Dairy Sci. & Technol.

(2015) 95:665–686
DOI 10.1007/s13594-015-0250-5
O R I G I N A L PA P E R

Effect of rennet on the composition, proteolysis

and microstructure of reduced-fat Cheddar
cheese during ripening

Kevany Soodam 1,2 & Lydia Ong 1,2,4 & Ian B. Powell 3 &
Sandra E. Kentish 1,4 & Sally L. Gras 1,2,4

Received: 30 January 2015 / Revised: 21 July 2015 / Accepted: 23 July 2015 /

Published online: 18 August 2015
# INRA and Springer-Verlag France 2015

Abstract Rennet is an important ingredient in Cheddar cheese manufacturing and both the
type used and the concentration applied can affect the ripening process. In this work,
reduced-fat Cheddar cheese was produced using different concentrations of microbial rennet
(Hannilase; 0.026, 0.052 and 0.150 international milk clotting units (IMCU).g−1 of milk) as
well as recombinant camel chymosin (0.052 IMCU.g−1 of milk). The composition of the
resulting cheeses was not statistically different, but the ratio of pH 4.6 soluble nitrogen/total
nitrogen (pH 4.6-SN/TN) increased significantly with increasing Hannilase rennet concen-
tration. This ratio was also significantly lower in the cheese made with recombinant camel
chymosin at a similar rennet concentration. Microstructural, biochemical and textural
changes in the cheese were also monitored during ripening. The gel made with a high
rennet concentration was qualitatively more porous but these changes in porosity were not
reflected in the freshly pressed cheese. After 31 weeks of ripening, the cheese made with
recombinant camel chymosin had a thicker protein network compared to the cheese made
with microbial rennet (Hannilase), possibly due to lower proteolysis. Most of the Hannilase
rennet was lost to the whey when the rennet was increased above the concentration of
0.052 IMCU.g−1 of milk. A lower concentration of Hannilase rennet may prove beneficial,
This paper is part of the Special issue dedicated to the 9th International Cheese Symposium held in Cork,
Ireland and organized by Teagasc in collaboration with University College Cork and INRA, 12th & 13th
November 2014.

* Sally L. Gras
[email protected]

1
Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville,
VIC 3010, Australia
2
Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville,
VIC 3010, Australia
3
Dairy Innovation Australia Limited, 180 Princes Highway, Werribee, VIC 3030, Australia
4
ARC Dairy Innovation Hub, Department of Chemical and Biomolecular Engineering, The
University of Melbourne, Parkville, VIC 3010, Australia
666 K. Soodam et al.

as the texture was not significantly affected at the end of the observed ripening period.
Recombinant camel chymosin may potentially be used as a substitute for products requiring
lower proteolysis during ripening. However, the texture of this cheese was harder than the
cheese made with Hannilase rennet at the end of the ripening period.

Keywords Cheddar cheese . Ripening . Cheese microstructure . Microbial rennet . Camel

chymosin . Proteolysis

1 Introduction

The term rennet is used to describe the wide range of coagulants that can be used for the
coagulation of milk into a gel structure. Bovine calf rennet, which consists primarily of
chymosin, has traditionally been used in cheese making, but a growing cheese industry,
combined with a limited supply of calf rennet, has led to the search for calf rennet
substitutes (Sousa et al. 2001). Rennet can originate from other animal sources (e.g.
porcine pepsin, bovine pepsin and chicken pepsin), microbial sources (e.g. Rhizomucor
(Mucor) miehei, Rhizomucor pusillus and Cryphonectria (Endothia) parasitica) or
plant sources (e.g. Cynara cardunculus) (Sousa et al. 2001). Inserting the coding
sequence for bovine chymosin into microorganisms has made fermentation-produced
chymosin (FPC) widely available (Kumar et al. 2010). More recently, recombinant
camel chymosin, also known as fermentation-produced camel chymosin (FCC)
(Kappeler et al. 2006), has also become commercially available.
The type of rennet can influence the primary proteolysis. For example, Sheehan et al.
(2004) showed that reduced-fat Mozzarella cheese made with R. pusillus proteinase
resulted in higher levels of pH 4.6 soluble nitrogen during ripening, when compared to
cheeses made with either FPC or R. miehei proteinase. The impact of FCC on the properties
of Cheddar and Mozzarella has been reported previously (Govindasamy-Lucey et al. 2010;
Bansal et al. 2009; Moynihan et al. 2014; Børsting et al. 2012). FCC has a much higher
ratio of milk clotting activity/general proteolytic activity, known as a C/P ratio, compared to
bovine chymosin. FCC produces harder and chewier full-fat Cheddar cheese after 150 days
of ripening, with a lower degree of primary proteolysis and less bitter and brothy flavours
(Bansal et al. 2009). Low-fat Cheddar cheeses made with FCC are similarly harder,
chewier and less bitter than cheeses made with FPC (Govindasamy-Lucey et al. 2010).
A previous study compared the effects of calf rennet with bovine pepsin and
reported that a more compact structure was usually observed in Cheddar cheese made
with the calf rennet (Stanley and Emmons 1977). No study appears, however, to have
compared the microstructural changes that may occur during Cheddar cheese making
when FCC or microbial rennet is used.
The rennet concentration applied may also affect the coagulation process and the
quality of the final cheese. Increasing the rennet concentration increases the rate of
enzymatic hydrolysis, with a resulting decrease in the time required for rennet-induced
coagulation (Spangler et al. 1991). The effect of rennet concentration on the ripening of
Cheddar cheese, however, has not been widely studied. Creamer and colleagues (1987)
found that rennet concentration did not majorly affect Cheddar cheese making, besides
producing a slightly soft gel when very low concentrations of rennet were added. There
was no difference in fat losses to the whey, resulting in a similar fat in dry matter
Effect of rennet during ripening 667

