LWT - Food Science and Technology 95 (2018) 99–106

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Effect of Lactobacillus rhamnosus on the antioxidant activity of Cheddar T

cheese during ripening and under simulated gastrointestinal digestion
Lu Liua,b, Xiuwei Qua,b, Qina Xiaa,b, Haixia Wanga,b, Ping Chena,b, Xiaodong Lia,b,∗, Lina Wanga,b,
Wanshuang Yanga,b
a
Food College, Northeast Agricultural University, No. 600 Changjiang St, Xiangfang Dist, 150030, Harbin, China
b
Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, No. 600 Changjiang St, Xiangfang Dist, 150030, Harbin, China

A R T I C LE I N FO A B S T R A C T

Keywords: This study was aimed at evaluating Lactobacillus rhamnosus for its ability to affect the antioxidant activity of
Lactobacillus rhamnosus Cheddar cheese during ripening and digestion. The effect of probiotic on the proteolytic patterns and antioxidant
Cheddar cheese activity of Cheddar cheese during ripening was analysed. Three fractions (> 10 kDa, 3–10 kDa, and < 3 kDa)
Antioxidant activity were separated at the end of ripening of cheese and after digestion. The results demonstrated that Lactobacillus
Ripening
sps dominated all stages of ripening cheese and maintained their viability at 8.44 log CFU/g at the end of
Simulated gastrointestinal digestion
ripening. The proteolysis concentrations were significantly higher in probiotic cheeses and the antioxidant ac-
tivity was at its maximum at the end of ripening. Cheeses made with added Lactobacillus rhamnosus had sig-
nificantly higher proteolytic activity and antioxidant activity (P < 0.05) than those without probiotics during
the entire ripening time. After digestion, the number of bacteria in cheese decreased significantly (P < 0.05), but
the polypeptide content was increased by 37.97% and the DPPH radical scavenging ability and reducing power
were increased by 7.46% and 17.58%. More small peptides were produced after digestion and the antioxidant
activity of cheese correlated with proteolysis and production of small molecule polypeptides that were further
enhanced upon incorporation of probiotics. The total acceptability of cheese was not affected by the addition of
probiotic bacteria.

1. Introduction
In recent years, there has been an increased scientific interest in
cheese, which is a rich source of essential nutrients such as proteins,
vitamins and short chain fatty acids that have a positive impact on body
functions, thereby improving health (Reale, Ianniello, & Ciocia, 2016).
Proteolysis is the major biochemical event during cheese formation,
which occurs during ripening via the action of proteases and peptidases,
such as cell wall-associated proteinases from lactic acid bacteria (LAB)
(Lu, Govindasamylucey & Lucey, 2016; Gobbetti, Minervini, & Rizzello,
2004). These proteinases break down main precursors proteins
(caseins) into shorter multifunctional peptides such as opioid peptides
(López-Expósito, Amigo & Recio, 2012), antihypertensives (Elvira et al.,
2010), antioxidant peptides (Chang, K-H, S-G & M-H, 2013; Chen et al.,
2012) and anti-bacterial peptides (Gobbetti et al., 2004).
Lactic acid bacteria (LAB) possess variable patterns of proteinases,
peptidases and peptide transport systems (Slattery, O'Callaghan,
Fitzgerald, Beresford, & Ross, 2010) that contribute to the development
of flavor and texture of cheese (López-Expósito et al., 2012; Law &
Haandrikman, 1997) and the release of bioactive peptides (Hayes, Ross,
Fitzgerald, & Stanton, 2007; Elfahri et al., 2014). Cell-envelope pro-
teases (CEP) break down milk proteins into peptides of about 5–30
amino acids. These peptides are then carried into the cell and further
hydrolysed by endopeptidases into shorter peptides and amino acids for
microbial protein synthesis (Lisa, Giuseppina Sefora & Davide, 2015;
Liu, Bayjanov, Renckens, Nauta, & Siezen, 2010). The acid-producing
starter cultures, such as Lactobacillus helveticus and Lactobacillus del-
brueckii ssp. bulgaricus strains can release bioactive peptides during milk
fermentation (Sadat-Mekmene et al., 2011; Zhang et al., 2015). In
contrast, a very small body of work deals with the potential of probiotic
strains to release such peptides (Fuglsang, Rattray, Nilsson, & Nyborg,
2003; Wang, Dong, Chen, Cui, & Zhang, 2010). Lactobacillus rhamnosus,
Lactobacillus plantarum and Lactobacillus casei are extensively used both
as probiotics and adjunct as cultures in different dairy products
(Aguilar-Toalá et al., 2017; Settanni & Moschetti, 2010). Milk-derived
peptides with different biological activities can be produced, when
probiotics are delivered with fermented dairy products (Hayes et al.,
2007). Research has found that the use of probiotics in dairy products

∗
Corresponding author. College of Food Science, Northeast Agricultural University, No. 600 Changjiang St, Xiangfang Zone, Harbin 150030, China.
E-mail address: hrblxd@163.com (X. Li).

