H y i NtJHI KtlUA MUU L-UINO i x o n t : ojMn n y .
ANTIMICROBIAL ACTIVITY AND CHARACTERIZATION OF CRUDE
BACTERIOCIN PRODUCED BY MARINE BACTERIA ISOLATED FROM VIETNAM
Nguyen Thi Hai Thanh, Nguyen Thi Hong Mai, Nguyen Van Duy InstibJte of Biotechnology and Environment, Nha Trang University
SUMMARY
Bacteriocins are antimicrobial peptides or proteins synthesized ribosomally by a bacterium that inhibits the growth of other bacteria, especially closely relative strains. As a result, bacteriocins can be used as potential drags for human and animal health. In dus study, we assessed the bactenocinogenic potenUal and antimicrobial spectrum of 12 manne bactena strains isolated from cobii (Rackycentron canadum), snubnose pompano [Trachinotus blochii), black tiger shrimp (Penaeus monodon) and ornate spiny lobster {Pamilirus omatus) in Nha Trang Bay and Cam Ranh Bay agamst together themselves and agamst fi)od-bome and animal pathogens.
TTie indicator bacteria, which were shown to be sensitive to at least one of crade bacteriocins in our study, included E. coli. Proteus mirabilis. Clostridium perfringens. Salmonella enterica. Vibrio owensii. V. parahaemolyticus. Staphylococcus aureus, and Bacillus cereus. In addition, die effect of temperature, pH and enzymes on the stability and activity of crade bacteriocin produced by lliEse strains was also detennined. Tlie results have indicated that they were completely inactivated \rfien treated with proteinase K, but stable to dTpsin and catalase treatment. Most of bacteriocin was resistent to the treatment at 60"C in 15 min, but lost 75% tlieir activity afer 30 min at 6Vi'C and conqiletely mactivated at 100°C fiir 30 min However, they remained active after incubation at pH 4- 10. The detenninarton of antimicrobial activity and charactenzation of crude bacteriocin contribute into studymg on bactcnocin diversity in marine bacteria isolated from VieflMam and fiirther their applications in medicine and agriculttire.
Keywords: antimicrobial activity, bacteriocin, marine bacteria, pathogen, physico-chemical characteristic
INTRODUCTION
The invasion development of aquaculture farming at the industrial scale, addition to climate changes increases associated infectious epizoobes (Desriac ef af., 2010). Prophylactic antibiotics can control epidemic diseases, but may induce an antibiotic-resistant mechanism in bacteria and an accumulation of unused residues of drugs in the environment which, in turn, results in longterm negative effects on human and animal health (Gillor ef al., 2008). Thafs why scientific communities have pnjposed friendly alternatives such as vaccines, antibiotic substitutes or use of probiotics. Bactenocinogenic bacteria stmlns appear to be an excellent candidate for a sustainable aitemative since bacteriocin would be used as an antibiotic substitute, whereas bacteria would be a potential pnsbiotic (Gillor ef al., 2008).
Bacteriocins are antimicrobial peptides or proteins synthesized rilx3Somally by a bacterium that inhibits the growth of other bacteria, especially closely relative strains. They have a relatively narrow killing spectrum (Riley, 2009). However, inhibitory spectra of bacteriocins or bacteriocin-like substance (BLIS) produced by Gram - positive bacteria are often much more bnsadly than most of bacteriocins produced by Gram - negafives. In general, tiiey tend to be active against a wide range of Gram-positive bacteria, and sometime inhibit Gram-negative species. Moreover, the degree of actiwty of bacteriocin-like agents against sensitive bacteria can sometimes be substantially increased by testing either at particular pH values or in the presence of chemical agents that weaken cell wall integrity (Al>ee efa/., 1995; Gillor efa/., 2008).
Bactenocins have been classified in four distinct classes on the basis of biochemical and genetic characterization: (1) lantibiotics, (11) small heat-stable, non-lanthionine peptides including (Ha) f./sferia-active peptides, (lib) incorporation complexes consisting of two peptides for activity and (He) thiol-activated peptides, (111) large heat-labile pnsteins, and (IV), complex bacteriocins, carrying lipid or carbohydrate moieties (Riley, 2009). Bacteria strains, which produce bacteriocins classes i and tl, have been suggested as promising probiotics. Bacteriocins pnsduced by lactic acid bacteria (LAB) have studied the most, showing their remarkable potential as food conservatives (Abee ef al, 1995), or as therapeutics for vetennary or medical uses (Riley, 2009). Nevertheless, only a few studies have focused on marine bacterium isolation (rom marine animals and tiie search for their ability to produce bacteriocins (Desriac ef al., 2010).