(FDM). It was also observed that as the ratio of rennet to casein increased, the amount
of residual rennet also increased, with a resultant increase in the breakdown of αs1-
casein (CN) during ripening.
A reduction of up to 40% of rennet concentration was found to only minimally affect
the composition of Mozzarella cheese, resulting in cheeses with similar protein, mois-
ture, salt and calcium concentration and pH, although the fat content did differ
(Kindstedt et al. 1995). During ripening, the rate of increase in the concentration of
12% trichloroacetic acid soluble nitrogen was slower for the cheeses made with reduced
rennet concentration. The concentration of αs-CN and the hardness or springiness of the
cheese, however, were not significantly affected. A study by Moynihan et al. (2014) also
found that low-moisture part-skim Mozzarella cheese had similar hardness when made
with different rennet concentrations. These studies are in contrast with a study on Gouda
cheese made with ultrafiltered milk that observed a significantly harder and less bitter
cheese with a twofold reduction in rennet concentration (Spangler et al. 1991). The
results also differ from a previous study of Meshanger cheese that showed a direct
relationship between the rennet concentration and the breakdown rate of αs1-CN in the
cheese, and a related decrease in cheese firmness during ripening (de Jong 1977).
A study of the microstructure of the gel or cheese can provide further insight into the
effect of rennet concentration or type during the processes of cheese manufacturing and
cheese ripening. These effects might include changes in fat loss during cheese making
or texture development during ripening. The effect of rennet concentration on the
microstructure of fat-free Feta cheese has been investigated in this manner using
TEM, where cheeses with a coarser protein network were observed with higher rennet
concentrations (Wium et al. 2003). An earlier study also investigated the microstructure
of Cheddar cheese with a reduced rennet-to-casein ratio; this was done, however, by
lowering the amount of rennet whilst increasing the concentration of the milk using
ultrafiltration (Green et al. 1981). There do not appear to be any studies that have
observed the impact of different concentrations of rennet on the microstructure of
Cheddar made with a standard concentration of milk protein, nor microstructure studies
that follow such cheeses during ripening.
In this study, we investigated the impact of the concentration of the microbial rennet,
Hannilase, on the composition, proteolysis and microstructure of reduced fat Cheddar
cheese during production and ripening. This microbial rennet from R. miehei is widely
used within the Australian dairy industry. We also prepared some cheeses with FCC
rennet (rarely used in Australia but yet of industrial interest) as a comparison. We chose
reduced-fat cheese to confirm whether microstructural changes during ripening would
be different to changes previously observed with Hannilase for full-fat Cheddar
(Soodam et al. 2014, 2015).

2 Materials and methods

2.1 Determination of cutting time of gel during cheese making

The storage modulus (G') of the milk was determined prior to cheese making, in order
to determine the cutting time of the gel, using an Advanced Rheometric Expansion
System rheometer (TA Instruments, New Castle, DE, USA), equipped with a 34-mm-
668 K. Soodam et al.

diameter cup and a bob 33 mm long and 32 mm wide. For this experiment,
milk was obtained from local suppliers, with a protein-to-fat ratio of 1.54±0.02,
a similar composition to the cheese milk used in later experiments. Starter
bacteria (0.05 g.L−1) were added to the warm (33 °C) cheese milk. Rennet
(Hannilase or Chy-Max M, Chr. Hansen) was added once the pH was ∼6.5, and
the sample (15 mL) was then immediately added to the cup. Hannilase rennet
concentrations of 0.026 international milk clotting units (IMCU).g−1 of milk
(low), 0.052 IMCU.g−1 of milk (control) and 0.150 IMCU.g−1 of milk (high) as
well as FCC rennet concentration of 0.052 IMCU.g−1 of milk were used. The
changes in the rheology of the milk during the coagulation process were then
monitored using a dynamic time sweep analysis (7200 s) with an angular
frequency of 0.8 Hz and a strain of 1%. The cutting time of the gel was taken
as the time taken to reach a G' of 50 Pa, based on the cutting time for the
control sample.

2.2 Cheddar cheese production

Eight batches of cheese were made in total over the course of three trials carried
out at laboratory scale using a method for manufacture described in a previous
study (Ong et al. 2013). Three batches of cheese were made in duplicate with
Hannilase, a microbial rennet (750 IMCU.mL −1 , Chr. Hansen, Bayswater,
Australia) from R. miehei, using concentrations of 0.026 IMCU.g−1 of milk
(low), 0.052 IMCU.g−1 of milk (control) and 0.150 IMCU.g−1 of milk (high).
Another batch of cheese was also made in duplicate with Chy-Max M (FCC,
1000 IMCU.mL−1, Chr. Hansen) using the same rennet strength (IMCU.g−1 of
milk) as the control treatment with Hannilase, i.e. 0.052 IMCU.g−1 of milk.
Rennet concentrations were supplied by the manufacturer.
The cheese was made with 20 kg of pasteurised (at 73 °C for 15 s) and
standardised cheese milk, obtained from a local dairy manufacturer, with a
protein-to-fat ratio of 1.54±0.02. The cheese milk was standardised by total
skimming and blending the milk with cream and ultrafiltration retentate obtain-
ed from whole milk. The cheeses were made in a randomised order. The cheese
milk was warmed to 33 °C before inoculation with freeze-dried direct vat set
(DVS) mesophilic/thermophilic blend lactic starter culture (0.05 g.kg−1 of milk,
Chr. Hansen), which contained strains of Streptococcus thermophilus,
Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis. Rennet
(Hannilase or Chy-Max M) was added once the pH was ∼6.5. The setting time
was varied (15–50 min) to ensure similar gel strength at cutting. Once the gel
coagulated, the curd was cut using a curd cutter with 1-cm-spaced wire blades
and cooked. Cooking was done by gradually increasing the temperature from
33 to 38 °C in a total of ∼40 min, while the stirring rate was gradually
increased from 12 to 40 rpm. The curd was then cooked at 38 °C with this
maximum stirring speed of 40 rpm, until the pH was ∼6.2. The sweet whey
was drained and the cheddaring step started. When the pH reached approxi-
mately 5.4, the curd was milled and salted with 2.5% (w/w) of salt (based on
the weight of the curd) before being pressed at ∼440 kPa overnight at room
temperature. The cheese was then stored at 8 °C for ripening and analysed at
Effect of rennet during ripening 669

weeks 1, 5, 18 and 31. The yield of cheese (Ya) was expressed as the mass of
the cheese per gram of cheese milk, measured using a weighing balance. The
dry matter cheese yield was calculated using the following equation:
 .  mass of cheese
Dry matter cheese yield %w w ¼
mass of cheese milk
100−moisture content
  100
100
On a separate day, gel freshly prepared at 33 °C using the same coagulation time as
during the cheese making was analysed within 20 min after coagulation, using confocal
laser scanning microscopy.

2.3 Residual rennet in the fresh cheese

An aqueous phase extract of each cheese sample was prepared (Hurley et al.
1999). The heptapeptide (30 μL, 1 mg.mL−1) Pro–Thr–Glu–Phe–[NO2–Phe]–
Arg–Leu (Bachem, Bubendorf, Switzerland) was mixed with sodium formate
buffer (200 μL, 0.1 mol.L−1, pH 3.2, 0.05% sodium azide (Chem-Supply,
Gillman, Australia)) and the aqueous cheese extract (70 μL). The sodium
formate buffer was prepared from 0.1 M formic acid (Ajax FineChem, Seven
Hills, Australia) and 0.1 M sodium formate (Chem-Supply). The sample was
then incubated at 37 °C for 5 h, before heating for 13 min at 70 °C and
freezing for future analysis.
The 300-μL sample described above was mixed with 300 μL of trifluoroacetic
acid (0.2%, Chem-Supply) and again incubated overnight at 4 °C, before centri-
fugation for 10 min at 16,000g using a bench top centrifuge (Eppendorf, Mac-
quarie Park, Australia). A sample of this solution was analysed using an Agilent
1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA), equipped with
an autosampler, UV detector and column oven. The column used was a 5 μm
Phenomenex column Jupiter 5U C18, with dimensions of 250 × 4.6 mm
(Phenomenex, Lane Cove West, Australia). The two solvents for the mobile phase
were (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile (Merck, Kilsyth,
Australia). The flow rate was set at 1 mL.min−1, the wavelength was 290 nm, and
the gradient applied was as follows: 0–25% B, 25 min; 25–50% B, 5 min; 50% B,
15 min; 50–0% B, 5 min; and 0% B, 10 min.
Control samples prepared with standard rennet concentrations (0–0.004 and
0–0.01 IMCU.mL−1 for Hannilase and FCC respectively) were used to develop
a calibration curve. The FCC rennet was observed to result in 3% of the
amount of peptide obtained from Hannilase, under the same conditions. The
results are expressed as the concentration of residual rennet in the cheese
(IMCU.g−1 cheese), or as the percentage of rennet retained in the cheese,
calculated as a percentage of the initial concentration of rennet added to the
milk. The concentration of residual rennet in the cheese can be used to indicate
the potential proteolytic activity during ripening while the percentage of rennet
retained is typically used as a comparative measure for different rennet types.
Analysis of residual rennet was conducted two times for each cheese.
670 K. Soodam et al.

