Journal Pre-proofs

Hydroxyproline determination for initial detection of halal-critical food ingre‐

dients (gelatin and collagen)

Mohd Hafis Yuswan, Nurul Hanani A. Jalil, Haslina Mohamad, Shamsidah

Keso, Nurhidayatul Asma Mohamad, Tengku Shahrul Tengku Md. Yusoff,
Nor Falahiah Ismail, Yanty Noorzianna Abdul Manaf, Amalia Mohd Hashim,
Mohd Nasir Mohd Desa, Yus Aniza Yusof, Shuhaimi Mustafa

PII: S0308-8146(20)31624-1
DOI: https://doi.org/10.1016/j.foodchem.2020.127762
Reference: FOCH 127762

To appear in: Food Chemistry

Received Date: 13 November 2019

Revised Date: 7 April 2020
Accepted Date: 2 August 2020

Please cite this article as: Hafis Yuswan, M., Hanani A. Jalil, N., Mohamad, H., Keso, S., Asma Mohamad, N.,
Shahrul Tengku Md. Yusoff, T., Falahiah Ismail, N., Noorzianna Abdul Manaf, Y., Mohd Hashim, A., Nasir
Mohd Desa, M., Aniza Yusof, Y., Mustafa, S., Hydroxyproline determination for initial detection of halal-critical
food ingredients (gelatin and collagen), Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.
2020.127762

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Hydroxyproline determination for initial detection of halal-critical food ingredients

(gelatin and collagen)

Authors: Mohd Hafis Yuswana,e,*, Nurul Hanani A. Jalila, Haslina Mohamada, Shamsidah

Kesoa, Nurhidayatul Asma Mohamada, Tengku Shahrul Tengku Md. Yusoffa, Nor Falahiah

Ismaila, Yanty Noorzianna Abdul Manafa,e, Amalia Mohd Hashima,d,e, Mohd Nasir Mohd

Desaa,b,e, Yus Aniza Yusofa,c,e, Shuhaimi Mustafaa,d,e

*Corresponding author. Email: hafisyuswan@upm.edu.my

Affiliation:
aLaboratory of Halal Services, Halal Products Research Institute, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor, Malaysia.

bDepartment of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

cDepartment of Process and Food Engineering, Faculty of Engineering, Universiti Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

dDepartment of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

1
eConsortium of Malaysia IPT Halal Institutes, Ministry of Higher Education, Complex E,

Federal Government Administrative Centre, 62604 Putrajaya, Malaysia.

Abstract

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from

either bovine or porcine animals. Current analytical methods for determining the sources of

gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide

matching. Thus, the aim of this study was to investigate the utility of monitoring

hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially

haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based

samples, one-way univariate analysis of variance followed by pair-wise comparison was used

to establish statistical significance. Multivariate chemometric analysis through principal

component analysis revealed a discrete distribution pattern among 59 samples due to

hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the

validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this

preliminary identification technique will aid the identification of potentially haram foodstuffs.

Keywords: hydroxyproline; gelatin; collagen; halal; amino acid profiling

1. Introduction

Gelatin is a water-soluble fibrous collagen hydrolysate with a high molecular weight ranging

from 97 to 250 kDa (Atma et al., 2018; Mulyani, Setyabudi, Pranoto, & Santoso, 2017;

Rakhmanova, Khan, Sharif, & Lü, 2018; Sanaei, Mahmoodani, See, Yusop, & Babji, 2013).

Collagen is a major structural protein of connective tissues present in blood vessels, cartilage,

2
skin, tendons, ligaments, and bones (Alves, Marques, Martins, Silva, & Reis, 2017; Hashim,

Mohd Ridzwan, Bakar, & Mat Hashim, 2015; Song & Li, 2017). Collagen is found in most

mammals, accounting for approximately 25 - 30% of their total protein (Alves et al., 2017;

Hashim et al., 2015; Song & Li, 2017). Since gelatin is produced through partial hydrolysis of

collagen, the quality of the gelatin depends on the source of the collagen. The special

physicochemical properties of gelatin and collagen have led to their widespread application in

a diverse range of fields. For instance, they are used as stabilizers, thickeners, and emulsifiers

in food, pharmaceuticals, cosmetics, and medicine (Azilawati, Hashim, Jamilah, & Amin,

2015).

However, the source of gelatin and collagen can be problematic. Most commercial

gelatin and collagen is manufactured from either bovine or porcine sources, specifically from

the skin and bone. A previous study reported that gelatin from porcine skin contributed to 41%

of the total world production, while bovine hide, bovine bone, and fish accounted for 28.5%,

29.5%, and 1%, respectively (Ali et al., 2018; Milovanovic & Hayes, 2018). This is problematic

for Muslim consumers owing to halal considerations around gelatin and collagen. Halal gelatin

and collagen are crucial as Muslims are predicted to account for 31% of the world population

by 2060 (Pew Research Center, 2017).