https://doi.org/10.1016/j.lwt.2018.04.053
Received 14 December 2017; Received in revised form 14 March 2018; Accepted 18 April 2018
Available online 19 April 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
L. Liu et al.
has an impact on their antioxidant properties and is even capable of
producing antioxidant polypeptides (Reale et al., 2016; Abadíagarcía
et al., 2013). The incorporation of probiotic bacteria into cheese can
also promote additional advantages (Lisa et al., 2015). Lactobacillus
rhamnosus strains have been noted for producing high levels of in-
tracellular aminopeptidase, predominantly in matured Cheddar cheese
(Sorayya, Byongh, Varoujan, & Kierann, 2010).
The specific peptidase activity of strains, pH, temperature and food
system can also affect the production of bioactive peptides (Lu et al.,
2016; Gupta, Mann, Kumar, & Sangwan, 2009; Algaron, Miranda, Le
Bars, & Monnet, 2003; Mettes et al., 2009; Meyer, Bütikofer, Walther,
Wechsler, & Sieber, 2009; Ong & Shah, 2008) and bioactive compo-
nents in food can exert their biological activities after digestion and
absorption by the body. Thus investigating the role of probiotics in
affecting the bioactive activity of a food during its processing, ripening
and digestion is necessary.
Therefore, this study was aimed to investigate the impact of
Lactobacillus rhamnosus on the antioxidant activity of Cheddar cheese
during ripening and simulated gastrointestinal digestion. Our findings
contribute to the understanding of how these probiotics could be
exploited to develop functional dairy products.
2. Materials and methods
2.1. Microorganisms and materials
Lactobacillus rhamnosus 6134 was obtained from the China Center
for Industrial Culture Collection. The strain was isolated from naturally
occurring human intestinal Lactobacillus sps of healthy subjects and
preserved in the Key Laboratory of Dairy Science, Food College,
Northeast Agricultural University in China. This strain can prevent the
growth of harmful bacteria and adjust the populations of other bacteria
groups forming various microecological systems in the human body and
promote defecation. We applied this strain to Cheddar cheese and
white-brined cheese in our previous study. (Liu et al., 2015, 2016,
2017).
The commercial strains Lactococcus lactis subsp. Cremoris and
Lactococcus lactis subsp. lactis were obtained from Chr. Hansen (Chr.
Hansen, Bayswater, Victoria, Australia).
All other chemicals were of food grade and from Sinopharm
Chemical Reagent Beijing Co., Ltd (Beijing, China).
2.2. Propagation of starter and probiotic organisms
Propagation of cheese starter organisms was performed according to
Liu et al. (2015). Lactobacillus rhamnosus 6134 was cultivated at least
two times in 12% (w/v) sterile reconstituted skim milk (RSM) at 37 °C
for 24 h under anaerobic conditions prior to inoculation (2% v/v) into a
bulk culture of the same medium.
2.3. Process of manufacture of Cheddar cheese
Two batches of cheddar cheese were manufactured according to the
method described in a previous experiment (Liu et al., 2015). Two
batches of Cheddar cheese were manufactured and all experiments in
each batch were done in triplicate. B-1: cheese with 1.5% (v/v) starter
culture; B-2: cheese with 1.5% (v/v) starter culture and 1.2% (v/v)
Lactobacillus rhamnosus. Each batch of cheese was ripened at 4 °C for
240 days.
2.4. Microbiological analysis
Viable counts of the organism were performed according to Pino
et al. (2017) with some modifications. 25 g of Cheddar cheese was
diluted in 225 mL of peptone water (1 g L−1) and macerated in a
homogenizer for 1 min at high speed. Then, 0.1 mL aliquots of each
100
LWT - Food Science and Technology 95 (2018) 99–106
dilution was either poured into a plate containing MRS agar mixed with
5% cysteine and incubated at 37 °C for 48 h for Lactobacillus rhamnosus
or into a plate containing LM17 agar with 0.17 g L−1 of cycloheximide
and incubated at 32 °C for Lactococci or into a plate containing. kana-
mycin Aesculin Azide Agar (Oxoid) and incubated at 32 °C for 48 h for
Enterococci. The deduced viable counts were expressed as log CFU/g.
2.5. Preparation of water-soluble extracts (WSE) of Cheddar cheese
Water-solution extracts were prepared according to Kuchroo & Fox
(1982) with a minor modification. 10 g of cheese and 100 mL of phy-
siological saline were homogenized at 1000 rpm for 5 min. The homo-
genate was centrifuged at 3000 × g for 30 min, after which it was fil-
tered through a 0.22 μm pore size filter to obtain the water-soluble
extracts (WSE). The WSE obtained were then lyophilized in a lyo-
philizer (Lab Tech Lyophilizer, Offenburg, Germany) at −20 °C and
−100 kPa for 72 h. The freeze-dried WSE were finally stored at −20 °C
for further analysis of antioxidant activity during ripening.
2.6. Determination of proteolysis of Cheddar cheese during ripening period
2.6.1. Estimation of water soluble nitrogen (WSN)
Estimation of water-soluble nitrogen in the extract was performed in
duplicate. 10 mL WSE was quantitatively estimated in a digestion bottle
by the Kjeldahl determination method. The results were expressed as
percentages of total nitrogen in cheese: pH4.6-SN = pH4.6 soluble ni-
trogen content/Total nitrogen content.
2.6.2. Estimation of tricholoracetic acid soluble nitrogen (TCA-SN)
To 16 mL of WSE, 4 mL of 12%TCA solution (w/v) was added, and
was left to stand for 1 h after mixing, The mixture was then centrifuged
(4000 × g, 20 min), and 10 mL of the middle of the supernatant was
aspirated out, which was determined quantitatively in a digestion bottle
by the Kjeldahl determination method. The results were expressed as a
percentage of the total nitrogen in cheese as: 12% TCA-SN = 12% TCA
soluble nitrogen content/Total nitrogen content.
2.6.3. Estimation of phospotungstic acid soluble nitrogen (PTA-SN)
From the pH4.6-SN filtrate, 5 mL, was mixed with 5 mL 5% PTA
solution (w/v) and was left to stand for 1 h, after which the mixture was
centrifuged (4000 × g, 20 min). A 10 mL aliquot from the middle of the
supernatant was taken in a digestion bottle and determined quantita-
tively by the Kjeldahl determination method. The results were ex-
pressed as percentages of total nitrogen in cheese as: 5% PTA-SN = 5%
PTA soluble nitrogen content/Total nitrogen content. This value was an
indicator of the extent of secondary proteolysis of cheese.
2.7. Simulated gastrointestinal digestion of cheese
Artificial gastric juice (AGJ) and artificial intestinal juice (AIJ) were
prepared according to Mozzi, Gerbino, Font de Valdez, & Torino
(2009). Cheddar cheeses were ground in a grinder and samples (10 g)
were placed in Erlenmeyer flasks containing 100 mL of AGJ (pH 2.0)
and incubated at 37 °C in a metabolic bath with reciprocal agitation of
120 rpm for 2 h. Samples of cheese were also treated similarly in AIJ for
4 h.
After simulated gastric and intestinal digestion, sample were pre-
pared according to the method of Chaves & Gigante (2016) with some
modifications. The digestive juices in cheese were centrifuged at
3000 × g at 4 °C for 30 min. The upper fat layer was discarded and the
supernatant was filtered through Whatman no. 2 paper to obtain di-
gestion extract as filtrate. The. digestion extracts were lyophilized in
lyophilizer (Lab Tech Lyophilizer, Offenburg, Germany) at −20 °C and
−100 kPa for 72 h, and then stored at −20 °C for further analysis of
antioxidant activity in simulated gastrointestinal digestion.
L. Liu et al. LWT - Food Science and Technology 95 (2018) 99–106

