Rennet, Home Made, Illustrated

Here are the results of an experiment at producing home made rennet.

Rennin is an enzyme which, in an acid environment, digests the water soluble milk protein casein into insoluble products. When these precipitate out of solution, the milk coagulates. The test is the famous “clean break” of cheese making.

Here, the abomasum of a suckling kid was cleaned, salted and dried. A small piece (0.75 gm) of it was suspended in warm water (30 C), and added to 1 gallon of inoculated milk. While a clean break was not achieved in three hours, by the evening (about 7 hours) the milk had formed a very firm coagulant.

This is my first attempt at using home made rennet.  I am sure that the process and conditions can be improved.  Let me know if you have suggestions.

See the bottom of the page for suggestions from Mr. Wolfgang Pachschwöll, of “Hundsbichler company Austria – producer of natural rennet.”

Here are some points of expert advice on making rennet from Wolfgang Pachschwöll of “Hundsbichler company Austria – producer of natural rennet”, sent in response to my initial posting of this page. (Thank you very much Wolfgang!)

1) Do not thoroughly clean out the inside of the abomasum. The “slime” inside contains rennin. Therefore, also no washing nor squeezing.

2) Lightly salt the abomasum, store undried with 30% salt in a closed container to activate the enzyme over three months. (Pepsin, another stomach enzyme, is also secreted in the inactive form (pemsinogen), and activated by acid or enzymatic action.)

3) The traditional way to then dry the abomasum is to inflate it like a balloon and dry by hanging in a cool dark place.

4) Dissolving and activation of rennin occurs best in acid conditions at a cool temperature.

 

Neufchatel Cheese

Beginning Cheese Making

Cheese Making for Beginners

While cheese making is theoretically a science, we also need to appreciate that it is an art.  Often cheese-making instructions appear simple, but there are skills and sensitivities which must be developed for successful cheese making.  Because it is beneficial to engage in projects from easy to challenging, I strongly suggest that you master the following projects in sequence before you progress to more difficult cheeses.

As an avid homesteader, I strive to keep the ingredients for these recipes relatively easily obtained from your local supermarket and to use the equipment commonly found in the kitchen. As you become more skilled and knowledgeable, you may well want to purchase more specific ingredients from the web (etc) to refine your procedures and finished products.

Discussion of Ingredients and Equipment

Ingredients:

Milk:
You can use a wide variety of milks, from commercially purchased pasteurized homogenized cow’s milk, TB certified raw cow’s milk, goat’s milk, sheep or even horse’s milk.  All will make cheese, each with its unique flavor.  Cheese from unpasteurized milk makes the best cheese, but should be cured for 2-4 months if there is any doubt about pathogens in the milk.  If you use pasteurized milk, you may need to add a little calcium chloride to firm up the curd because the heat makes the calcium unavailable.  Calcium is required for a good “clean break.”

Cheese can be made from whole milk (3.5%), 2% or from skimmed milk.  However, remember that the richness of flavor of the cheese is related to the amount of butterfat in the milk.  I do not recommend reconstituted powdered milk. It has not produced either a solid clean break, nor a good flavor of cheese.  Let me know if you do.

You should  get  1 to 1.5 pounds of cheese/gallon of milk.

For store-bought milk, because Pasteurization removes calcium from solution, you may need to add a small amount of calcium chloride to aid coagulation and form curd which does not fall apart when you stir it. The desired concentration of CaCl2 is usually specified as 0.02%. This would mean adding 3.6g CaCl2 to 5 gal of pasteurized milk. My measurements indicate that 3.6g of crystals = ~3/4 tsp. Thus 3/4 tsp crystals/5 gal = ~0.02%. You should completely dissolve the CaCl2 in about 1/4 cup water before adding it to the milk. Add it slowly with thorough stirring.  You should be able to purchase CaCl2 at brewer’s supply house.  Also, it is the non-NaCl deicer that is plant-friendly.  [You might be able to use CaSO4 (plaster of Paris) in a highly diluted state, tho it is much less soluble than CaCl2.]

Reconstituted powdered milk: I have not had good luck using powdered milk, but have heard some say that they have successfully used it.  I suggest you make friends with a local dairy person to get bulk milk.  (You will have better luck getting milk from homesteaders than with commercial operators.  Legally, you must purchase it as “pet milk.”

Ultra pasteurized milk: This is common on the shelves in Europe, but I find it VERY unsatisfying in taste, and, in my opinion, this milk is nearly worthless in making basic cheese. The protein strucure is radically altered, and calcium is chelated so that it is unavailable for coagulation. If you have success with ultrapasterized milk, let us know! (You CAN use it to make yogurt and labneh.)

Starter:
Bacteria must be added to acidify the milk so that the rennet will work, and to aid in the curing.  Cultured buttermilk can serve as a mesophilic starter (it likes room temperature), and yogurt can serve as a thermophilic starter (it prefers warmer temperatures).  You can also purchase pure cultures from cheese makers supply houses.  Some recipes call for addition of chemical acids to produce acidify the milk.  I prefer the flavor of cheese made from bacterially acidified milk.  Here is a detailed description of bacterial cheese starters from Peter Moller. Be aware that, especially with buttermilk, you should never add too much starter for too long a time such that the milk has even remotely thickened before you add rennet. With even slightly thickened milk, you will never get a “clean break.”

