The P-word

The European Commission set new limits for the use of nitrites and nitrates as food additives in 2008.  These chemicals are intended to protect against the growth of pathogenic bacteria such as Listeria, Salmonella, and Clostridium botulinum, while the EU limits are aimed at reducing exposure to nitrosamines.  We have long used nitrate and nitrite as preservatives in curing meat.  Nitrate and nitrite are permitted for use in foods in many countries such as Mainland China, the United States, the European Union, Canada, Australia and New Zealand.  The concern is that nitrate can be converted into nitrite in the gut and then form N-nitroso compounds such as nitrosamines, which may cause cancer in humans, though evidence as to whether nitrate or nitrite per se in food can cause cancer in humans is inadequate or limited.  We need to be very careful before branding food additives as causing human disease - we can find naturally occurring nitrates and nitrates in water and vegetables, especially in leafy and root vegetables such as lettuce, beets, celery, carrots, and more.  
There is a group of people who regard preservatives as being unhealthy and added by unscrupulous manufacturers.  They want their food to be “all-natural, with minimal processing and no chemicals”.  Apart from the fact that this description is meaningless, it got me thinking about additives and specifically preservatives.

As a first step in preparing this post, I followed standard research principles and surveyed the packaged foods in our kitchen cupboard and read all the ingredient labels - a challenge in itself for anyone with less than 20:20 vision.  It came as something of a surprise to find that few of the foods contained anything that I would regard as unnecessary in the food type.  Sure, there were acidity regulators and anti-caking agents, salt and the occasional antioxidant, natural colours and flavours, while some of the wines declared sulphur dioxide, but very few specific mentions of the dreaded preservatives.  A list of permitted additives can be found on the New Zealand Government food safety website.  [1]

From the microbiologist’s point of view, preservatives are added to prevent the growth of pathogens or spoilage organisms during the shelf life, reducing loss and increasing food safety.  Acidulants, organic acids and parabens have all been used, but ‘natural alternatives’ are being increasingly used.  Many leafy and root vegetables contain nitrate and nitrite and it is estimated that around 80% of the nitrates and nitrites we consume are naturally occurring from the plants we eat.  Nitrite may be formed by reduction of nitrate by bacteria in the food.

While I was at Hong Kong University, I co-supervised a PhD student who was studying the use of extracts of traditional Chinese herbs as food preservatives.  Several of the papers that came out of that work have been my most heavily cited, and it is clear that there is great interest in these natural chemicals in the preservation of meats and dairy products.  However, the potential for reliance on extracts may be limited because of source availability, and synthetic nature-identical versions of the active components may be necessary if natural preservatives are to be used extensively. 

Of course, manufacturers will often use chemical additives in conjunction with other preservation processes, such as refrigeration, freeze-drying or UV irradiation.  Boiling and addition of sugar, followed by sealing in airtight jars and packages, kills bacteria and prevents their regrowth and recontamination.  Canned foods are processed for long-term shelf stability and if the food is “low acid” i.e. with a pH greater than 4.5 and thus able to support the germination and growth of Clostridium botulinum spores, the heat process is designed to reduce the spore count by a factor of 1012, resulting in a sterile product that will keep for years, provided that the can remains sealed.

[1]  Identifying Food Additives.  ISBN No.: 978-1-99-004303-1 Online). https://www.mpi.govt.nz/dmsdocument/3433/direct



 

Germ Warfare

I’m sure that many people yearn for the glamorous life of the food microbiologist leading the war against microorganisms - yeah, right!  Perhaps those people visualise the microbiologist surrounded by modern laboratory equipment, such as PCR machines and gene sequencers, but the sad fact is that what is portrayed in films and television just isn’t reality.  It might be the case in forensic work or pathogen testing, but in the food industry, much of the work involves monotonous sampling and plate counting.

This is because there is a constant battle between food manufacturers and the microorganisms that can cause spoilage or food poisoning.  Food safety is assessed in relation to counts of specific bacteria, yeasts and moulds.  The International Commission on Specifications for Foods was formed in 1962 and wrote Microbiology of Foods Volume 2: Food Commodities in 1980.   This was updated in 1996 (1).  Our Microbiological Reference Criteria for Food were published by the Ministry of Health and Version 2 appeared in 1995.  The Reference Criteria are expressed in the ICMSF format as a guide to indicate when food can be considered unacceptable or unsafe.  

The result is that many food microbiologists and technicians spend a large part of their working day conducting plate counts on foods.  This is particularly so in the dairy industry, though large laboratories such as these may use automation for plating, such as spiral platers, robotic sample diluters, and image analysis for counting.  This is, of course, very expensive and involves a lot of plastic that probably cannot be recycled. Modern devices, such as the flow cytometer - a machine that counts particular cell types flowing through a tube by reflection of laser beams.  Against this background, it is disappointing that food-borne infections continue unabated in the USA, UK and Europe.  