2.8. Determination of antioxidant activity of Cheddar cheese during the Table 1

ripening period in simulated gastrointestinal digestion Viable counts (log CFU/g) of micro-organisms during ripening of Cheddar
cheese at 4 °C for 240 d.
2.8.1. DPPH radicals-scavenging activity Ripening period(d) Viable counts(log CFU/g)
The method of Timón, Parra, Otte, Broncano, & Petrón (2014) was
used to assess the DPPH radicals-scavenging activity. Freeze-dried WSE Lactobacilli Lactococci Enterococci
and digestion extracts were appropriately diluted in distilled water.
B-1 0 5.17 ± 0.04aA 9.40 ± 0.15aA 2.42 ± 0.08aA
0.1 mL of the sample solution (50 mg/mL) was mixed with 3.9 mL 30 5.41 ± 0.05bA 9.03 ± 0.02bA 2.58 ± 0.05aA
freshly prepared 60 μM methanolic solution of DPPH,. After 45 min, the 60 5.66 ± 0.02bA 8.53 ± 0.04bA 3.03 ± 0.02abA
absorbance of the resulting solutions was measured at 517 nm. Radical- 90 5.82 ± 0.05bA 7.25 ± 0.02cA 3.42 ± 0.03bA
120 5.53 ± 0.03bA 8.07 ± 0.14dA 4.22 ± 0.02bA
scavenging capacity was calculated as described in the equation below
180 5.46 ± 0.02bA 7.32 ± 0.22cA 5.37 ± 0.04cA
and the relative half inhibition concentration (IC50) was calculated 240 5.22 ± 0.04aA 7.11 ± 0.08cA 4.84 ± 0.05aA
during the ripening. IC50 is defined as the concentration of inhibitor
required to inhibit 50% of DPPH radical scavenging. To create a gra- B-2 0 7.92 ± 0.13aB 9.53 ± 0.11aA 4.17 ± 0.03aB
phical correlation between DPPH radicals-scavenging activity and 30 8.25 ± 0.08abB 9.30 ± 0.05abB 3.82 ± 0.23aB
60 8.36 ± 0.08bB 9.03 ± 0.08bB 4.02 ± 0.07aB
protein concentration, each sample was adjusted to at least five con-
90 8.42 ± 0.04bB 8.54 ± 0.12cB 3.94 ± 0.04aB
centration levels. The protein content of the samples was determined 120 8.73 ± 0.11bB 8.76 ± 0.14cB 3.42 ± 0.04bB
using the method of FolineLowry (Lowry, Resebrough, Farr, & Randall, 180 8.56 ± 0.04bB 7.63 ± 0.23dA 2.88 ± 0.02cdB
1951). The IC50 of the samples was then determined from the a linear 240 8.44 ± 0.05bB 7.50 ± 0.05dB 2.42 ± 0.13dB
regression plot as the concentration of protein in the sample required to
Results are expressed as mean ± standard error of means; n = 3 sets of data
inhibit 50% of DPPH radical scavenging.
analysed in duplicate. Means in columns with different small letters for same
Ablank − Asample strain and cheese during different ripening period are significantly different
Scavenging capacity (%) = × 100 (P < 0.05); Means in columns with different capital letters for same ripening
Ablank
period and strain between different cheeses are significantly different
where A blank is the distilled water was used instead of the WSE sample (P < 0.05).
and A sample is the absorbance of the sample after a specific incubation B-1: Cheddar cheese produced with only starter culture; B-2: Cheddar cheese
time. produced with starter culture and Lactobacillus rhamnosus.

2.8.2. Reducing power
The reducing power of the sample was investigated with the method
of Pepe et al. (2016). 2.5 mL of the sample solution (15 mg/mL) was
mixed with 2.5 mL of 0.2 M PBS (pH 6.6) and 2.5 mL of potassium
ferricyanide (10 mg/mL) and incubated at 50 °C for 20 min, followed by
cooling to room temperature. 2.5 mL of 10% trichloroacetic acid was
then added, and the mixture was centrifuged at 3000 × g for 10 min.
The supernatant was recovered and mixed with 0.5 mL of ferric chloride
solution (1 mg/mL). Absorbance of the supernatant was measured at
700 nm and the relative half inhibition concentration (IC50) was cal-
culated during ripening.
2.9. Changes in polypeptide content after simulated gastrointestinal
digestion
texture (1–10 points) and overall acceptance (1–10 points). Each batch
was evaluated in triplicate.
2.12. Statistical analysis
All experiments were done in triplicate and data were expressed as
mean ± standard error. One-way analysis of variance was used to
assay significant differences between the means (P < 0.05). The ana-
lyses were carried out using Statistix8 software. IC50 values were ana-
lysed by SPSS Statistix17.0.
3. Results and discussion
3.1. Microbiological analysis of cheese during the ripening period

The concentration of polypeptide contents was determined by biuret
method as described by Simonian & Smith (2006). 5 mL sample solution
was mixed with 5 mL 10% of trichloroacetic acid aqueous solution and
allowed to stand for 30 min. The mixture was then centrifuged at 4000
r/min for 20 min and the supernatant was aspirated.
2.10. Antioxidant properties in fractions separated according to molecular
weight by ultrafiltration
Fractions with molecular weight cutoffs of 3 and 10 kDa were se-
parated at the end of the ripening period of WSE and after digestion of
extracts using an ultrafiltration membrane system (Millipore, Billerica,
MA) in order to get three different fractions: Fraction 1: (> 10 kDa),
Fraction 2: (3–10 kDa) and Fraction 3: (< 3 kDa). The antioxidant ac-
tivity of the three fractions was also measured.
2.11. Sensory analysis
According to the method of Elsamani, Habbani, Babiker, & Ahmed
(2014). The sensory evaluation was performed by 15 professional pa-
nelists to assess the B-1 and B-2 at the end of the ripening by flavor and
taste (1–10 points), colour and appearance (1–10 points), body and
Results of microbiological analysis of cheese samples during ri-
pening were expressed as averages of three replicates with standard
deviation (Table 1). In the sample B-1, the Lactobacilli concentration
remained about 5 log CFU/g, which was quite constant during the ri-
peing period and was found to gradually decrease after 90 days of ri-
pening. There was a significant decrease in Lactococci concentration
(P < 0.05) up to 240 days of ripening, reaching a final value of 7.11 log
CFU/g at the end of ripening. On the contrary, the number of En-
terococci showed a trend towards increasing during ripening, achieving
a final value of 4.84 log CFU/g. In the sample B-2, Lactobacilli were
characterized by high cell density at the beginning of the ripening,
which significantly increased throughout the entire ripening period,
achieving a final value of 8.44 log CFU/g. On the other hand, Lacto-
cocci, and Enterococci exhibited a significant decrease during ripening.
Upon evaluating of the differences between control cheese (B-1) and
probiotic cheese (B-2), there was on significant effect on the level of
Lactococci upon addition of Lactobacillus rhamnosus, and the decreasing
trend may therefore be due to autolysis or unfavourable conditions in
the cheese (Ong, Henriksson & Shah, 2006). Moreover, addition of
Lactobacillus rhamnosus significantly influenced the Lactobacilli and
Enterococci count, with Lactobacilli showing higher values from the
beginning of the ripening and Enterococci exhibiting a significant

