CA2082324A1 - Arthrobacter nicotiana strain, activity of the strain, process for the isolation of the strain, sensor with the activity and use of the strain - Google Patents
Arthrobacter nicotiana strain, activity of the strain, process for the isolation of the strain, sensor with the activity and use of the strainInfo
- Publication number
- CA2082324A1 CA2082324A1 CA002082324A CA2082324A CA2082324A1 CA 2082324 A1 CA2082324 A1 CA 2082324A1 CA 002082324 A CA002082324 A CA 002082324A CA 2082324 A CA2082324 A CA 2082324A CA 2082324 A1 CA2082324 A1 CA 2082324A1
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- Prior art keywords
- strain
- acyl
- activity
- sensor according
- oxidase activity
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
- C12Y103/03006—Acyl-CoA oxidase (1.3.3.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/06—Arthrobacter
Abstract
Abstract The invention relates to an Arthrobacter nico-tiana strain having an acyl-CoA oxidase activity which is specific for C4-12-fatty acids, to this activity, to a process for the isolation of the activity or of a strain having this acitivty, to a sensor with the activity and to its use.
Description
2~8232~
GeSQ11ffChaft fiir siot0chnologl~ah2 Por~chung mbll ~GnF)~
Ma~cheroder weg 1, 330~ ~runswick Rancid or ~e~erlorated odor of milk and dalry products i~ caused by th~ liberatlon of short-cha1n C4,2-atty acld~ hy m~lk 11pasQ or by bactexi~ P~eD (1, 2). Th~ de~eation of ~hort-chdill ~atty acidY i 8 therefore hl~hly important in th~ q~alitative a~es~ment of d~iry product~ in the milk-collection method~ encountered in practice and for the prolonged ~toxage of ~aw ~ilk.
To date, th~ ~uantita~ive determination of free fatty acid~ h~6 be~n carried out by titrimetrlc detor-mlnation of the total aoid or by colorimetric analyses ~a~ed on the transfer of metal co~p~ from a cop~er nitr~t~ or cobalt ~ltra~e/trietha~ola~ine rea~ent after extraction o~ the free fatty aeid~ lnto a~ organic so1vent (3r 4)- The~e ~ethodJ are not specifia for the chain length in fatty ac1d~. ~inc~ short-chaln ~atty acid~ ~epresent appr~xLmately 5 to 6~ of the total ~atty acid6, their presence or ~b6enc~ only have a Minor effect on the end valu~. Equally, ~t 1~ ~o~sible to determine ~ree fatty acidY by ga~ chromato~raphy (5, 6). T~is method ~llow~ each f~tty acid to be d~ter~ined sepa-rately, but the qu~ntitative ~eparation of short-ch~in fatty ac id~ d2pends ta a hlgh deqree on the m~thod whiah ha~ been u~e~ for isolatin~ free fat~y aci~ fro~ a ~ple, ~akin~ the procedure g~nerally complicated. It has ~urthermore heen prapo~od to d~termlne ~ree fatty acids en2ymatically (7, 8)~ This method i~ ba~ed on the reeording of hydrogen peroxlde which ~ ~ormed in t~o ~ucces~ive reactlon~ by the enzymes acyl-CoA synthetaso and acyl-CoA o~i~a~e. Thlq method ha~ ~ome adv~ntag~
with regard to ~eleotivity ~nd speed, ~ ~en~or ~yEt~m ~ed on thi~ ~e~hod alre~dy h~ving heen propo~ed ~9).
When t~is method 1B u~ed for de~criblng the rancidity of dai.ry productK, however, spec1ficity i~ a proble~. The enzyme acyl-CoR ox~da~e, which i~ co~merci~lly av~ilable, i 8 ~enerally h~ghly ~peciflc with regard to long-chaln , 208232~
d5--hOJ-lq~2 1~:35 P.EI
~atty acid~, but ha~ only very poor ~poclflcity for ~hort-chain f~tty a~ids, in particu'lar butyric acid (7 a) .
~ ccorqing to the inv~ntion, it h~ now been found th~t the mi.croorgani~m Arthrobac~.er nicoti~na ha~ an acyl-CoA oxlda~ ~ctlvlty, whlch 1~ hlghly ~pecific fo~
~hort-chain fatty acid6, ~o that this microorg~ni6m can be u~od ~ucce~3f~11y ~or the dete~ninatlon of ~hort-chaln f~tty ~cid~.
~ hore i~ therefore propo~ed accordinq to the inve~tion a r~pi~, 6xped~ent ~nd ~electlve proc~66 fo~
tho determinasion of ~hort-chain fatty ~cid6 using the micrOor~AniY~ mentioned in the flow-in~ection analyslY
techniqu~, in p~rticular for u6e in th~ analysis of dalry products.
The sen~or accordin~ to the invention co~pri~e6 a microorg~ni~m membrane, a ~rotective membrane and an oxy~en electrvde.
~ ccordlng to the inYent~on, the ~ensor can be u~ed ~or the an~ly~l~ of d~iry products. D~lry prod~ats can be a co~plex mixture of a large number o~ compou~d~, it helng cha~cterlRtic th~t the casein content can be very high. ~n ~portant crit~rion ln th~ 6elect~0n of a s~itable protectiv~ membrane i6 ~herefore to be ~oen in tho fact tb~t the oore ~lze of the ~mbrane i~ Gm~ll enough to prevent c~ein mol~cule~ from coming into contact with the mlcroorgani~m membrane. ~ dlalysLs membran~ hav~ng a cutoff limit of approxLma~.ely 12,000 can there~ore be used ~ prot~ctlve m~n~ran~ ~rom thi~
point of view.
