Biotechnology Unit 1 : Dairy Industries i. Milk ii. Yoghurt iii. Cheese iv. Environmental Impact

i. Milk Milk is an important food for most British people. It is an important part of a balanced diet Milk contains: fat starch sugar protein minerals

Milk treatment All milk comes from dairy cows. It is treated in different ways to produce different types. Milk is available in forms such as: pasteurised skimmed UHT (ultra high temperature) powder

Heat treatment of milk Milk is an ideal place for bacteria to grow. Some bacteria are harmful so all milk is heat treated to kill them. Common methods of heat treating are by: pasteurisation Ultra High Temperature

Pasteurisation Most milk is treated by pasteurisation. Method: 1. Heat milk to 72ºC for 15 seconds. 2. Cool quickly to below 10ºC. 3. Pack in bottle, carton or container. Pasteurised milk will keep for up to five days in a fridge.

Ultra High Temperature UHT milk is heated to a higher temperature than pasteurised milk. UHT milk is heated to 135oC-142oC for 2-5 seconds. This process alters the taste of milk.

Resazurin Test Resazurin dye is a chemical that changes colour in response to the number of bacteria in a liquid. Can be used to tell us if milk is fit to drink.

Colour of sample Bacterial content Drinking quality of milk Blue-purple Very low Good mauve low Satisfactory pink medium Poor white high unsatisfactory

Experiment: resazurin test 5 day old milk + resazurin Fresh milk + resazurin 10 day old milk + resazurin

Results: Time (min) Fresh milk 5 day-old milk 10 day-old milk 5 10 15

Fat content of milk Milk can be graded by its fat content: Type of milk Fat removed Whole milk none Semi-skimmed half skimmed almost all Evaporated milk has ½ the water removed and is used like cream.

Content of milk (continued) Removing fat from milk reduces vitamin content e.g. Vitamin A Skimmed and semi-skimmed milk have fat removed so the vitamin content is reduced. Young children should be given whole milk which has more vitamins.

ii. Yoghurt Milk can be preserved (made to last longer) by changing it into yoghurt or cheese. Natural yoghurt can be used as ‘starter cultures’ to make yoghurt in the lab.

Making Yoghurt Starter cultures contain special bacteria that make lactic acid from the sugar (lactose) in the milk. lactose lactic acid (sugar in milk) (thickens and gives taste) Lactic acid thickens the milk and gives the yoghurt its taste.

Making yoghurt Method Heat milk to 43oC (helps bacteria grow) Add 1 teaspoon of starter culture (natural yoghurt) Cover with cling film. Incubate yoghurt at 43oC for 7 hours. When ready, place yoghurt in fridge for 4 hours.

Types of Yoghurt There are different methods for making yoghurts: Stirred yoghurts – bacteria is added to the batch. It is then put in to pots when ready. Set yoghurts – bacteria is added then the mixture is put straight into the pots where it sets.

Aseptic conditions Air contains many types of microbe. Many are also present in dust as tiny clumps called spores. During experiments, certain precautions should be taken to create sterile (aseptic) conditions.

This is done for two reasons: To stop unwanted microbes getting into the experiment and spoiling it To stop ant microbes used in the experiment escaping.

1. Hands should be washed, cuts should be covered. 2. Work surfaces should be disenfected. 3. Lab coats should be worn 4. All equipment should be sterilised in an autoclave (heated to 121C for 20 mins).

iii. Cheese Making Cheese Milk is pasteurised to kill most bacteria. Special bacteria are added to convert milk sugar (lactose) into lactic acid. Enzymes (rennet) are added to clot the proteins in milk to form solid cheese.

