By Eben van Tonder 
4 December 2022

Introduction

In an update I recently did to my review of curing systems, Bacon Curing – a Historical Review, I expanded the section on salt-only curing. Due to its relevance in terms of the recent emergence of “no nitrite” bacon that appeared on retail shelves around the world, including South Africa from the high-end retailer Woolworths, I also posted it as a stand-alone article, Evaluation of Woolworths “contain no nitrites” Bacon.

The question emerges how do bacteria penetrate meat? The relevance is that Richard Bosman and I are working tirelessly to create a curing system that uses bacterial fermentation. In order for meat to be cured, either an oxidation reaction of ammonium/ ammonia or L-Arginine is required or the reduction of nitrite is. (See my chapter in Bacon & the Art of living, The Curing Molecule) The focus of our work is on creating the former.

I posed a few initial questions about the relationship between the penetration of meat by bacteria and the percentage of salt used in a curing brine (with no nitrates and nitrites) to the inventor, scientist, entrepreneur and author, Greg Blonder. (For more on Greg visit Genuineideas) Greg set me on the path to the solution.

The pioneering work on the subject of the general penetration of bacteria in meat was done by the New Zealand researcher, C. O. Gill who was associated with the Meat Industry Research Institute of New Zealand in Hamilton. It is an area of meat science where remarkably little has been done since and I suspect, in light of the emerging new curing methods of meat fermentation which allows for an oxidation step to nitric oxide, will become a focus area over the next couple of years for researchers.

Preliminary Thoughts

Is Meat Intrinsically Sterile?

Meat is sterile if we exclude any bacteria contamination due to disease or injury. Gill (1979) writes that “it has been shown that muscle tissue from commercial carcasses is sterile if care is taken during sampling, the outer contaminated layers being first removed either by surgical techniques or by deep searing of the tissue with a hot template (Buckley et al. 1976; Gill et al. 1978).” He makes it clear this is the case across all species when he writes that “in addition to . . . work on mammals, there is also evidence that the flesh of fish and birds is usually sterile (Herbert el nl. 1971; Mead et al. 1973).” (Gill, 1979) Not only is the meat sterile, but “carcasses from normal healthy animals would appear to have considerable residual ability to maintain tissue sterility.” (Gill, 1979)

The clear fact is that meat is sterile on the inside and contamination with bacteria from the gut, post-slaughter is unlikely since “bacteria cannot pass across the intestinal wall nor penetrate muscle tissue until there is considerable breakdown of the tissue structure. Similarly, there is no movement of bacteria longitudinally within the intestinal wall until tissue breakdown is well advanced (Kellerman et al. 1976; Gill et al. 1976; Gill & Penney 1977). There is therefore no mechanism by which bacteria can pass from the intestine of dead animals to other tissues until at least several hours after death, the time involved being largely dependent on the temperature at which the carcass is stored.” (Gill, 1979)

Important Characteristics of Bacteria to Consider

We must be aware of the phases of bacterial growth.

We must also be aware of the fact that bacteria can be either proteolytic or non-proteolytic. Proteolytic bacteria is a type of bacteria that can produce protease enzymes, which are enzymes that can break down peptide bonds in protein molecules. The result of proteolysis is therefore the breakdown of proteins into smaller molecules catalyzed by cellular enzymes called proteases. (Shirai, 2017)

Proteolysis in dry-cured meat products has been attributed mainly to endogenous enzymes (Toldráet al. 1992a). On the other hand, Rodríguez (1998) found that “proteolysis on hams may be due not only to endogenous but also to microbial, enzymes.” Gill (1977) came to the same conclusion years earlier when they found that bacteria are confined to the surface of meat during the logarithmic phase of growth but when proteolytic bacteria approach their maximum cell density, extracellular proteases secreted by the bacteria apparently break down the connective tissue between muscle fibers, allowing the bacteria to penetrate the meat. Further, non-proteolytic bacteria do not penetrate meat, even when grown in association with proteolytic species. (Gill,1977)