In Islam, the consumption of porcine gelatin and collagen is prohibited. Furthermore,

although bovine animals are halal, they must be slaughtered according to Islamic law for their

gelatin or collagen to be considered halal (Khattak et al., 2011; Rakhmanova et al., 2018). The

source of gelatin and collagen is not only an issue for Muslims but also of concern to followers

of Judaism (kosher) and Hinduism (vegetarian). Jewish people are prohibited from consuming

porcine meat, while Hindus are prohibited from consuming bovine meat (Ardekani,

Mahmoodani, See, Yusop, & Babji, 2013). Moreover, certain denominations of Christianity

3
also prefer not to use porcine products (Ali et al., 2018). From a health perspective, porcine

meat can be considered a source of human parasitic infections involving protozoa and

helminths, thus increasing the risk of foodborne zoonoses (Baer, Miller, & Dilger, 2013;

Djurković-Djaković, Bobić, Nikolić, Klun, & Dupouy-Camet, 2013; McGlone, 2013; Rahman

et al., 2014). Furthermore, gelatin or collagen from bovine animals may be related to bovine

spongiform encephalopathy (BSE), commonly known as “mad-cow disease” (Abdullah et al.,

2016; Aisyah, Huda, Azhar, & Fazilah, 2014; Ali et al., 2018; Alves et al., 2017; Gorgieva &

Kokol, 2011; Milovanovic & Hayes, 2018).

Currently, most analytical methods for the detection of bovine and porcine gelatin and

collagen are based on quantitative polymerase chain reaction (qPCR) and liquid

chromatography-mass spectrometry (LC-MS) (Rakhmanova et al., 2018). However, qPCR is

not robust as it relies solely on the DNA. This is because gelatin and collagen are largely

polypeptide-based. Furthermore, the LC-MS method produces false positives in peptide

matching. This is due to the highly repetitive sequence motifs in gelatin and collagen molecules

combined with variability in hydroxylation sites and their relative abundance (Buckley, 2016).

A strategy that might be useful to detect the presence of gelatin and colagen in food is

through analyzing a molecule(s) that strongly associated with collagen or gelatin. In this regard,

hydroxyproline has been found to be a signature amino acid for gelatin and collagen (Stoilov,

Starcher, Mecham, & Broekelmann, 2018). Hydroxyproline is a major component of collagen,

accounting for approximately 13.5% of amino acids (Stoilov, Starcher, Mecham, &

Broekelmann, 2018). Previous studies have shown that hydroxyproline is vital for the gelling

of gelatin and collagen (Stoilov et al., 2018). The indispensable use of collagen and gelatin in

food or cosmetic industries is nonetheless of great concern among certain group of people with

specific diet preference or religious restrictions such as Muslim; therefore, a quick screening

4
method is necessary. We hypothesize that hydroxyproline, a signature amino acid of gelatin

and collagen can be used to preliminarily screen for the presence of potentially haram elements

in food products.

Therefore, the aim of this study was to evaluate the use of hydroxyproline, a signature

amino acid in gelatin and collagen, as a means to monitor the presence of animal-derived

collagen and gelatin as halal-critical food ingredients. The hydroxyproline monitoring can be

utilized as a first detection tool by halal analysts prior to further detailed halal testing; especially

for products containing undeclared collagen or gelatin that demand further scrutiny.

2. Materials and methods

2.1 Materials

L-2-aminobutyric acid (AABA) was purchased from Sigma-Aldrich Corporation (St. Louis,

USA). The high-performance liquid chromatography (HPLC) eluent (AccQ TagTM Eluent A),

a Waters AccQ Tag reversed-phase column (3.9 × 150 mm, 4 µm, 60 Å), and a derivatization

reagent (AccQ-FluorTM Reagent Kit) were purchased from Waters Corporation

(Massachusetts, USA). Amino Acid Standard H solution was purchased from Waters

Corporation (Maple Street, MA, USA). MS® PTFE syringe filters with a pore size of 0.45 µm

were purchased from Membrane Solutions (Shanghai, China). Sterile nontoxic and non-

pyrogenic syringes (3 mL) were purchased from Terumo® Corporation (Laguna, Philippines).

Acetonitrile (ACN) of liquid chromatography grade, 37% hydrochloric acid (HCl, EMSURE®

grade), and analytical grade L-hydroxyproline were purchased from Merck (Darmstadt,

Germany). Water was freshly obtained from a Sartorius Stedim Arium®611VF ultrapure water

system (Goettingen, Germany). Positive control samples (gelatin and collagen from porcine

skin type A, bovine skin type B, or fish skin) were purchased from Sigma-Aldrich Corporation

5
(St. Louis, USA). Negative control samples (carrageenan) and test samples (sea cucumber

extract, flour extract, dried dates, insect meal, crystal pure, chicken feed, bread, polymer cells,

algae, camel meat, edible bird’s nest, bean powder, jellyfish protein, herb powder, and

blackcurrant) were obtained from customers of the Laboratory of Halal Services, Halal

Products Research Institute, Universiti Putra Malaysia, who had requested amino acid

profiling. Samples for each positive and negative control was prepared 10 times, while, single

preparation was made for the test samples.

