua he vi sinh vat binh thu&ng moi tru&ng (nude bien, ao hd, song rach) Chiing dugc xem la tac nhan gay benh thu cap hoac gay benh ca hot Mot sir can bang cua he vi sinh vat ao nudi bi pha vd, cac vi sinh vat cd hai se phat trien d at va sdm cong hudng vdi cac yeu to cd hai khac de gay benh Sir d nhiem mdi trudng thdi gian ngan se tac dong tdi he than kinh, thdi gian dai se lam tang stress dan den giam kha nang tieu thu thirc an, giam tang trudng, lam tang man cam doi vdi cac tac nhan gay benh dac biet la cac virut Tat ca cac yeu to neu tren se lam cho thuy san chet hang loat chi mot thdi gian ngan Sir canh bao tir phia Nhat Ban cuoi nam 2006 khien chiing ta can phai xem xet lai mot each nghiem tuc ve ky thuat nudi de tir dua giai phap hgp ly, dam bao ve sinh an toan thuc pham neu nhu khdng mudn sau Nhat Ban, theo phan ung Domino se la cac nude khac Va nhu the khd Idng day manh xuat khau va thirc hien cdng nghi quyet cua Chinh phu Neu khdng sir dung hoa chat vd chat khdng sinh thi giai phap him hieu de giai quyet van de nhiem moi tru&ng ao nuoi chinh Id viec su dung cdc che pham sinh hgc Bien phap dd vd dang su dung bien tai cdc nude nuoi thuy san & Dong vd Tay Ban cdu nhu My, Braxin, Australia, Ixrael, Thdi Lan, Ddi Loan, Trung Quoc, Malaysia CHE P H A M SINH HQC PROBIOTIC Probiotic (trg sinh) la thuat ngir cd nguon gdc tir tieng Hy lap, bao gdm hai tir: Pro co nghla la vi va biosis co nghTa la sir song, la thuat ngir timg xuat hien nam 1965 nhung dugc nhac tdi nhieu nhung nam gan day tinh uu viet ciia chung Probiotic dugc dinh nghla la che pham chira cdc vi sinh vat song da chgn Igc ma dua vao ca the qua duang mieng vai lugng du lan se dnh hu&ng co lai den sire khoe vat chu Ddi vdi ngudi va dong vat (gia siic, gia cam), cac vi khuan probiotic thudng la vi khuan lactic, dugc tuyen chgn cong phu vi chung dac trung cho timg loai Tinh hieu qua cua probiotic da dugc chung minh bao gdm kha nang chdng nhiem triing vi khuan va virut (vi du virut rota gay tieu chay), kha nang chdng ung thu, kich thich hoat dong ciia he mien dich, lam giam cholesterol, cai thien sir dung lactoza d nhung ca the khong dung nap lactoza (khdng udng dugc sira), giam tac dung phu ciia chat khang sinh Sd dT probiotic cd dugc hieu qua nhu vay la vi chiing tac ddng lam on djnh khu he vi sinh vat dudng ruot, lam tang cac vi khuan cd ich (cac vi khuan sinh vitamin, sinh chat khang khuan, vi khuan phan giai dudng, tinh bgt., ), lam giam cac vi khuan co hai (cac vi khuan canh tranh thirc an, sinh chat doc ) Trong nudi thdy san dinh nghia ve probiotic dugc hieu vdi nghla rgng hon, lien quan den cd mdi tru&ng nuoi Khdc vai dong vat tren can, a ddng vat thiiy sinh, khu he vi sinh vat duang rugt khong ton tai nhu Id mot quhn the dgc lap dac thii cho timg loai, md cd su tuong tac thu&ng xuyen vdi mdi tru&ng va v&i ho^t dong cua vat chu Vi sinh vat cd mat mdi trudng nude anh hudng true tiep den phan khu he vi sinh vat dudng rugt va ngugc lai Cac vi sinh vat cd mat dudng ruot hau het co ngudn gdc tir moi trudng Thay cho muc dich chu yeu la tieu diet cac bao tu vi khuan, che pham sinh hgc dugc san xuat va dua vao sir dung vdi muc dich chu yeu la kich thich sir gia tang cua cac vi sinh vat CO Igi ao nudi Ngay nay, che pham sinh hoc dugc coi la cong cu him hieu, tao nen tang vung chac cho phan Idn hoat ddng nudi thuy san tren the gioi Che pham sinh hgc da dugc chap nhan rgng rai de khong che djch benh, tang sire de khang Khac vdi bien phap hda hoc va khang sinh, che pham sinh hgc cung cap mot phuong thirc an toan ben vung ddi vdi ngudi nuoi va tieu diing CHE P H A M SINH HOC HOAT DONG THEO PHUONG THlTC N A O ? Mac dii nhieu loai probiotic dugc dua vao sir dung nuoi trdng thuy san vai thap nien qua nhung viec sir dung cac che pham chu yeu theo kinh nghiem, mdi chi ap dung cac ket qua nghien cim ve probiotic tren ngudi va ddng vat d can cho ddng vat thiiy sinh ma chua thuc su hieu mot each tudng tan co che tac dong cua probiotic ddi vdi nhdm dong vat Tuy nhien ngudi ta cho rang, bat ky mot che pham sinh hoc nao cung phai dat dugc qua trinh sau: - Khdng che sinh hgc: nhimg ddng vi khuan cd ich che pham cd kha nang sinh cac chat khang khuan, vi du bacteriocin de tieu diet cac vi khuan gay benh ao - Tao sire song mdi: Cac vi khuan che pham dua vao ao se phat trien manh me ca ve so lugng va hoat tinh, cd kha nang tdn tai mdi trudng va dudng ruot, anh hudng co Igi doi vdi vat nudi - Xir ly sinh hgc: Kha nang phan giai cac chat hiru co nude giai phong cac axit amin, glucoza, cung cap thirc an cho cac vi sinh vat cd ich, giam thieu phan nito vd co nhu: NH3, NO2, NO3' , giam miii hoi thoi, cai thien chat lugng nude 8 VI SINH VAT Sir DUNG TRONG PROBIOTIC • • Viec sir dung cac vi sinh vat huu ich nham canh tranh vdi cac vi khuan gay benh da dugc img dung rgng rai chan nudi nhu la mot bien phap tdi uu thay thS cho viec sir dung khang sinh, va dang dan dugc ddn nhan nhu mot giai phap quan trgng kiem soat benh nuoi trdng thuy san Sir canh tranh cua vi sinh vat la mot cac qua trinh sinh thai cd the dan den sir thay ddi phan loai cua bat ky he sinh thai nao, cho dii la dat, nude, co the ddng vat hoac mdi trudng khac Nhung thay doi nhd co the anh hudng tdi toe phat trien hoac tir vong dan den sir thay doi ve loai uu the quan xa Mac dii chung ta chua biet het tat ca cac nhan td kiem soat toe tang trudng ciia mot loai nao dd nhung ciing du de co the thay ddi phan loai theo quy luat ire che canh tranh Vi sinh vat CO the tiet cac chat khang khuan, khong diet toan bg loai canh tranh nhung tieu diet dii de thiet lap lai the can bang, lam giam sd lugng loai canh tranh xuong dudi ngudng gay hai kinh te Thanh phan ciia che pham probiotic thudng la mot tap hgp cac chiing vi sinh vat song, dugc tuyen chon, tdi uu hoa, lam kho bang phun say, ddng khd hoac bgc alginat Moi nha san xuat co the chon cac loai khac nhau, nhien bien nhat van la cac loai sau day: - Vi sinh vat sinh phirc he enzym amylaza, proteaza, xenlulaza, lipaza phan giai chat hiru co khong mong muon cd ao nuoi nhu thuc an thira, v6 tdm, xac tao va ddng vat thiiy sinh Cac vi sinh vat bao gom: Bacillus subtilis, B licheniformis, B megaterium, B polymyxa - Cac vi sinh vat sinh chat khang khuan (bacteriocin, pediocin, ) gdm Lactobacillus plantarum, L acidophilus , Bifidobacterium sp., Pediococcus sp - Ngoai mot so che pham chiia nam men Saccharomyces cerevisiae, S boulardii, cac vi khuan nitrat hoa nhu Nitrosomonas, Nitrobacter Mot phan khac ciing thudng dugc thay che pham probiotic la tap hgp cac enzym cd nguon goc vi sinh vat nhu amylaza, proteaza, lipaza, xenlulaza, chitinaza, mot so vitamin thiet yeu hoac axit amin va chat khoang nham kich thich hoat tinh ban dau ciia vi sinh vat che pham va xiic tac cho sir hoat dgng cua enzym mdi trudng Cac vi sinh vat dugc lira chgn phai cd cac d^c dilm sau: - Khdng sinh dgc td, khdng gay benh cho vat chu va khdng anh hudng \ku d6n he sinh thai mdi trudng - Cd kha nang bam dinh len niem mac tieu hda va cac md khac ciia vat chii, canh tranh vi tri bam vdi cac vi sinh vat gay benh, khdng cho chiing tiep xiic true tiep vdi cac co quan cua co the - Co kha nang sinh cac ch§t ire ch6, ngan can su sinh trudng manh me ciia cac vi sinh vat gay benh Cac chat gdm nhieu loai cd the tac ddng don le hoac phdi hgp vdi nhau, bao gom cac chat khang sinh, bacteriocin, siderophore, lysozym, protease, hydroperoxit - Co kha nang sinh trudng nhanh, canh tranh thuc an, hda chat, nang lugng vdi cac vi sinh vat cd hai Vi du vi khuan probiotic sinh siderophore co kha nang lien k6t vdi ion sat, lam cho vi sinh vat gay hai khong sinh trudng dugc vi thieu sat - Tang cudng kha nang mien dich, tang cudng dap img mien djch tu nhien d tdm va kha nang tao khang the d ca - Co kha nang cai thien chat lugng nude ao nuoi sir hinh hang loat enzym phan giai cac chat hiru co, lam giam ham lugng BOD, giam cac dgc nhu H2S, NH3 Khong nhimg the, san pham trao ddi chat ciia vi sinh vat probiotic cdn cung cap cac enzym, cac nguyen td da, vi lugng cho vat chii, giiip chiing sir dung thirc an hieu qua hon va do tang trudng tot hon, KET LUAN Ro rang mdi lo lang ve sir xuat hien cac vi khuan khang thuoc tir ao nudi thiiy san sir dung chat khang sinh, cd the truyen gen khang thudc cho cac vi khuan gay benh d ngudi (ton tai ao nudi tdm), lam cho khang sinh khong cdn hieu nghiem de dieu tri benh cho ngudi nira se dugc giai tda neu thay the bien phap khang sinh bang sir dung che pham sinh hgc Trong thdi diem hien nay, viec sir dung che pham sinh hoc la giai phap uu viet nhat de cd dugc nang suat, chat lugng, hieu qua va sir phat trien ben virng ciia nganh nuoi trdng thiiy san ndi chung va nganh nudi tom ndi rieng 2.1 NGUYEN LIEU 2.1.1 Vi sinh vat - Cac chung vi khuan phan lap tir dat thugc mot sd tinh khu virc phia Bac, nude dua mudi, nude biin - Bg vi khuan kiem dinh: Proteus mirabilis Pseudomonas sp., Escherichia coli Vibrio parahaemolyticus, Aeromonas hydrophyla Bao tang Gidng chuan Vi sinh vat- Trung tam Cdng nghe Sinh hgc- Dai hgc Quoc gia Ha ndi cung cap 2.1.2 Hoa chat Cac hda chat lam moi trudng: -Pepton, cazein, gelatin, (Merck-Dire), Tinh bgt tan (Anh), CMC, Kitin Cac hoa chat diing phan loai va nghien ctm: Tris-HCl; EDTA; SDS; isoamyl alcohol; agaroza; ethilium bromid (Sigma), sorensen, axeton (Trung Qudc) 2.1.3 Thiet bj, dung cu May lac on nhiet (Nhat), noi len men (Nhat), may pH (My), may Ii tam, may OD, may GeneAmp PCR System, may dgc trinh tu ABI PRISM 3100 Avant Genetic Analyzer (My); may ly tam lanh (Sigma, Dire); may soi gel (My); may quang phd UV3000; may chay dien di 2.1.4 Moi tru*dng (xem phu luc) 2.2 PHUOTVG PHAP 2.2.1 Phirong phap phan lap va tuyen chon vi sinh vat - Phan lap vi sinh vat Can 10 g mau dat cho bao binh ndn dung tich 250 ml chira 90 ml nude vd triing, lac deu 30 phiit tren may lac Pha loang mlu vdi ndng giam dSn theo co sd 10 Hiit 0,05 ml djch da pha loang d tir 10^-10^ cSy gat d8u tren moi trudng Hutchinson-Clayton u d 30'^C Sau 48 gid, cSy vi khu4n da mgc vao mdi trudng thach NA de giir giong Doi vdi cac chung lactic, phan lap tren mdi trudng MRS, ii d 30V 48 gid - Tuyen chon Cac chiing vi khuan dugc so tuyen bang phuong phap cay vach tren moi trudng Hutchinson-Clayton cd bd sung cac co chat: CMC, tinh bot, kitin va tren moi trudng thach cazein/tween De tuyen chgn cac chiing sinh vi khu^n sinh khang sinh, vi khuan dugc nudi cSy tren mdi trudng thach NA Sau 72 gid, xac djnh hoat tinh khang sinh so rong tu ddi vdi cac chiing vi khuan lactic, kiem tra sir xuat hien cac khuan lac cd vdng phan giai CaCOa bao quanh - Phirorng phap xac djnh boat tinh enzym va hoat tinh khang sinh *Phuang phap dat thdi thach * Phuong phap nhd dich Hoat tinh enzym/hoat tinh khang sinh (HTE/HTKS) xac dinh theo cdng thirc: HTE/HTKS = D - d (mm) D: Dudng kinh vdng phan giai/vdng vd khuan + dudng kinh Id khoan/dudng kinh thdi thach d: Dudng kinh 16 khoan/dudng kinh thdi thach, - Xac djnh sinh khoi bang phuong phap mat quang hgc-OD d budc sdng 620nm - Phuong phap dem so lu-ong te bao Djch nudi cay vi khuan d cac dieu kien thich hgp dugc pha loang vdi mdi ndng ke ti^p giam 10 lan, Sau dd cay gat 0,05 ml djch pha loang tren dla thach mdi trudng, giir tii am 30^C Sau 24 gid d^m sd lugng khuin lac mgc tren dla Petri Cdng thuc tinh sd lugng t6 bao: So lu'ong te bao = ax 1/Kxl/V a: sd khuan lac trung binh moc tren dTa thach K: pha loang V: the tich dich pha loang dugc cay gat tren dTa thach - Phu-ong phap xac djnh vi khuSn G(+) hay G(-) Cd dinh tieu ban vi khuan bang ngon lua rdi nhudm b^ng dung dich tim kk tinh khoang mot phut Rira bang nude Nhudm tidp bang dung dich lot (dung djch Lugol) mot phut Rira b^ng nude Phii len \k boi dung dich etanol 95%: axeton (1:1) mot phut va lai rira bang nude Sau dd nhudm tilp b^ng thu6c nhudm mau dd Saft-anin 30-60 giay Rua qua nude, dh kho rdi soi kinh Nhom G(+) se khong bi dung moi hiru co (etanol, axeton) xky phirc ch^t mau giira tim kit tinh va idt nen se bat mau tim Nhom G(-) bj dung mdi tay mau thu6c nhudm mau diu nen se bit mau d6(Safranin)[15] - Phu-ong phap chuSn Therner De xac djnh ham lugng axit ciia cac chung vi khuAn lactic, \iy 10 ml djch sau nudi cky b6 sung 20 ml nude ck va 1-2 gipt chi thj phenolphtalein 1% Chuin bang NaOH 0,1N den xuat hien mau hong nhat b^n 30 giay thi dimg lai Xac dinh so ml NaOH da chuan Do axit dugc tinh theo cong thirc: °T = VwaOH X 10 "^rr^rxn AA ^ T 1Q Ar\ T h o r n * % Axit lactic - ^T x 0,009 "• ' '^o Therner tuong duong vdi mg axit lactic 10 bacterioxin Ly tam djch nudi cky vi khuSn lactic bang may ly tam lanh vdi toe 