In this study, we assessed bactenocinogenic potential and antimicrobial spectnjm of 12 marine bacteria strains isolated from cobia {Rachycentron canadum). snubnose pompano {Trachinotus blochii), black tiger shrimp (Pe/jaeus monodon) and ornate spiny lobster {Panulirus omatus) in Nha Trang Bay and Cam Ranh Bay against together themselves and against food-bome and animal pathogens, in addition, the sensitivity to heat, pH and enzymes of crude bacteriocins were also investigated and discussed. These knowledges are basis to apply bacteriocins as food preservatives or new dnjgs in medicine and aquaculture in Vietnam.
MATERIALS AND METHODS Strains and growth conditions
Total 12 bacteriocin producing strains were screened from hundreds of marine bacteria strains isolated from Uie gut of cobia {Rachycentmn canadum). snubnose pompano {Trachinotus blochii). black tiger shrimp (Penaeus monodon) and ornate spiny lobster {Panutinis omatus) in Nha Trang Bay and Cam Ranh Bay (Nguyen Van Duy et ai. 2012; Nguyen Van Duy and Pham Thu Thuy, 2012, Nguyen Van Duy and Nguyen Ihi Ngoc Thanh, 2012). The indicator strains include food-borne and animal pathogens supported from Nha Trang University (sti^ins Sail, 0 1 , B1.1), Institute of Veterinary Research and Development of Central Vietnam (strains GA, CP, SA), Nha Trang Pasteur Institute (strain VP2865) airf Australian InsUtuteof Marine Science (strain DY05) (Table 1). All bacteria were grown aerobically in trypticase soy broth (TSB) (HIMedia, India) supplemented with 2% NaCI at SO^C for 1 day on a rotary shaker at 180 rpm per min.
Chemicals and reagents
ProteinaseK 10lU mg-\ frypsin 10000 BAEE U mg\ChChymobypsm 10000 BAEEU mg\a-amylase200 lU mg"^ and lipase ^ 000 lU mg were purchased from Promega (USA). Other chemicals and reagents were fnam Merck (Germany).
Assay of antimicrobial activity
Antimicrobial activity was determined by agar-well difhjskm method as described by Todonav and Dicks (2009) with some moaiTi^tions Selected manne bacteria were inoculated into TSB medium and incubated at SO^C vwth agitation until mid logantnmic phage of grow*i (ODeoo = 0.8 - 1.S). Then ceil-free supematants were harvested by centrifugation at 6000 rpm/min Tor 30 mm at 4°C. Plates were overtakl vmth 3 ml soft agar containing 10* cells per ml of indicator bacteria. Wells
^ ^ ^ t * ^ ^ f-aJSr^' °^ '^® supernatant of 10* cells per ml of growing isolates was toaded into each well. Plates were incubated at 37"C for 24 - 36 h and then the diameter of inhibitory zone around the well was measured.
Assay of bacteriocin activity
Bactenodn activity was determined by agar-well diffusion method as described above with some modifications. The pH of the supernatant fluid was adjusted to 7.0 with IN NaOH CM" I N HCI to remove the effect of organic acid and tiien treated with catalase (Pramega, USA) at the final concentration of 0.5 mg ml"^ at Sy-C for 30 min to remove the effect of hydnagen peroxide. To check the protein property of bacteriocin, proteinase K or trypsin (Pramega, USA) at the final concentration of 1 mg ml were treated with supematant at 50°C for 3 hours.
