J Sci Food Agric 1997, 74, 199È208

Spoilage Processes and Proteolysis in Chicken asDetected by HPLCG-J E Nychas*

Agricultural University of Athens, Dept of Agricultural Industries, Lab of Food Microbiology &Biotechnology, Iera Odos 75, Athens 11855, Greece

and C C Tassou

National Agricultural Research Foundation, Institute of Technology of Agricultural Products, S Venizelou1, Lycovrisi 14123, Athens, Greece

(Received 10 April 1996 ; revised version received 10 September 1996 ; accepted 7 January 1997)

Abstract : Fresh poultry Ðllets inoculated or not with Pseudomonas fragi storedunder aerobic, vacuum and 100% packaging conditions to study the micro-CO2biological as well as the changes occurring in the concentration of glucose,lactate and water-soluble proteins, during the storage at 3 and 10¡C. It wasfound that lactic acid bacteria were the dominant organisms in inoculated oruninoculated poultry Ðllets stored under these conditions. The low-molecular-weight compounds such as glucose and L-lactate were found to decrease alwaysin these Ðllets during storage and the proÐle of water-soluble proteins as detectedby HPLC di†ered among the di†erent samples treated di†erently.

Key words : HPLC, proteolysis, microbiology of poultry meat, vacuum, modiÐedatmosphere.

INTRODUCTION

It is well known that several organisms growing onmuscle (meat or Ðsh) secrete a wide variety of extracellu-lar enzymes (Venugopal 1990) which are free to degradethe exogenous substrate thereby causing extensivedamage to muscle. In general, proteolysis and conse-quently slime production under aerobic conditions hasbeen attributed to pseudomonads (Gill and Newton1978 ; Nychas et al 1988 ; Schmitt and Schmidt-Lorenz1992a,b) and starts when the bacterial numbers reacharound 7È8 cfu g~1 (Dainty and Mackey 1992)log10and the concentrations of glucose and/or gluconate areexhausted (Nychas et al 1988 ; Lampropoulou et al1996). Dainty et al (1975) using gel electrophoresisreported that proteolysis in meat could not be detectedin either slime-inoculated with various bacteria (egAeromonas spp, Pseudomonas spp, Brochothrix ther-mosphacta, etc) or naturally contaminated beef untilspoilage odours and bacterial slime were evident.

* To whom correspondence should be addressed.

However, Tarrant et al (1973) and Porzio and Pearson(1980) employing SDS-PAGE techniques, demonstratedextensive degradation of myoÐbrillar proteins intoheavy meromyosin, light meromyosin and meromyosin.Recently, Schmitt and Schmidt-Lorenz (1992b) usingthe same technique in chicken carcasses stored at 4¡Cboth unpacked in desiccators as well as in the gas-permeable polyethylene foil, observed that there was anoticeable increase in low-molecular-weight peptides(less than 50 000 Da) and free amino acids. Theseauthors also noted that packed and unpacked carcassesspoiled in di†erent ways. The rate of growth and thecomposition of the developing microbial Ñora in themeat ecosystem during storage could account for thesedi†erences (Nychas 1994).

Reversed-phase HPLC (RP-HPLC) has been usedrecently to monitor casein hydrolysis from the action ofcell wall proteinases from L actococcus lactis (Reid et al1991) and cheese ripening (Gonzalez de Llano et al1995). Mottar et al (1985) applied HPLC analysis todetermine speciÐc proteolytic components producedfrom Gram-negative psychrotrophic bacteria in raw

199J Sci Food Agric 0022-5142/97/$17.50 1997 SCI. Printed in Great Britain(

200 G-J E Nychas, C C T assou

milk. The use of HPLC in food of animal origin islimited. In particular, HPLC has been used only eitherfor the identiÐcation of meats (Ashoor and Osman1988) and Ðsh species (Armstrong and Leach 1992) orfor the determination of various time/heated treatmentof muscle proteins (Davis and Anderson 1984).