10.000 vdng 15 phut, loai bd kk tiia, thu iky phkn dich Bo sung axetol lanh vao djch theo ty le thi tich 1:1 Sau giir lanh 4^C qua dem, ly tam thu ket tiia, lam khd tua d nhiet phdng va hda tan tua bang dem Sorensen 0,02M dSn pH Thir hoat tinh khang sinh theo phuong phap khuSch tan tren thach [58] - Phirong phap djnh lirong kitinaza Kitin la mot polyme dugc cku tir cac don phan la cac phan tu N-acetyl-Dglucosamin (GlcNAc) n6i vdi qua lien kk (3-1-4 Ham lugng kitinaza dugc xac dinh d budc song 585 nm thdng qua ham lugng N-acetyl-D-glucosamin (GlcNAc) giai phdng dudi tac dung ciia enzym Phuangphdp tien hanh: tron nhirng th6 tich bang (0,2 ml) djch enzym tho vdi 0,5% keo kitin dem axetat 0,05M, pH Phan img dugc thuc hien d 37^C 30 phut Ham lugng GlcNAc dugc giai phong dudi tac dung ciia enzym dugc d budc sdng 585 nm bang may quang ph6 GlcNAc (Sigma, My) dugc diing lam tieu chuan, vdi he sd hap phu la 2,4 x 10 [52] Dua vao dinh luat Lambert-Beer ta tinh dugc ndng ciia GlcNAc giai phong theo phuong trinh: Trong do: OD585 = KCul (*) K la he s6 hkp phu (K=2,4xl03) Cu la nong chat hap phu (GlcNAc) I la chieu dai anh sang qua dung dich (1=1 cm) - Phirotig phap xac djnh anh hudng cua cac dieu kien nuoi cay den kha nang tong hop enzym va khang sinh a Lua chgn moi tru&ng nuoi cay thich hgp Vi khuan dugc nudi tren mdi trudng thich hgp d 28-30^C hoac 40-45°C Sau 48 gid nudi cay xac djnh hoat tinh enzym, pH sau nuoi cay, va sinh khdi te bao Sau 72 gid nudi cay, xac dinh hoat tinh khang sinh, pH, va sinh khoi te bao b Anh huong cua thdi gian Vi khuan dugc nuoi cay tren mdi trudng thich hgp d 28-30'^C hoac 40-45V De xac dinh thdi gian thich hgp cho sir tdng hgp enzym, chat khang sinh va ddng thdi xac djnh sinh khdi Nuoi cay vong 72 gid, sau mdi 12 gid xac djnh hoat tinh enzym, sau mdi 24 gid xac djnh hoat tinh khang sinh va xac djnh sinh khoi bang phuong phap mat quang c Anh huong cua pH nuoi cay 11 mdi trudng thich hgp d 28-30°C hoac 40-45X, pH tir dfin Sau dd xac djnh pH sau nuoi cky, sinh khdi, hoat tinh enzym (sau 48 gid) va khang sinh (sau 72 gid) r d Anh hu&ng cua nhiet nuoi cay Vi khukn dugc nudi cky d cac nhiet do: 20; 25; 30; 35; 40; 45; 50^C, Sau 48 gid xac djnh pH sau nudi cky, sinh khdi, hoat tinh enzym Sau 72 gid xac djnh pH sau nudi cky, sinh khoi, hoat tinh khang sinh, e Anh hu&ng cua nguon cacbon vd nito Vi khuan dugc nudi cay tren moi trudng thich hgp cd bo sung 1% cac ngudn cacbon hoac nito hiru co, v6 co nhu: tinh bgt, glucoza, saccaroza, CMC, kitin, cam, hot dau tuong, cao men, pepton Sau 48-72 gid, xac djnh kha nang sinh trudng va kha nang sinh enzym, khang sinh ciia cac chiing - Anh hudng cua mot so yeu to den boat tinh enzym Cac chung vi khukn cd kha nang sinh enzym dugc nuoi cay lac 220 vong/phiit d diSu kien toi uu Sau 48 gid, ly tam 8000 vong/phiit 15 phiit d6 thu djch enzym tho a, Anh huong ciia nhiet dp den hoat tinh enzym TiSn hanh theo phuong phap nho djch Sau dd dat cac dTa Petri vao thang nhiet dp: 20^ 30^ 40^ 50^ 60^ Sau 24 gid, vdng phan giai co chkt b, Anh hu&ng cuapH den hoat tinh enzym Tikn hanh theo phuong phap nho djch, nhung tren co chat dugc pha dung djch dem tao gia trj pH 3, 4, 5, 6, 7, 8, Sau 24 gid vong phan giai co chat c, Anh huong ciia thdi gian bao quan den hoat tinh enzym Djch enzym tho dugc bao quan d nhiet -4^C tuan Tien hanh thir hoat tinh theo phuong phap nhd djch sau mdi tuan - Xac djnh kha nang chju man Vi sinh vat dugc cay tren mdi trudng thich hgp cd ndng mudi tang dan tir 0% den 12% Sau 48-72 gid, pha loang djch nudi d nong thich hgp xac djnh kha nang sinh trudng, hinh dang va kich thudc khuan lac theo phuong phap dem sd lugng te bao va xac djnh hoat tinh sinh hpc cua chiing, - Xac djnh tinh doi khang Cac chiing vi sinh vat dugc tien hanh thir tinh doi khang theo phuong phap nho djch tuong tu nhu xac djnh hoat tinh khang sinh - Phuong phap tao che pham a, Lua chgn chat mang cho len men xdp: Cay gidng cap hai vao cac moi trudng cd chira cac loai chat manp khac nhu: Bgt gao, bgt dau tuong, cam, bgt thoc mam 12 m Bd sung nude den dp am 45-50% U 48 gid d 30**C, sau dd xac djnh sd lugng ik bao ciia timg loai vi khukn b Ty le gidng cay thich hgp: Gidng ckp hai dugc bd sung vao mdi trudng chira chat mang thich hgp ddi vdi timg loai vi khuSn theo ty le khac nhau: 0,1-0,5-1-5-1013-15-20 % Bd sung nude d k im thich hgp va u ngay, sau dd dim sd lugng ik bao cua timg loai vi khuSn dk xac dinh kha nang sinh trudng cua cac chiing c Thai gian ii thich hgp: Chat mang sau bd sung gidng va lam am dugc u d nhiet 30V Cir sau 24 gid tiln hanh lay mlu xac dinh sd lugng te bao cua timg loai vi khuan d Thai gian bao quan cua chi phdm: Djch len men sau thdi gian thich hgp dugc trdn vdi chat mang theo ty le gidng cky thich hgp, sau u dem sky Trdn cac phan va nghien nhd, ddng vao cac gdi polyme hai Idp 250g Kiem tra bien dong s6 lugng timg loai vi khuan va hoat tinh sinh hgc cua che pham moi thang - Thir nghif m nuoi tom (xem chuong 3) a, Nuoi torn sd phdng thi nghiem Tdm su dugc nuoi be kinh, the tich mdi be 100 lit, nude lay tir ao lang nude nudi tom, vdi dp man 25%o, mdi be tha 100 tdm su vdi trgng lirgng ban dau 0,7- 0,8g va kich thudc 2,5-3cm Be thi nghiem bo sung 0,5gA che pham vi sinh, be ddi chung khdng bo sung che pham Hang cho tdm an thiic an cong nghiep va sue qua trinh nuoi Cir sau thay 1/3 nude va can trgng lugng ciia tom h, Khao nghiem che phdm VSV ngodi ao nuoi torn Thi nghiem dugc tien hanh d dia diem Nuoi tom cong nghiep: Xi nghiep nuoi thiiy san Dinh Vu, Hai Phdng: Dac diem ao nudi d vu thii 3, dp sau ciia nude dat tir 1,0-1,2m, cac ao tom xay dung tren nen rimg ngap man, trudc dd la bai cay sii vet day dac So bd tri thi nghiem nhu sau: Tdng ao nudi tdm, ao thi nghiem, ao sd CO dien tich 10.000m-, ao sd cd dien tich 5.000m^, ao sd ddi chung cd dien tich 5.000ml Ao Cong thuc Thi nghiem Ddi chiing Thi nghiem Cbe pham EMUNIV-S Khdng sir dung EMUNIV-S Mat (con/m^) 20 20 20 Nen day Dat Cling Dat Cling Dat cung + Tat ca cac ao deu dugc xir ly trudc thi nghiem theo dung quy trinh cua ao nudi cdng nghiep (xir ly phai day, xir ly nude bang chlorin, diet tap bang saponin, khir f r n n o K a n o v A i hAt^ 13 lay nude, han che sir dung hda chat chu ky nudi Trong cac ao deu cd he thdng quat nude, cung cac che cham sdc Theo ddi cac chi s6 mdi trudng thuy ly, thuy hda dinh ky 10 mot Ian + Trong qua trinh nudi tat ca cac ao deu sir dung cung thiic an tdm cdng nghiep + Ao sd va sd bo sung che pham vi sinh chu ky 10 mot lan Thang thii nhat 2,5kg/ha/lSn Thang thii hai 4kg/ha/lSn Thang thii trd len 6-7kg/ha/ian + Phan tich nude theo tai lieu "Hudng dSn phan tich mSu cho ao nudi thiiy san" cua Claude E Boyd & Craig Stueker vdi may quang UV/vis - Hitachi, Model: U-2010 + Xac dinh cac chi sd thuy ly, thuy hda theo tai lieu [26] -H Thu mau sinh vat ndi bang ludi vdt hinh chdp ndn, dudng kinh mieng ludi 25cm, chieu dai 90cm Vai ludi vdt thuc vat ndi cd 75 (75 sgi/cm^), vai ludi ddng vat ndi CO 49 + Mau sinh vat ndi dugc cd djnh dung dich formalin 5% + Kiem tra tdc dp sinh trudng cua tdm dinh ky 15 mot lan Danh gia ty le sdng va nang suat tdm tinh toan sau thu hoach + Kiem tra sinh trudng cua tdm bang phuong phap chieu dai tdm theo dinh ky bang thudc chia dp Can trgng lugng tom bang can dien tir cd dp chinh xaclO'^g, -h Ty le tdm sdng dugc xac dinh bang phuong phap "sir dung sang an" thang nuoi thii" nhat, phuong phap quang chai va xac dinh sd lugng ca the/m^ tir thang nudi thir hai den thu hoach, ket hgp vdi ket qua can dem sau thu hoach + Xac djnh nang suat nuoi tdm sau thu hoach, trir cac chi phi thii:c an, gidng, hda chat va cac chi phi khac + Thdi gian khao nghiem bat dau tir 28/03/2006 den 30/08/2006 - Phuong phap xac djnh mot so dac diem sinh hoc cua vi khuan Xac dinh khd ndng sinh bdo tu Cay vi khuan tren moi trudng thach thudng Sau tien hanh xir ly nhiet d 80^C Diing vi khuan da xir ly nhiet cay lai tren dTa thach thudng dat d 30^C Sau 24 gid neu xuat hien khuan lac thi ket luan chiing vi khuan cd kha nang sinh bao tir Neu khdng thi ket luan ngugc lai Xac dinh khd ndng hda long gelatin Cay vi khuan theo phuong thang dimg, sau vao ong nghiem moi trudng gelatin dung Sau 24 gid ii d 30V, lay ong nghiem giir d V gid Kiem tra ket qua neu thay mdi trudng van long thi ket luan co kha nang hoa Idng gelatin Ngugc lai la khdng cd kha nang 14 C02 cky vi khuSn vao dng nghiem chua ml mdi trudng thach thudng djch the da dat dng Duham Dat d 30°C, sau 24 gid kiim tra nSu thdy dng Duham cd noi len, kk luan cd kha nang sinh Ngugc lai ket luan khdng cd kha nang sinh CO2 Xac ^nh hoat tinh catalaza cky vi khudn vao dng nghiem chiia ml mdi trudng thach thudng djch the, dat d 30V Sau 24 gid, nhd gigt H2O2 vao djch nudi cay NSu siii bgt ta kk luan cd hoat tinh catalaza Ngugc lai khdng co hoat tinh - Phan loai theo sinh hoc phan tir O Phuangphdp tdch ADN vi khuan - Lay vong que cky vi khukn hoa vao 200 ^1 TE dng Eppendoft - Them lyzozym vao, trdn deu, sau dd u d 37°C 30 phiit - Them 100 |il SDS 10%, u d 37^C 30 phiit - Them 300 ^il PCI (phenol: chloroform: isoamyl alcohol) vao, trdn deu da lanh, sau dd ly tam vdi van toe 15.000 vdng/phiit Sau ly tam, \ky djch tren (Budc dugc lap lai lan) - Diing etanol lanh vdi the tich gap lan the tich mau de tiia ADN - Rira tiia bang etanol 70% - Lam khd ADN bang may co chan khdng - Them 30-50 ^il nude, bao quan de diing dan ODien di tren gel agaroza Day la ky thuat quan trpng vi la each chii yeu lam cho cac doan axit nucleic hien thj true tiep Phuong phap dua tren mot dac tinh cua axit nucleic la d pH trung tinh mang dien tich am nhd cac nhdm photphat nam tren khung photphodieste ciia cac sgi axit nucleic Dieu cd nghTa la cac phan tir se chay ve circ duong dat dien trudng Ky thuat dugc tien hanh tren mot dem gel cd tac dung phan tach cac axit nucleic theo kich thudc - Tien hanh: Dun tan 1% agaroza dung dich dem TAE Ix dd vao khuon, dgi cho ngudi va dat tam gel vao may dien di, ngap 300ml dung djch IX TAE Trpn deu 2|il dung djch loading buffer 6x vdi 5|il mau, nho vao gieng Chay dien di bang dong dien mot chieu vdi dien the lOOV, cudng ddng dien 80mA 30 phiit, bd ngam dung djch EtBr (nong dp 0,5 |il/ml) 20 phiit vdt Quan sat vach ADN tren may soi gel 15 The tich (%) Thanh phan 10 X buffer dNTP 1,25 mM Moi xuoi 10 16 Moi ngugc (10pmol/|il) Taq polymeraza Mau Nuoc 1,2 Du 100 (10pmol/^l) Mdi xudi: 5'- AGAGTTTGATCCTGGCTCAG -3' tuong img vdi vj tri nucleotit 27 den 47 cua E.coli Moi ngugc: 5'- AAAGGAGGTGATCCAGCC -3' tuong ung vdi vj tri nucleotit 1525 den 1507 cm E.coli - Chu trinh nhiet cho phan img PCR Buoc tien hanh Nhiet (°C) 94 lap lai 30 lan chu ky sau 94 55 72 72 Thai gian phiit 30 giay 45 giay phiit 30 giay phiit - Kiem tra cdc san phdm cua PCR bang dien di: Tien hanh tuong tu nhu doi vdi dien di genom O Phan ung khuech dgi ADN cho giai trinh tu Sir dung bp kit Cycle sequencing vdi hon hgp phan ung nhu sau : Terminator Ready Reaction Mix 8|il Mdi In! Mau l^il Nude cat dii den 20|il Chu trinh nhiet Bu-tfc tien hanh Nhiet ("C) Thoi gian 96 phiit lap lai 25 lan chu ky sau 96 10 giay 50 giay 60 phiit - 16 nucleic Do thuc tk thudng phai sir dung nhimg lugng axit nucleic rat nho (thudng la micro-, nano- hoac picogram) tien hanh cac thi nghiem tach ddng Khdng the xac dinh sd lugng mot each true tiep ma ndng ciia dung djch axit nucleic dugc xac dinh bang each do hap thu tai budc sdng 260 nm (A260) may quang phd kk Mot don vj (1,0) gia trj hap thu budc sdng 260 nm tuong duong vdi ndng 50|ag/ml cua ADN sgi kep, hoac tuong duong vdi ndng 40|ig/ml cua ADN hoac ARN mach don Ti so A260/A280 la chi sd cho thay nhilm cac chat nhu phenol hoac protein Ty sd A260/A280 la 1,8 ddi vdi mau ADN sach O Doc trinh tu ADN Trinh tu ciia rADN 16S cua cac chiing vi khuan dugc dgc true tiep tren may doc trinh tu tu ddng ABI 3100 Avant Sau dd ket qua trinh tu dugc so sanh vdi cac trat tu cua cac loai da co ngan hang gen qudc te de xac dinh den ten loai DAI HOC OUOC GIA HA NQl TPUNG TAM IHONG TIN THli VIEN 0006000006^ 17 KET QUA VA THAO LUAN Tir 47 mlu dat thu dugc tir Hda Binh, Ha Tay, Hai Phdng chiing tdi da phan lap dugc 130 chung vi khuan tren mdi trudng Hutchinson-Clayton d cac nhiet 30 C va 40*^C, tir dd so bg tuy^n chgn dugc 11 chiing cd kha nang sinh enzym phan giai protein, CMC, tinh bgt, kitin, lipit (trong dd cd chiing ua am: VI1, MIO, III, 112, V13 va chung ua nhiet: V20, CB51, L45, L50, T9, K8) va chiing cd kha nang sinh khang sinh manh: V72, V75, K9, K13 3.1 VI KHUAN PHAN GIAI HOP C H A T HLTU CO Thanh phan cac chat hiru co du thira ao nuoi vk da dang chira