foods

Article
Evaluation of Physiological Activity of Long-Term Ripened
Gouda Water Extract
Woojin Ki 1 , Gereltuya Renchinkhand 2 , Hyoungchurl Bae 1 and Myoung Soo Nam 1, *

1 Department of Dairy Science, College of Agriculture & Life Sciences, Chungnam National University,
Daejeon 34134, Republic of Korea; rl8917@naver.com (W.K.); hcbae@cnu.ac.kr (H.B.)
2 Department of Biology, School of Arts and Sciences, National University of Mongolia,
Ulaanbaatar 14201, Mongolia; handgai@yahoo.com
* Correspondence: namsoo@cnu.ac.kr; Tel.: +82-42-821-5782

Abstract: This study investigated peptide changes and their bioactive functions through the long-
term ripening of Gouda. Young Gouda (YG), medium Gouda (MG), and extra-sharp Gouda (EG)
water extracts were prepared and functional peptides were recognized using liquid chromatography–
high-resolution mass spectrometry. Two peptides with ACE-inhibitory effects (IQP and LQP) were
identified in YG, while in MG and EG were identified eight (EL, IVP, VP, LPP, VIP, IPP, VPP, and
VVPP) and six (EL, YL, VP, IR, YPEL, and DKIHPF) functional peptides, respectively. MG (70.26%)
and EG (46.81%) showed stronger antioxidant activity than YG (25.99%) in ABTS (2,2′ -azino-bis-(3-
ethylbenzothiazoline-6-sulfonic) acid) inhibition, though the DPPH (2,2-diphenyl-1-picrylhydrazyl)
inhibition rate decreased with ripening. The antihypertensive effect increased in MG (79.76%) and
EG (94.50%) due to ACE-inhibitory peptides. Measurements of inflammatory mRNA expression
levels and immunoblotting were conducted to assess the anti-inflammatory properties. MG and EG
suppressed the transcription of IL-1β and IL-6 mRNA. Immunoblotting indicated that EG suppressed
IκBα phosphorylation to 57%. The enhancement of bioactive function in the water-soluble part of
long-term ripened Gouda cheese may have affected identified peptides as well as unknown peptides.
Further studies are expected to aid in discovering these novel bioactive peptides.

Keywords: ripened Gouda; bioactive peptides; antioxidant; ACE inhibition; anti-inflammatory activity

Citation: Ki, W.; Renchinkhand, G.; 1. Introduction

Bae, H.; Nam, M.S. Evaluation of
Bovine milk is a complete food, containing almost all the nutrients that humans need,
Physiological Activity of Long-Term
and it is highly digestible. This food contains approximately 3.4% protein, including biolog-
Ripened Gouda Water Extract. Foods
ically active caseins and functional proteins (lactoferrin, transferrin, immunoglobulin, etc.).
2024, 13, 3446. https://doi.org/
Milk protein is highly nutritious and is currently recognized as the origin of peptides with
10.3390/foods13213446
significant physiological activity [1].
Received: 24 September 2024 Bioactive peptides are protein fractions that play diverse roles in physiological func-
Revised: 25 October 2024 tions and are beneficial for physical and mental health [2]. Peptides are involved in the
Accepted: 26 October 2024 cellular responses within the body by signaling biological processes, transporting trace
Published: 29 October 2024
elements, and inhibiting neurotransmitters and enzymes [3]. The physiological function
of peptides is rooted in the types and arrangement of amino acids [4]. Additionally, the
size of the peptide sequence also has an effect, and the size of multifunctional peptide
Copyright: © 2024 by the authors.
sequences has been seen to range from 2 to 20 amino-acid arrangements [3]. These peptides
Licensee MDPI, Basel, Switzerland. exhibit antioxidant, antihypertensive, anti-thrombotic, and immunomodulatory effects,
This article is an open access article and they have received significant interest owing to their possible biological benefits [5].
distributed under the terms and Milk protein-derived peptides can be obtained from popular sources of animal protein like
conditions of the Creative Commons yogurt, cheese, and kefir. Moreover, bioactive peptides have been confirmed to positively
Attribution (CC BY) license (https:// impact metabolic diseases like hypertension, obesity, and hyperlipidemia [6].
creativecommons.org/licenses/by/ Cheese is a long-standing food that has been made in human society for millennia,
4.0/). with more than 1000 types of cheese currently available [7]. Cheese is a popular dairy

Foods 2024, 13, 3446. https://doi.org/10.3390/foods13213446 https://www.mdpi.com/journal/foods

Foods 2024, 13, 3446 2 of 16

food that, depending on the type, is appreciated for its unique taste, pleasant texture, and
nutrient-rich content. Cheese is rich in protein and features numerous peptides derived
from milk. The level of protein breakdown and the different processing techniques applied
during production and aging determine the composition of these elements [8]. Milk protein
degradation is affected by various elements, including pH, chymosin, enzymes produced
by microbes, salt, storage duration, temperature, and humidity [9].
Enzymes in rennet or produced by microbes can decompose the casein in milk, leading
to the formation of peptides with physiological functions [9]. In particular, during the
ripening process, numerous fractions emerge from the caseins (αS1-, αS2-, β-, κ-casein),
mainly via cheese starters and foreign microbial-derived proteolytic enzymes [10]. There-
fore, even two different samples of the same type of cheese are likely to have bioactive
peptides that vary in type, amount, and function, depending on the degree of ripeness.
Gouda cheese originated in the town of Gouda, located between Rotterdam and
Utrecht in southern Poland in the western Netherlands, and has been produced since
the 6th century. Gouda is a round-wheeled, semi-hard type of cheese that is coated with
yellow-orange wax and aged for short or long periods to extend its shelf life [11]. Gouda
cheese’s physiologically active properties change significantly depending on the ripening
stage. A study of Gouda cheese’s antioxidant and ACE (Angiotensin Converting Enzyme)-
inhibitory effects [12] showed that the peptides present in the cheese, as well as their
bioactivity, continuously change depending on the ripening stage.
Therefore, studying the diversity and physiological role of peptides in Gouda cheese
at different ripening periods will likely uncover changes in composition and function and
reconfirm their value as fermented foods. This study investigated the potential of long-
ripened Gouda cheese as a health food by evaluating its antioxidant, immunomodulatory,
and antihypertensive activity at various stages of ripening.

