Luận án tiến sỹ nghiên cứu quá trình thủy phân protein cá bằng enzym protease từ b.subtilis s5

D~I HOC Quac GIA TP.HO CHi MINH
TRUONGD~I HOC KHOAHOCTV NHIEN

YO NGOC B{n
NGHIEN CUU QUA TRINH THUY PHAN PROTEIN CA BANG
ENZYME PROTEASE TU B.5UBTILI5 55
Chuyen nganh: Hml sinh
Mil s6: 1.05.10
TOM TAT LU!N ANTIEN si SINHHOC
THANHPHO HO CHI MINH-2004
Cong tr'inh du(jc hoan thanh t~i:
Tntong D<;tihQc Khoa hQc Tif nhien TP. H6 Chi Minh va Truong
D<;tihQcThuy san Nha Trang.
NguOi huang daD khoa hQc:
1. PGS.TS. D6ng Thi Thanh Thu
Truong Dc~lihQc Khoa hQc Tif nhien, D<;ti hQc Qu6c gla
TP.HCM
2. PGS. TS. Tr~n Thi Luye'n
Truong D<;tihQc ThUy san Nha Trang.
Philo bi~n 1: GS.TSKH. Nguy~n Van Thoa
Philo bi~n 2: GS.TSKH. Nguy~n TrQngC~n
Philo bi~n 3: PGS.TSKH. Ngo Ke'Sudng
Lu~n an dii du<;1cbao v~ truoc HQid6ng cha'm lu~n an ca'p Nha
nuoc hQp t<;tiTruong D<;tihQc Khoa hQc Tif nhien -D<;tihQc Qu6c gia
TP. H6 Chi Minh
Vao h6i 9 gio 00 ngay 15 thang 10Dam2004
Co th~ HIDhi~u luij.nan t~i:
Thu vi~n Truong D<;tihQc Khoa hQcTif nhien
- DHQGTP.HCM
ho<\icthu vi~n Khoa hQcT6ng h<;1pTP.HCM.
1

Md DAU
1. Ti'nh dIp thie't ciia lu~n an
Protease 1a mt)t nh6m enzyme du<;lcling d\mg trong nhi~u lInh vtfc
nhu: Nong nghi~p, cong ngM Thtfc phffm Trong thai gian gh day mt)t
s6 nha khoa hQcdii tie'n hanh nghien CUusit d\mg protease trong che'bie'n
Thuy san. Nhung, cac cong trlnh nghien cUusit d\mg protease trong lInh
vtfc nay chua dffydu va v§:ndn du<;lctie'pt\1Cnghien CUu.Bay la lInh vtfc
con mai me nhUngco ra'tnhi~u tri~n vQngd nuac ta.
Nguai ta co th~ thu nMn protease tit nhi~u ngu6n khac nhau nhu tit
thtfc v~t, tit nt)i tl:lngdt)ng v~t hay tit vi sinh v~t. Trong do ngu6n protease
tit vi sinh v~t nha't la tit vi khuffn Bacillus subtilis la huang nghien cUuco
nhi~u tri~n vQngvi B.subtilis co kha nang san xua't protease co hol:lttinh
cao va co nhi~u u'uthe'hon hh.
Trong che' bie'n ThUy san, protease co th~ du<;lcsit d\1ngtrong cong
ngM san xua't nuac mAmd~ rut ngAnthai gian che' bie'n ho~c sit d\1ngd~
san xua't bt)t dl:lmthuy phan tit ca tl:lp.Ca tl:lPla lol:lica kern gia tri kinh
te', tinh rieng tI:liKhanh Hoa, theo s6 li~u th6ng ke cua sd ThUysan, san
lu<;lngkhai thac ca m6i (mt)t lol:lica tl:lp)chie'm 20% -25% t6ng san lU<;lng
khai thac. Ca m6i co co thit khOngsan chAcnen gia tri kinh te'tha'p, trong
chuang trinh "chi biin cac san phdm co gia tri gia tang" cua Bt) ThUy
san co huang nghien cUusit d\1ngh<;lp19cac ngu6n ca tl:lp.Mt)t trong cac
huang "chi biin cac san phllm co gia tri gia tang" tit ca m6i do la sit
d\1ngca m6i d~ san xua't bt)t dl:lmthuy phan. Vi v~y, d~ tai "Nghien CIlu
qua trlnh thuy phtin protein ca bling enzyme protease ta B.subtilis S5" la
cffnthie't trong lInh vtfcche'bie'nThUysan.
2. Ml}cdich ciia lu~n an
Tham do cac di~u ki~n thich h<;lpd~ khai thac va thu nMn protease tit
canh tru'ang nuoi ca'yvi khuffnB.subtilis S5. Sau do sit d\1ngprotease nay
trong san xua't bt)t dl:lmthUyphan tit thit ca m6i thuang (Saurida tumbil)
va thit nghi~m san xua't nuac mAm tit ca com thuang (Stolephorus

commersonii).
2
3. D6i tu(jng va ph~m vi nghien CUll
3.1. D6i tu(jng nghien CUll
B.subtilis 55 la chUngvi khu~n co kha nang san xua'tprotease voi ho1;tt
tinh cao du<Jcphan l~p ttt ttf nhien. Ca m6i thuC1ng(5aurida tumbil) va ca
cdm thuC1ng(5tolephorus commersonii) du<Jcdaub bift t1;tivung bi€n
Khanh Hoa.
3.2. Ph~m vi nghien CUll:g6m 3 phh
1) Nghien cUuthu nMn che' ph~m protease ttt canh truC1ngnuoi ca'y vi
khu~n B.subtilis 55 va xac dinh m(}tso'd~c tinh cua enzyme nay.
2) Nghien cUuung d\mg protease B.subtilis 55 vao san xua't b(}td1;tm
thuy phan ttt ca m6i thuC1ng(5aurida tumbil).
3) Nghien CUu qua trlnh thUy phan ca cdm thuC1ng(5tolephorus
commersonii) b~ng protease B.subtilis 55 va d~ xua't huang ung d\mg
enzyme nay trong che' bie'n nuoc mifm.
4. Phudng phap nghien CUll
Lu~n an sil'd\mg cac phudng phap nghien CUucd ban cua Hoa sinh
hQc co stf h6 tr<Jcua cac 19thuye't loan hQCd€ phat hi~n ra cac tinh cha't
moi, cac m6i quail M gifi'acac d1;tilu<Jngva ki€m chung cac gia thuye't.
5. Cac dong gop mdi v~ mijt khoa hQc
-L~n dh lien nghien cUu m(}tcach hoan chlnh v~ cac d~c tinh cua
protease du<Jcsan xua't ttt chUng B.subtilis 55 phan l~p t1;tiVi~t Nam. Ke't
qua cua lu~n an gap ph~n lam phong phu them nhfi'ng hi€u bie't v~
protease ttt vi khu~n B.subtilis 55. .
- Dffnghien CUllsil'd\mg che' ph~m protease ttt B.subtilis 55 trong thuy
phan thit ca m6i d€ san xua't b(}td1;tmthuy phan. Ke't qua nghien CUucua
lu~n an khi du<Jcap d\mg trong san xua't se gap ph~n vao chudng trlnh
"chi biln cae san phdm co giLltrj gia tang" m(}ttrong b6n chudng trlnh
Ion cua B(}ThUysan.

