Food Biotechnology

ISSN: 0890-5436 (Print) 1532-4249 (Online) Journal homepage: https://www.tandfonline.com/loi/lfbt20

Effect of Free or Encapsulated Recombinant

Aminopeptidase of Lactobacillus rhamnosus S93 on
Acceleration of Cheddar Cheese Ripening

Sorayya Azarnia , Byong H. Lee , Daniel St-Gelais , Claude P. Champagne &

Kieran N. Kilcawley

To cite this article: Sorayya Azarnia , Byong H. Lee , Daniel St-Gelais , Claude P. Champagne
& Kieran N. Kilcawley (2010) Effect of Free or Encapsulated Recombinant Aminopeptidase of
Lactobacillus�rhamnosus S93 on Acceleration of Cheddar Cheese Ripening, Food Biotechnology,
24:2, 135-149, DOI: 10.1080/08905431003784853

To link to this article: https://doi.org/10.1080/08905431003784853

Published online: 27 May 2010.

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Food Biotechnology, 24:135–149, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 0890-5436 print
DOI: 10.1080/08905431003784853

Effect of Free or Encapsulated

1532-4249
0890-5436
LFBT
Food Biotechnology,
Biotechnology Vol. 24, No. 2, Apr 2010: pp. 0–0

Recombinant Aminopeptidase
of Lactobacillus rhamnosus S93
on Acceleration of Cheddar
Cheese Ripening
Lactobacillus
S. Azarnia et al.
rhamnosus S93 on Cheddar Cheese Ripening

Sorayya Azarnia1, Byong H. Lee1,2, Daniel St-Gelais2,

Claude P. Champagne2, and Kieran N. Kilcawley3
1
Departments of Food Science and Agricultural Chemistry, McGill University,
Ste-Anne-de-Bellevue, Quebec, Canada
2
Food Research and Development Centre, Agriculture and Agri-Food Canada,
St-Hyacinthe, QC, Canada
3
Department of Food Safety and Cultures, Moorepark Food Research Centre, Teagasc,
Moorepark, Cork, Ireland
The use of recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in
free or encapsulated form was investigated to shorten the duration of Cheddar cheese
ripening. Proteolysis was determined by measuring the soluble nitrogen as phospho-
tungstic acid (PTA-N) derivatives and free amino acids (FAA) over a 6-month period.
The experimental cheeses received higher scores for sensory properties than the control
cheese. The amounts of PTA-N and total FAA in the cheese with the encapsulated
enzyme after 2 months of ripening were close to those of the control cheese after 6
months, suggesting the acceleration in proteolysis by about 4 months.
Key Words: Cheddar cheese ripening; encapsulated enzyme; Lactobacillus rhamnosus
S93; recombinant aminopeptidase

INTRODUCTION
Cheese ripening is an expensive, slow, and lengthy process; therefore, reduc-
tion of ripening time has economic and industrial benefits for the cheese
industry. Of different methods employed to speed up cheese maturation,
addition of enzymes to milk seems to be the simplest and cheapest one. The

Address correspondence to Byong H. Lee, Departments of Food Science and

Microbiology/Immunology, McGill University, Montreal, QC, Canada H3A 2B4;
E-mail: byong.lee@mcgill.ca
136 S. Azarnia et al.

