Potential value of the Limulus lysate assay for the measurement of meat spoilage

Abstract

Using a microtitre plate method the Limulus lysate endotoxin assay was shown to be capable of detecting Pseudomonas spp. at viable cell concentrations of 102-l@1/ml. The changes in endotoxin concentration, microbial flora, pH and ERV of laboratoryproduced minced beef and minced pork stored at 4°C were monitored. The endotoxin concentration was shown to increase with the onset of spoilage. Factors were derived enabling an estimate of AMC to be made from the endotoxin concentration determined by the Limulus lysate assay. The use of the microtitre plate system significantly reduces the cost of the Limulus lysate assay.

Accumulation of Stimulants of Toll‐Like Receptor (TLR)‐2 and TLR4 in Meat Products Stored at 5 °C

“…The accumulation of TLR2‐ and TLR4‐stimulants was minimized by storing meats in the intact, rather than in the minced form, and by storage under a modified atmosphere, rather than exposed to air. Stimulants of both TLRs correlated well with bacterial counts, which were very similar to those reported in previous studies of microbial growth in minced meat products stored in air or under a modified atmosphere (Fallowfield and Patterson 1985; Lambropoulou and others 1996; Jay and others 2003). This information, taken together with the results from our previous studies that showed that meat‐derived TLR‐stimulants are not likely to be SFAs (Erridge and Samani 2009) and are inhibited by both polymyxin‐B and oxidized palmitoyl‐arachidonyl‐phosphatidyl choline (Erridge 2010), strongly suggest that the TLR2‐ and TLR4‐stimulants identified in meats are of bacterial origin, being presumably BLPs and LPS, respectively.…”

“…Finally, the limulus system is insensitive to BLPs, and so cannot be used for their quantification (Erridge and Samani 2009). Despite these limitations, however, the LAL assay has been used previously to demonstrate that the endotoxin content of spoiling meat correlates well with microbial load, a finding supported by the present study (Fallowfield and Patterson 1985; Jay and others 1979). The LAL assay has also been used to estimate the amount of endotoxin present in minced beef stored at refrigeration temperature for 6 d to range from approximately 32 μg/g to approximately 2.5 mg/g (Jay and others 1979; Fallowfield and Patterson 1985).…”

“…Despite these limitations, however, the LAL assay has been used previously to demonstrate that the endotoxin content of spoiling meat correlates well with microbial load, a finding supported by the present study (Fallowfield and Patterson 1985; Jay and others 1979). The LAL assay has also been used to estimate the amount of endotoxin present in minced beef stored at refrigeration temperature for 6 d to range from approximately 32 μg/g to approximately 2.5 mg/g (Jay and others 1979; Fallowfield and Patterson 1985). The present findings suggest that TLR4‐stimulants (presumably LPS) in minced meats reached a maximum of approximately 860 ng/g under a modified atmosphere and 7 μg/g when exposed to air for 8 d. For the reasons outlined above, it is likely that previous reports may have significantly overestimated the level of TLR4‐stimulating LPS present in meat products.…”

“…An alternative method that has been widely used for the quantification of LPS in foodstuffs is the limulus amoebocyte lysate (LAL) assay (Jay and others 1979; Fallowfield and Patterson 1985). As discussed previously, however (Erridge 2010), the LAL assay was found to be not suitable for quantifying the biological activities of TLR‐stimulants in foodstuffs for a number of reasons.…”

“…Total aerobic mesophiles (aerobic plate counts, APC), pseudomonads, and Enterobacteriaceae were quantified in meat extracts according to previously described protocols (Fallowfield and Patterson 1985; Lambropoulou and others 1996). Briefly, serial 10‐fold dilutions of each meat homogenate were prepared in sterile PBS and plated onto plate count agar (APC, for determination of total aerobic mesophile plate count), violet red bile glucose agar (VRBGA, for enumeration of Enterobacteriaceae), or pseudomonas agar with cetrimide‐fucidin‐cephaloridine supplement (Oxoid), selective for pseudomonads.…”