- Ke'tqua cua lu~n an la nhfi'ngdftn li~u moi v~ vi~c sil'd\mg protease
B.subtilis 55 d€ rut ngifn thC1igian che' bie'n nuoc mifm. Ke't qua nay la cd
sd d€ cac doanh nghi~p san xua't nuoc mifm cai tie'n qui trlnh cong ngh~
rut ngifn thC1igian che' bie'n.
3
6. Ke't dIU cua Iu~n an
Lu~n an g6m 156 trang, 88 trang ph\11\1c,14 trang li~t ke 142 tai Ii~u
tham khao. NQidung lu~n an g6m 3 ch11'dng,trong do co 30 bang va 70
d6 thi, hinh anh.
~ 'A
Chddng 1. TONG QUAN TAl LI~U
1.1. MQt s6 nghien cUu v~ protease
Protease la cac enzyme xuc lac s\f tMy phan lien ke't peptid (- CO -
NH -). Trong cac protease thi nhom protease lieu boa d11'c,1cnghien CUll
sdm va nhi€u hdn ca. Tit nam 1950 trd l<;tiday tren the' gWi co hang lo<;tt
protease dQng v~t, th\fc v~t va vi sinh v~t d11'c,1ctach chie't nghien CUll.
Trong thai gian g~n day cac nha khoa hQctren the' gidi t~p trung nghien
CUllv€ cac protease vi sinh v~t va dfi d<;ttd11'c,1cnhi€u thanh t\!Uto 1(1nv€
nnh v\fc nay. Hi~n nay, s6111'c,1ngcac enzyme d11'c,1csan xua't hang nam
tren the' gWi, d cac n11'dcpMt tri~n cM ye'u la cMu Au, My va Nh~t Ban
VaGkhoang 300.000 ta'n vdi doanh thu tit san xua't enzyme 11'dctinh VaG
khoang 500 tri~u USD. Trong do co khoang 600 ta'n protease tinh khie't
d11'c,1csan xua't tit vi sinh v~t baa g6m khoang 500 ta'n tit vi khua'n va 100
ta'n tit na'm m6c. Nhfi'ng n11'dcco cong ngM san xua't va ung d\1ng
protease lien tie'n tren the' gidi la: Dan M<;tch,Nh~t Ban, My, Anh, PMp,
Ha Lan, Trung Qu6c, Duc, Ao. Cac n11'dcnay dfi d~u t11'thich dang cho
cong lac nghien CUll,san xua't va ung d\mg protease tit vi sinh v~t. Chinh
vi the' nhip dQ san xua't enzyme d qui mo cong nghi~p t<;ticac n11'dcpMt
tri~nhang nam tang vao khoangtit 5%+ 15%.Ngaynay ng11'aita co th~
san xua't cac enzyme protease c6 dinh tren cac cha't mang khong tan cho

phep co th~ tai sa d\1ngl<;tienzyme nhi€u Mn. Vi v~y ma vi~c ung d\1ng
protease ngay cang gia tang.
d Vi~t Nam cling co nhi€u cong trinh cong b6 v€ vi~c nghien CUllsa
d\1ngprotease cM ye'u t~p trung vao cae protease th\fc v~t va dQng v~t
con protease tit vi sinh v~t mdi chi d11'c,1cnghien CUlltrong hdn ch\1cnam
trd l<;tiday. Vi v~y dn tie'p t\1Ctang c11'angnghien cUu v€ protease tit vi
sinh v~t noi chung va protease tit vi khua'nnoi rieng.
4
'1.2. Gioi thi~u v~ ntioe miim va phtidng phap su' d\mg protease
trong san xua't ntioe miim
Nuoc miim truy~n th6ng Hike't qua cua qua trlnh thuy phan protein thit
ca duoi tac dQngcua M protease nQitl;litrong di~u ki~n co mu6i theo co
che':
protease protease protease
Protein ~ ~ polypeptid .peptid ~ .axit alliin
Ciing voi qua trlnh thiiy phan protein thit ca con co hang lol;ltqua trlnh
bie'n d6i sinh boa sau sAcdo mc dQngcua nhi~t dQva h~ vi sinh v~t co
trong do ma hlnh thanh mall sAc, huang vi d~c tru'ng cua nuoc mAID.
Trong khoang 24 gia d~u tieD, duoi mc dQngcua M protease nQiti;licua
ca ma chUye'u la cac proteinase serine, proteinase kim 10l;liva proteinase
aspartic mQt ph~n protein thit ca bi thuy phan thanh peptid, polypeptid
boa tan trong nuoc b6i (nuoc ca). Sau do protein thit ca, polypeptid va
cac peptid du<;lcti;lothanh d tren Il;litie'p t1;1cbi thuy phan ml;lnhme bdi
cac proteinase kim 10l;litl;lothanh cac axit alliin t1!do, cac peptid ngiin va
da: co s1!hlnh thanh huang vi nuoc mAID.Tit giai dol;ln3 + 6 thang duoi
tac d1;1ngcua cac protease d~c bi~t Ia cac proteinase serine, cac peptid,
cac protein, polypeptid con Il;litie'p t1;1cbi thUy phan tl;lothanh cac axit
amiD t1!do. Khi do chU<;lpda:chin, nuoc mAmnit ra co mall va mlii vi d~e
tru'ng. Ch!t lu<;lngcua nuoe mAm ph1;1thuQe vao 10l;liea, t)11~mu6i sii'
d1;1ng,pH, nhi~t dQ,phudng phap ehe' bie'n Trongqua trlnh san xu!t ne'u

bie't ph6i h<;lphai boa cae ye'u to'nay thl nuoe mAmse dl;lteh!t lu<;lngcaD.
V~ nguyen tAethl t!t ea cae loai ea bi€n d~u co th€ sii'd1;1ngd€ san
K 'A J
Nh d
~
0
th
~. K
d
'A oS
th
d
A
xuat nuve mam. ltng e e e san xuat u<;lenuve mam dm ngon, Q
dl;lmcaDva mang Il;lihi~u qua kinh te' thl e~n chQnca co thit m~m, ham
lU<;lngprotein caD, de thuy phan, ti;lora so' lu<;lngnuoe miim nhi~u voi
huang vi thdm ngon. Cae 10l;liea thuang dlipg d€ san xu!t nuoe mAmla:
ea com, ea n1;1e,ea trieh, ea li~t, ea bo, ca m6i trong do ehu ye'u la ea
com va ea n1;1e.
Nhln chung cae phuong phap ehe' bie'n nuoe miim truy~n th6ng d nuoc
ta con tho so, thai gian san xu!t dai tit 6
+ 12 thang. D€ nit ngiin thai gian
san xu!t nuoe miim da:co nhi~u cong trlnh nghien CUllnh~m tang euang
5
ho~tdQngciia h~ protease co siintrongca va Mvi sinh v~t tl)'nhien bAng
cach tie'p nhi~t, cho mu6i nhi~u liin, nao daD. Nhu'ng thai gian che' bie'n
nudc mAm rhea cac phuong phap nay vh con dai. Vi v~y da:co mQtso'
cong trinh nghien cUu sit dl,mgprotease b6 sung them tit bell ngoai dS
tang cuang kha nang thuy phan protein ca thanh axit aminoPhuong phap
nay co ttu diSm la thai gian che' bie'n ngAn, luQng mAm c6t nhi~u hon