Rennet:
An enzyme rennin converts milk protein (casein) from a soluble to an insoluble material, causing the milk to gel (forming a clean break).  It will only work well in acidified milk.  The gelling process must be undisturbed to get a clean break.  Rennet is commonly available in supermarkets in the pudding section, or you can purchase liquid rennet from a cheese makers supply house. One tablet of Junket rennet is equivalent to 20 drops of fresh liquid rennet.

If you would like to try it, and have the materials available, I have successfully prepared home made rennet from the abomasum of a suckling kid.

Equipment:

Most decently equipped kitchens have the necessary equipment:

Heavy stainless steel pot with lid:  A non-reactive pot is important because the acidifying milk can dissolve aluminum.  Enamel pots would work as well.  A heavy bottom is important to evenly disperse the heat and prevent scorching.  Scorching affects the flavor and is a cleanup hassle.  The size of the pot should be large enough so that you have at least an inch of head space above the milk.  A cover is needed for the steps when the milk must sit for periods of time.  I bought a high quality five gallon pot like this years ago (expensive).  I have never been sorry.

Measuring cups  You may need measuring cups ranging from 1/4 cup to a quart.  Obviously you can improvise, but accurate measurements improve your success rate.  Measuring spoons are occasionally needed.

Thermometer  Very important is an accurate thermometer which reads in the range between freezing and boiling for water (~32-212 F, or 0 to 100 C).  A candy or meat thermometer can work.  There are several reasons that accurate temperature is important: the texture of the cheese depends a great deal on achieving a temperature to within onedegree.  Also, after milk for yogurt is scalded, itmust be cooled to 130 F for optimum bacteria action.

Whisk  Thorough mixing of starter and rennet is important.  A whisk is the obvioius choice for this step.

“Cheese cloth”:  The purpose of “cheese cloth” is to catch the curd and allow the whey to drain out.  What most people think of as “cheese cloth:” the very wide weave flimsy material is often useless for this purpose.  If your curd is fine, it passes through.  Even if it is large curd, the curd can become enmeshed in the coarse weave.  I use either large plain white cotton handkercheifs, or white non-terry cotton dish towels.  I recommend ignoring what is sold as “cheese cloth…”

Cheese press is required for the hard cheeses.  You can purchase them for a handsome price, or fashion one from materials at home.

Waxing your cheese:  This step is important so that your cheese does not dry out during aging, and to prevent mold from growing on the surface (mold needs air to grow).

Cheese Making, Course Syllabus

Setting Up a Home Made Cheese Press

How to Wax Cheese

Rennet for Making Cheese

Rennet, Home Made, Illustrated

Enzyme Assay: Lactase

Enzyme Assay: Lactase

[See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)]

hydrolysis_of_lactose

Lactose, (milk sugar) is a disaccharide formed from galactose and glucose. Its beta-galactosidic bond must be hydrolyzed to yield its component monosaccharides before they can be absorbed by the body. In humans, the enzyme lactase performs this task. Many individuals lose the ability produce lactase, and therefore “lactose intolarent” after they enter their teens. As a result, they suffer GI upset when they consume milk products (gas, abdominal pain, diarrhea). Lactase tablets may reduce this problem. We will be assaying the lactase content in commercial tablets, and use this enzyme as a typical enzyme in our subsequent studies.

ONPG_hydrolysis

The substrate used in the assay of this enzyme is o-nitrophenyl–D galactoside (ONPG), which, upon hydrolysis of the -galactosidic bond, yields galactose and o-nitrophenol, a yellow compound (absorption max = 450 nm) ( CRC Handbook: #p679, Merck Index, #6541).

Enzyme activity is proportional to the increase in A 450 during incubation.
Lactase_assay/ONPG hydrolysis

As in many enzyme assays, adjustments in concentrations and volumes may be needed for optimum results. Keep careful track of how you set up your experiment. This is often best accomplished by diagramming the procedure .

Materials and equipment: (per team of two students)

SUPPLIES: PER CLASS:
fresh lactase (suspend in buffer) (9000 unit tablet suspended in 90 mL buffer)
20 mM o-nitrophenyl–D galactoside (20 mL ONPG)
0.1 M PO 4 buffer, pH 5.5 (60 mL)
0.01 M PO 4 buffer, pH 5.5 (120 mL)
4% K 2CO3 (100 mL)

Distribute to each table:
20 mL dH2O in small flask
7 mL reaction mix* in 13 x 100 mm tube
1 mL 1:200 diluted enzyme (0.5 units/mL) in 13X 100 mm tubes
7 mL 4% K 2CO3 in 13 x 100 mm tube

