International Journal of Food Microbiology 128 (2009) 501–505

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International Journal of Food Microbiology

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Substrate utilization by Clostridium estertheticum cultivated in meat juice medium

Xianqin Yang, Sampathkumar Balamurugan ⁎, Colin O. GillAgriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C&E Trail, Lacombe, Alberta, Canada T4L 1W1

⁎ Corresponding author. Tel.: +1 403 782 8119; fax: +1E-mail address: balamurugans@agr.gc.ca (S. Balamur

0168-1605/$ – see front matter. Crown Copyright © 20doi:10.1016/j.ijfoodmicro.2008.10.024

a b s t r a c t

a r t i c l e i n f o

Article history:

Blown pack spoilage of vac Received 25 July 2008Received in revised form 8 October 2008Accepted 23 October 2008

Keywords:Clostridium estertheticumBlown pack spoilageMeat juice mediumSubstrate utilizationFermentation products

uum packaged beef, which results in packs being grossly distended with gas,is caused by the psychrophile Clostridium estertheticum. To determine what substrates are utilized byC. estertheticum during growth on beef, C. estertheticum subsp. estertheticum ATCC 51377, the type strain forthat organism, and two isolates from blown pack spoiled beef that were identified as C. estertheticum by 16 SrRNA gene sequencing were grown in meat juice medium at 10 °C for up to 14 days. Analysis of the growthmedia showed that all three organisms grew exponentially on glucose with simultaneous hydrolysis ofglycogen. Growth ceased when glucose in the media was depleted; but hydrolysis of glycogen continued at areduced rate, and lactate was consumed rapidly. The pH values of media fell during growth of the organisms,but rose as the concentrations of lactate subsequently decreased. The major products of fermentation duringutilization of glucose were butyrate and acetate, with butyrate greatly predominating. During fermentationof lactate the major products were butyrate and butanol, which were produced in similar amounts. Thefindings suggest that growth of C. estertheticum on vacuum packaged beef may be limited by the availabilityof glucose, as is the growth of other organisms that usually predominate in the flora of vacuum packagedmeat. However, production of gas by fermentation of lactate will likely continue after growth ceases.

Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.

1. Introduction

‘Blown-pack’ spoilage of vacuum packaged meats is characterizedby gross distension of the packs as a result of gas production bypsychrotolerant clostridia (Broda et al., 2003b). Although the involve-ment of psychrotrophic species in blown pack spoilage of processedand temperature abused meats has been reported (Broda et al., 1996,1999, 2000; Lawson et al., 1994), early blown pack spoilage of vacuumpackaged raw meats stored at near optimum chiller temperatures isapparently due to the psychrophilic species Clostridium estertheticumand Clostridium gasigenes. Spoilage by C. gasigenes, of chilled lamb andvenison, has been reported for only product from New Zealand (Brodaet al., 2002). However, C. estertheticum has been found to be the causeof blown pack spoilage of vacuum packed beef from South Africa,North America and northern Europe (Helps et al., 1999).

Blown pack spoilage of vacuum packaged beef occurs sporadically,with the condition developing in only a fraction of the packs in anyconsignment (Dainty et al., 1989). While the sporadic occurrenceof blown pack spoilage of beef may be due simply to variablecontamination of product with spores of C. estertheticum (Boeremaet al., 2003), the availability in themeat of substrates fermented by theorganism may play some part in determining whether or not blownpack spoilage develops. The availability of low molecular weight

403 782 6120.ugan).

08 Published by Elsevier B.V. All rig

substrates inmeat is known to affect the growth and spoilage activitiesof other meat spoilage bacteria (Nychas et al., 1998), but no study ofsubstrate utilization by C. estertheticum growing on beef has beenreported. Informationon thatmattermight be useful for explanation ofthe sporadic occurrence of blown pack spoilage, and might indicatepossible means for its control. Therefore, substrate utilization byC. estertheticum growing in a meat juice medium was investigated.Because of the possibility of variability in substrates utilization bydifferent strains of the organism, the utilization of substrates by boththe type strain and two isolates fromNorth America vacuumpackagedbeef recently spoiled by pack blowing was investigated.