nhung san phifm co mui vi va mall sAckern nudc mAm san xua't rhea
phuong phap truy~n th6ng. DS khAcphl,lchi~n tuQngnay nguai ta co thS
cho ch~y qua chuQp t6t dS la'y huang. Vi v~y, vi~c nghien cUu san xua't
nudc mAm rhea phuong phap sit dl,lngprotease con dn thie't phai duQc
tie'p tl,lCnghien cUu.
1.3. GiOi thi~o v~ b~t d~m va phu'dng phap su' dvng protease d~
san xoa't b~t d~m thuy phan tit ca
Trong qua trlnh khai thac danh bAt tbUy san d nudc ta, luQng ca t~p
kern gia tri kinh te'thuang chie'm ty tr<;}figIOn.Vi v~y, trong chuang trlnh
"chi biin cac san pMm co gid tri gia tang" cua BQTbUy san co phiin
nghien CUusit dl,lng hQp 19cac ngu6n ca t~p, mQt trong cac huang che'
bie'n do la san xua't bQtd~m tbUyphan. Thl)'ccha't cua qua trinh san xua't
bQt d~m ca b~ng phuong phap sit dl,lngprotease la qua trinh tbUyphan
protein ca t~o ra cac peptid va axit amin dudi tac dQngcua h~ protease
nQit~i va protease b6 sung tit bell ngoai. Trong qua trinh thuy phan thit
ca b~ng protease co nhi~u ye'u to'anh hudng tdi t6c dQthuy phan nhu: ty
l~ enzyme, nhi~t dQ,pH, ty l~ nudc b6 sung Nen yell ch d~t ra la phai
tim ra che'dQ thuy phan thich hQpdS san xua'tra bQtd~m ca d~t yell duo
Chudng 2.VOl TU(1NGvA PHUONGPHAp NGHIEN CUD
2.1. VO'itu'Q'ngnghien CUll
2.1.1. Ngo6n tho nh~n enzyme protease
B.subtilis S5 Ia chung vi khuifn co kha nang san xua't protease vdi ho~t
tinh cao duQc phan l~p tit tl)'nhien va lUll giii' t~i phOng Vi sinh - Vi~n
Sinh hQc Nhi~t ddi TP. H6 Chi Minh. ChUng B.subtilis S5 nay duQc sit
dl,lngdS nghien cUu san xua't protease b~ng phuong phap nuoi ca'y ban
rAn.Sau khi ditng qua trinh nuoi, thu canh trltang chua protease, sa'ykho,
xay min va sit dl,lngdS tach chie't protease.
6
2.1.2. Nguy~n li~u ea d~ san xudt nu'oe miim va bQt d~m
- Ca m6i nguyen li~u: Ca m6i thu'ang (hlnh 2.1) du'<;ledaub bAt t~i

vilng bi~n Khanh Hoa, ea tu'di du'<;leb,taehQntheo lieu ehu[{nTCVN 3250
- 88. Thit ea m6i thu'ang du'<;lesii'd\1nglam nguyen li~u d~ nghien eUusan
xua'tbQtd~m thuy pMn bAngprotease B.subtilis S5.
mnh 2.1. Ca mol thu'CIng(Saurida tumbil)
(Sau day gQitAt"ea m6i thu'ang" la "ea m6i").
- Ca edm nguyen li~u: ea edm thu'ang (hlnh 2.2) lam nguyen li~u san
xua't nu'demAmla lo~i ea n6i co mila V\1tir thang 2 + 5 va tir thang 7 + 10
hang Dam.San lu'<;lngea edm khai iliac (jNha Trang vao khoang 12.000-
15.000 ta'nInam.
II'mh 2.2. Ca cdm thu'CIng(Stolephorus commersonii)
(Sau day gQitAt"ea edm thu'ang" la "ea edm").
2.2. Phu'dng phap nghi~n CUll
-Xae dinh ho~t dQ protease theo phu'drig phap Anson.
- Dinh lu'<;lngprotein boa tan theo phu'dngphap Lowry.
- Xae dinh ham lu'<;lngnu'de, lipid, nitd t6ng so' (NTs),mtd NH3, mtd
axil amiD,tro, theo TCVN 3700, 3703, 3706, 3708, 5105
- 90.
- Xae dinh thanh ph~n va ham lu'<;lngaxil amiDbAngphu'dngphap sAc
ky khi teen may GC-17A SIllMADZU vdi dQnh~y 10-9.
- Xae dinh ham lu'<;lngkim lo~i bAngphu'dng phap quang ph6 ha'p th\1
nguyen tii'.
-Danh gia earn quaDtheo TCVN 3250-88.
- Tinh s~eh enzyme theo phu'dngphap sAcky lQegel sephadex G-75.
7
2.3. Phuong phap xii' Iy s61i~u: Xii Iy s6li~u nghien cUurhea phuong
phap th6ng ke sinh hQc.M6i thi nghi~m d~u tie'n hanh 3 Ian, m6i I&n3
mi1uva ke't qua la trung binh d)ng coa cac I&nthi nghi~m. s6 li~u duQc
xii Iy d~ ve d6 thi tren ph&nm~m Excel vdi h~ s6 tuong quail R ?:0,95.
Chu'o'ng3. KET QUANGHIEN CUU vA THAOLU!N
3.1. Nghien CUllsan xua't protease tit B.subtilis 55

3.1.1. Phuong phap giii'~m thich hQ'p
Thii nghi~m san xua't protease ttt B.subtilis S5 rhea phuong phap nuoi
ea'y ban rAntrong di~u ki~n co thong gio va giG'a"'mrhea 3 phuong phap
nhu san: phuong phap 1: b~ m~t khay nuoi khOng pho vai a"'m;phuong
phap 2: b~ m~t cae khay nuoi duQcphOvai a"'m;phu'ong phap 3: b~ m~t
khay nuoi pho vai a"'mva san 4 giOnuoi phon hoi nude qua may th6i khi
vao phong nuoi d~ t<.10de>a"'m.San 37 gio nuoi, tho canh tru'ong chua
enzyme va xae dinh ho<.1tde>protease. Ke'tqua th~ hi~n d bang 3.1.
Bang 3.1. Anh hudng cua phuong phap giii'~m toi ho~t dQprotease
B.subtilis 55 tho duQ'ctrong canh truong
Phu'ong phap
giG' a"'m
Ho<.1tde>protease
(UI/g canh
tru'ong)
1
20,2
2
21,1
3
28,2
Tr<.1ngthai eoa eanh tru'ong
B~ m~t eanh tru'ong bi thO, nUt,vi
khua"'nmQe trAngphia du'di con d
phia tren thl thong mQc.
Tu'ongtl! nhu'tren
B~ m~t canh tru'ong thOng bi thO,
kh6i canh tru'ong nong, a"'m,m~m,
vi khua"'nmQc trAng cii tren va
du'di.

