foods

Article
Extensive Chemometric Investigations of Distinctive
Patterns and Levels of Biogenic Amines in Fermented
Foods: Human Health Implications
Martin Grootveld 1, * , Benita C. Percival 1 and Jie Zhang 2
1 Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK;
[email protected]
2 Green Pasture Products, 416 E. Fremont Street, O’Neill, NE 68763, USA; [email protected]
* Correspondence: [email protected]; Tel.: +44-0-116-250-6443




Received: 19 October 2020; Accepted: 27 November 2020; Published: 5 December 2020


Abstract: Although biogenic amines (BAs) present in fermented foods exert important
health-promoting and physiological function support roles, their excessive ingestion can give rise to
deleterious toxicological effects. Therefore, here we have screened the BA contents and supporting
food quality indices of a series of fermented food products using a multianalyte-chemometrics
strategy. A liquid chromatographic triple quadrupole mass spectrometric (LC-MS/MS) technique was
utilized for the simultaneous multicomponent analysis of 8 different BAs, and titratable acidity, pH,
total lipid content, and thiobarbituric acid-reactive substances (TBARS) values were also determined.
Rigorous univariate and multivariate (MV) chemometric data analysis strategies were employed
to evaluate results acquired. Almost all foods analyzed had individual and total BA contents that
were within recommended limits. The chemometrics methods applied were useful for recognizing
characteristic patterns of BA analytes and food quality measures between some fermented food classes,
and for assessing their inter-relationships and potential metabolic sources. MV analysis of constant
sum-normalized BA profile data demonstrated characteristic signatures for cheese (cadaverine
only), fermented cod liver oil (2-phenylethylamine, tyramine, and tryptamine), and wine/vinegar
products (putrescine, spermidine, and spermine). In conclusion, this LC-MS/MS-linked chemometrics
approach was valuable for (1) contrasting and distinguishing BA catabolite signatures between
differing fermented foods, and (2) exploring and evaluating the health benefits and/or possible
adverse public health risks of such products.

Keywords: biogenic amines (BAs); fermented foods; chemometrics; multivariate (MV) statistical
analysis; liquid chromatographic triple quadrupole mass spectrometric (LC-MS/MS) analysis;
public health; lipid peroxidation; antioxidants

1. Introduction
Biogenic amines (BAs) may be biosynthesized and degraded via normal metabolic activities in
animals, plants, and micro-organisms. As such, these amines occur in a wide variety of foods, such as
fish, meat, and cheese products, and especially in fermented foods such as wines, and yoghurts,
etc. [1–3]. BA formation in foods usually occurs via the decarboxylation of amino acids [3], of which
there are rich sources in these matrices; for example, amino acids are present at very high levels in
grapes, and comprise ca. 30–40% of the total nitrogen content of wines [1–3].
Metabolic pathways available in lactic acid bacteria, which have the ability to grow and
thrive in foods and beverages, generate significant levels of BAs. Routes available for this are the
enzymatic production of putrescine from ornithine (catalyzed by ornithine decarboxylase) and/or from

Foods 2020, 9, 1807; doi:10.3390/foods9121807 www.mdpi.com/journal/foods

Foods 2020, 9, 1807 2 of 28

arginine via agmatine, a scheme involving prior conversion of the amino acid substrate to agmatine
with arginine decarboxylase, followed by transformation of agmatine to N-carbamoylputrescine
via the action of agmatine imino-hydroxylase, and then on to putrescine (a second route for
its generation involves the conversion of arginine to ornithine and then to this product via
the above ornithine decarboxylase-catalyzed route); putrescine to spermine, a process involving
the enzyme spermine synthase, and then spermine to spermidine via the actions of spermidine
synthase; cadaverine from lysine with lysine carboxylase and a pyridoxal phosphate co-factor;
2-phenylethylamine from phenylalanine catalyzed by aromatic amino acid carboxylases, including
tyrosine decarboxylase; tyramine from tyrosine via tyrosine decarboxylase action; histamine from
histidine with histidine decarboxylase; tryptamine from tryptophan with trypotophan decarboxylase,
another pyridoxal phosphate-dependent enzyme; and trimethylamine from trimethylamine-N-oxide
with a trimethylamine-N-oxide reductase (enzymes involved in the conversion of amino acids to BAs
are classified as decarboxylase deaminases) [4,5]. BAs may also be biosynthesized from the amination
and transamination of aldehydes and ketones [5], and this may be of some relevance to their detection
in marine oil products which have been allowed to autoxidize. Indeed, a range of aldehyde species
arise from the fragmentation of conjugated hydroperoxydienes, which are lipid oxidation products
resulting from the peroxidation of polyunsaturated fatty acids (PUFAs) [6].
Overall, microbial sources of BAs include yeasts, as well as gram-positive and -negative bacteria [7].
The physiological activity of BA synthesis in prokaryotic cells predominantly appears to be associated
with bacterial defense mechanisms employed to combat environmental acidity [8–10]. Hence,
amino acid decarboxylation in this manner enhances survival under harsh acidic stress states [9] via
proton consumption, and amine and CO2 excretion required to facilitate restorations of internal pH
values [11].
As with their biosynthesis, the catabolism of BAs is extensively outlined and reviewed in [5].
In view of their potentially toxic nature, fortunately humans have detoxification enzyme systems
which catabolically oxidize BAs in vivo. These enzymes principally comprise monoamine and diamine
oxidases (MAOs and DAOs respectively). MAOs are flavoproteins acting by the oxidative deamination
of BAs to their corresponding aldehydes, along with hydrogen peroxide (H2 O2 ) and ammonia.
Two different forms of MAO have been identified in humans [5]. DAOs are responsible for histamine
catabolism, as is histamine-N-methyltransferase, the latter catalyzing a ring methylation process [5].
Evidence available indicates that BAs may confer a series of human health benefits, which involve
their interactions with a wide variety of intracellular macromolecules such as proteins, DNA, and RNA.
Indeed, monoamines are typically precursors of neuromodulators and neurotransmitters [12]. Moreover,
evidence is accumulating that the polyamines spermine and spermidine are important for sexual
function and fertility [13], and polyamines in general are associated with cell growth and differentiation,
including protein biosynthesis [14]. Indeed, the generation of BAs in eukaryotic cells is essential, since
they are required for the critical biosynthesis of hormones, alkaloids, proteins, and nucleic acids [15].
One further plausible health benefit offered by both monoamine and polyamine forms of BAs is
their antioxidant potential [16], and recent studies have shown that they function efficiently in this
context, and protect against adverse unsaturated fatty acid peroxidation reactions when present in or
supplemented to culinary oils, and other foods rich in PUFAs [6] (details regarding the nature and
mechanisms of these antioxidant actions are provided in Section S1 of the Supplementary Materials).
Notwithstanding, the availability of these amines in the diet has not been without its problems.
Indeed, adverse toxicological events may be stimulated by the ingestion of foods which are known to
provide high concentrations of these agents, and one notable example is the provocation of deleterious
hypertensive events in patients receiving therapies with monoamine oxidase inhibitor (MAOI) drug
treatments [17]. A further problem is the depression of histamine oxidation, a process which arises from
the ingestion of putrescine and agmatine, which serve as potentiators of this process; this promotes
histamine toxicity episodes in humans [18]. Moreover, it has been reported that BAs such as putrescine
Foods 2020, 9, 1807 3 of 28

and agmatine give rise to their corresponding carcinogenic nitrosoamines from reactions with nitrite
anion, dietary or in vivo [19].
Human sensitivity to BAs is contingent on the availability and activities of detoxifying enzymes
featured in BA metabolism, i.e., specific ones such as histamine methyltransferase, and those less
specific such as mono- and diamine oxidases. However, since these enzymes are inhibited by different
classes of drugs, including neuromuscular blocking agents such as alcuronium, antidepressants [20],
and ethanol [21], the accumulation of BAs by the consumption of selected foods and beverages can,
at least in principal, give rise to clinical disorders, including the extremely hazardous serotonin
syndrome [22]. Further details regarding the adverse health effects associated with the excessive intake
of BAs are delineated in Section S2 of the Supplementary Materials.
Current consumer demands for safer and healthier foods has prompted a high level of research
investigations focused on BAs, although it should be noted that further studies are required to
expand this area. High levels of BAs can build up in fermented foods, including fish, fish sauce,
and cheese products. Their biosynthesis and accumulation therein are critically dependent on the
availability of bacteria with decarboxylase-deaminase enzyme activities, environmental conditions that
are unrestrictive towards their growth and propagation, and the efficient functioning of BA-generating
enzymes, together with the presence of sufficient amounts of the relevant amino acid substrates required.
Hence, supporting analytical methodologies for the identification and measurement of BAs are
of much importance to the food industry, and also from a public health perspective. Such methods
should ideally offer high levels of reliability in order to monitor the potential health benefits offered
by fermented food products, and also to circumvent any toxicological risks to consumers arising
from their excessive production therein; realistic estimates of their human consumption are also
major factors for consideration. To date, BA determinations in foods have represented a major
challenge for analytical chemists in view of their non-chromophoric nature, their natural occurrence
in complex multicomponent food and biological matrices, and high polarities, factors which are
further complicated by a requirement for high analytical sensitivity, potential interferences, and,
where relevant, chromatographic separation/resolution issues arising from the presence of many
structurally-related agents in samples requiring such analysis [23]. Methods previously available for
this purpose, and those for the screening of BA-producing bacteria, are outlined in Section S3 of the
Supplementary Materials.
Notwithstanding, in principle, the simultaneous and direct multicomponent determination of
BAs by the LC-MS/MS method described here, or a newly-developed strategy focused on largely
non-invasive high-resolution proton (1 H) nuclear magnetic resonance (NMR) analysis [6], serve as
valuable assets which, in combination with MV chemometrics strategies, may be employed for the
recognition of patterns of these bacterial catabolites which are characteristic of differential bacterial
sources of these agents.
Multivariate (MV) data analysis of multicomponent analytical datasets serves as an extremely
powerful means of probing and tracking metabolic signatures that are characteristic of differential
groups or classifications of samples, and when applied to explore the biochemical basis of human
disease etiology, this technique is commonly known as metabolomics [24]. Indeed, to date this
combination of multianalyte-MV analysis has been copiously utilized in many biomedical and clinical
investigations, mainly for the identification of diagnostic or prognostic monitoring biomarkers for
human diseases. However, when applied in a non-biomedical context, the technique can best be
described as chemometrics, a technology which also commonly employs many of the MV data analysis
strategies used in metabolomics experiments.
In view of the rich sources of BAs in fermented food products, in this study we determined the
contents of a total of 8 different BAs in a series of commercially-available fermented fish, fish sauce/paste,
vegetable sauce, cheese, wine/vinegar, and cod liver oil (FCLO) products. For this purpose, we employed
both univariate and MV chemometrics analysis techniques in order to recognize differential patterns
of these catabolites, which may be representative or characteristic of their food, bacterial, metabolic
Foods 2020, 9, 1807 4 of 28