2. Materials and Methods

2.1. Gouda Samples and Manufacturing Gouda Water Extracts (WEs)
The Gouda used in the study was classified into EG (extra-aged Gouda), MG (medium
Gouda), and YG (young Gouda) groups, and samples of extra-sharp (3-years ripened),
medium (6-months ripened), and young (less than 1-month ripened) Gouda, produced and
ripened at the Chungnam National University Animal Resources Research Center, were
used. Each Gouda was evenly ground, and 180 mL of sterilized distilled water (DW) was
blended with 20 g of Gouda. The mixture was extracted at 40 ◦ C for 1 h using an ultrasonic
extractor (POWERSONIC 420, HWASHIN, Seoul, Republic of Korea). After cooling, WE
was divided in layers by centrifugation (Supra R17, Hanil, Gimpo, Republic of Korea) at
6000× g for 20 min at 4 ◦ C. Then, the cooled lipid layer was taken off and the supernatant
was collected and sieved through Whatman No. 2 (Whatman, Maidstone, UK). In addition,
because long-term ripened Gouda contains decomposition products of various sizes and
has a high content of amino acids and short peptides, filtration according to molecular
weight was not performed to fully evaluate the physiological activity of the extract. The
obtained WE was freeze-dried and stored at −20 ◦ C.
Samples for mass spectrometry were modified from the method used in cheese peptide
determination [13]. The Gouda WEs extracted as described above were freeze-dried and
dissolved in deionized water (100 ppm).

2.2. Determination of Water-Soluble Nitrogen in Gouda

As an indicator of cheese ripeness, the quantification of nitrogen compounds was
carried out to observe variations of the nitrogen content in the water-soluble part.
The Gouda was finely ground, and 20 mL of sterilized DW was added to 5 g of the
Gouda. The mixture was homogenized for 5 min using an ULTRA TURRAX T25 (IKA
Co., Wilmington, NC, USA). It underwent centrifugation and was sieved, as described in
manufacturing Gouda WEs. This filtrate was used in the experiment below.
Foods 2024, 13, 3446 3 of 16

Following Hull’s method [14], 2.5 mL of the filtrate was mixed with 5.0 mL of reagent A
(12% trichloroacetic-acid aqueous solution). After leaving the mixture at room temperature
for 20 min, it was filtered through Whatman No. 42 (Whatman, Maidstone, UK). Then,
2.5 mL of the filtrate was taken, and 5.0 mL of reagent B (75 g of sodium carbonate and
10 g of hexametaphosphate dissolved in 500 mL of DW) and 1.5 mL of reagent C (50 mL of
Folin and Ciocalteu’s phenol (Sigma Aldrich, Saint Louis, MO, USA) mixed with 100 mL of
DW) were added and reacted in a heating bath at 30 ◦ C for 30 min. After the reaction was
completed, its optical density (OD) was measured at 570 nm with SpectraMax ABS Plus
(Molecular Devices, San Jose, CA, USA). The WSN content was calculated by Equation (1)
with tyrosine as the standard (0, 20, 40, 80 µg).

y = 0.0073x − 0.0012 (1)

R2 = 0.9989; y, OD at 570 nm; x, tyrosine content (µg).

2.3. Measurement of Protein and pH in Extracts

Protein and pH assessments were conducted to verify the alterations in the properties
of Gouda WEs. Protein concentrations of Gouda WE were measured using Bradford’s
technique [15]. The Gouda WE powder was dissolved in sterile DW (10 mg/mL) for
measurement. The dye solution was prepared by diluting dye reagent concentrate (Bio-rad,
Hercules, CA, USA) with DW at a 1:4 ratio. The measurement was performed by adding
10 µL of the Gouda WE solution and 200 µL of the dye solution to a microplate (96-well),
and the OD was then read at 595 nm with SpectraMax ABS Plus (Molecular Devices, San
Jose, CA, USA). After that, bovine serum albumin (BSA, Bio-rad, Hercules, CA, USA) was
used as a standard and expressed as BSA equivalent.
The alterations in pH were assessed at 25 ◦ C using an S-20K (Mettler Toledo Co.,
Columbus, OH, USA) following the combination of each freeze-dried WE (YG, MG, and
EG) with DW at a 2% (w/v) concentration.

2.4. Mass-Spectrometry Analysis of Gouda WEs

The functional peptides of Gouda WE were identified by LC-HRMS (ultra-liquid
chromatography–high-resolution mass spectrometry) analysis. The UHPLC system (1290 In-
finity II) was combined with a high-resolution mass spectrometer (TripleTOF 5600 plus)
equipped with Electrospray ionization (Duospray), both manufactured by SCIEX, Framing-
ham, MA, USA.
The Gouda WE solution (100 ppm) was filtered through a 0.2 µm syringe filter (hy-
drophilic PTFE, Hyundai Micro, Anseong, Republic of Korea). Twenty µL of WE solution
was injected and separated by passing through the column with a two-solvent gradient
elution. The operating time was 15 min, and the mobile phases used were A: deionized
water (v/v) with 0.1% formic acid, and B: acetonitrile (v/v) with 0.1% formic acid. The ratio
(A:B) of mobile phase started at 9:1 and ended at 1:9.
The components separated by the UHPLC system were ionized by electrospray and
analyzed by mass spectrometry. Mass spectrometry was performed in (+) ion mode. The in-
strument conditions were as follows: flow rate, 0.6 µL; source temperature, 100 ◦ C; collision
energy, 35 eV; desolvation gas flow, 30 L/h. Nitrogen gas was used. For mass spectrometry,
functional peptide information was collected from the Milk Bioactive Peptide Database
(MBPDB) [16] in advance, and the chemical formula of each peptide was calculated. Based
on this, expected m/z values were determined. Analyst TF 1.8.1 (SCIEX, Framingham,
MA, USA) software was used for the interpretation of mass-spectrometry results. The
recorded mass spectrum was analyzed to confirm the chemical structure, chemical reactions,
and molecular weight. Functional peptides were identified by comparing the measured
formula and m/z ratio based on the formula and predicted m/z ratio of the previously
investigated peptide.
Foods 2024, 13, 3446 4 of 16

2.5. Antioxidant Effect—2,2’-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonic Acid)

The 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) inhibition ability of
the Gouda WE was confirmed through decolorization [17]. The ABTS working solution
was created by mixing a 7 mM ABTS (Sigma-Aldrich, Saint Louis, MO, USA) with a 2.4 mM
potassium persulfate. The working solution was then stored without light at 25 ◦ C for
12~16 h before the experiment. The ABTS working solution was diluted with DW until
the OD measured at 734 nm reached 0.7 (±0.02). A mixture was prepared by combining
50 µL of Gouda WE solution (concentration of 5 mg/mL in distilled water), with 950 µL
of ABTS working solution. After a 10 min reaction, the OD at 734 nm was recorded using
a SpectraMax ABS Plus (Molecular Devices, San Jose, CA, USA). As a positive control,
L-Ascorbic acid (25, 50 µg/mL) was used. The inhibition rate was determined using
Equation (2). In Equation (2), the control was obtained by reacting distilled water with the
ABTS working solution instead of the sample (Gouda WE solution), and each blank was
treated with distilled water instead of the ABTS working solution.