ham lugng protein, tinh bgt, kitin, lipit kha cao, cd ngudn gdc tir thuc an thira, xac ddng thuc vat, chat thai tir tdm la mot nhung nguyen nhan chinh lam d nhilm mdi trudng ao nuoi Do dd, can ihik phai bd sung cac vi sinh vat cd kha nang phan giai cac chat hiru CO manh nham han che tinh trang nhiem hiru co ao, tir dd giam thieu benh tdm 3.1.1 Tuyen chon cac chung vi khuan co kha nang sinh enzym cao Bang Hoat tinh enzym ciia cac chiing phan lap Chiing vi khuan Hoat ti'nh enzym (D - d, mm) Kitinaza Amylaza Proteaza Lipaza CMC-aza V20 21 28 27 16 24 L45 16 21 15 21 L50 17 17 18 12 20 CB53 21 22 18 14 15 T9 23 22 22 22 K8 24 24 10 25 Vll 29 24 23 17 28 V13 19 21 17 13 18 MIO 20 17 18 10 17 111 20 20 16 10 17 112 26 20 21 24 Vi khuan dugc nuoi cay lac 220 vong/phiit tren mdi trudng HutchinsonClayton, pH d 30^C vdi cac chiing ua am va 40^C doi vdi cac chiing ua nhiet Sau 48 gid xac djnh hoat tinh enzym bang phuong phap khuech tan tren thach Ket qua ghi d hana nhr^ thaw hai ^hnr^rr voo va V11 CO hoat tinh phan giai liptit manh nhat Chiin oteaza manh, chung Vll lai cd kha nang sinh kitinaza va CMC-aza manh hon so vdi cac chung khac Chiing tdi lira chgn hai chiing V20 va VI1 dS ti6p tuc nghien cuu Do cac chung dugc lira chgn ch6 phkm phai cd nguon goc rd rang, nen chiing phai dugc xac dinh cac dac di6m hinh thai, dac dilm nudi cay, sinh ly, sinh hda vaphan loai 3.1.2 Phan loai cac chung vi khuan duac lua chon 5.7.2.7 Phan loai vi khuan den cdp dg chi theo phuong phap truyen thong Xac djnh cac dac dilm hinh thai, nhudm Gram, nhudm bao tu, kha nang di dong, kha nang chju mudi, kha nang ddng hda cac loai dudng cua hai chiing vi khuan Kit qua cho thay: Bang Dac dilm phan loai cua chiing vi khuan Vll va V20 • 3f Dac diem Ket qua , Ket qua Dac diem Giong non co hinh que + Hieu tiiy tien hoac vi hieu Dudng kinh > 2,5 jim - Kj bat buoc - Tiem mao 77 ^ - San pham len men toan lactat — Tiem mao co hinh que hoac cong - Khir sunfat sunfit'~ ^ Noi bao tir + Catalaza + Kha nang di dong -1- Oxidaza + Thuoc Gram (+) + Sinh axit tir glucoza - Hieu bat buoc + Khir nitrat nitrit - Nhu cau NaCI cho sinh trudng + Hoa long gelatin 2-10% 10 , ^ - - + Hinh Hinh dang khuan lac chung V20 Hinh Hinh dang khuan lac chung VI1 Chung ¥20: Khuan lac hmh trdn, cd mep viln xung quanh B I mat khuan lac khd, san, cd mau trSng nga anh vang Te bao hinh que, di ddng Gram duong va bao tu hmh trdn Chiing Vll: Hinh thai khuan lac trdn, be mat hoi xii xi, cd mep vien, mau trang dye Te bao hinh que, di dgng Gram duong va bao tir hinh trdn Theo md ta ciia Bergey (1994), chimg V20 va VI1 thugc chi Bacillus 20 intu Trong thirc tl, diing phuong phap phan loai truyen thdng cd the phan loai chinh xac din ckp dp chi nhung din ckp dp loai thi dp tin cay ddi khdng cao Vi vay, vi?c phan loai bang phuong phap sinh hpc phan tir la rat can thiet De djnh ten din loai cua chung vi khuan nay, trinh tu ADNr 16S dugc phan tich theo cac budc nhu d hinh Kilm tra san pham dien di tren gel agarose 1% cho thay tren ban gel thu dugc bang ADN ting sd nhat, dd cd ADNr 16S ADN dugc dung lam khudn cho phan ung PCR d l nhan gen len nhieu lan bang cac cap mdi da thilt ke dua tren trinh tu bao thu cua gen ARNr 16S ciia E.coli Nhu vay, theo ly thuyet thi san pham PCR thu dugc phai co chieu dai xap xi 1500bp Kit qua dien di d hinh cho thay chi xuat hien bang cd kich thudc 1500bp, rd net, khdng bj dut gay, cd dp tinh sach cao, cd thi sir dung cac phan img tiep theo San pham PCR sau tinh sach bang bd kit Vivapure dugc kiem tra tinh sach va ham lugng ADN Sau dd dugc khuyech dai tilp vdi cac mdi va hda chat cua bd kit Cycle sequencing de chuan bi xac djnh trinh tir tren may dpc trinh tu tu ddng ABI 3100 Avant Ket qua trinh tu dugc so sanh vdi trinh tu cua cac loai da cd ngan hang gen qudc te de djnh ten den loai va xay dung cay phat sinh chiing loai Tach ADN vi khuan i Dien di tren gel argarose Khuyech dai ADN bang phan img PCR i Kiem tra san pham phan img PCR bang dien di i ^ ' ôôã ^ M A IMMtt **"*•' ^ISOObp Khuyech dai ADN cho giai trinh tu i Xac dinh ham luong ADN i Doc trinh tu ADN tren may ABI 3100Avant Hinh Cac budc tien hanh xac djnh Hinh Kit qua dien di san pham PCR trinh tu ADN vi khuan - Marker 91 2,3 - V20 4,5 - VI1 ig V20: AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC GGACAGATGGGAGCTTGCTCCCTGATGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGC CTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTT CAATTATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTG AGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGAC TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAA CAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAACGCGCGCA GGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGA ACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG AACACCAGTGGCGAANGCGACTCTCTGGTCTGTACTGACGCTGAAGCGCGAAAGCGTGGGGAGCG AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCG CCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAG AACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGT GACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGG AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG GGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGA TCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG GTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAA GGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT Trinh tu ADNr 16S cua chung V l l : AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC GAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTG CCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGT TCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTG AGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGAC TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAA CAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGC AGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGA GACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATGGAGGA ACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCA ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGC CCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTC AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGA ACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTG ACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC AACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGA GGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG ACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATT GTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGT GGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAG iGATCACCTCCTTT 99 uih vdi trinh tu nucleotit cua ADNr 16S da cong )d tren ngan hang gen qudc tl (http://wv^^.ncbi.nlm.nih.gov) bang chuong trinh 3LAST Kit qua: • Chiing V20 cd dp tuong ddng so vdi chung 99% (1538/1543bp) B licheniformis • Chung VI1 cd dp tuong ddng 99,8% (1507/1510 bp) vdi chung B subtilis PHYLIP_1 Bacillus anthracisAYI 38383 Bacillus cereus An 38279 I I Bacillus thuringiensisAF 15595 ji Bacillus herbersteiniiAJ78102 I I ; I Bacillus flexus 50, 100 Bacillus megaterium 78 - Bacillus simplex 100 86 Bacillus asahii 99 100 Bacillus muralis ' 98 Bacillus psychrosaccharolyticus - Bacillus indicus AJ583158 100 Bacillus cibi AY550276 51 Bacillus firmus 52 Bacillus pumilus AB048252 Bacillus atrophaeus 100 " Bacillus mojavensis -53 100 : : 981 ! 90" V20 Bacillus licheniformis Bacillus vallismortis 55 Bacillus nematotocita 55 54 Bacillus subtilis Bacillus amyloliquefaciens 0.01 Hinh Cay phat sinh chiing loai ciia chung V20 so vdi cac loai gan gui dua tren trinh tu ADNr 16S 9^ Bacillus thuringiensis Bacillus anthracis 977 r- Bacillus cereus Bacillus herberstelnii 602 1000, ' • I 868 Bacillus flexus Bacillus megaterium 532i ! 978 Bacillus simplex Bacillus asahii 932 j - p Bacillus muralis Bacillus psvchrosaccharolvticus Bacillus rirmus 1000 Bacillus indicus Bacillus cibi 602 I, 'i - _ Bacillus pumilus Bacillus atrophaeus 999 I ^QQQ g43- ' Bacillus licheniformis Bacillus mojavensis j 8931 " Bacillus vallismortis Ii Bacillus nematotocita 700 V11 ^^^ Bacillus subtilis Bacillus amyloliquefaciens 0.01 Hinh Cay phat sinh chiing loai cua chung VII so vdi cac loai gan gui dua tren trinh tu ADNr 16S 3.1.3 Lua chon cac dieu kien nuoi cay thich hop cho sinh tong hop enzym 5.7.5.7 Lua chgn moi truong nuoi cay thich hgp Hai chung vi khuan V20 va VII dugc nuoi cay lac 220 vdng/phiit d tren loai mdi trudng khac ky hieu tir MTl den MT6, pH 7, d 30^C ddi vdi chiing VI1 va 40 C doi vdi chiing V20 Sau 48 gid xac djnh hoat tinh enzym, gia trj pH sau nuoi c^y va kha nang sinh trudng Ket qua dugc trinh bay d bang Chiing VI1 CO hoat tinh kitinaza va CMC-aza cao nhat tren mdi trudng MT3 vdi kich thudc vong phan giai kitin la cm, CMC la 2,8 cm Cdn chung V20 co vdng hoat tinh phan giai tinh bpt la 3,0 cm va protein la 2,9 cm Kha nang phan giai lipit cua ca hai chiing deu khong cao vdi vong phan giai 1,7 cm 24 Bdng Moi trudng nuoi cay thich hop ddi vol kha ndng sinh enzym ciia hai chiing vikhulnV20vaVll Hoat tinh enzym (D - d, mm) ChQng V20 Vll MT pH OD Kitinaza Amylaza Proteaza Lipaza CMC-aza MTl 7,8 0,85 15 16 21 10 18 MT2 8,4 0,98 19 23 25 13 23 MT3 8,6 0,94 22 30 29 17 25 MT4 8,2 0,91 16 20 23 18 MT5 8,6 0,89 20 22 19 12 20 MT6 8,0 0,85 19 18 19 18 MTl 8,1 0,92 27 23 26 13 25 MT2 8,2 0,98 24 22 24 13 27 MT3 8,4 0,97 30 25 26 17 30 MT4 8,3 0,86 18 18 21 11 16 MT5 8,5 0,81 18 18 15 17 MT6 8,1 0,87 20 22 16 15 20 Ket qua tren cho thay, kha nang sinh trudng va tong hgp enzym cua ca hai chiing vi khuan deu tot tren moi trudng MT3 Vi vay, MT3 dugc lira chgn cho nhung nghien ciru tiep theo 5.7.5,2 Lira chgn pH nuoi cay thich hgp Hai chung vi khuan dugc nuoi cay lac 220 vdng/phiit moi trudng MT3, d nhiet dp khoang 30^C vdi chiing VII va 40^C vdi chiing V20, pH dugc thay d6i khoang 4-9 Sau 48 gid, xac djnh hoat tinh enzym, pH, OD Kit qua dugc trinh bay bang 25 Bang pH thich hop d6i vdi kha nang sinh truong va sinh tdng hop enzym cua hai chung V20va Vll Hoat tinh enzym ( D - d , mm) Chung pH dau pH cuoi OD 6,3 0,63 8,1 Kitinaza Amylaza Proteaza a Lipaza CMCaza 12 10 15 13 18 0,74 17 25 26 15 22 8,2 0,84 20 27 27 16 25 8,3 0,97 22 31 30 18 27 8,4 0,95 18 29 27 18 26 8,0 0,80 10 24 20 14 25 4,0 0,42 10 12 10 7,8 0,85 16 18 25 12 25 7,8 0,98 29 21 23 16 32 8,1 0,99 31 27 28 19 33 8,2 0,91 27 23 26 15 24 8,7 0,83 17 20 18 15 V20 Vll Ket qua cho thay, ca hai chiing deu sinh trudng tot nhat dai pH tir den Song, vdi chimg V20 kha nang sinh enzym tot hon, cd the thich hgp vdi gia trj pH dao dong rong tir den 9, chung VI1 sinh enzym tot nhat d gia tri pH din Thdng thudng, gia tri pH ao nudi phai luon dugc tri d mire dn dinh tir 6,57,5 Trong trudng hgp ao co dau hieu nhiem, gia trj pH se cd the giam xuing hoac tang len khdng theo chieu hudng nhat djnh Khi dd, cac vi khuan nhu V20 se cd co hoi de phat buy kha nang cua minh 5.7.5.5 Lua chgn nhiet dp nuoi cay thich hgp Cac chiing chpn Ipc dugc nudi tren moi trudng MT3 tren he thdng may lac 6n nhiet 220 vong/phiit d 20, 25, 30, 35, 40, 45, 50°C, pH Sau 48 gid xac dinh cac thdng sd ve hoat tinh enzym, pH va OD 26 > doi vol kha nang sinh truong va tong hop enzym cua hai chiing V20 va Vll :hung V20 Vll Nhi^t d9 Hoat tinh enzym (D - d, mm) pH cuoi OD 20 5,4 25 Kitinaza Amylaza Proteaza Lipaza CMC-aza 0,56 13 14 12 16 6,7 0,78 18 19 21 13 23 30 7,0 0,90 22 23 24 18 25 35 8,0 0,91 23 26 27 20 26 40 8,3 0,97 23 32 31 21 27 45 8,2 0,96 20 31 29 21 28 50 8,4 0,93 16 27 25 19 28 20 7,8 0,82 22 20 15 18 20 25 7,9 0,96 29 26 25 18 28 30 8,2 0,97 32 28 29 20 33 35 8,1 0,92 27 23 24 16 27 40 7,8 0,79 16 18 15 12 23 45 7,7 0,71 10 18 18 16 50 6,9 0,43 12 10 12 10 Sir sinh trudng va kha nang tong hgp enzym ciia hai chiing vi khuan co khac biet ro ret ve nhiet Chiing V20 nhu la chung ua nhiet do, sinh khdi dat cue dai d nhiet dp nudi cay khoang 40-45*^C, boat tinh enzym the hien tdt nhat sinh trudng d nhiet dp tir 40^C Trong chiing Vll lai sinh trudng d nhiet dp thap hon tir 253 ^ , boat tinh phan giai cao nhat d nhiet dp 25-30^C (nhiet dp thich hgp ddi vdi su phat trien ciia tom) Sir phoi hgp ciia hai chiing vi khuan mot che pham se lam tang gidi ban ve pH va nhiet qua trinh xir ly d nhilm ao nudi Ben canh do, cac chiing thuan Igi xii ly bim day phoi day ao trudc ckp nude vao ao nuoi 3.L3.4 Lua chgn thai gian nuoi cay thich hgp Hai chiing vi khuan dugc tien hanh nghien cim anh hudng cua thdi gian din sir sinh trudng va sinh tong hgp enzym nham tim dugc thdi gian thich hgp ddi vdi qua trinh len men d quy mo Idn Sir dung mdi trudng MT3, pH 7, lira