pathway, and/or food processing technology sources. Such analytical information also serves to furnish
us with valuable information regarding the provision of these important nutrients in the human diet,
and to evaluate the toxicological/adverse health risks presented by the ingestion of fermented foods
containing portentously excessive levels of these agents. Currently, a total BA content of ca. 1000 ppm
is linked to toxicity, and in recommended manufacturing practices, 100 ppm histamine, or a total BA
content of 200 ppm, are considered acceptable levels which do not give rise to any associated adverse
health effects [25].
These studies were supported by the consideration of further food quality determinations on
these fermented food products, which consisted of pH values, titratable acidities (TAs), and total lipid
contents, along with an adapted method for determining lipid peroxidation status (thiobarbituric
acid-reactive substances (TBARS)).
With the exception of a small number of studies focused on BAs detectable in selected wine
products, e.g., [26], to the best of our knowledge this is the first time that MV chemometrics techniques
have been applied to explore potentially valuable “between-food classification: distinctions between
the concentrations and patterns of BAs in a series of different food products, albeit fermented ones.
Therefore, the aims of this investigation are to explore the abilities and reliabilities of LC-MS/MS-based
chemometrics analysis techniques to: (1) evaluate the possible public health benefits and/or risks
of BAs arising from the human consumption of fermented foods; and (2) effectively compare and
distinguish between differing patterns of BA molecules in different classes of fermented food products.

2. Materials and Methods

2.1. Fermented Food Products

Fermented food products (cheese, fish, fish sauce/paste, vegetable sauce, and wine/vinegar
classifications) were randomly selected and purchased from a variety of US retail outlets based in the
state of Nebraska. These comprised n = 4 fish samples, n = 9 fish sauce/paste samples, n = 4 vegetable
sauce samples, n = 5 cheeses, and n = 4 wine/vinegar samples (Table 1). Details of the fermentation
processes employed by the manufacturers involved were unavailable. Prior to analysis, all samples
were stored in a darkened freezer at a temperature of −20 ◦ C for a maximal duration of 72 h.

Table 1. Details of fermented food products investigated for each classification.

Fermented Food Classification Products Investigated

Full-fat pasteurized cow’s milk soft cheese (washed with
brandy); full-fat French cow’s milk soft-ripened cheese;
Cheeses
semi-soft washed rind Limberger cheese; full-fat pasteurized
cow’s milk soft cheese; French cow’s milk soft cheese.
Pickled mud fish; pickled gourami fish; dried gourami fish;
Fish
salted crab.
Loc fish sauce; scad fish sauce; anchovy fish sauce;
Fish Sauce/Paste Vietnamese fish sauce (×2); Thai fish sauce; standard U.S. fish
sauce; shrimp paste (×2).
Vegetable Sauce Bean curd; chili bean sauce; kimchi sauce; spicy tofu sauce.
Wine/Vinegar Balsamic vinegar (×2); red wine vinegar; Casella wine.

Fermented cod liver oil (FCLO) was a natural product that was manufactured and kindly donated
by Green Pastures LLC, 416 E. Fremont O’Neill, NE 68763, USA for this study. Separate batches
(n = 10) of this FCLO product were randomly selected by independent visitors to its manufacturing
site throughout a 6-month period, as noted in [6].
FCLO products were prepared from the fermentation of Pacific cod livers. Livers were frozen
(−20 ◦ C) within 40 min following their harvest from the Pacific Ocean, and then transported to a
Foods 2020, 9, 1807 5 of 28

preparation facility whilst remaining in the frozen state. Fermented CLO was produced from these cod
liver sources using a novel and proprietary fermentation technology. Briefly, cod livers were loaded
into a fermentation tank, and both salt and the fermentation starter agent were added to induce the
process. The tank was completely sealed during the fermentation and, following periods of 28–84 days,
the raw FCLO product accumulated and was then isolated from the tank. Following fermentation,
products were centrifuged, filtered to remove particulates, and then packed.
On arrival at the laboratory, FCLO product sample batches were de-identified through their
transfer to coded but unlabeled universal storage containers. Each sample was subsequently stored in
a darkened freezer at −80 ◦ C until ready for analysis (predominantly within 24 h of their arrival).

2.2. Analysis of BAs in Fermented Food Product Samples

A liquid chromatographic triple quadrupole mass spectrometric (LC-MS/MS) technique was
employed for the simultaneous analysis of up to 11 BAs in fermented food products using an adaption
of the LC-MS/MS method reported in [27]. A Shimadzu 8045 LC-MS/MS facility was used for this
purpose, the MS/MS detection system for the monitoring and molecular characterization of eluting BA
analytes. Primarily, pre-set accurately weighed masses of food samples were shaken with a 20.0 mL
volume of 70% (v/v) methanol/30% (v/v) water for 20 min, which were then centrifuged at 7000 rpm at
4 ◦ C for another 20 min period. The clear supernatant was subsequently transferred to 1.7 mL volume
amber auto-sampler vials for LC-MS/MS analysis. For wine/vinegar and FCLO samples, fixed aliquots
were filtered using a 0.45 µm filter paper prior to the above methanol/water extraction stage.
The LC facility comprised a pump, vacuum degasser, auto-sampler, and column compartment,
and finally a secondary variable wavelength spectrophotometric detection system was used for
these analyses. This system could operate up to 800 bar. The internal standard (IS) utilized
was tetra-deuterated histamine (histamine-α,α,β,β−d4 , (2HCl)), which was purchased from C/D/N
Isotopes Inc. (Pointe-Claire, Quebec, Canada). IS m/z values employed for quantification purposes
were 116.1 and 99.0 for precursor and product ions, respectively (112.1 and 95.1 respectively for
undeuterated histamine).
A 3-µm 50 × 2.1 mm Pinnacle® DB pentaflurophenyl (PFP) base with propyl spacer column
was employed for optimal BA analysis. Mobile phase 1 contained water solutions of the ion-pair
reagent trifluoroacetic acid (TFA) (either 0.05 or 0.10% (w/v)), and mobile phase 2 was acetonitrile
containing equivalent TFA concentrations. BA analytes were monitored in positive ion mode for
the MS/MS detection system. Reporting limit values for fermented food samples were 1 ppm for all
BAs determined.
Authentic BA calibration standards were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA) (histamine, H7125; cadaverine, 33220; putrescine, D13208; 2-phenylethylamine, P6513;
spermidine, 85578; tyramine, T2879; tryptamine, 193747), and Alfa Aesar Inc. (Heysham, UK)
(spermine, J63060). BA contents were determined from calibration curves developed with standard
solutions of concentrations 0.5, 1.0, 10.0, 50.00, 100.0, 200.0, and 400.0 ppb for each BA.

2.3. Total Lipid Analysis

Total lipid (fat) analysis was performed according to the AOAC 922.06 method. Briefly,
homogenized samples were treated with HCl, and then washed at least two-fold with both petroleum
ether and diethyl ether; solutions arising therefrom were then placed in pre-weighed beaker containers.
Subsequently, the lipid-containing ether solutions were evaporated, and the (w/w) % content of lipid
was determined directly from the weight gain of the container.

2.4. Determination of Thiobarbituric Acid-Reactive Substances (TBARS) Values

Primarily, accurately-weighed samples were digested with perchloric acid (HClO4 ),
and subsequently the resulting clear filtered supernatant solution was reacted with thiobarbituric acid
(TBA) for a period of 15–18 h at 27.5 ◦ C according to the method outlined in [28]. The absorbance value
Foods 2020, 9, 1807 6 of 28

at a wavelength of 532 nm was then determined, and TBA-reactive substance (TBARS) values were
reported as mg/kg (ppm) units following their quantification from a calibration curve developed with
MDA standards.