1 − (sample − sample blank)

InhibitionRate (%) = × 100 (2)
control − control blank

2.6. Antioxidant Effect—DPPH

The antioxidant evaluation method [18] confirmed the restraint of 2,2-diphenyl-1-
picrylhydrazyl (DPPH) by Gouda. A total of 20 µL of Gouda WE solution was added
to a microplate (96-well) that contained 180 µL of freshly made 0.2 mM 2,2-diphenyl-1-
picrylhydrazyl (Sigma-Aldrich, Saint Louis, MO, USA) solution in methanol. After reacting
in the darkroom for 15 min, the change in OD at 517 nm was measured with SpectraMax
ABS Plus (Molecular Devices, San Jose, CA, USA). The OD for blank was determined in the
same way using DW instead. The inhibition rate was determined using Equation (3).
 
S
InhibitionRate (%) = 1 − × 100 (3)
S0

S, the OD of the Gouda WE; S0 , the OD of the DW.

2.7. Antihypertensive Activity: ACE Inhibition

ACE inhibition was assessed using a modified spectrophotometric analysis technique
in vitro [19]. Rabbit-lung acetone powder (Sigma-Aldrich, Saint Louis, MO, USA) was
combined in a 1:10 ratio with a 0.1 M borate buffer solution containing 0.3 M NaCl. This
mixture was extracted by stirring at 4 ◦ C for 24 h. The enzyme solution was obtained by
collecting the supernatant following centrifugation at 15,000× g for 30 min. Hippuryl-
histidyl-leucine (HHL, Sigma Aldrich, Saint Louis, MO, USA) was prepared at a 5 mM
concentration by diluting it in a 0.1 M sodium borate buffer with 0.3 M NaCl, then utilized
as a substrate. Captopril (Sigma-Aldrich, Saint Louis, MO, USA) was chosen as a positive
control due to its excellent inhibitory activity. Next, a mixture of 100 µL volume of a 0.1 M
sodium borate buffer and 50 µL of enzyme solution was gently added to the 50 µL sample,
which had been pre-incubated at 37 ◦ C for 5 min. Subsequently, a 50 µL portion of the
substrate was introduced, and the reaction proceeded at 37 ◦ C for 30 min. Enzyme activity
was halted by adding 0.1 N HCl (300 µL). Following this, 1 mL of ethyl acetate was added
and vortexed for 15 s. It was then centrifuged at 960× g for 5 min at a temperature of 4 ◦ C.
The supernatant was gathered and dried using a Modulspin 31 (Hanil, Daejeon, Republic
of Korea), and then DW was added (1 mL) and dissolved well. The OD at 228 nm was
assessed using the SpectraMax ABS Plus (Molecular Devices, San Jose, CA, USA).
To prepare the sample blank, the reaction was halted using 0.1 N HCl (300 µL) before
the addition of the enzyme solution. Inhibition (%) was produced using Equation (1).
Foods 2024, 13, 3446 5 of 16

2.8. Immunomodulatory Function: Anti-Inflammation

2.8.1. Cell Cultivation
The experiment employed RAW 264.7, which was provided by the Korean Cell Line
Bank. Cells were kept at 37 ◦ C in a 5% CO2 . The medium utilized was Dulbecco’s modified
Eagle’s medium (DMEM, WELGENE, Gyeongsan, Republic of Korea), and it was enhanced
with 10% (v/v) FBS (fetal bovine serum, WELGENE, Gyeongsan, Republic of Korea) and
1% (v/v) penicillin and streptomycin (WELGENE, Gyeongsan, Republic of Korea).

2.8.2. Cytotoxicity Evaluation

We conducted a cell proliferation assay [20] to confirm the influence of Gouda WE
on the survival of RAW 264.7. RAW 264.7 cells were plated at a density of 3000 cells/well
in a microplate (96-well) and incubated for 24 h. Different concentrations of samples
were treated in each well and additionally cultured for 24 h. Next, the MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt)
solution (Promega, Madison, WI, USA) was added to each well and OD readings were
taken (490 nm) with a SpectraMax ABS Plus (Molecular Devices, San Jose, CA, USA). The
cytotoxicity of each Gouda WE was assessed by calculating it as a percentage relative to
the activity observed in the untreated control group.

2.8.3. Quantitative Real-Time PCR

RAW 264.7 cells were seeded in a 6-well format at a concentration of 1 × 105 cells/well
and allowed to incubate for 24 h. After that, the culture medium was substituted with
one containing 100 µg/mL of Gouda WE and cultivated for 24 h. Subsequently, cells
were placed for 6 h in the presence of 1 µg/mL lipopolysaccharide (LPS). The medium
was eliminated, cleaned with cold PBS, and the cells were processed with Ribo Ex (Gene
All, Seoul, Republic of Korea). Purified RNA was obtained using the Hybrid-R RNA
purification kit (Gene All, Seoul, Republic of Korea). To synthesize cDNA, RNA was
quantified with the Nabi UV/Vis Nano spectrophotometer (Micro Digital, Seongnam,
Republic of Korea). To the 1 µg of RNA, we added 1 µL of random hexamer (100 pmol/µL)
and 1 µL of a 10 mM dNTP mixture. The overall volume was then adjusted to 10 µL
using DEPC (Diethyl pyrocarbonate)-treated water. Subsequently, we incorporated an
M-MLV reverse transcriptase (1 µL; Promega, Madison, WI, USA), a 5X M-MLV RT reaction
buffer (4 µL; Promega, Madison, WI, USA), an RNase inhibitor (1 µL; Enzynomics, Daejeon,
Republic of Korea), and DEPC-treated water (4 µL). The mixture was kept at 25 ◦ C for
10 min followed by incubation at 50 ◦ C for 1 h to enable cDNA synthesis.
The expression of immunomodulatory cytokines was compared by quantitative real-
time polymerase chain reaction (qRT PCR) using an AriaMx (Agilent, Santa Clara, CA,
USA). The reaction was carried out under these conditions: denaturation at 95 ◦ C for 20 s,
annealing at 58 ◦ C for 20 s, and extension at 72 ◦ C for 20 s, repeated for 40 cycles. Table 1
shows the primer sequences used in the chain reaction.

Table 1. Primer sequence information of each target mRNA.

