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Unit I: Introduction to Biotechnology and Laboratory Procedures Biotechnology I/Zeiher Lab: Cheese Making Lab: Modified from Daughtery, E. 2012. Biotechnology Laboratory Manuel. Paradigm Publishing. pp. 8-11 and Brown, Judy. 2002 “Little Miss Muffet” in Shoestring Biotech. National Association of Biology Teachers. pp. 139-152. Introduction: The cheese-making industry is huge and is a good example of how biotechnology has improved an industrial process. Have you ever let milk stay too long in the refrigerator or on the kitchen counter? You will notice that the milk will begin to form clumps. This happens because the naturally occurring bacteria in the milk is causing the milk to turn sour. This was the original way that cheese was made This clumping occurs because milk bacteria have enzymes (protein that speed chemical reactions) that convert the milk sugar lactose to the product lactic acid. Lactic acid has a low acidic pH. This acidic pH causes the milk protein casein, to denature or break down and fall out of solution. Other enzymes, called proteases, may also act on casein. Proteases act as scissors cutting proteins, such as casein, into smaller fragments that will also fall out of solution. The lumps of denatured casein are called curds and are the clumps of milk observed when the milk sours. Curds, these solid clumps, are pressed together to form cheese. The liquid remaining after curdling is called whey as was mentioned in the nursery rhyme “Little Miss Muffet” Little Miss Muffet sat on her tuffett eating her curds and whey” Using early methods, cheese makers started new batches with a small amount of cheese (containing the enzyme-producing bacteria) they had saved from a batch of curdled milk that produced a good cheese. Although some cheese is still produced in this fashion, today, most commercially made cheese is produced in one of the four ways listed below. In each method, sterilized milk is used as a starting reagent. 1. Aging Method: The milk may simply naturally sour by exposing to air and naturally occurring bacteria. The curds that form are pressed and used to form cheese. 2. Bacterial-culture Method: New batches of cheese are started with specific cultures of Lactobacillus bacteria, bacteria known to make milk curdle found in, for example, buttermilk and yogurt. Unit I: Introduction to Biotechnology and Laboratory Procedures Biotechnology I/Zeiher 3. Calf Stomach Enzyme Method: One of the earliest methods for making cheese was accidently found when ancient people used animal stomachs as a canteen to store milk. They discovered that the milk stored in these stomachs became clumpy. It is now known that cells that line animal stomachs contain enzymes, specifically proteases. It was subsequently found that if we add a purified form of a protease, known as rennin, that it can stimulate the formation of curds by breaking down casein into small fragments. Rennin is isolated from the cells that line calves’ stomach. To retrieve the enzyme for commercial use, companies grind up calves’ stomach and separate and purify the rennin enzyme from other macromolecules (proteins, carbohydrates, nucleic acids). Because of the animal source of rennin, some vegetarians and vegans do not eat rennin cheeses (e.g. cheddar cheese, asiago cheese, blue cheese). 4. Recombinant Enzyme Method: Scientists isolated the gene that encodes for rennin from calves’ cells and inserted into a plasmid. The recombinant DNA molecule (Calf DNA + Fungal DNA) was inserted into yeast (a fungus) cells. The yeast cells produce rennin in larger amounts and faster than normally occurs in the calf’s stomach. Cheese makers use the genetically engineered enzyme known as chymosin to speed curdling process. chymosin cheeses include Jack, mozzarella, and most Swiss cheeses. Biological Question: Which curdling agent produces cheese at the fastest rate? Pre-Lab- In your lab notebook Write biological question Write a hypothesis as an If……. Then statement Answer Pre-Lab questions Summarize procedures in your own words (IYOW) Pre-Lab Questions 1. Explain the difference between curds and whey. 2. Why do the solids form in milk when cheese is made? In your answer describe the science behind the clumping. 3. List the four methods used to make cheese and briefly explain each method 4. What is the difference between rennin and chymosin? Supplies Needed per lab station Unit I: Introduction to Biotechnology and Laboratory Procedures 1 thermometer 1 hot plate 1 100 mL graduated cylinder 3 150 ml flask or beaker 1 clock or timer 1 1-mL pipette 1 bulb or pump 1 stirring rod 1 funnel 3 100-ml beaker 1 balance 1 permanent marker, such as a Sharpie or laundry marker 150-mL cultured milk 0.2-mL Chy-Max (0.1% w/v) 0.2 mL Rennin (0.1% w/v) 4 15 x 15-cm cheesecloth squares 1 weigh boat 5 highly absorbent paper towels plastic wrap Biotechnology I/Zeiher Materials: Procedure 1. 2. 3. 4. 5. 6. Each group will be doing the following treatments: buttermilk, rennin, chymosin, and scalded whole milk (negative control). Using a 10-mL pipet and pipet pump, transfer exactly 7 mL of scalded whole milk into a labeled 15 mL conical tube. (label needs to have the treatment name, your initials, the date, the period). Using a pre-set P1000, add 250 µL (0.25 mL) of the curdling agent. Cap the tube and mix gently by inverting three times. Record the "initial time". Place the milk-containing portion of the tube in 37oC water for at least 15 minutes. Check for curdling every 5 minutes, recording the time to curdle in minutes. To check for curdling, gently tilt the tube, being careful not to break up any curds. Curds are large lumps of solidified milk. After 15 minutes, place the tube upright at room temperature and check for curdling every 15 minutes for the next 2 hours. If curdling has not occurred within 2 hours, continue to check once every 4 hours. With the greatest accuracy possible, record the time, in minutes, until the milk curdles. 