2. Materials and methods

2.1. Maintenance and growth of bacteria

The bacteria used in the study were C. estertheticum subsp. es-tertheticum ATCC 51377, and two anaerobic, spore forming isolates froma vacuum pack of North American beef that had undergone blown packspoilage. The psychrophilic isolates were identified as C. estertheticumby 16S rRNA gene sequencing, using the primers of Broda et al. (2003a).The bacteria were maintained and cultured in prereduced media inHungate tubes thatwere flushedwith anoxygen free gasmixture of 80%N2, 10% CO2 and 10% H2 (Air Liquide Canada Inc., Red Deer, Alberta,Canada). Cultures were incubated and stored at temperature of 10 °C orless. The organisms were maintained in Reinforced Clostridial Medium

hts reserved.

Fig. 1. Values for the log of absorbance at 600 nm (log A600; ○) and the pH (●) of theculture of Clostridium estertheticum subsp. estertheticum ATCC 51377 growing in meatjuice medium incubated at 10 °C for up to 14 days. Data presented in the graph are themeans of duplicate determinations and their standard deviations were in the range of10–12% of the given values.

502 X. Yang et al. / International Journal of Food Microbiology 128 (2009) 501–505

(RCM; Oxoid, Mississagua, Ontario, Canada), and inocula for meat juicemediumwere cultivated in Peptone Yeast Extract Glucose Starch (PYGS)broth prepared according to Lund et al. (1990) using Difco materials(Becton–Dickinson, Sparks, MD, USA).

Meat juice medium (MJM; Gill, 1976) was prepared by pummelling100 g of extra lean beef with 100 ml of sterile, distilled water, in astomacher bag fitted with a filter, for 2.5 min, using a stomacheroperated at high speed. The stomacher fluid was withdrawn from thefilter sleeve of the bag and was transferred to a 500 ml flask. The fluidwas heated at 80 °C for 3 min and then was cooled on ice. The cooledfluid was filtered through a pad of cheese cloth to remove largeparticles, and then was filtered through a 0.2 μm membrane filter toobtain clear, sterile MJM.

Freshly prepared MJM was distributed in 7 ml volumes intoHungate tubes. Each tube of mediumwas supplemented with 0.15 mlof a 10% solution of bovine glycogen (Sigma–Aldrich, Oakville, Ontario,Canada) that had been sterilized by filtration through a 0.2 μmmembrane filter. The filled tubes were repeatedly flushed with theanaerobic gas mixture, and were repeatedly shaken, to obtain anoxicconditions in themedia. Initially, four groups of 6 tubeswere prepared.One group of tubes was not inoculated, one groupwas inoculated witha log phase culture of C. estertheticum subsp. estertheticum ATCC 51377,and the remaining groups were each inoculated with a log phaseculture of one of the presumptive C. estertheticum isolates. All groups oftubes were incubated at 10 °C. A tube from each group was withdrawnat the time incubation commenced and then at intervals of 2 days.Subsequently, four groups of 10 tubeswere prepared and inoculated asbefore. These groups of tubes were incubated at 10 °C for 14 days, witha tube being withdrawn from each group at times indicated by theresults from the initial groups of tubes.

When tubes were withdrawn from incubation, the absorbance at600 nm (A600) of each culture was determined using a spectro-photometer (Spectronic 20D; Thermo Electron, Pittsburgh, PA, USA)modified to accept Hungate tubes. Then, each tube was opened, thepH of the culture was determined using a pH electrode (AccumetAP61; Fisher Scientific, Ottawa, Ontario, Canada), and the culture wascentrifuged at 5000 ×g for 10 min at 4 °C. The supernatant wasdecanted and stored at −80 °C.

