Tgp chi Khoa hgc

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Tgp chi Khoa hgc - Cong nghe Thuy sdn So3/20lf

KET QUA NGHIEN CQU DAO TAO SAU DAI HQC

KHA NANG THUY PHAN PHYTATE VA CELLULOSE TRONG BA SAN CUA CHUNG BACILLUS SUBTILIS Cl

THE HYDROLYSIS ABILITY OF BACILLUS SUBTILIS C7 ON CELLULOSE AND PHYTATE IN CASSAVA RESIDUE

Mgch Tran Phinmg Thdo', Phgm Hong Nggc Thuy', Nguyen Minh TrP Ngay nhan bai: 21/11/2013, Ngay phaa bien thong qua: 21/02/2014; Ngiy duy?t dSng. 13/8/2014

TOM

TAT

Bacillus subtilis C7 co khd ndng sinh cdc enzyme cellulase. phylase de thuy phdn cellulose vd phytate trong bd sdn Nghien ciru tten hdnh khdo sal cdcyeu to dnh huang den hteu qud thiiy phdn cellullose vd phytate khi u ba san v&i chung vi khudn Bacillus subtilis Cl, tu do xdc dinh cdc thong so thich h(>p cho qud trinh H Ket qua cho thdy thai gian u thich hflp la 48 gia, nhiet dQ iJ 36 ± PC, ty lentt&c: honhfipld 2-J, ty le ddu nanh bo sung 10%. Hi^u sual ihuy phdn cellulose, phytate sau khi it ldn lu0 la 35,27±2.81% vd 39,48 ± 0,05%.

Til khoa: Bacillus subtilis 07, cellulose, phytate, ba sdn ABSTRACT

Bacillus subtilis Cl has ability to synthesize cellulase and phylase lo hydrolyze cellulose and phytate in cassava residue. This research studied factors that were influential in the productivity of cellulose and phytate hydrolysis in cassava residue with Bacillus subtilis Cl lo choose suitable factors for fermentation. Research results showed thai suitabie fermenting lime was 48 hours, fermenting temperature was 36 ± !"€, the ratio of water and mixture was 2' 1, the content of added soybean was 10%. The hydrolysis productivity of cellulose and phytate after the fermentation was 35,27 ± 2,81%

and 39.48 ± 0,05%o. respectively

Keywords: Bacillus subtilis Cl, cellulose, phytate, cassava residue

I. OAT VAN D&

San la cay luang thyc quan trpng, duoe trong d rit nhilu nudc trfin t h l gidi. To chdc Ndng Luang Lifin hifip quoc (FAO) xep san la cay luang thyc quan trpng d cac nudc dang phit tnln sau lua gao, ngd v i Itia mi. Ci Vifit Nam. cay s i n da chuyen doi vai tro tu eay luang thye thanh ciy cflng nghifip vdi toe dfl cao, nang suat va s i n luong s i n da tang nhanh d thfip ky d i u cua t h l ky XXI. Hifin nay, san dupc sd dyng chfnh trong che bien tinh bgt sin, san xuat ethanol, Ihdc an chan nufli v i an tuai Phan ba san cdn lgi tu qui trinh sin xuat tinh bpt l i nguon thdc an tilm ning cho gia sue, nhit l i Irong tinh trang hifin nay, nguon thuc an gia siic trong nudc khdng cung ung dO, Ihudng phai nhgp td nudc ngoai, chi phi thdc i n anh hudng Idn din thu nhgp cic hg gia dinh, ca sd chin nudi v i anh hudng d i n sy p h i l triln

cua nganh chin nudi. Tuy nhifin, Irong ba san c6n chua thinh phin khd hap thu dupe l i cellulose.

Ngoai ra, trong b i s i n chda axit phytic - yeu to khing dinh dudng - la ehat co kha nang tgo phdc chgt che vdi eie ion kim logi tgo nfin c i c muoi phytate {mu6i cua axit phytic vdi Na*), phytin (muli dta axil phytic vdi Ca=*, Mg^') v i c i c hpp chat khac nhu axit amm, protein, lam de e h l c i e enzyme tifiu hfla, ngan cin sy h i p thu c i c chit n i y v i o ca the song [8].