101
L. Liu et al.
Fig. 1. (A) Concentration of water-soluble nitrogen (WSN), (B) Trichloroacetic
acid-soluble nitrogen (TCA-SN) and (C) Phosphotungstic acid-soluble nitrogen
(PTA-SN) during the ripening of Cheddar cheese at 4 °C for 240 d; Results are
expressed as mean ± standard error of means; n = 3 sets of data analysed in
duplicate. Means among different ripening time with different small letters are
significantly different (P < 0.05); Means among different cheeses with capital
letters are significantly different (P < 0.05). B-1: Cheddar cheese produced with
only starter culture; B-2: Cheddar cheese produced with starter culture and
Lactobacillus rhamnosus.
decrease during ripening time. This may either be due to the compe-
tition between Lactobacillus rhamnosus and Enterococcus or due to poor
selectivity on SBM medium. The results of the microbiological analysis
thus reflected that Lactobacillus dominated all stages of ripening of
102
LWT - Food Science and Technology 95 (2018) 99–106
Cheddar cheese. This may due to it being enriched in proteases that are
required to degrade the milk protein and produce amino acids and
peptides for their own growth (Lisa et al., 2015).
3.2. Proteolysis of Cheddar cheese during ripening
Water soluble nitrogen (WSN), tricholoracetic acid soluble nitrogen
(TCA-SN) and phospotungstic acid soluble nitrogen (PTA-SN) leves
were analysed in the control and probiotic cheeses during ripening
period at 4 °C, which is shown in Fig. 1.
As shown in Fig. 1A, the pH4.6-SN content increased progressively
throughout the ripening period. The content of pH4.6-SN in all cheeses
was relatively low at the beginning of ripening and no significant dif-
ferences (P > 0.05) were observed between control and probiotic
cheeses up to the 90th day of ripening. After the 150th day of ripening,
the level of pH4.6-SN significantly increased (P < 0.05) with significant
differences (P < 0.05) between control and probiotic cheeses. This may
be due to WSN in cheese being produced mainly by primary proteolytic
action of chymosin and Lactobacillus rhamnosus, which has relatively
high hydrolysis ability and is retained in the curd (Ong et al., 2006).
Formation of TCA-SN was mainly due to the starter and non-starter
bacterial contents (Fox, 1993, pp. 343–367), which dictated the poly-
peptide content soluble in 12% TCA-SN. During the entire ripening
period, the level of TCA-SN in all cheeses increased progressively
(Fig. 1B) with no significant differences (P > 0.05) in the amount of
TCA-SN among all cheeses up to the 120th day of ripening. Higher levels
of TCA-SN were observed upon addition of probiotics after the 150th
day of ripening, which indicated that more products from primary
proteolysis became available as substrates for subsequent proteolysis by
Lactobacillus rhamnosus peptidases, thus increasing the levels of TCA-SN
of probiotic cheeses when compared with control cheese (Ong et al.,
2006).
5% PTA-SN can be used as an index of free amino acids in cheeses,
as shown in Fig. 1C. Only oligopeptides (< 600 Da) and free amino acid
are soluble in 5% PTA (Fox, 1993, pp. 343–367). The 5% PTA-SN
contents were similar in all cheeses until the 90th day of ripening. The
level of free amino acids in the sample B-2 was significantly higher
(P < 0.05) than in B-1 after the 120th day of ripening. This was prob-
ably due to increased peptidase activity of Lactobacillus rhamnosus that
resulted in enhanced activity of intracellular peptidases to hydrolyse
milk protein and produce more small molecular peptides and free
amino acids.
3.3. Antioxidant activity of Cheddar cheese during ripening period
The scavenging activity of the DPPH radical and the reducing power
were analysed in cheeses during the ripening period at 4 °C to estimate
the antioxidant activity of Cheddar cheese, expressed also as IC50 as
shown in Fig. 2.
Reduction in the concentration of DPPH was monitored by A de-
crease in the absorbance at a characteristic wavelength upon en-
countered with proton radical scavengers (Gabriela, Francisco, Adriana,
& Mónica, 2017). Addition of Lactobacillus rhamnosus had a significant
effect on the scavenging activity of DPPH radical of cheeses during ri-
pening (P > 0.05), which was higher than that of control cheese. The
IC50 values of cheeses decreased with an increase in ripening time and
cheeses with added Lactobacillus rhamnosus had significantly lower IC50
values (P < 0.05) than control cheese (Fig. 2A). DPPH radical scaven-
ging ability is correlated with the degree of proteolysis (Ren, Pan, Zeng,
Cao & Sun, 2013). Upon extension of ripening time of cheese, proteo-
lysis could result in the formation of various antioxidant peptides,
which increase the antioxidant activity of Cheddar cheese
(Abadíagarcía et al., 2013).
The reducing power is directly correlated with peptide cleavages,
which determines the ability of samples to donate electrons or protons
(Cumby, Zhong, Naczk, & Shahidi, 2008). The addition of Lactobacillus
L. Liu et al. LWT - Food Science and Technology 95 (2018) 99–106

Fig. 2. Antioxidant activity of control and probiotic

Cheddar cheese during the ripening at 4 °C for 240 d,
evaluated for (A) Scavenging activity of DPPH radical and
(B) Reducing power. Antioxidant activity presented as
percentage antioxidant activity ( ) and IC50 ( )
(concentration of WSE needed to inhibit 50% of
Antioxidant activity). Results are expressed as mean ±
standard error of means; n = 3 sets of data analysed in
duplicate. * indicate significant difference (P < 0.05)
from B-1; # means P < 0.05 respect to the previous time
in the same cheese. B-1: Cheddar cheese produced with
only starter culture; B-2: Cheddar cheese produced with
starter culture and Lactobacillus rhamnosus.