2.2. Analysis of supernatants

All analyses of each supernatant were performed in duplicate. A1ml portion of each thawed supernatant wasmixed with 5ml of 0.6 Nperchloric acid (Sigma–Aldrich) at 0 °C. A 0.2 ml portion of theacidified supernatant was neutralized with 0.02 ml of 3 M potassiumcarbonate, then incubated with 1 ml of amyloglucosidase solution(Sigma–Aldrich) at 40 °C for 2 h (Dalrymple and Hamm, 1973). Theenzymatic reactionwas stopped by addition of 0.2 ml of 3 N perchloricacid. After being held on ice for 10 min the mixture was centrifuged at12,000 ×g for 10 min at 4 °C. A 1.2 ml portion of the resultingsupernatant was withdrawn and neutralized by addition of 0.08 ml of3 M potassium carbonate. The preparationwas centrifuged at 3000 ×gfor 10 min at 4 °C and held overnight at 4 °C to allow potassiumperchlorate to precipitate.

The remaining acidified supernatant was centrifuged at 12,000 ×gfor 10 min at 4 °C. A 2.3 ml portion of the resulting supernatant waswithdrawn and neutralized by addition of 0.2 ml of 3 M potassiumcarbonate. The neutralized solution was centrifuged at 3000 ×g for10 min at 4 °C and then held overnight at 4 °C.

A 0.02 ml portion of the preparation obtained using amylogluco-sidase was used for determination of glucose, and 0.02 ml portion ofthe neutralized solution obtained from the acidified supernatantwas used for the determination of glucose and lactate, by means of aglucose and lactate analyzer (2300 STAT Plus™; YSI Life Sciences,Yellow Spring, OH, USA). Glycogen was calculated as glucose from thedifference between the amounts of glucose determined from the

amount found in the amyloglucosidase-treated preparation and theamount found in the neutralized supernatant.

Ammonia in the neutralized supernatant was determined by themethod of Mondzac et al. (1965) using a commercial kit (AmmoniaAssay Kit; Sigma–Aldrich) according to the manufacturer's instruc-tions. Total free amino acids were determined using a modifiedfluorometric method (Fisher et al., 2001). A 0.01 ml portion of theneutralized supernatant was mixed with 1 ml of a solution of o-phthaldialdehyde-β-mercaptoethanol (OPA-MET) reagent, which wasprepared by dissolving 100 mg of OPA in 10 ml of methanol, adding0.2 ml of MET to the solution, and then transferring the mixture to400 ml of 0.02 M borate buffer, pH 9.2. All components of the solutionwere obtained from Sigma–Aldrich. The fluorescence of the mixturewas measured using a luminescence spectrofluorometer (Model LS-50B; Perkin–Elmer, Norwalk, CT, USA) operated with excitation andemission detection wavelengths of 340 nm and 440 nm, respectively.

A further 1ml portion of each thawed supernatant wasmixed with0.2 ml of 25% (v/v) phosphoric acid then centrifuged at 14,000 ×g for5 min. The resulting acidified supernatant was used for determinationof volatile fatty acids (VFA) and alcohols by gas chromatography, at theDepartment of Agricultural, Food and Nutritional Science, Universityof Alberta, Edmonton, Alberta, Canada. For determination of fattyacids and alcohols, a 1 μl portion of the acidified supernatant wasinjected into a gas chromatograph (Model 3400; Varian Inc., WalnutCreek, CA, USA) fitted with Stabilwax-DA column (30 m×0.53 mID×0.5 μm film thickness; Restek Corporation, Bellefonte, PA, USA).The apparatus was operated in split mode with a split ratio of 1:20,with helium at a column head pressure of 7.5 psi. The injectortemperature was 170 °C. The initial column temperature of 35 °C wasmaintained for 3 min, and then the temperaturewas ramped to 190 °Cat 20 °C/min and held at 190 °C for 4 min. The flame ionizationdetector temperature was 190 °C. Peak integration was performedusing the Galaxie Chromatography Data system (Varian Inc.), withpeaks being identified by reference to external and internal standards.