disadvantages of direct addition of enzymes to milk are nonuniform distribu-

tion of enzymes in the curd, early proteolysis and texture defects, and a loss of
about 90% of enzymes in the whey during cheese making (Kirby et al., 1987).
Encapsulation technology could be an alternative to this method.
Gums, milk fat, and phospholipids have been used as encapsulating mate-
rials. Liposomes, which are made from phospholipids, have been used to acceler-
ate Cheddar cheese ripening (Kirby et al., 1987). Enzymes such as Flavorzyme
(a fungal protease/peptidase complex), neutral bacterial protease, acid fungal
protease, and Palatase (Kheadr et al., 2003) were encapsulated in liposomes
and added to cheese milk to develop Cheddar cheese flavor. However, liposome
technology is still too expensive to be used commercially for cheese ripening
(Azarnia et al., 2006).
Another alternative for enzyme encapsulation is using natural polysac-
charide or food-grade gums such as Na-alginate. This polysaccharide is a
polymer of mannuronic and guluronic acid residues and has the capacity to
bind divalent cations such as calcium resulting in the formation of a three-
dimensional gel network (Roberts, 1992).
However, alginate gels have large pores, which may result in enzyme
release during the operation (Tanaka et al., 1984). This problem could be
reduced by formation of a polyelectrolyte complex between alginate as an
anionic polymer and chitosan as a cationic polymer (Roberts, 1992). This
polymer has been used to immobilize biomolecules such as glucose oxidase
(Khani et al., 2006) and Flavorzyme (Kailasapathy et al., 2006). Alginate-pectin
capsules were used for the fortification of Cheddar cheese with folic acid
(Madziva et al., 2006).
Lactobacilli naturally contain intracellular proteinases, peptidases, and
esterases that can be used to accelerate cheese ripening (Lee et al., 2007).
However, some defects produced by their adjunct live or shocked cells led to
the use of their overexpressed enzymes in cheese ripening (Azarnia et al.,
2006). An aminopeptidase (PepN) from Lactobacillus rhamnosus S93 was
overexpressed up to a 1,000-fold in Escherichia coli; thus, the production cost
became cheap (Lee and Robert, 1997). It is not easy with the available commercial
enzymes (crude extracts) to demonstrate the effect of the pure aminopepti-
dase, and no pure enzyme study has been carried out. The heat shocked cells
of Lactobacillus casei (El Abboudi et al., 1991) and Lactobacillus helveticus
DPC 4571 (Lee et al. 2007) could accelerate the flavors of enzyme modified
cheeses, but little work has been performed on the encapsulated pure ami-
nopeptidase in the acceleration of cheese ripening. A more rational approach
in using the encapsulated pure enzyme was chosen in this study to accelerate
the Cheddar cheese ripening.
Alginate has been used as an immobilization matrix for biomolecules and
microorganisms (Champagne et al., 1992; Kailasapathy et al., 2006; Vårum and
Smidsrød, 2006), but no data are found on the application of the encapsulated
 Lactobacillus rhamnosus S93 on Cheddar Cheese Ripening 137

lactobacilli peptidases in alginate-chitosan particles to accelerate cheese rip-

ening. The objective of this study was to investigate the effect of recombinant
aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in free or encapsu-
lated form on the acceleration of Cheddar cheese proteolysis.

MATERIALS AND METHODS

Materials
All chemicals and media were purchased from Sigma (Sigma-Aldrich, Inc.,
St. Louis, Mo., USA) and Difco (Difco Laboratories, Detroit, Mich., USA),
respectively, unless otherwise mentioned. Recombinant plasmid B3-M (MluI)
consisting of aminopeptidase (PepN) gene from Lactobacillus rhamnosus S93
cloned in Escherichia coli was obtained from Agriculture and Agri-Food Canada,
Food Research and Development Centre (St. Hyacinthe, Quebec, Canada).
Starter strains, Lactococcus lactis ssp. cremoris (LL-74, LL-275 and LL-390)
were provided by Agropur (Granby, Quebec, Canada).

Preparation and Purification of Recombinant Aminopeptidase

Recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93
was prepared and purified as described by Azarnia et al. (2008).
Aminopeptidase (PepN) activity was determined spectrophotometerically
using leucine-p-nitroanilide (leu-pNA) as a substrate as described by Azarnia
et al. (2008). One unit of enzyme activity was defined as the amount of enzyme
required to release 1 μmol of p-nitroaniline/min under the defined conditions.
Protein concentration was estimated at 562 nm using a BCA™ (bicinchoninic
acid) Protein Assay Kit (Pierce Chemical Ltd., Rockford, Ill., USA). Specific
activity was measured in enzyme units per milligrams of protein.