Target Primer (Forward) Primer (Reverse)

5’-AGG TCA AAG GTT TGG AAG 5’-TGA AGC AGC TAT GGC AAC
IL-1β
CA-3’ TG-3’
5’-GTC CTT CAG AGA GAT ACA GAA 5’-AGC TTA TCT GTT AGG AGA GCA
IL-6
ACT-3’ TTG-3’
5’-CCA CCA CGC TCT TCT GTC
TNF-α 5’-AGG GTC TGG GCC ATA GAA CT-3’
TAC-3’
5’-CAG CTG GGC TGT ACA AAC 5’-CAT TGG AAG TGA AGC GTT
iNOS
CTT-3’ TCG-3’
GAPDH 5’-CCA TGG AGA AGG CTG GGG-3’ 5’-CAA AGT TGT CAT GGA TGA CC-3’
Note: IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; iNOS, inducible nitric oxide
synthase; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.
Foods 2024, 13, 3446 6 of 16

2.8.4. Immunoblotting
As performed in 2.8.3, RAW 264.7 was cultured in a 6-well format at 1.5 × 105 cells/well
for 24 h. After that, cells were treated with each Gouda WE (100 µg WE in 1 mL culture
medium) and cultured for 24 h. To induce an inflammatory response, LPS was applied
at a dose of 1 µg/mL for 30 min. Immediately after, the medium in each well was rinsed
with PBS. Cell lysates were obtained using a lysis buffer composed of 10 mM Tris-HCl
(pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 1% Triton X-100. The solution
underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for
one hour, followed by a transfer to a nitrocellulose membrane. Blocking was subsequently
carried out with 5% non-fat dry milk in PBST for one hour. The membrane was subjected
to antibodies specific to p-I κBα (1:1000), I κBα (1:1000), and GAPDH (1:2000), all from Cell
Signaling Technology, Danvers, MA, USA, for a duration of 12 h. Following the PBST wash,
the membrane was reacted with a horseradish peroxidase-conjugated secondary antibody
(Cell Signaling Technology, Danvers, MA, USA) for 1 h at 25 ◦ C. Membranes were utilized
with Supersignal (Thermo Fisher Scientific, Waltham, MA, USA) and luminescence was
identified with Luminograph I (ATTO, Tokyo, Japan). Photographed signals were edited
and calculated through ImageJ 1.8.0_172 software (NIH, Bethesda, MD, USA).

2.9. Statistical Processing

Each experiment was conducted in technical triplicate analysis (three measurements of
the same extract obtained from a few grams of a single cheese), and results were shown as
an average ± standard error. The dataset was processed by analysis of variance (ANOVA),
and the averages were confirmed with the Tukey test, applying a significance level of either
1% or 5%. A paired t-test was utilized to examine the cytotoxicity.

3. Results and Discussion

3.1. Variations in WSN of GUDA
The changes in WSN show that WSN increased with the ripening but decreased after
long-term ripening (Table 2).

Table 2. Variations in WSN during the ripening of Gouda.

Sample WSN (mg Tyrosine/kg)

Young Gouda 20.74 ± 0.19 a
Medium Gouda 149.24 ± 0.57 b
Extra-sharp Gouda 69.73 ± 0.58 c
Note: Each result is presented as the mean and standard error (3 repetitions). Matching letters exhibit no significant
difference (p < 0.01). WSN, water-soluble nitrogen.

During the ripening period, the rise in WSN of cheese is usually attributed to casein
degradation, which can differ based on the proteases originating from the rennet or starter
used in its manufacture [9]. Moreover, it has been established that the protease generated
by the starter leads to ongoing breakdown [21]. Thus, the rise in water-soluble nitrogens
serves as a marker for the progression of ripening.
In this study, WSN increased in medium Gouda but decreased in extra-sharp Gouda
compared to medium Gouda. This indicates that proteolysis occurred rapidly in the early
stages of ripening. Also, it shows that microbial metabolism and enzymatic action led to a
decline in the total amount of WSN during the long ripening period of three years.

3.2. Protein Quantification and pH of Gouda WEs

Table 3 presents the protein concentrations and pH values for each Gouda WE. Each
Gouda WE powder was entirely dissolved in distilled water at a specific concentration for
this analysis.
Foods 2024, 13, 3446 7 of 16

Table 3. pH and protein content of Gouda water extract.

Foods 2024, 13, x FOR PEER REVIEW Sample pH (2% in Distilled Water) 7 of 16
Proteins (mg BSAE/g DM)
YG 5.69 ± 0.02 a 20.01 ± 0.55 c
MG 6.01 ± 0.01 b 20.12 ± 1.31 c
EG ± 0.03 a
5.72WEs
3.2. Protein Quantification and pH of Gouda 12.09 ± 0.41 d
Note: Each result is shown as the mean and standard error (3 repetitions). Matching letters exhibit no significant
Table
difference (p <30.01).
presents
BSAE,the protein
bovine serumconcentrations and
albumin equivalent; DM,pH values
dry matter;for
YG,each
youngGouda WE. Each
Gouda extract; MG,
Gouda Gouda
medium WE powder wasextra-sharp
extract; EG, entirely Gouda
dissolved in distilled water at a specific concentration for
extract.
this analysis.
The protein measurement results were 20.01 mg BSAE/g DM for YG and 20.12 mg
BSAE/g
Table 3. pHDM,andfor MG, showing
protein content ofno notable
Gouda difference,
water extract. but that of EG decreased to 12.09 mg
BSAE/g DM. This appears to be due to the breakdown of proteins into small peptides and
Samplein other metabolites.
an increase pH (2% in Distilled
The pH of Water) Proteins (mg
Gouda WE significantly BSAE/g from
increased DM) YG
(5.69) toYGMG (6.01) but showed5.69 ± 0.02 20.01 ± 0.55
a c
no significant difference in EG (5.72).
MG 6.01 ± 0.01 b 20.12 ± 1.31 c
3.3. Identification
EG of Bioactive Peptides of
5.72 ± 0.03Gouda
a WEs 12.09 ± 0.41 d
Note:Based
Each result
on theis shown
MBPDB as data,
the mean
the and standard
bioactive error (3compositions
peptide repetitions). Matching lettersand
of YG, MG, exhibit
EG
no significant
were analyzed difference (p < 0.01). BSAE,
using LC-HRMS. bovine serum
The LC-HRMS albumin
results equivalent;
for YG, MG, and DM,EGdry
arematter;
shownYG,in
young Gouda extract;
Figures 1–3, respectively.MG, medium Gouda extract; EG, extra-sharp Gouda extract.
The peptides that exceeded the set threshold (1000) along with their known physiolog-
The protein
ical activity measurement
are shown in Tablesresults
2–4. IQP were
and20.01
LQP mg BSAE/g
(which haveDM for YG and 20.12
ACE-inhibitory mg
function)
BSAE/g DM, for MG, showing no notable difference,
were found based on matching from the database (Table 4). but that of EG decreased to 12.09 mg
BSAE/g DM. This appears to be due to the breakdown of proteins into small peptides and
an increase
Table in other
4. Discovery metabolites.
of bioactive TheinpH
peptides youngof Gouda WE significantly increased from YG
Gouda extract.
(5.69) to MG (6.01) but showed no significant difference in EG (5.72).
Protein
Peptide Intervals Expected m/z Found at m/z Activity Reference
Description 3.3. Identification of Bioactive Peptides of Gouda WEs
IQP αs2-CN Based on the357.2132
209–211 MBPDB data,357.2142
the bioactiveACE
peptide compositions
inhibition Jing of YG,
P. et al., MG, and EG
2014 [22]
LQP β-CN 103–105
were 357.2132
analyzed using LC-HRMS.357.2142
The LC-HRMS ACE inhibition
results for YG,Tonouchi
MG, andH.EG et al.,
are2008 [23]in
shown
Figures
Note: 1–3,
ACE, respectively. enzyme.
angiotensin-converting

Figure 1. Detection of young Gouda water extract by mass spectrometry.

spectrometry. The figure shows matched
peptides and
peptides and all
all peaks
peaks observed
observed during
during elution.
elution.
Foods 2024, 13, x FOR PEER REVIEW 8 of 16
Foods 2024, 13, 3446 8 of 16

Figure2.2.Detection
Figure of medium
Detection GoudaGouda
of medium water extract
waterbyextract
mass spectrometry. The figure shows
by mass spectrometry. matched
The figure shows
peptides
matchedand all peaks
peptides andobserved during
all peaks elution.
observed during elution.