2.4 Microscopy techniques—confocal laser scanning microscopy

and cryo-scanning electron microscopy of gel, curd and cheese

The gel, curd and cheese samples were analysed by confocal laser scanning microscopy
(CLSM; Leica TCS SP2 or Leica SP5; Leica Microsystems, Heidelberg, Germany) and
cryo-scanning electron microscopy of gel (cryo-SEM; Quanta; Fei Company, Hillsboro,
OR, USA), using methods described in a previous study (Ong et al. 2011). Stock solutions
of Nile Red and Fast Green FCF were prepared at a concentration of 1 mg.mL-1 in
dimethyl sulfoxide and water respectively and both stains were diluted tenfold in water
prior to staining. The emission wavelengths were set at 550–600 and 668–708 nm for Nile
Red and Fast Green FCF respectively. Analysis of the CLSM images was then carried out
using Imaris image processing software (Bitplane, South Windsor, CT, USA), as de-
scribed previously for the three-dimensional quantitative analysis of Cheddar cheese
microstructure (Ong et al. 2012), using a sample volume of ∼55,000 μm3 to normalise
parameters such as number of vertices and number of fat globules. Parameters obtained
using this method are described in a previous study (Soodam et al. 2014) and at least two
replicate analyses were performed for each parameter for each cheese treatment.

2.5 Analytical techniques

Hannilase was characterised by reverse phase high-performance liquid chromatography

and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry (MS) using an
Agilent 6200 Series ESI-TOF spectrometer to determine the molecular weight, as this
might be a factor in rennet retention. The charge series was deconvoluted to give a
molecular weight of 41.24 kg.mol−1. The apparent molecular weight of FCC is
available in the literature at ∼35.6–40 kg.mol−1 (Kappeler et al. 2006).
The fat and protein content of the milk used for cheese making was determined using a
Milko Scan FT120 (Foss, NSW, Australia). The fat and protein content of the cheese was
determined using the gravimetric method AS2300.1.3 (Australian Standard 2008) and the
Kjeldahl method AS2300.1.2.1 (Australian Standard 1991) respectively. The salt content
of the cheeses was determined using the Volhard method AS2300.6.5 (Australian Stan-
dard 1990). The moisture content of the cheeses was obtained by placing samples of
known mass (m1) in pre-weighed crucibles (m2) and then drying the samples at 100±3 °C
in an oven (S.E.M. Pty. Ltd., Magill, Australia) for at least 18 h. The weight of the samples
was recorded once stable (m3). The moisture content was then determined from the
weight loss from the original sample of cheese, as described in the following equation:
 .  m1−ðm3−m2Þ
Moisture content % w w ¼  100:
m1
The fat content of the sweet whey, collected during draining and cheddaring, and the
salty whey, collected during pressing, was determined using a modified version of the
method described by Atwood and Hartmann (1992). A ferric chloride–trichloroacetic
acid solution was obtained by mixing 10 mL of a solution containing 0.37 M FeCl3
(Ajax Finechem) and 0.1 M hydrochloric acid (HCl, Univar, Ajax Finechem, Austra-
lia), with 7.5 g trichloroacetic acid (TCA, Chem-Supply).
The whey samples were warmed to 37 °C and diluted twofold. The diluted sample
(50 μL) was then mixed with ethanol (600 μL, Merck), hydroxylamine hydrochloride
Effect of rennet during ripening 671

(100 μL of 2 M, Ajax Finechem) and sodium hydroxide (NaOH, 100 μL of 3.5 M,

Chem-Supply). The resultant mixture was incubated for 30 min at room temperature,
after which HCl (120 μL of 3.4 M HCl) was added to the mixture. Ferric chloride–
trichloroacetic acid solution (100 μL), prepared as described previously, was then
mixed into the acidified solution. The mixture was then centrifuged at 1500g for
5 min, and the supernatant was dispensed into a 96-well microplate (BD Falcon, North
Ryde, Australia). The absorbance of the solution was measured at 540 nm using a
microplate reader (Fluostar Optima, BMG Labtech, Ortenberg, Germany). The fat
concentration of the samples was determined by reference to standard triolein solutions
(Sigma-Aldrich, Castle Hill, Australia). Two replicate analyses were performed for
each cheese treatment.
A modified version of a method previously described (Pesce and Strande 1973) was
used to determine the protein content of the whey. TCA–Ponceau S working reagent
was prepared from TCA (Chem-Supply) and Ponceau S dye (Merck) as described by
Pesce and Strande (1973). The whey sample was diluted 20-fold and 100 μL of the
diluted sample was then mixed with 1 mL of the TCA–Ponceau S normal working
reagent. The resulting mixture was then centrifuged at 15,000g for 5 min. The resulting
precipitate was then dissolved in sodium hydroxide (NaOH, 0.2 mol.L−1, 700 μL). The
solution was dispensed into a 96-well microplate (BD Falcon), and the absorbance was
measured at 560 nm using a microplate reader (Fluostar Optima, BMG Labtech). The
protein concentration of the samples was determined by reference to standard bovine
serum albumin solutions (Thermo Scientific, Rockford, IL, USA). Two replicate
analyses were performed for each cheese treatment.
The pH of the cheese was obtained by homogenising 10 g of grated cheese with
10 mL of water. The pH of the slurry was then determined using an electrode pH meter
(Orion 720A, Orion Pacific, Frankston, Australia).
The pH 4.6 soluble extract was prepared based on the method of Sousa and
McSweeney (2001). Grated cheese (10 g) was homogenised with water (20 mL) and
the slurry adjusted to pH 4.6 using hydrochloric acid (HCl, 1.0 M). The sample was
then incubated at room temperature for half an hour, and the pH was readjusted to a
value of 4.6. The mixture was then incubated in a water bath at 40 °C for 60 min before
centrifugation at 3000g (4 °C, 30 min; Eppendorf). The soluble fraction found in
between the upper fat layer and lower casein precipitate was filtered through Whatman
No. 54 filter paper (GE Healthcare Life Sciences, Parramatta, Australia) to yield the pH
4.6 soluble extract. The nitrogen content of this extract was then determined using the
Kjeldahl method AS 2300.1.2.1 (Australian Standard 1991). The samples were
analysed once per cheese treatment.
The proteolytic pattern of the cheese during ripening was determined using sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cheese stock solu-
tion was prepared using the method of Ong et al. (2006), except that 25 mg cheese samples
were used. The stock solutions produced were then diluted approximately fourfold with
Tris (10 mmol.L−1)–EDTA (1 mmol.L−1) pH 8.0 buffer. Standards of α-CN, β-CN and κ-
CN (Sigma-Aldrich) were also prepared at a concentration of 0.25 mg.mL−1. An aliquot of
6.5 μL was mixed with 2.5 μL of Bolt LDS Sample Buffer (4×) and 1 μL Bolt Reducing
Agent (10×; Invitrogen, Mt Waverley, Australia). The solution was then heated at 70 °C
for 10 min and the resulting solution loaded into a 12% acrylamide Bis-Tris Plus gel
(Invitrogen). A molecular standard, SeeBlue Plus2 pre-stained (Invitrogen), was also
672 K. Soodam et al.