rhamnosus had no significant effect on the reducing power of cheeses at

the early 90th day of ripening (P > 0.05), and was subsequently sig-
nificantly higher than that of B-1 (Fig. 2B). The highest absorbance of B-
1 and B-2 were 0.445 and 0.592 at the end of the ripening time, re-
spectively. The IC50 values of cheeses ranged from 5.68 to 11.92 mg/mL
and from 5.13 to 11.54 mg/mL, while the reducing powers were ranged
from 0.122 to 0.441 and from 0.125 to 0.593 for B-1 and B-2, re-
spective. Lee, Yang, & Mau (2008) demonstrated that reduction ability
is closely related to the hydrogen-donating ability of the reducing
moieties. Therefore, proteolysis may have given rise to reductants that
reacted with free radicals to stabilize and terminate radical chain re-
actions, thereby resulting in high reducing power.
Fig. 3. Viable counts (log CFU/g) of micro-organisms in cheese after simulated
gastrointestinal digestion. Means among different samples with capital letters
3.4. Microbiological analysis of cheese after simulated gastrointestinal are significantly different (P < 0.05); Means among digestion stage with dif-
digestion ferent small letters are significantly different (P < 0.05). B-1: Cheddar cheese
produced with only starter culture; B-2: Cheddar cheese produced with starter
The viable counts of microbes in cheese after simulated gastro- culture and Lactobacillus rhamnosus.
intestinal digestion are shown in Fig. 3. The number of Lactobacilli,

103
L. Liu et al. LWT - Food Science and Technology 95 (2018) 99–106

Table 2 Table 3
Antioxidant activity of control and probiotic Cheddar cheese during simulated The antioxidant activity of polypeptides with different molecular weights.
gastrointestinal digestion.
Sample Fraction Antioxidant activity
Period Sample Antioxidant activity
DPPH radical Reducing power
DPPH radical scavenging Reducing power scavenging activity (OD700nm)
activity (%) (OD700nm) (%)

Before B-1 55.39 ± 1.86aA 0.445 ± 0.046aA Ripened B-1 Fraction 1 62.7 ± 1.72aA 0.387 ± 0.014aA
digestion B-2 74.69 ± 1.98aB 0.592 ± 0.023aB (< 3 kDa)
Gastric B-1 57.89 ± 2.34bA 0.486 ± 0.086aA Fraction 2 43.2 ± 1.03bA 0.523 ± 0.025bA
B-2 77.21 ± 1.66bB 0.625 ± 0.008aB (3–10 kDa)
Intestinal B-1 58.74 ± 2.57bA 0.503 ± 0.035bA Fraction 3 33.4 ± 0.56cA 0.322 ± 0.043cA
B-2 80.26 ± 1.97cB 0.696 ± 0.019bB (> 10 kDa)
B-2 Fraction 1 82.3 ± 0.93aA 0.592 ± 0.022aA
Results are expressed as mean ± standard error of means; n = 3 sets of data (< 3 kDa)
analysed in duplicate. Means among different samples with capital letters are Fraction 2 58.4 ± 1.22bA 0.712 ± 0.037bA
(3–10 kDa)
significantly different (P < 0.05); Means among digestion stage with different
Fraction 3 45.2 ± 0.64cA 0.389 ± 0.042cA
small letters are significantly different (P < 0.05).
(> 10 kDa)
B-1: Cheddar cheese produced with only starter culture; B-2: Cheddar cheese
produced with starter culture and Lactobacillus rhamnosus. Digestion B-1 Fraction 1 79.8 ± 0.62aB 0.401 ± 0.081aB
(< 3 kDa)
Fraction 2 62.3 ± 1.18bB 0.663 ± 0.022bB
(3–10 kDa)
Fraction 3 49.6 ± 0.52cA 0.341 ± 0.087cA
(> 10 kDa)
B-2 Fraction 1 90.1 ± 1.52aB 0.665 ± 0.094aB
(< 3 kDa)
Fraction 2 68.8 ± 1.21bB 0.793 ± 0.122bB
(3–10 kDa)
Fraction 3 50.8 ± 1.42cA 0.605 ± 0.086cB
(> 10 kDa)

Results are expressed as mean ± standard error of means; n = 3 sets of data

Fig. 4. Changes of polypeptide content of cheese after simulated gastro-
intestinal digestion. Means among different samples with capital letters are
significantly different (P < 0.05); Means among digestion stage with different
small letters are significantly different (P < 0.05). B-1: Cheddar cheese pro-
duced with only starter culture; B-2: Cheddar cheese produced with starter
culture and Lactobacillus rhamnosus.
Lactococci, Streptococci and Enterococci were significantly reduced after
digestion (P < 0.05). After simulated gastrointestinal digestion, the
number of Lactobacilli in B-1 and B-2 were 3.62 log CFU/g and 6.19 log
CFU/g, which were subsequently reduced by 31.03% and 26.66%, re-
spectively. Apart from Enterococci, all other bacteria had good survival
after the digestion, possibly owing to protection of the probiotics by the
cheese matrix (de Oliveira et al., 2014).
3.5. Antioxidant activity of Cheddar cheese after simulated gastrointestinal
digestion
As shown in Table 2, the antioxidant activity of cheese was sig-
nificantly improved after simulated gastrointestinal digestion
(P < 0.05). The DPPH radical scavenging activity in B-1 and increased
from 55.39% to 58.74% and in B-2 from 74.69% to 80.26%, with the
DPPH activity of probiotic cheese being significantly higher than that of
the control cheese (P < 0.05). The reducing power of B-1 and B-2 were
0.503 and 0.696, indicating an increase of 13.03% and 17.57%, re-
spectively. The improvement in reducing power maybe related to the
production of substances that supply hydrogen atoms and reduce Fe3+
into Fe2+, thus interrupting free radical chain reaction (Corrêa et al.,
2014).
In our study, the antioxidant capacity increased significantly after
simulated gastrointestinal digestion, but the number of probiotics in
analysed in duplicate. Means among different fraction in same simple with
different small letters are significantly different (P < 0.05); Means among dif-
ferent digestion periods in same simple with capital letters are significantly
different (P < 0.05).
B-1: Cheddar cheese produced with only starter culture; B-2: Cheddar cheese
produced with starter culture and Lactobacillus rhamnosus.
cheese was significantly reduced (P < 0.05) shown in Fig. 3. It could be
concluded that the viable counts and functional properties of probiotic
bacteria itself have little effect on the antioxidant activity of cheese
under the simulated gastrointestinal environment. The increase in an-
tioxidant activity of cheeses after simulated gastrointestinal digestion
may be due to degradation of casein in cheese by pepsin, trypsin, and
protease produced by the probiotics, thus conferring antioxidant pep-
tides (Abadíagarcía et al., 2013).
3.6. Polypeptide before and after digestion
As shown in Fig. 4, the polypeptide contents in the B-1 and B-2
cheeses were 1.56 mg/mL, and 1.87 mg/mL respectively at the end of
ripening. After digestion, the polypeptide contents increased sig-
nificantly (P < 0.05), reaching 1.83 mg/mL and 2.58 mg/mL, after an
increase of 17.31% and 37.97% respectively, indicating that pepsin and
trypsin could degrade cheese protein and produce more bioactive
peptides in the digested cheese. Indeed, a previous study had shown
that simulated gastrointestinal digestion produced more antioxidant
peptides and antimicrobial peptides (Sánchez-Rivera, Diezhandino,
Gómez-Ruiz, Fresno, Miralles & Recio, 2014). In this study, the poly-
peptide content in B-2, which contained Lactobacillus rhamnosus was
significantly higher than in B-1 (P < 0.05). Lactobacillus has been
shown to have the ability to produce two peptidase, carboxypeptidase,
three peptidase, aminopeptidase and endopeptidase (Peterson &
Marshall, 1990). Therefore, in this study, the proteases from Lactoba-
cillus rhamnosus may also play a role in proteolysis (Oh, Joung, Lee,
Kim, & Kim, 2016). In addition, studies have shown that the antioxidant
activity of casein peptides is not only related to the concentration of
polypeptides, but also related to the size of polypeptide molecules