I I
Ttt ke't qua bang 3.1 cho tha'y phu'ong phap phO vai a"'mtren b~ m~t
canh tru'ong nuoi va phon a"'mla phu'ong phap thich hQp nh~m giG'a"'mcho
canh tru'ong nuoi B.subtilis 55 d~ san xua't protease.
8
3.1.2. Xac dinh kha Dang san xua't protease cua B.subtilis 55 trong
di~u ki~n co thong gio va khOng thong gio
Tie'n hflOh san xua't protease tif B.subtilis S5 theo 2 phlidng phap:
phlidng phap 1: nuoi ca'y trong di~u ki~n khOngthong gio va phlidng phap
2: nuoi cffy trong di~u ki~n co thOng gio. Sau 37 gio, tho canh trliong
chua enzyme va xac dinh ho<:ltdQprotease. Ke't qua th~ hi~n d bang 3.2.
Bang 3.2. Anh hlfdng cua thong gio tOi ho~t dQprotease tho dlf(jc
Ke't qua nghien cUud bang 3.2 cho thffyphlidng phap nuoi ca'yban dn
co thong gio la thich h<;fpcho vi~c san xufft protease tit B.subtilis S5.
3.1.3. Xac dinh thai gian ke't thuc qua trinh nuoi B.subtilis 55
Tie'n hanh san xufft protease tit B.subtilis S5 theo cac di~u ki~n da
chQn, sau 17; 21; 25; 53 gio, tho canh trUong nuoi, sffykho va xac dinh
ho<:ltdQenzyme. Ke'tqua th~ hi~n d bang 3.3.
Bang 3.3. Anh hlfdng cua thai gian de'n ho~t dQprotease tho dlf(jc
Ke't qua nghien cUucho tha'y co th~ difng qua trlnh nuoi d thai di~m
41 gio d~ co th~ tho dli<;fccanh trUongco ho<:ltdQprotease cao.
3.1.4. D~ xua't quy trinh san xua't protease tit B.subtilis 85
Tif cac ke't qua nghien cUu d teen d~ xufft quy trlnh nuoi ban din san
xufftprotease tifB.subtilis S5 nhli sau:
Phlfdng phap Hot dQprotease
Trng thai canh trlfong
(UI/g canh trUong)
1
19,3
Nhit dQcanh trlfong nuoi co th6 leu

de'n 55°C, vi khuffn phat tri6n kern,
canh trlfong roi rc.
2
28,6
Nhit dQcanh trlfong nuoi t6i da leu
Wi 45°C, vi khuffn rnQctriing d. teen
va dlfdi, canh trlfong cleodinh.
Th<'1igian
17 21 25 29 33 37
41
45 49 53
(gi<'1)
Hot d(>
5,0 8,0 12,5 17,5 23,5 31,5
33,2
33,1 32,0 29,5
protease (DUg)
9
Nuoc 80% so voi nguyen lieu
BOt blip + kh6 d~u I,!c + cam
gaD
Khming bon h(ip
Giang B.subtilis 55
E>anhwi
1
ffinh 3.1. Sd d6 quy trlnh san xua't protease tt'iB.subtilis 85
* Thuye"tminh quy trlnh
+ Nhan gi6ng B.subtilis 85: gi6ng B.subtilis 85 phan l~p tu tt! nhien
va du'<;1ebaa quan (j 5°C tren moi tru'onggiG'gi6ng co thanh ph~n: agar:
2%, eao thit: 0,5%, peptone: 0,5%, NaCl: 0,25%.

Chuy~n gi6ng B.subtilis S5 vao moi tru'ong ho<;ttboa co thanh phh:
agar: 2%, eao thit: 0,5%, peptone: 0,5%, NaCl: 0,25%, tinh bQt: 1% va
nu'de ehie't gia d~u xanh (200glHt). Sau khi ho<;ttboa 48 gio, c1y gi6ng
10
sang m6i tnlong nhiln gi6ng e6 thanh phftn: djeh ehie't earn: 20%, djeh
thuy philo kh6 dftu I~e: 10%, NaCl: 0,36%, MgSO4:0,05%, CaCO3: 0,6%,
pH =7. Truac khi effy gi6ng, hffp va khi't trung m6i truong nhiln gi6ng
b~ng hdi nude bao hoa (j ap sufft latm trong thai gian 30 phut. Qua tdnh
nhiln gi6ng tie'n hanh tren may Hievdi t6e dQ200 vong/phut, sau 24 gio
ki€m ITagi6ng b~ng each Iffy gi6ng dang Hie effy tren m6i truong ho~t
hoa sau 24 gio thffy gi6ng mQe t6t, d~u kh6ng e6 khuffn l~e I~ Ia du<;le.
Sau khi nhan gi6ng Iy tam tho B.subtilis 55 dung d€ san xufftenzyme.
+ San xua't protease tit' B.subtilis SS
M6i truong nu6i B.subtilis 55 san xufft protease (152,19kg/me) e6
thanh phftn nhu sau: bQt belpxay mill: 70kg, earn g~o: 30kg, kh6 dh l~e
(45% protein): 50kg, NaCl: 540g, CaCO3: 900g, DAP: 750g, pH
= 7.
Khoang DAP pha trong 80% nude so vdi nguyen li~u, di~u ehinh pH =7,
trQnvdi nguyen li~usau d6 hffp(j 100°C trang 2gio, lam nguQide'n nhi~t
dQ 37°C va effygi6ng theo ty l~ 5% so vdi nguyen li~u, sau d6 philn d~u
ra cae khay m6i khay 12kg.
Trong qua trlnh san xufft b~ m~t cae khay nu6i du<;lephil vai ffm va
sau 4 gio nu6i beltdfiu phon hdi nude d€ t~o dQ ffmeho phong nu6i vao
khoang 90+100%. Nu6i trong di~u ki~n e6 th6ng gi6 vdi m\le dich d€
du<3iCO2,giai nhi~t va eung effpoxy eho vi khuffn.
Sau 41 gi<'1nu6i (j nhi~t dQphong tho eanh tru'<'1ngehua enzyme, sffy
kh6 (j nhi~t dQ50°C, xay mill va d6ng vao trong cae tui PE d€ baa qUaD.
3.2. Nghien cUu tach chie't va mQt s6 d~c tinh cua protease
3.2.1. Xac djnh dung djch chie't rut protease tit'B.subtilis SS
Protease B.subtilis 55 du<;leehie't rut ra kh6i bQt eanh truong ehua

enzyme bhg cae dung djeh: nude efft, dung dieh d~m Britton pH 6, dung
dieh d~m phosphat pH 6 va dung dieh mu6i sinh 1:9.Sau khi ehie't rut ly
tam tho djeh ehie't (DC) enzyme va xae dinh ho~t dQ protease, ke't qua
th€ hi~n (j hinh 3.2.
~
'0 .';:;'
;:,~. 90
" "
:0 ~.
'=' " 60
:-0.<6
;: ~ 30
". ~
0
::r:
11
120
100
%.3
0
.Nudc dlt . f)~m britton GI f)~m phosphat GI Mu6i sinh 19 I Dung djch chi"t enzyme
Hinh 3.2. AOOhuang ena dung dieh ehie't rut de'n ho~t dQprotease
Tli ke't qua hlnh 3.2 cho tha'ynudc ca'tchinh la dung moi thich hQpcho
vi~c chie'trut protease tli B.subtilis S5.
3.2.2. Xae djnh tae nhdn ke't tna protease B.subtilis SS
Tie'n hanh ke't tua protease B.subtilis S5 ra khai DC enzyme b~ng
aceton, ethanol va sulphat amon d cling n6ng de>70%. Sau do ly tam thu
che' ph£m enzyme (CPE), boa tan trong cling me>tth~ tich nudc ca'tva xac
dinh ho~t de>protease. Ke'tqua nghien cUuth~ hi~n d hlnh 3.3.
'0 :::;-120