The present work, which was part of the FLAIR89055 EU project, was undertaken to study the bac-terial breakdown of low-molecular-weight compoundsas well as proteins, in poultry Ðllets and also to examinethe possibility of using the HPLC for the evaluation ofspoilage processes in chicken during its storage invacuum, 100% or aerobic packaging condition.CO2

MATERIALS AND METHODS

Organism

Pseudomonas fragi, from the culture collection of theUniversity of Bath, UK, was used in this study. Pseudo-monas fragi was found to be the dominant organismamong other pseudomonads when meat (beef andpoultry) stored under vacuum or modiÐed atmospherepackaging (VP/MAP) conditions (Nychas 1994). Toprepare the inoculum, the organism was subcultured inappropriate broth medium (nutrient broth, Oxoid) at30¡C for 24 h ; an aliquot of the culture was diluted1 : 100 with quarter-strength RingerÏs solution to give acell density of c 7 cfu ml~1.log10

Meat

Poultry Ðllets without skin, were obtained from a localprocessing plant and transported under refrigeration toour laboratory within one hour. The Ðllets (c 70 g ;thickness c 0É5È0É6 cm) were fully immersed into anaqueous suspension of Pseudomonas fragi (log10 7É37

for 30 s and then drained for 5 s and storedcfu ml~1)at 0¡C for 1 h prior to packaging. For uninoculatedsamples the same procedure was followed replacing theaqueous suspension of P fragi with distilled sterilisedwater.

Packaging

After immersion, samples (inoculated with P fragi) insemi-rigid aluminium trays, were placed in polyethylenebags (Suprovac 90 supplied by Kempner, Witham, UK)with low oxygen, carbon dioxide and nitrogen (c 2É9,10É4 and 0É7 mm3 m~2 s~1 MPa~1 at 20¡C, 50% RH,respectively) permeability, the water vapour per-meability was c 3É05 ] 10~7 kg m~2 s~1 at 25¡C, 85%RH. The samples were stored either aerobically or invacuum or Ñushed with 100% The packs, in theCO2 .latter case, were evacuated and Ñushed twice before the

Ðnal treatment. Uninoculated samples were vacuumpacked in the same polyethylene bags. The bags fromall samples, were heat sealed with double seals. PoultryÐllets stored at 3 and 10¡C.

Microbiological examination

Samples (25 g poultry Ðllets) were weighed aseptically,added to sterile quarter-strength RingerÏs solution(225 ml), and homogenised in a stomacher (Lab Blender400, Seward Medical, London) for 60 s at room tem-perature. Decimal dilutions in quarter-strength RingerÏssolution were prepared and duplicate 1 ml or 0É1 mlsamples of appropriate dilutions poured or spread onthe following media : GardnerÏs (1966) STAA mediumfor Brochothrix thermosphacta (this medium was madefrom basic ingredients in the laboratory, and incubatedat 25¡C for 72 h) ; MRS pH 6É2 (Oxoid) for lactic acidbacteria, overlaid with the same medium and incubatedat 25¡C for 96 h under anaerobic conditions ; Pseudo-monas agar (Oxoid) medium supplemented withcetrimideÈfucidinÈcephaloridine (CFC) for Pseudomonasspp, incubated at 25¡C for 48 h.

Low-molecular-weight determination

Poultry Ðllets (25 g) was reduced to a Ðne suspension in225 ml cold water (3È5¡C) in a stomacher for 60 s. Aportion was Ðltered (Whatman No. 1) and the clear Ðl-trate stored at [80¡C before chemical analysis.

Glucose was assayed enzymatically by the GOD-PERID kit (Boehringer, Mannheim GmbH). L-Lactatewas assayed enzymatically by the method of Gutmannand Wahlefeld (1984). The colorimetric method ofSedmak and Grossberg (1977) was used for the determi-nation of water-soluble nitrogen substances.

HPLC analysis

From the above-mentioned Ðltrate (sample prepara-tion), a 5 ml portion was treated with triÑuoroaceticacid (TFA, 20 g litre[ 1, Ðnal concentration) and cen-trifuged for 5 min at 4000] g. Three millilitres ml ofthe TFA-soluble proteins were Ðltered (0É2 km;Millipore) and sodium azide (Ðnal concentration 2 glitre~1) was added to the clear Ðltrate to preserve thesample which was stored at [80¡C before HPLCanalysis.

Apparatus

The soluble-TFA protein proÐles were analysed in aSpectra Physics HPLC consisting of a Spectra PhysicsP2000 two-pump system, with a Rheodyne 7125 injec-tor (Ðtted with a 20 kl loop), connected with a Spectra

Detection of proteolysis in chicken by HPL C 201

Focus UV/Vis detector using low-inertia scanning tech-nology (similar to a photodiode array), supplementedwith the appropriate Spectra focus software running inan IBM 80386 OS/2 computer, with a 250 ] 4 mmMZ-SIL (MZ-Analytical, D-6500 Mainz) 300 7 kmC18column, using bu†er A (10 mM potassium phosphate,pH 7É2, in distilled water) and bu†er B(acetonitrile] bu†er A (60 : 40, v/v)). A linear gradientwas used from 0 to 7% bu†er B in 20 min with a Ñowrate of 0É5 ml min~1. Peak width was 12, peak thresh-old 600 and 0É068 AUFS at 280 nm. Spectroscopic datawere collected from 190 to 360 nm, and chromatogramswere monitored at 210, 220 and 280 nm. The reagentswere water ; pass distilled and deionised water through a0É45 km Ðlter membrane ; acetonitrile, analytical grade ;TFA, anhydrous, analytical grade.