loaded in each gel. Bolt 2-(N-morpholino)ethanesulphonic acid SDS Running Buffer

(1×; Invitrogen) was used as buffer, and the system was run for 1 h and 15 min at 125 V.
The protein bands were then stained and then destained as described by Ong et al. (2006)
except that 7.5% v/v acetic acid was used and the de-staining solution did not contain
methanol. The gel was then scanned using a Fujifilm Intelligent Dark Box II with LAS-
3000 Lite V2.2 software and LAS-3000 Image Multi Gauge (Fujifilm, Brookvale,
Australia) to quantify the intensity of α- and β-CN bands during ripening. The data
presented are representative of two gels.
The hardness of cheese samples was determined using a two-bite compression test on
a TA-XT Plus texture analyser (Stable Micro Systems, Surrey, UK) as the force required
to compress the cheese to 50% original height at 2 mm.s−1. A modification of the method
of Ong et al. (2012) was used with cylindrical samples of 2.0 cm in diameter and 2.0 cm
in height. Texture analysis was performed at least three times for each cheese treatment.

2.6 Statistical analysis

All cheese treatments were performed in duplicate, and the results presented are the
mean of the two data points (n=2), where all replicate analyses were averaged for each
cheese. The cheese composition and processing time, the whey composition, the yield,
the residual rennet analysis, the pH 4.6 soluble nitrogen and the image analysis of the
gel and curd were analysed using one-way analysis of variance and Tukey’s paired
comparison, with a significance level of α=0.05. For all other analyses, a parameter ID
(ID=1, 2, 3, 4 5, 6, 7, 8) was assigned to each of the eight batches of cheese. This
parameter helped to link the original cheeses to the cheeses during ripening. The
experimental results were then analysed using “General Analysis of Variance” in
GenStat (VSN International, Hemel Hempstead, UK). If changes due to time or rennet
were significant, the data were further analysed using Tukey’s paired comparison, with
a significance level of α=0.05. A Tukey’s paired comparison, with a significance level
of α=0.05, was used to analyse the residual rennet treatment (low, high Hannilase
rennet concentration, FCC rennet) with respect to the control. Multiple linear regression
(Microsoft Excel) was also used to analyse the texture during ripening.

3 Results and discussion

3.1 The effect of rennet on the storage modulus (G'), cutting time of the gel
and processing time of cheese making

A rheological experiment was carried out prior to the cheese making to determine the
required cutting time for the gel. The effect of rennet concentration and type on the gel
strength is shown in Fig. 1. As the Hannilase rennet concentration increased from
control to high (from 70 to 200 μL.kg−1 of milk), the gel strength and rate of gel
strength development increased due to increased enzymatic hydrolysis. FCC also
induced a faster change in gel strength when compared to Hannilase (control),
despite the similar rennet strength used, resulting in a decrease in cutting time with
this enzyme. This may be due to the higher milk clotting activity of FCC with respect to
the proteinase from R. miehei (Kappeler et al. 2006).
Effect of rennet during ripening 673

250

High
FCC
200
Control

150
G' (Pa)

100
15 19 30
50

0
0 10 20 30 40 50 60 70 80
Time (min)
Fig. 1 The storage modulus (G') measured from the time of rennet addition for milk with control (black solid
line) or high (light grey solid line) Hannilase rennet concentration or fermentation-produced camel chymosin
(dark grey solid line). Arrows indicate the time when the G' measured reached 50 Pa (defined as the cutting
time) and the shaded region indicates the error bars. The results are expressed as the mean±the standard
deviation of the mean (n=2)

The cutting time was defined here as the time required to reach G' of 50 Pa (Fig. 1).
The control, high and FCC samples were cut at 30, 15 and 19 min during the cheese
making. The gel made from low rennet treatment was too soft to be measured using the
rheological test and was instead determined during the actual cheese making. The time
required for the gel with low rennet concentration to reach a medium firmness, as
determined by an experienced cheese maker, was found to be 50 min.
Increasing the rennet concentration or using FCC resulted in a decrease in coagu-
lation time, as discussed above. All four treatments, however, had a statistically similar
(P>0.05) total processing time (from starter addition to milling; Table 1), showing that
a change in rennet concentration or rennet type will not affect plant productivity.

3.2 Fat and protein loss, yield and composition of the cheeses

The protein and fat lost to the sweet and salty whey were not significantly different
between treatments, (Table 2). It should be noted that while the fat and protein loss to
the sweet whey of the low rennet treatment appears lower than the other three
treatments, the results are not significant due to large standard deviations. The compo-
sition of the cheese (fat, protein, moisture, salt, FDM, salt in moisture (S/M), moisture
in fat-free substance (MFFS)), shown in Table 1, were all statistically similar (P>0.05)
between treatments. Similar observations were made in previous studies; S/M, MFFS
(Creamer et al. 1987), protein, moisture and salt (Kindstedt et al. 1995) were similar for
cheeses made with different rennet concentrations. The results are also consistent with
previous studies where a similar composition was reported for full-fat or low-fat
Cheddar cheese made with FCC and FPC (Bansal et al. 2009; Govindasamy-Lucey
et al. 2010). The fat content was observed, however, to be significantly affected by
coagulant concentration in Mozzarella cheese (Kindstedt et al. 1995), in contrast to this
study, although the cause of this difference is not known.
 674

Table 1 The composition of Cheddar cheese made with different rennet concentrations (low, control or high Hannilase rennet concentrations) or different types of rennet (Hannilase
and FCC)

Rennet Cheddaring time (min) Processing time (min)* Protein (% w/w) Fat (% w/w) Salt (% w/w) Moisture (% w/w) FDM (% w/w) S/M (% w/w) MFFS (% w/w) YDM (% w/w) Ya (% w/w)

