CLAYTON STATE UNIVERSITY

A Comparison of the Curd Yield and Rate of

Fermentation Produced Between Chymosin
(CHY-MAX) and Vegetable Rennet
Biology 3250L Microbiology Lab, Clayton State University

Blake Anderson, Alonza Lewis, Quatarsha Magee, and Hong-An Vo
11/12/2013















A comparison of the curd yield and rate of fermentation produced chymosin (CHY-
MAX) and vegetable rennet
Blake Anderson, Alonza Lewis, Quatarsha Magee, and Hong-An Vo
Biology 3250L: Microbiology Lab, Clayton State University
Dr. Furlong








Abstract
CHY-MAX claims to have the advantages that will enable it to surpass other rennet in
flavor, time saving, coagulant efficiency, etc. This report represents an attempt to
compare CHY-MAX to vegetable rennet in focus on time for curds formation and
amount yield in order to test CHY-MAXs assertion. As an approach to this, two mugs of
milk were used; one was supplemented with CHY-MAX, the other with vegetable rennet.
Both of them were incubated in the appropriate temperature to initiate the development of
curds. The results obtained show CHY-MAX did boost the milk to produce more curds in
a shorter amount of time compared to vegetable rennet. It yielded 3.19 times higher in
quantity and five minutes faster. Although it is challenging to draw a definite conclusion
from this experiment, the end results pointed out CHY-MAX as a more efficient
supplement to produce more curds in less time.

Introduction
The production of cheese via milk fermentation is an ancient practice predating
written history (Cheese Lab). According to Jannette Roeder (2013), cheese was likely an
accidental discovery resulting from the routine practice of storing milk in vessels made
from animal stomachs. Rennet, found in stomach lining, contains chymosin and other
proteases, which, combined with the heat from the sun, caused the stored milk to curdle
and eventually form a primitive cheese product. After tasting the curds, word of mouth
caused the discovery to spread and thus the process of making cheese was developed.
Since this discovery, cheese makers have created various ways of producing curds
and influencing the type and amount of curds produced. The simplest method used to
make cheese requires only two ingredients: milk and time. Overtime, milk left at an
ambient temperature will curdle on its own due to the metabolic processes of bacteria that
naturally exist in milk. However, the amount of time required for such process can be
long; thus it is common for a coagulating agent or starter culture to be added to milk as an
order to boost the formation of curds.
There are several coagulating agents used by cheese makers but the agent of
interest in this study is chymosin present in rennet. Besides the aforementioned animal
rennet, two other alternatives exist: vegetable rennet and CHY-MAX. The enzymes of
vegetable rennet are biochemically similar to chymosin but plant coagulants tend to be
more proteolytic (Esteves et. Al., 2003). CHY-MAX is essentially chymosin that has
been produced by inserting bovine chymosin genes into the chromosomes of bacteria or
yeast. These vectors are then placed in large fermentation vessels, where they produce
large amounts of chymosin, which can then be purified.
The objective of this study was the production of edible cheese from cow milk
while discerning the optimal coagulating agent for milk curdling. CHY-MAX is being
advertised to produce higher cheese yields and faster curdling time. Past studies have
compared both vegetable rennet (Esteves et al., 2003) and CHY-MAX (Barbano et. Al.,
1992) to animal rennet individually and found that each alternative to animal rennet
produces curds at a higher yield and faster rate. However, few studies were found
comparing these two alternatives. In order to determine the better alternative, separate
portions of whole milk will be curdled using CHY-MAX and vegetable rennet to test the
hypothesis that CHY-MAX will produce curds faster and at a higher yield than vegetable
rennet. CHY-MAX is a purified sample of chymosin, whereas vegetable rennin is an
extract containing only a small concentration of the desired enzyme. As a result, the
higher concentration of the desired enzyme of CHY-MAX will result in a higher curd
yield and faster rate of curdling.

Methods
Cheese making is considered a delicate process involving several steps and often
extended waiting periods. The process begins by first inoculating whole milk with the
appropriate starter culture. After a brief incubation time, 100mL of the whole milk is
transferred to two appropriate containers. In the case of this experiment, two mugs were
used as the containers. The milk of each container will be inoculated with a coagulating
agent; CHY-MAX in one and vegetable rennet in the other. It is important that the
containers are labeled with the date, rennet used, and researcher initials. Before
inoculation of the milk portions, two spoons were obtained and labeled to prevent cross
contamination. Use separate spoons for each condition and be careful not to cross
contaminate. Next, two rennet solutions were prepared and 1000uL of each was
transferred to the appropriate container. After the addition of the rennet, the containers
were placed in a 32
o
C water bath incubator. Immediately after the placement of the
containers in the water bath, a timer was set and the solutions were checked for curd
formation every minute. The time at which curdling began and finished was recorded for
each condition.
After curd formation was completed, the whey and curds of each condition were
separated by lining two funnels with cheesecloth (pre-weighted), one for each condition.
The contents of each container were slowly poured through one of the funnels into two
separate graduated cylinders. Be sure to mass each cheese cloth and record their mass in
an appropriate lab journal. Because each cloth will be used for a different treatment, be
sure to label which cloth will be used for which treatment. After draining for a few
minutes, each cheese cloth was slowly lifted from the funnel and gently squeezed to
remove any excess whey from the curds now present in the cheesecloth. The cheesecloths
containing the curds were then weighted and the mass of each empty cheesecloth was
subtracted from the total mass to give the mass of curds produced from each treatment.
The volume of whey produced from each condition was measured using the graduated
cylinders in which it was drained into.
After obtaining the appropriate measurements, the curds were then cut into
smaller pieces and recombined with the whey into the original containers. The curds were
then cooked at 37 38
o
C in a water bath for fifteen minutes. Gentle stirring of the
mixture was applied so that the curds did not mat. The curds were then transferred to a
cheese mold lined with cheesecloth to create an optimal cheese shape. Next, the cheese
was drained, salted, and pressed by the instructor. Finally, the cheese was allowed to age
for one week at 16
o
C in an appropriate cheese cave.