'" ".
gp~1O0
:0 B' 80
:a
:g'~ 60
~ 0 40
~. ~ 20
0
100 99.9
98
.Ethanol
II Sulphat amon
GIAceton
Tac nhan k"t IDa
Hinh 3.3. AOOhuang ena tae nhdn ke't tna tOiho~t dQprotease CPE
Tli hlnh 3.3 cho tha'y ethanol chinh la tac nhan thich hQpcho vi~c ke't
tua d~ thu protease CPE.
E>~chQn duQc n6ng de>tac nhan ke't tua thich hQp tie'n hanh ke't tua
protease B.subtilis S5 bhg ethanol d cac n6ng de>khac nhau: 60%,
65%, ,80% ke't qua duQCth~ hi~n d hlnh 3.4.
~
~
~
'<O~
'0 '0
'" "
" ",.
~ "
<0'
'0

'i<.
0
:I:
120
100
80
60
40
20
0
100
99,89
93,1
83,5
60 ~ m ~ w
Ndngd~Ethanol(%)
Hinh 3.4. AOOhuang ena n6ng dQethanol de'n ho~t dQprotease CPE
12
Ke't qua a hinh 3.4 cho tha'y ethanol
an6ng dO75% la thich hQpcho
vi~c ke't tua thu protease CPE.
3.2.3. Anh hu'dng cua pH de'n ho~t dQprotease B.subtilis SS
Ke't qua nghien CUuduQcth~ hi~n a hinh 3.5.
L :~L : :
li~ /- ~
0 2 ~ 3 U 4 U 5 U 6 ~ 7 U 8 U 9 ~
I -DC -o-CPE I pH
Hinh 3.5. Anh hu'dng cua pH de'n ho~t dQprotease B.subtilis SS
Ke't qua nghien cUua hinh 3.5 cho tha'ykhoang pH thich hQpcho ho<.tt
dOprotease B.subtilis 55 la 5,5 - 7 va pH t6i thich la 6.

3.2.4. Anh hu'dng cua nhi~t dQde'n ho~t dQprotease B.subtilis SS
3.2.4.1. Xac dinh nhi~t dQthich hf/p cho ho~t dQ enzyme
Ke't qua nghien cUuanh huang cua nhi~t dOde'n ho<.ttdOprotease DC
va CPE duQcth~ hi~n a hlnh 3.6.
~ 150
j
'E~
"" "" 100
g,"g.
D "
13 <6 50
'0- >
~. ~. 0
0
x
Ii g g 0 t'-~
~
. . . . . . . . .
0 30 35
40
45 50 55 60 65
70 75
Nhi~t dQ
-DC -0- CPE
Hinh 3.6. Anh hu'dng cua nhi~t dQ de'n ho~t dQ protease B.subtilis SS
Tit hinh 3.6 cho tha'yca protease DC va CPE d€u co nhi~t dOtmch
hQpla 55°C.
3.2.4.2. Xac dinh dQb~n nhi~t cua protease B.subtilis SS
Xii' 19 nhi~t protease DC va CPE acac nhi~t dO 60°C va 65°C trong
cac khoang thC1igian 0; 5; 10; ;35 phUt.5au do nhanh chong lam l<.tnhva

xac dinh ho<.ttdOprotease. Ke'tqua th~ hi~n a hinh 3.7.
13
:g;g 150
~
",,'0
.§~IOO
'=: .D
:;;.;s 50
'0 >
5; 0
o. ~
:r:~ 0 5 10
-
-0
-
=t
.
0
.
t
.
0
.
'9'
. .
0- -0
~ $
15 20 25 30 35
Thai gian xii' 19 (pMt)
- DC65 CPE65I

I -DC60
-0-CPE60
IDnh 3.7. DQb~n nhi~t cua protease B.subtilis S5
Ke't qua hlnh 3.7 cho tha'yprotease B.subtilis 55 kern b~n voi nhi~t so
voi cac protease tu tht!c v~t va b~n hdn protease tu na'mm6c.
3.2.5. Aoo hudng cua n6ng dQmu6i an t8i ho~t dQprotease
Ke't qua nghien CUllanh hudng cua NaCl toi hol;ltdQ protease CPE
duejcth~ hi~n d hlnh 3.8.
~ 150
:g ,~
g,o~ 100
"0 '"
J:! :<:.
:g- ~ 50
~ 0
'" '"
o.
:r:
17 19 21 23
N6ng dONaCI (%)
IDnh 3.8. Anh hudng cua n6ng dQNaCl t8i ho~t dQprotease CPE
Ke't qua th~ hi~n d hlnh 3.8 cho tha'y cang tang n6ng dQmu6i an b6
sung vao phiin ung enzyme hol;ltdQ protease B.subtilis 55 cang giam.
Nhu' v~y d~ co th~ sa dl;mgmQtcach co hi~u qua protease B.subtilis 55
trong che' bie'n thuy san nen sa dl;mgtrong cac di~u ki~n cang co it mu6i
an cang t6t ho~c ne'u phiii b6 sung them mu6i an vao h6n hejpthuy phan
co protease B.subtilis 55 thl nen cho mu6i nhi~u l~n.
3.2.6. Anh hudng cua cae ion kim lo~i hoa trf 2 va mQt so' cha't d~c
hi~u OOomtm ho~t dQprotease B.subtilis S5
Tie'n hanh nghien CUllanh hu'dngcua cac ion kim 10l;liboa tri 2 va mQt

s6 cha't d~c hi~u nh6m thu'ong co vai tro quan trQng d6i voi hol;ltdQ
protease. Ke'tqua du'ejCth~ hi~n d hlnh 3.9.
0
0
3 5 7 9 11 13 15
14
~ 120
01)
:g <S 100
01) -8 80
c
"0 \0
::: "" 60
~. ~ 40
~. ~ 20
::r::~ 0
~
~
(, ~< x x x x x x x x x x,,>,' '1'
O
t\."-$'
~ -# cP "',;' ~:;: p"i # "lY 0~ 0~ ",-<"'i ~:;: 0"- <i,'Y<' ~'Y o,,>i}~
Cha't them vao ptian ti'ng
Ifmh 3.9. Anh hu'dng cua ion kim lo~i va mQt s6 cha't ~c hi~u
nhom de'n ho~t dQprotease B.subtilis 85
Tu hlnh 3.9 cho tha'y ta't ca cac cha't them vao phan ftng (trir 2-
Mercaptoethanol va L-cystein) d~u lam giam ho;:ttd(>protease CPE va
DC. Cac ion Cuz+,Pbz+,Hgz+,HzOzva EDTA lam giam m;:tnhho;:ttd(>cua
protease CPE va mftc d(>lam giam nhi~u hdn so vdi protease Dc. Trong
khi do 2-Mercaptoethanol va L-cystein l;:ti lam tang r5 r~t ho;:tt d(>

protease CPE nhU'nganh huang ra't it de'n ho;:ttd(>protease DC.
3.2.7. Tach protease CPE qua cQt IQcgel sephadex G-75
Tach protease CPE qua c(>tIQcgel sephadex G-75, c(>t(1O0x2cm),
din b~ng c(>tb~ng d~m Britton pH 6,0; t6c d(>qua c(>t18ml/giCf,m6i phan
do;:tn 3m!. Sau khi qua c(>t thu cac phan do;:tn d~ xac dinh ho;:tt d(>
protease va dinh luQngprotein; ke't qua th~ hi~n a hlnh 3.10.
1.5
II
c ~
c
~ 8'
1
8 "0
"" c
<0- ""
"0 c
OJ).eo 0.5
<= OJ)
'<0 a
z '-'
0
b
q ~ ~ ~ 0/ ~ ~ ~ ~ ~ ~
-0- N6ngdOProtein(mg)
- Ho~tdOenzyme(VI)
~
1.2
<I)~
1 a <=
;., "'.