Effetrt of physiochemical factors on the activity of crude bacteriocin The effect of enzymes
The supematant was first adjusted pH to 7.0 and treated with catalase as described above. To chedc the chemical property of bacteriocin, proteinase K, trypsin, a-chymotrypsin and lipase or a-amylase (Promega, USA) at a final concenb^tion of 1 mg ml" were added. Aftenward, Uie supematant were incubated at Uieir optimal temperatures as recommended by tiie manufacture. In particular, ti^psin and proteinase K were incubated at 50°C for 3 hours, and lipase and a-amylase at 37^C and 20°C for 2 hours, respectively. The residual adiwty after enzyme treatment was detemiined as described previously.
f/earancfp//sfai)r//f/
For tiie deterniination of heat stability, the cell-free neutralized supematant of bacteriocin was prelncubated at different temperatures eO-IOO-C for 15 - 30 min and 1 2 r c for 15 min. The remaining bactenocin activity was checked by agar- well diffusion method as described above. Slmilariy, the effect of pH on the bacteriocin was determined by adjusting the cell-free supernatant to pH 2.0-12.0 vwth 1 N HCI or 1 N NaOH. After 30 min of incubation at 30''C. the samples were readjusted to pH 7.0 and the bacteriocin activity was determined (Todorav and Dicks, 2009).
RESULTS AND DISCUSSIONS
Antimicrobial spectra of 12 marine bacteria strains
The results from Table 1 have shown that the cell-free supematants from all 12 isolates exhibited antimicrobial activity against at least one of selected indicators. Among them, the culture extracts of bacteria isolated from snubnose pompano (T. b/oc/i/)such as D9.1, 010 and 015 inhibited the growth of most of indicators with the sfrongest activity (20- 40 mm in inhibitory zone diameter) against Proteus spp. (N1.4, T9, T14, B3.7.1), V. owensii DY05, Bacillus pumilus B3.10.2B, B. cereus B1.1 and Staphylococcus aureus SA. In addition, the sfrains L5B and M2, which originated from spiny lobster (P. omatus), showed their strong antimicrobial activity against Profeus spp. (CT1.1, T9, T14)and E. co//GA with the inhibitory zone diameter more than 30 mm. Nevertheless, the sfrain N1.4 was indicated as the weakest inhibitory agent that only slightly prevent 6/21 indicator strains from the growth with relatively small inhibitory zone diameter Antimicrobial activity plays an important rale for a bacterium in community to exclude or inhibit competitive bacteria, which enable to be performed thraugh producing non-specific antimicrobial substances, such as organic acids or hydrogen peroxide, and target-specific toxins such as bacteriocins or BLIS, and bacteriophages (Riley, 2009; Desriac ef al.. 2010). In this research, 12 manne bacteria sfrains showed their wide antimicrobial spectra against several important food-borne and animal pathogens. For example, E. coli strains can cause serious food poisoning in humans and gut diseases in animals, while S. aureus Is a common cause of skin infections, respiratory disease and also food poisoning in humans. Some sfrains of B. cereus and Salmonella are also harmful to humans and cause foodbome illness (Abee ef al., 1995).
In addition, the Vibrio strains, one of the most important pathogens recognized in larval cultures, provoking a high mortality in marine animals (Desriac ef al.. 2010) were inhibited significanUy by cell-free supematants of strains D9.1, DIO, 015 and L5B. Interestingly. Vibrio owensii strain DY05 has just recently been demonstrated as an agent of disease causing rapid and high mortality in phyllosoma larva of ornate spiny lobster {P. omatus) cultured in Australia (Goulden ef a/., 2012) In our research, Proteus spp. strains isolated fram snubnose pompano (strains DIO and 015) and lobster (strains T9 and T14) expressed their strong antagonistic activity against the lobster pathogen DY05, indicating the potential of these bacteria for the development as probiotics in lobster culture. However, ftjrther study on the pathogenicity ofthese potential probiotics is required, in addition, some Proteus sfrains {CTI.1, GI, N1.4. T9and B3 7.1) with antimicrobial activity found in this study could belong to novel species with undetermined toxicity (Nguyen Van Duy and Pham Thu Thuy, 2012; Nguyen Van Duy and Nguyen Thi Ngoc Thanh, 2012).