Nhieu nghien cuu da chi ra Bacillus spp. cfl khi ning sinh l i n g hpp enzyme cellulase, phylase [1], [2], [3], [10] de thuy phan cellulose v i phytate. Do dd, vific nghifin cdu kha nang Ihuy phan cellulose v i phytate trong b i s i n td chung Bacillus subtilis 07 i i d n thilt, gdp phin ning cao g i i trj sd dyng. giS tri kinh t l eho ba sin v i hgn c h l d nhilm mfli Uudng do q u i Irinh san xult linh bfll sin g i y ra.

' Mgch Trin Phuong Thdo: Cao hpc Cong ngh^ sau thu ho^ch 2011 - Tnr&ng Dgi hpc Nha Trang 2 ThS Phgm Hing Ngpc Thiiy, =TS Nguyin Mmh Tri: Khoa Cong ngh# thyc phim - Tnr&ng Dgi hpc Nha Trang

180 • TRUONG OAI HQC NHA TRANG

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Tgp chi Khoa hoc - Cong nghe Thdy sdn So 3/2014

II. D 6 | T U O N G , V A T LI6U VA PHU'O'NG P H A P NGHIEN CIJU

1. D l i t u y n g nghien CIJIJ

ChOng vi khuin Bacillus subtilis C7 dupc lly tu Phflng Thf nghifim Vi sinh, Khoa Cflng nghfi thye phim, Trudng Bgi hgc Nha Trang.

2. V | t li$u nghien ciru

B i s i n : Sau qua trinh che biln tinh bpt s i n , ba sin dupc thu nhgn va s l y khfl d i n him lupng I m ti> 5 - 6% de l i l n hinh nghifin cuu. Ba sin mua Id Cflng ty Trich nhifim huu hgn Anh Tuyel, Cam Hii Tiy, Cam Lim, Khanh Hoa.

Dfiu nanh: Dgu ninh dupe dupc tiln hinh phin kigi d l logi bo c i c hgt bj hu hdng, s i u mpt, sau dd tien hinh s l y khd d i n ham lupng am khoang 5 - 6%, xay nhd d l su dyng cho nghien cdu. D i u ninh dupc mua tgi chp^ Bam (Nha Trang, Khinh Hda).

Cim ggo: Sd dung cam mdi, khdng cd du vj chua hay ding, khong cd mCii hdi. Bd I m cOa c i m khflng q u i 15%.

Ri dudng: Ri dudng dupe mua d Cdng ty C l phin Dudng Ninh Hda cd dp am 48%, bao quan noi kho rao d^ su dung cho nghifin cuu.

3. PhiPcmg phap nghifin ciJu 3.). Phinmg phap phin tich

3.1.1. X i c djnh h i m lupng cellulose theo TCVN 4329-93 [9]

Nguyen lac: Dung dung djeh axit va kilm vdi nong dfl n h l l djnh thOy phan va tach khoi mSu thu cic chit bflt dudng, protein, dau md, mflt phin hemicelulose va lignin con Igi l i cellulose thd.

3.1.2. Xic djnh him luang phytate theo phuang phip Wade [4]

Nguyfin tie: Su dyng dung djch HCI 2,4% d l chill axit phytic ra khoi mau, sau do bo sung dung djch Wade (0,03% FeCI^ v i 0.3% axit sulfosalisylk:) de phin dng vflt axit phytk:, lupng dung djch Wade ktiflng tic dyng vdi axil phytic dupc xac dinh bing cich so miu d budc song 500 nm, Id dd tinh dupc ham lupmg axit phytic d i t i e dgng vdi dung djch Wade.

III. K £ T Q U A NGHIEN CI>U VA T H A O LUAN

1. Anh hirdng cua ty )$ d$u nanh den kha nang thuy phan cellulose va phytate

3.1.3. X i e djnh hogt dfl enzyme bing phuang phip do dudng kinh vflng thuy phin [5], [6]

Nguyen tic: Cho enzyme l i c dyng Ifin ca ehit d i e hifiu cho tdng enzyme (cartwxy metyl cellulose (CMC) d l i vdi cellulase, phytate d l i vdi phytase) trong mfli trudng Ihgch, ca diat bj phin giii, dfl dye ciia mdi trudng bj giim. mfli trudng trd nfin trong suit, Bfl ldn cOa vdng phan glai phin i n h mdc dfl hogt dflng cOa enzyme.