chgn gia trj nhiet trung binh la 40V ddi vdi chiing V20 va 28-30^C ddi vdi chiing VI1 Hai chiing dugc nuoi cay lac 220 vong/phiit tren may lac on nhiet Cir sau 12 gid tiln hanh kilm tra boat tinh enzym, pH sau nuoi cay va OD Ket qua dugc trinh bay bang 97 inh hudng den sinh khdi va boat tinh enzym ciia ca hai chiing Cac chung sinh trudng manh bat dau sau 24 gid den 48 gid Sinh khdi tang cue dai d khoang 48 gid va giam dan theo thdi gian Hoat tinh enzym cung tang dong thdi qua trinh sinh trudng, manh nhat d 48 gid va tuong ddi dn djnh khoang tir 36 den 60 gid Nhu vay, thdi gian thich hgp de thu dugc lugng sinh khdi Idn nhat va boat tinh cao nhat la 48 gid Bang Thoi gian nuoi cay thich hop doi vol sinh truong va sinh tong hop enzym cua hai chung V20 va Vll Chung Hoat tinh enzym ( D - d , mm) Thoi gian (gifr) 12 pH cuoi OD Kitinaza Amylaza Proteaza Lipaza CMCaza 7,6 0,67 13 18 16 11 21 24 8,0 0,89 18 23 19 12 26 36 8,1 0,95 22 31 30 19 27 48 8,2 0,97 25 33 32 22 29 60 8,4 0,93 23 30 27 18 26 72 8,1 0,85 20 24 26 16 20 12 7,7 0,55 18 12 19 20 24 8,0 0,96 25 21 25 14 28 36 8,2 0,98 30 28 29 19 33 48 8,3 0,99 33 29 29 21 35 60 8,4 0,95 32 26 28 18 29 72 8,0 0,83 26 26 28 25 V20 Vll Cd the nhan thay kha nang sinh lipaza ciia ca hai chiing deu kem hon so vdi kha nang sinh cac enzym khac Trong kha nang sinh kitinaza va CMC-aza ciia chiing Vll manh hon so vdi chung V20 Ngugc lai, chiing V20 cd kha nang sinh proteaza va amylaza cao va tuong ddi dn dinh hon chiing VI1 Sir kit hgp ciia hai chung se lam tang kha nang phan giai phan Idn cac loai co chat thudng co cac ao nudi tdm bj nhiem 5.7.5.5 Li4a chgn ngudn cacbon vd nito thich hgp Chiing tdi tien hanh nghien ciru anh hudng ciia nguon cacbon va nito tdi kha nang sinh trudng va sinh tdng hgp enzym ciia cac chiing vi khuan ngoai muc dich tim kiem chat mang hgp ly xac djnh dugc co chat thich hgp de san xuat chi pham tir cac vi khuan Cac chiing dugc nuoi cay tren mdi trudng MT3 cd bd sung 1% co chat cacbon/nito nuoi d dieu kien tdi uu Sau 48 gid kiem tra kha nang sinh trudng va M/^r»f +«*^K A £^ ll TO r»r>/-vr» r\ m i r ^ n n ^'^^'on/nito thich hgp 28 :bon den kha nang sinh truomg va sinh tong hop enzym cua hai chung V20 va Vll V 20 V 11 Hoat tinh enzym (D - d, mm) pH cuoi OD Kitinaza Amylaza Proteaza Lipaza CMCaza Tinh hot 8,1 0,96 24 31 32 18 26 Glucoza 8,0 0,85 20 17 29 16 Saccaroza 7,6 0,78 18 15 16 16 CMC 8,0 0,92 21 30 28 19 27 Kitin 8,4 0,97 22 23 29 10 20 Cam 8,5 0,90 25 27 32 13 21 Tinh bpt 7,7 0,92 23 25 16 10 29 Glucoza 8,2 0,87 16 21 15 20 Saccaroza 8,2 0,85 17 17 17 - 18 CMC 8,4 0,96 30 28 19 13 34 Kitin 8,3 0,93 32 29 19 20 32 Cam 8,3 0,96 14 30 A- r^ /to/ \ NgUun \^ V* /07 21 28 24 Bang Anh huong ciia nguon nito* huu ca den kha nang sinh trudng va sinh tong hop enzym ciia hai chiing V20 va Vll Hoat tinh enzym (D - d, mm) Nguon N(l%) pH cuoi OD Kitinaza Amylaza Proteaza BDT 8,0 0,99 25 34 33 15 25 Cao men 8,1 0,95 24 26 30 21 29 Pepton 7,3 0,96 20 30 29 19 28 BDT 8,7 0,98 34 28 29 17 33 Cao men 8,4 0,94 28 28 21 22 29 Pepton 8,6 0,88 28 20 12 20 23 V 20 V 11 Lipaza CMCaza BDT: Bgt dau tuong Hai chiing vi khuan deu sinh trudng manh tren cac nguon cacbon nhu tinh bgt, CMC, cam va kha nang sinh tdng hgp enzym cao hon mdi trudng xuat hien CO chat cam irng Vi du, chiing VI1 co kha nang tdng hgp enzym manh nhat tren ngudn cacbon la CMC, kitin, cam nhung cd mat kitin moi trudng thi boat tinh kitinaza cao hon Tuong tu nhu vay co mat CMC, hoat tinh CMC-aza cao '"•^ ' *^ * ' ' " - ^ ' ' k h a n g djnh qua rat nhieu thi nghiem) Trong do, 29 TUdng va sinh tdng hgp enzym cua chiing V20 la tinh bgt, CMC hoac cam Bgt dau tuong, cao men, pepton dIu la nhimg ngudn nito hiru co tdt nhat ddi vdi sinh trudng va tong hgp enzym cua ca hai chiing Ca hai chiing deu sinh tdng hgp enzym sir dung ngudn nito la bgt dau tuong Day la mot yeu td quan trgng san xuat d quy md nho va len men cong nghiep Tuy nhien, boat tinh lipaza lai the hien manh nhat tren ngudn nito la cao men Bang Anh huotig ciia nguon nita v6 cc den sinh truong va sinh tong hgp enzym ciia hai chiing V20 va Vll Hoat tinh enzym (D - d, mm) Nguon N ( l % ) pH cuoi OD Kitinaza Amylaza Proteaza Lipaza CMC-aza Ure 8,3 0,82 23 24 30 12 27 (NH4)2S04 8,0 0,90 26 30 31 17 29 NaN03 8,3 0,87 24 29 21 15 26 NaN02 8,6 0,83 20 21 24 13 23 Ure 8,9 0,96 20 26 16 28 (NH4)2S04 8,2 0,88 29 27 25 20 34 NaNOs 7,9 0,91 34 25 27 19 35 NaN02 8,5 0,83 22 22 23 13 19 V 20 V 11 Tir kit qua tren ta thay, moi chiing khac cd kha nang sir dung nguon nito khac Chung V20 sinh trudng va tong hgp enzym manh vdi ngudn nito vd co la ure hoac (NH4)2S04, chiing Vll lai la (NH4)2S04hoac NaNOs- Nhin chung thi ca hai chiing deu co kha nang sir dung tdt cac nguon nito vd co va hiru co 3.L3.6 Anh huong cua nhiet dp den hoat tinh cda enzym Vi khuan dugc nudi cay lac 220 vong/phiit mdi trudng thich hgp d 2830V vdi chung VII va 40V vdi chung V20 Sau 48 gid, ly tam 8000 vdng/phiit 15 phiit dl loai bo sinh khoi, thu djch enzym tho Tien hanh thir boat tinh enzym theo phuong phap nhd djch Dat cac dTa thu boat tinh d cac nhiet khac tir 20 den 60V Sau 24 gid, xac djnh boat tinh enzym thu dugc ket qua nhu bang 10 Enzym hai chiing V20 va VI1 sinh la nhimg enzym kha ben nhiet Gidi ban nhiet tdt nhat ddi vdi enzym ciia chung V20 la tir 30-50^C Trong gidi ban nhiet hoat tinh enzym thay doi khong dang ke Dac biet d 60 C enzym amylaza ciia chung V20 van cdn hoat tinh manh Enzym lipaza chiing sinh dudng nhu 30 a thap (20V) hoac qua cao (60°C) dIu lam mat boat tinh lipaza ciia chiing Hoat tinh enzym ciia chung VI1 on dinh hon dai nhiet tir 30-40 C CMC-aza va kitinaza ciia chiing VI1 tuong ddi dn dinh dudi tac ddng ciia nhiet do, bin d nhiet 30-40^C, d 50°C boat tinh cua hai enzym giam khdng dang ke nhung tren 50V boat tinh enzym cua chung giam manh Bang 10 Anh huong cua nhiet den hoat tinh enzym cua hai chiing V20 va Vll Hoat tinh enzym (D - d, mm) Chung V20 Vll Nhiet ("c) • Kitinaza Amylaza Proteaza Lipaza CMC-aza 20 15 19 17 - 13 30 24 33 31 20 29 40 24 32 30 18 27 50 23 31 27 17 22 60 14 28 22 - 16 20 18 23 12 25 30 33 29 28 21 35 40 32 21 23 19 35 50 30 16 17 15 31 60 25 16 11 20 5.7.5.7 Anh huong cua pH den hoat tinh enzym Djch enzym tho dugc tien hanh thir boat tinh enzym theo phuong phap nho dich tren cac dTa mdi trudng co chat cd pha vdi dung dich dem de tao cac gia trj pH khac tir den Dat cac dTa thir boat tinh d tu am 30^C Sau 24 gid, xac djnh boat tinh enzym thu dugc ket qua nhu bang 11 31 Bang 11 Anh huong ciia pH den hoat tinh enzym ciia hai chiing V20 va Vll Hoat tinh enzym (D - d, mm) Chung V20 Vll pH Kitinaza Amylaza Proteaza Lipaza CMC-aza - - 13 14 24 19 14 26 12 10 21 20 26 26 11 24 26 32 30 16 28 25 31 29 15 29 16 22 18 22 - 16 - - 10 - 16 11 - 17 18 18 14 12 26 22 23 25 12 33 33 27 28 21 30 33 28 24 16 25 17 14 13 15 pH cung phan nao anh hudng den boat tinh enzym ciia ca bai chiing Hau het cac enzym deu boat ddng d pH trung tinh, manh dan len tir pH va manh nhat d gia trj pH 7-8 CMC-aza chiing VII sinh lai boat ddng manh nhat d pH kitinaza lai thich hgp vdi gia trj pH 7-8 Enzym amylaza va proteaza cua chiing VI1 boat ddng tdt nhat d pH 7, d gia trj pH boat tinh hai loai enzym cd giam nhung khong dang ke Hoat tinh Lipaza ciia ca hai chiing deu manh d gia trj pH 7, pH qua axit hoac qua kiem deu lam giam boat tinh enzym 3.L3.8 Anh huong cua th&igian den hoat tinh enzym Djch enzym tho dugc bao quan d - 4^C, cii sau tuan tien hanh kiem tra boat tinh theo phuong phap nho djch Cac thi nghiem cho thay chung V20 va VI1 co hoat tinh enzym kha cao Chiing tdi tien hanh nghien ciru anh hudng cua thdi gian den boat tinh enzym nham muc dich cho cac nghien cuu khac ve enzym hoac tao mot san pham enzym co gia trj kinh te ddi vdi nuoi trdng thuy san Ham lugng enzym che pham rat can cho sir phan ? i I J i -:.'.„ ^u "hh vat sinh trudng tha xudng ao Khi nghien ciru -^9 lb cac loai enzym cua hai chung boat tinh enzym cua ca hai chiing deu tuong ddi dn djnh thdi gian tir den tuan Sau tuan, hoat tinh cd giam nhung khdng dang ke Dac biet phai ke den kitinaza ciia chiing Vll kha ben sau thdi gian 10 tuan Bang 12 Anh huong ciia thoi gian bao quan din ben enzym ciia hai chiing V20, Vll Chung V20 Vll Hoat tinh enzym (D - d, mm) Thoi gian (tuan) Kitinaza Amylaza Proteaza Lipaza CMC-aza 26 32 32 21 29 26 30 32 20 27 26 29 30 16 26 21 23 25 12 26 10 19 20 19 12 23 34 29 28 22 35 33 27 26 13 35 33 22 25 11 28 30 19 25 27 10 30 15 21 26 5.7.5.9 Dinh lugng kitinaza cua chung Vll Do chung VII CO kha nang sinh kitinaza manh nen chiing tdi tien hanh djnh lugng kitinaza nham xac djnh kha nang chuyen hda kitin ciia chimg Trdn nhirng the tich bang (0,2 ml) djch enzym tho vdi 0,5% keo kitin dem axetat 0,05M, pH Phan img dugc thuc hien d 37°C 30 phiit Ham lugng GlcNAc dugc giai phdng dudi tac dung ciia enzym dugc d budc song 585 nm bang may quang phd GlcNAc (Sigma, My) dugc dimg lam tieu chuan, vdi he so hap phu la 2,4 X 10^ Ket qua dugc bang 13 Bang 13 Djnh luong ham luotig kitinaza chiing Vll tao OD585 ChungVll 0,155 GlcNAc chuan 0,21 Dua vao djnh luat Lambert-Beer ta tinh dugc ndng ciia GlcNAc giai phong theo phuong trinh: OD585 = KCul (*) Trong dd: K la he sd hap phu (K=2,4xl0^), Cu la ndng chat hap phu (GlcNAc), I la chieu dai anh sang qua dung djch (1=1 cm) -^^ 0,155 / (2,4x10^1) = 0,0646 xlO'^ So sanh vdi GlcNAc chuan d budc sdng 585 ta cd: Cs = OD585/ Kl = 0,21/ (2,4x10^1) = 0,0875 x 10'^ Ndng dp kitinaza tuong duong vdi ndng dp GlcNAc dugc giai phdng nhu vay ta se tinh dugc ndng moi cua kitinaza dugc tao dich nudi la: CM = n/v = 0,0646 xl0'V0,2 = 0,323mM Va lugng kitinaza cd kha nang chuyen hoa den 73,8% (Cu/Cs) lugng kitin GLcNAc Day la mot ket qua dang nghi nhan ve kha nang san sinh kitinaza cua chiing vi khuan VI1 nham ung dung xir ly d nhiem mdi trudng nudi hai san, 3.1.3.10 Khd ndng sinh chat khdng khuan cda hai chung V20 vd Vll Bang 14 Kha nang khang mot so vi sinh vat thuong gap ciia V20 va Vll Vi sinh vat kiem djnh Hoat tinh khang sinh (D-d, mm) Vll V20 Vibrio parahaemolyticus - Escherichia coli + + Pseudomonas sp 17 11 Sarcina lutea 14 16 Staphylococcus aureus - Salmonella typhi - - Klebsiella sp - 18 Fusarium oxysporum 12 16 Candida albicans - - Proteus mirabilis 13 +: cd uc che - : khdng uc che Ngoai kha nang sinh enzym phan giai hgp chat hiru co manh, cac chung V20 va VII dugc khao sat kha nang ire che mot sd loai vi khuan gay benh thdng thudng, dac biet la Vibrio Djch nudi cay ciia hai chung dieu kien toi uu sau ly tam loai bo sinh khdi te bao dugc tien hanh kiem tra boat tinh khang sinh bang phuong phap khuech tan tren thach thong thudng Bang 14 cho thay ca hai chiing deu cd kha nang sinh chat khang khuan chong lai nhieu loai vi khuan c6 ao nudi nhu Vibrio Pseudomonas, Proteus tham chi ca nam Fusarium Mac dii d mdi trudng nude cac chat khang khuan ton tai khdng lau bj pha loang hoac bj phan giai bdi cac vi sinh vat khac, nhung chiing lien tuc dugc tao nen van ire che dugc cac vj khuan gay benh lien ke ^4 SINH Bdn chiing vi khuan V75, V72, K9, K13 cd kha nang sinh khang sinh dugc nudi cky lac 220 vdng/phiit tren mdi trudng thach dinh dudng, d pH trung tinh, nhiet dp 2830^C Sau 72 gid, xac djnh boat tinh khang sinh ddi vdi mot sd chiing vi khuan kiem dinh quan tam nhu: V parahaemolyticus E coli Proteus mirabilis, Pseudomonas sp., Aeromonas hydrophyla thu dugc ket qua nhu d bang 15 Ddi vdi cac vi khuan gay benh cho tdm, chi cd chung V75 phd khang sinh rong va boat tinh manh nhat Do sd lugng cac loai mot che pham khdng nen qua nhieu, cho nen budc dau chiing toi lira chgn chiing V75 lam chiing sinh khang sinh phan che pham Bang 15 Hoat tinh khang sinh sa bo ciia cac chiing vi khuan nghien ciru Hoat tinh khang khuan (D-d, mm) K13 V75 V72 K9 24 11 28 17 14 26 26 13 12 16 15 18 17 22 10 20 Vi sinh vat kiem djnh V parahaemolyticus E coli A hydrophyla Pseudomonas sp Proteus mirabilis 3.2.1 Phan loai vi khuan V75 Vi khuan V75 cd khuan lac trdn, mau trang sira, nhay, bat mau Gram duong, hieu khi, cd kha nang di dong va hinh bao tir, phan img catalaza va oxidaza duong tinh Theo phan loai cua Bergey [I7]chiing V75 thudc chi Bacillus Khi phan tich trinh tu nucleotit ciia doan ADNr 16S, so sanh vdi cac trinh tu ADNr I6S da dugc cdng bo ngan hang gen cho thay tuong ddng ve trinh tu nucleotit gen ma hoa cho ARNr 16S cua chung V75 dugc xac djnh la B subtilis vdi dp tuong d6ng 100% (I500/I500bp) # > f •.