Figure3.3.Detection
Figure Detectionofof
extra-sharp Gouda
extra-sharp water
Gouda extract
water by mass
extract by spectrometry. The figure
mass spectrometry. Theshows
figure shows
matched
matchedpeptides
peptidesand allall
and peaks observed
peaks during
observed elution.
during elution.

In the case of MG (aged for 6 months) several peptides with notable biological activities
The peptides that exceeded the set threshold (1000) along with their known physio-
were identified. EL is known to have antioxidant activity. IVP, VP, LPP, VIP, and VVIP have
logical activity
the effect are shown
of inhibiting in Tables
ACE, while 2–4.VPP
IPP and IQPare
and LQP (which
recognized havemultifunctional
for their ACE-inhibitory func-
tion) wereincluding
properties, found based on matching from
immunomodulatory, the database
antioxidant, (Table
and ACE 4).
inhibition (Table 5).
In EG (3-year ripening), some peptides disappeared with long proteolytic periods, and
Tablephysiologically
new 4. Discovery ofactive
bioactive peptides
peptides (YL,in
IR,young
YPEL,Gouda extract. were detected (Table 6).
and DKIHPF)

Expected Found at
Peptide Protein Description Intervals Activity Reference
m/z m/z
IQP αs2-CN 209–211 357.2132 357.2142 ACE inhibition Jing P. et al., 2014 [22]
LQP β-CN 103–105 357.2132 357.2142 ACE inhibition Tonouchi H. et al., 2008 [23]
Note: ACE, angiotensin-converting enzyme.
Foods 2024, 13, 3446 9 of 16

Table 5. Discovery of bioactive peptides in medium Gouda extract.

Protein
Peptide Description Intervals Expected m/z Found at m/z Activity Reference

54–55, 156–157,
EL αs1-CN 261.1445 261.1444 Antioxidant Suetsuna K. et al., 2000 [24]
163–164
IVP αs1-CN 86–88, 126–128 328.2231 328.2228 ACE inhibition Jing P. et al., 2014 [22]
23–24, 99–100,
VP β-CN 188–189, 215.1390 215.1388 ACE inhibition Norris R. et al., 2014 [25]
193–194
LPP β-CN 166–168 326.2074 326.2073 ACE inhibition Norris R. et al., 2014 [25]
VIP αs2-CN 215–217 328.2231 328.2228 ACE inhibition Jing P. et al., 2014 [22]
Antioxidant Chakrabarti S. et al., 2017 [26]
IPP κ-CN 129–131 326.2074 326.2073 Anti-inflammatory Adams C. et al., 2020 [27]
ACE inhibition Adams C. et al., 2020 [27]
Antioxidant Chakrabarti S. et al., 2017 [26]
VPP β-CN 99–101 312.1918 312.1919 Anti-inflammatory Adams C. et al., 2020 [27]
ACE inhibition Adams C. et al., 2020 [27]
VVPP β-CN 98–101 411.2602 411.2600 ACE inhibition Wang Z-L. et al., 2011 [28]
Note: ACE, angiotensin-converting enzyme.

Table 6. Discovery of bioactive peptides in extra-sharp Gouda extract.

Protein
Peptide Description Intervals Expected m/z Found at m/z Activity Reference

54–55, 156–157,
EL αs1-CN 163–164 261.1445 261.1447 Antioxidant Suetsuna K. et al., 2000 [24]

106–107,
YL αs1-CN 109–110 295.1652 295.1653 ACE inhibition Mullally M. et al., 1996 [29]

23–24, 99–100,
VP β-CN 188–189, 215.1390 215.1382 ACE inhibition Norris R. et al., 2014 [25]
193–194
IR β-LG 163–164 288.2030 288.2038 ACE inhibition Murakami, M. et al., 2004 [30]
YPEL αs1-CN 161–164 521.2606 521.2602 Antioxidant Suetsuna K. et al., 2000 [24]
DKIHPF β-CN 62–67 756.4039 756.4045 ACE inhibition Gobbetti M. et al., 2000 [31]
Note: ACE, angiotensin-converting enzyme.

According to our findings, we discovered peptides with antihypertensive potential

in YG and several peptides with antioxidant, immunomodulatory, and antihypertensive
potential in MG. Similarly, we identified peptides with antioxidant and ACE-inhibitory
properties in EG. Among the three experimental groups, the MG contained the most diverse
types of functional peptides. In addition, it has been observed that different types were
produced at each ripening stage. These results confirm that various bioactive peptides are
produced owing to the breakdown of proteins during the ripening process, and that the
peptide composition changes over time.

3.4. ABTS Inhibition of Gouda WEs

The inhibitory effect on ABTS radicals was conducted by treating each sample with
ABTS solution, as shown in Figure 4. YG showed a radical inhibition rate of 25.99 ± 0.18%.
The scavenging activity increased to 70.26 ± 0.18% due to the change caused by ripening
in MG. However, the scavenging activity for EG was 46.81 ± 0.44%, which was higher
than that of YG but lower than that of MG. The decrease observed after the initial increase
aligned with findings from earlier research [32], which evaluated antioxidant activity
according to the ripening stage of Cheddar cheese.
 ABTS solution, as shown in Figure 4. YG showed a radical inhibition rate of 25.99 ± 0.18%.
The scavenging activity increased to 70.26 ± 0.18% due to the change caused by ripening
in MG. However, the scavenging activity for EG was 46.81 ± 0.44%, which was higher than
that of YG but lower than that of MG. The decrease observed after the initial increase
Foods 2024, 13, 3446 aligned with findings from earlier research [32], which evaluated antioxidant activity
10 ofac-
16
cording to the ripening stage of Cheddar cheese.