2.2 Amino acid standard preparation

A 1000-pmol/µL amino acid standard solution was prepared by transferring 400 µL of Amino

Acid Standard H solution (consisting of aspartic acid, serine, glutamic acid, glycine, histidine,

arginine, threonine, alanine, proline, tyrosine, valine, methionine, lysine, isoleucine, leucine,

and phenylalanine, each at a concentration of 2.5 mM, and cysteine at 1.25 mM) to a 2-mL

Eppendorf tube. Hydroxyproline was spiked into the standard solution by adding 400 µL of 2.5

mM hydroxyproline solution. Subsequently, 40 µL of the 2.5 mM internal standard AABA was

added to the standard solution prior to adding 160 µL of ultrapure water. A serial dilution of

amino acid standards (L1: 25 pmol, L2: 100 pmol, L3: 250 pmol, L4: 500 pmol, and L5: 1000

pmol) was prepared 10 times for method validation. The amount of each amino acid (%, w/w)

was normalized to the internal standard AABA from a single point calibration amino acid

standard solution.

2.3 Amino acid hydrolysis and derivatization

Each sample was weighed to approximately 0.2 g for hydrolysis prior to derivatization as

described previously with some modifications (Raja Nhari, Che Man, Ismail, & Anuar, 2011).

6
Briefly, the weighed sample was incubated with 5 mL of 6 N HCl at 110 °C for 24 h in a

Memmert 100-800 oven (Schwabach, Germany). Then, the hydrolysate was transferred into a

100-mL volumetric flask and 4 mL of 2.5 mM AABA was added and the volume was made up

with ultrapure water. Next, 2 mL of the hydrolysate was filtered through an MS® PTFE syringe

filter (0.45 µm) into an Eppendorf tube. For derivatization, 70 µL of borate buffer was added

to a new clean Eppendorf tube followed by the addition of 10 µL of filtered hydrolysate. The

mixture was vortexed immediately for 5 s. Then, the mixture was derivatized by adding 20 µL

of AccQ-FluorTM Reagent and vortexed again for 5 s. The mixture was transferred into an

HPLC insert vial prior to chromatographic separation.

2.4 Chromatographic separation

HPLC separation was performed as described previously with some modifications (Raja Nhari

et al., 2011). The HPLC system consisted of a Waters e2695 separation module, a Waters

column compartment, and a Waters 2475 multi λ fluorescence detector (Maple Street Milford,

MA, USA). The sample was injected into a Waters AccQ Tag reversed-phase column (3.9 ×

150 mm, 4 µm, 60 Å) at a flow rate of 1 mL/min at 36 °C for 50 min. Mobile phases A, B, and

C were AccQ TagTM Eluent A (1:10), 100% ACN, and 100% ultrapure water, respectively. The

column was equilibrated for 10 column volumes prior to sample injection at a volume of 10

µL. The gradient for the chromatographic separation was initially set to 98% A: 0.8% B: 1.2%

C for 0.5 min, then 92% A: 3.2% B: 4.8% C from 0.5 to 15 min, then 85% A: 6% B: 9% C

from 15 to 19 min, then 65% A: 14% B: 21% C from 19 to 32 min, maintained for 32 to 33

min, then 40% B: 60% C from 33 to 35 min, maintained from 35 to 38 min, then back to the

initial conditions from 38 to 39 min prior to equilibration for the next injection from 39 to 50

7
min. The excitation and emission wavelengths fluorescence detection were set at 250 and 395

nm, respectively. Each sample was injected by single injection.

2.5 Method validation

The validation parameters, including linearity, recovery, precision, limit of detection (LOD),

and limit of quantification (LOQ) were evaluated according to the Center for Drug Evaluation

and Research (CDER, 1994) and European guidelines (European Commission, 2017). To

ensure the validity of the hydroxyproline determinations, inter- and intra-laboratory

comparisons were conducted in accordance with the International Organization for

Standardization (ISO) 17025, clauses 7.7.1 and 7.7.2 (Anonymous, 2017).

2.6 Data analysis

All data (positive control, negative control, and test samples) were subjected to analysis of

variance (ANOVA) analysis, wherein the null hypothesis (Ho) was ‘all the samples have the

same hydroxyproline mean value', and the alternative hypothesis (Ha) was ‘at least one of the

samples has a different hydroxyproline mean value'. A pairwise comparison test was conducted

using Tukey’s honest significant difference (HSD) only if the Ho was rejected, wherein the

hydroxyproline mean difference for each sample was considered significant at p <0.05. The

ANOVA and pairwise comparison tests were performed using SPSS® Statistics software,

version 25 (IBM, USA). Subsequently, the raw data for all samples were subjected to principal

component analysis (PCA) by setting the levels of hydroxyproline and the other 17 amino acids

as variable (K), and each sample was designated as an observation (N) to construct a PCA

dataset. The scaling parameter for the PCA was unit variance. The PCA was performed using

MKS Umetrics AB SIMCA software, version 14.1.0.2047 (Umea, Sweden). For inter- and

8
intra-laboratory comparisons, z-score (z) was calculated for hydroxyproline assessment using

the following formula:

𝑥―𝑥
𝑧=
𝜎

where 𝑥 = observed value, 𝑥 = reference value, and 𝜎 = standard deviation of target samples.