3.2. B6 tli thi nghidm xic dmh anh hifdng cua ty 1$

d$u ninh, nhi^t dC 0, ty 1$ nu-drc bo sung, thin gian vi thinh phin moi tnfdng den khi ning thtiy phin cellulose vi phytate

B l tri thi nghifim x i c dinh anh hudng cua ty lfi d i u ninh, nhifit dp u, ty lfi nudc bo sung, Ihdi gian va thinh phin moi trudng d i n qua trinh thuy phin cellulose v i phytate vdi khoang biln Ihien cua cac y l u t l nhu sau:

- Ty lfi dfiu nanh: t u 0 d i n 20% vdi budc nhiy l i 5%.

- Nhifit dp u: khio s i t d ba muc nhifit dfl l i 24 ± I^C, 31 ± I^C, 36 ± I^C.

- Ty lfi hon hpp (ba s i n + dgu ninh): nuo^:

1:1; 1:1,5; 1:2; 1:2,5.

- Thdi gian: khio sat Ihdi gian Ci Id 0 gid din 72 gid vdi budc nhiy l i 12 gid.

- Thinh phan mfli trudng: khio s i t bin edng thdc mfli trudng:

Cflng thuc 1 (CTI): 5% D i u ninh + 5% c i m ggo + 90% ba sin;

Cdng thdc 2 (CT2): 5% Bau nanh + 5% ri dudng + 90% ba san;

Cdng thuc 3 (CT3): 5% Bgu ninh + 5% c i m ggo + 5% ri dudng + 85% b i s i n ;

Cflng Ihdc 4 (CT4): 10% £)au ninh + 90% ba san Cfl djnh mfit dfl vi khuan l i 10*CFU/g.

3.3. Phin tich so lieu

MOI thi nghifim dupc tien hanh 3 l l n , kit q u i trinh biy l i trung binh epng cua 3 l l n thi nghifim ± dfl tin cgy 95%. So lifiu dupc phin tich phuang sai (ANOVA), sy khic bifil co y nghla vdi p < 0,05.

IMM

10 I

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SK lOH H% 20S

'J : 1

0 I

OHifo suit Hray (ddo ptayUte Oi) Hottd^ceUiiltie

Hinb 1. Ank hudng caa ty 1$ d|g n^oh aia kbi Dang thuy pbio ceUuiose vi phyUte

TRUONG OAI H p c NHA TRANG • 181

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Tgp chi Khoa hoc - Cong nghe Thuy sdn So3/20U

Tu d l thj hinh 1 cho thiy, khi tJ lfi dgu ninh b l sung l i 10%, dudng kinh vdng phin giii CMC v i phytate dgi cyc dgi, l l n luvt la 9,08 ± 0,95 mm v i 7,08 ± 0,96 mm. Hifiu suit thuy phin phytate v i cellulose ciJng t i n g d i n khi ting ty lfi dfiu ninh ty 0% den 10%, cy the khi khflng bo sung d i u ninh Ihi hifiu suit thuy phin phytate v i cellulose l i 9.36 ± 0,35% v i 25,6 ± 2,90%, khi IJ tfi dgu nanh l i 10% thi hifiu suit thuy phan phytate va cellulose dgt

cao nhit, lln lu^t l i 39.48 ± 0.05% v i 35,27 ± 2,81%, Nlu Hep lyc ting ty lfi d|u ninh Ifin 15% v i 20% thi hifiu suit thuy phin phytate v i cellulose giim din.

Do dd, chpn ty lfi dgu ninh b l sung l i 10%, k i t q u i niy luang l y vdi k i t q u i nghifin euu aia L Noubivics(1995)[7].

2. Anh hirdng cua nhi^t dp u din khi ning thuy phan cellulose v i phytate

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• I m ^{^^1} ^{s t} ^{a I} ^{, 1}

• H i ^ s o k d i d y p h i n cc1hi](Me(H)

O H t ^ w i l M y i i U D pfayttfe(H>

AHoftd^ceUDlase

• Hoftd^I^iytHe

Nlilftd§(»C) IDnh 2. Anh hudng ciia ohi^ 3^ sin kha Nghifin cdu lya chpn 3 muc nhiet dp 24 - 30 - 36°C nhim theo Ihang nhiet dp thyc t l d cae dia phuang nhung viing hay Irlng va ehl biln tinh bfll san nhu Quing Trj, Quang Binh, Khinh Hda... Khi ting nhifit dfl li t d 24 ± r C Ifin 36 ± 1"C thi dudng kinh Ihuy phin CMC va phytate tang; hifiu suit thiiy phin cellulose v i phytate cung tang tuong dng. 0 nhifit dfl li l i 36 ± 1°C thi dudng kfnh thuy phan CMC v i phytate l i 9,17± 0,72 mm va 7.75 ± 0,5 mm;