Types of rennet Calf rennet calves Fungal rennet fungus Milk clotting enzymes (rennet) can come from different sources. Type of rennet Source Advantage Disadvantage Calf rennet calves Original source, used for centuries. Animals must be killed, risk of disease Fungal rennet fungus Cheap, large amounts, OK for vegetarians. taste GM yeast rennet yeast No animals involved, OK for vegetarian, same as animal rennet. Public concern about genetically modified foods

iv.Environmental Impact: Monitoring Waste Cheese making uses the enzyme rennet which makes the milk proteins clot to form curd. The liquid left is called whey. milk + rennet curds (solid) whey (liquid) cheese waste product

Whey and pollution Whey contains sugar. What would happen if whey was released into rivers? 1. Bacteria would use the whey sugars as food and reproduce. 2. As the number of bacteria increased it would use up the oxygen so oxygen levels would decrease in the water. 3. Fish and other living organisms would start to die as the oxygen level decreased.

Pollution prevention To prevent pollution whey can be:- a. treated before release b. upgraded (used for something else)

Treatment of whey Add bacteria which feed on whey and turn it into carbon dioxide and water. Remove bacteria and release cleaner water into river. Test water oxygen level to make sure it is OK.

Upgrading whey Waste whey used as food for growing some types of yeast. In the right conditions these yeast strains produce alcohol from the sugars in the whey. Alcohol produced is creamy (found in Baileys Irish Cream)

Preventing pollution

Biotechnology Yeast Industries i. Bread ii. Beer iii. Fermented milk drinks iv. Flavouring and food colouring v. Environmental impact

i. Bread Yeast: a single-cell fungus (plant) used in bread-making for 1000’s of years is added to flour to make bread rise (dried or fresh yeast – activity 2.1)

Yeast (continued) Yeast is a living organism. It respires to release carbon dioxide. It is the carbon dioxide that makes bread rise.

Growing yeast Huge numbers of pure yeast can be grown in large vessels called fermenters. This yeast can be used in the baking or brewing industry to make bread or beer. Cultures of pure yeast can be grown on an agar plate.

Beer is an alcoholic drink made from: water ii. Beer Beer is an alcoholic drink made from: water barley sugar hops yeast

The role of yeast in making beer sugar alcohol + carbon dioxide + energy Yeast uses sugar to release energy. During this process, called fermentation, alcohol and carbon dioxide are released. The gas carbon dioxide is what makes the beer fizzy. yeast

Ales and lagers Different strains of yeast give different ales and lagers. These yeasts use the sugars at different rates and at different temperatures.

Ale and lager yeasts Type of yeast Growth temp Time to grow Position of yeast in vessel Ale 12-18ºC 6 days Rise to top Lager 8-12ºC 21 days Sink to bottom Ale yeasts

Making beer There are over 1200 different brands of beer in Britain each with its own flavour. Around half the beer is lager, the rest is bitter, ale and stout. Different beers are brewed in different ways and have different alcohol contents. Traditional beers have around 4% alcohol (activity 2.3)

Alcohol contents of beer (Activity 2.4) Drink Alcohol Content Lager Barley ale Low alcohol beer Alcohol free beer Wine Vodka Whiskey

Alcohol contents of beer (Activity 2.4) Drink Alcohol Content Lager 4% Barley ale 11% Low alcohol beer 1% Alcohol free beer 0.05% Wine 12% Vodka 40% Whiskey

Maturing the beer Beer must be matured before it can be drunk. Maturing beer: Improves flavour Removes any solids Gives ‘sparkle’ (activity 2.6)

Beer can be: cask conditioned brewery conditioned (often called real ale) brewery conditioned (kegs, bottle, cans)

Cask conditioned beer Cask conditioned beer is put into casks (huge containers made from wood or steel) Sugar is added to the cask. Yeast still producing carbon dioxide which makes the beer ‘sparkle’. Beer produced is dark with a strong flavour.

Brewery conditioned beer Stored in large tanks Sold in kegs, bottles or cans. Remains of yeast and other solids removed. Beer (e.g.) lager is clear and bright. Long shelf life (keeps for a long time)

Differences : cask conditioned and brewery conditioned beers (Activity 2.6) Cask conditioned beer Brewery conditioned beer Example of type of beer produced Storage conditions Description of beer

Differences : cask conditioned and brewery conditioned beers (Activity 2.6) Cask conditioned beer Brewery conditioned beer Example of type of beer produced Real ale e.g. Lager Keg beers Bottles/cans Storage conditions Casks Sugar added Yeast present Large tanks Yeast removed Description of beer Dark colour High flavour Clear/bright Lasts longer

iii. Fermented Milk Drinks In many countries it is difficult to keep milk and yoghurt fresh. The milk can be fermented slightly to make it alcoholic. Yeast is used to turn the sugars in milk into alcohol.