Of course, some food microbiologists do lead an interesting life.  These are the Special Agents investigating food contamination, poisoning and spoilage.  While it might be argued that there is nothing new under the Sun, each case is different - human error, mechanical breakdown, packaging failures and poor equipment design may all figure in the systematic examination, leading to an understanding of what has gone wrong and a suggested solution.  Investigation often involves looking closely at the process and interviewing operators.  Access to a suitable microbiological testing laboratory is often essential in identifying the problem.

Looking back over many years as a consultant microbiologist, I can think of lots of investigations where the explanation was a surprise: 

A dried vegetable processing operation in which the product had spikes of contamination.  On this occasion, I was fortunate to see the problem immediately.  The vegetables passed through a steam blancher which was located under a cold air trunking.  Steam condensed on the trunking, which was covered in a thick black tarry layer, and every so often, condensate would fall onto the vegetables being conveyed to the drier.  A factory design fault.

Staphylococcus aureus contamination of canned product.  This was not a low acid food and therefore was not subject to a 12-D process.  The immediate suggestion was that the cans were leaking during cooling, sucking in cooling water.  However, it was found that the cans were dump loaded into a lidded vessel and hot water was introduced and held for the duration of the process schedule.  With a temperature above 75oC for a suitable time, the Staphylococci should have been destroyed.  We eventually discovered that the vessel was overloaded and some of the cans were above the water level, so did not receive the scheduled process.

In a small pie shop, minced meat for pie filling was cooked on a stove in large pans of about 35 L capacity, which were then put into a chiller to cool.  On occasion, the pies spoiled after the growth of Clostridial spores activated by the heating.  I found that the pans of meat took over 48 hours for the centre to cool to about 12oC.  The solution was simple - put the filling into shallow trays for cooling.

 Listeria monocytogenes was found in sliced meat.  The slicer was a standard design, but was very difficult to clean properly and dried meat residue was found in the transfer mechanism.  The slicer was mounted on a stainless steel table, and the whole area was meticulously cleaned.  However, the table surface was heavily scratched and two feet mounting spikes penetrated the stainless steel.  Unfortunately, the stainless steel was only a couple of millimetres thick and was laid on Medium Density Fibreboard (MDF).  The result was that the MDF got wet every time the table was cleaned and became colonised by L. monocytogenes.

These examples show that those working in processing facilities may be essentially blind to potential failures and a fresh set of eyes with appropriate experience may recognise the problems quickly. 



References

1.  ICMSF. (1996) Microoganisms in Foods 6:  Microbial Ecology of Food Commodities.
2.  Ministry of Health (1995)  Food Administration Manual S. 11: Microbiological Criteria

Still want to drink raw milk?

 I hesitate to post this and it may offend some readers, but the message is, in my opinion, entirely valid.  And, hey, we microbiologists have to have a little fun every now and again.

https://youtube.com/shorts/CuSwN2CYylQ?si=DYigRj0-nNJ2Kcu1

 

To those who followed this yesterday, I'm sorry that the link was wrong.  This one should direct you to the intended short vid. 

Germ of a new food microbiology

 Think of a food microbiologist you know.  I’m guessing that you now have in mind someone wearing a white laboratory coat, surrounded by food samples, Petri dishes and agar slopes, using an inoculating loop and making smears on microscope slides.  This might be accurate in most cases.

In the 80s, I read an article entitled “Germ of a New Food Microbiology”.  The author’s argument was that the so-called “Standard Plate Count” gives us less information on food safety than just about any other analysis.  There is no indication of how the microbial population will perform in the hands of the consumer;  the SPC is anything but total - many of the bacteria in the sample may not be able to grow on the count medium or at the incubation conditions; single cells and clumps will both be counted as one cell; if we use selective media to count specific types of bacteria, perhaps pathogens, they may not grow if they have been stressed during processing or storage; sample preparation, incubation and transfer to selective media may involve several days, meaning that the count may be obtained only after a significant proportion of the shelf life is over.  We also get no information on how the consumer might react after eating the food

The situation hasn’t changed very much.  Any modern analytical technique still has to be able to correlate with the plate count because of the way food safety is measured..  

The development of rapid microbiological methods now has a long history.
In many cases, the development has involved reducing the number of steps in the process, automating manipulative procedures and reducing the scale of operations to reduce costs.

Many so-called ‘rapid methods’ will give a result within hours of being set up.  However, they may involve significant technician time.