2.5. Titratable Acidity (TA) and pH Value Determinations

Titratable acidity values were determined using the AOAC 947.05 method [29], and pH
measurements were made using a modified FO PROC 31 protocol which is based on the USDA
PHM method. The latter approach is based on the formation of a homogenized food/water slurry
which was allowed to stand prior to pH determination with a probe.

2.6. Experimental Design and Statistical Analysis

2.6.1. Univariate Statistical Analysis

The experimental design for univariate analysis of the individual BA, TA, pH, and further variable
dataset involved an analysis-of-variance (ANOVA) model, which incorporated 1 prime factor and 2
sources of variation: (1) that “between-fermented food classifications”, a qualitative fixed effect (FFi );
and (2) experimental error (eij ). The mathematical model for this experimental design is shown in
equation 1, in which yij represents the (univariate) BA or alternative analyte dependent variable values
observed, and µ their overall population mean values in the absence of any significant, influential
sources of variation.
yij = µ + FFi + eij (1)

ANOVA was conducted with XLSTAT2016 and 2020 software. Datasets were autoscaled
(i.e., the mean value of each parameter monitored was subtracted from each entry, and the residual then
divided by food class standard deviation, which was computed with an (n − 1) divisor) prior to analysis.
In view of heterogeneities between the intra-sample variances of fermented food classifications,
i.e., heteroscedasticities, the robust Welch test was employed to determine statistical significance of
differences observed between the mean BA and other food quality variable values for each fermented
food group. post-hoc ANOVA evaluations of the statistical significance of differences between the mean
values of individual fermented food groups were performed using the Bonferroni test.
A similar ANOVA-based experimental design was applied to additional design models selected
to determine the statistical significance and food class specificities of BA analytes only. For these
purposes, the 8 BA dataset, which included those determined in the n = 10 batches of the FCLO
product, was either constant sum (CS)-normalized or not, and then generalized logarithmically
(glog)-transformed, and finally autoscaled prior to analysis. The CS normalization data preparation
task was applied in order to evaluate the significance of fermented food classification-dependent BA
profile patterns. The non-CS-normalized dataset also included total BA level as a further possible
explanatory variable. MetaboAnalyst 4.0 (University of Alberta and National Research Council, National
Institute for Nanotechnology (NINT), Edmonton, AB, Canada) was utilized for the analysis of these
data. Probability values obtained from post-hoc ANOVA comparisons of individual BA levels between
fermented food classes were false discovery rate (FDR)-corrected.
Tests for the heteroscedasticity of ANOVA model residuals (Levene’s test) were performed using
XLSTAT2020 (Addinsoft, Paris, France).

2.6.2. Multivariate Chemometrics and Algorithmic Computational Intelligence (CI) Analyses

Principal component analysis (PCA), partial least squares-discriminatory analysis (PLS-DA),
correlation, and agglomerative hierarchical clustering (AHC) analyses of the combined BA dataset
were performed using XLSTAT2016 and 2020 and MetaboAnalyst 4.0 [30] software module options.
The dataset was generalized glog-transformed, and autoscaled prior to MetaboAnalyst 4.0 analysis,
but only autoscaled for XLSTAT2016 and 2020 analyses. All these MV analysis strategies were primarily
performed on non-CS-normalized data. For the PCA and PLS-DA analyses, limits for significant
Foods 2020, 9, 1807 7 of 28

explanatory variable loadings vectors/coefficients were set at ≤−0.40 or ≥0.40. Validation of PLS-DA
models was performed by determining component number-dependent Q2 values (predominantly for
two classification comparisons), and permutation testing with 2000 permutations. The significance of
variable contributions to these models was determined by the computation of variable importance
parameter (VIP) values (values >0.90 were considered significant).
Additional PCA analysis was performed in order to explore associations or independencies of
individual BAs and other active variables considered, e.g., pH and TA values, total lipid contents,
etc. For this purpose, a maximal 5 PC limit was applied, and PCA was then conducted on autoscaled
data using varimax rotation and Kaiser normalization. The loadings of each analytical variable on
successive orthogonal PCs was then sequentially evaluated. Similarly, this form of PCA was employed
to investigate possible inter-relationships and orthogonalities between BA variables analyzed in FCLO
batches sampled from the same manufacturing source specified above.
A further PCA model involved its application to the 8 BA dataset alone, which was either
CS-normalized or not, glog-transformed, and autoscaled prior to analysis. As noted above,
the CS-normalization data preparation step was utilized in order to evaluate the significance of
any differential patterns or distributions of BA analytes which may be characteristic of fermented food
classifications. This analysis was performed using MetaboAnalyst 4.0.
The random forest (RF) machine-learning algorithm approach was also utilized for classification
and discriminatory variable selection purposes (MetaboAnalyst 4.0 Random Forest module), with 1000
trees (ntree) and 4 predictors selected at each node (mtry) subsequent to tuning. The dataset was
randomly split into training and test sets containing approximately two-thirds and one-third of entries
respectively. The training set was employed to construct the RFs model, and an out-of-the-bag
(OOB) error value was determined to evaluate the classification performance of this. Again, this
analysis was performed on the glog-transformed and autoscaled dataset, either with or without prior
CS-normalization as specified in the manuscript.
Missing data, specifically total lipid and (TBARS):(total lipid) ratios for 2 × fish sauce/paste, 1 ×
vegetable sauce, 1 × wine/vinegar, and 1 × cheese samples, were estimated by the support vector
machine (SVM) impute technique [31] (MetaboAnalyst 4.0), or supplementation with the explanatory
variable column mean values, along with a corresponding reduction in degrees of freedom available
for parametric univariate statistical testing (XLSTAT2016 or 2020).

3. Results and Discussion

3.1. BA Levels and Food Quality Indices in Fermented Food Products, and Univariate Analysis of These
Analytical Data
Mean ± SEM values for the individual and total BA contents of the FF products investigated
are provided in Table 2. The major contributors towards the relatively high BA levels observed in
fermented cheese samples were cadaverine (mean 60% of total) and tyramine (mean 21.5% of total).
Although three of the cheese products analyzed had total BA concentrations of 30–63 ppm, two of them
were found to be as high as 666 and 780 ppm, which were markedly above the recommended 200 ppm
content limit. The ANOVA Welch test demonstrated that there were highly significant differences
between these total BA values (Table 3), as expected (p = 2.84 × 10−4 ); such differences were largely
explicable by those observed between the cheese and wine/vinegar product classifications investigated.
Hence, characteristic “markers” of fermented cheese samples appeared to be cadaverine
and tyramine, which had contents markedly elevated over those of the other fermented food
products evaluated, although there were very high intra-fermented food classification variances
for these estimates.
Foods 2020, 9, 1807 8 of 28

Table 2. Biogenic amines (BA) contents and quality indices of fermented foods investigated. Mean
± SEM BA levels, and titratable acidity (TA), pH, total lipid, thiobarbituric acid-reactive substances
(TBARS) and (TBARS):(total lipid) ratio values, for five classes of fermented food products (cheese,
fish, fish sauce/paste, vegetable sauce, and wine/vinegar) purchased at a range of U.S. retail outlets
(bracketed numbers represent the number of different products analyzed for each classification).

Fish Sauce Vegetable Wine/Vinegar

BA Variable/ppm Cheese (5) Fish (4)
(9) Sauce (4) (4)
Cadaverine 191.6 ± 99.8 30.7 ± 5.6 45.2 ± 8.2 30.6 ± 14.0 0.7 ± 0.7
Histamine 5.7 ± 1.6 10.6 ± 2.9 20.0 ± 5.8 17.7 ± 9.5 1.9 ± 1.9
2-Phenylethylamine 11.1 ± 6.8 13.1 ± 8.0 8.3 ± 4.2 5.00 ± 5.00 nd
Putrescine 21.2 ± 14.9 14.9 ± 7.2 18.9 ± 5.1 18.4 ± 7.7 3.3 ± 0.15
Spermidine 9.6 ± 3.2 10.9 ± 2.5 15.0 ± 1.9 24.5 ± 10.1 4.4 ± 1.5
Spermine 3.5 ± 2.2 12.6 ± 4.5 18.7 ± 3.0 11.4 ± 4.3 1.5 ± 1.5
Tryptamine 7.0 ± 5.8 2.0 ± 0.7 5.6 ± 1.7 3.4 ± 1.0 nd
Tyramine 69.4 ± 42.5 8.9 ± 3.7 17.2 ± 4.0 36.4 ± 20.5 0.6 ± 0.4
Total BAs 322.2 ± 166.0 103.8 ± 12.7 155.9 ± 18.7 147.9 ± 56.2 12.4 ± 5.5
Titratable Acidity
1.3 ± 1.1 0.6 ± 0.2 0.6 ± 0.1 0.7 ± 0.2 3.6 ± 1.2
(g acid/100 g)
pH 6.09 ± 1.38 6.33 ± 0.62 5.47 ± 0.28 5.18 ± 0.53 2.99 ± 0.20
Total Lipid (% w/w) 23.3 ± 2.0 6.9 ± 2.7 4.2 ± 1.4 5.1 ± 2.8 1.1 ± 0.2
TBARS Value (ppm) 0.07 ± 0.05 0.35 ± 0.14 0.47 ± 0.27 0.09 ± 0.03 0.83 ± 0.51
102 .(TBARS):(Total
Lipid)
0.5 ± 0.4 10.6 ± 6.85 33.1 ± 22.3 5.6 ± 3.55 98.3 ± 45.7
Ratio (ppm(% w/w)−1 )
nd: not determined.