In terms of the penetration of bacteria into fresh meat, Gill (1977) found that the “penetration of meat by nonmotile bacteria (i.e. not mobile) and the rapid rate of advance of invading microorganisms indicate that physical forces are involved in the movement of bacteria through meat. Non-proteolytic species do not invade in company with proteolytic species probably because, with mixed cultures, penetration originates in the area of growth of a microcolony of the proteolytic species so that the non-proteolytic bacteria are excluded. Protease production by bacteria does not occur until the end of logarithmic growth when the meat is in an advanced stage of spoilage. Therefore, unless the meat has been treated with a protease preparation to cause breakdown of the muscle structure, there should be no penetration of bacteria into organoleptically sound meat.” Gill (1977)

Also, “the proteolytic species were present between the muscle fibers throughout the meat, and some degradation of muscle fibers occurred. . . . Penetration of meat by bacteria apparently results from the breakdown of the connective tissue between muscle fibers by proteolytic enzymes secreted by the bacteria.” Gill (1977) Shirai (2017) quotes Gill (1984) when he stated that bacteria migrate into meat via gaps between muscle fibers and endomysia. Gill did not salt the meat as part of their experiments.

Is Bone-Taint Evidence of Intrinsic Bacteria?

Bone-taint is often given as evidence for the existence of intrinsic bacteria in the deep tissue regions of fresh meat. When discussing this, Gill (1979) says that “in hams which have been injected (pumped) with brine, any deep spoilage is likely to result from the injection of extrinsic bacteria.” (Gill, 1979) He rules out that bone-taint is evidence of intrinsic bacteria when he writes “‘bone-taint’ of hams is not unequivocal evidence for the occurrence of intrinsic bacteria.”

For all the possible causes for bone-taint considered by researchers by the 1970s, I give the complete paper by Gill (1979) below where he concludes that “it is clear that more than one condition is encompassed by this term (bone-taint), and it is possible that with beef carcasses a considerable proportion of the conditions so described are not the result of bacterial growth in deep tissues.” (Gill, 1979)

How Would Starter Culture Bacteria Enter Salt-Only Dry-Cured Meat

The question is relevant because I suggested in my comments on salt-only curing methods that bacteria play a role in oxidising nitrogen-containing elements to nitric oxide and that it is possible to have a curing system where no nitrite is used.

How Bacteria Can Enter Dry-Cured Meat

Gill did not dispute the fact that bacteria from the surface are able to penetrate meat. More recent studies have, however, shown this to be the case even for non-proteolytic bacteria also. Bosse (2015) studied the kinetics of migration of colloidal particles in meat muscles in the absence and presence of a proteolytic enzyme to simulate non-motile bacteria penetration. They concluded that “particles are able to diffuse into the densely packed fiber structure of meat muscles, which is contrary to the long-held belief that such penetration may not occur in the absence of extensive proteolysis or mechanical damage of tissue.” (Bosse, 2015)

Water and Salt: Changes to Microstructure of Meat

To develop a possible model of vectors facilitating the migration of bacteria to the deep tissue parts of meat, we consider the combined effect of water and salting.

Thorarinsdottir (2011) investigated the effects of salting and different pre-salting procedures (injection and brining versus brining only) on the microstructure and water retention of heavy salted cod products. They found that “salting resulted in shrinkage of fibre diameter and enlargement of inter-cellular space. Water was expelled from the muscle and a higher fraction became located in the extra-cellular matrix. These changes were suggested to originate from myofibrillar protein aggregation and enzymatic degradation of the connective tissue. During rehydration, the muscle absorbed water again and the fibers swelled up to a similar cross-sectional area as in the raw muscle. However, the inter-cellular space remained larger, resulting in a higher water content of the muscle in the rehydrated stage.” (Thorarinsdottir, 2011) Such water would undoubtedly contribute to the migration of bacteria during a starter culture containing brining of meat. Their observation is that when salt is rubbed on the meat surface and migrates into the meat, water is expelled from the muscle and a higher fraction which becomes located in the extra-cellular matrix will undoubtedly aid the migration of bacteria into meat in a salt-only curing system. The inter-cellular space is also enlarged during dry salting, believed to result from enzymatic degradation of structural components in the muscle during the first days of dry salting.