Figure 4. Inhibitory
Figure Inhibitoryeffect
effecton
onthe
the2,2’-azino-bis
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic
(3-ethylbenzothiazoline-6-sulfonic acid) by by
acid) water
waterex-
tract of of
extract Gouda. Each
Gouda. result
Each is shown
result as the
is shown as mean and standard
the mean errorerror
and standard (3 repetitions). Matching
(3 repetitions). letters
Matching
exhibitexhibit
letters no significant difference
no significant (p < 0.01).
difference ASC, ASC,
(p < 0.01). L-ascorbic acid; acid;
L-ascorbic YG, young Gouda
YG, young extract;
Gouda MG,
extract;
medium Gouda extract; EG, extra-sharp Gouda extract.
MG, medium Gouda extract; EG, extra-sharp Gouda extract.

3.5. DPPH
3.5. DPPH Inhibition
Inhibition of
of Gouda
Gouda WEs
WEs
The inhibitory effect of
The of Gouda
Goudaon onDPPH
DPPHgradually
graduallydecreased
decreased during
during thethe
ripening,
ripening, as
displayed
as displayed in in
Figure 5. At
Figure thethe
5. At firstfirst
part (YG),
part thethe
(YG), inhibition raterate
inhibition waswas36.77 ± 0.14%.
± 0.14%.
36.77 In MG,In
MG,
it wasit 30.58
was 30.58 ± 0.42%,
± 0.42%, and
and for EG for(ripened
EG (ripened for 3 years)
for 3 years) it wasit28.60
was 28.60 ± 0.20%.
± 0.20%. ContraryContrary
to the
to the findings
findings from ABTS,
from ABTS, the inhibitory
the inhibitory effecteffect on DPPH
on DPPH was was not increased
not increased by ripening
by ripening and
and
was was
the the lowest
lowest in in Ripening
EG. EG. Ripeningof of Gouda
Gouda did didpositively
not not positively
affectaffect
the the DPPH
DPPH radical
radical scav-
Foods 2024, 13, x FOR PEER REVIEW 11 of 16
scavenging,
enging, and and as observed
as observed in the
in the previous
previous ABTS ABTS data,
data, the the activity
activity tended
tended to decline
to decline as
as the
the ripening
ripening proceeded.
proceeded.
The results of the inhibition of the DPPH radical and the ABTS assay suggest that
Gouda cheese is expected to show the best antioxidant activity when it undergoes an ap-
propriate level of ripening rather than long-term ripening. There is a need for further re-
search to compare the antioxidant activity by subdividing the ripening stages of Gouda.
However, while the activity decreased in DPPH, the ABTS radical inhibition was still sig-
nificantly higher compared to the early stage of ripening. The value of long-term ripened
Gouda was positively evaluated in light of this, along with other functionalities.

Figure 5.
Figure 5. Inhibitory
Inhibitory effect
effect on
on the
the 2,2-diphenyl-1-picrylhydrazyl
2,2-diphenyl-1-picrylhydrazyl by by water
water extract
extract of
ofGouda.
Gouda. Each
Each
result is shown as the mean and standard error (3 repetitions). Matching letters exhibit no significant
result is shown as the mean and standard error (3 repetitions). Matching letters exhibit no significant
difference (p
difference (p << 0.01).
0.01). ASC,
ASC, L-ascorbic
L-ascorbic acid;
acid; YG,
YG, young
young Gouda
Gouda extract;
extract; MG,
MG, medium
medium Gouda
Gouda extract;
extract;
EG, extra-sharp Gouda extract.
EG, extra-sharp Gouda extract.

3.6. ACE Inhibition Potential

The ability of Gouda cheese WE to lower high blood pressure was assessed by exam-
ining its ability to inhibit the activation of ACE. The ACE converts angiotensin I into an-
giotensin II. This conversion simultaneously deactivates bradykinin, a vasodilator,
thereby producing a potent vasoconstrictor effect and increasing blood pressure [27].
Foods 2024, 13, 3446 11 of 16

The results of the inhibition of the DPPH radical and the ABTS assay suggest that
Gouda cheese is expected to show the best antioxidant activity when it undergoes an
appropriate level of ripening rather than long-term ripening. There is a need for further
research to compare the antioxidant activity by subdividing the ripening stages of Gouda.
However, while the activity decreased in DPPH, the ABTS radical inhibition was still
significantly higher compared to the early stage of ripening. The value of long-term
ripened Gouda was positively evaluated in light of this, along with other functionalities.

3.6. ACE Inhibition Potential

The ability of Gouda cheese WE to lower high blood pressure was assessed by exam-
ining its ability to inhibit the activation of ACE. The ACE converts angiotensin I into an-
giotensin II. This conversion simultaneously deactivates bradykinin, a vasodilator, thereby
producing a potent vasoconstrictor effect and increasing blood pressure [27].
In the present study, ACE inhibition increased with longer ripening periods (Table 7).
As a raw Gouda, YG demonstrated relatively low ACE-inhibitory activity, measured at
47.02%. In contrast, MG and EG presented significantly high ACE inhibition, which
was related with the emergence of various peptides with ACE inhibition that have been
previously analyzed. MG showed a high inhibition rate of 79.76%, whereas EG showed the
highest inhibition rate with an increase of 94.50%.

Table 7. ACE inhibition of Gouda water extracts.

Compound (25 mg) ACE Inhibition Rate (%)

Captopril (12.5 mg) 95.83 ± 0.24
YG 47.02 ± 0.02 a
MG 79.76 ± 0.05 b
EG 94.50 ± 0.05 c
Note: ACE, angiotensin-converting enzyme; YG, young Gouda extract; MG, medium Gouda extract; EG, extra-
sharp Gouda extract; Data represented by the mean and standard error (3 repetitions). Matching letters exhibit no
significant difference (p < 0.01).

Results from previous studies on the peptides of ripened cheese [33] have reported
that ACE suppression function depends on the extent of proteolysis, and that this ability
gradually decreases as a result of extensive proteolysis after peaking. The study results
indicate that inhibitory ability tends to enhance with ripening. However, the ACE-inhibitory
ability of Gouda did not decrease after 6 months (medium) and was stronger at 3 years
(extra-sharp). Additionally, goat cheese shows higher ACE-inhibitory activity compared
to cheeses like Emmental, Camembert, and Edam [34]. It is expected that, with long-term
ripening, it could be noted for its excellent functionality in addition to flavor compared to
other cheeses.