3. Anh hirfl'ng cua ty lfi nudc bo sung din kha nang thOy phan cellulose va phytate nfing thiiy ph^n cellulose vJi phytate

hifiu suat thuy phin cellulose dgt 33,30 ± 0,57% vi phytate dgt 39,66 ± 0.02%; ting 19% v i 21% sovM khi li d nhiet dg 24 ± I^C, Neu tiep tyc ting nhifit d$

len 40''C v l n l i dieu kifin vi khuin Bacillus subtilis C7 thfch nghi v i sinh enzyme phin dng tilp tyc Uiiiy phan tuy nhifin mdc nhifit dfl niy khflng thl duy tri mflt each ty nhifin ma chi rai v i o Irong khoing 24 - sa^C, Vi vgy, nhifit dfl 36 ± 1"C thfch hpp cho q u i trinh thuy phin cellulose v i phytate

• Hifn n i t 6iif {Alio celhili»e(H) DHi^iuitllidyiriiiD [diyltte(K) AHoitdQcdhibae

Hinh 3. Anh h u d n g ciia ty 1^ n u m : ho sung deo k b i n i n g thiiy p h a n cellulose v i phytate

T d hinh 3 eho Ihly, khi t^ le hdn hpp: nudc t i n g dan t d 1:1 Ifin 1: 2 thl dudng kinh thiiy phin CMC vJ phytate l i n g Id dd hifiu suit thiiy phin cellulose v i phytate cOng tang d i n . Cf mde ty lfi h i n hpp: nudc IS 1 : 2, hifiu suit thuy phan cellulose l i 31,54 ± 1,32% va hifiu suit tiiiiy phan phytate l i 37,01 ± 0,67%. Tuy nhien khi tilp tye tang ty lfi h i n hpp:nudc Ifin 1:2,5 thi hifiu suit thiiy phan cellulose v i phytate khdng khic bi^l (p > 0,05). Vi t h l chpn ty lfi hon hpp- nudc la 1 : 2 (dfl i m tuang dng l i 70%) eho q u i trinh li.

182 • TRL/ONG OAI HOC NHA TRANG

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Tgp chi Khoa hgc - Cong ngh^ Thuy sdn So 3/2014

T h i V i g l u

m n h 4. Anh huihig cua thM gian u skn k h i nang thuy phSn cellulose v i phytate Tu hinh 4 eho thiy. khi tang thdi gian li t d 12 gid

den 48 - 60 gid thi dudng kinh CMC va phytate t i n g td do hifiu suit thuy phan cellulose v i phytate eung tang. Hifiu suit thiiy phin cellulose sau khi li 48 gid.

Sat 31,54 ± 0,82%; neu tiep tuc l i n g Ihdi gian li Ifin 60 gid v i 72 gid Ihi hifiu suit thuy phan cellulose

khdng cfl sy khic bifit (p > 0,05). Thdi gian u la 48 gid v i 60 gid Ihi hifiu suit thiiy phin phytate lan luat l i 36,98 ± 0,47% v i 40,06 ± 1,71%. Sau 48 gid Cl Ihl hifiu suit tiiuy phin cellulose v i phytate deu eao han 30%, do do chpn thdi gian li b i sin vdi chiing vi khuin Bacillus subtilis C7 l i 48 gid.

5. Anh hirdng ciia t h i n h phan moi trirdng den kha nang thuy p h i n cellulose va phytate

' i

^ •Hi$DSDitlii^ptalnc^hilose<K) M OHi^inittfa^fliinpIiytde(K>

* i AHoftd^celtalise 3 M *Hoft(t4idiytise

g d i n k h i n i n g thuy p h i n cellulose v i phytate

khdng cd sy khic bifit (p > 0,05), trong khi d CT4 dudng kinh CMC dgt cao nhit 8,58 ± 0,36 mm hifiu suit thiiy phin cellulose cao nhit d 31,54 ± 0,82.

Hifiu suit thiiy phan phytate d CT3, CT4 cOng cao hon d CTI v i CT2 (p < 0,05), dudng kfnh thiiy phytate dgt eao nhit d CT4 l i 6,08 ± 0,15 mm va hifiu suit dgt 36,74 ± 1,49. VI v i y cd I h l b l sung d i u ninh nhim l i n g him lupng chit dinh dudng v i lang hifiu suit thiiy phin cellulose v i phytate.

