BTE1014 Week 5 Notes PDF

BTE1014_Week_5_Notes Learning Objectives: Gain a general understanding of the methods used in microbiology Understand what microbiological methods are used in brewing and why. Learn about beer spoilage microorganisms and how to detect them. Understand how to prevent beer spo...

BTE1014_Week_5_Notes Learning Objectives: Gain a general understanding of the methods used in microbiology Understand what microbiological methods are used in brewing and why. Learn about beer spoilage microorganisms and how to detect them. Understand how to prevent beer spoilage. Methods in Microbiology Since the start of your degree you have used microbiological methods in your practical’s and learned about them in lectures. There is a vast array of methods to choose from. What types of microorganisms are used in industry? We will have the desirable microorganisms which are the production microbes that are used to make the product. However, you can also have undesirable microbes which can lead to contamination of your product. Remember back to our earlier lectures. Microbiological methods can be used to assess the quality and quantity of your inoculum but also to assay for contamination. The methods you use will depend on the question you want answered. 1. Qualitative methods will allow you to test for the presence or absence of a microbe. The amount of microbe present is not important or the identity of the microbe. 2. Quantitative methods will tell you how many microorganisms are present but still no identity. 3. Identification methods will tell you what the microbe is. Some methods can do all three and detect, quantify and identify the microorganisms present. What/where do you test? Production strain – remember inoculum development -> important to make sure your inoculum is active and pure and the correct concentration to inoculate the fermenter. Raw material – is this contaminated? In-process and finished product evaluation – is there any contamination? Pre- and post- sterilisation bioburden analysis - is there any contamination? Preservative efficacy testing – is the preservative working and preventing microbial growth? Environmental monitoring (air, surfaces, personnel) -> continual testing of an industrial setting to prevent contamination. Water testing – ensure the water used in the process or even for cleaning is not contaminated. 1 Qualitative and Quantitative Methods These are methods that can detect the presence of microbes and can also quantify the number of microorganisms present in the sample. These methods can be split into two groups: 1. Classical methods 2. Rapid Microbiological Methods (RMMs) Classical Methods Some methods can directly measure cell number: – Direct microscopic count – Viable plate count / CFU/ml count Other methods are indirect measurements of cell number. These methods measure cell mass: – Dry weight – Turbidity / Optical Density (OD) Direct microscopic count: This method uses a hemocytometer, which is a counting chamber that can be viewed using a microscope. A known volume of culture is placed in the hemocytometer chambers, viewed using the microscope, cells are counted, and an equation is used to calculate the total number of cells. Viable plate count / Colony Forming Unit per ml (CFU/ml) count. You have all carried out this experiment in your undergraduate labs. Prepare 10 fold serial dilutions of your sample, plate 0.1 ml per agar plate, incubate plates for a set period of time, count the colonies, and calculate the colony forming units/ml. 2 Dry Weight Measurement. This is useful for measuring the growth of samples that cannot be separated out into single colonies, example mycelia of fungi. The samples are first grown in liquid. Use a pre-weighed piece of filter paper to filter the sample. The filter paper is allowed to dry overnight to remove liquid and the filter paper is then weighed again. The dry weight of the sample is calculated as follows: Dry weight (mg) = weight of filtered sample (mg) – filter paper with no sample (mg) Turbidity / Optical Density (OD). It is important to understand that it is an OPTICAL DENSITY (OD) measurement and NOT absorbance. The optical density can be defined as a measurement of a component within the sample that can slow or delay the transmission of light, example is cells in a culture. Whereas absorbance is the measurement of a component in the sample that can absorb light. When you measure the OD of a sample the cells in the culture do NOT absorb the light but instead DEFLECT or SCATTER light. Therefore, the OD reading is a measurement of unscattered light – as only that light that is NOT deflected is measured by the spectrophometer. OD measurements are also affected by cell size -> not all microbe cells are the same size. There are several advantages to classical methods. They are simple to carry out and relatively inexpensive. The main disadvantages are these methods can be time consuming as they are all cultivation based. You need to wait for the microbes to grow and results can take 2-14 days to acquire which may not be quick enough in some settings. 