Experimental set up and statistics

This type of experiment was carried out twice. In everysampling day two packages were drawn and the micro-biological and physiochemical analysis performed asdescribed above. The coefficient of variation betweensamples from the same experiment or between samplesfrom di†erent experiment was calculated with theSYSTAT (Systat Inc, Evanston, IL, USA).

RESULTS AND DISCUSSION

Microbiological changes

The initial microÑora of fresh poultry Ðllets (Table 1)comprised, in decreasing order of magnitude, Pseudo-monas spp, Brochothrix thermosphacta and lactic acidbacteria. The extent of the contribution of these to theÐnal Ñora depends on the packaging system used.

At 3 and 10¡C there was signiÐcant di†erence in thenumbers of these bacteria between any of the atmo-spheres. At 3¡C the 100% atmosphere increasedCO2the lag phase of all bacteria in either inoculated or notinoculated samples. Moreover, their numbers werealways lower at the end of storage period comparedwith the numbers of bacteria in samples stored undervacuum or aerobically. The numbers of Br ther-mosphacta and Pseudomonas spp generally increasedfaster than those of the lactic acid bacteria, and attainedthe highest numbers in the aerobic atmosphere, while in100% the numbers remained static or showed aCO2slight increase only. Growth of Br thermosphacta, andPseudomonas spp on vacuum-packed stored poultryÐllets was intermediate between those on the aerobicand 100% stored poultry. In contrast, lactic acidCO2bacteria grew faster under vacuum and 100% andCO2become the dominant organisms in either inoculated oruninoculated samples. It needs to be mentioned,

however, that despite inoculation of meat Ðllets withrelatively high numbers of P fragi, no major di†erencesin the developing and dominant microÑora were appar-ent at all three temperatures of storage, vis a� vis those ofthe uninoculated packs. Similar results have beenreported when meat (beef, pork and lamb) was inocu-lated with relatively high numbers of L isteria mono-cytogenes, Salmonella typhimurium, Salmonellaenteritidis, Staphylococcus aureus or Y ersinia entero-colitica and stored under VP/MAP at 0, 3, 5 and 10¡C(Nychas 1994). The developing and dominant micro-Ñora at all temperatures used in this study were appar-ent the same in both inoculated and uninoculatedpacks.

In general, it needs to be stressed that each of theatmospheres (VP/MAP) noted above selected a micro-bial Ñora dominated by Gram-positive bacteria(principally lactic acid bacteria and Brochothrixthermosphacta) rather than the Gram-negative micro-Ñora ones that develop on meat stored under chill con-ditions in a normal atmosphere (air). This is due to theinhibitory e†ect of on Gram-negative aerobic psy-CO2chotrophic bacteria (Nychas 1994 ; Davies 1995).

The microbiological results obtained in this study aresimilar with those reported in literature concerninguninoculated meat (beef, pork, minced beef) stored undersimilar conditions (Dainty and Mackey 1992 ; Drosinos1994 ;KakouriandNychas1994 ;Lampropoulouetal1996).Nychas(1994)foundthatthepreservativeactionofVP/MAPis not so much due to the control of the total microbialpopulationbuttotherestrictionofgrowthofthoseorganismshavingthepotentialtocausethemostrapiddeteriorationofmeat.Itisworthtonotethatnotonlythecontributionofeachmicrobialgroupdependsontheconcentrationof intheCO2gasmixturesbutalsotheselectionofstrainsÐnallygrownonmeat (Dainty and Mackey 1992).

Physicochemical changes

The changes in the concentration of glucose, L-lactateand in water-soluble proteins, are given in Table 2. Theconcentration of glucose decreased progressively almostin all samples. This decrease mainly occurred by the endof storage and was greater in higher temperatures.Similar observations were made for L-lactate. Indeed,the Ðnal L-lactate content in samples stored underaerobic conditions was signiÐcantly lower than in thosepacked under vacuum or with 100% Similarly,CO2 .the concentration of glucose and L-lactic decreased pro-gressively when samples inoculated or uninoculated,stored at 10¡C. It needs to be noted that the decrease ofthese two low-molecular-weight compounds (glucoseand lactate) was always delayed in samples Ñushed with100% Similar results have been reported fromCO2 .Nychas and Arkoudelos (1990), Kakouri and Nychas(1994), Drosinos (1994), Drosinos and Board (1995) and

202 G-J E Nychas, C C T assou

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Lampropoulou et al (1996) in uninoculated meat (meatjuice, minced beef, lamb, pork and poultry Ðllets)samples stored under VP/MAP conditions. Thedecrease of glucose and/or lactate could be due to theirutilisation from pseudomonads or lactic acid bacteria(Gill and Molin 1991 ; Kakouri and Nychas 1994 ; Dro-sinos and Board 1995), while the production of aceticacid have been attributed to lactic acid bacteria.