0.8
i;j .g
<I) <=
0.6 <0- ~
"0 ""
0.4 ';;j. ;::;
o.z ~ 2-
0
Philo do~n
Ifmh 3.10. siic ky d6 protease CPE qua cQt IQcgel sephadex G-75
Ke't qua a hlnh 3.10 cho tha'y sau khi qua cQt IQcgel sephadex G-75
thu duQc4 dinh protein theo thftt11qua c(>tla I, II, III, IV. Trong do chi co
15
2 dinh co ho~t dOprotease cM ye'u 1adinh I va II (ki hi~u la P-I va P-II).
P-II co ho~tdO protease chie'm toi 84,3% ho~t dO len cOt va co dOtinh
s~ch ga'p 15l~n so voi protease DC ban dffu(bang 3.4).
Bang 3.4. Tom tiit qua trinh tinh s~ch protease
Sail khi tach protease CPE qua cOt lQcgel, tie'n hanh thu cac phan
do~n co P-I (phan do~n tit 6 - 12) va P-II (phan do~n tit 13 - 24) d~ tie'p
tl,lCnghien CUllmQts6 tinh cha'tcua cac protease nay.
3.2.8. MQt s6 tInh chift cua cae P.I va P.II
3.2.8.1. Anh htidng cua mQt s6 ion kim lo~i hoa trf 2 va mQt s6 chift
d~c hi~u nhom tOiho~t dQP-I va P-II
Ke't qua nghien CUllth~ hi~n d hinh 3.11.
~ 250
55 200
""
,":::00 150
;g <§
"" -5 100

"
~:g 50
<0'
'"
~
~
';j.
0
:r:
0
L ~< x x x x x x x x x x.>.: 'l-
o
(\,.~
"'- of d' <0;'; ~!i ~~ if <t.P G~ d' q,o'Y ~!i (y {Y~ ~'Y tI<}~
V
Chit'l them vito phao «fig
IDnh 3.11. Anh htidng cua cae ion kim lo~i hoa trf 2 va mQt s6
chift d~c hi~u nhom de'n ho~t dQP-I va P-II
Ke't qua nghien CUllcho tha'y ion Caz+va MgZ+d n6ng dO1O-3Mlam
giam ho~t dOP-I; nh11ngCaz+l~i khOnganh hudng de'n ho~t dOP-II. Mnz+
khong anh hudng toi ho~t dOP-I nh11ngl~i lam tang ho~t dOP-II. Cac ion
Znz+, Cdz+,Baz+,Cuz+,Hgz+,CN-, Fez+, HzOz va EDTA d~u lam giam
Miiu Protein
Ho<).tdQ protease
DQs<).ch
(mg) (ill)
(%)
Ho<).tdQ rieng
(Ifill)
(ill/mg protein)

DC
192,55 8,28
100,00
0,043
1,0
CPE
21,33
6,63
80,07
0,311
7,2
P- I
1,48 0,18 2,17
0,122
2,8
P- II
8,69 5,59 67,51 0,643
15,0
16
ho<:ttdQP-II; trong d6 Hg2+,H2O2va EDTA uc che' m<:tnhme ho<:ttdQP-II
va lam giam tu'dng ung tdi 100%,97,9% va 98,6% ho<:ttdQP-II. D~c tinh
nay kern theo kha nang du'<;1cho<:tthoa du'ditac d\mg cua cac chtt khii'2-
Mercaptoethanol va L-Cystein (2-Mercaptoethanol va L-Cystein lam
tang tdi 200%va 189,7%ho<:ttdQP-II) la OOungtinhchtt d~ctru'ngcua
protease c6 chua nh6m thiol (-SH) trong trung tam ho<:ttdQng cua
enzyme. Ke't qua nay chung to P-II thuQc nh6m proteinase thiol
(proteinase cystein) theo cach phan lo<:ticua Hartley.
D~ tie'p h,lCtlm hi~u v~ d~c tinh cua P-II, till nghi~m tie'nhaOOxii'19P-
II b~ng EDTA vdi n6ng dQtrong h6n h<jpla 6 x 10.3M(j 30°C trong thai
gian 10 phut. Sau khi xii' 19 tai ho<:tt hoa enzyme b~ng: 2-

Mercaptoethanol, Mg2+,Mn2+va h6n h<;1pMg2+va Mn2+.Ke't qua nghien
CUuth~ hi~n (j hlnh 3.12.
tOO
0:0
':8] 75
"
.§ '0
-= '" 50
,; <6
'" >
'OJ.;;; 25
:ij ~
0
DC A B c
D E
M~u thi nghi~m
Hinh 3.12. Anh htfdng cua 2-MercaptoethanoI, Mg2+va Mn2+de'n
sf/.h6i ph\lC ho~t dQP-II (dii bi xii'Iy trtfoc voi EDTA)
DC: mfiu d6i chung. A: mfiu xii'I9 vdi EDTA.
B : mh tai ho<:tth6a vdi 2-Mercaptoethanol
C : mh tai ho<:tthoa vdi Mg2+. D: mfiu llii ho<:tthoa vdi Mn2+.
E : mh tai ho<:tthoa vdi Mg2+va Mn2+.
Ke't qua hlnh 3.12 cho thty sau khi xii'19 vdi EDTA ho<:ttdQcua P-II
giam g~n OOu'hoan toan. Nhu'ngne'u sau d6 l<:tithem 2-Mercaptoethanol
thl ho<:ttdQcua P-II h6i phl,lctdi 75%, con ne'u them Mn2+ho~c h6n h<;1p
Mg2+va Mn2+thl ho<:ttdQenzyme chi h6i phl,lc25% ho<:ttdQban d~u. Ke't
qua tren mQt l~n nua l<:tikh~ng dinh P-II la mQt proteinase thiol. M~t
17
khac ke't qua nay con cho tha'yP-II ding dn ion Mn2+dS h6 tr<jcho ho<;lt
dOngcua mlnh.