*•* • T* 500bg Hinh Ket qua dien di san pham PCR ' Marker 2,3 Chiing V75 35 AGAGTTTGATTCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACA GATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAA GACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACA TAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAAC GGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACA CGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAG CAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCC GTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTT TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAG TGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCA GTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGG ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTT AGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAG GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT TACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAG GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC CTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAA GGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAG AACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCA GTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATA CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAG GTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTAAGTCGTAACAAGGTAG CCGTATCGGAAGTGCGTGGGGTTCCCCTCCTTT Xay dung cay phat sinh chiing loai cua chiing V75 va cac loai gan gui chi Bacillus cho thay V75 nam ciing mot nhanh vdi B subtilis PHYLIP_1 Bacillus Bacillus I 97 I Bacillus ' g2 j Bacillus flexus Bacillus megaterium — 86 anthracis Bacillus cereus herberstelnii 100 i thuringiensis 53 Bacillus simplex Bacillus asahii 97 Qo Bacillus Bacillus muralis psychrosaccharolyticus Bacillus firmus 52 100_ Bacillus - ! _- _ indicus Bacillus cibi Bacillus pumilus Bacillus 99 ^ QQ g^ atrophaeus Bacillus Bacillus 93 70 97 „ „ Bacillus Bacillus V75 Bacillus licheniformis mojavensis vallismortis nematotocita subtilis Bacillus amyloliquefaciens Hinh Cay phat sinh chiing loai ciia chiing V75 vdi cac loai co quan he gan gui dua vao '-* *'r 1500 bazo cua ADNr 16S 36 nuoi cay den kha nang sinh chat khang sinh 3.2.2J Lua chpn moi tru&ng nuoi cay thich hgp Chung V75 dugc nudi cay lac 220 vdng/phiit tren loai mdi trudng khac ki hi?u la MT2, MT3, MT4 d diSu kien 28-30^C Sau xac dinh boat tinh khang sinh va kha nang sinh trudng cua cac chung tren cac mdi trudng khac Bang 16 Lua chon moi truong nuoi cay thich hop doi kha nang sinh khang sinh ciia chiing V75 Hoat tinh khang sinh (D-d, mm) Moi E coli 20 A hydrophyla - PseudomoP, mirabilis nas sp 18 17 MT2 8,4 0,95 V parahaemolyticus 12 MT3 8,0 0,98 29 27 15 18 25 MT4 8,4 0,89 - 11 12 13 16 truong pH OD Chung V75 cd kha nang sinh trudng tot tren ca mdi trudng, nhung boat tinh khang sinh cao nhat d moi trudng MT3, thuc te day cung la mdi trudng kha don gian ve phan nen nen cd the chgn cho cac nghien ciru tiep theo 3.2,2.2 Anh hu&ng cda pH den hoat tinh khdng sinh Chiing V75 dugc nudi cky lac 220 vdng/phiit mdi trudng MT3 d 28-30 C, pH tir din Sau ngay, xac djnh boat tinh khang sinh, kha nang sinh trudng va pH sau nudi cay Ket qua dugc trinh bay bang 17 Bang 17 Anh hudng ciia pH den hoat tinh khang khuan cua chiing V75 Hoat tinh 1ihang sinh (D-d, mm) A Pseudomo- E coli hydrophyla nas sp mirabilis 13 12 - - + 0,88 17 15 10 14 8,0 0,93 27 23 15 16 18 8,1 0,98 29 28 16 20 24 8,4 0,91 28 19 12 19 16 8,0 0,84 16 11 pH pH dau cuoi OD haemolyticus 4,8 0,65 7,8 K para- (-):k lone CO hoat tinh (+), (++): h oat tinh veu K6t qua d bang 17 cho thay chung V75 sinh trudng va cho boat tinh khang sinh d pham vi rgng (pH 5-9), nhien boat tinh manh nhat nudi d pH ban dku la ^7 hoat tinh khdng sinh Nuoi cay chung V75 tren may lac 220 vdng/phiit mdi trudng MT3, pH d cac nhiet tir 20-50V Sau ngay, xac dinh boat tinh khang sinh, kha nang sinh trudng va pH sau nudi cay Ket qua dugc trinh bay bang 18 cho thay: Kha nang sinh khang sinh cua chiing V75 chju anh hudng kha nhieu cua nhiet Hoat tinh cao nhat nudi d 30°C Nhiet cao tren 40°C hau nhu khdng cdn kha nang sinh khang sinh, ngoai trir kha nang khang Pseudomonas sp vdi boat tinh trung binh Bang 18 Anh huong ciia nhiet den hoat tinh khang sinh ciia chiing V75 Hoat tinh khang sinh (D-d, mm) Nhiet ("C) pH cuoi OD 20 7,6 25 V parahaemolyticus E coli A hydrophyla Pseudomonas sp P mirabilis 0,81 22 24 11 ++ 8,4 0,95 28 25 14 14 15 30 8,3 0,99 30 28 17 21 24 35 8,3 0,92 25 23 19 19 15 40 8,6 0,87 10 12 15 45 8,1 0,62 ++ ++ - 12 13 + 50 7,9 0,59 + + - - - 3.2.2.4 Anh huong cua th&igian den hoat tinh khdng sinh Chiing vi khuan V75 dugc nudi cay d cac dieu kien nhu tren Cir sau 24 gid tien hanh kiem tra boat tinh khang sinh va kha nang sinh trudng va pH sau nudi cay Ket qua dugc trinh bay bang 19 Thdi gian dong vai trd quan trpng ddi vdi hieu suat san sinh cac san pham bac hai ciia vi sinh vat Chiing V75 cd kha nang sinh khang sinh manh sau 48 gid nuoi cay va dat cue dai d 72 gid, tire la sau pha logarit Bang 19 Anh huong ciia thoi gian nuoi cay den hoat tinh khang sinh ciia V75 Hoat tinh khang sinh (D-d, mm) Thoi gian (gi6) pH cuoi 24 OD V parahemolyticus E coli A hydrophyla Pseudomonas sp P mirabilis 7,97 0,93 10 15 ++ 18 48 8,35 0,98 28 23 12 17 20 72 8,44 0,95 32 29 20 23 25 96 8,41 0,80 24 28 16 19 20 ++: CO ire che •^8 8ACTERIOXIN Ngoai cac chiing da lira chpn chiing tdi tien hanh phan lap va tuyen chpn mot sd chung vi khuan lactic tir biin va nude dua mudi tren mdi trudng MRS Ket qua thu dugc chung vi khuan lactic lira chgn dugc chiing L40 la L 1.1 cd kha nang khang vi khuan gay benh d tom manh nhat Bang 20 Kha nang khang khuan cua mot so chiing vi khuan lactic Hoat tinh khang khuan (D-d, mm) Chung V parahaemolyticus E coli P mirabilis 1101 11 13 16 T9 - + + SP4 17 13 10 L6 10 16 L22 12 14 14 Ll.l 24 20 17 L40 26 23 18 3.3.1 Phan loai chung L40 Dac diem hinh thdi Tte bao chung L40 cd dang hinh que, bat mau Gram duong, khdng co kha nang di ddng, khong sinh bao tir, khuan lac trdn, nhan, mau trang sira Phan ung catalaza am tinh, CO kha nang dong hoa cacbonhydrat amygdalin, arabinnoza, xenlobioza, fructoza, glucoza, lactoza, maltoza, manitol, melezitoza, saccaroza, nhung khdng sir dung sorbitol, CO kha nang sinh axit manh Can cu vao cac dac diem hinh thai, sinh ly, sinh hoa, dua theo khoa phan loai Bergey's chung L40 dugc xac djnh thudc chi Lactobacillus 39 sinh ly sinh hoa ciia chiing L40 D^c diem Ket qua Dac diem Ket qua Gi6ng non co hinh que + Dudng kinh > 2,5 jam - Arabinnoza +++ Hinh bao tu - Xenlobioza + Kha nang di dong - Fructoza + Hidu tiiy tien hoac vi hieu - Glucoza 4-+ Kj bat buoc - Lactoza + Thuoc Gram + + Maltoza ++ Hieu bat buoc - Manitol + Melezitoza + Saccaroza + Sorbitol - Kha nang sinh trudng d nong mudi 2-12% +++: Dong hoa tot Kha nang dong hoa dudng + ++: Dong hoa trung binh Dong hoa yeu Khong dong hoa Phan loai theo phuong phap sinh hgc phan tu Viec dua cac chimg vi sinh vat sdng vao mdi trudng la mot viec lam he trpng, phai dam bao chac chan rang chiing khong gay hau qua xau ddi vdi sue khoe dong vat nudi va moi trudng, vay chiing toi tien hanh xac djnh trinh tu ADNr 16S de djnh danh chinh xac den loai Sau tien hanh phan img PCR, trinh tu ADNr I6S dugc dpc tren may dpc trinh tu tu dong 3100 Avant bang doan mdi dac hieu 500bp Hinh Kk qua dien di san pham PCR chung L40 Marker 2,3 L40 40 GAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTA ACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAAC AACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGC GTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTA ATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCC ACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACT CTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGC CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGG GCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTG CATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATG CGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTC GAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAA GTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACG GCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA TTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTT CCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGAC TGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCT ACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAG CCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGG ATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTT GTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTA GGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT So sanh vdi dij lieu ngan hang gen Quoc te xac djnh dugc chiing L40 co dp tuong dong 100% (1380/1380 bp) vdi loai L plantarum Xay dung cay phat sinh chung loai cua chung L40 va cac loai gan gui chi Lactobacillus cho thay L40 nam ciing mot nhanh vdi L plantarum PHYLIH Lactobacillus versmoldensis ^ QO , i 57 Lactobacillus suebicus Lactobacillus vaccinostercus Lactobacillus hammesii 59 Lactobacillus paracollinoides 55 Lactobacillus manihotivorans Lactobacillus paraplantarum ^00 66 Lactobacillus arizonensis Lactobacillus pentosus 56 80 L40 Lactobacillus plantarum 0.01 Hinh 10 cay phat sinh chiing loai ciia chiing L40 vdi cac loai co quan he gan gui dua vao tu 1380 bp ciia ADNr 16S 41 ic Ll.l D^c ^int hinh thdi - Khuan lac: Tren moi trudng MRS, sau nuoi cky di§u kien vi hieu khi, khuan lac co hinh tron, hoi loi, c6 mau trang due Dudng kinh khuin lac tir 1-1,5mm - ik bao: Dudi kinh hien vi dien tu (vdi dp phong dai 20.000 \kn) cho thiy it bao cua chiing Ll.l tir hinh cau den hinh ovan, xep cac chu6i ngan - Nhupm Gram: Chiing Ll.l la Gram (+), khong sinh bao tir 3.2.2 D^c diem sinh ly, sinh hoa cua chiing Ll.l Bang 22 Dac diem sinh ly, sinh hoa ciia chung Ll.l Dac diem Nhiet dp sinh trudng pH sinh trudng Ket qua 10-45*^C 3-9,5 Dac diem Sinh CO2 tir Glucoza Ket qua +/- Kha nang di dong - Sinh proteaza - Kha nang sinh bao tir - Hoat tinh catalaza - Kha nang chju NaCl 0-6,5% Sinh axit tir len men cac co* chat L- Arabinoza + Lactoza + D- Xyloza - Trehaloza + Rhamnoza - Melibioza + Riboza + Rafinoza + Glucoza + Melezitoza + Mannoza + Tinh bpt - Fructoza + Manitol + Galactoza + Sorbitol - Sacaroza + Esculin - Mantoza + Salixin + Xenlobioza + Amygladin - Dua theo Bergey's Manual, chiing Ll.l thuoc chi Enterococcus 3.2.3 Phan loai bang sinh h9c phan tu Khi so sanh trinh tu ADNr 16S cua chung Ll.l vdi cac trinh tu ciia cac chiing vi khuAn lactic ngan hang gen qudc t^ cho thay: Trinh tu nucleotit chiing Ll.l co dp tuong dong 100%) (1412/1412 bp) so vdi loai Enterococcus lactis 42 :TGGCGGCGTGCCTAATACATGCAAGTCGTACGCTTCT 1, ^^ .^^ ^ w^« J^^AGAAGAGTGGCGAACGGGTGAGTAACACGTGGGTA ACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCG CATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAG TTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACAT TGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGA AAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAG AAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA GCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACT GGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATAT GGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGG GGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGG GTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGT TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAA CGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGG GCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCAAGACTGCCGGTGAC AAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTG CTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCA GTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACG CCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCC GAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTC GTAACAAGGTAGCCGTATCGGAAGGTGCGGCTG Enterococcus la vi khuan hinh cau Gram duong, khong sinh bao tir, kj tiiy tien, CO the phat trien d dai nhiet dp rong tir 10-45**C va moi trudng co nong dp muoi cao den 9,6% Loai vi khuan lactic Enterococcus lactis ciing la mot nhCmg loai da dugc img dung de san xuat che pham probiotic tren the gidi Loai ciing phan bo vdi mat dp kha cao he dudng ruot cua vat nuoi Vi vay chiing co y nghia thuc tien tao che pham probiotic de tang cudng cac VSV co ich he tieu boa cua dong vat Xay dung cay phat sinh chiing loai giira Ll.l va cac loai co quan he hp hang gan chi Enterococcus Ket qua cho thay chiing Ll.l nam ciing mot nhanh vdi loai Enterococcus lactis 43 cams Enterococcus villorum 53 ~ Enterococcus mundtii Enterococcus sanguinicola Enterococcus durans Enterococcus