Understanding Whisky Production · Unit 7: Sensory Assessment & Microbiology

Chapter 17: Microbiological Control: Contaminants, Detection, and Sanitisation

Identifying wild yeasts, acetic and lactic acid bacteria, ATP bioluminescence, culture media, and chemical sanitisers in the distillery.

Warm, sugary cereal wort is an irresistible feast not just for the distiller’s pitched yeast, but for an unruly ecosystem of airborne bacteria and wild yeasts waiting in every unwashed valve and pipe joint.

Uncontrolled bacterial infections can devastate distillery yields: acetic acid bacteria convert ethanol into vinegar, while excessive lactic acid bacteria can consume sugars before yeast can multiply. Maintaining microbiological control relies on rapid hygiene verification, such as ATP bioluminescence swabs that detect organic residue within seconds, combined with scheduled steam and chemical sanitisation regimes.

Health Risks and Implications

There are two broad types of microbiological contamination: fungi and bacteria. Bacteria are microscopic unicellular (single cells) or filamentous (grouped cells that stop dividing and grow longer) organisms. Bacteria exist in a range of shapes and sizes, and inhabit a wide variety of environments. Fungi exist as microscopic cells; they include yeasts and moulds or macroscopic bodies (which the eye can see) such as mushrooms. Let us first take a closer look at bacteria.

Bacteria

Bacteria are prokaryotic, which means that they do not have a nucleus and rarely have membrane bound organelles.

Organelles are the equivalent of the organs of the body which are found in microscopic cells. They carry out the functions essential to the life of the cell.

Only a few bacteria can survive and multiply in products containing alcohol, and these are non-pathogenic (which means that they do not cause disease). The main sources of bacteria in the distillery are the raw materials, yeast, the buildings (floors and drains), and our process equipment. The main species found in distilleries are Lactobacillaceae (lactic acid producing). They may contaminate fermentations and can drastically reduce the alcohol yield of fermentations by competing for the culture yeasts’ food source (fermentable sugars). Unless we control them effectively, Lactobacillus spp. and Pediococcus spp. are the most common contaminants in the raw material and fermentation. They also affect the final product flavour by reducing the pH and producing off-flavours.

There are other types of bacteria present in the distillery which may not contaminate the product but do have the potential to harm us humans. Escherichia and Enterobacter are found in the intestines of animals (including us!) and indicate that the water supply is contaminated with untreated surface water, or even sewage. Both micro-organisms can survive in unfermented wort and impart a parsnip type off-flavour. Escherichia coli . can cause vomiting and diarrhoea if consumed in food or drink.

Enterobacter will not grow at a pH lower than 4.3. During alcoholic beverage production, the Enterobacter species can infect the mash and wort. They are particularly problematic as they can change nitrates from raw materials into nitrosamine compounds (e.g. NDMA: N-nitrosodimethylamine) which are carcinogenic.

Legionella pneumophila can cause Legionnaires disease, which is a type of pneumonia that is spread by airborne water droplets. It is found in a variety of water systems such as hot tubs, showers, air conditioning units, and cooling towers. In many countries, the law requires businesses to adopt measures to prevent the occurrence of Legionella pneumophila in water systems.

Fungi

Fungi are eukaryotic, which means that they have both a nucleus and membrane-bound organelles. They exist as two main kinds: yeast and fungi. Yeast are single-celled microbes, whereas fungi tend to exist as chains of elongated cells. Fungi are extremely important for producing alcohol and making bread. We choose the yeast type (the strain) specifically to provide the desired characteristics of the alcoholic beverage that we are producing. The yeast we use for fermentation is called our culture yeast. All other yeast types are then classed as wild yeasts. Like all other fungi, they must be kept out of our product and plant.

Fungal mould can be present on the raw materials such as malted barley and other cereal grains. Mouldy grains can be a source of aflatoxins, which can cause cancer. Fungal mould and slime on plant and building has the potential to contaminate the product. This mould and slime can also harbour spoilage bacteria. So, to summarise, if we find it in our distillery, it is a problem!