The lnvention therR$ore r~late~ to a ~l~w in~ec-tion analy~ 8 ~yflt~m for short-chain fatty acl~, ln which a microbia~ fflectrode iB employed whlch ha~ the microorq~niem Arthrobacter nicotlana, a dialysis membrane and an o~ygen electrode. The respon~e of thi~ sy~t~m show~ high ~pecificity for ~hort-ch~in C~l2-fatty ~alds and a linear function of the concentratlon of ~utyr~a acid ln the rnn~e from O .11 to 1.7 mM. ~he ~mple . . . _ .
20~232~
C~ 492 18: 36 P.06 ~requency wa~ appro~imately 2n sa~p1es/h at a flow ~at~
of 1.0 ml~min. The microbial electrode i6 highly ~e1ec-tive and stable. The invention 1 juitab1~ for r~co~ding 11poly~L~ of raw miIk ~ring storage.
A further embQdimen~. of the lnvention re1ate~ to ~he u~e of a ~on~or acaor~in~ to the inventlon for the deCection of mast1ti~. Thih i~ becAu~e lt has been sh~l in lar~e-scale tri~l~ that ~he car~oxyl estera~e activity o m~Lstitis milk increa~o~ with the degree o~ ~ev~r1ty of the di~ea~e and that the correlation with m~ti~i~ d~t~
i~ high. ~he aCtivity of the carboxyl estera~e i~
~enerally re~tric~d to short-ch~in ~atty ac~d esterR
(12, 13). These rel~tion~h1ps are utili~ed by ~he u~e ~ccording to the i.nven~ion.
Opt~zation of ~n FIA ~ynte~. The pH-dependency of a microbial electrode i~ tested ln the pH range ~rom 6.~ to 7.5 (~lg. 2). ~ven ~hou~h, at the heginnlng, t~e r~tcs grcLdua11y increa~e a~ the p~ drop~, the reaction b~ffer i~ kept at approximately pH 7.0 in further experiment~, thi~ being wi~h a v10w to the ~ability of the microorgani~m ~ployed. If the flow rate is alte~ed in the range from 0.5 to 2.0 ml/min, both the ~e~ponae ~nd the base 1ine rever~ion time (90~) drop a6 the flow rate increa~es (~g. 3). The sele~tion of the flow rates i8 a compromi~e be~ween sensitivity and frequency of ~ampl~ng. If, for example, the flow rdto i6 l.O ml~m1n, the re~ponse of the ~en~or i~ .linear in the range from o 1.7 mM-n-butyric acid tPig. 4) ~ the accuracy of fi~ ~ucc~s~ive measurementu being better than l.~S. The frequcncy oi ~a~pling i~ ~pproxi~tely 20 samp1es/h.
Opt~ imm~ilizatLon condi~ions. P;.gu~ 5 ~hows the effect of the cell amount in ~ PVA membrane on th~
ro~ponso. ~aximum r~spon~e L~ found ~ cell ~mountA in the rAn~ fro~ 2 ~o 4 mg~om2. The re~ult o~ high con-cerItration~ o~ th~ ~icroorgani~ ~s very little re~pon~.
Speci~iclty o~ ~he r~pon~e for fatty ~cit~. It ~ known to provld~ standard Rol-ltion~ for a hu~n ~1Ood Rampl~ by mixing fro~ fat~y aoLd~ ~nd a buff~r con~inin~
20~232~
-lc~ ]U-lyg~ t~:36 F'.~3'?
bovine ~erur~ ~Ibumin (9~ 11). A solutien iu provided according ~o the invention hy mixing fatty acid~ ~nd n bu~fer containing c~sein, ~ince ~a~ein iu a maln protein in dairy p~odllctn. Table 1 shcws ~5 comparlson of the respon~ to v~riou~ speclos of fatty ~cid~ Ln milk. The ~onnur ~h~ws th~ hLgh ~p~clfloity ~o~ ~hort-chnLn fntty acid~ in th~ rang~ from but~ric acid ~C~0) to lauric acid (clzu)~ The6~ fatty asid~ ~re li:~erated by ~ilk lip~ o~
by b~cterial llpa~es and contribut~ ~ainly to the rancid odor of dalry product~ hes~ re~ults ~ug~e~t th~t tho Hen~or aoaording to tne invention i~ sui~ble for the d~te~min3t~0n of th~ r~ncid odor of dairy product~.
Table 1~ Compar~on of re~ponuec to v&riou~ fatty ~cid6 __ _ _ _ ~atty nc~.d Reco~ding Relative vallle ___ . _ ~ .. . .