Preparation of Freeze-dried Chitosan-coated Alginate Capsules

The alginate capsules coated with chitosan were prepared according to the
modified method of Azarnia et al. (2008). The mixture of purified recombinant
PepN and the cooled Na-alginate (Sobalg FD 126, Grindsted Products, Inc.,
Rexdale, Ontario, Canada) solution (1.6%) at a ratio of 1:3 was extruded through
a 300-μm nozzle of an encapsulator (Inotech Labor, IE-50 R, Eulerstrasse,
Switzerland) into a cold (4°C) gelling solution-containing chitosan (0.1%)-CaCl2
(0.1 M). All capsules hardened in the gelling solution for 10 min under agitation
(70 rpm). The capsules were harvested by filtration and washed with deionized
distilled water. All experiments were carried out in triplicate. The encapsulation
efficiency was calculated as described by Azarnia et al. (2008).
The free or encapsulated enzyme was frozen at −80°C and dried using a
freeze drier (FTS System, Inc. Biopharm Division, N.Y., USA) as described by
138 S. Azarnia et al.

Azarnia et al. (2008). Dried capsules (0.1 g) were dissolved in release buffer,
30 mL of trisodium citrate solution (2%, wt/vol), at room temperature and gen-
tly stirred (70 rpm) to release the entrapped enzyme from the capsules. The
aminopeptidase activity was measured after the complete dissolution of the
capsules into the release buffer.

Cheddar Cheese Manufacture

Starter strains Lactococcus lactis ssp. cremoris (LL-74, LL-275 and
LL-390) were separately grown at 21°C for 16 h in pasteurized (85°C for
10 min) reconstituted low heat skim milk powder (12%, wt/vol). They were
used at the same ratio (1: 1: 1) in cheese-making trials. Control and experi-
mental cheeses were made in triplicate in vats containing 200 L milk at the
pilot plant of Agriculture and Agri-Food Canada, Food Research and Develop-
ment Centre (St. Hyacinthe, Quebec, Canada), by an experienced cheese master.
The same cows’ milk was used for all batches on each day of manufacture.
Milk was pasteurized at 73°C for 16 s. Calcium chloride (25 g/100 kg milk) and
starter culture (1.5%, vol/vol) were added to the milk (elapsed time 60 min).
Double strength chymosin (0.02%, vol/vol; Maxiren®, DSM Food Specialties
USC Inc., Parsippany, N.J., USA) was added at 32°C. After about 30 min, the
curd was cut and heated to 38°C. After drainage (elapsed time 150 min), ched-
dar processing occurred at 38°C. The curd was milled (pH 5.15), salted (2.0%, wt/
wt, elapsed time 108 min), placed in 10-kg hoops, pressed (1 h, 2.46 kgf/cm2),
packed under the vacuum condition, and stored at 4°C for 6 months. Different
batches of the encapsulated enzyme were used for different cheese-making tri-
als. The same amount (20,000 units/200 L milk) of the freeze-dried encapsu-
lated or free form of the purified recombinant PepN was added at the rennet
treat mentor salting stage, respectively. Sampling was carried out at 15 d, 2, 4
and 6 months of ripening. All analyses were carried out in duplicate.

Estimation of the Encapsulated Enzyme Entrapment in the Curd

The incorporation rate of encapsulated recombinant PepN into the curd
was indirectly estimated by measuring the aminopeptidase activity in the
whey obtained after drainage and pressing steps of the cheese manufacturing.
The aminopeptidase activity in the whey of the cheese with encapsulated
enzyme was compared to that of the control cheese.

Compositional Analysis
The total nitrogen was determined by the macro-Kjeldahl method (IDF,
1986). Moisture was determined by drying to a constant weight at 102°C (IDF,
1982). Salt content of cheese was measured by a Corning chloride analyzer 926
(Nelson-Jameson, Inc., Marshfield, Wisc., USA). Fat content was determined
 Lactobacillus rhamnosus S93 on Cheddar Cheese Ripening 139

by the Mojonnier extraction procedure and ash by heating samples at 550°C

for 16 h in a muffle furnace. The pH of cheese (10 g) macerated in 10 mL dis-
tilled water was measured (Hannon et al., 2005) using a pH meter (Ross® pH
electrodes, Thermo Orion, Beverly, Mass., USA). Milk was analyzed for fat,
protein, lactose, and total solid on a Milkoscan FT 120 (Foss North America,
Brampton, Ontario, Canada). All analyses were carried out in duplicate.

