Understanding Whisky Production · Unit 7: Sensory Assessment & Microbiology

Chapter 17: Microbiological Control in the Distillery

Wild yeasts, lactic acid bacteria and other contaminants, ATP bioluminescence, culture media, chemical sanitisers, and how distillery hygiene is verified.

From the perspective of a wild microorganism, a whisky distillery is essentially paradise with a leaky roof. There are vast vats of warm, sugary porridge, gentle ambient temperatures, and an abundance of cozy microscopic real estate tucked into timber staves, pipe bends, and valve seals.

The central paradox of malt distilling is that it operates under a state of perpetual biological truce. Unlike commercial brewing, where wort is rigorously boiled in a copper kettle to eradicate every living spore before fermentation begins, Scottish malt distilling intentionally skips the boiling stage. To boil the wort would deactivate the delicate starch-converting enzymes (like limit dextrinase) required for secondary conversion in the washback. Consequently, every litre of cereal wort enters the fermenter alive with agricultural hitchhikers. The entire craft comes down to giving our chosen cultured yeast a massive numerical head-start, while keeping unwanted microbial opportunists from staging an unscripted rebellion.

Maintaining microbiological control relies on rapid hygiene verification, such as ATP bioluminescence swabs that detect organic residue rapidly, combined with scheduled steam and chemical sanitisation regimes.

Health Risks and Biological Realities

Distillery microbiology divides broadly into two competing empires: fungi (which include our beloved single-celled yeast) and bacteria. Bacteria are microscopic unicellular or filamentous organisms that inhabit a wide variety of environments. 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. The main sources of bacteria in the distillery are the raw materials, yeast, the buildings (floors and drains), and our process equipment. The main bacteria found in whisky fermentations are lactic acid bacteria, chiefly Lactobacillus species. If large numbers enter the fermentation, they compete with the culture yeast for nutrients, reducing yeast growth and alcohol yield; in well-run fermentations they grow mainly late in the fermentation (van Beek and Priest (2000)). Lactic acid bacteria can also affect flavour: van Beek and Priest found that lactobacilli isolated from whisky fermentations could transform compounds in wort into flavour-active products.

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 coli is a faecal indicator organism: finding it in a water supply indicates faecal contamination and the possible presence of pathogens (Drinking Water Quality Regulator for Scotland). E. coli is also one of the causes of food poisoning, whose symptoms include diarrhoea and vomiting (NHS).

Enterobacteria can also contaminate wort (Priest, Cowbourne and Hough 1974, Journal of the Institute of Brewing, 80(4), 342 to 356).

Legionella pneumophila can cause Legionnaires’ disease, a potentially fatal form of pneumonia that people contract by inhaling small droplets of water containing the bacteria (Health and Safety Executive). Common sources include hot and cold water systems, spa pools, cooling towers and evaporative condensers. In the UK, the Health and Safety Executive advises dutyholders to assess and manage the risk in water systems, including cooling towers and systems that can spread breathable droplets.

Fungi

Fungi are eukaryotic, which means that they have both a nucleus and membrane-bound organelles. They include yeasts and moulds. Yeasts are single-celled microbes, whereas moulds tend to grow as chains of elongated cells. Yeasts 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. Moulds on cereals can produce mycotoxins; aflatoxins, the most harmful type, can potentially cause cancer (Food Standards Agency). 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 is a water-borne parasite that causes the diarrhoeal disease cryptosporidiosis. Its oocysts are not inactivated by chlorination at the levels used in water supply, so physical treatment such as filtration is used to remove them (Drinking Water Quality Regulator for Scotland).

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. 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 them 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.

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.

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 aim to keep unwanted microbes out of the product; no cleaning or sanitising step is perfectly effective every time. If contamination occurs, the cleaning regimes must be revised to make a repeat as unlikely as possible.

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 (Kozulis and Page 1968, Proceedings of the American Society of Brewing Chemists, 26, 52 to 58).

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

Different microbes grow best at different temperatures, and the appropriate incubation conditions depend on the organism being sought.

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 2⁰, 2¹, 2², 2³ … 2ⁿ, 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:

The diagram is a qualitative illustration rather than a set of measured counts. During the exponential phase, a small initial population can increase rapidly under favourable conditions.

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.

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 (known as spread plates), or simply added to a liquid broth.

Adding samples to media is called inoculation.

We can also incubate liquid samples at a set temperature 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 shown below:

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. Incubation typically takes several 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. 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 several days for a result, as 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. PCR can give a result much faster than culturing.

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

Ultraviolet (UV) light can inactivate microorganisms in water. The dose required depends on the organism and the desired level of inactivation; the dose delivered also depends on the equipment, flow and how well the water transmits UV. The US Environmental Protection Agency guidance expresses UV dose in millijoules per square centimetre (mJ/cm²). UV leaves no disinfectant residual, so contamination can recur after treatment. Particles and turbidity can shield microorganisms and reduce effectiveness.

Ozone

Ozone can be used to disinfect water. Its effectiveness and suitability depend on the system, water quality, materials and operating conditions.

Chlorine dioxide

Chlorine dioxide is another disinfectant used in water treatment. Its use depends on the system and applicable requirements, including the control of residues and by-products. Neither treatment removes the need to manage contamination risks elsewhere in the plant.

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.

Cleaning removes soil from equipment; disinfection reduces microorganisms. Some products combine cleaning and disinfecting functions, but their effectiveness depends on how they are used. The Food Standards Agency explains the importance of cleaning first and following the product’s dilution and contact-time instructions. Sanitisation is not the same as sterilisation, and a treated surface can become contaminated again.

Sterilisation aims to destroy all forms of microbial life, including spores. Steam sterilisation uses a validated combination of temperature, exposure time and steam contact; the appropriate cycle depends on the load and equipment. The US Centers for Disease Control and Prevention describes common steam sterilisation temperatures of 121°C and 132°C and explains why exposure times vary.

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.

Peracetic acid, chlorine-based products, quaternary ammonium compounds and aldehydes are among the disinfectants used in food and drink settings. The choice depends on the surface, the organisms of concern, the product’s approved use and the process. Dilution, contact time and any rinsing requirement must follow the specific product instructions.

Disinfectants can also present hazards to workers and react with other chemicals. The Health and Safety Executive guidance for the food and drink industry explains exposure risks, precautions and why some products need to be kept apart.

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 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.

Steam can also be used where the equipment and materials are suitable. Heat transfer, temperature and exposure time all matter; the cycle must be appropriate to the equipment and validated for its purpose. The CDC steam sterilisation guidance explains how steam contact and load affect a sterilisation cycle.

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. Bacterial activity can influence fermentation and flavour, so its timing and extent matter.

Sources and review

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Want to test the whole course rather than one chapter? The whisky production quiz asks 12 questions from cereals and malting through to oak maturation.

About the author

John Fegan · Editor

Hailing from the land of haggis and deep-fried Mars bars, John was always going to find his way to whisky. He has run Uisge Beatha since 2015 and Whiskipedia.com since 2020.