Table 3. Statistical significance and nature of differences between the mean BA contents and other
food quality indices for fermented food products. Both robust Welch and Bonferroni-corrected post-hoc
ANOVA test significance (p) values are provided. Abbreviations: ns, not statistically significant. * These
values were close to statistical significance, but did not attain a p value of ≤0.05 with the robust
Welch test.

Welch Test Post-hoc Significant Differences

BA/Index
(WT) p Value (All p < 0.05: Bonferroni Test)
Cheese > Wine/Vinegar;
Cheese > Fish Sauce/Paste;
Cadaverine (ppm) 0.0016
Cheese > Fish;
Cheese > Vegetable Sauce
Histamine (ppm) 0.087 * All ns
2-Phenylethylamine (ppm) ns All ns
Putrescine (ppm) 0.068 * All ns
Vegetable Sauce > Wine/Vinegar;
Spermidine (ppm) 0.029 Vegetable Sauce > Cheese;
Vegetable Sauce > Fish
Fish Sauce/Paste > Wine/Vinegar;
Spermine (ppm) 0.010
Fish Sauce/Paste > Cheese
Tryptamine (ppm) ns All ns
Tyramine (ppm) 0.021 Cheese > Wine/Vinegar
Total BAs 2.84 × 10−4 Cheese >> Wine/Vinegar
Foods 2020, 9, 1807 9 of 28

Table 3. Cont.

Welch Test Post-hoc Significant Differences

BA/Index
(WT) p Value (All p < 0.05: Bonferroni Test)
Wine/Vinegar > Fish;
Wine/Vinegar > Fish Sauce/Paste;
Titratable acidity (g acid/100 g) 0.024
Wine/Vinegar > Vegetable Sauce;
Wine/Vinegar > Cheese
Wine/Vinegar < Fish;
Wine/Vinegar < Fish Sauce/Paste;
pH 6.91 × 10−4
Wine/Vinegar < Vegetable Sauce
Wine/Vinegar < Cheese
Cheese > Wine/Vinegar;
Cheese > Fish Sauce/Paste;
Total lipid (% w/w) 1.09 × 10−3
Cheese > Fish;
Cheese > Vegetable Sauce
TBARS value (ppm) ns ns
Wine/Vinegar > Fish;
102 .(TBARS):(Total Lipid) ratio
ns Wine/Vinegar > Vegetable Sauce;
(ppm(% w/w)−1 )
Wine/Vinegar > Cheese
ns: not statistically significant.

Univariate statistical analysis performed by ANOVA (robust Welch test derivative), and also
post-hoc Bonferroni test values, demonstrated that the mean values of each food classification examined
were significantly or highly significantly different for 7 and 9 of the marker index variables respectively
(p values ranging from <0.0003 to 0.04 for the former test, Table 3). Figure 1 shows a heatmap of the
mean BA contents, and further variables included in this analysis; this clearly displays significantly
higher tyramine, cadaverine, putrescine, and tryptamine levels in the fermented cheese products;
higher histamine concentrations in the fish sauces/pastes explored, as expected (although vegetable
sauces also had quite high levels of this BA); and also greater spermine contents in the fish paste/sauce
products (ca. 1.5-fold greater than the mean value found for the fish classification, the next highest
concentration). The vegetable sauce products had the highest mean spermidine levels, whereas the
fermented fish group contained the largest amounts of 2-phenylethylamine detectable.
As expected, mean TA values were significantly greater for the wine/vinegar products than they
were for all the other fermented food classes investigated, and correspondingly the mean pH value
for the former group was significantly lower than those of all the other fermented foods. Of course,
the mean total lipid content of the cheese group (23.3%) was significantly greater than all other food
classifications tested (p ca. 10−3 ), although no significant differences were found for the secondary lipid
peroxidation TBARS marker. However, an examination of the mean ratio of TBARS index to total
lipid content revealed that this value was markedly greater for the wine/vinegar group than that of
all other food product types (Bonferroni-corrected post-hoc ANOVA tests), and significantly so over
that of the cheese samples analyzed, as might be expected in view of the very low fat contents of
fermented wine/vinegar samples (for example, it varies from 0.15–0.44% (w/v) in Zhenjiang aromatic
vinegar samples [32]), and potentially substantially inflated TBARS levels resulting from quite high
levels of TBA-reactive acetaldehyde and acrolein, amongst other aldehydes, present in such fermented
products [33–36]. Indeed, many other aldehydes are reactive towards the TBA reagent, and also form
chromophoric products on reaction with it [28]. Estimates for acetaldehyde in vinegar products can be
as high as 1.0 g/kg respectively [33], but such levels are highly variable, with much lower levels being
found, e.g., 2.6 mg/L (ca. 60 µmol/L) [37].
Foods 2020, 9, 1807 10 of 28
Foods 2020, 9, x FOR PEER REVIEW 11 of 28

1. Heatmap
FigureFigure diagram
1. Heatmap displaying
diagram the nature,
displaying extent and
the nature, ANOVA-based
extent and ANOVA-based significance of univariate
significance of
differences between
univariate mean values
differences betweenof mean
all 8 BA andoffurther
values all 8 BAchemoanalytical food quality food
and further chemoanalytical variables (near
quality
right-hand
variables y-axis)right-hand
side (near for the fermented
side y-axis)cheese (red),
for the fish (green),
fermented cheesefish sauce/paste
(red), (dark
fish (green), fishblue), vegetable
sauce/paste
(darkblue),
sauce (pale blue), and
vegetable sauce (pale
wine/vinegar blue), and
(mauve) wine/vinegar
products. (mauve)dataset
The complete products.
wasThe complete datasetand
glog-transformed
was glog-transformed and autoscaled prior to analysis, but not CS-normalized.
autoscaled prior to analysis, but not CS-normalized. Transformed analyte intensities are shown Transformed analyte
in the
intensities are shown in the far right-hand side y-axis: deep blue and red
far right-hand side y-axis: deep blue and red colorations represent extremes of low and high contents colorations represent
extremes of low and high contents respectively. The left-hand side of the plot shows results arising
respectively. The left-hand side of the plot shows results arising from an associated agglomerative
from an associated agglomerative hierarchical clustering (AHC) analysis of these variables, which
hierarchical clustering (AHC) analysis of these variables, which reveals two major analyte clusterings,
reveals two major analyte clusterings, with three sub-clusterings for one of these. The top right-hand
with three sub-clusterings for one of these. The top right-hand side major cluster comprises TBARS
side major cluster comprises TBARS level, (TBARS):(total lipid) ratio and TA value, whereas the
level, (TBARS):(total
second containslipid) ratio
all other and variables,
analyte TA value,including
whereasall theBAsecond contains
contents. allsecond,
The first, other analyte
and thirdvariables,
sub-
including
clusters within the bottom right-hand side major cluster feature spermine, spermidine, and histamineside
all BA contents. The first, second, and third sub-clusters within the bottom right-hand
major cluster
(the firstfeature
two ofspermine, spermidine,
these arising from the sameand histamine
putrescine (the
and first two of these
metabolically arising
upstream from the
ornithine same
and
putrescine and metabolically
agmatine/arginine upstream
sources ornithine and
respectively); agmatine/arginine
tyramine, cadaverine, sources respectively);
and total lipid; and tyramine,
2-
phenylethylamine,
cadaverine, and total lipid; putrescine, tryptamine, and pH respectively.
and 2-phenylethylamine, putrescine, tryptamine, and pH respectively.

Prior to the
Acetaldehyde, performance
a volatile flavor ofcomponent
MV statistical of analysis
a varietyofofthe dataset
foods andacquired,
beverages simple
suchPearson
as cheese,
correlations were explored between all explanatory variables considered, and
yoghurt, and wines [34], represents one of the most abundant carbonyl compounds detectable in wine, Figure 2 shows a
correlation heatmap for these relationships. Clearly, there were moderate
and typically accounts for ca. 90% of the total aldehydes present; its concentrations therein usually to strong positive
correlations observed between all fermented food BAs present, the strongest observed between 2-
range from 10 to 200 mg/L (predominantly, it is generated as a yeast by-product during alcoholic
phenylethylamine and tyramine (both aromatic BAs), tryptamine and spermine, and most notably,
fermentation processes [35], or from the chemical oxidation of ethanol [36]). However, very high levels
between cadaverine and histamine. Food pH values were found to have the strongest positive
of the unsaturated aldehyde
correlations with tyramine acrolein are also>present
> putrescine in redalthough
tryptamine, wine products
spermidine[33].was
Furthermore, a wide
predominantly
range of further aldehydes
uncorrelated with this have
index.been found as
Moreover, to anticipated,
serve as major flavor
TA was constituents
strongly of traditional
negatively Chinese
correlated with
rose vinegar,
pH value and> these include
putrescine aliphatic≈ n-alkanals
> tyramine such as in
histamine contents heptanal,
that order.hexanal,
TBARSnonanal, and dodecanal
level, however, was
(ranging fromindependent
largely 6–147 µg/kg), with
of all BAslarger amounts
and their of benzaldehyde
concentrations, with the(851 µg/kg)of[38].
exception spermidine, which
exhibited
Hence, a weak
overall positive
these datarelationship with this variable.
clearly demonstrated Similarly,
that, in total lipid
a univariate level was
context, therealso mainly
were indeed
uncorrelated with all BA contents but was quite strongly anti-correlated
significant differences between the mean contents of BAs and further parameters considered with (TBARS):(total lipid)
for the
ratio and
five classes non-lipid-normalized
of fermented food products TBARS value (both expected). The (TBARS):(total lipid) ratio was
studied.
either strongly or moderately anti-correlated with all BA levels, and this may provide an indication
Prior to the performance of MV statistical analysis of the dataset acquired, simple Pearson
correlations were explored between all explanatory variables considered, and Figure 2 shows a
correlation heatmap for these relationships. Clearly, there were moderate to strong positive correlations
observed between all fermented food BAs present, the strongest observed between 2-phenylethylamine
and tyramine (both aromatic BAs), tryptamine and spermine, and most notably, between cadaverine
Foods 2020, 9, 1807 11 of 28