Analysis of Proteolytic Indices

The water-soluble nitrogen (WSN) and the total nitrogen soluble in 5%
phosphotungstic acid (PTA-N) were determined by the methods described by
Christensen et al. (1991). All results obtained from the measurement of differ-
ent nitrogen fractions were expressed as a percentage of total nitrogen and all
determinations were made in duplicate.
Individual free amino acids (FAA) were determined on 24% trichloroacetic
acid filtrates prepared from the water-soluble nitrogen fraction using a Jeol
JLC-500/V Amino Acid Analyzer (Jeol Ltd, Garden City, Herts, UK) fitted
with a Jeol sodium high performance cation exchange column (Kilcawley et
al., 2006) and the results expressed as μg/g cheese. All analyses were carried
out in duplicate.

Sensory Evaluation
Sensory properties of the cheeses were evaluated after 2, 4 and 6 months
of ripening by nine trained panelists at Agriculture and Agri-Food Canada,
Food Research and Development Centre (St. Hyacinthe, Quebec, Canada).
After cutting the cheeses into small cubes (about 5 g) and keeping them at
room temperature for about 2 h, four portions (5 g) of each sample were put in
an amber-glass bottle. The bottles were capped and coded with a 2-digit num-
ber. Samples were presented in random order to the panelists. Palate cleans-
ing between samples were carried out using deionized water. Samples were
evaluated, without swallowing, considering three attributes, that is, texture,
flavor, or aroma using a five-point hedonic/quantitative scale as very good
(+2), good (+1), acceptable (0), bad (−1), and very bad (−2).

Microbiological Analysis
After 15 d, 2, 4 and 6 months of storage, the cheese packages were opened
and cut aseptically. Samples (10 g) and a 90 mL sterile peptone solution (0.1%,
wt/vol) were blended in a Stomacher 400 circulator (Seward Lab., London,
UK) for 3 min at 260 rpm. Bacterial numbers were determined according to
the method described by the American Public Health Association for dairy
products (APHA, 1992). All plates were made in duplicate. Lactococci and
lactobacilli were plated on M17-Agar and Rogosa Agar (adjusted pH 5.5 with
140 S. Azarnia et al.

acetic acid 96%), respectively, and incubated at 30°C for 4 d. Plated lactoba-
cilli were incubated in an anaerobic incubator (Thermo Fisher Scientific, Fisher
Canada, Nepean, Ontario, Canada). After incubation at 22°C for 4 d, yeasts
and molds were enumerated on Rose Bengal Agar supplemented with chloram-
phenicol (Bishop Canada Inc., Burlington, Ontario, Canada). Enumeration of
total coliforms were carried out on Violet Red Bile Agar after incubation at
37°C for 24 h. Staphylococci were plated on Staphylococcus Medium 110 and
incubated at 37°C for 48 h.

Statistical Analysis
The data from WSN, PTA-N, concentration of total FAA, microbial counts;
cheese and milk compositions and sensory properties were evaluated accord-
ing to a split plot design with three replicates using the PROC GLM procedure
of SAS software (SAS Institute Inc., Cary, N.C.). Principal Component Analy-
sis (PCA) (Johnson, 1998) was carried out on individual FAA data to reduce
the dimensionality of the data. Statistically significant differences between
different treatments were determined by Fisher’s least significant difference
(F-value). The Tukey’s test using SAS software was carried out to compare the
means among the proteolytic indices and sensory properties of cheeses. Means
were compared at a significant level of 5%.

RESULTS AND DISCUSSION

Enzyme Preparation and Purification

The purification of crude enzyme extract containing the recombinant
aminopeptidase of Lb. rhamnosus S93 showed that the enzyme was purified
by about 12-fold compared to the crude extract with a recovery of about 29%,
and the molecular mass of the enzyme was about 90 kDa (data not shown).

Encapsulated Enzyme Incorporation

The encapsulation efficiency was about 90%, which is consistent with our
previous result (Azarnia et al., 2008). No aminopeptidase activity was observed
in the whey obtained from the cheese with encapsulated enzyme or from the
control cheese. Almost the entire encapsulated enzyme was entrapped into the
cheese matrix, indicating the stability of the capsules during Cheddar cheese
manufacturing.

Compositional Analysis
The same cows’ milk was used for all batches on each manufacturing day
so no significant differences (p > 0.01) were observed in milk compositions
 Lactobacillus rhamnosus S93 on Cheddar Cheese Ripening 141

Table 1: Composition of control and experimental chesses at 15 days of ripening.