Table 1. Antimicrobial activity of cell-free supamatants from 12 marine bacteria strains of Vietnam origin against together themselves and against f b o d - b o m e & animal pathogens
Animal hosts Snubnose pompano Ornate spiny k)bster Black tiger shrimp
Isolates CTI.1 D9.1 D10 015 {CR9.1){CR10)(CR15)
G I LSB M2 (R2)
N1.4 T9 T14 B3.10.2B B3.7.1
Proteus sp. CTI.1 Bacd/us cereus 09.1 P. mirabilis DID P. mirabilis D15 Proteus sp. G1 Enteroaxxus faecaBs LSB Klebsiella pneumoniae M2 Proteus sp. N1.4 Proteus ^ . T 9 P. mfraW//sT14 B. pi//nnlusB3.10.2B P/Ofei/ssp. B3.7.1
I f
Escherir^ia txii GA + Staphylococcus aureus SA 0 Cloetridium perfringens C P 0 Salmonella enterica Sail 0 Vibrio parahaemolyticus C1 0 V. parahaemolyticus VP2865 0 V. oivens//DY05 + 8. cereus B1.1 ++
^, >30 mm; 0. no inhibition zone
¥
('} The diameter of InNbHory zone
B a c t e r i o c i n a c t i v i t y o f 1 2 m a r i n e b a c t e r i a s t r a i n s
The most sensitive indicators of 12 stiBins of research interest w e r e selected for screening bacteriocin activity of marine l>acteria. After neubalized to p H 7 ti3 exclude Uie effect of organic acids, a n d then treated with catalase to remove the effect of hydrogen peroxide, cell-free supematants of 12 marine bacteria strains w e r e spotted onto soft a g a r plates seeded wiUi selected sensitive indicators. The results w e r e presented in T a b l e 2 .
Table 2. Bacteriocin actlWty of 12 marine bacteria of Vietnam origin against sensitive indicators No. Isdates Inhlbitoiy zone diameter (D-d. mm) Sensitive indicators
LSB 015 010
P. mirabilis T14 P. miratttiis T14 P. rrorab/7/sT14 Proteus sp. T9 P. mirabilis^^4 P. mirat^ils TtA S. enterica Sail S. enterica Sail S. enterica Sail K. pneumoniae M2 Pmteus sp. T9 Proteus sp. CT1.1
The results fttim Table 2 have indicated Uie cell-free neutralized s u p e m a t a n t s o f 6 sfrains {B3.7.1 B3 I D 2 B T9 T14 M2 and N I .4) m u c h smaller in inhibitory z o n e diameter ttian ttieir natural s u p e m a t a n t s , suggesting ttiat their antimic'roblai activities could result from organic acid production. In ttie confrary, the activity of 6 remaining strains w e r e found to be relatively stable after ttieir s u p e m a t a n t s were neutralized. Even no inhibitory activity of ttiese sttains w a s observed after proteinase K tieattnent. indicating the prateinaceous nature of inhibitory c o m p o u n d s , which may b e bacteriocins or BLIS, i h e s e results are similar to tiie bacteriocin activity reported in 5 Pseudoaltemmonas isolates and 3 unidentified isolates fiom marine animals in Austtalia (Wilson ef al., 2 0 1 0 ) a n d in Vibrio s b a i n s isolated from seahorses (Balcazar etal., 2010). Thus, 6 ttiese s t i ^ i n s w e r e selected for further study on physico-chemical characteristics
E f f e c t o f e n z y m e s , p H a n d t e m p e r a t u r e o n b a c t e r i o c i n a c t i v i t y
Crude bactenocins from 6 selected sfrains were tested for their sensitivity to various e n z y m e s (Table 3 and Figure 1).
r r , ! c ! ! l T ' * ' u ^ ^ ^ remained or little decreased after catalase treatment, indicating ttiat inhibitory activity was not i n h ! ^ ^ ! l ^ ^ ' 7 " " ° " 9 ' ^ ^^'^^ bacteriocins of all of 6 sfrains w e r e completely inactived by proteinase K treatment, inhibitory activity of s u p e m a t a n t s produced by 6 sti:ains w a s different after ttie e x p o s u r e to frypsin and chymofrypsin. In particular, a complete inachvation w a s o b s e n ^ w h e n bacteriocin exfracts o f t w o sfrains C T 1.1 a n d G I w e r e traated by these two enzymes, while ttie activity of bacteriocins p r o d u c e d by four remaining sfrains s e e m e d to b e unchanged
- C C O N G N G H E S I N H H p c T O A N Q U 6 C 2 0 1 3
TaUe 3. Effect of enzymes, pH and temperatim o n a i l M c R M r i ac&vi^ of cnxle bacleriodris
Treatment
Contiol (•)
Inhibitory zone CT1.1 13.5 Enzyme treatment Catalase Proteinase K Trypsin a-Ctiymotrypsin a-Amylase Upase p H l r e a b n e n f p H 2 p H 4 p H 6 p H 7 p H B p H I O p H 1 2 Heat beatment eO'C, 15 min 60°C. 30 min 1 0 0 ^ , 15 min 100°C, 30 min 121''C, 15 min
13
13.5 13.5
4 11 13.5 12 6
13.5 10 8
G I 14 12
4 8 13 5 14 7.5
13 11 9 4
diameter (D-d, mm) D9.1 12 12 12 11 12 11
4 11 12 8 6 4 11
DID 14.5 14 8 9 6 8
14 14 14.5 7 5
13 D15 15 8 8 8 10 9.5
12 15 15 7 6
6 4
LSB 23 21 23 21 23 23
9.5 18 23 13 8 4 18
C) Contml: cell-free neutralized supematants without any tmatmenls, -: no antimicrobial activity
Figure 1. Effect of enzymes on antimicrobial activity of cmde bacteriocins produced by bacteria strains D9.1 (CR9.1) {A), CT1.1 (B) and DIS {CR15} (C). OC: control: sanples treated widi enzymes presented on pkies.