Modern methods often involve molecular techniques, particularly the polymerase chain reaction (PCR) and sequencing.  PCR relies on the ability of DNA polymerase to replicate a portion of a DNA molecule, using specific primers that bind to complementary strands of the target DNA.  The laboratory process usually involves a thermal cycler.  The replication of regions between the primer binding sites results in an exponential amplification of the target, so that within a few hours, millions of copies are produced.  

Recent developments have enabled real-time detection of the products by means of fluorescent reporter molecules that bind to the amplified products.  The progress of the amplification can be followed by monitoring the increase in fluorescence as the number of cycles increases.  The larger the number of target molecules in the sample, the fewer numbers of cycles are required to reach a detection threshold, so the number of cycles required indicates the level of contamination of the sample.

Though the PCR technique can detect a single molecule of DNA, there are some hurdles to overcome.  If we are looking to detect numbers of bacteria in the range <3 cfu/g to 10^2 cfu/g  either large samples or enrichment of the samples is required.  However, if we want to retain the relationship between the initial numbers in the food sample and enumeration by PCR, enrichment cannot be used.  In addition, the food matrix itself may interfere with the replication process.   

Taking this a step further, we can now analyse the population in particular parts of the processing equipment, using small portable real-time sequencers that allow microbiome-based monitoring of surfaces within the plant.  In turn, this may enable the identification of sources of the contamination and allow timely intervention by suitable control measures.

In order to get away from assessment of food safety based on plate counts, we need to formulate microbiological reference criteria based on these new methods and introduce them to our microbiological food standards.

The material presented here represents a great simplification of the techniques involved.  If the article has inspired you to seek further information, you will find hundreds of explanatory articles on the Internet - just search for RT-qPCR, and Microbiome-based environmental monitoring etc.

More on antibiotic resistant bacteria in natural waters and wild-harvested foods.

I wrote an article on 05/12/2012 about our isolation of antibiotic-resistant genes in bacteria in water and in stream and river muds, comparing pristine waters with polluted ones along a stretch of a river in the Waikato region of New Zealand.

I recently read another article by Jack Heinemann and Sophie Joy van Hamelsveld from University of Canterbury in Stuff:  https://www.stuff.co.nz/pou-tiaki/300930926/antibioticresistant-bacteria-in-wild-cockles-and-watercress-putting-people-at-risk-of-serious-illness.  It's worth a read.

The article raises yet another concern about antibiotic resistance.  The testing of water for recreational use does not guarantee that mahinga kai, wild-harvested foods, such as shellfish, are safe to eat.  Shellfish can concentrate bacteria from the water to high levels, even when the tested water appears to be safe.

Is the food still safe to eat?

 For the past few days, New Zealand has suffered from high winds and extensive flooding as a result of Cyclone Gabrielle, which in some coastal areas has coincided with high tides.  Many areas have been without power for extended periods and thousands of people have had to leave their homes, in some cases being rescued from the roofs of their houses.

I was interviewed by a radio journalist who wanted to ask questions about the safety of food and water. Any food that has been contaminated by the flood waters is, of course, not fit to eat.  But what of foods that were in refrigerators and freezers?

This is a difficult question to answer.  If the power has been off for a few hours and the refrigerator has not been opened, the food will probably be safe to eat, however, some areas are not likely to have power restored for several days.  Some foods, such as yoghurt are naturally preserved by their acid content and will be shelf-stable even at room temperature for a few days.  Hard cheeses will also be safe.  Similarly, milk in sealed bottles will be OK for a couple of days.  Vegetables are also shelf-stable for several days at room temperature, provided that they have not been in the water.  Eggs have their shells to protect the contents, but it would be worth washing the shells to prevent contamination of the contents when they are cracked open.  Perhaps a good guide is to think of supermarket displays - if the food such as fruit, potatoes, tomatoes and cabbages is sold from open counters, it can reasonably be expected to last for several days.

The big concern will be fish and meat.  If the frozen food has thawed out completely, it may still be safe to eat if it is thoroughly cooked.  if the food still has ice crystals in it, refreezing is possible.  Beef and pork steaks are essentially sterile inside, so it is only necessary to cook the outside, remembering to cook the edges too.  Fish is normally sold in fillets, so again, frying or barbecuing will render it safe, however, the food cannot be stored for another few days before consumption.  I would be more concerned about chicken, which is always difficult to cook throughout because of the uneven thickness of drumsticks etc.  In all cases, the critical thing is to get the temperature of the food above about 75C throughout.  This will kill all vegetative (non-sporulated) bacterial cells.  If a thermometer is used, it must be clean and inserted into the thickest part of the food.  More care is required with minced meat.  During the mincing operation, the outside of the meat is mixed in with the interior portions, so thorough cooking is required - medium-rare hamburgers are out! 