Making fermented milk drinks (activity 2.7/2.8) Kefir is a refreshing, fizzy, slightly alcoholic, yoghurt drink. This drink can be made by a method called immobilisation.

Making Kefir Step 1 Sodium alginate + lactase (enzyme that breaks down lactose) Add wine yeast Add calcium chloride drop by drop (hardens beads) Immobilised beads

Step 2 Warm milk Add live yoghurt Add Immobilised beads Leave at 43ºC for 5 hours Filter mixture to give fermented milk drink Beads can be re-used kefir

Immobilisation Immobilisation can be used to trap an enzyme and some yeast into a jelly bead. Advantages: After the reaction the beads can be washed and re-used. Saves money (enzymes are expensive) Bead easily separated from product (e.g. by filtering) jelly coat yeast + enzyme

iv. Food flavouring Yeast can be used for: Making bread Alcoholic drinks Flavouring food Foods with yeast flavouring: Meat flavoured crisps e.g. chicken,bacon Oxo cubes Marmite (activity 2.9)

Food colourings (activity 2.10) Wild salmon and trout have pink flesh. This colour comes from the pink coloured prawns and shrimps they eat. Farmed salmon would have grey flesh but they are fed red dye just before they are killed which makes their flesh pink. Red dye

Feeding dye doesn’t affect the flavour but makes their flesh more appealing to eat. Now red yeast can be fed to the fish. This gives a pink colour to their flesh. Red yeast

v. Environmental Impact Waste from yeast industries should not be dumped in rivers. Yeast would act as food for bacteria which would cause pollution.

Getting rid of waste Waste can be upgraded and used for animal feed. Treated water should be tested before releasing into rivers.

The methylene blue test We are going to test some water samples to see if they could be put into a river. Collect: samples A, B and C 3specimen tubes+lids 3 labels dropper measuring cylinder methylene blue dye

Testing water samples for pollution: The Methylene Blue Test Time Colour change Water condition Pollution scale Immediate 2-3 days 4-5 days

The methylene blue test Time Colour change Water condition Pollution scale Immediate Blue to clear Dangerous for river Very polluted 2-3 days 4-5 days Needs more treatment Slightly polluted Still blue Safe for release Not polluted

Detergent Industries

Enzymes in washing powders The word detergent means ‘something that cleans’ e.g. soaps washing up liquid washing powder

Biological washing powders Biological washing powders contain enzymes. Enzymes are chemicals that improve the way in which the powder cleans.

What is biological washing powder made of? Biological washing powder is made up of: 1% enzymes 99% water softeners bleach other chemicals (to help water get into the clothes)

Where do the enzymes in washing powder come from? Bacteria are tiny organisms found almost everywhere on Earth. Scientists found bacteria that were harmless and produced enzymes that could be used in washing powders. Large numbers of these bacteria grow (cultured) very quickly in huge industrial fermenters that give the best conditions for growth. Enzymes produced are then separated from the bacteria and used to make biological washing powder.

The use of enzymes in washing powders Enzymes in washing powders digest the stains on clothes like enzymes in the gut digest food. Different enzymes digest different stains. Fat digesting enzymes digest fatty stains. Starch digesting enzymes digest fatty stains. Enzymes make up a small part of powder but a large part of the cleaning power!

Activity 3.1: To show how an enzyme can remove a stain Stains from food like eggs contain protein. Photographic film has protein on its surface. In the following experiment, enzymes like those in biological washing powder are used to remove the ‘stain’ on a piece of photographic film Protein ‘stain’

Expt. : To investigate the effect of enzymes on a protein stain Method: Collect 2 test tubes. Label test tubes A and B Add 1 piece of film to each test tube. Half fill tube A with enzyme Half fill tube B with water Shake each tube Put tubes in water bath at 50oC for 30 mins – shake tubes every 5 mins Remove film, dry and examine.