104
L. Liu et al.
Table 4
Sensory analysis for control and probiotic Cheddar cheeses.
Samples Sensory parameters
Flavor and Colour and Body and Overall
taste appearance texture acceptance
B-1 8.9 ± 0.8b 8.8 ± 0.6a 6.7 ± 0.4a 8.6 ± 0.9a
B-2 8.1 ± 0.3a 8.5 ± 0.7a 7.1 ± 0.8 a 8.2 ± 0.7a
Results are expressed as mean ± standard error of means; n = 3 sets of data
analysed in duplicate. Means in columns with different small letters are sig-
nificantly different (P < 0.05).
B-1: Cheddar cheese produced with only starter culture; B-2: Cheddar cheese
produced with starter culture and Lactobacillus rhamnosus.
(Timón et al., 2014). Therefore, it is necessary to study the antioxidant
properties of peptides with different molecular weights.
3.7. Antioxidant activity of polypeptides with different molecular weights of
Cheddar cheese before and after digestion
At the end of the ripening period, the < 3 kDa fraction showed the
highest free radical scavenging capacity, with and the DPPH radical
scavenging activity of Fraction 1 of B-1 and B-2 being 62.7% and 82.3%
respectively (Table 3). These observations were similar to the results of
K.H. Sabeena Farvin, who found that the lower the molecular weight,
the stronger the reducing ability of the fragments, and the stronger the
antioxidant activity (Sabeena Farvin, Carolinep, Ninaskall & Charlotte,
2010). Casein hydrolysates showed that antioxidant peptides (< 3 kDa)
have a stronger hydroxyl radical scavenging activity (Timón et al.,
2014). The reducing power of the < 3 kDa and 3–10 kDa fractions were
higher than that of > 10 kDa fraction, and the fraction containing
peptides with a molecular mass of 3–10 kDa demonstrated the strongest
reducing power (B-1: 0.523; B-2: 0.712). The antioxidant activity of
cheeses could be further enhanced by incorporation of probiotic bac-
teria. These results indicated that most of the biologically active pep-
tides accumulated at the early stage of ripening, and some peptides are
hydrolysed into shorter peptides as the proteolysis proceeds further.
The fraction containing peptides with a molecular mass between < 3
kDa and 3–10 kDa demonstrated a strong antioxidant activity.
There were significant differences in the antioxidant activity of
cheese before and after digestion (P < 0.05). The DPPH radical
scavenging activities of B-2 were significantly increased (P < 0.05),
going up from 82.3% to 90.1% for Fraction 1 and from 58.4% to 68.8%
for Fraction 2. The trend of change in the reducing power of cheeses
before and after digestion was the same as the trend of DPPH radical
scavenging activity. The antioxidant activity of different B-2 fractions
was significant higher than those of B-1, to which Lactobacillus rham-
nosus was not added (P < 0.05). These observations suggest that the
increased antioxidant capacity of cheese after simulated gastro-
intestinal digestion is likely to be related to small molecular weight
polypeptide from casein with high antioxidant activity, which are de-
graded by pepsin, trypsin, and protease produced by probiotics.
3.8. Sensory analysis
Sensory scores of cheeses after 240 days of storage are given in
Table 4. No differences were found between the B-1 and B-2, regarding
appearance and overall acceptance. The texture of B-2 with probiotic
was significantly higher than B-1 (P < 0.05), but the flavor and taste of
B-2 was slightly lower than B-1. Probiotic bacteria could promote
proteolysis and broke the initial network structure of casein in cheese to
make it a little bitter and softer than control cheese (Lannes, 2004). For
the panel group, the presence of probiotic was not found to be a limiting
factor to the total acceptability of cheese. The higher content of acid in
105
LWT - Food Science and Technology 95 (2018) 99–106
probiotic cheese make it more acidic in taste and more fragile in texture
(Buriti, Rocha & Saad, 2005).
4. Conclusion
In this study we report the use of Lactobacillus rhamnosus as func-
tional starter culture to improve the antioxidant activity of Cheddar
cheese during ripening and simulated gastrointestinal digestion. We
found that Lactobacillus dominated all ripening stages of cheese and
maintained high viability. The antioxidant activity of cheeses was the
highest at the end of ripening. After simulated gastrointestinal diges-
tion, the antioxidant activity and the polypeptide content of cheeses
increased significantly (P < 0.05) with more shorter peptides being
produced. Our results indicate that Lactobacillus rhamnosus could pro-
mote the proteolysis of cheeses to form small molecular peptides and
free amino acids. After simulated gastrointestinal digestion, the anti-
oxidant activity of cheeses is mainly due to the interaction of probiotics
protease, pepsin and trypsin, which converts casein to shorter peptides
with antioxidant activity. The total acceptability of cheese was not af-
fected by the addition of probiotic bacteria.
Acknowledgement
This research was supported by Applied technology research and
development project of Harbin in 2017 (2017RAQXJ087).
References
Abadíagarcía, L., Cardador, A., St, M. D. C., Arvízu, S. M., Castañotostado, E.,
Regaladogonzález, C., et al. (2013). Influence of probiotic strains added to cottage
cheese on generation of potentially antioxidant peptides, anti-listerial activity, and
survival of probiotic microorganisms in simulated gastrointestinal conditions.
International Dairy Journal, 33, 191–197.
Aguilar-Toalá, J. E., Santiago-López, L., Peres, C. M., Peres, C., Garcia, H. S., Vallejo-