Results
After performing the procedure illustrated in the previous section and weighing
the curds, it was observed that the artificial chymosin (CHY-MAX) produced curds faster
and at a higher yield than vegetable rennet. The yield of curds produced by CHY-MAX
was 3.19 times that of the vegetable rennet; 60.7 grams compared to 19.0 grams,
respectively. CHY-MAX started to develop curds at the twenty-third minute after
placement while vegetable rennet began at the eighteenth minute. Despite this fact, CHY-
MAX completed this coagulating process sooner than vegetable rennet; thirty-five
minutes compared to forty minutes. Therefore besides producing a higher yield, CHY-
MAX also generated the curds at a slightly faster rate.

Discussion
After evaluating the results of this study, it can be stated that the initial hypothesis
that CHY-MAX will produce curds faster and at a higher rate than vegetable rennet is
supported by the findings. However, due to a lack of successive trials, it is difficult to
statistically analyze the results from the study. Therefore, it is difficult to draw any
definitive conclusions from this study. In future studies, it will be wise to perform a
minimum of ten trials for each of the two conditions. By increasing the sample size, it
will be possible to use a two-population t-test comparing the mean curd weight of each
condition. The rate of curd production can be statistically analyzed via the same t-test. A
separate p-value can then be found using each t-score, which can then be compared to a
critical p-value of 0.05 to test for significance.
Another issue that arose in the study was a failure for CHY-MAX to produce
curds in the first trial. The whole milk containing the CHY-MAX was allowed to
incubate at 35
o
C for over seventy minutes with no sign of curd production. This can be
due to any of several factors. The most probable cause may be that the vial of CHY-
MAX was not sufficiently mixed prior to use, resulting in its concentration being too low
to effectively curdle the milk in an observable length of time. Another concern was the
difference between the two milk containers. The mug used for CHY-MAX floated during
the incubating process while the mug containing vegetable rennet did not, which
indicates the difference in their materials. This can be one of possible issues causing
CHY-MAX failed to form curds. Despite an inability of the trial to produce curds, it was
possible to use the data of other researchers in lieu of the failed trial, which was omitted
from the results.
Though the results support the hypothesis, they do not provide evidence regarding
the reasoning behind it. In order to test this, future experiments should be performed
comparing the curd formation and rate from milk coagulated with varying concentrations
of CHY-MAX. Three conditions can be established, one with no CHY-MAX, one with a
concentration of CHY-MAX equivalent to the concentration of the related enzyme in
vegetable rennet, and the final using a pure, undiluted CHY-MAX. If the concentration of
chymosin plays a role in curd development, curd production and rate should be similar
for milk treated with vegetable rennet and milk treated with the solution of CHY-MAX
with a (concentration equivalent to the related enzyme in vegetable rennet).
In conclusion, though the results of this study support the initial hypothesis, in
order to draw further support, it is essential that future studies be conducted using a
sufficient enough sample size to allow for accurate statistical analysis of the data.
Furthermore, care must be taken in ensuring proper mixing and preparation of the CHY-
MAX solution to ensure a high enough concentration of CHY-MAX to facilitate milk
curdling.



Bibliography
Barbano, D.M., Rasmussen, R.R. (1992). Cheese yield performance of fermentation-
produced chymosin and other milk coagulants. Journal of Dairy Science. 75(1), 1
12. doi: 10.3168/jds.S0022-0302(92)77731-5
Esteves, C.L.C., Lucey, J.A., Hyslop, D.B., Pires, E.M.V. (2003). Effect of gelation
temperature on the properties of skim milk gels made from plant coagulants and
chymosin. International Dairy Journal, 13(11), 877 885. doi: 10.1016/S0958-
6946(03)00114-6
Roeder, Jeannette. (2013). History of Cheese. International Dairy Foods Association.
Retrieved November 13, 2013, from http:// http://www.idfa.org/news--
views/media-kits/cheese/history-of-cheese/