and histamine. Food pH values were found to have the strongest positive correlations with tyramine
> putrescine > tryptamine, although spermidine was predominantly uncorrelated with this index.
Moreover, as anticipated, TA was strongly negatively correlated with pH value > putrescine > tyramine
≈ histamine contents in that order. TBARS level, however, was largely independent of all BAs and
their concentrations, with the exception of spermidine, which exhibited a weak positive relationship
with this variable. Similarly, total lipid level was also mainly uncorrelated with all BA contents but
was quite strongly anti-correlated with (TBARS):(total lipid) ratio and non-lipid-normalized TBARS
value (both expected). The (TBARS):(total lipid) ratio was either strongly or moderately anti-correlated
Foods
with all BA 2020, 9, x FOR
levels, PEER
and REVIEW
this may provide an indication of their potential antioxidant functions. 12 ofIn
28view

of the complexity of these inter-relationships, the MV PCA and PLS-DA techniques were employed to
of their potential antioxidant functions. In view of the complexity of these inter-relationships, the MV
explore them further.
PCA and PLS-DA techniques were employed to explore them further.

Figure 2. Correlation
Figure 2. Correlation heatmap
heatmapdisplaying
displaying positive and negative
positive and negativeinter-relationships
inter-relationships between
between BA BA
concentrations,
concentrations, pH and
pH and TA TA values,
values, total
total lipidcontents,
lipid contents, TBARS
TBARS indices
indicesand
and (TBARS):(total lipid)
(TBARS):(total ratiosratios
lipid)
(TBARS/total
(TBARS/total lipid).
lipid). TheThe left-hand
left-hand ordinateand
ordinate andtop
top abscissa
abscissa axes
axesshow
showAHC AHC analysis based
analysis on these
based on these
Pearson correlations (as a similarity criterion). From the top abscissa axis, of the two major
Pearson correlations (as a similarity criterion). From the top abscissa axis, of the two major clusteringsclusterings
revealed, that on the right-hand side contains all BA variable levels with the exception of spermidine,
revealed, that on the right-hand side contains all BA variable levels with the exception of spermidine,
together with positively-correlated pH values, whereas the left-hand side one consists of all lipid- and
together with positively-correlated pH values, whereas the left-hand side one consists of all lipid- and
lipid peroxidation-based variables, spermidine concentrations, and TA values.
lipid peroxidation-based variables, spermidine concentrations, and TA values.
3.2. Principal Component Analysis (PCA) of the Multivariate Fermented Food Dataset
PCA was primarily conducted in order to acquire an overview of the degree of distinctiveness
between, i.e., clustering of, the fermented food classifications investigated, and also to identify any
potential data outliers. An examination of two-dimensional (2D) scores plots from this analysis
demonstrated that no significant outliers were detectable, and that PCs 1, 2, and 3 accounted for 41.5,
16.4, and 11.1% of the total variance respectively for the complete dataset which was glog-
transformed and autoscaled. 2D and three-dimensional (3D) scores plots featuring these two most
important PCs revealed that there was a reasonable level of distinction between the wine/vinegar and
Foods 2020, 9, 1807 12 of 28

3.2. Principal Component Analysis (PCA) of the Multivariate Fermented Food Dataset
PCA was primarily conducted in order to acquire an overview of the degree of distinctiveness
between, i.e., clustering of, the fermented food classifications investigated, and also to identify any
potential data outliers. An examination of two-dimensional (2D) scores plots from this analysis
demonstrated that no significant outliers were detectable, and that PCs 1, 2, and 3 accounted for 41.5,
16.4, and 11.1% of the total variance respectively for the complete dataset which was glog-transformed
and autoscaled. 2D and three-dimensional (3D) scores plots featuring these two most important PCs
revealed that there was a reasonable level of distinction between the wine/vinegar and all other food
product groups, and also between the cheese and fish classifications (Figure 3a); however, distinctions
between the fish, fish sauce/paste, and vegetable sauce groups were not found, there being a significant
degree of overlap between them. Notwithstanding, the sample sizes of the fermented fish and vegetable
sauce groups involved were quite limited. A corresponding preliminary correlation circle diagram is
shown in Figure 3b. Clear observations from this diagram are that (1) 2-phenylethylamine, tyramine,
and cadaverine, and to a lesser extent, putrescine and tryptamine, are all correlated with PC1, and this
observation indicates their communality in this model; (2) food pH values are also strongly correlated
to PC1, and this indicates that higher values of this parameter may arise from the basicity of the above
BAs (gas-phase primary amine basicity values increase with the length of its carbon chain substituents
in view of their electron-donating positive charge-stabilizing effects—such values also increase with
progression from primary to secondary to tertiary alkylamines [39]); (3) an at least partial correlation
of histamine contents with PC2, which indicates distinction of this BA from those aligned with PC1;
(4) an inverse correlation (anti-correlation) of total lipid level with the (TBARS):(total lipid) ratio
index, as might be expected; and (5) a strong anti-correlation of TA value with BA levels, particularly
Foods 2020, 9, xand
tryptamine FORputrescine,
PEER REVIEW 14 of 28
and this suggests that these amines serve to offer neutralization potential
against acidic fermented food products. Also notable from this Figure are very strong correlations
per orthogonal PC was sufficient to provide acceptable levels of distinction between different sub-
between the fermented food supplementary variable cheese and total lipid content, and between
classes of such wines.
wine/vinegar and TA value, as indeed expected.

(a)

Figure 3. Cont.

1
Histamine

0.75
TBARS Value
Spermine
[TBARS]:[Lipid]
0.5 Spermidine
Tryptamine

0.25
Foods 2020, 9, 1807 13 of 28

(a)

1
Histamine

0.75
TBARS Value
Spermine
[TBARS]:[Lipid]
0.5 Spermidine
Tryptamine

0.25
Putrescine
PC2 (19%)

2-Phenylethylamine Tyramine
0 pH
Titratable Acidity
Cadaverine
WINE/VINEGAR
-0.25

CHEESE
-0.5
Total Lipid

-0.75

-1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
PC1 (38%)

(b)

Figure 3. (a) 3D PCA scores plot of PC3 vs. PC2 vs. PC1, showing some degrees of distinction
between different fermented food classes, i.e., those of cheese, fish, fish sauce/pastes, vegetable
pastes, and wines/vinegars (particularly that between the wine/vinegar classification and all others).
(b) Preliminary correlation circle diagram displaying correlations between all explanatory variables
considered, and PCs 1 and 2 in a PCA model applied to the complete autoscaled (standardized) dataset.
Active variables are depicted in red, whereas two of the supplementary variable classifications (cheese
and wine/vinegar) are shown in blue. Variance contributions for PC1 and PC2 are indicated.

A more detailed analysis of these PCA loadings was made with the application of varimax rotation,
Kaiser normalization, and a maximal number of 5 PCs considered. For this model, such variable
loadings, and the percentage of total variance accounted for by each PC are available in Table 4.
This analysis revealed that cadaverine, tryptamine, 2-phenylethylamine, and tyramine all strongly and
positively loaded on PC1, spermidine and histamine strongly and positively loaded on PC3 (along with
a more minor contribution from 2-phenylethylamine), and putrescine and spermidine loaded strongly
and positively on PC5, albeit also with histamine to a much lesser extent. Interestingly, all aromatic
BAs strongly loaded on PC1, as observed above (Figure 3b), whereas spermidine and its metabolic
precursor putrescine both co-loaded onto the same PC (PC5).
Foods 2020, 9, 1807 14 of 28

Table 4. PCA loadings vectors for BAs and additional fermented food analyte parameters (including
total lipid contents, and pH and TA values) for a 5 PC-limited model performed with varimax rotation
and Kaiser normalization. Percentage variance contributions for PCs 1–5 and their (unrotated) analysis
eigenvalues are also listed. Bold numbers are for a purpose specified in the Figure legends.