3.2.8.2. Anh htidng cua pH va nhi~t dQde'n ho~t dQP-II
Do P-II 1a dinh ho<;ltdOprotease chinh khi tach protease CPE qua cOt
1Qcgel sephadex G-75 nen tie'p tt;lcnghien CUllky hdn v€ protease nay.
Ke't qua nghien CUllcho tha'y P-II co pH thich h<jp6,0 va nhi~t dOthich
h<jp60°c.
3.2.9. Nghien cUu bao qUail protease CPE cua B.subtilis S5
Tie'n hanh nghien CUllbaa qUail protease CPE CJ4°C cho tha'y hoan
roan co thS bao qUail protease CPE cua B.subtilis S5 CJd<;ingdong kho
ho~c boa tan trong dung dich sorbitol co n6ng dOtren 10%. Ke't qua nay
mCJra mOthuang san xua'tprotease B.subtilis S5 duai d<;lngdich long ra't
ti~n l<jicho vi~c ung dt;lngtrong tht!c te'san xua't.
3.3. Nghien cuu qua trinh thuy phdn thjt ca m6i biing protease
B.subtilis S5 va san xuilt bQt d~m
3.3.1. Thanh ph§n boa hQccua ca m6i
Ke't qua xac dinh sd bOthanh phftn kh6i lu<jngcua ca m6i daub bAt
vao thang muCfiCJvung biSn Khanh Hoa dti<jctrlnh bay CJbang 3.5.
Bang 3.5. Thanh ph§n kh6i lti(/ng cua ca m6i (%)
Thit ca fillet
f)~uca
Xudngca Yayvavffy
NOit<;lng
56,23 18,46 13,69 5,64 5,98
Ke't qua phan rich cho tha'y ca m6i co ty l~ ph~n an du<jckha cao
(56,23%), ham lu<jngprotein t6ng s6 cao (19,7%) va ham lu<jnglipid tha'p
(1,4%) Den xe'p vito IO<;lica g~y ra't thich h<jpcho vi~c san xua't bOtd<;lm
va dam bao bOtd<;lmthanh phffm it bi oxy boa bCJilipid. Ngoai ra ke't qua
phan rich thanh ph~n axit amiDcua thit ca m6i con cho tha'y thit ca chua
kha d~y du cac axit amin thay the'va khong thay the'. Vi v~y, ca m6i la
d6i tu<jngdn du<jcnghien CUlldS san xua't thanh bOt d<;lmb6 sung vito
bOtdinh du6ng cho nguCfi.

18
3.3.2. Nghien euu cae di~u ki~n thieh h(jp eho qua trinh thuy phan
thjt ea m6i biing protease B.subtilis 55
3.3.2.1. Anh hu'dng eua ty 1~CPE tOi qua trinh thuy phan
Ke't qua phan rich cho tha'y khi tang t)11~protease CPE b6 sung vao
h6n h<;1pthuy phan thit ca m6i trong khoang 0,1%
- 0,3% thi t6c dQ thuy
phan t'.lOthanh protein boa tan, peptid va nitd axil amiD (N ) tang t)11~
thu~n voi 1U<;1ngenzyme b6 sung them vao h6n h<;1pthuy phan. Nhung
ne'u tang t)11~enzyme b6 sung vao h6n h<;1pthtiy phan 1en0,4% thl t6c dQ
thtiy phan khOng tang d6u nhu trll'ocma ham 1u<;1ngnitd NH3 (NNH3)t'.lO
thanh l'.litang cao. Nhu v~y, t)11~protease CPE thich h<;1pcho qua trinh
thtiy phan thit ca m6i 1a0,3%.
3.3.2.2. Anh hu'dng eua nhi~t dQ !Oi qua trinh thuy phan thjt ea
m6i biing protease B.subtilis 55
Ke't qua nghien CUllthuy phan thit ca m6i b~ng protease CPE C1cac
nhi~t dQ khac nhau cho tha'y khi thtiy phan thit ca m6i b~ng protease
B.subtilis 55 C150°C thl t6c dQ thuy phan nhanh hdn bi~u hi~n 1a ham
1U<;1ngprotein boa tan, ham 1u<;1ngpeptid va ham 1u<;1ngN t'.lOthanh theo
thCfigian thtiy phan cao hdn.
3.3.2.3. Anh hu'dng eua pH !Oiqua trinh thuy phan thjt ea m6i
Nghien CUllthuy phan thit ca m6i b~ng protease B.subtilis 55 C1cac pH
khac nhau cho tha'ypH t1,1'nhien 1athich h<;1pcho qua trlnh thuy phan.
3.3.2.4. Anh hu'dng eua lu'(jng nu'de b6 sung tOi qua trinh thuy phan
thjt ea m6i biing protease B.subtilis 55
Qua ke't qua phan rich qua trlnh thtiy phan thit ca m6i b~ng protease
B.subtilis S5 trong di6u ki~n co b6 sung nuoc voi cae t)11~khac nhau cho
tha'y t)11~nuoc thich h<;1pb6 sung vao h6n h<;1pthtiy phan thit ca m6i b~ng
protease B.subtilis S51A20%.
3.3.2.5. Anh hu'dng eua ty l~ mu6i an tOi qua trlnh thuy phan thjt

ea m6i biing protease B.subtilis 55
Ke't qua nghien CUlleho tha'y b6 sung 3% mu6i an vao qua trlnh thuy
phan thit ca m6i b~ng protease B.subtilis S5 1athich h<;1pcho vi~c uc che'
19
vi sinh v~t gay thoi ma v~n dam baa eho qua trlnh thuy phan di~n ra
m~nh.
3.3.2.6. Anh htfdng eua ty I~ ethanol tOj qua tdnh thuy philo thit
ea m6i biing protease B.subtilis 55
Cae ke't qua phan tich eho tha'yb6 sung 8% ethanol VaGh6n heJpthuy
philo thit ca moi bAngprotease B.subtilis 55 la thleh heJpeho vi~e lie ehe'
vi sinh v~t gay thoi ma vh dam bao eho qua trlnh thuy philo di~n ra
m~nh.
3.3.2.7. Anh htfdng eua ty I~ sorbitol t8i qua tdnh thuy phan thit
ea m6i biing protease B.subtilis 55
Ke't qua thi nghi~m eho tha'y d~ lie ehe' ho~t di)ng eua vi sinh v~t gay
thoi trong qua tdnh thuy philo thit ca m6i b~ng protease B.subtilis 55 co
th~ b6 sung sorbitol vdi ty l~ 4%.
3.3.2.8. Dng d\mg protease B.subtilis 55 d~ san xua't bQt d~m tit ea
m6i
* Xae dinh thOi gian thleh hqp eho vi~e ue ehe'vi sinh v~t gay th6i
dieh thuy philo biing mu6i an, ethanol va sorbitol
Ke't qua cho tha'y doi vdi cae m~u b6 sung mu6i an 3% va b6 sung
sorbitol 4% san 16 giC1thuy philo co hi~n tueJngbi hu hOng bi~u hi~n la
ham lueJngpeptid, Naat~o thanh Mt d~u giam con ham lueJngNNH3t~o
tMnh beltd~u tang cao. VI v~y doi vdi hai m~u nay phiii dung qua trlnh
thuy philo (Jgiai dol,ln16giC1.Con mh b6 sung 8% ethanol thl san 18 giC1
tbUyphilo m~u nay beltd~u co hi~n tueJngbi hu hong. Do v~y, phiii dung
qua trlnh thuy philo (Jgiai dol,lnnay.
* San xua't thii' bQt d~m tit thit ea m6i
Thii'nghi~m thii'san xua't bi)t d~m theo cae thOng sO'tlm dueJe(Jtren.