3.7. Anti-Inflammatory Effects of Gouda WEs

3.7.1. Effects of Gouda WEs on Cell Viability
Macrophages were exposed to YG, MG, and EG, and cell proliferation analysis was
performed to assess the cytotoxicity of the samples. The concentration range was 500 to
50 µg/mL, and each group was cultured for 24 h after treatment. The cytotoxicity of the
sample was evaluated by comparing the cell viability with that of the untreated group.
Cell viability in the YG- and MG-treated groups at all concentrations was higher than
control, whereas EG showed lower cell viability at high concentrations (Figure 6). When
samples were treated for 500 µg/mL, the cell survival rate was 152.65 ± 3.80% for YG,
165.25 ± 3.03% for MG, and 70.34 ± 0.57% for EG. The impact of the sample was lessened
as the concentration decreased, and no cytotoxicity was found at 100 µg/mL, with YG
showing 115.32 ±0.90%, MG 115.16 ± 3.53%, and EG 106.81 ± 5.12%.
 samples were treated for 500 μg/mL, the cell survival rate was 152.65 ± 3.80% for YG
165.25 ± 3.03% for MG, and 70.34 ± 0.57% for EG. The impact of the sample was lessened
as the concentration decreased, and no cytotoxicity was found at 100 μg/mL, with YG
showing 115.32 ±0.90%, MG 115.16 ± 3.53%, and EG 106.81 ± 5.12%.
Foods 2024, 13, 3446 As a result, the treatment concentration of Gouda WEs to RAW 264.7 was
12 ofestablished
16
at 100 μg/mL, as it did not notably impact cell viability.

Figure6.6.Effects
Figure Effectsof of
Gouda water
Gouda extracts
water on theon
extracts survival of RAWof
the survival 264.7
RAW cells. Eachcells.
264.7 resultEach
is shown as is shown
result
the mean
as the meanand standard error (3error
and standard repetitions). *, difference
(3 repetitions). from the nontreated
*, difference from the group at 5% significance
nontreated group at 5% signif
level;
icance***,level;
difference from the nontreated
***, difference from thegroup at 0.1% group
nontreated significance (paired t-test).
levelsignificance
at 0.1% ■, YG
level (youngt-test). ■
(paired
Gouda extract); •, MG (medium Gouda extract); ▲, EG (extra-sharp Gouda extract).
YG (young Gouda extract); ●, MG (medium Gouda extract); ▲, EG (extra-sharp Gouda extract).
As a result, the treatment concentration of Gouda WEs to RAW 264.7 was established
3.7.2.
at Changeas
100 µg/mL, initInflammatory
did not notablymRNA
impact Levels
cell viability.
IκBα phosphorylation triggers the activation of NF-κB, resulting in the secretion o
3.7.2. Change in Inflammatory mRNA Levels
inflammatory mediators like IL-6, IL-1β, TNF-α, and iNOS [35]. To evaluate the influence
IκBα phosphorylation triggers the activation of NF-κB, resulting in the secretion of
of Gouda WE on the LPS-induced inflammation in macrophages, changes in mRNA tran
inflammatory mediators like IL-6, IL-1β, TNF-α, and iNOS [35]. To evaluate the influ-
scription
ence wereWE
of Gouda compared in Figure 7.
on the LPS-induced inflammation in macrophages, changes in mRNA
The expressions
transcription were comparedof inflammation
in Figure 7. related to mRNA, including IL-1β and IL-6, de
creased with maturation.
The expressions Specifically,
of inflammation relatedIL-1β expression
to mRNA, includingdecreased to 2.36%
IL-1β and IL-6, and IL-6 ex
decreased
with maturation.
pression Specifically,
decreased to 19.04% IL-1β expression
in the decreased
EG treatment to 2.36%
group. On and IL-6 expression
the other hand, TNF-α ex
decreased to 19.04% in
pression decreased to the EG treatment
66.20% compared group.
to theOn the other
TNF-α hand, TNF-α
expression of the expression
control, but it wa
decreased to 66.20%
slightly higher thancompared to the
that of MG, TNF-αdecreased
which expressiontoof45.06%.
the control,
We but
foundit was slightly
that iNOS expres
higher than that of MG, which decreased to 45.06%. We found that iNOS expression
sion increased by 187.25% compared with that in the control plot. Regarding the func
increased by 187.25% compared with that in the control plot. Regarding the functional
tional peptide profile in this study, it appears that the immunomodulatory peptides pro
peptide profile in this study, it appears that the immunomodulatory peptides produced
duced Gouda
during duringripening
Goudacontributed
ripening contributed
to the noted to the noted anti-inflammatory
anti-inflammatory properties. As a properties.
result, A
not all mRNA levels decreased, but IL-1β and IL-6 levels reduced, with the effect becoming
more evident as ripening advanced.
Contrary to the fact that YG increased the immune response of macrophage, both MG
and EG significantly inhibited mRNA expression excluding iNOS, which is not unrelated
to the appearance of immunomodulatory peptides. These results seem to be influenced
by the emergence of peptides such as IPP and VPP in MG. Additionally, although no
anti-inflammatory peptides were identified in the case of EG, it may be due to the influence
of previously unknown peptides, and interesting results may be obtained through further
detailed analysis.
x FOR PEER REVIEW 13 of 16

Foods 2024, 13,a3446

result,
not all mRNA levels decreased, but IL-1β and IL-6 levels reduced, with the effect 13 of 16
becoming more evident as ripening advanced.

Figure 7. Impact ofFigure

Gouda 7. WEs
Impact onofthe transcription
Gouda WEs on theoftranscription
inflammatory mRNA. Each
of inflammatory resultEach
mRNA. is shown
result is shown as
as the mean and standard error (3 repetitions). Matching letters above each bar suggest
the mean and standard error (3 repetitions). Matching letters above each bar suggest no signifi-
no significant
cant difference (p <difference
0.05). WE, (p <water
0.05). extract;
WE, waterLPS, lipopolysaccharide;
extract; IL-6, interleukin-6;
LPS, lipopolysaccharide; IL-1β,
IL-6, interleukin-6; in-interleukin-
IL-1β,
terleukin-1β; TNF-α,1β; tumor
TNF-α, necrosis factor-α;
tumor necrosis iNOS,
factor-α; inducible
iNOS, induciblenitric
nitric oxide synthase;YG,
oxide synthase; YG, young
young Gouda extract;
Gouda extract; MG,MG, medium Gouda extract; EG, extra-sharp Gouda
medium Gouda extract; EG, extra-sharp Gouda extract. extract.