3. Results and discussion

3.1 Profiling of hydroxyproline

The levels of 17 amino acids in the positive controls, negative controls, and test samples were

investigated chromatographically. Figure 1 shows the results of chromatographic separation

for all 17 amino acids present in the porcine gelatin, porcine collagen, and carrageenan samples.

The signal intensity for hydroxyproline in porcine gelatin is higher than that in porcine

collagen, although the amino acid profile is similar. This is due to additional hydroxyproline

formation through proline hydroxylation, which is an inevitable consequence of gelatin

formation from the hydrolysis of collagen (Shoulders & Raines, 2009). No hydroxyproline was

detected in the carrageenan samples.

For this section, only hydroxyproline is discussed in detail, as shown in Table 1, after being

subjected to ANOVA. The results for the other 16 amino acids are shown in Table S1

(Supporting Information). As shown in Table 1, the presence of hydroxyproline is significantly

different among the positive and negative control samples (p <0.05). Generally, the amount of

hydroxyproline is higher in the gelatin samples than in the collagen samples, except for the fish

skin gelatin samples. This is again explained by the increase in hydroxyproline signal intensity

due to proline hydroxylation (Shoulders & Raines, 2009). However, the amount of

hydroxyproline is higher in the fish skin collagen samples (5.18 ± 0.06% w/w) than in the fish

9
skin gelatin samples (3.82 ± 0.04% w/w), as shown in Table 1. Fish gelatin has been obtained

in various yields from many species, including Epinephelus sexfasciatus, Lutjianus

argentimaculatus, Rastrelliger kanagurta, Pristipomodes typus, and Clarias gariepinus

(Sanaei et al., 2013). In the current study, the low gelatin yields for fish skin samples may have

been influenced not only by the fish species, but also by the extraction conditions, such as pH

and incubation period (Ardekani et al., 2013; Sanaei et al., 2013).

Moreover, hydroxyproline is not detected in the negative control sample (carrageenan)

(Table 1). Carrageenan is a polysaccharide extracted from seaweed, which explains the absence

of hydroxyproline (Necas & Bartosikova, 2013; Noor, 2018). Carrageenan was chosen as a

negative control sample because it can also be used as a stabilizer, thickener, or emulsifier

(Necas & Bartosikova, 2013; Noor, 2018). Hydroxyproline has previously been demonstrated

as an amino acid marker for fibrillar collagens, which are commonly found in members of the

kingdom Animalia (Stoilov et al., 2018).

The confirmation of hydroxyproline as an amino acid marker for gelatin and collagen was

subjected to a multivariate chemometric analysis by PCA instead of one-way univariate

analysis by ANOVA. PCA was used in a previous study to overview the patterns and grouping

of bovine and porcine gelatin sources in processed products (Nur Azira, Amin, & Che Man,

2012). However, the potential of hydroxyproline for the differentiation of gelatin- or collagen-

based samples has not been analysed using PCA before.

3.2 Chemometric analysis of hydroxyproline

Chemometric analysis is a technique used to treat the complex information obtained from

samples produced by an analytical instrument. This technique is usually performed by PCA

through dimensional reduction, wherein the original variability of the data set is retained

10
(Osorio et al., 2013). In the current study, a data set for the PCA model was constructed from

59 samples with the amounts of hydroxyproline (% w/w) and 16 other amino acids as variables.

The dataset for the PCA model is shown in Table S2 (Supporting Information).

Figures 2(a) and 2(b) show the PCA score plot and PCA loading plot, respectively. The

distribution patterns of the samples are explained by the PCA score plot, whereas the PCA

loading plot describes the relationship among the 17 amino acids, which correlate to the

samples as tabulated in the PCA score plot. The PCA score plot contains two principal

components (PCs), with PC1 and PC2 accounting for 45.4% and 18.8%, respectively. The first

two PCs indicate a total variance (R2) of 64.3% with a prediction (Q2) of 54.7%. By the ellipse

Hotelling’s T2 (T2 critical 95%), the negative control samples (carrageenan) were observed as

an outlier. This observation is due to the absence of all amino acids (except for arginine and

proline) from the carrageenan samples, as shown in Figure 2(b). Nevertheless, the arginine and

proline in the carrageenan samples are considered to be false positives, as carrageenan is a

sulfated polygalactan (not a polypeptide) formed by alternating units of D-galactose and 3,6-

anhydro-galactose joined by α-1,3 and β-1,3-glycosidic linkages (Necas & Bartosikova, 2013).

According to Figure 2(a), all the positive control samples are grouped together, as are the

negative control samples. A previous study showed that samples close to each other exhibit

similar properties, while separated samples are dissimilar in terms of variables (Wold &

Sjöström, 1998), in this case hydroxyproline. Moreover, there is an intra-variation for the test

samples. This is due to the variability in the amount of hydroxyproline and other amino acids

in the PCA dataset (Table S2).

Furthermore, the farthest amino acid from the origin indicates a strong contribution to the

PCA model, while the amino acids with highly correlated variables were clustered together

(Yuswan et al., 2019). Figure 2(b) indicates that hydroxyproline and histidine correlate with

11
the gelatin and collagen samples, as they are situated in the same quadrant. Nevertheless,

hydroxyproline is preferred as a signature amino acid for halal-critical ingredients because it is

regularly found in collagen and many gelatin products.