Protozoa

In addition to bacteria and yeast, it is also worth mentioning protozoa, which can be found in distillery water systems. Cryptosporidium is a protozoan, which like yeast, is a eukaryote. Protozoa tend to be larger than bacteria and yeast. Cryptosporidium are common water-borne parasites, which cause either diarrhoea (intestinal cryptosporidiosis) or coughing (respiratory cryptosporidiosis). They can survive in wash, so we must remove them from any water used in the distillery.

Bacteria are prokaryotic, which means that they do not have a nucleus and they rarely have membrane bound organelles.

Fungi and protozoa are eukaryotic, which means that they have both a nucleus and membrane-bound organelles.

Microbiological Control Methods

Microbiological control is not only essential for employee health and safety, but it also ensures product quality and food safety (which is, of course, essential for the success of our businesses). There are five methods that we can employ for microbiological control:

1. Hygienic design of plant and processes:

Micro-organisms flourish on rough and dirty surfaces, and in dead-legs in pipework that cannot be cleaned effectively. We must design production equipment so that the surfaces are smooth, easy to clean and drain, and resistant to heat and cleaning chemicals. We will cover this in more detail in the Plant Cleaning lesson. If we do not design equipment according to these criteria, microbes can form layers known as biofilms. In a biofilm, the upper layer protects the lower layers of growth from cleaning chemicals, making then hard to clean and sanitise.

2. Effective plant cleaning and sanitisation:

Good housekeeping, general cleaning, strict and regular process equipment cleaning, and sanitisation, are all very important. Used frequently and with rigour, these practices can work together to prevent microbial contamination. Adequate cleaning can take the form of manual cleaning, or fully automatic Cleaning-in-Place (CIP), which uses detergents for cleaning and sanitisers or disinfectants to kill microbes. Cleaning must also effectively remove scale, as it shields microbes from the CIP chemicals. In the Plant Cleaning lesson, we will cover cleaning methods comprehensively.

We can use soak baths filled with a sanitiser to sanitise hoses, portable pipes, and fittings. For effective soaking, items must be fully submerged (must be free of air bubbles) and undamaged. Effective soaking also takes time, so we need to have duplicate sets of equipment to use while soaking occurs. In addition, we need to ensure the efficacy of the sanitiser used in the soak bath by measuring its strength and replacing it when necessary. We also need to regularly inspect portable equipment, which can become damaged through high usage and constant soaking.

3. Raw material and product sanitisation:

We can also carry out raw material and product sanitisation treatments. These range from grain drying and fumigation, to the process of distillation itself. We can also pasteurise our product to ensure a specific kill rate of potential microbiological contamination.

4. Creating conditions to inhibit microbiological growth:

Another method involves maintaining an environment that is not conducive for microbiological growth. The presence of alcohol, lack of oxygen, low temperatures, low pH and a lack of nutrients that could support growth all help to create a challenging environment for organisms to thrive. Think about how the liquids that we ferment change in composition. Before fermentation they are full of sugar and nutrients, oxygen is present, the temperatures are moderate and there is no alcohol. After fermentation most of the nutrients are used up, no oxygen is present and the pH has decreased and become more acidic. It should be obvious that the biggest microbial contamination risk is the unfermented liquid.

5. Monitoring microbiological quality:

Monitoring microbiological quality helps us to detect microbial contamination. It tells us whether we have contamination, where it is, and how severe it is. All raw materials are inspected and will be rejected if they show signs of contamination. If contamination occurs, all affected plant and equipment must be cleaned and sanitised after the contamination source is eliminated. This may mean that we need to carry out additional cleaning, over and above the standard cleaning and sanitising regime.

Standard cleaning regimes should ensure that there are zero unwanted microbes in the product. If contamination occurs, the cleaning regimes must be revised to ensure that repeat contamination is impossible.

Plant maintenance also plays a vital role in microbiological control, as any leaking pumps, pipes, or valves are potential sources of contamination.

Let us now apply the above theories of microbiological control to our yeast handling systems.