PC (Unrotated
PC1 (4.58) PC2 (2.26) PC3 (1.78) PC4 (1.28) PC5 (0.89)
Eigenvalue):
% Variance
26.6 16.9 12.2 16.1 11.2
Contribution
2-Phenylethylamine 0.71 −0.23 0.43 0.03 −0.06
Cadaverine 0.90 −0.07 −0.19 0.21 −0.04
Histamine 0.25 0.45 0.63 0.025 0.32
Putrescine 0.57 −0.04 −0.19 0.42 0.50
Spermidine 0.14 −0.19 0.20 0.06 0.78
Spermine −0.08 −0.06 0.85 0.22 0.12
Tryptamine 0.79 0.07 0.11 0.13 0.30
Tyramine 0.88 −0.11 0.03 0.15 −0.03
Titratable acidity (TA) −0.11 0.17 −0.30 −0.86 −0.09
pH 0.29 −0.10 0.04 0.92 −0.07
TBARS value −0.08 0.95 0.08 −0.05 −0.05
Total lipid 0.45 −0.31 −0.21 0.37 −0.61
(TBARS):(Lipid) ratio −0.14 0.92 −0.07 −0.23 −0.08

The TBARS secondary lipid oxidation index, along with its value normalized to total food lipid
content, both loaded strongly and positively on PC2, as might be expected, although histamine also
contributed somewhat towards this PC. Moreover, TA and pH values powerfully loaded on PC4
negatively and positively respectively, as would be expected from their anticipated negative correlation
in fermented food products (putrescine also made a moderate positive contribution towards this
component). Total lipid content was found to load significantly on PCs 1 and 5, positively and
negatively so, respectively.
In a related study focused on PCA of both BAs and polyphenolics in Hungarian wines,
Cosmos et al. [26] found that PC scores successfully clustered differential groups of these product
classes, and that PC loadings vectors displayed significant patterns of BA and polyphenol levels.
However, it should be noted that for this analysis, spermidine, and tyramine strongly loaded on PC1
(positively and negatively, respectively), agmatine and the sum total BA concentration loaded strongly
and positively on PC2, spermine and cadaverine both strongly and negatively loaded on PC3, and that
histamine loaded strongly and positively on PC4 alone. These associations between the BA analytes
tested did not correspond to those found in the present study, although in the above MV analyses
we elected not to include the total summed BA concentration value. Furthermore, our study also
included the determinations of 2-phenylethylamine and putrescine, and not agmatine, but that reported
in [26] monitored the latter BA but not 2-phenylethylamine and putrescine. However, as noted by the
authors of [26], these PC loadings are only applicable to one region of Hungarian wine production,
and their results will not be readily transferable to others, let alone other classes of fermented foods,
especially in consideration of the often highly variable methods of fermentation, sources of fermentative
micro-organisms, and conditions employed for these purposes. Notwithstanding, these researchers
also concluded that in view of the loading patterns of BAs observed, it was unnecessary to measure all
BA variables for quality assessments, and that only one per orthogonal PC was sufficient to provide
acceptable levels of distinction between different sub-classes of such wines.
Foods 2020, 9, 1807 15 of 28

From this analysis, the unambiguously strong loadings vectors of the aromatic BAs
2-phenylethylamine and tyramine on PC1 provide evidence that they may indeed arise from the same
biological and/or metabolic sources; however, this observation may also be rationalized by the natural
production of tyrosine from phenylalanine, i.e., that involving the possible hydroxylation of the latter
substrate to the former catalyzed by the enzyme phenylalanine hydroxylase (PAH) potentially available
in fermentative lactobacilli employed for the production of fermented food products, followed by
enzymatic transformation of the tyrosine product to tyramine by fermentative bacteria. To date, PAH
is the only known aromatic amino acid hydroxylase found in bacteria [40].
The loadings of spermine and spermidine on different orthogonal PCs (PC3 and PC5, respectively)
is not simply explicable, although the co-loading of spermidine’s metabolic precursor putrescine on
PC5 is consistent with them being featured in the same metabolic pathway. However, the co-loadings
of BAs on differential PCs, particularly PC1, may reflect their engenderment from identical or related
bacterial sources.
Notably, PC2 was dominated by powerful loading contributions from TBARS level and
(TBARS):(total lipid) ratio (both positive), and PC4 by strong loadings from TA and pH values
(negative and positive loadings vectors, respectively). These inter-relationships are, of course, expected,
and are consistent with the data presented in Figure 3b. PC5 was retained in the model since it was the
only one available which had a strong loading contribution from spermidine.

3.3. Distinction of Fermented Food Classifications Using PLS-DA

Similarly, PLS-DA of the dataset revealed an effective discrimination between the cheese and
wine/vinegar classifications, although the fish, fish sauce/paste and vegetable sauce sample PC score
datapoints were again unresolved; however, a visualized combination of these three fermented food
classifications was at least partially resolved from the fermented cheese group (Figure 4). Permutation
testing of the PLS-DA model confirmed its ability to distinguish between all the differing fermented
food classifications evaluated (p = 0.022). For this model, key discriminatory variables were selected
on the basis of their variable importance parameters (VIPs), and these were total lipid content
(1.81) > cadaverine content (1.61) > (TBARS):(total lipid) ratio (1.36) > TA value (1.24) > histamine
content (1.14) > 2-phenylethylamine content (0.78); data were glog-transformed and autoscaled prior
to analysis. The top three discriminators largely arise from differential levels of lipids, cadaverine,
and (TBARS):(total lipid) ratio between each of the fermented food groups, e.g., for the total lipids and
cadaverine variables, the cheese content was significantly greater than that of all other fermented food
groups, and for the above ratio, its value was significantly greater in the wine/vinegar group than it
was in all other groups.
The quite strong distinctions observed between the cheese, wine/vinegar, and fish-fish
sauce/paste-vegetable sauce composite products is readily explicable by significant or even substantial
differences between the higher contents of cadaverine, tyramine, and, to a lesser extent, tryptamine in
cheese, than those of the four other fermented food product classes. Further key discriminators are TA,
pH, and total lipid contents, the latter of which is, of course, much higher in the cheese group.
content (1.14) > 2-phenylethylamine content (0.78); data were glog-transformed and autoscaled prior
to analysis. The top three discriminators largely arise from differential levels of lipids, cadaverine,
and (TBARS):(total lipid) ratio between each of the fermented food groups, e.g., for the total lipids
and cadaverine variables, the cheese content was significantly greater than that of all other fermented
Foods
food2020, 9, 1807and for the above ratio, its value was significantly greater in the wine/vinegar 16
groups, of 28
group
than it was in all other groups.

(a)

(b)

Figure 4. (a,b). 2D and 3D PLS-DA scores plots (PC2 vs. PC1, and PC3 vs. PC2 vs. PC1, respectively)
revealing strong distinctions between the cheese, wine/vinegar, and a considered combination of fish,
fish sauce/paste and vegetable sauce fermented food groups ((a) also shows 95% confidence ellipses for
each fermented food classification). Little or no distinction between the latter three fermented food
groups were discernable using this MV analysis approach.

3.4. RF Modelling of Fermented Food Classifications

Application of the RF CI classification technique was found to be only partially successful for
the classification of the different fermented food groups investigated. Using the models described in
Section 2.6.2, this approach correctly classified 4/4 wine vinegar, 6/9 fish sauce/paste, and 3/5 cheeses,
but 0/4 for both fish and vegetable sauce products.
Foods 2020, 9, 1807 17 of 28

3.5. PCA of FCLO BAs

The FCLO product considered was primarily investigated separately since only BA contents,
and not parameters such as pH and TA were available for it. Moreover, its total lipid content is,
of course, not far removed from a value of 100%, and therefore it would be inappropriate to test this
index in the above MV analysis models (similarly, total lipid level-normalized TBARS values would
also be inappropriate to test in these systems). However, it was possible to explore inter-relationships
between FCLO BA concentrations and/or their orthogonality status using a rigorous PCA approach
featuring varimax rotation and Kaiser normaliszation in order to maximize success with the assignment
of individual BA variables to PCs.
Table 5 lists the BA contents of n = 10 FCLO product batches. The total concentrations of BAs in
these samples was higher than the recommended ”limit” of 200 ppm in only two out of ten batches
of the samples tested, albeit marginally so (only 14 and 20% higher). Similarly, bioactive histamine
was completely undetectable in this product. As noted in [6], all BAs monitored were completely
undetectable in three other natural, albeit unfermented, CLO products included for comparative
purposes. All BAs tested were found to be reasonably soluble in FCLO lipidic matrices, and also in 1/3
(v/v) diluted solutions of this product in deuterochloroform (C2 HCl3 ), presumably as the uncharged
species with their amine functions deprotonated (solubility in these media is expected to increase with
increasing amine function substituent chain length and hydrophobicity).

Table 5. (BA concentrations (ppm) of n = 10 separate batches of a FCLO product. Total BA and
corresponding mean ± SEM values are also provided. Histamine and spermine were undetectable in
all samples analyzed.