Cheese with
Composition Control cheese Cheese with free enzyme encapsulated enzyme

Salt (%) 1.51 ± 0.03 1.50 ± 0.03 1.50 ± 0.02

Moisture (%) 38.76 ± 0.27 38.73 ± 0.29 39.05 ± 0.22
S/M1 (%) 3.89 ± 0.06 3.87 ± 0.04 3.84 ± 0.06
F/DM2 (%) 51.26 ± 1.27 51.28 ± 1.27 51.39 ± 1.15
Protein (%) 23.73 ± 0.09 23.75 ± 0.08 23.48 ± 0.10
Ash (%) 3.21 ± 0.10 3.23 ± 0.08 3.19 ± 0.07
pH 5.05 ± 0.06 5.07 ± 0.07 5.04 ± 0.05
1
Saltin Moisture.
2
Fat in Dry Matter.
The results shown are the average of triplicate trials and expressed as mean ± standard deviation.

used for cheese-making trials (data not shown). The compositions of the con-
trol cheese with the free enzyme and cheese with the encapsulated enzyme at
15 d of ripening time are shown in Table 1. No differences (p > 0.05) were
observed among the cheeses for the compositions.

Microbiological Analysis
The changes in the mean populations (from the three trials) of lactococci
and lactobacilli in the cheeses during ripening time of 6 months at 4°C are
presented in Table 2. Lactococcal population decreased significantly (p < 0.05)
during storage time in all cheeses. The mean population of starter lactic acid
bacteria declined 1.76, 1.73, 1.83 log cycles after 6 months of ripening in the
control cheese, cheeses treated with the free or encapsulated enzyme, respec-
tively (Table 2). The results showed that the encapsulated materials did not
affect the cell counts. No pathogens, yeasts, and molds were found in all
cheeses in this study, which means that the cheeses were prepared and stored
in a good condition.
Low pH, low temperature, high concentration of salt and lack of lactose
result in the reduction of the starter’s cell count during the ripening period.
This causes an increase in the lactococci intracellular enzymes such as
peptidases into the cheese matrix, which is important in flavor generation in
the cheese during maturation (Law, 2001; Lortal and Chapot-Chartier, 2005;
Kenny et al., 2006).
Conversely, lactobacilli grew in all cheeses during the ripening period and
their population increased from about 1 log10 cfu/g of cheese to 6 log10 cfu/g of
cheese after 6 months of maturation (Table 2).
Nonstarter lactic acid bacteria (NSLAB) such as lactobacilli grow in
Cheddar cheese during maturation. They could originate from milk, cheese
making equipment, air, or personnel (Fox et al., 1998).
142 S. Azarnia et al.

Table 2: Changes in microbial counts during ripening period.

Microbial counts Cheese with

(log10 cfu g−1 Ripening time Cheese with free encapsulated
cheese) (Days) Control cheese enzyme enzyme

Lactococci 15 7.81 ± 0.17 7.77 ± 0.16 7.86 ± 0.16

60 7.33 ± 0.25 7.16 ± 0.17 7.26 ± 0.12
120 6.40 ± 0.19 6.51 ± 0.11 6.37 ± 0.13
180 6.05 ± 0.18 6.04 ± 0.16 6.03 ± 0.21
Lactobacilli 15 1.00 ± 0.09 1.06 ± 0.06 1.04 ± 0.06
60 3.38 ± 0.12 3.29 ± 0.11 3.27 ± 0.14
120 5.64 ± 0.13 5.54 ± 0.10 5.58 ± 0.12
180 5.70 ± 0.11 5.81 ± 0.12 5.71 ± 0.16

The results shown are the average of triplicate trials and expressed as mean ± standard deviation.