T h e s e results are in a g r e e m e n t viith results recorded by previous studies, e.g.. nisin, plantaricin C a n d plantaricin D a s f o o d biopreservatives a p p l i e d all over t h e w o r i d ( A b e e et al.. 1 9 9 5 ; Gillor ef al., 2 0 0 8 ) . A l s o , bacteriocin G 2 isolated Lactobacillus plantarum G 2 w a s inactivated b y trypsin ta«abnent but remained inhibitory activity after proteinase K a n d chymofrypsin t r e a t m e n t {Seatovic ef ai, 2 0 1 1 ) . This can b e explained by different cleavage mechanisms of types of proteases resulting f r o m t h e diversity of a m i n o acid s e q u e n c e s in polypeptides. In addition, freatment with a - a m y l a s e a n d lipase d i d not significanUy a f f e c t t h e antimicrobial activity, except tat t h e sfrain G 1 , suggesting that ttie bacteriocins f r o m these strains w e r e not a t t a c h e d to a c a r b o h y d r a t e or lipid moiety (Riley, 2 0 0 9 ) .
Moreover, inhibitory activfty o f bacteriocins p r o d u c e d b y 6 isolates w a s f o u n d to be stable in the p H range 4 . 0 to 10.0 (Table 3), e v e n the activity of bacteriocins f r o m D9.1 a n d LSB still remained 5 0 % at p H 2. T h e s e results s u g g e s t j h e potential application of t h e s e m a r i n e bacteria a s probiotics o r multi - d m g s in aquacultijre because bacteriocins p r o d u c e d by ttiem c a n act in fluctuating p H e n v i r o n m e n t s .
Finally, bacteriocins f r o m 6 m a r i n e strains w e r e heat-labile bacteriocins (Table 3). M o s t of t h e m w e r e resistent to ttie heat . treatinent at eO^C in 15 m i n . b u t lost 7 5 % ttieir activity afer 3 0 m i n at 6 0 ° C a n d completely inactivated at lOO^C for 3 0 min or at 121''C for 15 m i n . R e m a r k e d l y , antimicrobal activity o f bacteriocins fram LSB, D9.1 a n d D I O w a s lost completely after incubation at eO^C f o r 3 0 min only. T h e s e results a r e similar to date recorded by bacteriocin class 111, including large, heat-labile b a c t e r i o c i n s (Riley, 2 0 0 9 ) , suggesting that t h e bacteriocins from ttiese isolates in o u r research c o u l d belong to Class III.
Overall, t h e p h y s i c o - c h e m i c a l characteristics of bacteriocins f r o m 6 marine bacteria support important data for further studying o n bacteriocin diversity in marine bacteria of V i e t n a m origin a n d applying these bacteria as potential probiotics in a q u a c u l t u r e a n d f o o d t e c h n o l o g y .