faecium 54 I Enterococcus ratti 50 Enterococcus faecalis 54 58 59 L1.1 97 Enterococcus lactis Enterococcus azikeevi 99 Enterococcus pseudoavium 77 Enterococcus hirae 0.01 Hinh 11 Cay chiing loai phat sinh cua chung Ll.l co quan he hp hang gan dua vao trinh tuADNr 16S ,.x 3.3.2 Anh hudng cac dieu kien nuoi cay den kha nang sinh axit va khang sinh 3.3.2.1 Lua chgn mdi tru&ng nuoi cay thich hgp Cac chiing vi khuan lactic dugc nuoi cay tTnh tren loai moi trudng khac d 30^C, pH trung tinh Sau 48 gid, xac djnh kha nang sinh trudng, djnh lugng axit sau nuoi cay theo phuong phap chuan dp Therner Xir ly djch sau nudi cay de xac djnh boat tinh bacterioxin Ket qua dugc trinh bay bang 23 Bang 23 Kha nang hinh axit va bacterioxin tren cac moi truong khac • Chiing L40 Ll.l Moi truong pH cuoi So lu-ong te bao (xlO'CFU/ml) Ham lirong axit (mg/l) - — • ( Hoat tinh kliang khuan (D-d, mm V paraltaemolyticus E coli P mirabil MRS 3,7 132 36 26 22 19 MT5 3,2 83 26 13 12 KT-A 4,2 69 17 11 MRS 3,7 97 32,1 17,5 17 15 MT5 3,3 72 22,3 14 11 10 KT-A 4,3 58 13,56 10,3 ' Tir ket qua tren chiing tdi chpn moi trudng MRS de nuoi cay vi khuan L40, Ll.l cho cac nghien ciru tiep theo 44 Chung L40 va Ll.l dugc nudi cay tinh mdi trudng MRS d pH ban dau thay ddi tir din 9, nhiet nudi cay tri d 30^C Sau 48 gid xac djnh kha nang sinh trudng, boat tinh bacterioxin va kha nang sinh axit cua chiing Bang 24a Anh huong cua pH tdi su hinh axit va bacterioxin ciia chiing L40 pH ban dau pH sau 3,7 So lu-ffng te bao (xlO^CFU/ml) 97 3,6 113 34 3,8 120 3,7 Hoat tinh khang sinh (D-d, mm) Ham luong axit (mg/l) 32 K parahaemolyticus 21 E.coli P mirabilis 19 14 27 18 15 33 28 23 21 115 31 25 18 12 4,5 65 28 16 15 11 5,7 14 23 11 Chung L40 sinh trudng va tdng hgp bacterioxin tot nhat d pH Kha nang hinh axit ciia chiing vi khuan kha cao, tang dan va dat cue dai d pH img vdi pH sau nudi cay la 3,64 va giam dan den pH Bang 24b Anh huong ciia pH toi su hinh axit va bacterioxin ciia chiing Ll.l Hoat tinh khang khuan (D-d, mm) pH ban dau pH sau So iiroTig te bao (xlO^CFU/ml) Ham luong axit (mg/l) 3,7 65 31 3,6 73 32 25 18 15 3,8 89 33 27 20 15 3,7 85 31 22 17 12 4,4 65 29 17 14 10 5,6 14 22 V parhaemolyticus 19 E.coli 17,5 P mirabilis 13 pH ban dau co anh hudng d6n kha nang sinh axit cua Ll.l Gia tri pH nuoi cay ban dau tir d6n thich hgp vdi sinh tdng hgp chat khang khuan Kha nang hinh axit cua chiing vi khuan kha cao, tang dan va dat cue dai d pH img vdi pH sau nuoi c^y la 3,84 va giam dan den pH 3.3,2.3 Anh hu&ng cua nhiet dp Nhiet nudi c^y dugc nghien ciru d nhirng gia trj khac tir 20 den 45^^C, ~ " Kk qua thu dugc d bang 25 45 den kha nang sinh axit va bacterioxin cua L40 Nhi^t ("C) 20 So lu-ong te bao (xlO'CFU/m 1) 36 Ham lirong axit (mg/l) 25 y parahaemolyticus 12 25 93 27 30 116 35 Hoat tinh khang sinh (D-d, mm) E.coli P mirabilis 10 26 20 17 33 29 25 24 122 31 40 115 29 28 23 26 15 23 45 71 17 15 11 ,« ^A Bang 25b Anh huong ciia nhiet den kha nang sinh axit va bacterioxin cua Ll.l Hoat tinh khang khuan (D-d, mm) So luong te bao (xlO^CFU/ml) Ham lu-ong axit (mg/l) 20 36 22 25 82 25 30 96 35 Nhiet P mirabilis 21 10 12 29 25 13 13 99 32 27 14 13 40 95 27 19 12 45 71 15 13 ("c) • V parahaemolyticus 10 E.coli K6t qua cho thay: nhiet dp thich hgp doi vdi sinh tong hgp axit la khoang 30 C Trong dd hgat tinh khang khuan cung dao dong gidi han nhiet dp 25-40^C va cao nhat d khoang 30^C Day cung la nhiet dp phii hgp vdi nhiet dp ciia ao nuoi d ca ba mi^n Bac, Trung, Nam nude ta Kha nang sinh trudng ciia hai chiing tot nhat khoang 30-40*^C Tuy nhien, d nhiet dp 25°C hai chiing van sinh trudng kha t6t 3.3.2,4, Anh hu&ng cua th&igian nudi cay Chiing L40 va Ll.l dugc nudi c^y tren mdi trudng MRS ddieu kien toi uu Sau m6i 12 gid, xac djnh kha nang sinh trudng, ham lugng axit va boat tinh bacterioxin (Bang 26) 46 n den kha nang sinh bacterioxin va axit ciia L40 Thdi gian (gifr) So lu-ong te bao (xlO^*CFU/ml) H^m lirong axit (mg/l) 12 26 24 Hoat tinh kh^ng sinh (D-d, mm) V parahaemolyticus E.coli P mirabilis 17 - - - 53 20 12 15 36 89 23 20 16 12 48 134 35 32 27 25 60 147 31 31 26 26 72 139 29 29 19 14 Bang 26b Anh huong ciia thoi gian den kha nang sinh bacterioxin va axit ciia Ll.l Hoat tinh khang khuan (D-d, mm) Thoi gian So lu'ong te bao (xlO^CFU/ml) H^m lu-ong axit (mg/l) 12 26 12 24 53 17 36 89 48 V parahaemolyticus P mirabilis 13 11 21 21 15 12 100 31 25 17 13 60 107 29 26 16 15 72 97 23 23 14 11 E.coli Ham lugng axit tang theo thdi gian, dat cue dai d khoang 48 gid va giam dan Kha nang sinh trudng va sinh bacterioxin khang vi khuan gay benh tot nhat khoang thdi gian nudi cay tir 48 din 60 gid Sau 60 gid, kha nang sinh trudng bat dau giam Vay, thdi gian thich hgp nhat de len men thu sinh khoi, axit hoac khang sinh ciia chiing L40 va L1.1 la tu khoang 48-60 gid 3,3.2,5, Anh huong cua nong dp mudi Do phan Idn tdm dugc nudi nude bien hoac nude Ig nen cac vi khuan bo sung vao ao nudi phai co kha nang chju muoi Chiing L40 dugc nuoi cay moi trudng MRS d cac ndng mu6i khac tir den 12% Sau 48 gid, xac djnh kha nang sinh trudng va boat tinh khang sinh cua chung nay, ket qua d bang 27 47 muoi den kha nang sinh trudng ciia chung L40 Hoat tinh khang sinh (D-d, mm) (%) So lu-ong te bao (CFU/ml) Ham lu-ong axit (mg/l) 181X10^ 28 V parhemolyticus 31 60 X 10^ 27.5 21 X lO*" Nong dp mu6i E.coli P mirabilis 25 27 29 26 24 19 19 12 14 X 10^ 5 + - 10 52 X 10-^ - + - 12 15 X 10^ - - - +: boat tinh yeu -: khong cd boat tinh Nhan thay, cac chung co kha nang sinh trudng tdt nhat d nong muoi tir 2-4% nam pham vi nong dp mudi cho phep cua cac ao nuoi (2-5%) Tuy nhien, ndng dp muoi cao hon 6% thi kich thudc khuan lac nho, tren 8% vi khuan khdng kha nang sinh axit va boat tinh bacterioxin thi hau nhu khdng cd Bang 27b Anh huong cua nong muoi den kha nang sinh trudng ciia chung Ll.l t Nong So luong te muoi bao (%) (CFU/ml) Hoat tinh khang khuan (D-d, mm) P mirabilis E.coli Ham lu-ong axit (mg/l) parahaemolyticus 25,3 X 10^ 32 22,1 15,2 13 12,1 X 10' 29 19 11 11 3,2 X 10" 16 11 8 1,6 X 10' + - 10 0,32 X 10' - - - 12 - - - - +: hoat tinh yeu; -: khong co boat tinh 3.4 NGHIEN CUU TAO CHE PHAM 3.4.1 Tinh doi khang cua cac chiing Cac chiing du dinh chpn lam ch8 pham dugc nuoi cay lac tren mdi trudng thich hgp d^ thu djch nudi cd chira khang sinh Lan lugt thir kha nang doi khang theo phuong phap khuyech tan Kk qua cho thay, cac chung khdng doi khang lan manh dd thuan Igi d^ nuoi cky hon hgp hoac trpn mot che pham 48 khang ciia cac chung nghien cuu Chung V20 V20 Vll V75 L40 Ll.l - - - - - + - + - Vll - V75 - - L40 - + + Ll.l - - - ^ ^ ^ ^ ^ ^ ^ ^ + + ^ ^ ^ ^ (-) khong doi khang (+): Co uc ch6 khong dang kk Sd lieu tren bang 28 cho thay, chiing V75 khdng cd kha nang kim ham vi khuan V20 va V l l , song cd kim ham khdng dang ke chiing vi khuan lactic L40, chiing Ll.l va L40 CO ire che nhung vong ire che md va dudng kinh nhd ( P Qui •5 X • • V v p v.»Tha- S.^rt., ^^ ~ -"^ ^;^;^ / ^ -•J ;#!; - /• — *\-r(v>r'tA Dong Rhuong • V • Tnudn , o >;Mh.6n^Ha.BangV ,V;-" ,Ar^^»i\ ^5\ \ -Jv-*- r ;Thaoh' •;• 03nq Hoo NqhfX^ > ^ „ ^ , _ J NUipOl'-'il.nhTan ' / u CAT \ Tan • „ Thanh KlENlTHOY \ ._ ^ I \ ' —^Son ' ^ , • Hop Dac V ' * ^ ^ AaTh^o ^ft^^^ ^^^r,^ ^ Ngu 'TJn Phong-, V N^c,^ ,.\ >->43u«^^- > b £ » n ; Doan ; "^ N ^ , , > ^ ^ ' ^ - > N^C \ ' ^- ^ - ^ - gQ^ s^'^enTnarg W / ^ - ' - - HungTh^ng "'^i^^ln'o' • H a V ^ - - N^San '3arg La ^^.r'^, v>.i Qu*n/Uuc DO SON ' P ^'nr^ 8>iND40 D SON * f \ y^n^sr " , ' ^on D^u Hmh 12 Ban d6 cac khu nuoi trdng diuy san Hai Phong Chuan bf ao tru&c nuoi tom 3.5.2.1 Ve sinh ao nudi Bat dau vy nudi mdi nude ao dugc thao can, nao vet, rira sach day ao (co the diing vdi bom xa nude, rua that sach Idp miin ba huu co ling dpng d day ao) Sau dd tien hflnfi rin /4^ii A/Ai ffor» r«5+ AA^Trt/^+^-^0 cdng thuc sau: 57 Lugng voi (kg/ha) 5,1-5,5 800-1000 5,6-6,0 500-800 6,1-6,5 200-500 6,6-7,0 100-200 Giii: ao kho vong - Lay nude da xu ly lang Ipc tir ao chira vao ao nuoi qua ludi Ipc co kich thudc mat ludi 2° = 5mm, giir d mire nude ban dku khoang 0,5 - 0,6m Ngam - r5i thao nude 3.5.2.2 Xu ly che phdm vi sinh vat De dam bao khu he VSV co ich ao phat trien va ton tai n^n day ao chiing toi bon vdi lieu lugng 15kg/ha Hoa che pham vao 100 lit nude sach, khuay deu sau tat deu tren nen day ao, diing cao cho che pham trpn vao nen day mot Idp khoang lOcm, phoi - cho VSV phat trien 3.5.2.3 Xu ly vd cdp nu&c vao ao nudi + Xir ly nude de cap vao ao nuoi: Trong thdi gian chuan bj ao nuoi va trudc tha tom giong phai lay nude vao ao chira lang de xir ly bang chlorin vdi nong dp 1530kg/1000m^ nude 24 gid hoac formol 30kg/1000m^ nude roi cap vao ao nuoi + Diet ca tap: Sau xir ly chlorin tien hanh diet ca tap bang Saponin li^u lugng 15kg/1000m^ nude Saponin duoc ngam vao nude 12 -24 gid sau mdi tat xu6ng ao, chay quat 30 phut cho thudc trpn deu vao nude va ngam, sau cir quat nude gid/ + Gay mau nude: Diing urea 20kg/ha, lan lOkg/ha, chia lam lan bon lien tiSp Cung cap them gidng tao N I U ao kho gay mau nude thi chiing ta ii cam ket hgp vdi 1% chk phkm vi sinh dang bpt va ri mat 2%, nude mam 2% bo sung nude dk dat dp am 45-50% li 48 gid sau bon xudng ao vdi lieu lugng 20kg/ha + Quan ly mdi trudng nude ao nudi: Sau ki6m tra cac chi tieu mdi trudng dat yeu cau mdi tiln hanh tha gidng tdm Dinh ky kilm tra cac chi tieu sau: Oxy boa tan, dp man, dp kilm, dp trong, NO2, NH3, H.S, pH, COD, BOD5 58 /SV: Cir theo chu ky 10 sir dung chi phim sinh hpc vdi lilu lugng: Thang thu nhit 2,5kgAia/ian Thang thir hai 4kg^a/ian Thang thir3trdlen6-7kg/ha/ian - X 3.5.3 Cac chi so thuy ly, thuy hoa nu-dc ao nuoi torn Cac chi sd thiiy ly, thuy boa nude ao nudi tdm duoc theo ddi hang tuan va kit qua dugc trinh bay d bang 41 Kit qua trinh bay d bang 41 cho thay: - Ao sd va sd bd sung chi phim vi sinh thi biin ddng cac chi sd mdi tnrdng nude sau thang nudi dIu dn djnh va nam gidi han cho phep - Nhiet dp d tat ca cac ao thi nghiem va ddi chimg tuong ddi ddng dIu, dao ddng tir 28-33^C - pH d ao ddi chimg len cao hon cac ao TN, cd din 8,8 nen da cd anh hudng nhat djnh tdi tdm nudi, cdn cac ao TN chi dao ddng tir 7,5- 8,5 Bang 4L Cac chi so ly hoa nirdc ao nuoi tom Chi so t^C pH Do man (S%o) Do (cm) TB Min Max TB Min Max TB Min Max Min Max Dp sau (cm) H2S (mg/i) TB TB TB TB COD (mg/l) TB BOD5 (mg/i) Do cumg TS/ CaC03(mg/i) TB TB DO (mg02/l) NO2 (mg/l) NH4(mg/i) Aol 30 28 32 8,0 7,5 8,5 20 10 25 30 45 130 0,12 1,86 0,05 12,5 Ao2 30 28 32 8,0 7,4 8,8 20 11 25 30 50 120 0,16 2,97 0,27 Ao3 31 29 33 9,1 17,3 14,3 8,1 7,5 8,7 20 12 25 30 50 120 5,5 0,13 2,01 0,13 15,6 9,8 125 136 132 Chii thich: Min: Tdi thilu, TB: Trung binh, Max: Tdi da 59 I tir 10-25%o, nen rat phii hgp cho tdm nudi Nam thdi tiet khac nghiet, it mua nen ndng dp mudi it dao ddng, cac nam trudc trdi mua nhilu thi cd thdi diem dp man xudng 6-7%o hoac cd trdi ban ban thi dp man len cao tren 25%o - Ham lugng dxy la ylu td kha quan trpng bilu thj chat lugng mdi trudng nude Cac qua trinh dxy boa sinh hpc va boa hpc xay cang manh se can mot lugng Idn dxy hoa tan nude din din ham lugng dxy hoa tan d dd giam di Nhu vay, mot ao nudi lugng dxy hoa tan nude thap hay cao se la chi sd gian tilp bilu thj lugng vat chit huu CO dl bi phan buy sinh hpc d nhilu hay it, qua dd cd thi thay ao dd bj d nhiem d mire dp cao hay thap - Ndng dp dxy hda tan d cac ao sir dung chi phdm sinh hpc thudng dn djnh d mire binh thudng, dd d ao ddi chimg cd sir dao ddng khdng dIu Dilu chimg td viec sir dung che pham sinh hpc da cd tac dung lam giam cac phkn chat thai him co nhiem va lam tang ham lugng dxy hda tan ao nudi tdm - Ham lugng NO2 cac ao thi nghiem trung binh la 0,12 va 0,l3mg/l cdn d ao ddi chimg trung binh la 0,16mg/l Ham lugng NH4 va H2S d cac ao thi nghiem deu d mire dp cho phep, dd a ao ddi chimg ndng dp NH4 va H2S trung binh thudng cao ban cac ao thi nghiem - Nhu ciu vl dxy hda hpc (COD) d ao sd I la 12,5mg/l, d ao sd la 17,3mg/l va d ao sd la I5,6mg/I - Nhu cau dxy sinh hda (BOD5) trung binh d ao thi nghiem deu nam gidi ban cho phep, BOD5 tai ao ddi chimg vugt qua pham vi cho phep trung binh dat 14,5mg/l Gidi ban tieu chuan ddi vdi nude phuc vu nudi trdng thiiy san theo tieu chuan Viet Nam la dudi lOmg/1 Nhin chung cac chi sd NH4, NO2, H2S, COD, va BOD5 d cac ao TN cd bd sung chi pham sinh hpc ludn thlp hon ao ddi chimg va nam pham vi cho phep cua mot ao nudi trdng thuy san Nhu vay, viec sir dung chi pham sinh hpc vao nudi trdng thuy san la mot hudng dung dan chuong trinh nudi tdm sach khdng diing hoa chat va chat khang sinh 3.5.4 Bien dpng cac chi so ly, hoa cua bim