Low 94±2a 309±4a 30.1±0.6a 23.7±0.7a 1.5±0.0a 39.1±2.0a 38.9±0.1a 3.8±0.2a 51.3±2.1a 5.7±0.1a 9.4±0.1a
b a a a a a a a a a
Control 129±1 325±22 29.3±1.5 23.5±0.4 1.7±0.1 40.2±1.1 39.2±1.3 4.2±0.2 52.5±1.7 5.5±0.0 9.2±0.2a
b a a a a a a a a a
High 128±3 329±6 30.6±1.3 22.4±1.4 1.7±0.0 40.5±1.5 37.6±1.5 4.2±0.2 52.1±0.9 5.3±0.0 8.9±0.3a
b a a a a a a a a a
FCC 140±14 316±27 30.1±0.1 24.5±0.2 1.5±0.2 38.1±0.5 39.5±0.0 3.8±0.6 50.5±0.5 5.6±0.3 9.1±0.4a

The results are expressed as the mean±the standard deviation of the mean (n=2). Superscript letters a and b: means in a single column with the same superscripts are not significantly
different (P>0.05)
FDM fat in dry matter, S/M, salt in moisture, YDM yield in dry matter per gram of cheese milk, Ya total yield of cheese per gram of cheese milk, MFFS moisture in fat-free substance
*Processing time=total time from starter addition to milling
K. Soodam et al.
Effect of rennet during ripening 675

Table 2 The fat and protein composition of whey made with different rennet concentrations (low, control or
high Hannilase rennet concentrations) or different types of rennet (Hannilase and fermentation-produced
camel chymosin, FCC)

Rennet Sweet whey Salty whey

Weight of Fat Protein FL PL Weight of Fat Protein FL PL

whey (kg) (% w/w) (% w/w) (% w/w) (% w/w) whey (kg) (% w/w) (% w/w) (% w/w) (% w/w)

Low 17.33± 0.16± 0.86± 5.13± 17.67± 0.19± 0.16± 0.86± 0.06± 0.20±
0.05a 0.07a 0.25a 2.17a 4.97a 0.02a 0.05a 0.00a 0.02a 0.02a
Control 17.28± 0.23± 1.23± 7.40± 25.25± 0.15± 0.19± 0.69± 0.05± 0.13±
0.10a 0.07a 0.23a 2.23a 4.69a 0.03a 0.01a 0.15a 0.01a 0.05a
High 17.37± 0.22± 1.19± 6.86± 24.51± 0.16± 0.12± 0.64± 0.03± 0.12±
0.00a 0.01a 0.16a 0.51a 3.40a 0.03a 0.04a 0.08a 0.02a 0.01a
FCC 17.36± 0.25± 1.21± 8.06± 25.11± 0.14± 0.22± 0.70± 0.06± 0.12±
0.00a 0.00a 0.02a 0.16a 0.84a 0.03a 0.06a 0.05a 0.03a 0.04a

Results are expressed as mean±standard deviation of mean (n=2). Superscript letter a: means in a single
column with the same superscripts are not significantly different (P>0.05)
FL fat lost per kilogram of fat in the cheese milk, PL protein lost per kilogram of protein in the cheese milk

The yield (Ya) and yield in dry matter (YDM) were also not significantly different
between cheese treatments here (P>0.05) when all four treatments were analysed
together. Statistical analysis of only the cheeses made with Hannilase, however, showed
a decreasing trend in the YDM as rennet concentration increased (P<0.05). It is not
known why increasing Hannilase concentration should result in a decrease in the YDM,
and further work is needed to elucidate this mechanism. The yield value of 8.9–9.4% w/
w was slightly higher than that observed in a previous study investigating reduced-fat
Cheddar cheese (8.09% w/w) (Fenelon and Guinee 1999). However, the cheese milk
used in our study was standardised using ultrafiltration and also adjusted to a lower
protein-to-fat ratio, meaning that simple yields (calculated as mass yield per volume of
milk) are not directly comparable.
Generally, cheese made with rennet with a more specific enzyme activity (high C/P
ratio) should result in higher cheese yields due to less loss of protein to the whey, an
observation made in studies of R. miehei coagulants and bovine chymosin (Ustunol and
Hicks 1990). It is unknown why similar yields were obtained in this study. It is possible
that the different variants of the R. miehei microbial rennet used in the different studies
might account for the results obtained. Alternatively, differences in yields are usually
less than 1%, and it is possible that the scale of the experiment is too small for these
differences to be observed. Furthermore, statistically similar cheese yields were also
obtained when FCC and FPC were compared (Bansal et al. 2009), even though FCC
has higher selectivity (C/P) than FPC. The current study provides further evidence that
Hannilase and FCC can be used interchangeably without large changes in cheese
composition.

3.3 Residual rennet in cheese

The concentration of residual rennet activity in the cheeses made with a low Hannilase
rennet concentration was significantly lower than in the control cheese (Table 3), as
676 K. Soodam et al.

Table 3 Residual rennet and rennet retention in Cheddar cheese made with different rennet concentrations
(low, control or high Hannilase rennet concentrations) or different types of rennet (Hannilase and
fermentation-produced camel chymosin, FCC)

Rennet Rennet added to milk (IMCU.g−1 of milk) Residual rennet (IMCU.g−1 of cheese) Rennet retention (%)a

Low 0.026 0.0044±0.0005 * 1.59±0.16

Control 0.052 0.0066±0.0001 1.16±0.04
High 0.150 0.0063±0.0001 0.38±0.02 *
FCC 0.052 0.1891±0.0053 * 32.60±0.66 *

The results are expressed as the mean±the standard deviation of the mean (n=2)
*Results are statistically different based on pairwise comparison with respect to the control cheese (P<0.05)
a
Results for rennet retention are expressed as the percentage of rennet present in the total amount of cheese
produced over the total amount of enzyme added to the milk

might be expected. The percentage of the rennet activity retained, however, was similar.
Past studies have all shown that increasing the calf rennet concentration will increase
the residual rennet concentration in the cheese (Dulley 1974; Creamer et al. 1987), in
agreement with these results. When the Hannilase concentration was increased beyond
the control value (>0.052 IMCU.g−1 milk), however, the residual rennet concentration
did not increase further (Table 3). As a result, the percentage retention of rennet was
lower (P<0.05). These results suggest that there is a critical Hannilase concentration
above which further retention of rennet activity in the curd does not occur, with a
subsequent decrease in the percentage of residual rennet activity in the cheese.
The FCC rennet clotting activity when added to the milk was the same as that used
for the control cheese made with Hannilase rennet (Table 3), yet the residual rennet
activity in the FCC cheese was significantly higher, with a difference of ∼29-fold. It
should be noted, however, that the sensitivity of the two assays differed for the two
enzymes. Previous studies have found the retention of microbial rennet to be typically
low, at ∼2–3% (Holmes et al. 1977), broadly consistent with the fraction of the milk
aqueous phase entrapped in the curd. On the other hand, retention of FCC activity was
found to be ∼20% within the pH range of 6.65–6.00 (Børsting et al. 2014), consistent
with the retentions found in this study.
The residual rennet activity is a function of both the physical retention of rennet in
the curd and the loss of rennet activity due to denaturation, proteolysis or other damage
to the enzyme during cheese manufacture. The apparent molecular weight of FCC is
∼35.6–40 kg.mol−1 (Kappeler et al. 2006), and the molecular weight of the proteinase
from R. miehei was found here to be ∼41.24 kg.mol−1 by ESI-TOF mass spectrometry.
It is therefore unlikely that the retention was influenced by the size of the rennet
enzyme but other possible factors may be responsible, such as electrostatic interactions
with caseins or the enzyme surface hydrophobicity. For the pH of 6.2 for whey draining
used here, the net charge, calculated based on known pKa values and the Henderson–
Hasselbalch equation (Moore 1985; Berg et al. 2007) for the FCC and R. miehei, would
be approximately −8 and −19. This may contribute to the difference in retention
observed due to different interactions with the negatively charged caseins and at higher
concentrations of Hannilase rennet this may also potentially result in more expulsion
into the whey due to these repulsive forces.
Effect of rennet during ripening 677