T U a h p k i a b i m c HJnh 5. Anh h u o n g cfia t b i n h p h i n mSi t n h Cong Ihdc 1 (CTI): 5% dgu nanh + 5% c i m ggo + 90% ba s i n ;

Cflng Ihdc 2 (CT2)- 5% dfiu ninh + 5% ri dudng + 90% b a s i n ;

Cflng Ihuc 3 (CT3): 5% dfiu ninh + 5% c i m ggo + 5% ri dudng + 85% b i san;

Cflng thdc 4 (CT4): 10% d^u ninh + 90% b i sin.

K i t q u i trong hinh 5 cho Ihly, dudng kfnh CMC v i hifiu s u l l thiiy phin cellulose d C T I , CT2, CT3

T R U C N G D A I H p c NHA TRANG • 183

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Tgp chi Khoa hgc - Cong nghj Thuy sdn So 3/2014

IV. K^T LUAN VA KltN NGHj I . K l t l u I n

Xie d|nh dupc eie thflng s l eiia q u i trinh <i ba s i n vdi chiing Bacillus subtilis C7 d l q u i trinh Ihiiy phan cellulose v i phytate dgt hifiu qua cao.

K i t qua eho thiy thdi gian li thfch hpp l i 48 gid, nhifit dgii 36 ± I^C, tJ lfi nudc: h i n hpp l i 2:1, tJ lfi dfiu ninh b l sung 10%. Hifiu s u l l thuy phin

cellulose, phytate sau khi li l l n lugi l i 35,27 ± 2,81%

v i 39,48 ±0,05%, 2. Kiln ngh|

Nghifin cdu su dyng k l l hpp gida chiing Bacillus subtilis C7 v i cac chiing vi sinh vgt khic d l l i n g hifiu q u i thiiy phin cellulose va phytate, giim him lupng dflc l6 cyanua d l dng dyng trong s3n xult thdc i n chin nudi.

TAI LIEU THAM KHAO Tieng Vif I

Nguyen Minh Tri, Mach Tran Phucmg Thao, Nguyen Thj Thanh Hai, Ph;im Hong Ngpc Thiiy, 2013. Tuyen chgn chiing Bacillus spp. sinh enzyme v^ nhan sinh khoi tren moi trudng bi san. Tap chi Ndng nghi^ va Phat tnen Nong thon, 223: 84-SE, 2. TCVN 4329-93 Xac dinh chat xo tho.

Tieng Anh

Crispen Mawadza. Rajni Hatti-Kaul, Remigio Zvauya, Bo Mattiassoo, 2000. Purification and characterization of celluloses produced by two Bacillus strains. Journal of Bioteclmology, 83: 177 - 187.

Deepmoni Deka, P.Bhai^avi, Ashish Sharma, Dinesh Goyal, and M Jawed, and Anm Goyal, 2011. Enhancement of Cellulase Activity from a New Stram ot Bacillus subtilis by Medium Optimization and Analysis with Various Cellulosic Substrates.

Enzyme Research.

Fruhbeck G, Alonso R, Marzo F, Santidrian S., 1995. A modified method for the indirect quantitative analysis of phyt^e in foodstuffs. Anal biochem.

6. Jacob, M.B. and Gerstein, M.J., 1960 Handbookof Microbiology. D. Van Nostrand Co. Inc. Princeton. New Jersey, USA, 61, 7. Ji Hwan Yoon, Ji Eun Park, Dong Yeon Suk, 2007. Comparision of Dyes for easy detection of Extracellular Cellulase in

Fungi. Mycobiology, 35

Noubi, Festus A. Numfor and L.. 1995 Effect of fiill-fat soya bean flour on the quality and acceptability of fermented cassava flour. The United Nations University Press, Food and Nutrition Bulletin, 16.

Oboh, Ganiyu, 2006. Nutnent enrichment of cassava peels using a mixed culture of Saccharomyces cerevisae and Lactobacillus spp, solid media fermentation techniques. Electromc Joumal of Biotechnology: 46-49.

10 Thuy Thl Tran, Suhaila Omar Hashim, Yasser Gaber, Gashaw Mamo, Bo Mattiasson, Rajm Hatti-Kaul, 20U. Themiostable alkaline phytase from Bacillus sp MD2: Effect of divalent metals on activity and stability. Joumal of Inorganic Biochemistry, 105: 1000-1007,

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