day ao nudi tom Biin day ao nudi tdm dugc lay mau d thdi diem dau vu va cudi vu, mau dugc bao quan lanh din di phan tich Cac so lieu d bang 29 cho thay tir thang nudi dau tien cho din cudi vu, biin ddng N va P tdng sd khdng nhieu, chi sd OC thay ddi nhilu d ao ddi - -' - • " • Jd d ao TN chi 8.0 va 8,5% Dac biet ham lugng H2S 60 on d ao TN va TN tuong img la 407mg/kg va 432mg/kg d thdi diem cudi vu Sau thu hoach, quan sat mau sic bun d cac ao nudi chiing tdi nhan ihky: d ao ddi chimg biin cd mau den va thdi, cdn cac ao TN xu ly chi pham sinh hpc thi biin cd mau xam hoac sang va khdng thay cd mui thdi ma cd miii biin binh thudng Nhu vay, su dung chi pham sinh hpc da tang cudng sir phan buy chat hiru co va giam thieu H2S nen day ciia ao nudi la rat cao Bang 42 Bien dgng cac chi so ly hoa ciia bun day ao tir thang nuoi den thu hoach Chii so S N (%) Z P(%) OC (%) H2S (mg/kg) AolTN Ao2DC Ao3TN Dau vu 0,091 0,073 0,086 Cuoi vu 0,089 0,098 0,095 Dau vu 0,071 0,073 0,069 Cuoi vu 0,086 0,136 0,141 Dau vu 3,3 3,5 3,4 Cuoi vu 8,0 10 8,6 Dau vu 197 186 177 Cuoi vu 407 730 432 3.5.5 Bien dong cac chi so thuy sinh vat cac ao nuoi torn 3,5,5,1 Bien dpng vi sinh vat hieu hoai sinh tong sd vd Vibrio Sd lugng vi sinh vat hieu hoai sinh tdng sd va Vibrio dugc trinh bay d bang 30 Vi sinh vat bilu tdng sd d cac ao TN cd bd sung chi pham vi sinh tang manh tir 2,5 lO'* CFU/ml din 3,29.10^ CFU/ml vi qiia trinh nudi chung ta thudng xuyen bd sung dinh ky chi pham vi sinh va cac thang cudi lugng thirc an thira va phan tdm cang nhieu nen sd lugng VSV cang tang manh, cdn d ao ddi chimg thi tang khdng dang ke Bang 43 Bien dong vi sinh vat tong so va Vibrio Vi sinh vat • X i' Thang nuoi AolTN A o l DC Ao3TN 2,5.10' 2,7.10' 3,0.10' 7.10^ 10^ 5.10^ 9,1 10' 1,3.10' 2,6.10' 3,29 10^ 3,67.10' 1,65.10* 1,37.10^ 1,43.10' 1,76.10^ 2,05.10^ 4,15.10- 2,37.10- 2,12.10^ 3,63.10^ 3,01.10^ 2,02.10^ 7,38.10^ 2,89.10^ tong so I VSV hilu (CFU/ml) S Vibrio (CFU/ml) 61 > sung che pham vi sinh dao ddng nhe tir 1,37.10^ CFU/ml d thang nudi thir len 3,01.10^ CFU/ml d thang nudi thir va giam xudng din 2,02.10^ CFU/ml d cudi vu Dilu chimg td cac chung vi sinh vat cd ich chi phlm da canh tranh thirc an va sinh cac chat khang sinh ire chi vi khukn Vibrio gay benh cho tdm CJ ao ddi chung khdng bd sung chi phkm thi sd lugng vi khuan Vibrio tang nhanh theo thdi gian nudi, sd lugng tang tir 1,43.10^ CFU/ml len din 7,38.10^CFU/ml Kit qua sd lugng Vibrio tang rat cao d ao ddi chimg va thap d cac ao thi nghiem, dilu cho thSy cac chiing vi sinh vat hiru hieu chi pham cd kha nang uc chi manh vi khuan gay benh cho tdm 3.5.5.2 Bien dpng so lugng thuc vgtphii du cdc ao nuoi Thuc vat phii du da dugc thu tir cac ao thi nghiem va ddi chumg theo djnh ky mdi thang mot lan, ket qua dugc trinh bay d bang 44 Bang 44 Bien dQng so lirgng thuc vat phu du cac ao nuoi torn Loai tao (xlO^ tb/l) Tao luc Chlorella AolTN Ao2DC Ao3TN Min 0,735 1,24 1,18 Max 14,2 6,12 13,5 Min 2,13 0,21 2,76 Max 5,76 0,35 6,01 Min 4,78 0,23 4,15 Max 9,56 0,37 8,95 Min 1,26 0,012 2,14 Nitzchia Max 5,87 0,15 6,18 Vi khuan lam Cyanobacter Min 0,036 0,97 0,089 Max 0,046 4,36 0,092 Min 0,011 1,02 0,018 Max 0,017 6,21 0,127 Min 8,952 3,682 10,337 Max 35,453 17,56 34,859 Tao khue Skeletonema Chaetoceros Tao giap Ceratium Tdng T Chu thich: Min: Tdi thieu, Max: Tdi da Ket qua bang 44 cho thay: - Tao luc chu ylu la chi Chlorella phat triln manh d ao sd va ao sd 3, mat dp dat 0,735.10^ -14,2.10^ tb/l Trong khi, d ao ddi chimg sd lugng tl bao thap hon nhieu so vdi cacao thi nghiem 62 h phan loai va phat trien manh nhat d cac ao thi nghi?m Chi Skeletonema d ao thi nghiem sd lugng la 6,01 10^ tb/I nhung ngugc lai chiing phat triln ylu d ao ddi chung va chi dat 0,35 10^ tb/l Chi Chaetoceros va chi Nitzchia d cac ao thi nghiem cung dat sd lugng cao hon ban so vdi ao ddi chimg Cac loai tao khue rSt cd ich cho tdm nudi, la thirc an tdt cho tdm nhirng thang dku mdi tha - Ngugc lai vdi cac loai tao khac, vi khuin lam phat triln manh d ao ddi chimg, cdn a cac ao thi nghiem thi lai phat trien ylu - Tao giap phat triln manh d ao ddi chimg, dat 6,21 10^ tb/I nhung lai phat triln ylu dcac ao thi nghiem chi dat tir 0,017.10^ - 0,127.10^ tb/l Nhu vay, d ao ddi chung, tao lam va tao giap phat triln rat manh, dd d cac ao thi nghiem chiing phat trien rat yeu Dac biet tao khue va tao luc phat triln rat tdt d cac ao thi nghiem (hai loai tao rat cd Igi cho tdm nudi d hai thang dau) va phat triln kem d ao ddi chimg Ket qua cho thay chi phkm vi sinh EMUNIV-S da cd sir kich thich sir phat trien cua tao khue va tao luc, ban che sir phat trien ciia tao lam va tao giap 3,5.5.3 Bien dpng sd lugng dpng vat phii du ao nudi tdm Ddng vat phii du la co sd thirc an cua thiiy vuc tiep sau nhdm thuc vat ndi Thanh phan va sd lugng ddng vat phii du cac ao thi nghiem va ao ddi chimg dugc theo ddi hang thang, cac sd lieu dugc xir ly thdng ke va trinh bay d bang 45 Chiing tdi nhan thay: - Nhdm Chan mai cheo (Copepoda) cd mat dp dao ddng tir 215 den 587 conlm^ d cac ao thi nghiem, ao ddi chimg dat 198 den 602 con/m\ - Nhdm Rau nganh (Cladocera) cd mat dp dao ddng tir 35 den 127 con/m\ d cac ao thi nghiem, d ao ddi chimg la 41 den 103 con/m^ - Nhdm Triing banh xe (Rotatoria) d cudi vu tang cao gap bdn lan so vdi dau vu tai cac ao thi nghiem, cdn d ao ddi chimg thi cudi vu tang gap hon ba so vdi dau vu - Nhdm Giap xac (Crustacea) cd sd lugng rat it, dao ddng tir 19 - 42 con/m^ d tat ca cac ao nudi tdm Trong dd nhdm Than mim va nhdm Vd bao lai xuat hien it va khdng dang kl chi dat tir 1-5 con/m\ Nhdm giun nhilu to dao ddng khdng nhilu d tat ca cac ao nudi tdm tir dkw vu din cudi vu va d mat dp thap tir 2-15 con/m 63 ; dong vat phii du cac ao nuoi torn Chan Rau Triing Giap Than V6 bao Giun mai cheo nganh banh xe xac mem (Ostrac nhieu to (Copepo (Cladoce Rotator (Crusta (Melius oda) (Polych Ao da) ra) ia) ceria) ca) TNI 236-573 35-122 51-203 20-38 1-2 1-4 3-11 347-953 DC 198-602 41-103 47-169 19-42 1-3 1-5 6-15 313-939 TN3 215-587 38-127 43-195 22-39 1-4 2-4 2-8 323-064 Tong (con/m^) acta) Qua ket qua bang tren cho thay ddng vat phii du cac ao nudi cd mat dp tuong ddi dIu d tat ca cac ao nudi tdm Dilu cd the nit ket luan che pham vi sinh khdng cd anh hudng gi tdi sir phat trien ciia ddng vat phii du ao nudi tdm 3.5.6 Danh gia su* tang trudng ciia tom cac ao nuoi Tat ca cac ao dIu tha gidng tdm sii PL 15 khde manh, khdng bi benh, chieu dai tmng binh dat l,5cm/con va cd trpng lugng trung binh tuong duong dat 0,025g/con cung thdi dilm tha tdm gidng Tdm sii gidng dugc lay tir trai gidng Binh Thuan va dugc kilm tra ty le nhilm MBV d ca xuat va nhap vl nudi, Ty le tdm nhiem MBV ia 20%, mat dp nudi la 20 con/ml Tdc dp tang trudng ciia tdm nudi dugc theo ddi cir theo chu ky 15 chilu dai va can trpng lugng tdm, kit qua dugc trinh bay d bang 46 Bang 46 Tdc tang trudng cua tom cac ao nuoi theo thoi gian T h a n g nuoi T h a n g nuoi T h a n g nuoi T h a n g nuoi Wtb Ltb Wtb Ltb Wtb Ltb Wtb Ltb (g) (cm) (g) (cm) (g) (cm) (g) (cm) TNI 0,75 3,60 3,10 6,05 14,7 10,2 21 14,5 DC 0,78 4,01 2,30 4,90 12,5 9,76 18,5 11,0 TN3 0,80 4,31 3,06 5,97 13,9 10,0 20,0 13,7 Ao Chu thich: W.^: Trong luong trung binh; L,b: Chieu dai trung binh Tir kk qua bang 46 cho thdy, a ao d6i chimg torn sinh truong cham hon so voi cac ao CO b6 sung cha ph4m sinh hoc, Khi thu hoach tom co kich thuoc nho va khong d6ng d6u, chiSu dai chi dat 11 cm, lugng trung binh chi dat 18,5g/con cac ao co b6 sung ch^ phim sinh hoc thi kich thuoc torn thu hoach Ion hon, chik dai dat 13,7 - 14,5 cm, ty le torn d6ng d^u cao, lugng trung binh cua tom dat 20-21 g/con 64 tdi nhan thay nhung ao cd bd sung chi phdm sinh hpc thi mau sic nude rat it biin ddi va cd mau xanh vang, cdn d ao ddi chimg thi mau nude cd nhung biin ddng that thudng, it dn dinh horn Hon niia, tdc dp sinh trudng ciia tom nudi da tang ban bom ban so vdi tdm d ao ddi chung, dac biet tdm cd mau sac sang, khdng bi ban va vd tdm chac 3.5.7 Ty 1? song va nang suat dat du-ffc cac ao nuoi Ty le sdng va nang suat tdm dugc tinh toan theo kit qua thu hoach cudi vu bang each dim thi dilm tdm sdng va can trpng lugng, kit qua trinh bay d bang 47 Bang 47 Ty le song va nang suat tom Ao thi Mat Thoi gian nghifm (con/m^) nuoi (ngay) AolTN 20 Ao DC Ao3TN > Trong lu-ong Nang suat (%) torn (g/con) (kg/ha) 120 70 18-25 3.010 20 120 41,2 16-22 1,525 20 120 64 17-24 2,624 Ty le song - Ty le sdng: d ao thi nghiem sd I xir ly che pham EMUNIV-S cd ty le sdng tir 6075%, ao sd cd ty le sdng 60-65% Cd tdm thu hoach tuong ddi ddng deu, tdm khde manh, khdng thay cd dau hieu benh ly cho den thu hoach Trong dd d ao ddi chung khdng sir dung chi pham sinh hpc thi tdm cd ty le sdng thap horn, chi dat 40-42%, dac biet tdm cd mau hai xam, vd tdm bj ban, va tdm bj mdn rau, mdn dudi nhieu, kich cd torn khdng ddng dIu Hien tugng duoc giai thich nhu sau: d ao ddi chimg khdng sir dung chi pham sinh hpc nen ndng dp cac chat dpc ao cao lam cho ty le tdm chet cao ban d cac ao ddi chirng va cang vl cudi vu thi tdm bi dau hieu benh ly cao, tdm khdng khde manh, - Nang suat: Ao thi nghiem sd nang suat thu hoach dat 3,01 tan/ha, ao sd nang suat dat 2,624 tan/ha Cdn ao ddi chimg nang suat chi dat 1,525 tan /ha Kit qua tren cho thdy, viec diing chi phim sinh hpc da cd tac dung lam sach mdi trudng nude ao nudi tdm, giiip tdm sdng khde va tang ty le sdng, vay nang suat thu dugc tang cao ban viec khdng sir dung che pham vi sinh 3.6 SO SANH CHE PHAM EMUNIV-S v O l MOT SO LOAI TREN T H | TRU^CJNG MQT SO AO NUOI THUOC DIA BAN TINH NAM DINH Khi khao sat mdi trudng ao nudi tdm su d Ddng Nam Diln cua cac hd dan, chung tdi nhan thay mire nude ao nudi tdm rat ndng (0,5 - 0,7m), va dp cao v.v nen Idp giam nhdm chat chira nito dang ke (de lam tuomg la 65 ao tdm cd Idp thuc vat day dky dac thi ham lugng DO trung binh dugc la 2,5 mg/l (dao ddng - 3,2 mg/l); Ham lugng B0D5 rit cao, tmng binh 24 mg/l (biin dpng - mg/l) va tri sd COD trung binh 29 mg/l (biin dpng 22 - 35 mg/l) Ngoai ra, biin ao sau vu nudi tdm su dku tien d Ddng nam Diln cung cd IUi^ng chat hihi ca, nita tdng sd va photpho tdng sd cao ban dat day ao dang kl Dieu dang luu y nu:a la mot sd ao nudi tdm su d Ddng Nam Diln chi vu nudi (nam 2004) - da dimg mot lugng trung binh clorin la 32 - 36 ppm, formalin 10 - 11,5 ppm va BKC 1,3-1,5 ml/m3 (formalin va BKC da diing dl xir ly benh tdm va vi tao) Rieng lugng BKC da diing gap ddi lilu hudng din cua cac nha san xukt Vi vay, chiing tdi mudn thu nghiem mot sd chi pham vi sinh vat diing dl xir ly cac ao nudi tdm su cdng nghiep d khu vuc Ddng Nam Diln, huyen NghTa Hung, tinh Nam Djnh hy vpng khac phuc dugc mot sd ban chi da neu 3.6.1 NQI DVNG THlT NGHIEM a, Che pham Sir dung cac chi pham vi sinh vat qua dudng thuc an (Lactovet ciia Cdng ty CP Phat Triln Ndng Thdn; Biof cua Vien Cdng Nghe Sinh Hpc) va xOr ly mdi trudng ao nudi (BRF-2 cua Cdng ty Cdng Nghe Viet-My, Biochie ciia Vien Sinh Hpc va EMUNl-S GS Pham Van Ty nghien ciru) Che pham vi sinh diing cho cac ao dugc tiln hanh theo phuong phap rut the, bao dam tinh ngau nhien va khach quan b, Bo trithd' nghiem Bang 48 So* thir nghiem Chi tieu theo doi Ten sd ao Di^n tich ao, m^ Mat dp tha, PL/m^ Che pham vi sinh Ao nuoi tom su ban tham canh W (Ao doi chimg I) 12 14 3358 17 3010 17 3246 17 3095 17 Biof Biof BRF-2 Biof BE Biof ES Ao nuoi cong nghiep 20 (Ao doi 17 chimg 2) 4200 4800 23 23 ES LT ES Biochie: B:, EMUNI-S: ES : Lactovet: LT Lilu lugng xir dung theo hudng din cua nha san xuat va nha nghien ciru Biof va Lactovet bd sung vao thirc an cho tdm sau tha ; BRF-2, Biochie va EMUNI-S 66 c 30 d ao nudi cdng nghiep va 45 d ao nuoi ban tham canh; khdng xir ly mdi trudng ao nudi tdm su trudc thu hoach 10 Mong mudn mdi trudng mat nude ao nudi tdm su sau thir nghiem cd dp va mau nude dat tieu chuin; lugng oxi hoa tan, dp nhilm bin (BOD5, COD, Nito tdng sd, NO3, NO2 v.v.