EQUIPMENT
25 mL grad cylinder
100 mL grad cylinder
test tubes: five 13×100 in rack
200 and 1000 micropipets and tips
vortex
Eppendorf Repipeter with 10 mL syringe
37 C hot block, 13 mm holes
stopwatch
spectrophotometer
2 cuvettes in rack at spectrophotometer

01_suspend_lactase_Pa172076md
Suspending lactase in buffer

1. a: Record the brand of lactase, labeled number of units of lactase/tablet and the expiration date.
b: Weigh one lactase tablet, note whether 9,000 FCC or 3,000 FCC units/tablet.
c: Grind in a mortar and pestle until finely ground.
d: Suspend/dissolve to 100 units/mL: Grind a tablet in about 5 mL of chilled 0.01 M PO4 buffer, pH 7.
For 9,000 unit tabs, q.s. to 90 mL with same buffer, including mortar and pestle rinses.
For 3,000 unit tab, q.s. to 30 mL (Solution will be cloudy because of undissolved binder.)
e: Dilute 1:200: Add 0.1 mL of enzyme suspension into 19.9 mL 0.01 M PO4, pH 5.5, in a 25 mL grad cylinder.
Most enzymes should be kept on ice until ready to use, this may not be necessary for lactase.

*2. Prepare reaction mix (Rxn Mix): Per desk: Per class of 20:
0.1 M PO4 pH 5.5 buffer 5.6 mL 56 mL
20 mM ONPG 1.4 mL 14 mL

3. a. Copy the following table into your notebook.
b. Then set up a series of numbered 13×100 mm test tubes as follows.
c. Add water to the tubes first, then RxnMix with the repeater pipetter. (Not the enzyme yet

09.5_prewarm_tubes_Pa172074

4. Vortex and pre-warm these tubes in a 37 C hot block for two minutes.

5. At 30 second intervals, add listed uL of enzyme, vortex, start a stopwatch with 1st tube, place in 37 C hot block.

10_finished_assay_close_Pa172073md
Lactase assay, after incubation

6. After exactly 15 minutes, add 1.0 mL 4% K2 CO3 down the side of the first tube, mix and remove from hot block. At 30 second intervals, repeat 4% K2 CO3 addition for each of the successive tubes, mix and set aside.

7. Read the absorbency at 450 nm, record in your notebook table, graph and discuss results.

8. Calculate the number of units of lactase (1.000 OD unit/15 min) in the original tablet. (See following protocol). Compare with other brands of lactase.
CALCULATION OF LACTASE ACTIVITY/TABLET:
If 1 unit of lactase produces an OD of 1.000/15 min., and the assay was run for 15 mins:
units/tablet = A 450 x 100 mL/tablet x dilution factor x 1/(aliquot in mL

REAGENTS, MATERIALS AND CALCULATIONS FOR LACTASE ASSAY page 26

19 September 1993. rvsd 25 October 1994, 18 Sept 95, 20 Sept. ’96
0.1 M PO 4 pH 7.0 BUFFER:
For 200 mL, weigh out: 1.0 g KH2PO 4
1.8 g Na 2HPO4
dissolve in 200 mL H 2O, check pH, adjust to 7.0 if nec. with either H 3PO4 or NaOH. Store at 4C.

0.01 M PO 4 pH 7.0 BUFFER : (for suspension and dilution of enzyme)
Q.s. 50 mL of pH 7.0 0.1 M PO 4 buffer to 500 mL with dH 2O.

20 mM o-nitrophenyl–D galactoside (ONPG): (chromogenic substrate)
Weigh out: 602 mg ONPG
dissolve in about 80 mL 0.01 M PO4 buffer, pH 7.0 with swirling and slight warming.  q.s. with buffer to 100.0 mL.]
REAGENT TO HALT REACTION:
4% K 2CO3 : dissolve 8 g K 2CO3 in 200 mL dH2 O, stir to dissolve.
MATERIALS AND EQUIPMENT for team of four assaying given brand of lactase:
(two sub teams each perform an assay) 10/25/94, rvsd 18 Sept ’95, 20 Sept. ’96

EQUIPMENT:
mortar and pestle
100 mL graduated cylinder
ice bath
5.0 mL pipet (for dH 2O)
pipet bulb or helper
2 x 200 lambda micropipettes
(for ONPG and enzyme)
2 x 1000 lambda micropipettes
(for buffer and 4% K2CO3 )
2 16 x 150 mm test tubes
10 13 x 100 mm tubes
two test tube racks for 13×100 tubes
37C hot block for 13 x 100 mL
2 stopwatches
spectrophotometer, warmed up
at spectrophotometer:
cuvettes in rack
wipettes
SUPPLIES:
lactase tablets
100 mL 0.01 M PO 4 buffer
(to suspend and dilute enzyme)
30 mL dH 2O in 125 mL flask
(to make up assay set)
3 mL 20 mM o-nitrophenyl–D galactoside
(ONPG)
15 mL 0.1 M PO 4 buffer, pH 7.0
(for assay tubes)
15 mL 4% K 2CO