3.3 Validity of the hydroxyproline assay

Method validation for hydroxyproline determination as a method to identify halal-critical

ingredients was performed for several validation parameters, including linearity, recovery,

precision, LOD, and LOQ. Table 2 shows the results for each validation parameter. The

linearity value (R2) for hydroxyproline is higher than 0.999 in the concentration range 32.783–

1311.3 ng/µL on the calibration curve. This is an appropriate value according to the CDER.

For recovery evaluation, two amino acid standards with concentrations corresponding to

the lowest (L1) and highest (L5) concentrations of the linear range of the calibration curve. As

shown in Table 2, the recovery rates for L1 and L5 are 85.104% and 99.945%, respectively. A

recovery of 90%–110% is recommended for good accuracy (CDER, 1994; European

Commission, 2017). The low recovery for L1 might be due to the low sensitivity, which is in

turn due to the low concentration of spiked hydroxyproline.

Precision was evaluated by measuring the repeatability of analysis at L1 and L5 from 10

replicates under the same analytical conditions. Precision is expressed as relative standard

deviations (RSDs), as shown in Table 2. These are 15.435% and 1.796% for L1 and L5,

respectively. Different RSD values for precision are recommended by different regulatory

bodies. For instance, the European guidelines recommend ≤20% (European Commission,

2017), whereas ≤1% is recommended by the CDER (1994).

The LOD and LOQ values for hydroxyproline are shown in Table 2. Both LOD and LOQ

were determined according to the relationship a × SDL ÷ m, where a is equal to 3 in the case of

12
LOD and 10 for LOQ, SDL is the residual standard deviation of the linear regression, and m is

the slope of the linear regression (Shrivastava & Gupta, 2011). In this study, the LOD and LOQ

are 2.017 and 6.722 ng/µL, respectively.

According to ISO 17025, clauses 7.7.1 and 7.7.2, “the testing and calibration laboratories

shall monitor the validity of results and the laboratory performance” (Anonymous, 2017). The

validity of hydroxyproline as a signature amino acid for gelatin and collagen determination was

demonstrated by inter- and intra-laboratory comparison. The inter- and intra-laboratory

comparison is mandated for the accredited ISO 17025 laboratory to ensure the validity of the

test method, in addition to demonstrating the competency of the analyst (Allard & Amarouche,

2017; Softić, Zaimović-Uzunović, & Basić, 2012; Szewczak & Bondarzewski, 2016). Table 3

shows the inter- and intra-laboratory comparison results for the bovine gelatin skin sample. For

the inter- and intra-laboratory comparison analyses, the validity for hydroxyproline was

evaluated based on the z-score. The z-score values for z ≤ 2, 2 ≤ z ≤ 3, and z ≥ 3 are considered

satisfactory, questionable, and unsatisfactory, respectively (Allard & Amarouche, 2017;

Szewczak & Bondarzewski, 2016; Visser, 2006). According to Table 3, all the inter- and intra-

laboratory comparison values are satisfactory, as the z-score is ≤ 2. This result demonstrates

the reliability of the test method for profiling the amino acids, specifically hydroxyproline. A

z-score ≤ 2 also demonstrates the accuracy of hydroxyproline amount, which only deviates

within 95% of the reference value (𝑥).

Moreover, the presence of hydroxyproline was verified for the 15 test samples, as shown

in Table 4. Of the 15 test samples, in seven samples (algae, bean powder, sea cucumber extract,

bread, polymer cells, blackcurrant, and edible bird's nest), hydroxyproline is not detected. Five

samples (flour powder, dried date, insect meal, herb powder, and camel meat) have

hydroxyproline levels below the LOD and LOQ, on the other hand, two samples (jellyfish

13
protein and crystal pure) show clear detection of hydroxyproline. As shown in Table 3, the

‘below LOD and LOQ’ statuses for the five samples are due to baseline noise in the

chromatographic profile, which contributes to false positives. Jellyfish protein samples clearly

show positive detection status, and previous studies have proposed jellyfish as an alternative

source of collagen (Alves et al., 2017; Cheng et al., 2017; Gorgieva & Kokol, 2011; Hashim et

al., 2015; Wichuda, Sunthorn, & Busarakum, 2016). The high levels of hydroxyproline in

jellyfish protein powder and crystal pure show that they likely contained collagen or gelatin,

which prompted further investigations to determine the origin of the substances.

4. Conclusions

A unique signature amino acid marker for gelatin and collagen, known as hydroxyproline, was

validated through stepwise statistical analysis. One-way univariate ANOVA revealed that

hydroxyproline is significantly present in the gelatin and collagen from 44 samples (Table 1).

This observation confirmed the uniqueness of the hydroxyproline profile in different gelatin

and collagen samples. Subsequently, the effect of hydroxyproline among the 59 samples on the

discrete distribution pattern in the PCA was observed (Figure 1). This discrete distribution is

due to the variability in the hydroxyproline values in the PCA dataset. To ensure the validity

of the hydroxyproline determination, method validation was conducted using several validation

parameters, including linearity, recovery, precision, LOD, and LOQ. All the validation results

complied with CDER (1994) and European Commission (2017) recommendations.