„) dudi ngudng cho phep c, Cdc thong so khao sat vd phuong phap phan tich Bang 49 Thiet bi va phuotig phap Chi tieu Phep pH PHmetre Dp man Chuan dp hoa hpc CaCOj Chuan dp hoa hpc H2S Chuan chiet va N03-, N02- Chuan quang NH4^ N tdng sd P tdng sd P04^' Bj chii Phoi hgp v&i: - Phong thi nghiem hod phan tich Trwigtdm phan tich - Vien Hod cdng nghiep S04^Fe tdng sd Yt'\ Fe'" Mn, Zn, Cu, Pb Cue phd Mg, AI va km loai nang Phd hap thu nguyen tir Cloramin Chuan dp hoa hpc DO Dien hoa COD Chuan dp BOD5 Sinh boa Biin day va dat day ao nudi cudi vu Chuan dp hoa hpc 67 - Bo mdn Hod phan tich (Khoa Hod Tru&ng Dgi hoc Khoa hoc Tic nhien, Hd Noi) a^ Mhu nu&c ao nuoi torn su Khi dimg che pham vi sinh vat dl xix ly nude ao nudi tdm su ban tham canh da t^o n&i s\r khac rd r^t ve mau nude ao nudi; Che phkm Biochie va EMUNl-S td ban han BRF-2; D ^ bi?t, mau nude ao nudi dimg EMUNI-S cho mau nude d^p (mau non chuoi) va bin mau ban so vdi cac chi pham khac MAU NU3C AO DOI AO DUNG BIOCHIE ^BA a* AO DUNG EMUNIV.S AO DUNG BRF - Hinh 13 Mau nude ao nudi tdm su (ao doi chimg va ao diing BRF-2, Ip rd mau nen day ao) Nhin chung, dp d cac ao nudi (tir 45 din 90 nudi tha) sir dung che pham EMUNI-S d^t kit qua tdt nhat, roi din Biochie (Hinh 14) Nhung quan sat cua chiing tdi ve mau nude va dp d cac ao nudi tdm su rat phii hgp vdi nhan xet cua 68 /i sinh EMUNIV-S da kich thich su phat trien tao khue va tao luc (rdt cd ich cho tdm nudi thang dau nudi tha) va ban chi sir phat trien cua vi khuan lam va tao giap %giitriBQ cuanh6ni 160 HlNH 14 ANH H U N G CUA CHfi PHAM VI SINH D^N Dp TRONG Ci CAC AO NUOI TOM SU TB (45-90 ngiynufti) b, Kiem tra moi tru&ng mgt nu&c ao nuoi vd l&p biin day ao Kit qua phan tich (bang 50) cho thay, viec xir ly cac che pham vi sinh chua lam thay ddi pH, oxy boa tan, BOD5, COD, H2S, NO'", NO^' cd d tang nude mat ciia cac ao nudi tdm su; Dilu cd thi mdi trudng mat nude ao nudi tdm su d khu v\rc chiing tdi thu nghiem chua bi d nhiem tdi muc ma cac che pham vi sinh da sir dung cd the gay nen su thay doi ro ret dugc Tuy nhien xir ly ao nudi bang EMUNl-S da cd xu hudng lam tang ham lugng N tong so, NH4", P04^" ban cac che pham khac- dieu cd thi chi pham EMUNI-S cd cudng dp phan giai chat hiru co va nito manh ban (Hinh 15) Dilm dang lim y nira la hinh nhu chi pham Biochie cd the lam giam lugng chit ran la lung cd nude ao nudi tdm - neu dugc nhu vay thi rat quy 69 ir ly che pham vi sinh den moi trudng mat nu-ffc ao nuoi torn sii Chi tieu theo doi Ten sd ao Che pham sinh hpc Ao nuoi torn su ban tham canh 10 (Ao doi chimg 1) Biof 12 14 Biof BRF-2 Biof BE Biof ES CHI TIEU PHAN TICH pH 9,93 8,40 8,14 8.21 Oxy hoa tan, mg/l 4,40 4,55 4,35 4,60 BOD5, mg/l 3,35 3,10 3,40 3,10 COD, mg/l 5,15 5,20 5,20 5,10 H2S, mg/l 0,05 0,05 0,05 0,05 N tong so, mg/l 0,12 0,13 0,13 0,22 NO3", mg/l 0,50 0,50 0,50 0,50 NH4\ mg/l 0,05 0,05 0,05 0,28 NO2", mg/l 0,02 0,01 0,03 0,03 P04'-, mg/l 0,60 0,58 1,05 1,09 Fe tong so, mg/l 0,28 0,36 0,12 0,24 Al tong so, mg/l 0,70 0,40 0,45 0,80 Chat ran la lung, mg/l 249 224 125 238 Thir nghiem cdn cho thay: d hai ao nudi cdng nghiep (ao 17 va ao 20 DC2) mac dii dIu diing EMUNI-S dl xir ly nude ao, nhung ao sd 17 cd sir dung them Lactovet (bd sung qua thuc an), ao sd 20 (khdng dugc bd sung Lactovet) da cd sir chenh lech dang ke vl ham lugng nita tdng sd, NH4\ photpho tdng sd, Fe tdng sd va Al tdng sd nude ao nudi tdm su Xac nhan cd thi khuyin cao, nhirng ao nudi tdm sii cdng nghiep nen sir dyng ca hai dang chi pham sinh hpc (qua thirc an va xu ly ao nudi) se cho kit qua kha quan ban Nhan xet tren cung phii hgp vdi kit qua thir nghiem cua cac tac gia san pham Biof (Vien cdng nghe sinh hpc), Lactovet (Cdng ty cd phan phat trien cdng nghe ndng thdn), EMAS (Cdng ty cd phan Vi sinh Ung dung) v.v 70 LN B U N D A Y A O N U O I TOM sC (sau 90 nuCi tha) 25 20 15 10 ''^-OM(ts),%xlO P205,% xIOO OCl Emuniv.s •N(ts),% xlOO K2O,%xl0 BRF-2 Lactovet + Emuniv.s 002 Ddng thdi, tien hanh xac dinh mot sd phan boa hpc cua Idp biin day /hinh 15/ (d ao nudi tdm, sau nudi tha 90 ngay) nhan thay, ciing sir dung Lactovet va EMUNI-S thi Idp bun day cd lugng chat him ca tdng sd, nita tdng sd la thap nhat; Xac nhan cung phii hgp vdi ket qua nghien cuu cua nhdm tac gia ciia che pham Biochie va Biof ThOr nghiem cua chung tdi budc dau cho thay, hai che pham (EMUNI-S va Lactovet) cd kha nang phan giai chat him ca va nita kha thich hgp ddi vdi cac ao nudi tdm su khu vuc Rang Ddng, huyen NghTa Hung, tinh Nam Djnh (nam 2005) c, Su thay doi khoi lugng tom nuoi Kilm tra sir thay doi khdi lugng tdm su sau nudi tha 30, 45, 60 va 90 va nlu tinh theo ty le % gia tri binh quan d ao nudi (Hmh 16) cho thay tdc dp tang trudng tuong ddi cua tdm su d ao xir ly EMUNI-S hoac Biochie nudi 30 va 60 cho ket qua kha quan; Tuy nhien, dp tang trudng tuong doi cua tdm sii (tir 30 - 90 nudi) chi cd ao diing EMUNI-S cho kit qua tdt bom ca; chi pham Biochie tuong tu doi chimg va BRF-2 la kem nhat 71 Iciia ao nudi ninn lo AINH rtuuiNG CUA CHE PHAM VI SINH DfisF T A N G T R U N G T U D N G DOI CUA TOM SU 30 nu6i 45 ng^y nu6i DD^ichOng m BRF-2 M Biochie ^Emuniv.5 60 nu6i TB(30-90 nudi) Tuy nhien, chung tdi chua ly giai dugc tai tdm nudi dugc 45 (khi quang chai kilm tra cac ao nudi tdm dang thu nghiem) thi ca ao nudi tdm sii da dugc xir ly cac chi pham sinh hpc (Biochie, EMUNI-S va BRF-2) cd tdc dp tang trudng tuong ddi deu kem ao tdm sii nudi ddi chung? d, Ket luan so bd Mot vai thir nghiem cua chung tdi chua dugc nhac lai va thir nghiem d nhieu noi, vay chi nen coi nhu nhung thdng bao la: * Khi diing chi pham vi sinh dl xir ly ao nudi tdm su d khu vuc Ddng Nam Dien cho mau nude dep, bin m^u hon va cd dp tot hon so vdi ao nudi ddi chimg (tir 45 den 90 nudi tha) * Xir ly ao nudi bang EMUNI-S cd xu hudng lam tang ham lugng N tdng sd, NH4+, P043- ban cac chi pham vi sinh khac ciing thir nghiem * Su dung Lactovet ciing vdi EMUNI-S nhan thay lap biin day ao nudi tdm su cd lugng chat huu co tdng sd, nito tdng sd thap nhat 72 KET LUAN Da su dung chung vi khuin dl san xuat chi phkm sinh hpc xir ly d nhiem moi trudng nude nudi tdm, dd cd chiing vi khuin Bacillus va chiing vi khuin lactic Da xac dinh dugc cac dac diem sinh ly, sinh hda va phan loai cac chung bang phuong phap sinh hgc phan tu Chiing V20 dugc xac dinh la Bacillus licheniformis, cac chiing V l l , V75 la Bacillus subtilis, chiing L40 la Lactobacillus plantarum, chiing Ll.l la Enterococcus lactis Ket qua nghien cim cho thiy cac loai khdng anh hudng xau tdi mdi trudng va ddng vat nudi nen dugc sir dung phd biin cac cbl pham probiotic dimg chan nudi va xir ly d nhiem mdi trudng Da nghien cuu anh hudng cua mot sd yeu td din kha nang sinh trudng, sinh tdng hgp enzym, chat khang sinh, axit va bacterioxin ciia cac chiing sir dung san xuat che pham Da chgn dugc chat mang thich hgp la cam va bpt dau tuong vdi ty le thich hgp va cac dieu kien anh hudng den qua trinh len men xdp tao che pham Thdi gian bao quan che pham tdt nhat la thang ke tir san xuat Da tien hanh thi nghiem ddi vdi xu ly mdi trudng ao nudi tdm va anh hudng ciia che pham ddi vdi sir phat trien cua tdm nudi cdng nghiep: - Trong phdng thi nghiem: Che pham vi sinh cd kha nang lam sach nude va giam thilu d nhiem mdi trudng nude va nen day ao nudi tdm cdng nghiep, giam cac chi tieu nhu chat thai hiru co, cac dpc NH3 va H2S - Khao nghiem ngoai ao nudi: + Viec bd sung chi phim VSV vao mdi trudng ao nudi da lam tang kha nang phan buy va chuyin hda cac hgp chit hiju co nude ao nudi, tang cudng kha nang lam sach va giiJ* dn dinh mdi trudng nude va nen day ao nudi tdm + Sir dung chi phim VSV da lam tang ty le song cua tdm va nang suat tdm thu hoach tang len gip so vdi khdng su dung che pham Da chuyin giao cdng nghe cho Cdng ty Cd 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Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon, Life science building-University of Madras, India 61 Yang R., Jonhson M.C (1992), "Novel method to extract large amounts of bacteriocins from lactic acid bateria" Applied and Environmental Microbiology, 58(10), p.3355-3359 77 Phu luc Thanh phan moi tru-dng dung nghien ciru Moi tru&ng thach dinh dudng NA (Nutrient agar) (gA) Cao thjt 10 Thach 17 Pepton 10 NaCl 0,5 KH2P04 MgS04.7H20 0,5 NaN03 0,5 NaCl FeClj 0,01 glucoza 10 Thach 17 Moi tru&ng MTl Mdi tru&ng MT2- Hutchinson-Clayton KH2P04 CMC 10 NaNOj 2,5 Djch vi luong ml Djch vi lugng gdm: CaCb: 0,1 MgS04.7H20: 0,3 NaCl: 0,1 - Moi tru&ng MT3 Cao thit Nude chiet nam Pepton 10 men 10 Nude chiet nam ml Moi tru&ng MT4 Nude chiet ca men Pepton KH2PO4 Moi tru&ng MT5-Gid Gia 100 Duong kinh 20 KH2PO4 0,5 MgS04.7H20 0,2 20 Tinh bot Moi tru&ng MT6 Bgt dau tuong 78 FeCb: 0,01 Cao men CaCOs MgS04.7H20 0,2 Pepton Mdi tru&ng MRS Glucoza 20 Pepton 10 CaCOj 10 Cao men Cao thjt K2HPO4.3H2O Tween 80 ml CHjCOONa MgS04.7H20 0,58 MnS04 0,28 100 Glucoza 20 - Moi tru&ng KT~A Ca chua Moi tru&ng thach bo sung them 17 g agar 1000 ml 79 PHU LUC Hinh d^ng khuan lac chiing V20 Hinh d^ng khuan I9C chung VII Hinh dang khuan lac va te bao chiing vi khuan chung V75 Hinh d ^ g khuan lac va te bao chung L40 Hinh dang tl bao chung Ll.l KHA NANG PHAN GIAI CAC CHAT HQU CO COA CAC CHONG Kha nang phan giai protein cua chung V20 Kha nang phan giai tinh bgt ciia chung V20 VI SINH VAT Kha nang phan giai lipit ciia chung Vll Kha nang phan giai xenluloza cua chiing V20 Kha nang phan giai chitin ciia chiing V20vaVll MOT SO HINH A N H THUG TE Hinh Gidng tdm su nudi thi nghiem Hinh Thi nghiem b^ kinh Hinh Ao chu&i bj tha tdm gidng NUOI TOM Hinh Mau nude ao ddi chiing FTinh Mau nude ao thi nghiem Hinh C!ho tdm gidng lam quen vdi mdi trudng nude nudi Hinh Tha tdm su gidng vao giai Hinh Ao mdi tha tdm gidng Hinh Chum bi thiic an cho tdm Hinh 10 Ki^m tra su tang trudng cua tdm Hinh 11 Thu hoach tia tdm Hinh 12 Tdm sii thuong phim PHAN PHU LUC 3NG HOA XA HOI CHU NGHIA VIET NAM Doc lap - Tg - Hanh phuc ^ ^ S S t ^ B I E N BAN KIEM TRA TIEN DO THL/C HIEN % - : ^ ^ i ^ ^ E TAI TRONG DIEM/OAC BIET CAP DHQGHN Ten dg tai.- jCm ^(^^^ Cc/cfiurj itn/i noc LQ^o ^Kcirn Masd: ahnfr 'icc^ ({)' Mrry ^^x ,c^ Aoai hrn ^^^^ U^i ri-^oc n^b' ^ 7^ ^^^^" ^'^" Chu nhiem de tai: Co quan chii tri: P6^T^ Phdm Vah Tu Tru&rig Dgi hgc Khoa hpc Tu nhien Thdi gian thirc hien de tai: V «L CH! 5~ - Ngay kiem tra tien do: / / / S.Ut - (z ^^ Bang Anh hudng cua thdi gian den kha nang sinh axit va bacterioxin cua chung L40 Thfri g i a n (giir) S6 Iirgrng te bao (xlO'CFU/ml) H a m lu-Qrng a x i t (mg/l) 12 26 17 - • - 24 53 20 12 15 36 89 23 20 16 12 48 134 35 32 27 25 60 147 31 31 26 26 72 139 29 29 19 14 12 24 36 48 60 Hogt t i n h b a c t e r i o x i n (D-d, m m ) V parahaemolyticus Kcoli P mirabilis 72 ThMgianlgid^ n m v parahaemolyHcus ' - - S4 li/png \i bdo - * - Hdm lirg-ng aiit 1E coli CIZ] P mirabilis Hinh Anh hudng cua thdi gian nuoi cay den kha nang sinh axit va bacterioxin (HTKK- hoat tinh khang khu^n) (trai) va kha nang sinh axit cua chung L40 tren moi trUdng MRS (phai) • Anh hu&ng cda nong dp muoi Do p h i n Idn torn dUdc nuoi nUdc bien hoac nUdc Id nen cac vi khuan bo sung vao ao nuoi phai c6 kha nang chiu mudi Chung L40 dUdc nuoi cay moi trUdng MRS d cac nong dp muoi khac t\i den 12% Sau 48 gid xac dinh kha nang sinh trUdng va hoat tinh bacterioxin cua chung (Bang 6) Nong So lugrng te muoi (%) bao (CFU/ml) Ham luong axit (mg/l) Hoat tinh bacterioxin (D-d,mm) V E coli p muahdts parahaemolMicus 181 X10' 32 31 IS 80 X 10' 29 29 26 24 21 X 10' 16 19 12 14 X 10^ + 10 52 X 10- - + 12 15 X 10- ! ' 27 - i +): boat tinh yei (-): khono co hoat tfnh Nhan thay chiing L40 co kha nang sinh iruang tot nhat a nong Oio muoi lu 2-4'^ n^m pham vi cho phep cua cac ao nuoi Tuy nhien nong muoi cao ho^n 6' < thi kha nang sinh trudng thap bie^u hien d kfch thudc khu^n lac nho Ndng muoi tren 8S \i khuain-khong kha nang sinh axit va hoat tinh bacterioxin hau nhu khong o:o IV KET LUAN Tren co sd nghien cuu ve dac diem hinh thai, sinh ly, hoa sinh va phan tich tiinh tir AND 16S, chiing L40 duoc xac dinh la Lactobacillus pUmtarum Day la chung cd kha nang chiu mudi cao, kha nang sinh axit va bacterioxin cao chdng lai mot sd \ i khuan ga> benh o torn (dac biet la vi khuan gay benh Vibno paialiaemolyiicus) da dupe tkra \ao che pham xir ly benh torn diJng nuoi trdng thuy san Kha nang tong ht^p bacterioxin loi nhai duac nuoi cay tren moi tnrdng MRS d :^v) BJMMARY SELECTING BACTERIA WITH ANTAGONISTIC ACT | VITY AGAINST COMMON PATHOGENS IN SHRIMP I Tran T h i T h a n h Huyen, P h a m Van Ty, Dao Thi Luong j Faculty of biology, Hanoi University of Science Strain L40 isolated from slime of shrimp pond was identified as Lactobacillus ^tarum based on cultural physiological, chemical and analysis of 16S rDNA sequence The krain exhibited strongly antagonistic activity against some pathogenic bacteria, especially ^ibrio parahaemolyticus The opt cultivation condition for growth and bacteriocin production raBonMHS medig at 30°C, pH and 48-60 hours time course JD^ HQC QUOC GIA HA NQI ! JTap chi Khoa hoc / ; Tl: 84-4-7547902 CQNG HOA XA HQI CHU N G H U VIET NAM PQC lap • Tu • Hanh phuc Ha Ngi, Ngay 18 thdng nam 2007 GIAY NHAN BAI Kinh gui Quy tdc gid: •.JmAjiii D.MA Li^hL^:&^ j]ui±iId'^ ^ 'rfH' f L
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