3 Rapid Microbiological Methods (RMMs) Overall, these methods are faster to complete than classical methods. They can sometimes be cheaper and easier to use. They can also be less labour intensive. These methods can be growth based in that you still need to grow the sample. Some may require a growth-based step. These methods include: 1. ATP-Bioluminescence 2. Viability tests 3. Endotoxin testing 1. ATP Bioluminescence. This method is based on measuring the emission of light from living organisms. It is dependent on the presence of ATP in cells. ATP is only found in living cells, therefore dead cells will not emit light and will not be measured. In the presence of ATP, the enzyme luciferase can break down the substrate luciferin to produce light. Therefore, if we add luciferin and luciferase to a sample, the ATP in the living cells will allow the reaction to occur and light will be emitted. The amount of light will be a measurement of the number of viable microbes present in the sample. One disadvantage to this method is the lack of sensitivity. It can only detect cells numbers of 103 or higher. Therefore, a pre-enrichment growth step may be needed. This could increase the time it takes to get the result. 2. Viability technologies. These methods do NOT rely on growth of microbes. They can differentiate living from dead cells and have the potential to detect low levels of contamination. They involve the direct labelling of a viability marker within the cell. One method involves using viability stains. These stain target metabolically active cells. Only living (viable) cells with intact membranes will retain the fluorescent dye. Dead cells in the sample will not fluoresce. Example, LIVE/DEAD stain. Green and red dyes are added to the sample. Green dye can penetrate ALL membranes; therefore, it will measure live and dead cells. However, the red dye can only penetrate dead cells. When you view the sample under the microscope, the number of green cells will correspond to viable cells and the red cells will be dead. 4 3. Endotoxin Test. This is also called the pyrogen test. A pyrogen is a bacterial toxin that may produce fever, shock, and death. If you are using a recombinant bacterial strain to produce a product it must be tested. All pharmaceutical products, cosmetics, biotherapeutics and medical devices need to be tested. The original test was the Rabbit test but it was not a very ethical test. The Limulus Amoebocyte Lysate (LAL) test now used. LAL comes from the blood of Limulus polyphemus. The blood will coagulate in the presence of endotoxin. Methods to identify microorganisms. Why would we want to identify a microbe? – Characterise a microbe with important industrial applications. – Relate individual cases to an outbreak. – Associate a specific outbreak with a specific food. – Trace the source of contaminants in a manufacturing process. Identification tests will get you to the species level. You can also type microbes to beyond the species level- Sub-species Strain Sub-strain Example: Escherichia coli. Not all strains of E. coli cause a problem but some such as E. coli O157:H7 can cause severe food poisoning. Therefore if E. coli is identified in food linked to a food poisoning outbreak, you would need to type the E. coli strain. Methods to identify microbes can be classified into two groups: 1. Classical Methods 2. Rapid Methods 5 Classical Methods These methods are based on tests that look at the phenotypes of a microorganism. 1. Morphology: a. Shape and size of colonies on agar plates. b. Gram stain: i. Gram positive or Gram negative 2. Temperature profiles 3. Coagulase test 4. Oxidase test 5. Vogues Proskauer test 6. Indole test 7. Ability to ferment certain carbon sources. 8. Ability to ferment certain nitrogen sources. 9. Oxygen requirements 10. Antibiotic susceptibility tests 11. Presence/absence of surface antigens The main disadvantage of these methods is that they are growth dependent and take time to carry out. Analytical Profile Index (API) strips. The API strip is made up of a series of cupules containing various freeze-dried reagents and colour indicators designed for biochemical tests. Each cupule is inoculated with the unknown microbe and incubated for a set period of time at the correct temperature. Tests are determined to be positive or negative based on colour changes, this produces a profile number which is looked up in a code book/online and can identify the unknown microbe. 6 Rapid Methods for Species Identification and Typing These methods can look at phenotypic or genotypic properties. The main difference to classical methods is the speed at which you get results. Phenotypic Rapid Methods. 1. Automated API systems: The Vitek instrument (bioMerieux) reads the barcode. The card contains 64 chemical tests – many different types available. There is an integrated transfer tube that will inoculate each well with the test microbe. Card is incubated in the machine and the machine will read the results and identify the unknown microbe. Rapid test with very little hands-on labour needed. 2. Fatty Acid Profiling The cellular fatty acid composition of microorganisms is very stable and highly conserved. These profiles can be used to identify microbes. However, it is important to know that how the organisms are grown and how samples are prepared may change the fatty acid composition. 7 Genotypic Rapid Methods. 