Thus, in this study, we evaluated the use of hydroxyproline as a primary screening tool

to detect the presence of animal-derived collagen and gelatin in various food products. This

may help halal authority bodies to ascertain whether certain foodstuffs require further

investigation for gelatin and collagen, either originating from bovine or porcine animals.

14
Further study into differentiating bovine and porcine gelatin as well as bovine and porcine

collagen based on hydroxyproline is warranted. We are currently focusing on an isotopomics

approach for the aforementioned proposed work.

Funding: The study was supported by the Geran Putra – Inisiatif Putra Muda [GP-

IPM/2019/9676200] from Universiti Putra Malaysia, Serdang, Selangor, Malaysia, and the

Consortium of Malaysia IPT Halal Institutes (KIHIM) [no. 63900911-10205] from the

Ministry of Higher Education, Malaysia.

Compliance with Ethical Standards

Conflict of Interest: Mohd Hafis Yuswan declares that he has no conflicts of interest. Nurul

Hanani A. Jalil declares that she has no conflicts of interest. Haslina Mohamad declares that

she has no conflicts of interest. Shamsidah Keso declares that she has no conflicts of interest.

Tengku Shahrul Tengku, Md. Yusoff declares that he has no conflicts of interest. Nurhidayatul

Asma Mohamad declares that she has no conflicts of interest. Nor Falahiah Ismail declares that

she has no conflicts of interest. Yanty Noorzianna Abdul Manaf declares that she has no

conflicts of interest. Amalia Mohd Hashim declares that she has no conflicts of interest. Mohd

Nasir Mohd Desa declares that he has no conflicts of interest. Yus Aniza Yusof declares that

she has no conflicts of interest. Shuhaimi Mustafa declares that he has no conflicts of interest.

Ethical approval: This article does not contain any experiments with human participants or

animals performed by any of the authors.

15
Informed Consent: Informed consent is not applicable in this study.

References

Abdullah, M. S. P., Noordin, M. I., Nyamathulla, S., Ismail, S. I. M., Jasamai, M., Wai, L. K.,

… Shamsuddin, A. F. (2016). Physicochemical evaluation and spectroscopic

characterisation of gelatine from shank and toes of Gallus gallus domesticus. Sains

Malaysiana, 45(3), 435–449.

Aisyah, N., Huda, N., Azhar, M., & Fazilah, A. (2014). Poultry as an alternative source of

gelatin. Health and the Environment Journal, 5(1), 37–49.

Ali, E., Sultana, S., Hamid, S. B. A., Hossain, M., Yehya, W. A., Kader, A., & Bhargava, S.

K. (2018). Gelatin controversies in food, pharmaceuticals, and personal care products:

authentication methods, current status, and future challenges. Critical Reviews in Food

Science and Nutrition, 58(9), 1495–1511.

Allard, A., & Amarouche, S. (2017). Analysis of interlaboratory comparison when the

measurements are not normally distributed. In C. Corletto (Ed.), 18th International

Congress of Metrology (Vol. 12003, p. 12003). Les Ulis, France: EDP Sciences.

https://doi.org/10.1051/metrology/201712003

Alves, A., Marques, A., Martins, E., Silva, T., & Reis, R. (2017). Cosmetic potential of marine

fish skin collagen. Cosmetics, 4(39), 1–16.

Anonymous. (2017). General requirement for the competence of testing and calibration

laboratories (ISO/IEC 17025).

Ardekani, V. S., Mahmoodani, F., See, S. F., Yusop, S. M., & Babji, A. S. (2013). Processing

16
 optimization and characterization of gelatin from catfish (Clarias gariepinus) skin. Sains

Malaysiana, 42(12), 1697–1705.

Atma, Y., Lioe, H. N., Prangdimurti, E., Seftiono, H., Taufik, M., Fitriani, D., & Mustopa, A.

Z. (2018). The hydroxyproline content of fish bone gelatin from Indonesian Pangasius

catfish by enzymatic hydrolysis for producing the bioactive peptide. Bioinformasi Journal

of Natural Product Biochemistry, 16(2), 64–68.

Azilawati, M. I., Hashim, D. M., Jamilah, B., & Amin, I. (2015). RP-HPLC method using 6-

aminoquinolyl-N-hydroxysuccinimidyl carbamate incorporated with normalization

technique in principal component analysis to differentiate the bovine, porcine and fish

gelatins. Food Chemistry, 172, 368–376. https://doi.org/10.1016/j.foodchem.2014.09.093

Baer, A. A., Miller, M. J., & Dilger, A. C. (2013). Pathogens of interest to the pork industry: a

review of research on interventions to assure food safety. Comprehensive Reviews in Food

Science and Food Safety, 12(2), 183–217.

Buckley, M. (2016). Species identification of bovine, ovine and porcine type 1 collagen;

comparing peptide mass fingerprinting and LC-Based proteomics methods. International

Journal of Molecular Sciences, 17(4), 445.

Center for Drug Evaluation and Research (CDER). (1994). Reviewer Guidance’ Validation of

Chromatographic Methods. Rockville, MD, USA.: FDA.