In the study by Holmes et al. (1977), it was shown that the activity of calf rennet
decreased during cheese making but that the activity of the Mucor miehei rennet was
not affected at the point of whey removal. The strain of enzyme used in this prior study,
however, is different to the enzymes typically used commercially, which are often
chemically modified to reduce their heat stability. We have no data on the relative
stability of the commercial Hannilase enzyme free in solution versus the enzyme when
associated with caseins or other curd components but our results lead us to hypothesise
that there may be a stable enzyme fraction (stabilised by specific binding to caseins)
and that the upper boundary for that fraction is determined by the number of available
binding sites. If so, any free enzyme may be more readily heat-inactivated during
cheese making and this effect will be more pronounced for the samples with higher
concentrations of Hannilase.

3.4 Effect of rennet concentration and type on the microstructure of the gel

The CLSM and cryo-SEM images of the freshly formed gels are shown in Fig. 2. From
the images, the gel structure appears as a porous protein network, with spherical fat
globules dispersed throughout. Qualitatively, increasing the concentration of rennet per
mass of protein from low to high (Fig. 2a–c, e–g), appears to increase the porosity of
the gel. This was not observed in the quantitative data from image analysis due to high
variability in the data (P>0.05, Table 4). A more porous gel made with a higher
concentration of rennet could contribute to a higher loss of rennet from this gel,
resulting in the lower rennet retention for these samples (Table 3). Any difference in
gel structure porosity also has the potential to affect fat loss to the whey (Ong et al.
2015), as well as the texture of the resultant cheese; a denser network may then result in

Low Control High FCC

a b c F d
P
F
P
F
P
P
F

e f g h F
P F
P P
F
F
P

Fig. 2 Three-dimensional (3D) confocal microscopy images (a–d) and cryo-scanning electron microscopy
images (e–h) of the microstructure of gels made using low (a, e), control (b, f) or high (c, g) Hannilase rennet
concentration or fermentation-produced camel chymosin (d, h). Protein is stained green (FCF) and fat is
stained red (Nile Red). The confocal images are 3D reconstructions built from 40 layers, adding up to a total
depth of 10 μm. Cryo-scanning electron microscopy images were captured using a solid state detector at a
magnification of ×4000. The scale bars are 10 μm (a–d) or 20 μm (e–h) in length. Images can be viewed in
colour in the electronic version of this manuscript. F indicates fat globules and P indicates protein
678 K. Soodam et al.

Table 4 Porosity determined from 3D confocal microscopy images of the gel made with low, control or high
concentrations of Hannilase rennet concentration or fermentation-produced camel chymosin, FCC

Rennet Porosity

Low 0.41±0.05a
Control 0.48±0.03a
High 0.52±0.07a
FCC 0.52±0.04a

The results are expressed as the mean±the standard deviation of the mean (n=2). Superscript letter a: means in
a single column with similar superscripts are not significantly different (P>0.05)

a harder cheese (Ong et al. 2013). Similar fat and protein losses in the whey were
observed in this study (P>0.05, Table 2), however, suggesting that the enzymes can be
interchanged without undesirable changes in cheese properties.
The microstructure of the control gel (Fig. 2b, f) and the gel made with FCC treatment
(Fig. 2d, h) did not differ significantly. The porosity obtained was similar (Table 4),
despite the difference in gelation obtained for these two treatments (Fig. 1). The similar
porosity of the control and FCC treatment but higher retention of rennet observed in
FCC cheese suggests, however, that factors other than porosity also influence the
retention of rennet in cheese, as discussed in the previous section (Sect. 3.3).

3.5 The effect of rennet concentration and type on the microstructure of cooked
curd, milled curd, fresh cheese and cheese during ripening

The structural differences visible in the gel due to the different rennet treat-
ments are less obvious in the cooked and milled curd. No significant differ-
ences in porosity were found by qualitative observation (Fig. 3) or quantitative
analysis of images (P>0.05; data not shown). It is possible that the difference

Low Control High FCC

a b c d P
F
F
Cooked
P P
F F curd
P

e F f g h
F
F
P
Milled
F
curd
P
P P

Fig. 3 Cryo-scanning electron microscopy images of cooked (a–d) and milled curd (e–h) made using low (a,
e), control (b, f) or high (c, g) Hannilase rennet concentration or fermentation-produced camel chymosin
rennet (d, h). Cryo-SEM images were captured using a solid state detector at a magnification of ×8000. The
scale bars are 10 μm in length, F indicates fat globules, P indicates protein, and the arrows indicate
micropores
Effect of rennet during ripening 679

in cheddaring time for the different treatments may have played a role in these
results (Table 1). Micropores are visible in the cryo-SEM images of the cooked
and milled curd across the treatments (Fig. 3). Generally, the rennet concentra-
tion used did not significantly affect the number and diameter of fat globules in
the cooked curd or milled curd (P>0.05).
The microstructure of the fresh cheese observed by cryo-SEM is shown in
Fig. 4a–d. The micropores that were previously visible in the cooked and milled
curd (Fig. 3) are now all pressed closed. Qualitatively, the microstructure of the
four different types of fresh cheeses was similar, irrespective of the treatment used.
After ripening for 31 weeks, however, the microstructure of the cheeses as
observed using cryo-SEM appeared visually different (Fig. 4e–h), with a thicker
protein network in the FCC cheese compared to the cheeses made using
Hannilase. This difference may arise as a result of the lower general proteolytic
activity of FCC compared to Hannilase (Kappeler et al. 2006; Martin et al. 1980).
Although determined under different experimental conditions, Kappeler et al.
(2006) indicated that the R. miehei proteinase is approximately six times more
proteolytic than FCC, based on the nonspecific hydrolysis of casein.
The number of fat globules as well as number of vertices, a measure which
may indicate the branching of the rendered protein surface (Soodam et al.
2014), determined by quantitative CLSM image analysis, was not significantly
affected by rennet or ripening time (data not shown). In a previous study, a
significant decrease in the number of vertices was observed with ripening
(Soodam et al. 2014). The trend observed in this study may be due to either
the shorter ripening period, the use of reduced fat cheeses instead of full fat
cheeses, or the different magnifications used in capturing the CLSM images, as
images were captured at ×100 instead of ×63 as was previously used, to allow
a better resolution of the structure.