Butyric ~scid 4:D 59 Caproic nci~ 650 62 Caprylic aci~ B:0 100 Capric aci.d 10;0 B0 LQ~r~c ~cid 12~0 4~
~yrl~tic ac~d 14-0 9 Palmitlc acid 16:0 Ste~r$c ~cid 18:D
Oleic A~id 18~1 1 Linolelc acid 18t2 6 SelectiYit~. To test the ~olectj.Yity of the microbial electrcde accordin~ to the invention, the re~ponseu to the following co~pound~ were dete~mirled:
c~u~in (3&), bovine uerum ~lbumin (~.04~ to~e ~4.6~), galac~e ~10 ~g~ lucoRe ~10 mg-~, L-glycine (3 mg-~), choline (30 mg-~), cre~tine t3 mg-%), urea (30 mq-~), L-lactate ~3 ~g~ citr~te (0.2~ cto-globulin (0.5~) and trioleln (1~). Each concentr~tion i~
hrought to a level cuRt.om~ry for ~lk according to the 2 ~ 2 ~
G'~ U/- 1 4Y~ I E: 3~7 P . E~8 broug~lt to a level cu~tom~ry for milk according to ~.he 1iterature (1). The~e compound~ give no re~ponRR. The method a~cording to the ~nvent.ion i~ b~ed on the appll-catlon of the mirobi~1 electrode accordinq to the invention which is co~ered wlth a dialysi6 membrane hn~lng a Rmflll por~ size.
In additlon, the ~lect~od~ is p1ac~d lnto a l~no with continuou~ ~low, which mcan~ that the re~id~nce tim~
of the in~ectod sampl~ tn the flow ~ell, which is prD-~ided with th~ microbial alectrode, i~ very short. If a compound with high diffu~ion rate thro~gh the di~1ysi6 ~e~br~n~ and hi.gh a~imilibility by thc immobiliæed microorgani~ Bhows a re~pon~e in the sy~tem i~ ~u~t be a 6pec lfLc aompo~nd.
Applioatien of thu microb~1 el~utrode. The microbi~l electrod~ according to the inv~ntlon L~ em-ployed for ~h~ d~termination of short-chain fatty aclds in milk. Pr~deter~nod amountR of butyrio acid/ or ~apric acid, are added to a commerclally available milk. A$ can be ~een from T~ble 2, ~ti~ctory dstect10n da-ta are obtained (95 to 1~ Yigure 6 shows the relationship be~ween the storage time of ~tirr~d raw mllk at 25C and the Ben~or re~pDn~e~ l~ha sen~or re~pon~Q in~ es~ wlth the ~tor~ge tlme of the raw mil~, ~hich #ugg~st~ thAt the system according to the inventi.on is suitab1e for recor~-in~ lipolysi~ of raw ~ilk during ~torage.
2Q8232~
`IC~ q~' I8: :~7 P. 09 Table 2~ ~termination of fatty ~cidR which hav~ ~e~n ad~ed to mil~
_ _ _ _ _ _ ~atty ~ddition Content Found RQC~V~
acid C~ncentr~ti~n (mM~
.
Butyrlc acid 0 0.65 0.65 0.28 O.g3- 0.8~ 9 0.56 1.21 1.2~ g~
1.12 1.7~ 1.69 ~5 2~80 3.4~ 3.39 9 CHpr~c 4cid 0 0.41 0.41 0.29 0.70 0.71 101 .58 0.99 0.9~ 99 1.45 1.86 1.86 100 ~ .
Shelf life aud operational sta~ility. The shel~
ll~e of the ~nobilL~ed mlcroorgani~m and the operationAl ~tability of the mlc~obial electro~e according to the invention i8 exa~ined. ~or thl~ purpn~, three microblal electrodeR are produced under identical condltion~
(Pig~re 7). The respon6e o~ the first eleotrode i~
~etermined on the d~y of product~on. s~foro the re~ining elect_ode~ ~re used, they are ~torcd at 5'C in a nutrient m~dium which contain~ butyric acid. No reduction in compari~on with ~he respon~e on d~y 1 1 R observed when th~ re~pon~e~ o~ the ~econd and the thlrd electrode ~xe determined on d~y 3 and ~y 7 after production.
Th~ operational stabili~y of t~e microbial electrod~ iY determined by u~in~ the third electrode for the continuou~ de~ermination of 10 sample~ in one day, op~rating time 5 h. AB can be ~een from Figure 7, ~o ~gnificant decrea6e in respon~ found, even after eight d~ys. The~e re~ults ~ow that tha microbial eleotrode accordin~ to the ~n~ention has ~ood Rt~billty, ~ven t~o weeks a~t~r production.
2082~2~
1 99~ ] ~ 8 The invention will be illu~trated in greater det~il hereinAfter with refersn~e to the fi~ures and experumental data.
~`igure 1: SchemQtic diagr~m of n ~low sy~tem~; C =
c~rrier ~o.llltion; P = pun~p; S = ~ample solu~ion; MC
mixirl(3 ~oll; ~'C = ~low cell ~lth mlcroblal ~lec~rod~
W = wa~te.
Figur~ 2: pH-d~p~ndency of 8 microbi~l elec~rode; butyric acid concantration 1.68 m~ flow rate 1.0 ~l/uin.
Fiquro 3: Effect of th~ flow rat~ on th~ ru~ponso t-~ ~nd ba~ line rever~ion time (o); bu~yric acid concantratlon 1.68 mM.
F~ure 4~ ~pioal courRe of a re~pon~e to butyric acids a = 0.11 r~s; b - 0.28 mM; c = 0.56 mM; d= 0.84 mM~ e -1.12 m~ f = 1.6R m~; g = 2.24 mM; h ~ 2.80 mM; i 3.36 m~.
Figure 5: ~ffect of the cell ~mount in the PVA membrane Dn the reRpon~e~ the condition~ corr~sponded to those in Figure 2.
Figure 6: Relation~hip between the ~tor~ge t~me of the ~tirred rnw mil~ ~t 2sC ~nd the sen~or re~pon~; the ~ensor re~pon~e~ ~e repre~ented in the form of the correJponding butyric ~a id conc~ntr~tion.