Microbiological Control of Yeast Handling Systems

As we discovered earlier, our beloved culture yeast is actually a fungus. We need to manage our yeast handling systems to ensure that our yeast is protected whilst also eliminating any invading fungi and bacteria.

To eliminate wild yeast, or at least keep their numbers low, we need to use clean pitching yeast. We can achieve this through careful purchasing of our yeast from reputable suppliers, and rigorous CIP and hygienic yeast handling practices, including housekeeping and plant maintenance.

The best way to keep pitching yeast free of contamination is to ensure that we use rigorous CIP and hygienic yeast handling practices, including regular housekeeping, plant maintenance, and restricting the amount of times that yeast is reused.

Thankfully, most modern plant and equipment where yeast can be present are constructed of stainless steel, which is easy to clean and sanitise.

Now that we have described how to control microbes in the production facility, let’s move on to cover how we check how well these controls are working.

Methods of Detecting Microbiological Contamination

As we are a food manufacturer, we must be able to check the cleanliness of everything within the distillery. When we say cleanliness, we are referring to the absence of dirt, soil, and any microbes that can contaminate raw materials, equipment, and product (causing changes that may be dangerous to consumers).

We must be aware of two important parameters when dealing with microbiological contaminations:

1. The type of contamination - the type refers to the name (genus and species) of the organism.
2. The amount of contamination - this indicates the concentration of the microbes and how widespread the problem is.

If contamination is discovered, we must react quickly to eliminate it and prevent its growth and rapid spread.

Detection is inherently difficult. As micro-organisms are very small (microscopic in fact), they are not visible to the naked eye. The initial contamination can also begin at a very low level in large volumes of product. Both sampling and detection can thus miss these initial contaminations.

So how do we sample our product? Well, most microbiological sampling is manual. We thus only take a ‘snap shot’ of the sampled population at set frequencies. These frequencies are set to make the sampling as representative as possible. Sampling is undertaken using aseptic (sterile) technique. Aseptic technique reduces the chances of the sampling operation contaminating (introducing microbes that weren’t there before) the sample.

Let us quickly look at some common distillery microbiological sampling methods:

• Sample tap (from a vessel or pipe): an on-line sample tap is sanitised by heat (usually a flame) or a sanitiser, and the sample is collected into a sterilised sample bottle. In-line sample taps are cleaned with Cleaning-in-Place (CIP), together with the vessel or pipe, before the sample is taken into a sterilised sample bottle. The sterile bottle is sealed after filling and taken to the lab for further processing.

• Any surface: swabs are taken by removing the swab from its sterile tube and rubbing the tip of a swab over the surface being tested. The swab is then returned to the tube and sealed ready for culturing in the lab.

Now that we know how to take an aseptic sample, let us now move to the lab and explore the next step in the process of microbial detection - culturing.

Culturing

Culturing is the controlled growth of microbes. This is achieved using four criteria: nutrients, temperature, the presence or absence of oxygen, and time. Let us now look at each of these in a bit more detail.

Nutrients - media

A medium refers to a food source that the microbes of interest can grow on. Some media are designed to allow the growth of several different organisms; these are called nutrient media. An example of nutrient media for growing distillery yeast and bacteria is the Universal Beer Agar.

Different micro-organisms need different nutrients to grow. We thus need to select the right medium for the organism of interest.

There are two main types of media:

• Agar - a solid nutrient medium that is usually used in petri dishes (plates).
• Broth - a liquid medium that contains the required nutrients.

Media that only support the growth of specific organisms are called selective or differential media.

There are two ways in which we can make these media selective. One is to ensure that they contain the key nutrients that the micro-organisms of interest need in order to grow. The other is to add a biocide (antibiotic) to help prevent the growth of other types of micro-organisms. For example, if we are detecting bacteria in a sample of yeast, we want to inhibit yeast growth so that we can better detect the bacteria.

Temperature

As with nutrients, different microbes grow best at different temperatures. Pathogenic bacteria like E. coli tend to grow best at body temperature (37°C), whereas Lactobacillus prefers 44°C. Most spoilage organisms are cultured in the lab at 25-30°C. Generally speaking the lower the temperature, the slower the growth.