Tube contents Protein digested? Film + Enzyme Film + water

Comparing biological and non-biological washing powders Biological washing powders contain enzymes. Non-biological washing powders do not contain enzymes. This experiment compares the ability of these two types of washing powder to remove different stains

Comparing bio and non-bio washing powders Warm water + bio powder Warm water +non- bio powder Stained cloth Stained cloth

Stain Removed with bio powder? Removed with non-bio powder?

Disadvantage of biological washing powders Original biological washing powders sometimes caused an allergic reaction in some people. This caused skin rashes, eczema and asthma. New powders are now produced with enzymes enclosed in a harmless waxy coating. This helps to prevent allergic reactions. (Activity 3.3)

Advantages of biological washing powders (Activity 3.4) Adding enzymes to biological washing powders means cleaner clothes (stains are digested). 2. Work best at low temperatures 40oC - 55oC (temperatures above 60oC destroy enzymes) so need to heat clothes to high temperatures to get them clean-saves energy and money. 3. Lower temperature used with biological washing powders reduce damage to delicate fabrics.

Environmental Impact : Monitoring Waste Making detergent uses energy for: Production Packaging Transporting But most energy is used in the home for: The wash cycle Tumble drying Ironing

Detergents and energy Activity 3.5 To provide all the energy needed power stations burn coal, oil or gas. This gives off carbon dioxide and sulphur dioxide that pollute the atmosphere. Low temperature wash = less energy good for public and environment!!

One manufacturer of detergents has set targets for waste management: Reduce weight and volume of packing Use reusable materials Encourage recycling programmes Encourage safe disposal practises New ‘Micro’ powders and liquids use less packaging and powder for each wash.

Detergents and the Environment (Activity 3.6) Detergents are flushed away as waste water and can pollute the environment. Detergents can be toxic (poisonous) to wildlife. Manufacturers test products to check they won’t harm fish or other living organisms in rivers.

Detergents containing phosphates and sulphates pollute river Tiny plants (algae) reproduce quickly to form ‘bloom’ Algae die Bacteria feed on dead algae Number of bacteria increases Bacteria use up oxygen in water Fish and animals die

Reducing Environmental Impact Detergents in water tested at sewage works in mini sewage plants Sewage plants could remove the phosphates and sulphates to reduce environmental impact Using low phosphate detergents can also reduce environmental impact

Washing clothes in other countries (Activity 3.7) Most people in the world wash clothes by hand Washing machines in other countries are different from those in the UK In the USA washing machines are bigger and use more water In Japan and Taiwan washing machines do not heat the water. Clothes are pre-soaked and washed more often

Biotechnology Unit 4 : Pharmaceutical Industries i. Antibiotics ii. Antifungals

Antibiotics ~ discovered in London in 1928 by Alexander Fleming (Scottish scientist) ~ He was growing bacteria on agar plates to study. ~ One of his plates had been contaminated with a fungal spore and he noticed no bacteria would grow near it ~He found out that fungi produce chemicals to stop growth of competing bacteria

~These chemicals are called antibiotics ~The antibiotic Fleming had discovered was penicillin and had been produced by the fungi penicillium ~Different types of antibiotics kill different bacteria – but not viruses like those causing flu or the cold.

Other scientists developed ways of extracting peniciliin fron the fungus and purifying it The first man to be treated with penicillin was a policeman. He was ill from blood poisoning and not expected to live more than a few hours. When injected with penicillin there was a huge improvement in his condition. The man only died when the antibiotic ran out after ten days.