P~gur~ 7I Shelf life of an Lmmob$1ized m$croorganl~m () and operntlonal ~tability of ~ microblal electrode (o).
Mat~i~lu. Cn~ein (00dium s~lt from bovlne mllX) and dialy~ls membr~ne (cellulo~e tube from Slgma Chem~cal Co.; the c~llulo~e ~ube would retaln 90 to 99~ of cyto-ohrome C of MW 12,400 ~f a cytochrome C ~olutlon over 10 hours. Polyvinyl alcohol (PVA of MW ~pprox. 2~,000) by Serva Feinchemlca Co. The other reagents were u3ed wit~
analytlcal grade. ~s~llled water wa~ u~ed ln all proce-d4re~.
Culture and i~mobili~ation of the m~croorganism.
Arthrooacte~ nicotiana was grown for 18 hour~ under ~ro~lc condltion6 ~t 30'C ln a ~00 ml ~lask containing 25 ml of a medium ~ompo~ed of 0.3~ of yea~t ext~t, 0.5%
of peptone, 0.5& o~ dipDtaa~ium hydrogen pho~ph~te, 0.5~
_ _ ... .
' ' ' ' 2082~
Lt~-N3~)-19YB 18: ~8 P. 11 o~: kadlum [~ic] dihyd~oqen phosphate (in each ca~e weight~olume) anA 1% of butyri~ Acid (volume/volu~e).
The medium wa~ ~entrifuged for 10 minute~ ~t 4B00 rpm, and the cells which had been ~eparated by centri$~g~tion were washed with approxi~ately lno ml of a 0.~ percent:
~odlum c~lorld~ solution. A pred~termfned amo~t or the cell~ wa6 mixed with pota~l~m phouphate bu~fe~ (0.~ M;
pH 7.0) contain~nq 5~ of PVA.-The cell ~u~penslon wa~
~prayed on ~ ~la~ plate ~5 cm x 2 cm) ~nd dried for 3 hours at room t~mperature and then at 5C overnl~ht.
Construction of a microblal electrode. The mlcrobi~l slectrode employed had a mla~oorganl~m me~Prane, ~ Teflon membrane, a dialysis membrane and an oxy~en electrode ~Schott Ger~te GmbH ) . T~e PVA m~mbrane with the i~mob~lized mLcroorganism wa~ cut out in the ~hape of di~k~ (5.9 m~ d~amete~) and attached on tep of the Teflon membrane of the oxygen electrode. rhe dialy~i~
m~mbrane was uRed a~ a pr~te~tiv~ membrane to cover the miaroo~gani~m membrsne on the electrode and to hold it in place.
Fr~o fatty acld~ wers emulslf~ed in the buffer ~olut~on by u~ing ca~ein; a pota~iu~ pho~phate buf~er (0.1 M) containing 3~ of case~n (welght~vol~e) wau used aA the reac~ion ~uf fer. Free fatty aclds were added to thifi solution and emulsified by ult~onic tre~tment (10 minute~); the ~mul~io~ form~d in thi6 mannor wau u~ed as eample ~olution. The e~ul6ion was ~table for one day.
FIA ~yBtQII. Flgu:re 1 ~hows a ~c~lematic diagram of the flow ~stem. PotaGoium pho~phate buffer (0.1 M, pH
7.0) wa~ u~ed a~ oarri~r solution. ~he carrie~ ~olution togethor with ln~ected sample ~ol~tion t35.5 ~1) wa~
pumped through a mixing coil (internal di~meter 0.8 mm and length 79 cm) and transpor~ed to a flow oell equippGd wi~h the microbial electrode. When the flow rate wa~ uet At 1 . O ml/min, tho disperhion of the ~ample ~olution injectud w~ ~pprDximately ~ a~urument~ were taken at ~OUC, the peak size being recorded a~ the re~pon~e.
GeSQ11ffChaft fiir siot0chnologl~ah2 Por~chung mbll ~GnF)~
Ma~cheroder weg 1, 330~ ~runswick Rancid or ~e~erlorated odor of milk and dalry products i~ caused by th~ liberatlon of short-cha1n C4,2-atty acld~ hy m~lk 11pasQ or by bactexi~ P~eD (1, 2). Th~ de~eation of ~hort-chdill ~atty acidY i 8 therefore hl~hly important in th~ q~alitative a~es~ment of d~iry product~ in the milk-collection method~ encountered in practice and for the prolonged ~toxage of ~aw ~ilk.
To date, th~ ~uantita~ive determination of free fatty acid~ h~6 be~n carried out by titrimetrlc detor-mlnation of the total aoid or by colorimetric analyses ~a~ed on the transfer of metal co~p~ from a cop~er nitr~t~ or cobalt ~ltra~e/trietha~ola~ine rea~ent after extraction o~ the free fatty aeid~ lnto a~ organic so1vent (3r 4)- The~e ~ethodJ are not specifia for the chain length in fatty ac1d~. ~inc~ short-chaln ~atty acid~ ~epresent appr~xLmately 5 to 6~ of the total ~atty acid6, their presence or ~b6enc~ only have a Minor effect on the end valu~. Equally, ~t 1~ ~o~sible to determine ~ree fatty acidY by ga~ chromato~raphy (5, 6). T~is method ~llow~ each f~tty acid to be d~ter~ined sepa-rately, but the qu~ntitative ~eparation of short-ch~in fatty ac id~ d2pends ta a hlgh deqree on the m~thod whiah ha~ been u~e~ for isolatin~ free fat~y aci~ fro~ a ~ple, ~akin~ the procedure g~nerally complicated. It has ~urthermore heen prapo~od to d~termlne ~ree fatty acids en2ymatically (7, 8)~ This method i~ ba~ed on the reeording of hydrogen peroxlde which ~ ~ormed in t~o ~ucces~ive reactlon~ by the enzymes acyl-CoA synthetaso and acyl-CoA o~i~a~e. Thlq method ha~ ~ome adv~ntag~
with regard to ~eleotivity ~nd speed, ~ ~en~or ~yEt~m ~ed on thi~ ~e~hod alre~dy h~ving heen propo~ed ~9).