Presence or absence of oxygen

Anaerobic bacteria grow when no oxygen is present, whereas aerobic bacteria require oxygen for growth. If we are trying to detect anaerobic bacteria, we must exclude all oxygen, or they will not grow.

Time

When we talk about microbial growth, we are not talking about them getting bigger (although they do up to a point), we mean that they are increasing in number. During this growth, their cells divide and grow in number.

Under favourable conditions, a growing bacterial population doubles at regular intervals. Growth occurs as geometric progression: 1, 2, 4, 8, etc. or 2 0 , 2 1 , 2 2 , 2 3 … 2 n : where n = the number of generations. This is called exponential growth.

When a fresh medium is inoculated (samples are added to the medium) with a given number of cells, and the population growth is monitored over a period of time, we can yield a typical bacterial growth curve by plotting the number of living cells against time, as shown below:

It is important to note that the scale here is logarithmic. This means the numbers on the vertical axis are actually 20, 400, 6,000, 80,000 and 100,000 per mL. As we can see, as most micro-organisms multiply exponentially, a small initial contamination can rapidly lead to a very large contamination (within a very short period!).

Microbial Detection Techniques

Now that we know the elements we need to control in order to restrict or eliminate microbial growth, we will now stay in the lab and discuss how to process these samples. These processes must ensure that we detect (grow) the microbes that are in the sample and exclude any other microbes that may be in the lab environment.

Liquid samples

Liquid samples can be filtered through plastic membranes that catch any microbes. The membranes are then transferred onto an appropriate medium for culturing. These are called membrane plates. This process is shown in the video below:

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Liquid samples can also be mixed with liquid agar that is poured onto plates and subsequently sets (known as pour plates). They can also be spread over the surface of the set agar, or simply added to a liquid broth (known as spread plates).

Adding samples to media is called inoculation.

We can also incubate liquid samples at a set temperature (usually 25-30°C) for several days; this will allow any contaminating microbes to grow. This process is called forcing. We can then either inoculate media with the forced samples, or we can simply assess their aroma for off-notes.

Swab or solid samples

Swab or solid samples from plant or media can be streaked across a plate, as

The streak technique is designed to spread out the sample so that individual organisms grow and we can hence identify them. One cell forms a single colony.

Colonies are groups of cells that have grown from a single microbe, on or in the media.

Incubation

All inoculated media are stored in an incubator. The times and temperatures of incubation can vary depending on the type of micro-organism under investigation. However, most media are held at 25-30 ° C for two to five days.

After incubation, the number of colonies growing on the plates are counted and the result is reported as colony forming units (CFUs). For membrane and pour plates where we know the sample’s volume, this will be quoted as CFU/mL. Using a microscope to count the colonies shortens the amount of time required for incubation to as little as 24 hours. This gives us an idea of the amount of microbes present in the sample. For broths the presence of the organism of interest is often indicated by a colour change and increase in turbidity (cloudiness) in the broth.

We can use a variety of methods to identify micro-organisms. When using a microscope, we primarily use morphology, observing the bacteria’s size, shape, and motility (if and how it moves). We can also stain them with special dyes, or investigate what chemicals they produce as they grow. The figure below shows some forms of bacterial morphology:

Rapid Detection Techniques

We need to know the microbiological status of our product and plant but we don’t always have time to wait for the incubation period to elapse. Rapid detection techniques can be used instead of waiting two to five days for a result as it is the case with the traditional microbiological detection techniques.

These rapid tests are based on physical, biochemical or molecular methods. Some of the more commonly used rapid detection tests are:

• Micro-colony - Although growth of colonies on a solid culture medium is a traditional method, detection can be speeded up by observing growth on a microscope slide containing culture media, or by specialist optical detection after a shorter culturing period.

• ATP bioluminescence - This is a widely used, relatively inexpensive portable method that uses the chemicals that make fire flies glow. These chemicals react with adenosine tri-phosphate (ATP), a substance found in all living cells, to produce light. A swab is used to sample a surface or rinse water. The swab containing the sample is then inserted into a plastic tube and mixed with the chemicals. The plastic tube is then inserted into a luminometer which measures the amount of light produced. The amount of light is proportional to the amount of ATP in the sample and hence the level of contamination.