Low Control High FCC

a b c d
P
P P
F
Week
F
F 1
F P

F F
e f P
g h P

F P Week
P 31
F

Fig. 4 Cryo- scanning electron microscopy images of the microstructure of freshly pressed (a–d) and mature
cheeses (e–h) made using low (a, e), control (b, f) or high (c, g) rennet concentration or fermentation-produced
camel chymosin (d, h). Cryo-scanning electron microscopy images were captured using a solid state detector
at a magnification of ×8000. The scale bars are 10 μm in length. F indicates fat globules, P indicates protein,
and the arrows indicate the protein network
680 K. Soodam et al.

3.6 Proteolysis during ripening

The level of proteolysis in the cheese was assessed by comparing the ratio of pH 4.6
soluble nitrogen/total nitrogen (pH 4.6-SN/TN) at the end of the ripening period
(Fig. 5a). This ratio quantifies primarily the peptides released due to the action of
rennet (O'Keeffe et al. 1978), although whey proteins and proteins from plasmin
activity such as proteose peptones may also contribute to the pH 4.6-SN/TN to a small
extent (O'Keeffe et al. 1978).
This ratio of pH 4.6-SN/TN increased significantly with increasing Hannilase rennet
concentration (P<0.05). Given the estimated amount of residual rennet in the cheeses
(Table 3), it would be expected that the pH 4.6-SN/TN ratio for the control and high
rennet concentration cheeses would be similar. The S/M and MFFS of the cheeses were
similar, suggesting that factors other than composition may be affecting proteolysis. On
the other hand, unknown factors might be affecting the estimation of residual rennet in
the different cheeses. Higher proteolysis may be obtained over time (31 weeks) when
more rennet is added, as observed for the high rennet treatment compared to the control
rennet treatment, due to the presence of more proteinases in the cheese. This difference
in rennet concentration may, however, not be significant enough to be observed in the
enzyme assay described in Sect. 3.3 above.
During ripening, the pH increased significantly (P<0.05, Fig. 5b); the pH during
ripening is usually affected by the formation of organic acids such as lactic acid and the
buffering capacity of the cheese. A similar rise in pH has been observed before for
reduced fat Cheddar cheese (Fenelon and Guinee 2000). In Fig. 5b, the control cheese
appears to have a slower rate of pH increase with time, compared to the other cheeses;
the cause of this difference is not currently known. The S/M ratios for the cheeses were
similar, as was the pH at draining. A difference in residual lactose may influence the
changes in pH but similar milk was used for all cheeses. Similarly, the calcium and
phosphorus content may affect the pH during ripening (Upreti and Metzger 2007),
although the pH at coagulation and draining were not statistically different (P>0.05). A
further study on the sugar and mineral content of the cheese may bring more insight on
the subject.

a 40 b 5.6
pH 4.6-SN/TN (% )

35 5.5
30
5.4
25
pH

20 5.3
15 5.2
10
5.1
5
0 5
Low Control High FCC 0 5 10 15 20 25 30 35
Rennet Ripening time (weeks)
Fig. 5 a The concentration of pH 4.6 soluble nitrogen/total nitrogen (pH 4.6-SN/TN) in cheese at week 31 for
cheeses made with different rennet concentrations (low, control or high Hannilase rennet concentration) or
different types of rennet (fermentation-produced camel chymosin, FCC). b Changes in the pH of the cheese
made using low (blue diamond), control (red square) or high (green triangle) Hannilase rennet concentration
or FCC (black circle) rennet at weeks 1, 5, 18 and 31 of ripening. The results are expressed as the mean±the
standard deviation of the mean (n=2). Images in colour can be viewed in the electronic version of this
manuscript
Effect of rennet during ripening 681

Despite a greater retention of rennet, the pH 4.6-SN/TN in the FCC cheese was
significantly lower than in the control cheese (Fig. 5a). This can only be explained by
the lower general proteolytic activity of FCC (Kappeler et al. 2006). Previous studies
have similarly reported lower proteolysis when Cheddar cheese was made with FCC
instead of FPC (Bansal et al. 2009; Govindasamy-Lucey et al. 2010) but these results
were attributed to a lower strength of FCC rennet; the rennet strengths used in the first
study were ∼0.039 IMCU.g−1 of milk) of FCC and ∼0.058 IMCU.g−1 of milk of calf
chymosin and in the second study 0.039 IMCU.g −1 of milk of FCC and
0.0485 IMCU.g−1 of milk of calf chymosin respectively. Moynihan et al. (2014),
however, also observed lower levels of proteolysis when cheeses were made with
FCC added at the same rennet concentration as FPC, consistent with this study. The
findings of this study confirm that the proteolytic activity in cheese is reduced even
when FCC is used at a similar rennet strength, consistent with reports of the lower
proteolytic activity of the isolated enzyme.
The proteolysis within each of the cheeses was also monitored using SDS-PAGE
(Fig. 6a). The proteolytic pattern of Cheddar cheese made with FCC and bovine
chymosin produced by fermentation has been compared previously using urea-PAGE

Week 1 Week 5 Week 31 MW

a STD
High
Control
High
Control

FCC
Low
FCC
-CN
Low

High
Control
-CN

FCC
Low

188

98

62
49

38
s2-CN
s1-CN
-CN 28
-CN
Casein degradation
products including 1-CN

-lactoglobulin A
para- -CN 14
-lactalbumin
Low molecular weight products
including 2-CN and 3-CN
B 6

b c
(expressed as a % of

Remaining -CN (expressed as a % of -

100 100

80 80
at week 1)

CN at week 1)

60 60
s1-CN

40
s1-CN

40
Remaining

20 20

0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Ripening time (weeks) Ripening time (weeks)
Fig. 6 a Image of SDS-PAGE gel (12%) of ripened cheeses made using low, control or high rennet Hannilase
concentration or fermentation-produced camel chymosin rennet at week 1, week 5 or week 31 of cheese
maturation. STD is the standard molecular weight marker and α-CN and β-CN are pure standards of these
compounds. The various protein bands have been identified based on their relative positions on the SDS-
PAGE gel, with reference to literature (Basch et al. 1989). The black rectangles indicate two regions of
interest, named region A or region B. Changes in the proportion of (b) αs1-CN and (c) β-CN, expressed as a
percentage of the concentration present in the cheeses at week 1, for cheeses made with low (blue diamond),
control (red square) or high (green triangle) Hannilase rennet concentration or fermentation-produced camel
chymosin (black circle) rennet. Protein concentration was measured using the intensity of the αs1-CN and β-
CN protein bands from the SDS-PAGE gel in Fig. 5, at weeks 1, 5 and 31. The results are expressed as the
mean±the standard deviation of the mean (n=2). Images in colour can be viewed in the electronic version of
this manuscript
682 K. Soodam et al.