When t~is method 1B u~ed for de~criblng the rancidity of dai.ry productK, however, spec1ficity i~ a proble~. The enzyme acyl-CoR ox~da~e, which i~ co~merci~lly av~ilable, i 8 ~enerally h~ghly ~peciflc with regard to long-chaln , 208232~
d5--hOJ-lq~2 1~:35 P.EI
~atty acid~, but ha~ only very poor ~poclflcity for ~hort-chain f~tty a~ids, in particu'lar butyric acid (7 a) .
~ ccorqing to the inv~ntion, it h~ now been found th~t the mi.croorgani~m Arthrobac~.er nicoti~na ha~ an acyl-CoA oxlda~ ~ctlvlty, whlch 1~ hlghly ~pecific fo~
~hort-chain fatty acid6, ~o that this microorg~ni6m can be u~od ~ucce~3f~11y ~or the dete~ninatlon of ~hort-chaln f~tty ~cid~.
~ hore i~ therefore propo~ed accordinq to the inve~tion a r~pi~, 6xped~ent ~nd ~electlve proc~66 fo~
tho determinasion of ~hort-chain fatty ~cid6 using the micrOor~AniY~ mentioned in the flow-in~ection analyslY
techniqu~, in p~rticular for u6e in th~ analysis of dalry products.
The sen~or accordin~ to the invention co~pri~e6 a microorg~ni~m membrane, a ~rotective membrane and an oxy~en electrvde.
~ ccordlng to the inYent~on, the ~ensor can be u~ed ~or the an~ly~l~ of d~iry products. D~lry prod~ats can be a co~plex mixture of a large number o~ compou~d~, it helng cha~cterlRtic th~t the casein content can be very high. ~n ~portant crit~rion ln th~ 6elect~0n of a s~itable protectiv~ membrane i6 ~herefore to be ~oen in tho fact tb~t the oore ~lze of the ~mbrane i~ Gm~ll enough to prevent c~ein mol~cule~ from coming into contact with the mlcroorgani~m membrane. ~ dlalysLs membran~ hav~ng a cutoff limit of approxLma~.ely 12,000 can there~ore be used ~ prot~ctlve m~n~ran~ ~rom thi~
point of view.
The lnvention therR$ore r~late~ to a ~l~w in~ec-tion analy~ 8 ~yflt~m for short-chain fatty acl~, ln which a microbia~ fflectrode iB employed whlch ha~ the microorq~niem Arthrobacter nicotlana, a dialysis membrane and an o~ygen electrode. The respon~e of thi~ sy~t~m show~ high ~pecificity for ~hort-ch~in C~l2-fatty ~alds and a linear function of the concentratlon of ~utyr~a acid ln the rnn~e from O .11 to 1.7 mM. ~he ~mple . . . _ .
20~232~
C~ 492 18: 36 P.06 ~requency wa~ appro~imately 2n sa~p1es/h at a flow ~at~
of 1.0 ml~min. The microbial electrode i6 highly ~e1ec-tive and stable. The invention 1 juitab1~ for r~co~ding 11poly~L~ of raw miIk ~ring storage.
A further embQdimen~. of the lnvention re1ate~ to ~he u~e of a ~on~or acaor~in~ to the inventlon for the deCection of mast1ti~. Thih i~ becAu~e lt has been sh~l in lar~e-scale tri~l~ that ~he car~oxyl estera~e activity o m~Lstitis milk increa~o~ with the degree o~ ~ev~r1ty of the di~ea~e and that the correlation with m~ti~i~ d~t~
i~ high. ~he aCtivity of the carboxyl estera~e i~
~enerally re~tric~d to short-ch~in ~atty ac~d esterR
(12, 13). These rel~tion~h1ps are utili~ed by ~he u~e ~ccording to the i.nven~ion.
Opt~zation of ~n FIA ~ynte~. The pH-dependency of a microbial electrode i~ tested ln the pH range ~rom 6.~ to 7.5 (~lg. 2). ~ven ~hou~h, at the heginnlng, t~e r~tcs grcLdua11y increa~e a~ the p~ drop~, the reaction b~ffer i~ kept at approximately pH 7.0 in further experiment~, thi~ being wi~h a v10w to the ~ability of the microorgani~m ~ployed. If the flow rate is alte~ed in the range from 0.5 to 2.0 ml/min, both the ~e~ponae ~nd the base 1ine rever~ion time (90~) drop a6 the flow rate increa~es (~g. 3). The sele~tion of the flow rates i8 a compromi~e be~ween sensitivity and frequency of ~ampl~ng. If, for example, the flow rdto i6 l.O ml~m1n, the re~ponse of the ~en~or i~ .linear in the range from o 1.7 mM-n-butyric acid tPig. 4) ~ the accuracy of fi~ ~ucc~s~ive measurementu being better than l.~S. The frequcncy oi ~a~pling i~ ~pproxi~tely 20 samp1es/h.