Another, though not infallible guide is to look at the food and smell it - if it smells off, then don't eat it.  Things like bacon sometimes become a bit slimy as a result of bacterial growth on the surface.  If it is fried properly, it will be safe to eat, but may have off-flavours.

Of course, canned foods are safe, provided that there are no holes in the can. Similarly, sealed packages of snack foods, such as 'chippies' will be OK to eat and may make the kids feel a bit happier.  It is a good idea always to have a supply of canned foods in the cupboard so that they are available in emergency situations like ours.

Another major concern is water.  Some water treatment plants have been out of action for a couple of days as a result of power outages and some watersheds may have been swamped by flood waters.  A number of water supply authorities have said that the tap water is safe to drink.  However, if there is any doubt about its safety, it should be boiled.  I was asked about water for making up baby formula.  In that situation, I would always recommend using boiled water.  Rural households often collect water from the house roof and store it in large tanks.  In my opinion, this water will be safe, provided that the roof and tanks have not been inundated, but once again, if there is any doubt and the power is on, it should be boiled.

All of these questions will become more relevant when people are allowed to return to their dwellings. There may be a temptation to try to salvage food from refrigerators and cupboards.  If there is any doubt, the safest thing to do is to dump it, as food poisoning will put even more stress on health services at this time.

Our next concern will be resupply.  Many roads are impassable, leaving some towns completely cut off.  Supermarkets are very short on stock, even if they have power to operate the tills.  Foods may be in short supply for a while, but in addition, orchards and vegetable farms have been severely damaged, so shortages will continue for months.  There may be more reliance on imports.

Odd Spot:  if the cyclone were not enough to challenge the population, we also had a magnitude 6.3 earthquake yesterday.  It was centred about 50km NE of Paraparaumu in the North Island, so it was under the sea.  There have been no reports of injuries or damage, but it's something we could all have done without!

Will in vitro meat be the new SCP?

 In the 1960s, several companies began development of Single Cell Protein (SCP) as a protein source for human and animal feed.  SCP refers to protein produced by microorganisms, such as bacteria, yeasts and unicellular algae.  This was not a new concept and can be traced back at least as far as 1936.

My first job was as a fermentation experimental officer at ICI in the UK, helping to develop the ICI Single Cell Protein Process.  Several other companies were developing SCP for human and animal consumption.  ICI intended to produce 1m te/yr by the 1990s; Shell was working on producing SCP as a by-product of gas oil dewaxing.  Several other processes were based on mycoprotein from Fusarium sp.- ‘Quorn’, and algae.  These projects involved very significant effort and investment; it has been estimated that by 1983, the main players had invested in R&D a staggering $US 2.9 x 10⁸ in today’s terms.

Image shows the ICI production fermenter for SCP.  60m tall with a capacity of 3,000m³.   I do not own the copyright of this image.

The new processes were beset with problems.  Bacterial cells contain relatively large amounts of RNA, which can result in gout and kidney stones in consumers, as purines increase plasma uric acid, so RNA reduction was required.  Some products had undesirable taste, and considerable downstream processing was required to produce acceptable texture and mouthfeel. Consumer acceptance of food made from bacteria was also difficult to achieve.  However, one of the greatest hurdles was the scale of operation required and the resulting cost of the product.  Most of these processes, while being technological marvels, were not commercially successful.  In fact,  as far as I am aware, the only product now on the market is Quorn.

Against this background, we see in vitro meat (IVM) or cultured meat, being developed as a sustainable food.   It is also referred to as ‘Lab-grown meat’, illustrated by lumps of tissue apparently grown in Petri dishes, though this is perhaps somewhat misleading, as the scale of operation to produce sufficient product to be economically viable would require a facility similar in size to a modern dairy plant. To produce IVM, stem cells are collected from living animals and cultured in a reactor, using suitable culture media, which must be made up of food-grade components.  Typically, vitamins, antibiotics, growth factors, such as calf serum, horse serum and chicken embryo extract are included, (though I have seen a recent report stating that calf serum is not necessary). The stem cells will differentiate into muscle cells under the influence of certain hormones.  Obviously, strict sterility must be maintained, or contaminating bacteria will grow in the highly nutritious medium.  The developing cells will require a supply of oxygen, carbon dioxide will be produced in the metabolic processes, and heat will have to be removed.

Merely getting the muscle cells to grow is only part of the process - Since consumers want something that resembles  meat as closely as possible, some form of three-dimensional scaffolding is usually required for the cells to fuse together to form organised tissues.

For many people, the decision to purchase IVM will come down to taste, texture, appearance and cost.  Estimates suggest that IVM will be twice the price of farmed meat.   I saw recently a report that a burger pattie could cost around $11.35  I will be happy to eat IVM, but not if the product costs a lot more than my favourite grass-fed beef fillet steak.