Bacterial spores especially endospores have been a major issue in food safety because they have a particular resistance to most antimicrobials. The most effective form of inactivation is to exploit thermal processing but the key issue is that whilst there may be a successful reduction in the spore count the quality of the products suffers as a consequence. Not only that but unwanted and even harmful products such as acrylamide can form as well as generating a host of other food processing contaminants.
Over the last few decades there has been a steady demand for milder, less abrasive processing technologies to deal with low acid foods for example. As a consequence spore inactivation has involved technologies such as electro-technologies including ohmic heating and pulsed electric field processing (PEF). Another technology of high regard is high-pressure thermal sterilization (HPTS). At the moment the mechanisms of inactivation of spores in particular have lagged very seriously behind the knowledge behind thermal pasteurisation and sterilization.
One of the main differences in thermal sterilization is the difference between the use of wet and dry heat technology in killing spores. Wet heat has the capacity to kill spores but differs from dry heat in the mechanism of killing.
The Nature Of Spores
The classic spore formers of note in food products are the Bacillus and Clostridium species which have always been problematic.
The spore itself is a very well protected structure made up of many layers. It has a core which contains all the genetic material it needs to direct protein synthesis surrounded by an inner membrane and then a germ cell wall. A larger cortex layer surrounds this germ cell wall. The cortex is enclosed by an outer membrane with a protective coat and then a structure called the exosporium completely surrounds that.
The coat contains a wide range of very interesting proteins which deserve discussion elsewhere. The cortex is made up of peptidoglycan layers. Generally, it is a very well protected structure and appears to be unique to bacterial spores.
In terms of resistance to wet heat, the exosporium and the coat show very little activity. Its not very clear at the moment whether the outer membrane has any role in offering resistance to wet heat. When we come to the cortex then we discover the bacterial spore has at its disposal the most resistance layer. It appears to operate in conjunction with the inner membrane which has all kinds of unusual, almost unique properties. For example this inner membrane has a low lipid mobility and low permeability. The core has many unique features too. It contains compounds such as dipicolinic acid and EPA along with a very low water content – as low as 25% of the wet weight of spores generally. This cortex too serves up considerable resistance to wet heat.
Dipicolinic Acid
A compound in the spore’s core which is a pyridine derivative. It has two carboxylic acid groups in the 2′- and 5′ position of a pyridine ring. At physiological pH, the two carboxyl groups are ionised. It’s importance is such that it makes up 20% of the core’s dry weight.
DPA is synthesized in the mother cell as it surrounds the developing spore which then absorbs it or transports it into the central core region. The compound is chelated with divalent cations especially calcium to form CaDPA (calcium dipicolinate). It functions in reducing the core water level which allows for the perceived level of dryness. When the spore starts to germinate, all the dipicolinic acid is released.
Wet Heat Resistance
It has been a long road of research to understand what has been happening here but most of the factors were discovered by the scientists Phil Gerhardt and Bob Marquis. They are:
- Sporulation temperature – a higher temperatures produces more resistant spores.
- The divalent cations interacting with dipicolinic acid are very important especially that of calcium ions.
- The growth temperature optimum for bacterial cells is critical. Apparently, cells with a higher growth temperature optimum produce more heat resistance spores. A case bacteria species would be Geobacillus stearothermophilus which is the most renowned for this.
- The core water content – an inverse correlation exists between it and spore wet heat resistance. This is the most important factor. It seems the lower this water content the better the level of resistance.
The nature of the cortex along with the level of calcium dipicolinate in the core determine this core water content and to a lesser level, the sporulation temperature.
The DNA is protected by interactions with what are called alpha/beta-type small, acid soluble spore proteins.
In 2016, Kuipers and Wells-Bennik at the University of Groningen in the Netherlands found some extremely wet heat resistant bacterial spores from the Bacillus subtilis family in some commercially processed foods. These bacteria were resistant to wet heat at 100ºC and this was considerably much higher than that for wild-type B. subtilis spores which were killed at 85ºC.
A Transposon For Wet-Heat Resistance
The wet-heat resistance but not dry-heat resistance imparted to these bacteria was due to a transposon. The transposon has an operon called spoVA 2mob. The operon is only expressed in the developing spore. The operon has many genes associated with it. One in particular encodes a protein with two domains of unknown function called 2DUF. Deletion of the gene called 2duf almost eliminates this high spore heat resistance. The 2DUF protein is most likely a spore inner membrane protein but how it works is a complete unknown
There is a key figure in a paper by Berendsen et al., (2016) which looks at the decimal reduction time in minutes at 112.5 ºC (i.e. a time to achieve a ten-fold reduction in spore viability) versus the presence of operon in the B. subtilis spore. They looked at the decimal reduction time for a variety of different strains which contained no, 1,2, and 3 copies of the spoVA 2mob operon. If no operon is present, then it takes about ten or twelve seconds at 112.5 ºC to achieve a kill of 90%. As soon as the operon is present, the time to achieve a 90% kill is 1 minute, with two copies of the operon it is 10 minutes and with three copies of the operon, it becomes a staggering 20-30 minutes. Three copies give a five hundred fold level of resistance compared to spores without the operon. It shows how important the operon is to spore heat resistance.
What is fascinating here is what does that 2duf gene actually produce.
The Five Ways That Kill Spores
- DNA damage caused by dry heat and ultraviolet radiation
- Inner membrane damage where the spores are ruptured on germination. Hypochlorite and chlorine dioxide are effective agents for this.
- The rupture of the dormant spore’s inner membrane with the leaching out of the core contents. This includes especially the calcium dipicolinate. A strong acid will achieve this.
- Very strong alkali will chemically destroy an essential component of the germination apparatus. It may not kill the spores completely.
- Damage to a protein essential for spore viability and usually wet heat and probably hydrogen peroxide are effective for this.
Protein Damage Caused By Wet Heat Kills Spores
There is evidence for wet heat damage of critical proteins from a few studies by Peter Setlow.
Any survivors of spore populations from wet heat treatment which showed a 90% reduction in their numbers had no mutations or DNA damage. There is a release of calcium dipicolinate (CaDPA) from wet-heat treated spores that is quite some time after they are actually dead.
Wet heat killed spores can germinate relatively normally. If there is too much heat then CaDPA is released and there is no germination. Also, germinated wet-heat killed spores do not lyse but can no longer make any ATP which is the main reason why they die. It is reckoned this leaves only protein damage which are most likely core proteins. Small amounts of damaged proteins have been identified in heat-killed spores. It seems though that the specific protein that is damaged producing spore death remains to be identified. It may be involved in cell metabolism and/or ATP production.
References
Berendsen, et al. (2016) ISMEJ 10 pp. 2633-2642
Leave a Reply