Cheng, X., Shao, Z., Li, C., Yu, L., Raja, M. A., & Liu, C. (2017). Isolation, characterization

and evaluation of collagen from jellyfish Rhopilema esculentum Kishinouye for use in

hemostatic applications. PLOS ONE, 12(1), e0169731.

Djurković-Djaković, O., Bobić, B., Nikolić, A., Klun, I., & Dupouy-Camet, J. (2013). Pork as

17
 a source of human parasitic infection. Clinical Microbiology and Infection, 19(7), 586–

594.

European Commission. (2017). Guidance document on analytical quality control and method

validation procedures for pesticides residues analysis in food and feed.

SANTE/11813/2017. European Commission Directorate-General for Health and Food

Safety, 1–46. https://doi.org/10.13140/RG.2.2.33021.77283

Gorgieva, S., & Kokol, V. (2011). Collagen- vs. gelatine-based biomaterials and their

biocompatibility: review and perspectives. In P. R. Pignatello (Ed.), Biomaterials

Applications for Nanomedicine (pp. 1–36). InTech. https://doi.org/10.5772/24118

Hashim, P., Mohd Ridzwan, M. S., Bakar, J., & Mat Hashim, D. (2015). Collagen in food and

beverage industries. International Food Research Journal, 22(1), 1–8.

Khattak, J. Z. K., Mir, A., Anwar, Z., Wahedi, H. M., Abbas, G., Khattak, H. Z. K., &

Ismatullah, H. (2011). Concept of halal food and biotechnology. Advance Journal of Food

Science and Technology, 3(5), 385–389.

McGlone, J. (2013). The future of pork production in the world: towards sustainable, welfare-

positive systems. Animals, 3(2), 401–415.

Milovanovic, I., & Hayes, M. (2018). Marine gelatine from rest raw materials. Applied

Sciences, 8(12), 2407.

Mulyani, S., Setyabudi, F. M. C. S., Pranoto, Y., & Santoso, U. (2017). Physicochemical

properties of gelatin extracted from buffalo hide pretreated with different acids. Korean

Journal for Food Science of Animal Resources, 37(5), 708–715.

Necas, J., & Bartosikova, L. (2013). Carrageenan: a review. Veterinarni Medicina, 58(4), 187–
18
 205.

Noor, H. M. (2018). Potential of carrageenans in foods and medical applications. Global Health

Management Journal, 2(2), 32–36.

Nur Azira, T., Amin, I., & Che Man, Y. B. (2012). Differentitation of bovine and porcine

gelatins in processed products via sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE) and principal component analysis (PCA) techiques.

International Food Research Journal, 19(3), 1175–1180.

Osorio, M. T., Downey, G., Moloney, A. P., Röhrle, F. T., Luciano, G., Schmidt, O., &

Monahan, F. J. (2013). Beef authentication using dietary markers: chemometric selection

and modelling of significant beef biomarkers using concatenated data from multiple

analytical methods. Food Chemistry, 141(3), 2795–2801.

Pew Research Center. (2017). The changing global religious landscape.

Rahman, M. M., Ali, M. E., Hamid, S. B. A., Mustafa, S., Hashim, U., & Hanapi, U. K. (2014).

Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog

meat adulteration in meatball formulation. Meat Science, 97(4), 404–409.

Raja Nhari, R. M. H., Che Man, Y., Ismail, A., & Anuar, N. (2011). Chemical and functional

properties of bovine and porcine skin gelatin. International Food Research Journal, 18,

813–817.

Rakhmanova, A., Khan, Z. A., Sharif, R., & Lü, X. (2018). Meeting the requirements of halal

gelatin: a mini review. MOJ Food Processing & Technology, 6(6), 477–482.

Sanaei, A. V., Mahmoodani, F., See, S. F., Yusop, S. M., & Babji, A. S. (2013). Optimization

of gelatin extraction and physico-chemical properties of catfish (Clarias gariepinus) bone

19
 gelatin. International Food Research Journal, 20(1), 423–430.

Shoulders, M. D., & Raines, R. T. (2009). Collagen structure and stability. Annual Review of

Biochemistry, 78(1), 929–958.

Shrivastava, A., & Gupta, V. (2011). Methods for the determination of limit of detection and

limit of quantitation of the analytical methods. Chronicles of Young Scientists, 2(1), 21.

https://doi.org/10.4103/2229-5186.79345

Softić, A., Zaimović-Uzunović, N., & Basić, H. (2012). Proficiency testing and interlaboratory

comparisons in laboratory for dimensional measurement. Journal of Trends in the

Development of Machinery and Associated Technology, 16(1), 115–118.

Song, H., & Li, B. (2017). Beneficial effects of collagen hydrolysate: a review on recent

developments. Biomedical Journal of Scientific & Technical Research, 1(2), 1–14.

Stoilov, I., Starcher, B. C., Mecham, R. P., & Broekelmann, T. J. (2018). Measurement of

elastin, collagen, and total protein levels in tissues. In Methods in Cell Biology (1st ed.,

Vol. 143, pp. 133–146). Elsevier Inc.

Szewczak, E., & Bondarzewski, A. (2016). Is the assessment of interlaboratory comparison

results for a small number of tests and limited number of participants reliable and rational?