~Different types of antibiotics kill different bacteria – but not viruses like those causing flu or the cold. Penicillin now cures diseases such as pneumonia and diptheria

Antibiotic action If an antibiotic can inhibit growth of a species of bacteria we say the bacteria is sensitive to that antibiotic If an antibiotic has no effect we say that species of bacteria is resistant to the antibiotic. There is no one antibiotic that works against all species of bacteria

+ + + - + + + Antibiotic + + + = very effective + + = effective Pneumonia Tuberculosis Typhoid Diptheria penicillin + + + - Streptomycin Tetracycline + + + Cloram-phenical + + + = very effective + + = effective + = slightly effective - = no effect

Different antibiotics have different methods of destroying bacteria: Some destroy bacterial cell walls Some burst the cell membrane Some interfere with the bacterial cell’s chemical reactions

Different types of antibiotics (Activity 4.3) Different antibiotics are effective against different infections. Antibiotic Infection which it treats penicillin respiratory infections (and many others) aminoglycosides Eye and skin infections fluoroquiniles Gonorrhoea (STD) cephalosporinc Urinary infections tetracyclines acne

Choosing the correct antibiotic When a patient has an unknown bacterial infection, a sample of body fluid taken so that the bacteria can be grown on nutrient agar. A multidisc is placed on the agar surface.

It is important to have a choice of antibiotics because: The person may be allergic to an antibiotic Bacteria may become resistant to an antibiotic

Antibiotic production Antibiotics produced in large fermenters holding 200,00 litres Growth conditions are controlled by computer to provide correct Temperature pH Oxygen concentrations Food supply Sterile conditions Antibiotic purified by filtering and solvent extraction

Genetic modification Genetic modification is a new technology. It changes the genes found in living things. The penicillin gene can be taken from the fungus and put into bacteria. These ‘genetically modified’ bacteria can then produce very large quantities of penicillin. New, more effective antibiotics can also be produced to help fight disease.

Antibiotic production Activity 4.2 Antibiotics were first produced in ___________ by ______________ ____________. They are chemicals which kill ____________. They do not kill _________. Antibiotics are produced in huge ____________. Growth conditions inside the fermenters are controlled by ____________. If the glucose level in the vessel falls then the ________ will detect this _____________and more ____________ will be added. The antibiotic is _________________ by filtering and _______________ extraction. ______________ modification is a new ______________ which can alter the _____________ of living organisms. This new technology may be used to produce new _____________ which will be better at fighting_________

Antibiotic production Activity 4.2 Antibiotics were first produced in London by Alexander Fleming. They are chemicals which kill bacteria. They do not kill viruses. Antibiotics are produced in huge fermenters. Growth conditions inside the fermenters are controlled by computers. If the glucose level in the vessel falls then the computer will detect this change and more glucose will be added. The antibiotic is purified by filtering and solvent extraction. Genetic modification is a new technology which can alter the genes of living organisms. This new technology may be used to produce new antibiotics which will be better at fighting disease.

Antibiotic resistance Some infections have become resistant to antibiotics. This means that the antibiotic is no longer effective. Staphylococcus aureus is a bacteria that causes abscesses and boils 1940’s this bacterium was sensitive to penicillin so it could be used to treat Staphylococcus aureus infections.

Now some strains of this bacteria are resistant to penicillin and it is no longer effective. These strains have also become resistant to other antibiotics and are known as MRSA. These strains are prevalent in hospitals where infections are easily spread. This has happened because because antibiotics have been over-used.

Public concern People are concerned about the over-use of antibiotics in agriculture and by vets. The same antibiotics are used in animals and humans. It is thought that this might result in more resistant strains of bacteria. New antibiotics to which bacteria are not resistant are continually being looked for.

Anti-fungals Some infections are caused by microbes called fungi They are spread from person to person by tiny groups of fungi called spores. Drugs used to treat these infections are called anti-fungals. Anti-fungal treatment slows down or stops fungal growth.

Athlete’s foot This fungi likes to grow areas of the skin which are warm and moist and get little fresh air. Forms an itchy rash between the toes. Transferred in shared dressing areas or showers. Flakes of skin from an infected person are enough to pass the infection on Treated with antifungal creams or powders.

Oral thrush Fungal infection of the mouth. Seen as white spots in the mouth. Common amoung: Babies People with ill fitting dentures Chemotherapy patients Drug users Treated with antifungal mouth washes or pastilles

Ringworm Begins as a patch of itchy skin Spreads to form spiral shapes Caused by a fungus not a worm!