C O N C L U S I O N S
The cell-free s u p e m a t a n t e f r o m 12 m a r i n e bacteria exhibited antimicrobial activity against together themselves a n d against f b o d - b o m e a n d a n i m a l pathogens s u c h as B. coli Proteus mirabilis, Clostndium perfringens. Salmonella enterica Vibrio owensii, V. parahaemolyticus. S. aureus and B. cereus. fi^mong them, 6 strains L5B, D15, D I O . D 9 . 1 , G I
H O I N G H ! K H O A H O C C O N G N G H ^ S I N H H O
vi C T I 1 w e r e ftiund to e x p r e s s ttieir stable and sfrong antimicrobial activfty a g a i n s t sensitive indicators P r o f e u s Sf^"
T14 and T 9 Bacteriocins pnDduced b y Uiese stiains w e r e stable to c a t a l a s e freatinent but c o m p l e t e l y inactivated |tf)en freated witti proteinase K. T r y p s i n o r a - c h y m o t i y p s i n freatinent inactived bacteriocins o f bwo s t i 3 i n s C T 1.1 a n d G 1 white Uie activity s e e m e d to b e s t e b l e i n bacteriocins p r o d u c e d b y LSB. D 1 5 , 0 1 0 a n d D 9 . 1 after ttie e x p o s u r e to tttose enzymes Most o f bacteriocins w e r e resistent to t h e freatment at 6 0 C for 15 m i n , but lost 7 5 % ttieir activity after 30 mki at eO^C a n d completely inactivated a t lOO^C ft)r 3 0 m i n . However, ttiey r e m a i n e d active after incubation a t p H 4 - 1 0 . A c k n o w l e d e e m e n t : Ihe research was financMfy supported from NAFOSTED for dte Prrgea No. 106 03-2011 34 Authors are gralefid », students Di^g TW TTumh Bmk and Pham Thi Ihu Thuy at Nha Trang Vntversityfor their excellent techmad assistant.
R E F E R E N C E S
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XAC DINH HOAT T ( N H K H A N G K H U A N V A T I N H C H A T H C A L V C O A DICH
BACTERIOCIN T H O TO VI K H U A N B I ^ N V I ^ T NAM
N g u y £ n T h j Hai T h a n h , N g u y S n T h j H o n g M a i , N g u y e n V ^ n D u y ' Viin Cing nghi sinh hgc vi MOi tnrimg. Tnrdng D^i hgc Nha Trang
T 6 M TAT
Bacteriocin li cic pei^tide khing khuin dugc san sinh tfr vi khuin niy dk iic ch€ sinh truong cua c&c vi khuin khic, nliSt la cic chikg cd quan h$ t i ^ hoa gJin gui. Do d6, chiing li nhiing dugc phim tiem ning bio v^ cho sue khde con ngu6i v i d$ng v|t. Trong nghien cihi niy, chung t&i xic dtnh khi nSng sinh bacteriocin vk pb.6 khing khuin ciia 12 chiing vi khuin bien du^c phan lip ti> ci giA {Rachycentron canadum), ci chim vay ving {Trachinotus blochii), tom sii {Penaeus monodon) vk torn him b6ng {Panulirus omatus]
d V)nh Cam Ranh va Vjnb Nha Trang Cic thu nghiSm bao gom xic djnh hoat tinh kliing tin nliau ciing nhu khing cac tic nhin gIV ibhi thvrc phim va gay t > ^ cho d^ng v i t Cac vi khuin chi thj nh^y cim vdi it nhit mpt bacteriocin thd trong nglii^n cuu niy bao gom E coll. Proteus mirabilis, Clostridium perfringens. Salmonella enterica, Vibrio owensii. V. parahaemolylicus, Staphyiococcis aureus, vi Bacillus cereus. Dong th£ri inh huon^ cua ntii^t dp, pH vi enzyme tdi dg t)£n vi hoat tinh ciia djch bacteiiociii di6 tu ck chiing niy cung duqrc xac djnh. K6t qua chi ra rang hiu het cic bactenocin dku bj bit hogt khi xu ly vdi proteinase K nhung bfe vfti tripsin vi catalase. Cac bacteriocin ben 6 60°C trong 15 phiit, nhung hogt tinh giam 75% sau 30 phut cr fXfC vi bat hogt hoin toin is 100°C trong 30 phiit. Tuy nhifin, cic bacteriocin niy vln flie hi?n hogt tinh sau khi xur ly 6 pH 4-10. Ket qua xic djnh hoat tinh khing khuin vi tinh chat h6a ly ciia cac bacteriocin tho giiip nang cao hiiu biSt vi da dang sinh hpc ciia bactenocin tii vi khuan bien Vi(l Nam, cung nhu nhimg iing d\ing xa hon tiong y dugc va nong nghi§p.
Tu khoa: bacteriocin, hogt tinh khing khuan, tac nhan gay b?nh, tinh chit h6a ly, vi khuin biln
• Author for correspondence: E-mail: duvnv&intu.edu vn