Proteolysis Analysis
The results obtained from the assessment of WSN, which was expressed
as a percentage of total nitrogen, are shown in Table 3. The level of nitrogen
fraction as primary proteolysis product increased significantly (p < 0.05) in all
cheeses with storage time. No differences (p > 0.05) were observed between
the control and experimental cheeses in this fraction at each stage of ripening.
Increasing the amount of WSN during ripening time is due to the degra-
dation of casein to low molecular weight of water-soluble peptides and amino
acids by milk enzymes, residual coagulant, starter lactic acid bacteria (LAB),
and NSLAB (Sousa et al., 2001; Dabour et al., 2006).
The other proteolytic indices, that is, PTA-N and total FAA, also increased
in all cheeses during the ripening time (Table 3). The amounts of these sec-
ondary proteolytic indices were significantly (p < 0.01) higher in the cheese
with encapsulated enzyme and in cheese with free enzyme than those of the
control cheese at each sampling time (Table 3), indicating the acceleration of
the ripening process. The amounts of PTA-N and total FAA in the cheese with
encapsulated enzyme after 2 months of ripening was close to the control
cheese after 6 months, indicating the acceleration of about 4 months in the
proteolysis. According to the Tukey’s test, there were significant differences at
the level of 5% among the mean levels of the PTA-N as well as of the total
FAA. As presented in Table 3, cheese with the encapsulated enzyme showed a
significantly higher mean level of PTA-N and total FAA compared to those of
other cheeses over a 6-month ripening period. These results are in agreement
with the other research such as using liposome (Kirby et al., 1987) or gellan
and k-carrageenan (Kailasapathy and Lam, 2005) as materials for enzyme
encapsulation leading to an increase in proteolysis rates.
In our study, both free and encapsulated forms of the recombinant PepN
led to enhancement of secondary proteolysis compared to that of the control.
However, an important advantage of encapsulated enzymes is that they can
 Table 3: Changes in proteolytic indices during ripening period.

Ripening time (d)

60 120 180

Parameters C1 F2 E3 C F E C F E

WSN/TN (%)4 16.28 ± 0.20 16.63 ± 0.20 16.37 ± 0.24 20.42 ± 0.29 20.62 ± 0.30 20.59 ± 0.28 23.28 ± 0.26 23.73 ± 0.27 23.88 ± 0.28
PTA/TN (%)5 1.21c ± 0.10 1.87b ± 0.08 2.17a ± 0.12 1.64c ± 0.17 2.45b ± 0.14 2.97a ± 0.22 2.00c ± 0.10 3.10b ± 0.14 3.60a ± 0.28
Free amino acids (μg/g cheese)
ASP 49 ± 7 142 ± 15 247 ± 83 79 ± 12 263 ± 77 370 ± 105 114 ± 27 423 ± 8 505 ± 7
THR 30 ± 17 76 ± 6 124 ± 22 56 ± 33 145 ± 10 181c ± 8 227 ± 17 197 ± 5 185 ± 18
SER 36 ± 7 43 ± 10 67 ± 11 60 ± 20 99 ± 11 115 ± 10 102 ± 14 170 ± 5 184 ±10
GLU 387 ± 61 605 ± 35 708 ± 150 583 ± 97 986 ± 91 1133 ± 120 764 ± 42 1146 ± 40 1277 ± 68
PRO 31 ± 19 19 ± 3 8±1 23 ± 21 0 0 0 0 0
GLY 21 ± 6 37 ± 12 45 ± 10 33 ± 8 64 ± 13 69 ± 13 54 ± 8 99 ± 4 114 ± 21
ALA 41 ± 8 64 ± 11 82 ± 6 65 ± 8 102 ± 8 124 ± 20 96 ± 8 159 ± 7 174 ± 22
CYS 14 ± 4 35 ± 11 52 ± 25 22 ± 1 59 ± 26 82 ± 13 79 ± 7 94 ± 11 129 ± 21
VAL 85 ± 19 310 ± 37 372 ± 29 124 ± 22 488 ± 42 519 ± 51 183 ± 34 661± 14 717 ± 39
MET 70 ± 10 81 ± 11 97 ± 21 127 ± 13 155 ± 19 175 ± 28 192 ± 43 252 ± 10 272 ± 21
ILE 13 ± 4 63 ± 11 73 ± 26 20 ± 4 95 ± 10 114 ± 37 29 ± 6 132 ± 5 157 ± 18
LEU 242 ± 54 723 ± 25 863 ± 132 449 ± 52 1157 ± 72 1211 ± 124 624 ± 40 1599 ± 32 1670 ± 56
TYR 83 ± 24 209 ± 10 249 ± 47 169 ± 47 324 ± 21 357 ± 34 218 ± 50 435 ± 27 508 ± 30
PHE 348 ± 53 547 ± 18 624 ± 47 577 ± 35 824 ± 62 910 ± 23 807 ± 28 1099 ± 72 1158 ± 97
HIS 142 ± 8 159 ± 6 178 ± 15 234 ± 23 246 ± 33 282 ± 23 285 ±19 327 ± 17 385 ± 32
LYS 94 ± 16 222 ± 22 259 ± 57 122 ± 25 333 ± 47 439 ± 85 156 ± 12 478 ± 14 521 ± 42
NH3 37 ± 3 67 ± 4 92 ± 5 47 ± 5 101 ± 20 129 ± 9 73 ± 11 144 ± 8 155 ± 7
ARG 216 ± 26 304 ± 14 375 ± 40 328 ± 33 467 ± 23 485 ± 22 443 ± 22 614 ± 27 647 ± 25
Total free amino
acids (μg/g
cheese) 1939 ± 346 3706 ± 261 4515 ± 727 3118 ± 459 5908 ± 585 6695 ± 725 4446 ± 388 8029 ± 306 8758 ± 534
1
Control cheese; 2Cheese with free enzyme; 3Cheese with encapsulated enzyme.
4
Water Soluble Nitrogen/ Total Nitrogen (%)
5
Phosphotungstic acid Soluble Nitrogen/ Total Nitrogen (%)
The results shown are the average of triplicate trials and expressed as mean ± standard deviation. Values within a row with different letters differ
(p < 0.05).