Opt~ imm~ilizatLon condi~ions. P;.gu~ 5 ~hows the effect of the cell amount in ~ PVA membrane on th~
ro~ponso. ~aximum r~spon~e L~ found ~ cell ~mountA in the rAn~ fro~ 2 ~o 4 mg~om2. The re~ult o~ high con-cerItration~ o~ th~ ~icroorgani~ ~s very little re~pon~.
Speci~iclty o~ ~he r~pon~e for fatty ~cit~. It ~ known to provld~ standard Rol-ltion~ for a hu~n ~1Ood Rampl~ by mixing fro~ fat~y aoLd~ ~nd a buff~r con~inin~
20~232~
-lc~ ]U-lyg~ t~:36 F'.~3'?
bovine ~erur~ ~Ibumin (9~ 11). A solutien iu provided according ~o the invention hy mixing fatty acid~ ~nd n bu~fer containing c~sein, ~ince ~a~ein iu a maln protein in dairy p~odllctn. Table 1 shcws ~5 comparlson of the respon~ to v~riou~ speclos of fatty ~cid~ Ln milk. The ~onnur ~h~ws th~ hLgh ~p~clfloity ~o~ ~hort-chnLn fntty acid~ in th~ rang~ from but~ric acid ~C~0) to lauric acid (clzu)~ The6~ fatty asid~ ~re li:~erated by ~ilk lip~ o~
by b~cterial llpa~es and contribut~ ~ainly to the rancid odor of dalry product~ hes~ re~ults ~ug~e~t th~t tho Hen~or aoaording to tne invention i~ sui~ble for the d~te~min3t~0n of th~ r~ncid odor of dairy product~.
Table 1~ Compar~on of re~ponuec to v&riou~ fatty ~cid6 __ _ _ _ ~atty nc~.d Reco~ding Relative vallle ___ . _ ~ .. . .
Butyric ~scid 4:D 59 Caproic nci~ 650 62 Caprylic aci~ B:0 100 Capric aci.d 10;0 B0 LQ~r~c ~cid 12~0 4~
~yrl~tic ac~d 14-0 9 Palmitlc acid 16:0 Ste~r$c ~cid 18:D
Oleic A~id 18~1 1 Linolelc acid 18t2 6 SelectiYit~. To test the ~olectj.Yity of the microbial electrcde accordin~ to the invention, the re~ponseu to the following co~pound~ were dete~mirled:
c~u~in (3&), bovine uerum ~lbumin (~.04~ to~e ~4.6~), galac~e ~10 ~g~ lucoRe ~10 mg-~, L-glycine (3 mg-~), choline (30 mg-~), cre~tine t3 mg-%), urea (30 mq-~), L-lactate ~3 ~g~ citr~te (0.2~ cto-globulin (0.5~) and trioleln (1~). Each concentr~tion i~
hrought to a level cuRt.om~ry for ~lk according to the 2 ~ 2 ~
G'~ U/- 1 4Y~ I E: 3~7 P . E~8 broug~lt to a level cu~tom~ry for milk according to ~.he 1iterature (1). The~e compound~ give no re~ponRR. The method a~cording to the ~nvent.ion i~ b~ed on the appll-catlon of the mirobi~1 electrode accordinq to the invention which is co~ered wlth a dialysi6 membrane hn~lng a Rmflll por~ size.
In additlon, the ~lect~od~ is p1ac~d lnto a l~no with continuou~ ~low, which mcan~ that the re~id~nce tim~
of the in~ectod sampl~ tn the flow ~ell, which is prD-~ided with th~ microbial alectrode, i~ very short. If a compound with high diffu~ion rate thro~gh the di~1ysi6 ~e~br~n~ and hi.gh a~imilibility by thc immobiliæed microorgani~ Bhows a re~pon~e in the sy~tem i~ ~u~t be a 6pec lfLc aompo~nd.
Applioatien of thu microb~1 el~utrode. The microbi~l electrod~ according to the inv~ntlon L~ em-ployed for ~h~ d~termination of short-chain fatty aclds in milk. Pr~deter~nod amountR of butyrio acid/ or ~apric acid, are added to a commerclally available milk. A$ can be ~een from T~ble 2, ~ti~ctory dstect10n da-ta are obtained (95 to 1~ Yigure 6 shows the relationship be~ween the storage time of ~tirr~d raw mllk at 25C and the Ben~or re~pDn~e~ l~ha sen~or re~pon~Q in~ es~ wlth the ~tor~ge tlme of the raw mil~, ~hich #ugg~st~ thAt the system according to the inventi.on is suitab1e for recor~-in~ lipolysi~ of raw ~ilk during ~torage.
2Q8232~
`IC~ q~' I8: :~7 P. 09 Table 2~ ~termination of fatty ~cidR which hav~ ~e~n ad~ed to mil~
_ _ _ _ _ _ ~atty ~ddition Content Found RQC~V~
acid C~ncentr~ti~n (mM~
.
Butyrlc acid 0 0.65 0.65 0.28 O.g3- 0.8~ 9 0.56 1.21 1.2~ g~
1.12 1.7~ 1.69 ~5 2~80 3.4~ 3.39 9 CHpr~c 4cid 0 0.41 0.41 0.29 0.70 0.71 101 .58 0.99 0.9~ 99 1.45 1.86 1.86 100 ~ .