1. Analysis of ribosomal RNA genes: In earlier lectures we talked about using the sequence of ribosomal RNA genes to identify microorganisms from an environmental sample. In that example you will have a mix of microbes. You can also use this method to identify pure cultures of unknown microbes. To identify a bacterium, you analyse the sequence of the 16S rRNA gene. For fungi you look at the sequence of the 18S rRNA gene or The Internal Transcribed Spacer (ITS) sequence. ITS1 5.8S rRNA ITS2 18S rRNA gene gene 28S rRNA gene 2. PCR based methods. What if you are looking for a specific microorganism? What if you are not trying to identify an unknown microorganism but you are looking for a specific microbe in your sample. In the food industry you routinely check food for specific pathogens that are known to cause food poisoning. In the health industry you might need to confirm the presence of a specific pathogen to determine the infection a patient may have. In these scenarios looking at the rRNA is a more roundabout way to get your result. Instead you can use PCR to target genes that you know are present in the microbe you are trying to identify. Conventional PCR / Endpoint PCR. You should all be familiar with this method. To identify a specific microorganism, you would extract DNA from the sample, use primers for the microbe of interest and run a PCR. The final reaction is then run on a gel. If you get a band on the gel that confirms the microbe of interest is present. Conventional PCR works well but you only see the result at the end. A PCR reaction usually take 40 cycles. 8 Real Time PCR. This method allows us to observe the PCR reaction over time (all cycles of the PCR). It does this by using fluorescence technology. The components of the PCR reaction are the same as conventional PCR but you also add a fluorescent dye. This dye can bind to DNA. After each PCR cycle, the machine take a fluorescence reading, if PCR product is present you will get fluorescence. The amount of fluorescence should increase after each cycle as more PCR products are made. The computer programme linked to the real time PCR machine will plot the number of cycles against fluorescence reading for each cycle. You always include a no template control sample never goes above the baseline. This control contains no DNA and there should be no fluorescence. In your test sample, if the microbe DNA is present, the primers will bind and after each cycle the fluorescence should increase. How to choose which method to use? 1. Assay throughput: how many samples need to be tested per day, week, month. 2. How quick is it to complete? How soon do you need the result? 3. Cost of consumables? 4. Labour requirements? 5. Database available for the system? 6. Facility requirements? Microbial Spoilage of Beer Wort can be considered a good medium for microbes to grow in. Beer is thought to be microbiologically stable due to its ethanol content, hop bitter compounds, high carbon dioxide content and low pH. Beer also has very few nutrients available to microorganisms as they were used up by the fermentative yeast to produce the beer. What types of microbes can contaminate beer? – Gram-positive bacteria – Gram-negative bacteria – Wild yeast 9 Gram Positive Bacteria They are often called the Lactic Acid Bacteria (LAB). They mainly produce lactic acid from glucose. LAB are not always a problem as some can be used as probiotics. Lactobacillus and Pediococcus spp. are important for beer spoilage. Hop resistance is another major problem associated with bacteria that spoil beer. Some bacteria can contain the Hop resistance genes, horA and horC. These bacteria are unaffected by the presence of hop compounds and are a major threat to beer quality. They have the ability to survive in beer and multiply. Gram Negative Bacteria There are two categories: obligate anaerobes and aerobic/facultative anaerobic gram negative bacteria. 1. Obligate anaerobes – mainly from the genera Pectinatus, Megasphaera and Zymophilus. If found in beer the spoilage problem must be addressed. 2. Group of aerobic and facultative anaerobic gram negative bacteria: – Acetic acid bacteria: This group of bacteria can oxidise ethanol to produce acetic acid. Examples: Acetobacter -> sour, vinegary taste and Zymomonas -> fruity, rotten apple, rotten egg – some Enterobacteriaceae. Normally cannot grow in finished beer. They can be found in the initial stages of beer production and lead to unwanted flavours in final product. An example is Obesumbacterium spp: can be found in pitching yeast and fermenting wort. Parsnip flavour and sulphur smell. Wild Yeast A wild yeast is a yeast that is not deliberately used and under full control of the brewer. Not all wild yeast harm beer BUT their presence indicates a sterilisation issue in the brewery. There are two groups: – Non-Saccharomyces yeasts – Saccharomyces yeasts Non-Saccharomyces yeasts – Brettanomyces: can survive high alcohol levels, low oxygen and low pH – Candida spp. – Pichia spp. Most of these species can be found throughout the brewery – dirty sampling ports or other surfaces. They can lead to off-flavours and haze sediment. Saccharomyces yeasts There are the most widespread. They are the greatest threat since they are so similar to production strains. Saccharomyces cerevisiae var. diastaticus is a variant of S. cerevisiae. It is a super-attenuating yeast due to its ability to ferment residual carbohydrates in beer. These residual carbohydrates are not normally metabolised by pure culture yeast strains. The main problem they cause is an increase in carbon dioxide (CO2) caused by secondary fermentation and the consequences include gushing of beer and bottle bursting. Contamination with S. cerevisiae var. diastaticus can cause economic losses, occasionally expose the consumer to risk of injury, and lead to the recall of products. 