143
144 S. Azarnia et al.

be added directly to milk at the renneting stage rather than to the curd at the
salting stage, so that enzyme can be distributed uniformly in the curd. There-
fore, study of the cheese microstructure could be useful to find the effects of
two methods on the uniform distribution of the enzyme in the cheese matrix.
The FAA assessment can be used as an indicator of proteolysis activity
during ripening (Hannon et al., 2005). Table 3 compares the concentration
of individual FAA in the control and the experimental cheeses at 2, 4, and
6 months of the ripening time. The levels of individual amino acids were
higher in the experimental cheeses at all sampling times compared to those of
the control cheese, except for proline which decreased at 2 months and disap-
peared at 4 months in the experimental cheeses. Aminonopeptidase of Lb.
rhamnosus had a weak activity for proline production (Arora and Lee, 1994),
but proline may have been metabolized by proline specific peptidases such as
x-prolyldipeptidyl peptidase (Habibi-Najafi and Lee, 1994) and proline imi-
nopeptidase (Habibi-Najafi and Lee, 1995), derived from lactic flora in the
cheese. Ammonia (NH3) was also present in all samples at all sampling times
and its concentration increased with the ripening time and was higher in the
experimental cheeses than the control (Table 3). This finding is in agreement
with the other studies (Wallace and Fox, 1997; Hannon et al., 2005). The
major free amino acids in all cheeses during the ripening time were Leu, Glu,
Phe, Val, Arg, and Lys (Table 3). Leucine was the most abundant amino acid
in the experimental cheeses (Table 3). These are attributed to the property of
the recombinant aminopeptidase used in this study, which has the high
affinity for Leu-, Lys-, Glu-, Val-, and Arg-peptides, and it has preference for
dipeptides containing Leu as the N-terminal residue (Arora and Lee, 1994). It
has been reported that aminopeptidase N or C (PepN or C) released Lys, Leu,
Arg, Met, or Phe from the N-terminal position and has been shown to be effec-
tive in reducing levels of bitter peptides in cheese (Habibi-Najafi and Lee,
1994; Swearingen et al., 2001). The release of Phe, Tyr, Leu, Ileu, Val, and
Met was coincident with good cheese flavor (Hannon et al., 2005). These
results are also in agreement with findings of other studies such as Puchades
et al. (1989), Wallace and Fox (1997), and de Wit et al. (2005), who found that
Leu, Phe, and Glu were the most abundant in Cheddar cheese. Glu, Met, Leu,
Lys, and Val were higher in the Cheddar cheeses with added lactobacilli as an
adjunct culture than in control cheese (Gardiner et al., 1998). A large quantity
of Leu has also been reported in lactobacilli-added Cheddar cheeses (Puchades
et al., 1989).
The PCA was carried out on the raw data of the individual FAA. Principal
component 1 (PC1) and principal component 2 (PC2) significantly separated
the cheeses on the basis of ripening time and the treatments accounted for a
cumulative variation of 90.34% (Fig. 1). The cheese with encapsulated enzyme
was separated from the others with the high amounts of the individual free
amino acids during the ripening process (Fig. 1).
 Lactobacillus rhamnosus S93 on Cheddar Cheese Ripening 145
Principal component 1 (85.13%)