Polymerase chain reaction (PCR) - This method detects small amounts of DNA from the target microorganism (living or dead) in order to identify their presence. Modern PCR machines are able to process a sample in between 30 minutes and four hours to obtain a result.

Real time RT-PCR is a standard method used for virus detection, including the SARS-CoV-2 virus.

Although rapid methods overcome the time delays using traditional detection there are some downsides:

• They may require culturing up of sample to a level where contaminants can be detected.
• They may not differentiate between dead or alive cells.

Now we have discovered the ways in which we detect microbes we can now move on to see how we control them in the plant. Let’s start at the beginning with the microbiological control of product and process water.

Microbiological Control of Product and Process Water

Wherever we get our water from, it is unlikely to be low enough in microbes to be used directly. Most plants will undertake some form of microbiological control on the water supply. This can involve UV light, ozone, a chlorinated disinfectant such as chlorine dioxide, or indeed a combination of these treatments. Let us explore each of these in a little more detail.

UV light

Ultra-violet (UV) light, in the range of 240-280 nm, kills bacteria, fungi, and viruses by destroying their DNA. The UV radiation causes a photochemical reaction, killing up to 90% at a dose of 2,000-8,000 µW/s/cm². For higher kill rates, we would need a dosage of 60,000-80,000 µW/s/cm². UV light has no residual effects, thus re-infection after treatment can occur. UV light is only effective on water that has been filtered to remove particles and turbidity. Any particles left in solution can shield the microbes from the effect of the UV light.

Ozone

Ozone gas (O 3 ) is generated from oxygen on demand and injected into the cold/ambient water circulating through the line. As well as using it for water sanitisation, we can use ozone as a sanitiser for the main CIP processes. It is stable at pH 6.5-8, and has a high kill rate for bacteria, fungi, and spores. Ozone is safe to use with stainless steel but it degrades certain rubbers and plastics. It breaks back down into oxygen after around 10 minutes; therefore, it cannot be stored.

Chlorine dioxide

Stabilised chlorine dioxide (ClO 2 ) is becoming a popular choice amongst food manufacturers. It is less corrosive to steel and does not taint the product like other chlorine sanitisers. Chlorine dioxide can be used to sanitise clean water at a concentration of 0.2 - 0.5 mg/L. It is a very strong oxidising agent that causes a high kill rate of bacteria, fungi, and viruses. It also helps to combat biofilms. Chlorine dioxide has a residual effect, thus re-infection after treatment is unlikely.

Sanitisers

We should now have a fundamental understanding of the types of micro-organisms that can be present. We have explored sampling and identification requirements, as well as some of the implications and health risks that occur if micro-organisms do infect our plant and/or product. We should also now have a basic understanding of their control. This next section introduces the importance of sanitisation.

Sanitisation, or disinfection, is simply the act of killing microbes on the surfaces of plant and equipment so that they are safe to use. Logically, we call the chemicals that we use to do this disinfectants or sanitisers. Most sanitisers cannot clean surfaces, so we use them after our detergents have cleaned the plant. There is, after all, little point in sanitising equipment that is still dirty! (We explore how to select and use detergents in the lesson on Plant Cleaning.)

Although sanitisers kill microbes this is rarely 100%. However, when used correctly, sanitisers will prevent microbial contamination. To kill all microbes, we must use stronger chemicals and very high temperatures. It is therefore incorrect to call a sanitiser a sterilant, or to claim that they sterilise equipment.

Sterilisation is the act of killing all microbes, including the spores. This is commonly undertaken in the pharmaceutical manufacturing industry. Steam at a minimum temperature of 122°C for 15 minutes must be used.

We will now explore some of the more commonly used sanitisers within the distillery.

Chemical Sanitiser Types

Several types of chemical sanitisers are used in the distillery. Let us explore the most commonly used types.