They state that the microstructural changes in dry-cured ham and these “have been related to proteolysis (as we developed above) and have been described as degradation of the proteins in the costamere and in the cell membrane. After curing, the Z-disks are no longer in line. It has also been observed that the myofibrillar bundle becomes more compact with a large number of empty spaces or gaps in between neighbouring myofibrils.” (Larrea et al., 2007).

Conclusion

Changes to the microstructure of dry-cured meat which results in water being expelled from the muscle to become located in the extra-cellular matrix is one of the likely routes for the migration of bacteria during salt only curing from the surface into the deeper tissue regions. Further, there is an increase in the inter-cellular space that was believed to result from enzymatic degradation of structural components in the muscle during the first days of dry salting. Besides this Staphylococcus xylosus, known for its ability to oxidise nitrogen and form Nitric Oxide is a proteolytic bacterium.

Further Reading

Hansen CL, van der Berg F, Ringgaard S, Stødkilde-Jørgensen H, Karlsson AH. Diffusion of NaCl in meat studied by (1)H and (23)Na magnetic resonance imaging. Meat Sci. 2008 Nov;80(3):851-6. doi: 10.1016/j.meatsci.2008.04.003. Epub 2008 Apr 11. PMID: 22063607.

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Reference

Bosse, R., Gibis, M., Schmidt, H., Jochen Weiss, J. (2015) Kinetics of migration of colloidal particles in meat muscles in the absence and presence of a proteolytic enzyme to simulate non-motile bacteria penetration. Food Research International, Volume 75, 2015, Pages 79-88, ISSN 0963-9969, https://doi.org/10.1016/j.foodres.2015.05.054. (https://www.sciencedirect.com/science/article/pii/S0963996915300429)

CALLOW EH. The action of salts and other substances used in the curing of bacon and ham. Br J Nutr. 1947;1(2-3):269-74. doi: 10.1079/bjn19470037. PMID: 18907930.

Gill CO, Penney N. Penetration of bacteria into meat. Appl Environ Microbiol. 1977 Jun;33(6):1284-6. doi: 10.1128/aem.33.6.1284-1286.1977. PMID: 406846; PMCID: PMC170872.

Gill C. O.. (1979) A Review Intrinsic Bacteria in Meat. Journal ofApplied Bacteriology 1979, 47,367-318

Rodríguez, M., Núñez, F., Córdoba, J. J., Bermúdez, M. E., Asensio, M. A.. (1998) Evaluation of proteolytic activity of micro-organisms isolated from dry cured ham. Applied Microbiology, Volume85, Issue5, November 1998, Pages 905-912, https://doi.org/10.1046/j.1365-2672.1998.00610.x

Shirai, H., Datta, A. K., Oshita, S.. (2017) Penetration of aerobic bacteria into meat: A mechanistic understanding,
Journal of Food Engineering, Volume 196, 2017, Pages 193-207, ISSN 0260-8774, https://doi.org/10.1016/j.jfoodeng.2016.10.012.
(https://www.sciencedirect.com/science/article/pii/S0260877416303739)

Thorarinsdottir, K. A., Arason, S., Sigurgisladottir, S., Gunnlaugsson, V. N., Johannsdottir, J., & Tornberg, E. (2011). The effects of salt-curing and salting procedures on the microstructure of cod (Gadus morhua) muscle. Food Chemistry, 126(1), 109-115. https://doi.org/10.1016/j.foodchem.2010.10.085