Shelf life aud operational sta~ility. The shel~
ll~e of the ~nobilL~ed mlcroorgani~m and the operationAl ~tability of the mlc~obial electro~e according to the invention i8 exa~ined. ~or thl~ purpn~, three microblal electrodeR are produced under identical condltion~
(Pig~re 7). The respon6e o~ the first eleotrode i~
~etermined on the d~y of product~on. s~foro the re~ining elect_ode~ ~re used, they are ~torcd at 5'C in a nutrient m~dium which contain~ butyric acid. No reduction in compari~on with ~he respon~e on d~y 1 1 R observed when th~ re~pon~e~ o~ the ~econd and the thlrd electrode ~xe determined on d~y 3 and ~y 7 after production.
Th~ operational stabili~y of t~e microbial electrod~ iY determined by u~in~ the third electrode for the continuou~ de~ermination of 10 sample~ in one day, op~rating time 5 h. AB can be ~een from Figure 7, ~o ~gnificant decrea6e in respon~ found, even after eight d~ys. The~e re~ults ~ow that tha microbial eleotrode accordin~ to the ~n~ention has ~ood Rt~billty, ~ven t~o weeks a~t~r production.
2082~2~
1 99~ ] ~ 8 The invention will be illu~trated in greater det~il hereinAfter with refersn~e to the fi~ures and experumental data.
~`igure 1: SchemQtic diagr~m of n ~low sy~tem~; C =
c~rrier ~o.llltion; P = pun~p; S = ~ample solu~ion; MC
mixirl(3 ~oll; ~'C = ~low cell ~lth mlcroblal ~lec~rod~
W = wa~te.
Figur~ 2: pH-d~p~ndency of 8 microbi~l elec~rode; butyric acid concantration 1.68 m~ flow rate 1.0 ~l/uin.
Fiquro 3: Effect of th~ flow rat~ on th~ ru~ponso t-~ ~nd ba~ line rever~ion time (o); bu~yric acid concantratlon 1.68 mM.
F~ure 4~ ~pioal courRe of a re~pon~e to butyric acids a = 0.11 r~s; b - 0.28 mM; c = 0.56 mM; d= 0.84 mM~ e -1.12 m~ f = 1.6R m~; g = 2.24 mM; h ~ 2.80 mM; i 3.36 m~.
Figure 5: ~ffect of the cell ~mount in the PVA membrane Dn the reRpon~e~ the condition~ corr~sponded to those in Figure 2.
Figure 6: Relation~hip between the ~tor~ge t~me of the ~tirred rnw mil~ ~t 2sC ~nd the sen~or re~pon~; the ~ensor re~pon~e~ ~e repre~ented in the form of the correJponding butyric ~a id conc~ntr~tion.
P~gur~ 7I Shelf life of an Lmmob$1ized m$croorganl~m () and operntlonal ~tability of ~ microblal electrode (o).
Mat~i~lu. Cn~ein (00dium s~lt from bovlne mllX) and dialy~ls membr~ne (cellulo~e tube from Slgma Chem~cal Co.; the c~llulo~e ~ube would retaln 90 to 99~ of cyto-ohrome C of MW 12,400 ~f a cytochrome C ~olutlon over 10 hours. Polyvinyl alcohol (PVA of MW ~pprox. 2~,000) by Serva Feinchemlca Co. The other reagents were u3ed wit~
analytlcal grade. ~s~llled water wa~ u~ed ln all proce-d4re~.
Culture and i~mobili~ation of the m~croorganism.
Arthrooacte~ nicotiana was grown for 18 hour~ under ~ro~lc condltion6 ~t 30'C ln a ~00 ml ~lask containing 25 ml of a medium ~ompo~ed of 0.3~ of yea~t ext~t, 0.5%
of peptone, 0.5& o~ dipDtaa~ium hydrogen pho~ph~te, 0.5~
_ _ ... .
' ' ' ' 2082~
Lt~-N3~)-19YB 18: ~8 P. 11 o~: kadlum [~ic] dihyd~oqen phosphate (in each ca~e weight~olume) anA 1% of butyri~ Acid (volume/volu~e).
The medium wa~ ~entrifuged for 10 minute~ ~t 4B00 rpm, and the cells which had been ~eparated by centri$~g~tion were washed with approxi~ately lno ml of a 0.~ percent:
~odlum c~lorld~ solution. A pred~termfned amo~t or the cell~ wa6 mixed with pota~l~m phouphate bu~fe~ (0.~ M;
pH 7.0) contain~nq 5~ of PVA.-The cell ~u~penslon wa~
~prayed on ~ ~la~ plate ~5 cm x 2 cm) ~nd dried for 3 hours at room t~mperature and then at 5C overnl~ht.
Construction of a microblal electrode. The mlcrobi~l slectrode employed had a mla~oorganl~m me~Prane, ~ Teflon membrane, a dialysis membrane and an oxy~en electrode ~Schott Ger~te GmbH ) . T~e PVA m~mbrane with the i~mob~lized mLcroorganism wa~ cut out in the ~hape of di~k~ (5.9 m~ d~amete~) and attached on tep of the Teflon membrane of the oxygen electrode. rhe dialy~i~
m~mbrane was uRed a~ a pr~te~tiv~ membrane to cover the miaroo~gani~m membrsne on the electrode and to hold it in place.