Accreditation and Quality Assurance, 21(2), 91–100.

Visser, R. G. (2006). Interpretation of interlaboratory comparison results to evaluate laboratory

proficiency. Accreditation and Quality Assurance, 10, 521–526.

Wichuda, J., Sunthorn, C., & Busarakum, P. (2016). Comparison of the properties of collagen

extracted from dried jellyfish and dried squid. African Journal of Biotechnology, 15(16),

642–648.
20
Wold, S., & Sjöström, M. (1998). Chemometrics, present and future success. Chemometrics

and Intelligent Laboratory Systems, 44(1–2), 3–14.

Yuswan, M. H., Aizat, W. M., Desa, M. N. M., Hashim, A. M., Rahim, N. A., Mustafa, S., …

Lamasudin, D. U. (2019). Improved gel-enhanced liquid chromatography-mass

spectrometry by chemometrics for halal proteomics. Chemometrics and Intelligent

Laboratory Systems, 192, 103825. https://doi.org/10.1016/j.chemolab.2019.103825

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Figure 1. Chromatographic separation of 17 amino acids in (a) porcine gelatin, (b) porcine

collagen, and (c) carrageenan samples.

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Figure 2. Principal component analysis (PCA) results. (a) PCA score plot for the distribution

pattern of positive control samples (porcine gelatin N = 7, bovine gelatin N = 6, fish gelatin N

= 8, porcine collagen N = 8, collagen fish N = 8), negative control samples (carrageenan N =

7), and 15 test samples (sea cucumber extract, flour extract, dried date, insect meal, crystal

pure, chicken feed, bread, polymer cells, algae, camel meat, edible bird’s nest, bean powder,

jellyfish protein, herb powder, blackcurrant); and (b) PCA loading plot for the distribution

pattern of 17 amino acids correlated to the type of samples as tabulated in the score plot.

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Table 1. Amounts of hydroxyproline among positive control and negative control samples.

Sample Amount of sample (N) Amount of hydroxyproline1 (% w/w)

Positive Control
Collagen porcine skin
Collagen fish skin
Gelatin porcine skin
Gelatin bovine skin
Gelatin fish skin
8 5.29 ± 0.05 b
8 5.18 ± 0.06 b
7 6.35 ± 0.08 c
6 6.52 ± 0.99 c
8 3.82 ± 0.04 a

Negative Control
Carrageenan 7 Not Detected

Total samples 44
1 Values are the means from replicates (N), with the corresponding standard deviation. Means

with different superscripts are significantly different at p-value <0.05 by one-way univariate

ANOVA.

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Table 2. Validation parameters for hydroxyproline determination

Recovery (%)b Precision (% RSD)b

Amino acid Linearity LODc LOQd
(R2)a
L1 L5 L1 L5

Hydroxyproline 0.9997 85.104 99.945 15.435 1.796 2.017 6.722

aLinearity was determined from five concentration levels of serial amino acid standard solution

(L1: 25 pmol, L2: 100 pmol, L3: 250 pmol, L4: 500 pmol, and L5: 1000 pmol).
b3.27 and 131.13 ng/µL of hydroxyproline are spiked into LI and L5, respectively.

Measurements were taken from 10 replicates under the same analytical conditions.
cLOD = limit of detection in ng/µL.
dLOQ = limit of quantification in ng/µL.

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Table 3. Inter- and intra-laboratory comparisons (ILC) for hydroxyproline determination

Amount of hydroxyproline (% w/w)

Comparison z-score Assessment
Reference value1 (𝑥) Observed value2 (𝑥)
Our lab
laboratory

Analyst 1 11.51 -0.06 Satisfactory

Intra-

Analyst 2 11.63 13.73 1.19 Satisfactory

Analyst 3 10.44 -0.67 Satisfactory
Analyst 4 10.82 -0.46 Satisfactory
laboratory

Participating lab
Inter-

Replicate 1 11.63 14.38 1.68 Satisfactory

Replicate 2 14.32 1.64 Satisfactory

1 Reference value is a mean of observed value from four analysts.

2 Observed value is an experimental value from amino acid profiling.

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Table 4. Amounts of hydroxyproline in the 15 test samples

Amount of hydroxyproline
Test Sample Assessment
(% w/w)
Algae 0.00 Not detected
Bean powder 0.00 Not detected
Sea cucumber extract 0.00 Not detected
Flour powder 0.63 Below LOD and LOQ
Dried date 0.22 Below LOD and LOQ
Insect meal 0.06 Below LOD and LOQ
Herb powder 0.62 Below LOD and LOQ
Camel meat 0.51 Below LOD and LOQ
Chicken grower pellet 0.00 Not detected
Bread 0.00 Not detected
Polymer cells 0.00 Not detected
Blackcurrant 0.00 Not detected
Edible bird's nest 0.00 Not detected
Jellyfish protein 5.84 Clearly Detected
Crystal pure 10.21 Clearly Detected

Highlights:

 Hydroxyproline is present in gelatin and collagen, not in plant-based samples.

 Plant- and animal-based samples can be distinguished based on hydroxyproline.

 The hydroxyproline assay can be used to identify halal-critical ingredients.

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