10 Microbiological Methods in Brewing You will need to use microbiology methods in brewing in two scenarios. Firstly, when you are preparing your yeast inoculum to inoculate the fermentation tank. You will need to be able to count the number of yeast cells you have in your culture. Secondly, you will need to monitor your brewing process for contamination. Remember – desirable and undesirable microbes. How to measure the quantity of cells in a yeast culture prior to inoculation of the fermentation tank. You can use any of the following: 1. CFU/ml count 2. Direct microscopic count with a haemocytometer 3. Turbidity / OD How to measure the viability and vitality of a yeast culture – Viability is the percentage of live cells in a population – Vitality will measure the overall health of your yeast cells. A cell could be viable but still have reduced vitality. It is important that the yeast you use in your fermentation are both viable (living) and actively growing well (vitality) for an efficient fermentation. 11 Viability tests – basically checking if your yeast cells are alive 1. Plate count method (CFU methods) 2. Spotting test 3. Zone of growth inhibition 4. Culture in liquid medium 5. Viability staining methods. – Dyes are dependent on the integrity and functionality of the cell membrane. Some stains cannot enter a living cell with a fully functional membrane or are pumped back out. If the stain is present in the cell, then the cell is dead e.g. methylene blue (blue), propidium iodide (red) Other stains have to be activated inside a living cell e.g. fluorescein diacetate (green) Taken from: Comparison of methods used for assessing the viability and vitality of yeast cells 12 Vitality methods – determine the overall health of the cells. 1. Determine the amount of cellular ATP based on the luciferin reaction -> see above notes on ATP bioluminescence. 2. Determine certain enzyme activity -> you use a dye that is only broken down by metabolically active cells. There will be a colour change. - MTT assay : living cells will turn the yellow MTT to purple. Measure the intensity of purple to measure vitality of cell. - XTT assay: living cells change from yellow to dark orange. 13 Methods to identify spoilage microorganisms in beer. The factors you need to consider: How quickly do you need the results?: – this will depend on the stage of brewing you take your sample for testing How specific do you need your test to be? – what do you want to know? do you have a contamination do you need to know the type of microorganism? What level of sensitivity do you need? – limit of detection: lowest concentration of contamination microorganism that can be detected Do you have facilities, staff….? – do you have space? – do you have equipment? – can your staff carry out these tests? Non-specific Tests. They can flag potential problems but not tell you what microorganism is causing the contamination but will not identify the actual microbes. (1) ATP bioluminescence (2) Culture based methods: – Universal Beer Agar: Supports the growth of Lactobacilli, Pediococci, Acetobacter, Zymomonas spp. and wild yeast strains. You can use it with either direct surface plating or pour plate techniques with serial dilutions of the sample can be employed. Plates can be incubated aerobically or anaerobically depending on the type of microbes you are trying to detect. – Fast Orange Wild Yeast Agar: PIKA FastOrange Wild Yeast Broth (Bouillon) is a PCR-grade culture medium for the visible or PCR detection of wild yeasts in the brewing process & packaged products. FastOrange Wild Yeast grows wild yeasts such as Diastaticus and Brettanomyces while suppressing growth of brewers yeasts and bacteria. Samples are added to Fast Orange Wild Yeast and incubated at 25 °C. Samples can be processed by PCR after 2 days enrichment, or growth can be detected by increased turbidity or sediment formation after 3-5 days. Specific Tests. These will detect AND identify the beer spoilage microorganisms. PCR technology and using primers specific for beer spoilage microorganisms: 1. Primers for Hop resistance genes -> horA and horC 2. Primers for S. cerevisiae var diastaticus -> presence of the STA1 gene 14 6-in-1 Beer Spoilage PCR Micro Test Kit (Microbiologique) The Beer Spoilage PCR Micro Test kit was developed to rapidly detect major spoilage microorganisms within 2 hours directly from a spoiled beer sample or following a 24-48 hour enrichment step (recommended for clear beer). This kit is intended for the detection of specific spoilage-causing wild yeasts and bacteria in beer. Simultaneous detection of wild yeasts (Saccharomyces diastaticus, Brettanomyces) and bacteria (hops-resistant Lactic acid bacteria, Acetic acid bacteria, Megasphaera and Pectinatus) Detection of spoilage organisms in spoiled products in 2 hours: Limit of detection: 10 to 100 cells/mL Detection of spoilage organisms by enrichment: Limit of Detection: 1 - 10 cells/mL Sample enrichment: 24 - 48 hr PCR operation and detection: 2 hr 15 Fluorescence microscopy: VIT® test kits: Rapid analysis of microorganisms using fluorescence microscope. 16 How to protect beer from spoilage The food/beverage industry use hurdle technology to protect from contamination. This is when you use a combination of small hurdles instead of one single big hurdle to extend the shelf life/preserve food/drinks. 17 Factors that can control microbial growth in beer: Intrinsic factors: due to the beer itself – Ethanol – Low pH – Hops – Carbon dioxide – Low oxygen levels – Lack of nutrients – Sulphur dioxide (not all beers) Extrinsic factors: environment the beer is made in or stored – Mashing – Kettle boil – Pasteurisation (not all beers) – Filtration (not all beers) – Bottle conditioning (not all beers) 18