Fr~o fatty acld~ wers emulslf~ed in the buffer ~olut~on by u~ing ca~ein; a pota~iu~ pho~phate buf~er (0.1 M) containing 3~ of case~n (welght~vol~e) wau used aA the reac~ion ~uf fer. Free fatty aclds were added to thifi solution and emulsified by ult~onic tre~tment (10 minute~); the ~mul~io~ form~d in thi6 mannor wau u~ed as eample ~olution. The e~ul6ion was ~table for one day.
FIA ~yBtQII. Flgu:re 1 ~hows a ~c~lematic diagram of the flow ~stem. PotaGoium pho~phate buffer (0.1 M, pH
7.0) wa~ u~ed a~ oarri~r solution. ~he carrie~ ~olution togethor with ln~ected sample ~ol~tion t35.5 ~1) wa~
pumped through a mixing coil (internal di~meter 0.8 mm and length 79 cm) and transpor~ed to a flow oell equippGd wi~h the microbial electrode. When the flow rate wa~ uet At 1 . O ml/min, tho disperhion of the ~ample ~olution injectud w~ ~pprDximately ~ a~urument~ were taken at ~OUC, the peak size being recorded a~ the re~pon~e.
Claims (16)
1. Arthrobacter nicotiana strain with acyl-CoA
oxidase activity which is specific for C4-12-fatty acids.
oxidase activity which is specific for C4-12-fatty acids.
2. Arthrobacter nicotiana strain DSM 6707.
3. Acyl-CoA oxidase activity which is specific for C4-12-fatty acids and which can be isolated from an Arthro-bacter nicotiana strain according to Claim 1 or 2.
4. Process for the isolation of an Arthrobacter nicotiana strain according to Claim 1 or 2 or for the isolation of an acyl-CoA oxidase activity according to Claim 3, characterised in that - with the aid of a sensor comprising an oxygen electrode and any desired Arthrobacter nicotiana strain immobilized on a carrier, the strain is tested for acyl-CoA oxidase activity which is specific for C4-12-fatty acids, in the presence of an aqueous medium containing C4-12-fatty acids, and - a strain for which such an activity is detected is isolated.
5. Sensor for detecting fatty acids, comprising an oxygen electrode and an acyl-CoA oxidase activity, characterized by an acyl-CoA oxidase activity which is specific for C4-12-fatty acids.
6. Sensor according to Claim 5 or 6, characterized in that it furthermore comprises an acyl-CoA synthetase activity.
7. Sensor according to Claim 5 or 6, characterized in that the acyl-CoA oxidase activity and/or the acyl-CoA
synthetase activity is immobilized.
synthetase activity is immobilized.
8. Sensor according to one of Claims 5 to 7, charac-terized by an acyl-CoA oxidase activity in the form of an Arthrobacter nicotiana strain according to Claim 1 or 2.
9. Sensor according to Claim 8, characterized in that the Arthrobacter nicotiana strain is immobilized with the aid of polyvinyl alcohol, preferably on a polyvinyl alcohol carrier.
10. Sensor according to one of Claims 5 to 9, charac-terized in that the sensor is provided with a protective membrane which protects the acyl-CoA oxidase activity.
11. Sensor according to Claim 10, characterized by protective membrane made of cellulose.
12. Sensor according to Claim 10 or 11, characterized by a cellulose protective membrane having a cutoff limit of approximately 12,000.
13. Use of the sensor according to one of Claims 5 to 12 for determining the content of C4-12-fatty acids in liquid media, in particular in dairy products.
14. Use of the sensor according to one of Claim 5 to 12 for the detection of mastitis.
15. Use according to Claim 13 of 14 in flow injection analysis (FIA).
16. Use according to Claim 13 or 14, where the system in which the FIA is carried out provides the delivery (C) of a carrier solution, the delivery (S) of the sample solution, a pump (P), a mixing zone (MC), for example a mixing coil, a flow cell (FC) with a sensor according to one of Claims 5 to 11, and a discharge (W).
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4136844.4 | 1991-11-08 | ||
| DE4136844A DE4136844A1 (en) | 1991-11-08 | 1991-11-08 | Arthrobacter nicotiana strain with shorter-chain acyl-coA-oxidase activity |
| DEP4211046.7 | 1992-04-02 | ||
| DE4211046 | 1992-04-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2082324A1 true CA2082324A1 (en) | 1993-05-09 |
Family
ID=25908931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002082324A Abandoned CA2082324A1 (en) | 1991-11-08 | 1992-11-06 | Arthrobacter nicotiana strain, activity of the strain, process for the isolation of the strain, sensor with the activity and use of the strain |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0541108A1 (en) |
| JP (1) | JPH05260956A (en) |
| CA (1) | CA2082324A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4257730B2 (en) * | 2003-08-25 | 2009-04-22 | キッコーマン株式会社 | Acyl CoA oxidase, its gene, recombinant DNA, and method for producing acyl CoA oxidase |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5915625B2 (en) * | 1979-03-01 | 1984-04-10 | 東洋紡績株式会社 | Novel acyl coenzyme A oxidase and its production method |
| JPS5840466B2 (en) * | 1979-10-26 | 1983-09-06 | 東洋醸造株式会社 | Production method of acyl-CoA oxidase |
-
1992
- 1992-11-06 EP EP92119033A patent/EP0541108A1/en not_active Withdrawn
- 1992-11-06 CA CA002082324A patent/CA2082324A1/en not_active Abandoned
- 1992-11-09 JP JP4323632A patent/JPH05260956A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0541108A1 (en) | 1993-05-12 |
| JPH05260956A (en) | 1993-10-12 |
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