Principal component 2 (5.21%)

Figure 1: Principal component analysis of the individual amino acids in Cheddar cheeses.
a: Control cheese at 60 d; b: Control cheese at 120 d; c: Control cheese at 180 d; d: Cheese
with free enzyme at 60 d; e: Cheese with free enzyme at 120 d; f: Cheese with free enzyme at
180 d; g: Cheese with encapsulated enzyme at 60 d; h: Cheese with encapsulated enzyme
at 120 d; i: Cheese with encapsulated enzyme at 180 d.

Sensory Evaluation
The Tukey’s test showed differences (p < 0.01) for the three attributes of
texture, flavor, and aroma among the cheeses. According to the Tukey’s grouping,
cheese with the encapsulated enzyme received significantly higher mean
scores of flavor and aroma among the cheeses at 2 and 6 months of the ripen-
ing time (Table 4). No differences were observed between the mean hedonic
scores of texture, flavour, and aroma for cheese with the encapsulated enzyme
and cheese with the free enzyme at 4 months of the ripening period. There were
146
Table 4: Sensory scores of cheeses ripened for 180 days

Ripening time (d)

60 120 180
Sensory
attributes C1 F2 E3 C F E C F E

Texture 0.19b ± 0.79 0.33ab ± 0.88 0.48a ± 1.01 0.19b ± 0.62 0.70ab ± 0.72 0.704a ± 0.87 0.04c ± 0.73 0.44b ± 0.75 0.78a ± 0.85
Flavor −0.15b ± 0.91 0.11b ± 1.05 0.44a ± 1.15 0.07b ± 0.92 0.59ab ± 0.84 0.704a ± 0.91 0.26c ± 0.81 0.59b ± 0.80 1.00a ± 0.73
Aroma 0.19b ± 0.76 0.44b ± 0.80 0.55a ± 0.85 0.22b ± 0.85 0.74ab ± 0.76 0.815a ± 0.88 0.26c ± 0.69 0.67b ± 0.55 0.96a ± 0.76
1
Control cheese; 2Cheese with free enzyme; 3Cheese with encapsulated enzyme.
The results shown are the average of triplicate trials and expressed as mean ± standard deviation.
Values within a row with different letters differ (p < 0.05).
 Lactobacillus rhamnosus S93 on Cheddar Cheese Ripening 147

no significant differences between the mean scores of the three attributes for
the cheese with free enzyme and control cheese at 2 and 4 months of the
ripening period (Table 4). Enhancement of the sensory properties in experi-
mental cheeses, particularly in the cheese with the encapsulated enzyme, is
consistent with our results obtained from the proteolysis evaluation. The
panelists did not recognize any defect due to the presence of alginate capsules
in the samples.

CONCLUSIONS
This study presents a method to enhance the proteolysis of Cheddar cheese
using the purified recombinant aminopeptidase of Lactobacillus rhamnosus
S93 in the free or encapsulated form. The use of encapsulated enzyme
resulted in an acceleration of about 4 months in proteolysis compared to the
control cheese. Efficient incorporation of enzymes in milk before cheddaring
can be achieved using this encapsulation technique. Although this study was
aimed to develop an encapsulation method, which can affect Cheddar cheese
ripening, it would be useful for other type of cheeses.

ACKNOWLEDGMENTS
This work was supported by an operating fund of Agriculture and Agri-Food
Canada and NSERC grant awarded to Byong H. Lee. We thank Normand
Robert and Gaétan Bélanger for their technical supports and collaboration in
sensory assessment. We also thank Jacinthe Fortin, Dr. Hassan Sabik, and
Dr. Ali Taherian for their collaboration in sensory assessment.

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