Non-rinse sanitisers

Peroxyacetic acid (PAA) is a combination of hydrogen peroxide and acetic acid. At 200-400 mg/L, it is considered safe to use without subsequent rinsing. It has good residual activity, which prevents re-infection before the plant is used. It has a broad spectrum of bactericidal activity and it does not corrode stainless steel. When used, peracetic acid breaks down into hydrogen peroxide and acetic acid, which will further degrade into water, oxygen, and carbon dioxide. Hydrogen peroxide on its own will break down into water and oxygen. None of the breakdown products are hazardous.

PAA is effective over a wide temperature range, although it tends to be used cold. However, it typically takes a long time for PAA to work (around 20 minutes) at low temperatures.

PAA can be hazardous to humans at much higher concentrations than those used for sanitisation. It can affect the flavour of the product if the concentration is high, or if a large volume is left in the tank. Because of this, some plants rinse equipment after using it. Of course, if you do, the water used to rinse the plant must itself have been sanitised (usually with chlorine dioxide).

The Soil Association in the UK and the Organic Consumers Association in the USA both require companies to rinse off PAA residues with ‘potable’ water if producing organic products.

Chlorine based sanitisers

Chlorine dioxide can be used as a sanitiser at 5 mg/L. If used at this strength, it must be thoroughly rinsed. If left unrinsed at this concentration, it can cause pitting corrosion in stainless steel.

Hypochlorites can be used as a chlorine sanitiser, but their use is uncommon. They must be kept at low concentrations, typically at 150-200 mg/L chlorine, with a high pH, to reduce the evolution of free chlorine (which is a highly toxic gas). It is essential to rinse off residues with sanitised water.

Environmental sanitisers (detergents/disinfectants)

Environmental sanitisers are generally high foam producing detergents. They are used for sanitising floors, walls, external surfaces of tanks and pipes, and for cleaning drains. For these sanitisers to work effectively, they must be able to cling to the surface for a specific amount of time. They must also be easily removed by rinsing.

Quaternary ammonia compounds are non-corrosive compounds. They are effective at low concentrations and low temperatures. They are prone to foaming, which helps them to cling.

Gluteraldehydes can also be used for environmental cleaning. They are commercially available as acidic solutions that are activated on-site by making them alkaline. They have a wide spectrum of use as they are effective against most bacteria and fungi.

Sanitising Chemical Selection Factors

The table below details the parameters to consider when selecting a sanitiser:

Heat Sanitisation

Our final sanitisation method does not require chemicals. Instead, we use heat for a specific length of time to kill any microbes present and prevent contamination.

We often use hot water to sanitise mains and pipework, including the associated valves and pumps. We must include all areas of the pipework in the hot rinse and it must reach the minimum temperature for the minimum amount of time. When undertaken on large pipe diameters and long pipe runs, we should only begin timing the sanitisation once all parts of the system have reached the target temperature. We can establish this target temperature by measuring the temperature of the water returning to the source of hot water.

The flow rate of the water must be such that the temperature is maintained in all areas; pumps should thus be sized accordingly. A high water velocity (2 m/s) and turbulent flow will also improve heat transfer from the water to the surface being sanitised. Many plants recover the heat at the end of the clean using heat exchangers. Hot rinse water is often reused as pre-rinse or make-up water for subsequent cleans.

We can also use steam to sanitise pipework and small vessels (e.g. wooden vessels). Steam is more commonly used in yeast handling equipment (smaller vessels only), and product mains (especially those containing trap filters). The steam used is called wet steam. The presence of liquid in the wet steam helps transfer the heat from the steam to the surfaces, and most importantly, to any microbes! The table below shows the difference in the disinfection effectiveness of wet and dry steam:

It is important that the steam we use is free of particles and chemicals, as they could potentially taint the product.

Key Takeaways

Preventing premature microbial contamination protects fermentable sugars for the pitched yeast. Controlled bacterial activity is beneficial only in late-stage fermentation after yeast has achieved primary alcohol conversion.

About the author

John Fegan · Editor

John has run Uisge Beatha since 2015 and Whiskipedia.com since 2020. He owns whisky casks and writes from direct experience of their storage, insurance and bottling decisions.

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