The changes of Coomassie blue compounds (nitrogencompounds with a molecular mass higher than[3000 Da) were measured throughout the storageperiod in this study. It was found that these compoundswere increased progressively in most samples storedunder all atmospheres tested with di†erent rate (Table2).

The changes in the concentration of the free aminoacids in Ðllets samples stored under vacuum pack oraerobic conditions was used from Schmitt and Schmidt-Lorenz (1992a,b) to monitor the microbial spoilage.They found that free amino acids increased in propor-tion to the colony counts. De Castro et al (1988) report-

ed that Gram-negative bacteria in refrigerated beefinvariably secrete aminopeptidases which could be mea-sured (free amino acids) for rapid bacteriological qualityof meat.

The above-mentioned water-soluble nitrogen com-pounds were further analysed with the HPLC. Theinitial day of storage, these were separated into sixpeaks (Table 3 ; Fig 1A), where the most hydrophilicproteins were Ðrst eluted followed by the less hydro-philic ones (Corran 1989). These peaks were presentalso at the end of experiment in all samples inoculatedor not with P fragi (Table 3) at both temperatures. Thenumber of the peaks increased in some cases with threenew ones (Table 3). In particular, the appearance of thenew hydrophilic peak number 1 (RT\ 4É33) was evi-dently only at the end of storage at 10¡C in both unin-oculated or inoculated with pseudomonads sampleswhile the hydrophobic peak number 8 (RT \ 12É36)progressively appeared in all samples stored at bothtemperatures (Table 3).

The retention times of the peaks, their concentration

TABLE 3The contribution (area %) of triÑuoroacetic acid-soluble nitrogen compoundsa elutedfrom a 250 ] 4 mm MZ-SIL 300 7 km column (j \ 215 nm), found to be presentC18initially and at the end of the storage of poultry Ðllets inoculated or not with Pseudo-

monas fragi, under di†erent packaging conditions at 3 and 10¡C

Peak no RT Days of storage

0 12 at 3¡C 7 at 10¡C

Air VP co2 Air VP co2Inoculated samples

1 4É33 Èa 4É8 È È 6É3 2É7 2É52 4É71 8É7 13É8 13É7 11É6 11É1 10É9 11É93 5É03 5É1 8É1 È 1É9 10É7 6É2 5É14 5É45 3É9 10É2 11É7 8É8 16É1 11É4 15É05 6É71 17É3 8É6 15É6 17É3 1É65 8É5 9É66 7É27 È 11É1 5É5 5É0 17É2 8É0 8É47 9É75 38É3 25É0 33É5 33É2 18É4 19É4 27É38 12É36 È 1É4 3É0 1É2 2É5 2É3 2É59 13É70 È È È È 2É1 È È

Uninoculated samples1 4É33 È È È È 2É9 2É2 0É22 4É71 8É7 8É0 6É5 9É8 5É9 8É1 7É13 5É03 5É1 2É0 3É0 È 10É3 2É0 2É14 5É45 3É9 4É8 5É5 4É5 8É7 9É5 9É45 6É71 17É3 19É1 17É1 29É6 9É0 19É0 21É66 7É27 È 5É2 5É4 È 5É5 È È7 9É75 38É3 43É5 49.5 41.0 42É9 45.9 44.18 12É36 È 0É8 3É3 È 1É4 È 2É39 13É70 È È È È 2É1 È È

a Each number is the mean of two samples taken from di†erent experiments (coefficientof variation of mean of samples taken from di†erent experiments \5%). Each samplewas analysed in duplicate (coefficient of variation of samples from the same experiment\0É7%).b È peak was not found.

Detection of proteolysis in chicken by HPL C 205

Fig 1. Chromatographic proÐle of soluble nitrogen compounds in triÑuoroacetic acid (20 g litre~1), at 215 nm, of chicken thighinoculated with Pseudomonas fragi, on day 0 (A) and after 12 days under aerobic (B), vacuum (C) and 100% (D) storageCO2conditions at 3¡C.