Cordoba, B., et al. (2017). Assessment of multifunctional activity of bioactive pep-
tides derived from fermented milk by specific lactobacillus plantarum strains. Journal
of Dairy Science, 100, 65–75.
Algaron, F., Miranda, G., Bars, D. L., & Monnet, V. (2003). Milk fermentation by lacto-
coccus lactis with modified proteolytic systems to accumulate potentially bio-active
peptides. Dairy Science & Technology, 84, 1217–1224.
Buriti, F. C. A., Rocha, J. S. D., & Saad, S. M. I. (2005). Incorporation of Lactobacillus
acidophilus, in Minas fresh cheese and its implications for textural and sensorial
properties during storage. International Dairy Journal, 15(12), 1279–1288.
Chang, O. K., K-H, S., S-G, J., M-H, et al. (2013). Casein hydrolysis by bifidobacterium
longum kacc 91563 and antioxidant activities of peptides derived therefrom. Journal
of Dairy Science, 96, 5544–5555.
Chaves, K. S., & Gigante, M. L. (2016). Prato cheese as suitable Carrier for Lactobacillus
acidophilus La5 and Bifidobacterium Bb12. International Dairy Journal, 52, 10–18.
Chen, M., Jing, A. O., & Bo, L. I. (2012). Effect of molecular weight on the antioxidant
activity of casein peptide. Science & Technology of Food Industry, 33, 95–99.
Corrêa, A. P. F., Daroit, D. J., Fontoura, R., Meira, S. M. M., Segalin, J., & Brandelli, A.
(2014). Hydrolysates of sheep cheese whey as a source of bioactive peptides with
antioxidant and angiotensin-converting enzyme inhibitory activities. Peptides, 61,
48–55.
Cumby, N., Zhong, Y., Naczk, M., & Shahidi, F. (2008). Antioxidant activity and water-
holding capacity of canola protein hydrolysates. Food Chemistry, 109, 144–148.
Elfahri, K. R., Donkor, O. N., & Vasiljevic, T. (2014). Potential of novel lactobacillus
helveticus, strains and their cell wall bound proteases to release physiologically ac-
tive peptides from milk proteins. International Dairy Journal, 38, 37–46.
Elsamani, M. O., Habbani, S. S., Babiker, E. E., & Ahmed, I. A. M. (2014). Biochemical,
microbial and sensory evaluation of white soft cheese made from cow and lupin milk.
LWT-Food Science Technology, 59, 553–559.
Elvira, M. H., Lucila, S., & Pasquale, F. (2010). Bioactive peptides derived from casein and
whey proteins. Biotechnology of Lactic Acid Bacteria: Novel Applications, 233–249.
Fox, P. F. (1993). Cheese: Chemistry, physics and microbiology (2nd ed.). London, UK:
Chapman & Hall.
Fuglsang, A., Rattray, F. P., Nilsson, D., & Nyborg, N. C. (2003). Lactic acid bacteria:
Inhibition of angiotensin converting enzyme in vitro and in vivo. Antonie van
Leeuwenhoek, 83, 27–34.
Gabriela, F. R., Francisco, K., Adriana, M. R., & Mónica, G. P. (2017). Potential anti-
oxidant peptides produced from whey hydrolysis with an immobilized aspartic pro-
tease from Salpichroa origanifolia fruits. Food Chemistry, 237, 350–355.
Gobbetti, M., Minervini, F., & Rizzello, C. G. (2004). Angiotensini-converting-enzyme-
inhibitory and antimicrobial bioactive peptides. International Dairy Journal, 57,
173–188.
Gupta, A., Mann, B., Kumar, R., & Sangwan, R. B. (2009). Antioxidant activity of cheddar
L. Liu et al.
cheeses at different stages of ripening. International Journal of Dairy Technology, 62,
339–347.
Hayes, M., Ross, R. P., Fitzgerald, G. F., & Stanton, C. (2007). Putting microbes to work:
Dairy fermentation, cell factories and bioactive peptides. Part i: Overview.
Biotechnology Journal, 2, 435–449.
Kuchroo, C. N., & Fox, P. F. (1982). Soluble nitrogen in cheddar cheese: Comparison of
extraction procedures. Milchwissenschaft Milk Science International, 37, 331–335.
Lannes, S. C. D. S. (2004). Cheese rheology and texture. Revista Brasileira de Ciencias
Farmaceuticas, 40(2) 269–269.
Law, J., & Haandrikman, A. (1997). Proteolytic enzymes of lactic acid bacteria.
International Dairy Journal, 7, 1–11.
Lee, Y. L., Yang, J. H., & Mau, J. L. (2008). Antioxidant properties of water extracts from
monascus, fermented soybeans. Food Chemistry, 106, 1128–1137.
Lisa, S., Giuseppina Sefora, R., & Davide, T. (2015). Impact of non-starter lactobacilli on
release of peptides with angiotensin-converting enzyme inhibitory and antioxidant
activities during bovine milk fermentation. Food Microbiology, 51, 108–116.
Liu, M., Bayjanov, J. R., Renckens, B., Nauta, A., & Siezen, R. J. (2010). The proteolytic
system of lactic acid bacteria revisited: A genomic comparison. BMC Genomics, 11,
36–42.
Liu, L., Chen, P., Zhao, W., Li, X., Wang, H., & Qu, X. (2017). Effect of microencapsulation
with the maillard reaction products of whey proteins and isomaltooligosaccharide on
the survival rate of lactobacillus rhamnosus, in white brined cheese. Food Control,
2017(79), 44–49.
Liu, L., Li, X., Bi, W., Zhang, L., Ma, L., Ren, H., et al. (2015). Isomaltooligosaccharide
increases the lactobacillus rhamnosus viable count in cheddar cheese. International
Dairy Journal, 68, 389–398.
Liu, L., Li, X., Zhu, Y., Bora, A. F. M., Zhao, Y., Du, L., et al. (2016). Effect of micro-
encapsulation with Maillard reaction products of whey proteins and iso-
maltooligosaccharide on the survival of Lactobacillus rhamnosus. Lebensmittel-
Wissenschaft und -Technologie- Food Science and Technology, 73, 37–43.
López-Expósito, I., Amigo, L., & Recio, I. (2012). A mini-review on health and nutritional
aspects of cheese with a focus on bioactive peptides. Dairy Science & Technology, 92,
419–438.
Lowry, O. H., Resebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement
with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.
Lu, Y., Govindasamylucey, S., & Lucey, J. A. (2016). Angiotensin-i-converting enzyme-