Contrary to 3.7.3. Suppression

the fact that YG of IκBα Phosphorylation
increased the immune response of macrophage, both
MG and EG significantly Our inhibited
research showed
mRNAthat properlyexcluding
expression matured Gouda
iNOS,WE effectively
which inhibited IL-1β
is not unre-
and IL-6 signaling in the macrophage cell line. Based on this fact,
lated to the appearance of immunomodulatory peptides. These results seem to be influ- it was hypothesized that
Gouda WE would inhibit the phosphorylation level of IκBα
enced by the emergence of peptides such as IPP and VPP in MG. Additionally, although in the NF-κB pathway that
induces inflammation. Accordingly, we performed an immunoblot of IκBα and p-IκBα to
no anti-inflammatory peptides were identified in the case of EG, it may be due to the in-
determine its effect on NF-κB pathway activity, which primarily regulates inflammatory
fluence of previously unknown
responses. peptides,
Figure and interesting
8 demonstrates results
that YG caused may
IκBα be obtained through
phosphorylation 9% more than LPS
further detailed analysis.
alone. On the other hand, MG reduced the LPS-triggered phosphorylation of IκBα to 92%.
Furthermore, in EG treatment, IκBα phosphorylation was inhibited to 57% and exhibited
3.7.3. Suppressionstrong
of IκBα Phosphorylation
anti-inflammatory effects. From these results, inhibitory activity was not evident in
Our researchYG, but it seems
showed to inhibit the
that properly inflammation
matured Gouda by WEreducing the phosphorylation
effectively inhibited IL-1βof IκBα by
peptides formed during the ripening period in MG and EG.
and IL-6 signaling in the macrophage cell line. Based on this fact, it was hypothesized that
We confirmed that extra-sharp Gouda WE effectively inhibited inflammatory re-
Gouda WE would inhibit the phosphorylation level of IκBα in the NF-κB pathway that
sponses both in the nucleus and in the cytoplasm based on its impact on inflammatory
induces inflammation.
mRNAAccordingly,
expression and we performed
IκBα an immunoblot
phosphorylation. oflong-term
Therefore, IκBα andripened
p-IκBαGouda
to could
determine its effect on NF-κB pathway activity, which primarily regulates inflammatory
create high value as an anti-inflammatory functional food. However, as mentioned earlier,
responses. Figurea 8detailed
demonstrates that
analysis of the YG caused IκBα
undiscovered phosphorylation
peptides 9% more
must be conducted first. than
LPS alone. On the other hand, MG reduced the LPS-triggered phosphorylation of IκBα to
92%. Furthermore, in EG treatment, IκBα phosphorylation was inhibited to 57% and ex-
hibited strong anti-inflammatory effects. From these results, inhibitory activity was not
evident in YG, but it seems to inhibit the inflammation by reducing the phosphorylation
of IκBα by peptides formed during the ripening period in MG and EG.
Foods 2024,
Foods 2024, 13,
13, 3446
x FOR PEER REVIEW 14
14 of 16
of 16

Figure 8.
Figure 8. Suppression
Suppression of
of IκBα
IκBα phosphorylation
phosphorylation by
by Gouda
Gouda WE.
WE. LPS,
LPS, lipopolysaccharide;
lipopolysaccharide; GAPDH,
GAPDH,
Glyceraldehyde-3-phosphate dehydrogenase; IκBα, nuclear factor of κ light polypeptide gene
Glyceraldehyde-3-phosphate dehydrogenase; IκBα, nuclear factor of κ light polypeptide gene en-
en-
hancer in B-cells inhibitor α; WE, water extract; YG, young Gouda extract; MG, medium Gouda
hancer in B-cells inhibitor α; WE, water extract; YG, young Gouda extract; MG, medium Gouda
extract; EG, extra-sharp Gouda extract.
extract; EG, extra-sharp Gouda extract.

We confirmed that extra-sharp Gouda WE effectively inhibited inflammatory re-

4. Conclusions
sponses both
In this in the
study, nucleus
water andofinyoung,
extracts the cytoplasm based
medium, and on its impact
extra-sharp Gouda onwere
inflammatory
analyzed
for their bioactive peptide composition and various health properties. The Gouda
mRNA expression and IκBα phosphorylation. Therefore, long-term ripened could
study found
create high value as an anti-inflammatory functional food. However, as mentioned earlier,
that different ripening periods resulted in the emergence of different bioactive peptides
a detailed
in analysis
each ripening of theThe
stage. undiscovered peptides
activity of Gouda WEmust beantioxidant
as an conducted first.
was evaluated, and
long-term ripening was not positive for antioxidant activity. In terms of antihypertensive
4. Conclusions
properties, it was shown that ACE inhibition increases with ripening. In particular, the
In this study,effects
anti-inflammatory water revealed
extracts of young, medium,
extra-sharp and extra-sharp
Gouda effectively Gouda
suppresses were ana-
inflammatory
lyzed for at
responses their
bothbioactive peptide and
gene-expression composition and various
protein levels. health suggest
These findings properties.
that The study
long-term
found that
ripened different
Gouda can beripening
valuedperiods resultedfood
as a functional in the emergence
that may offerofhealth
different bioactive
benefits pep-
related to
high
tides blood
in eachpressure
ripening and inflammation.
stage. The activityThis study did
of Gouda WE not perform
as an peptide
antioxidant waspurification
evaluated,
and quantification
long-term ripeningof thewasextract. Furtherfor
not positive analysis and purification
antioxidant of theof
activity. In terms peptides,
antihyper-as
well as properties,
tensive biological experiments
it was shownto support
that the research
ACE inhibition findings,
increases with need to beInconducted.
ripening. particular,
Furthermore, due to the
the anti-inflammatory limitations
effects revealedof the scale of this
extra-sharp study,
Gouda the reproducibility
effectively among
suppresses inflam-
multiple cheeses with
matory responses the same
at both stage could and
gene-expression not be confirmed.
protein levels.Research on this suggest
These findings is necessary
that
to support ripened
long-term the reliability
Goudaofcan thebe
results
valuedof as
this experiment.
a functional food that may offer health benefits
related to high blood pressure and inflammation. This study did not perform peptide pu-
Author Contributions:
rification Conceptualization,
and quantification M.S.N.;
of the extract. methodology,
Further W.K.,
analysis and G.R., H.B. of
purification andtheM.S.N.;
pep-
software, W.K.; validation, M.S.N.; formal analysis, W.K., G.R., H.B. and M.S.N.; investigation, W.K.,
tides, as well as biological experiments to support the research findings, need to be con-
G.R., H.B. and M.S.N.; data curation, W.K. and M.S.N.; writing—original draft preparation, W.K.;
ducted. Furthermore, due to the limitations of the scale of this study, the reproducibility
writing—review and editing, W.K., G.R., H.B. and M.S.N.; visualization, W.K.; supervision, M.S.N.;
among multiple cheeses with the same stage could not be confirmed. Research on this is
All authors have read and agreed to the published version of the manuscript.
necessary to support the reliability of the results of this experiment.
Funding: This research received no external funding.
Author Contributions:
Institutional Conceptualization,
Review Boarding Statement: M.S.N.; methodology, W.K., G.R., H.B. and M.S.N.; soft-
Not applicable.
ware, W.K.; validation, M.S.N.; formal analysis, W.K., G.R., H.B. and M.S.N.; investigation, W.K.,
Informed
G.R., H.B. Consent Statement:
and M.S.N.; Not applicable.
data curation, W.K. and M.S.N.; writing—original draft preparation, W.K.;
Data Availability Statement: The originalH.B.
writing—review and editing, W.K., G.R., and M.S.N.;
contributions visualization,
presented in theW.K.;
studysupervision,
are includedM.S.N.;
in the
All authors have read and agreed to the published version of the manuscript.
article; further inquiries can be directed to the corresponding author.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflicts of interest.
Institutional Review Boarding Statement: Not applicable.
References
Informed Consent Statement: Not applicable.
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