(area %) as well as their ultra violet (UV) spectra(results not shown), were used to evaluate the changesof these water soluble nitrogen compounds during thestorage (Table 3 ; Figs 1 and 2).

It needs to be noted that the retention times of peaksfrom 1 to 9 shown in Table 3, were very reproducible,although their UV spectra di†ers signiÐcantly (resultsnot shown), among the chromatographs of samplesstored under di†erent packaging conditions. Moreoverthe Ðnal concentration of the peaks present initially,

varies signiÐcantly among all the samples tested in thisstudy (Table 3). For example, the increase of concentra-tion of the hydrophilic (compared with the peaknumbers 7, 8, 9) peak number 4 (RT\ 5É45), at the endof storage period in inoculated samples, was 2È3 timeshigher, than the increase of this peak in uninoculatedsamples treated identically at 3¡C. Similarly, the inocu-lation of samples with pseudomonads inÑuenced theproteolysis of poultry Ðllets. Indeed the contribution ofthe most hydrophobic peak number 7 (RT\ 9É75)

206 G-J E Nychas, C C T assou

Fig 2. Chromatographic proÐle of soluble nitrogen compounds in triÑuoroacetic acid (20 g litre~1), at 215 nm, of chicken thighinoculated with Pseudomonas fragi, on day 0 (A) and after 12 days under aerobic (B), vacuum (C) and 100% (D) storageCO2conditions at 10¡C.

increased only in uninoculated samples (Table 3) whiledecreased in inoculated ones regardless the packagingand the temperature conditions applied.

These di†erences in the proÐle of water-soluble pro-teins may be due not only to the indigenous proteolyticmeat enzymes (autolysis ; Schmitt and Schmidt-Lorenz1992a,b) but also to reÑection of a di†erent degradationmechanism caused by the di†erent type and thenumbers of microbial Ñora which dominated in poultryÐllets as a consequence of the manmade ecosystemapplied in this study. For example, in this study at theend of storage period, lactic acid bacteria were the pre-

dominant organisms (c 108) in samples stored undervacuum pack and 100% while pseudomonads inCO2 ,poultry meat stored aerobically, regardless the inocu-lation or not with P fragi. It needs to be mentioned thatlactic acid bacteria although can produce extracellularproteinases, considered to be weakly proteolytic (Lawand Kolstad 1983) when compared with other groups ofbacteria, especially Pseudomonas spp.

The hypothesis of microbial proteinase involvementmust take into account factors controlling the inductionof such enzymes in proteolytic bacteria. It is well recog-nised that synthesis of microbial proteases is inÑuenced

Detection of proteolysis in chicken by HPL C 207

by the availability of nutrients. Synthesis of proteasesduring growth in simple nutrients suggests that thepresence of protein is not required for protease synthe-sis (Venugopal 1990). Indeed, Harder (1979) proposedthat (i) organisms produce very low basal levels ofextracellular enzymes in the absence of an inducer, and(ii) the regulation and extracellular production of pro-teinases are based on induction and end-product and/orcatabolite repression. For example, it is reported thatglucose inhibited proteinase production by a milkisolate of P Ñuorescens (Ju†s 1976). According to Gill(1976) and Nychas et al (1988), as long as low-molecular-weight componentsÈespecially glucoseÈareavailable, meat proteolysis is inhibited. Dainty et al(1975) and Gill and Newton (1980), reported also thatthe levels of protein or fat do not change during theonset of rigor nor are they substrates for microbialattack prior to the onset of spoilage.

In our samples there were always signiÐcant amountsof glucose and lactate (Table 2) while the proteolysiswas evident regardless the microbial level. This is inaccord with the Ðndings of Schmitt and Schmidt-Lorenz(1992a,b) who demonstrated protein breakdown withgel electrophoresis, concluded that the breakdown ofproteins could occur in Ðllets at relatively early stage ofstorage regardless of microbial numbers. In contrast,Dainty et al (1975) have reported that proteolysis startswhen the bacterial numbers reach around 107È108 cfu g~1.

The Ðndings of this study add some information onproteolysis and may extend the use of HPLC on theunderstanding of meat proteolysis. It needs to bestressed however that more studies are needed to correl-ate microbial spoilage and proteolysis on the meatsurface.

ACKNOWLEDGEMENTS

This work was funded by the EEC DGXII in theFLAIR framework programme (FLAIR 89055 project).Technical assistance by Mrs A Kakouri for microbio-logical analysis is gratefully acknowledged. The authorswish to thank Dr E Drosinos for critical comments.

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