FCLO Batch
Biogenic Amine (ppm) 1 2 3 4 5 6 7 8 9 10 Mean ± SEM
2-PE 86 103 50 17 76 0 1.4 0 1.9 1.3 33.7 ± 13.0
Tyramine 70 88 43 8 32 0 1.8 0 1.1 0 24.4 ± 10.3
Tryptamine 35 24 26 3 8 0 0 0 1.7 1.5 9.9 ± 4.2
Cadaverine 23 11 25 0 7 0 0 0 0 0 6.6 ± 3.1
Putrescine 14 10 14 0 0 0 0 0 0 0 3.8 ± 2.0
Spermidine 0 4 0 0 0 0 0 0 0 0 0.4 ± 0.4
Total 228 240 158 28 123 0 3.2 0 4.7 2.8 78.8 ± 31.3

PCA performed on the FCLO BA dataset revealed that cadaverine, putrescine, and tryptamine
all loaded strongly and positively on the first of two automatically-selected PCs (PC1), whereas the
aromatic BAs 2-phenylethylamine and tyramine loaded strongly and positively on the second (PC2),
along with spermidine (Table 6). These data displayed some consistency with PC loading values
obtained on the full fermented food dataset (Table 4), which had 2-phenylethylamine and tyramine
both strongly loading on one PC (PC1). However, such levels will, of course, be critically dependent
on the microbial fermentation sources, parameters employed for fermented food production, and
production conditions for these processes.
Foods 2020, 9, 1807 18 of 28

Table 6. PCA loadings vectors for FCLO BAs in a two PC-limited PCA model performed with varimax
rotation and Kaiser normalization. Percentage variance contributions for these PCs and their (unrotated)
analysis eigenvalues are also listed. Bold numbers are for a purpose specified in the Figure legends.

PC (unrotated Eigenvalue) PC1 (4.03) PC2 (1.58)

% Variance Contribution 52.8 40.6
2-PE 0.35 0.84
Tyramine 0.54 0.84
Tryptamine 0.93 0.33
Cadaverine 0.99 −0.01
Putrescine 0.95 0.23
Spermidine −0.11 0.93

3.6. MV Chemometric Analysis of BA Data Only: Recognition of Fermented Food Class-Distinctive BA

Patterns Using CS-Normalization
Additionally, we conducted univariate and MV analyses of datasets which were restricted to
the BA profiles only, but also included the n = 10 FCLO samples reported above. Additionally,
these analyses were performed with and without application of constant sum (CS) normalization.
The CS-normalized data format was employed in order to facilitate the recognition of fermented food
class-specific BA patterns. For the non-CS-normalized format, the total BA content value was also
included as an explanatory variable, as indeed it was in [26].
Firstly, ANOVA performed on the CS-normalized, glog-transformed, and autoscaled dataset
found very highly significant, albeit FDR-corrected p values for three of the sum-proportionate mean
BA concentration differences observed between the fermented food classifications explored in this
manner. Notably, these differences were observed for cadaverine, 2-phenylethylamine, and tryptamine
(Table 7), and post-hoc testing revealed that for cadaverine, the cheese products had significantly greater
proportionate levels than three others, and for both 2-phenylethylamine and tryptamine, FCLO had
significantly higher ones than all other products examined. These differences in CS-normalized values
are readily visualizable in the form of an ANOVA-based heatmap (Figure 5a), which revealed
characteristic BA signatures for three of the fermented food product classifications. Clearly,
the cheese, FCLO, and wine/vinegar sampling groups have high proportionate levels of cadaverine,
2-phenylethylamine/tyramine/tryptamine (all aromatic BAs), and metabolic pathway-associated
putrescine/spermidine/spermine, respectively. However, when evaluated in this univariate system,
”between-fermented food class” mean differences observed for putrescine, spermine, spermidine,
histamine, and tyramine were not found to be statistically significant.
Secondly, both PCA and PLS-DA models were employed, and these approaches were successful
in providing evidence for the MV distinctiveness of the FCLO, cheese, and wine/vinegar groups;
however, as noted for the analyses conducted on the combined BA/further food quality parameter
dataset, unfortunately no distinctions were observed between the fermented fish, fish sauce/paste,
and vegetable sauce products (Figure 5b,c).
For the CS-normalized dataset (without total BA concentrations as an additional variable),
PLS-DA variable importance parameter (VIP) values were in the order spermidine (1.48) > putrescine
(1.34) > spermine (1.20) > histamine (1.06) > 2-phenylethylamine (0.94), whereas those for the
non-CS-normalized dataset were spermidine (1.56) > spermine (1.35) > 2-phenylethylamine (1.32) >
putrescine (0.84) (total BA level was a very poor predictor variable for the latter). As expected, there were
significant differences between the sequential orders of these values when prior CS-normalization
was implemented.
Foods 2020, 9, 1807 19 of 28

Table 7. Univariate statistical significance and nature of differences observed between the mean
CS-normalized, glog-transformed, and autoscaled BA contents of fermented food samples (cheese,
FCLO, fish, fish sauce/paste, vegetable sauce, and wine/vinegar products) in a completely randomized,
one-way ANOVA model. The significance of FDR-corrected post-hoc ANOVA tests are also provided
(significant differences are ranked in order of their decreasing statistical significance, i.e., increasing p
value). The “between-fermented food class” source of variation was not statistically significant for
putrescine, spermidine, spermine, histamine, or tyramine when tested in this model.

BA FDR-Corrected p Value Significant post-hoc ANOVA Differences

Cheese > FCLO; Cheese > Vegetable Sauce; Cheese >
Wine/Vinegar; Fish > FCLO; Fish Sauce > FCLO;
Cadaverine 1.49 × 10−5 Vegetable Sauce > FCLO; Fish > Wine/Vinegar; Fish
Sauce > Wine/Vinegar; Vegetable Sauce >
Wine/Vinegar.
FCLO > Cheese; FCLO > Fish; FCLO > Fish Sauce;
2-Phenylethylamine 8.25 × 10−4 FCLO > Vegetable Sauce; FCLO > Wine/Vinegar; Fish
> Vegetable Sauce.
FCLO > Cheese; FCLO > Fish; FCLO > Fish Sauce;
Tryptamine 2.93 × 10−2
FCLO > Vegetable Sauce; FCLO > Wine/Vinegar.

Moreover, for the PLS-DA model adopted without CS-normalization, histamine, spermidine,
and spermine contents all loaded significantly on component 1 (loading vector coefficients 0.48, 0.57,
and 0.47 respectively); 2-phenylethylamine, cadaverine, tyramine, and total BA levels on component
2 (loadings vector coefficients 0.42, −0.61, −0.57, and −0.61 respectively); 2-phenylethylamine and
tryptamine levels on PC3 (loadings vector coefficients 0.50 and 0.57 respectively); and putrescine
and spermine on PC4 (loadings vector coefficients 0.75 and −0.73 respectively). For this dataset,
a four-component model was found to be most effective (permutation p value 0.0055)
Importantly, it should be noted that one now common issue in chemometrics/metabolomics
experiments is the occurrence of a univariately-insignificant variable which remains multivariately-
significant. Such observations are readily rationalized, firstly by the complementation (i.e., correlation)
between explanatory variables, i.e., separately they do not, but when combined together as a MV
composite (e.g., as a sufficiently-loading PC variable), they do serve to explain “between-classification”
differences detected; secondly, consistency effects arising from the “masking” of potential
univariately-significant differences by high levels of biological source sampling and/or measurement
variation may be responsible (such variation may be averaged out via the conversion of datapoints to
orthogonal component scores as in the PCA and PLS-DA models applied here); and thirdly, relatively
small sample sizes for each classification involved (fermented foods in this case)—unfortunately,
strategies applied to correct for FDRs promote the risk of statistical type II errors (i.e., false negatives) [24].
The PLS-DA evaluation was then extended and performed for pairwise comparisons of the
differing fermented food classifications (CS-normalized dataset only). Firstly, as expected, Q2 values
for the fish vs. fish sauce/paste, fish sauce/paste vs. vegetable sauce, and fish vs. vegetable sauce
comparisons were all moderately negative, and p values for associated permutation tests were all >0.10.
However, these values for the wine/vinegar vs. FCLO, and FCLO vs. cheese two classification model
comparisons revealed that Q2 (permutation p values) indices for these comparisons were 0.71 (0.059)
and 0.72 (0.090), but only 0.38 (0.16) for the wine/vinegar vs. cheese one (values were based on models
containing two, five, and one components respectively). Hence, these results provide some evidence
for the success of this strategy in distinguishing between the FCLO product, and both the cheese and
wine/vinegar ones, although permutation test p values obtained for these models were a little higher
than the 0.05 significance level, i.e., they were close to statistical significance.
Foods 2020, 9, 1807 20 of 28
Foods 2020, 9, x FOR PEER REVIEW 19 of 28

(a)

(b)

(c)