3. Results

The data presented for substrate or fermentation productconcentrations are the means of duplicate determinations of eachconcentration. Growth of C. estertheticum subsp. estertheticum ATCC51377 in meat juice medium ceased after 4 days incubation at 10 °C(Fig. 1). The pH of the medium decreased progressively until that time,and then increased.

Fig. 2. Values for the concentrations of glucose (○), glycogen (●) and lactic acid (□) inmeat juice medium used for the cultivation of Clostridium estertheticum subsp. es-tertheticum ATCC 51377 at 10 °C for up to 14 days. Data presented in the graph are themeans of duplicate determinations and their standard deviations were in the range of10–12% of the given values.

Fig. 4. Values for the concentrations of acetic (○) and butyric acids (●), and ethanol (□)and butanol (■) in meat juice medium used for the cultivation of Clostridiumestertheticum subsp. estertheticum ATCC 51377 at 10 °C for up to 14 days. Data presentedin the graph are the means of duplicate determinations and their standard deviationswere in the range of 10–12% of the given values.

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After 4 days incubation glucose was not detectable in the medium(Fig. 2). The rate of utilization of glycogen was rapid between days 2and 4, but it was relatively slow before and after that. Lactate was notutilized before day 4, but subsequently it was utilized rapidly until day8, after which the rate of utilization progressively declined. Theconcentration of ammonia in the medium increased progressivelyduring incubation although no change in the concentration of freeamino acids was detected (Fig. 3).

The concentrationof acetate in themediumincreasedduring thefirst2 days of incubation but subsequently remained at a constant, low level(Fig. 4). The concentration of butyrate increased rapidly between days 2and 6, but subsequently the rate of increase was slower. Ethanol andbutanol were detected in themedium after 3 days. The concentration ofethanol did not increase until after 4 days, and the subsequent rate ofincreasewas slow. The concentration of butanol increased slowly atfirst,

Fig. 3. Values for concentrations of ammonia (○) and total free amino acids (●) in meatjuice medium used for the cultivation of Clostridium estertheticum subsp. estertheticumATCC 51377 at 10 °C for up to 14 days. Data presented in the graph are the means ofduplicate determinations and their standard deviations were in the range of 10–12% ofthe given values.

but then rapidly to reach a relatively high concentration after 14 days.There were no changes in the concentrations of any of the substancesthat were assayed during incubation of the uninoculated medium.

The two presumptive C. estertheticum isolates utilized substancesand produced fermentation products similarly to the type strain andthe results from one of the isolates is presented in Fig. 5. However,with the isolates, attainment of the maximum rate of growth wasdelayed, glucose was still detectable in the medium when growthceased, and the rates of utilization of lactate and pH increase after thecessation of growth were not so great as with the type strain. Theconcentration of butanol, acetic acid and butyric acid was 0.035 mM,3.23 mM and 2.970 mM, respectively, at 104 h at which time glucosewas almost depleted and lactate was beginning to be metabolised,leading mainly to an increase in butanol (1.682 mM) and butyric acid(18.2 mM) and very little increase in acetic acid (4.0 mM).

Fig. 5. Values for the log of the absorbance at 600 nm (A600;○) and pH (●) of cultures ofa presumptive Clostridium estertheticum isolate growing in meat juice medium, and theconcentration of glucose (□) and lactic acid (■) in the medium, when cultures wereincubated at 10 °C for up to 14 days. Data presented in the graph are the means ofduplicate determinations and their standard deviations were in the range of 10–12% ofthe given values.