7. If curdling has not occurred by the end of the lab period, place on the lab bench and let Unit I: Introduction to Biotechnology and Laboratory Procedures Biotechnology I/Zeiher it incubate overnight (at room temperature) and check the next day. Keep the tube upright so any curds will fall to the bottom. 8. When you return to lab the next day, measure the volume of curds (solids) and whey (liquid) in the tube. You may be able to read the volume of each directly from the tube. If not, filter the curds as described below: Pour the whey and curds through cheese cloth in a funnel into a 10-mL graduated cylinder. Determine the volume of when collected in the graduated cylinder. By subtraction, determine the volume of curds as described in calculations section. 9. In your lab notebook, make a data table similar to Table 1.2 and record the data for your sample plus one each of the other variable groups. This will give you data for one experimental trial of each curdling agent. Record the name of the person from whom you obtained the data. As a class, we will average the data for each experimental group. Table 1.2 The Effects of Cheese-Curdling Agents on Curdling Time and Volume Curdling Agent Time to Curdling Volume of Whey Volume of Curds (min) (mL) (mL) buttermilk rennin chymosin Distilled water Calculations Volume of Curds (mL) = Total Volume of starter milk culture – Volume of Whey (mL) Data Analysis and Results 1. Draw two graphs: a. Average time to curdling for each treatment. b. Average volume of curds produce by each treatment. 2. How well did the single trial your group did support the hypothesis? Explain. Unit I: Introduction to Biotechnology and Laboratory Procedures Biotechnology I/Zeiher 3. Averaged data are the best way to answer an experimental question. Explain why. 4. Does it appear that the number of trials for each curdling agent sufficient? Yes or no? Explain your answer. 5. Do you think the whey-o-meter was adequate for an accurate determination of whey volume and, indirectly, curd volume? Why or why not? If yes, explain why. If no, suggest a better system to determine the volume of curds or the volume of whey. Extension Activity Scenario Assume you are a member of a team of biotechnicians from a cheese making company. The president of the company offers a significant bonus to the team that designs the most costeffective protocol to increase the yield of cheese. You and the members of your team decide to accept the challenge. How will your team organize themselves to accept this challenge? Your task is to develop a protocol that will increase cheese-yield based on what you learned from the previous experiment and then test that protocol to determine if your ideas are correct. Review the cheese-making protocol you previously did, then choose a variable that you could modify that might enhance curd formation. Brainstorm what variables you could vary In developing your protocol (Complete this in your Lab Notebook) 1) 2) 3) 4) 5) 6) Define your biological question Based on your biological question, develop a hypothesis Provide your experimental design – Experimental Group, Control Group, Fixed Variables List the materials required Outline the procedure you will use After this is completed get the instructors initials. After completion of experiment 1) 2) 3) 4) Organize your data in well-constructed table Show patterns observed in the data graphically Summarize the patterns observed in the graph but do not interpret the results Draw conclusions Unit I: Introduction to Biotechnology and Laboratory Procedures Biotechnology I/Zeiher a. b. c. d. e. State claims Use evidence from data to support your conclusions Reject/Accept hypothesis Describe possible errors and its effect on results Make a recommendation to the cheese company supervisor about which variable should be considered to improve production. f. State any new questions you might ask based on the conclusions drawn from this experiment Technical Standards Lab addresses: 3.0 Demonstrate Critical Thinking and Scientific Problem-Solving Skills 3.1 Identify and use industry-recognized observational methods and skills. 3.2 Identify and structure tractable, easily managed and controlled questions showing evidence of observation and connection to prior knowledge. 3.3 Develop and test hypotheses utilizing experimental design distinguishing between controls and variables and use experimental, analytical, and statistical design. 8.0 Demonstrate Basic Lab Skills in the Use of Equipment and Instrumentation 8.1 Use software/hardware for scientific analyses and documentation (e.g. Excel, PowerPoint, Word). 8.2 Identify and demonstrate proper use of laboratory glassware. 8.3 Identify and demonstrate proper use of laboratory balances. 8.4 Identify and demonstrate proper use of micropipettes. Unit I: Introduction to Biotechnology and Laboratory Procedures Biotechnology I/Zeiher Teacher Notes Chemical Preparation DIRECTIONS FOR SETTING UP THE LAB One day ahead of the lab Chymosin Bioengineered Enzyme Solution Prepare stock solution of selected enzyme at manufacturer’s suggested concentration. For example, Chy-Max is prepared at a 1.0g per 60-ml deionized water. Store in tightly capped 100ml storage bottle. The liquid product contains approximately 0.1% active chymosin. E mporase comes ready to use. Follow the manufacturer’s recommendation for refrigeration. Chymosin recombinant rennin can be ordered from Sargent Welch cat# WLB1030 Preparation of scalded milk Heat whole milk to 82oC and leave at that temperature for 5 min. Store in sterilized bottle.