(Bansal et al. 2009); however, Fig. 6a reports a comparison of Hannilase and FCC
rennet using SDS-PAGE. Protein bands were identified by comparison with α-CN and
β-CN standards, as well as molecular weight standards.
Hydrolysis of α-CN and β-CN can clearly be observed during ripening as the
intensity of the bands decreases between week 1, 5 and 31 for all treatments
(Fig. 6a). The breakdown of αs1-CN and β-CN, with respect to week 1, was further
quantified (Fig. 6b, c) and the percentage of αs1-CN and β-CN left was statistically
similar for the three Hannilase treatments at both weeks 5 and 31. This result was
somewhat surprising. Differences were apparent, however, in the concentration of
casein degradation products, as shown in Fig. 6a. These differences are consistent with
differences in the ratio of pH 4.6 soluble nitrogen/total nitrogen (pH 4.6-SN/TN),
which measures the pH 4.6 soluble fraction affected by proteolysis.
The protein bands in region marked A (∼17–24 kg.mol−1), for weeks 5 and 31,
appear fainter in the cheese made from low rennet concentration compared to
cheeses made from control or high concentrations. These regions include the
products from the degradation of αs1-CN, αs2-CN and β-CN (Basch et al. 1989),
which could indicate proteolytic differences as a result of different rennet concen-
trations. Proteolytic products can also be generated by plasmin or can be used by
bacteria within the cheese, so these patterns could also reflect different rates of
metabolism in the cheese.
The FCC cheeses had significantly higher residual αs1-CN and β-CN at week 5
compared to the control cheese (Fig. 6b, c), which can be attributed to slower
proteolysis in these cheeses. The data collected at week 31, however, suggest that the
type of rennet does not significantly affect the degradation of αs1-CN and β-CN if the
cheeses are given sufficient time to ripen. Previously, the literature has reported both
slower initial proteolysis and less final degradation of αs1-CN in Cheddar cheeses made
with FCC, with respect to those made from FPC (Bansal et al. 2009; Børsting et al.
2012). The difference between the results obtained here and previous studies may be
due to differences in FCC concentration, as the previous studies used a lower rennet
concentration (discussed above).
The proteolytic pattern for cheese made from FCC observed here was overall similar
to the pattern for the cheeses made from microbial rennet (Fig. 6a), except for a few
distinct differences. Comparing the control cheese and the cheese from FCC at weeks 5
and 31, it can be seen that FCC has less intense protein bands in the region marked A
and has only two protein bands in the region marked B (molecular weight in range of
∼8–12 kg.mol−1) instead of the three observed for the control cheese. These protein
bands most likely belong to proteolytic products from α-CN and β-CN. Such differ-
ences in proteolytic specificity and total proteolytic activity can affect levels of
bitterness or ‘brothy’ characteristics and also alter the availability of precursors for
other flavour compounds.

3.7 Texture profile during ripening

The cheese hardness was significantly affected by time, as assessed by the texture
analysis (P<0.05, Fig. 7). The rennet concentration, however, did not significantly
affect cheese hardness. Moynihan et al. (2014) also showed that low-moisture part-
skim Mozzarella cheese made with high concentrations of bovine chymosin had higher
Effect of rennet during ripening 683

160

140

120

Hardness (N)
100

80

60

40

20

0
0 5 10 15 20 25 30 35
Ripening time (weeks)
Fig. 7 Changes in the hardness of cheeses made using low (blue diamond), control (red square) or high
(green triangle) Hannilase rennet concentration or fermentation-produced camel chymosin (black circle)
rennet at weeks 1, 5, 18 and 31 of ripening. The results are expressed as the mean±the standard deviation
of the mean (n=2) (Colour figure online)

proteolysis after 28 days, as indicated by pH 4.6 soluble nitrogen, than the cheese made
with low concentrations of bovine chymosin. In the same study, however, it was also
observed that the hardness of cheeses made with high and low bovine chymosin did not
differ significantly during ripening. This suggests that the hardness may not be
determined solely by proteolysis and may be affected by other factors such as calcium
phosphate solubilisation (O'Mahony et al. 2005).
The FCC cheeses were slightly harder than the cheeses made with Hannilase at
weeks 18 and 31 (Fig. 7). The difference in hardness between rennet treatments was not
significant (P>0.05) when analysed independently of time. Multiple linear regression
of the combined data at weeks 18 and 31, however, showed that the rennet type
significantly affected the texture (P<0.05). This result parallels previous studies that
found that cheeses made with FCC were significantly harder than cheese made with
FPC (Bansal et al. 2009; Govindasamy-Lucey et al. 2010). When Mozzarella cheese
was made with different concentrations of recombinant chymosin (0.06–0.1 mL.kg−1 of
milk), the texture was not significantly different (Kindstedt et al. 1995), similar to the
results shown with Hannilase here. These studies illustrate that the three enzymes from
calf, camel and microbial sources behave differently and may differ in their influence
on texture during ripening.

4 Conclusion

Reduced fat Cheddar cheese was produced using different concentrations of microbial
rennet as well as an alternative enzyme, recombinant camel chymosin. Increasing the
concentration of rennet or using a different rennet type (Hannilase versus FCC) altered
the milk coagulation time but did not affect the overall processing time. The compo-
sition and yield of the cheese were similar, as was the loss of protein and fat to the
whey.
The rennet concentration affected the gel microstructure, which was more porous
qualitatively when a high concentration of rennet was used. The residual rennet in the
684 K. Soodam et al.

cheese increased when the amount of rennet was increased from low to medium concen-
trations, as expected. Increasing the rennet concentration used above the control value,
however, yielded cheeses with a statistically similar amount of residual rennet to the
control. These results show that most of the rennet was lost to the whey when high
concentrations were employed, which may have been accentuated by the increased
porosity of the gel at higher rennet levels. The residual rennet in the cheese was
significantly higher in the cheeses made from camel chymosin compared to Hannilase,
despite the similar rennet strength used in both treatments. At the end of the observed
ripening period, the microstructure of the cheeses as shown by cryo-SEM was
different, with a thicker protein network observed for the cheese made with recombinant
camel chymosin, which may be indicative of the slower proteolysis shown by this cheese
during ripening.
The results of this study suggest that at high concentrations of Hannilase rennet,
most of the rennet was lost to the whey and the cheese was not significantly texturally
different. Conversely, the use of a lower concentration of rennet resulted in slower
initial proteolysis but the texture was not significantly affected, suggesting that manu-
facturers could adopt lower concentrations of rennet. The recombinant camel chymosin
could also potentially be used as a substitute for Hannilase, especially for products that
require a lower degree of proteolysis.

Acknowledgments The authors acknowledge the Australian Government for providing the Australian
Postgraduate Award (APA) scholarship and Dairy Innovation Australia for financial support (08209C). We
thank Murray Goulburn, Lion and Warrnambool Cheese and Butter for their involvement in the project. We
also thank the Particulate Fluids Processing Centre (PFPC), the Bio21 Institute, the Electron Microscopy Unit
and the Biological Optical Microscopy Platform for equipment access and Mr Roger Curtain for his help in
operating the cryo-SEM.

Conflict of interest Kevany Soodam, Lydia Ong, Ian B. Powell, Sandra E. Kentish and Sally L. Gras
declare that they have no conflict of interest.

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