inhibitory peptides in commercial Wisconsin cheddar cheeses of different ages.
Journal of Dairy Science, 99, 41–52.
Mettes, N., Torben, M., Bénédicte, F., Kimi, S., & Jeanette, O. (2009). Peptide profiles and
angiotensin-i-converting enzyme inhibitory activity of fermented milk products:
Effect of bacterial strain, fermentation ph, and storage time. International Dairy
Journal, 19, 155–165.
Meyer, J., Bütikofer, U., Walther, B., Wechsler, D., & Sieber, R. (2009). Hot topic: Changes
in angiotensin-converting enzyme inhibition and concentrations of the tripeptides
val-pro-pro and ile-pro-pro during ripening of different swiss cheese varieties. Journal
of Dairy Science, 92, 826–836.
Mozzi, F., Gerbino, E., Font de Valdez, G., & Torino, M. I. (2009). Functionality of exo-
polysaccharides produced by lactic acid bacteria in an in vitro gastric system. Journal
of Applied Microbiology, 107(1), 56–64.
Oh, N. S., Joung, J. Y., Lee, J. Y., Kim, S. H., & Kim, Y. (2016). Characterization of the
microbial diversity and chemical composition of Gouda cheese made by potential
probiotic strains as an adjunct starter culture. Journal of Agricultural and Food
Chemistry, 64(39), 7357–7366.
de Oliveira, M. E. G., Garcia, E. F., de Oliveira, C. E. V., Gomes, A. M. P., Pintado, M. M.
E., Madureira, A. R. M. F., et al. (2014). Addition of probiotic bacteria in a semi-hard
goat cheese (coalho): Survival to simulated gastrointestinal conditions and inhibitory
106
LWT - Food Science and Technology 95 (2018) 99–106
effect against pathogenic bacteria. Food Research International, 64, 241–247.
Ong, L., Henriksson, A., & Shah, N. P. (2006). Development of probiotic cheddar cheese
containing lactobacillus acidophilus, lb. casei, lb. paracasei and bifidobacterium spp.
and the influence of these bacteria on proteolytic patterns and production of organic
acid. International Dairy Journal, 16, 446–456.
Ong, L., & Shah, N. P. (2008). Release and identification of angiotensin-converting en-
zyme-inhibitory peptides as influenced by ripening temperatures and probiotic ad-
juncts in cheddar cheeses. LWT-Food Science Technology, 41, 1555–1566.
Pepe, G., Sommella, E., Ventre, G., Scala, M. C., Adesso, S., Ostacolo, C., et al. (2016).
Antioxidant peptides released from gastrointestinal digestion of “stracchino” soft
cheese: Characterization, in vitro intestinal protection and bioavailability. Journal
Functional Foods, 26, 494–505.
Peterson, S. D., & Marshall, R. T. (1990). Nonstarter lactobacilli in cheddar cheese: A
review. Journal of Dairy Science, 73(6), 1395–1410.
Pino, A., Van, H. K., Pitino, I., Russo, N., Carpino, S., Caggia, C., et al. (2017). Survival of
potential probiotic lactobacilli used as adjunct cultures on pecorino siciliano cheese
ripening and passage through the gastrointestinal tract of healthy volunteers.
International Journal of Food Microbiology, 252, 42–51.
Reale, A., Ianniello, R. G., & Ciocia, F. (2016). Effect of respirative and catalase-positive
Lactobacillus casei adjuncts on the production and quality of Cheddar-type cheese.
International Dairy Journal, 63, 78–87.
Ren, X. F., Pan, D. D., Zeng, X. Q., Cao, J. X., & Sun, Y. Y. (2013). Effects of enzymatic
hydrolysis with cell wall proteinase (cep) on structural and functional properties of
casein. Modern Food Science & Technology, 29, 2643–2648 +2585.
Sabeena Farvin, K. H., Carolinep, B., Ninaskall, N., & Charlotte, J. (2010). Antioxidant
activity of yoghurt peptides: Part 1-in vitro assays and evaluation in ω-3 enriched
milk. Food Chemistry, 123, 1081–1089.
Sadat-Mekmene, L., Jardin, J., Corre, C., Mollé, D., Richoux, R., Delage, M. M., et al.
(2011). Simultaneous presence of prth and prth2 proteinases in lactobacillus helve-
ticus strains improves breakdown of the pure alphas1-casein. Applied and
Environmental Microbiology, 77, 179–186.
Sánchez-Rivera, L., Diezhandino, I., Gómez-Ruiz, J.Á., Fresno, J. M., Miralles, B., & Recio,
I. (2014). Peptidomic study of Spanish blue cheese (Valdeón) and changes after si-
mulated gastrointestinal digestion. Electrophoresis, 35(11), 1627–1636.
Settanni, L., & Moschetti, G. (2010). Non-starter lactic acid bacteria used to improve
cheese quality and provide health benefits. Food Microbiology, 27, 691–697.
Simonian, M. H., & Smith, J. A. (2006). Spectrophotometric and colorimetric determi-
nation of protein concentration. Current Protocols in Molecular Biology, 10–11.
Slattery, L., O'Callaghan, J., Fitzgerald, G. F., Beresford, T., & Ross, R. P. (2010). Invited
review: Lactobacillus helveticus - a thermophilic dairy starter related to gut bacteria.
Journal of Dairy Science, 93, 4435–4454.
Sorayya, A., Byongh, L., Varoujan, Y., & Kierann, K. (2010). Proteolysis development in
enzyme-modified cheddar cheese using natural and recombinant enzymes of lacto-
bacillus rhamnosus. Food Chemistry, 120, 174–178.
Timón, M. L., Parra, V., Otte, J., Broncano, J. M., & Petrón, M. J. (2014). Identification of
radical scavenging peptides (< 3 kDa) from Burgos-type cheese. LWT-Food Sci
Technol, 57(1), 359–365.
Wang, H. K., Dong, C., Chen, Y. F., Cui, L. M., & Zhang, H. P. (2010). A new probiotic
cheddar cheese with high ace-inhibitory activity and γ-aminobutyric acid content
produced with koumiss-derived lactobacillus casei zhang. Food Technology and
Biotechnology, 48, 62–70.
Zhang, S., Zhang, L., Jiao, Y., Li, H., Shigwedha, N., Zhang, Y., et al. (2015). Lactobacillus
delbrueckii subsp. Bulgaricus proteinase: Purification by ion-exchange and hydro-
phobic interaction chromatography. International Journal of Food Properties, 18,
1560–1567.