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4. Discussion

Clostridium estertheticum ATCC 51377, the type strain isolated fromblown vacuum packs of South African beef, was originally described asa psychrophilic Clostridium that ferments glucose with the productionof butyrate and acetate (Collins et al., 1992). It was noted that whengrown on meat in vacuum pack, butyrate and acetate were producedin the ratio of four to one; and that butanol and butyl esters weredetected in the head space gas. Subsequently the organism wasrenamed C. estertheticum subsp. estertheticum, and the most abundantproducts of fermentation were identified as butyrate, acetate andformate (Spring et al., 2003). Another organism, isolated from blownpacks of North American beef and initially named Clostridiumlaramiense (Kalchayanand et al., 1993) was renamed C. estertheticumsubsp. laramiense. This organism was distinguished from C. esterthe-ticum subsp. estertheticum by several characteristics, including theability to utilize glycogen, which is present in most meat of normal pH(Immonen and Puolanne, 2000) and production of butyrate, lactate andbutanol as themost abundant fermentation products. The findings of thisstudymust raise some doubts about the current classification, as the typestrain of C. estertheticum subsp. estertheticum and the recent isolates fromthe blown vacuum packs of beef all utilized glycogen, produced mainlybutyrate and acetate during growth on glucose, but produced mainlybutanol and butyric acidwhenmetabolizing lactate.Whether or not othercharacteristics distinguish between two subspecies of C. estertheticumwould then seem to require further investigation.

All three organisms grew exponentially when utilizing glucose andglycogen simultaneously, but ceased growing when glucose wasexhausted and the rate of glycogen utilization decreased. The decreasein the rate of glycogen utilizationwas probably due to the extracellularamylase that must mediate its hydrolysis acting less effectively on theα-1,6 branching linkages of the glucose residues than on the α-1,4linkages of the initially exposed, unbranched chains (Maitin et al.,2001). Evidently, as glucose became undetectable in growth mediadespite the continuing decrease of glycogen concentrations, therelatively slow release of glucose from glycogen was inadequate tomeet the glucose fermenting capacities of the cultures at maximumnumbers.

Despite ceasing growth, C. estertheticum clearly initiated the rapidfermentation of lactate as the availability of glucose became limited,with the resultant reduction in the organic acid content of themedium reversing the pH decrease that occurred during glucosefermentation. Other clostridia that ferment lactate, such as Clostri-dium tyrobutyricum which causes late blowing spoilage of low acidcheeses (Ingham et al., 1998), can grow on that substrate only with thesimultaneous utilization of acetate or pyruvate (Rosenberger, 1956);and only when acetate at least is present at relatively highconcentrations (Bhat and Barker, 1947). Whether those organismsferment lactate without growth when the required amount of acetateor pyruvate is not available does not seem to have been ascertained(Senyk et al., 1989). None the less, the data suggest that might be sowith C. estertheticum in meat juice medium, in which the organismproduced little acetate during growth on glucose. That matter willrequire further investigation. However, it seems likely that themaximum numbers of C. estertheticum in the spoilage flora of vacuumpackaged meat are determined by the amount of glucose available forits growth, as is the case with the bacteria that usually predominate inmeat spoilage flora (Gill and Gill, 2005).

Further investigation of the utilization of amino acids will also berequired. Descriptions of the odours of, and analysis of the volatiles inthe head space gases of blown packs of beef indicate that break downof amino acids occurs during the development of blown packsspoilage (Dainty et al., 1989; Kalchayanand et al., 1989). The increasingconcentrations of ammonia with time of incubation of culturesobserved in this study indicate a continuous, low level utilization ofone or more amino acids. Small amounts of break down products

from, particularly, sulphur containing amino acids can contributegreatly to spoilage odours (Edwards and Dainty, 1987). Therefore,determination of the specific amino acids degraded by C. estertheticumduring its growth on meat would be of interest, as such activities maycontribute to spoilage odours that develop in otherwise normalvacuum packs as well as in blown packs of meat.

Acknowledgements

We thank Ms. F. Costello, I. Larsen and R. Thacker for theirassistance with glucose and lactic acid analyses, and Dr. K. Lien forperforming the analysis of volatile fatty acids and alcohols. Funding forthis study was provided by Alberta Livestock Industry DevelopmentFund.

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