Sau khi thuy philn co d~e dieh thuy philo bAngphuong phap co quay chao
thong r6i sa'ytho (Jnhi~t di) 70°c. Ke't qua danh gia earn quan va philn
20
tich thanh ph~n hoa hQcco ban cua b9t d:;tmcho tha'ycac m~u thuy phan
d~u co tqng thai dm quail g~n tuong tlfnhau, trong do m~u thuy phan b6
sung 3% mu6i an co m~u sAckern ha'p dh hon so voi hai m~u con h~iva
co vi kha m~n. v~ thanh ph~n boa hQccua b9t d:;tmthl m~u thuy phan b6
sung 4% sorbitol cho bOtd:;tmtho duQcco nhi~u I1udi€m hon nhu co ty l~
ph~n bOt d:;tmboa tan cao bon, ham luQng Naacao hon ding nhu ham
luQngprotein boa tan cao bon. M~t khac khi h:;tchloan gia thanh bQtd:;tm
thuy phan tu ca m6i bing protease B.subtilis S5 con cho tha'y m~u thuy
phan b6 sung 4% sorbitol cho san phfim b9t d:;tmco gia thanh tha'phon d
(30.220dlkg thanh phfim).
Ke't qua phan tich thanh phh khoang cha't, ki€m ITacac chi lieu vi
sinh v~t va thanh ph~n axil amin tlf do (bang 3.6, 3.7, 3.8) cua m~u bOt
d:;tmsan xua't theo phuong phap sU'd\)ngsorbitol d€ ticche'vi sinh v~t gay
th6i cho tha'y b9t d:;tmsan xua't theo phuong phap nay hoan loan d:;ttlieu
chufin vi sinh thlfc phfim va co ham htQng axil amin tlf do cao (toi tren
23,6%).
Bang 3.6. Thanh ph~n khoang cha't cua bQt d~m tit' ca m6i (~g)
Bang 3.7. Ke't qua ki~m nghi~m vi sinh bQt d~m thuy philn tit'ca m6i
Ca2+
Mg2+
Mn2+ Pb2+ CU2+
Hg2+
As
53,0
43,0 6,3
0,12
2,4

Ve't Ve't
SIT
Ten chi lieu xet nghim
Ke't qua
1
T6ng so'vi sinh vt hie'u khf Ig
6,8.102
2 Escherichia coli
Am tfnh
3
Staphylococcus aureus
Am dnh
4
Salmonella Am tfnh
5
Clostridium perfringens
0
21
Bang 3.8. Thanh ph~n axit alliin tll do cua bc}td~m tit ca m6i
3.3.2.9. D~ xua't quy trlnh san xua't bc}td~m thuy philn tit ca m6i
Tit cae ke't qua nghien cU'ud tren cho phep rut ra quy trlnh san xua't
bQt d~m tMy phan tit thit ca m6i thu'C1ng(Saurida tumbil) bhg protease
B.subtilis S5 du'<;1cth€ hit;n tren hlnh 3.13.
STT
Ten axit amin
Ham 1u'<Jng(g/g bQtdm)
1
Alanine
10.241,0
2

Glycine
19.894,0
3
Aminobutyric axIl
1.202,0
4
Valine
11.006,0
5 Leucine
10.951,0
6 Isoleucine
501,0
7 Norleucine
161,0
8 Threonine
7.271,0
9 Serine
6.314,0
10
Aspartic axIl
3.243,0
11 Methionine
3.728,0
12
Hydroxyproline
1.631,0
13
Phenylalanine
4.368,0
14

Glutamic axil
4.563,0
15
Ornithine
2.830,0
16
Lysine
31.900,0
17
Histidine
6.269,0
18
Tyrosine
108.513,0
19
Tryptophan
1.553,0
20
T6ng cc}ng
236.139,0
B6 sung nude
I
B6 sung CPE
Bii
H Thi1can gia sue I
I Nghi~n mjn I
.
I Bao g6i bao qmin I
Hinh 3.13. Quy trinh san xua't bQt d~m tit ca mo'i
* Thuye't minh quy trinh

. Nguyen li~u: ca m6i tlidi d<;lttieu ehu£n TCVN 3250 - 88.
- Xllly: lo<;libi) cae ph~n khong sa d1,mgdu'<;fenhu' d~u, vay, vay,
xu'dngva thu phh thit ea sa d\mg eho vi~e san xufftbQtd<;lm.
. Xay nho: d~ lam tang di~n tieh tie'pxue gifi'aenzyme va thit ca.
. Thuy philo: thit ea sau khi can dinh lu'<;fngdu'<;fcph6i trQnvdi 0,3%
ehe' ph£m protease B.subtilis S5, 20% nu'de va 4% sorbitol r6i trQn d~u.
Thuy philn (j nhi~t dQ50°Cva pH tl,fnhien eua thit ea trong 16 gio.
22
-
Nguyen lieu (ea meJi)
'j
I
Thitea
.
I
Xaynhi)
.
B6 sung sorbitol
H
Thuyphiln
I:
t
I
Dun soi
I
1
.
Ly tilm tach m{J
I
.

23
-Dun soi: ke't thuc qua trinh thuy phan dun soi trong 15 phut d~ vo
hoi;ltenzyme va die:;tvi sinh v~t.
- LQc: d~ 10i;libOph~n xuong, vay, vay con Ifilltrong h6n hc;fP.
- Ly Him tach ma: tach miJ co trong san ph~m nhAm hi;lnche' qua
trinh oxy boa lipid d~n de'n vie:;cbie'n mh, mui va lam giam chfft 1uc;fng
cua bQt di;lmthanh ph~m. Ly tam tach miJ bAngmay 1ytam 1i;lnhvoi t6c
dQ 7000 vong/phUt trong 15 phUt. Sau khi Iy tam IOi;libO lOpmiJ tren b~
m~t va tach thu ph~n dich thUyphan. Sau do co d~c bAngphuong phap co
quay chan khOng, sffykhOd nhie:;tdQ70°C, nghi~n nhO,baa goi bQtdi;lm
thanh ph~m bAngtui PE trong di~u kie:;nchan khong d~ bao quan.
3.4. Sd' d1)ngprotease B.subtilis 85 trong san xua't mfoc miim
3.4.1. Thanh phdn boa hQccua ca cdm
Thanh phffn boa hQCco ban cua ca com duc;fcth~ hie:;nd bang 3.9.
Bang 3.9. Thanh phdn boa hQc cd ban cua ca cdm (%)
Ke't qua nghien CUllcho thffyca com co ham Iuc;fngprotein kha cao toi
18,7% va ham Iuc;fnglipid thffp. Ngoai ra khi phan tich thanh ph~n axil
amin cua ca com con cho thffy ca com co kha d~y du va can d6i axil
amino Chinh VIthe' ca com la d6i tuc;fngthich hc;fpcho vie:;csan xufft nuoc
mcim.
3.4.2. Xac dinh di~u ki~n thfch hqp cho qua trinh thuy philo ca
cdm bhng protease B.subtilis 85
3.4.2.1.Xac dinh ty I~ CPE thfch h«jpcho qua trinh thuy phdn
Ke't qua nghien CUllthUyphan ca com bAngprotease B.subtilis 55 voi
cae ty Ie:;CPE b6 sung khae nhau cho thffy ty Ie:;protease CPE thich hc;fP
b6 sung vao qua trinh thUyphan ea com Ia 0,2%.
Nuoc
Protein
Lipid Khoang
NNH3 NTs

Naa
78,3 18,7 1,7 1,2 0,025 3,7
0,308