FigureFigure 5. (a) Heatmap

5. (a) Heatmap diagram
diagram displaying
displaying themost
the mostunivariately-significant
univariately-significant differences between
differences mean
between mean
valuesvalues of eight
of eight BA explanatory
BA explanatory variables
variables (near
(near right-handside
right-hand side y-axis)
y-axis) for
forthe
thefermented
fermentedcheese (red),
cheese (red),
FCLO (green), fish (dark blue), fish sauce/paste (pale blue), vegetable sauce (purple), and wine/vinegar
(yellow) products. The complete BA dataset was CS-normalized, glog-transformed, and autoscaled
measurement variation may be responsible (such variation may be averaged out via the conversion
of datapoints to orthogonal component scores as in the PCA and PLS-DA models applied here); and
thirdly, relatively small sample sizes for each classification involved (fermented foods in this case)—
unfortunately, strategies applied to correct for FDRs promote the risk of statistical type II errors (i.e.,
false negatives) [24].
Foods 2020, 9, 1807 21 of 28
The PLS-DA evaluation was then extended and performed for pairwise comparisons of the
differing fermented food classifications (CS-normalized dataset only). Firstly, as expected, Q2 values
for the fish
prior vs. fish sauce/paste,
to analysis. AHC analysisfish shownsauce/paste vs. vegetable
on the left-hand sauce,
side ordinate axisand fish vs. vegetable
demonstrated two majorsauce
analyte clusterings,
comparisons the upper one
were all moderately consisting
negative, andofp values
putrescine, spermidine,permutation
for associated and spermine pathway
tests were all >
biomolecules (and histamine), whereas the lower one features all aromatic
0.10. However, these values for the wine/vinegar vs. FCLO, and FCLO vs. cheese two classification BAs, along with cadaverine.
model (b)comparisons
3D PCA PC3 vs. PC2 vs. that
revealed PC1 scores plot for the same
Q2 (permutation CS-normalized
p values) indices for dataset
these shown in (a), showing
comparisons were 0.71
(0.059)reasonable
and 0.72 or(0.090),
strong distinctions
but only 0.38 between
(0.16)thefor
cheese, wine/vinegar and
the wine/vinegar vs. FCLO
cheese fermented food were
one (values classes.
based
(c) 3D PLS-DA PC3 vs. PC2 vs. PC1 scores plot for the corresponding
on models containing two, five, and one components respectively). Hence, these results provide some non-CS-normalized dataset,
which also incorporated total BA content as a potential explanatory variable (again, effective distinctions
evidence for the success of this strategy in distinguishing between the FCLO product, and both the
between the cheese, FCLO, and wine/vinegar classes were notable).
cheese and wine/vinegar ones, although permutation test p values obtained for these models were a
littleWehigher
thenthan the 0.05
elected significance
to statistically level, i.e.,
combine thethey
fish,were
fish close to statistical
sauce/paste, significance.
and vegetable sauce groups,
We then the
and repeated elected to statistically
PLS-DA modellingcombine in orderthe to fish, fish sauce/paste,
compare the sauce/fish and vegetablecheese,
composite, sauce groups,
FCLO,
and wine/vinegar
and repeated the PLS-DA groups usingmodelling in order to compare
the CS-normalized dataset.the This
sauce/fish
analysiscomposite,
exhibitedcheese,
a quiteFCLO,
high
and wine/vinegar
level of classification groups using
success (Figure 6a); Q2 for this
the CS-normalized dataset. This analysis
comparative exhibited a quite
four-classification high level
analysis was
0.44, and a PLS-DA permutation test confirmed its significance (p = 0.031). The loadings of each BA
of classification success (Figure 6a); Q 2 for this comparative four-classification analysis was 0.44, and

a PLS-DA
variable onpermutation test confirmed
PLS-DA components 1 andits2 significance (p = 0.031).
is shown in Figure The this
6b, and loadings of each BA
demonstrates variable
three groupson
PLS-DA
of componentsthe
these predictors: 1 and
first 2with
is shown
highlyinpositive
Figure component
6b, and this 1demonstrates
and highly negative three groups of these
component 2
predictors:
loadings (allthe first with
aromatic BAs,highly positive component 1tyramine,
i.e., 2-phenylethylamine, and highly andnegative component
tryptamine); 2 loadings
the second (all
with low
aromatic
to intermediateBAs, positive
i.e., 2-phenylethylamine,
component 1 but highly tyramine,
positiveand tryptamine);
component the (metabolically-related
2 loadings second with low to
intermediate
putrescine, positive component
spermidine, and spermine, 1 buttogether
highly positive component
with histamine); and2 loadings
the third(metabolically-related
with highly negative
putrescine,
loadings on spermidine,
component 1, and
butspermine,
negligibletogether
loadingswith histamine);2and
on component the thirdonly).
(cadaverine with highly negative
These grouped
loadings
BA loadingson component
vectors were1,very but negligible
consistent withloadings
otheronobservations
component made 2 (cadaverine
from theonly). These grouped
MV analysis of these
BA loadings vectors were very consistent with other observations made from
data as a full six fermented food classification dataset. Specifically, they are completely reflective of the the MV analysis of
these data
patterns of BA as “markers”
a full six found
fermented food classification
in fermented dataset. Specifically,
FCLO, wine/vinegar, they arerespectively
and cheese products completely
reflective
(Figure 5a).of the patterns of BA “markers” found in fermented FCLO, wine/vinegar, and cheese
products respectively
Finally, RF analysis (Figure
of this5a).
revised dataset showed that this approach had an at least reasonable
level Finally, RF analysis
of classification of thiswith
success, revised datasetFCLO
all (10/10) showed and that
88% this approach
(15/17) of thehad an at least
fish/sauce reasonable
combination
level of classification
samples being correctly success, withnotwithstanding,
classified; all (10/10) FCLOonly and60 88%and(15/17)
50% ofofthe thecheese
fish/sauce combination
and wine/vinegar
samples being
fermented foodcorrectly
products,classified;
respectively, notwithstanding,
were. only 60 and 50% of the cheese and wine/vinegar
fermented food products, respectively, were.

(a)
Figure 6. Cont.
Foods2020,
Foods 9, x1807
2020, 9, FOR PEER REVIEW 22of
22 of28
28

(b)
Figure
Figure 6.
6. PLS-DA
PLS-DAevaluation
evaluationofofrevised
reviseddataset
datasetwith
withcombined
combinedfish, fish, fish
fish sauce/paste,
sauce/paste, and
and vegetable
vegetable
sauce
sauce classifications
classifications (abbreviated
(abbreviated COMBO);
COMBO); CS-normalization
CS-normalizationwas wasapplied
appliedto to the
the dataset
dataset prior
prior to
to
analysis.
analysis.(a)(a)3D3D
PLS-DA
PLS-DA component
component 3 vs. component
3 vs. component 2 vs.2component
vs. component 1 scores plot revealing
1 scores some
plot revealing
clustering of the of
some clustering fermented food classifications
the fermented (i.e., cheese,
food classifications wine/vinegar,
(i.e., cheese, wine/vinegar,FCLO, and COMBO).
FCLO, and COMBO).(b)
(b) Corresponding
Corresponding component
component 2 vs.2 component
vs. component 1 loadings
1 loadings plotplot for this
for this PLS-DA
PLS-DA analysis.
analysis.

3.7. Scientific
3.7. ScientificSignificance
Significance and
and Human
Human Health
Health Implications
Implications of
of Results
Results Acquired
Acquired
Results acquired
Results acquiredfrom from thethecombined
combined applications of univariate
applications and MV and
of univariate chemometrics techniques
MV chemometrics
in this study
techniques in clearly
this study demonstrated that the latter
clearly demonstrated that strategy
the latterwas valuable
strategy for distinguishing
was valuable between
for distinguishing
between fermented wine/vinegar products and cheeses, and the discrimination between both ofclasses
fermented wine/vinegar products and cheeses, and the discrimination between both of these food these
from either fish, fish sauce/paste, or vegetable sauce products (or a statistical
food classes from either fish, fish sauce/paste, or vegetable sauce products (or a statistical combination of them)
was possible of
combination on them)
the basis
was of possible
their BA, on total lipid,
the pH,
basis ofand TABA,
their values;
totalnevertheless,
lipid, pH, and suchTA techniques
values;
were not readily able to distinguish between the latter three fermented
nevertheless, such techniques were not readily able to distinguish between the latter three fermented food classes. However,
a rigorously-constrained
food classes. However, aunivariate analysis method
rigorously-constrained selected analysis
univariate to overcome complications
method selected toarising
overcomefrom
intra-food classification heteroscedasticities and FDRs was able to successfully
complications arising from intra-food classification heteroscedasticities and FDRs was able to distinguish between
the vegetable
successfully sauce andbetween
distinguish fish groups through significantly
the vegetable sauce and fish higher
groups andthrough
lower levels of spermidine
significantly higher
and lower levels of spermidine and 2-phenylethylamine, respectively, present in the formerresults
and 2-phenylethylamine, respectively, present in the former class. Moreover, experimental class.
indicated that
Moreover, cadaverine,
experimental tyramine,
results putrescine,
indicated and tryptamine
that cadaverine, concentrations
tyramine, putrescine,may andalltryptamine
contribute
significantly towards
concentrations may all food pH values
contribute in view of their
significantly strong
towards positive
food correlations
pH values in view withofthis parameter
their strong
found, together
positive correlationswithwith
corresponding
this parameter negative
found,ones with TA
together withvalues (Figure 3b).negative ones with TA
corresponding
values Moreover,
(Figure 3b). BA-targeted univariate and multivariate analyses of CS-normalized data was found
to beMoreover,
valuable for providing univariate
BA-targeted useful discriminatory information,
and multivariate analyseswhich highlighted the
of CS-normalized datacharacteristic
was found
patterns of BA biomolecules, which may be valuable for further investigations
to be valuable for providing useful discriminatory information, which highlighted the characteristic of the particular nature
and/or geographic origins of fermented foods, and the mechanisms involved in
patterns of BA biomolecules, which may be valuable for further investigations of the particular nature their formation. Indeed,
the present
and/or study found
geographic that of
origins such patterns comprised
fermented foods, andcadaverine only for cheese
the mechanisms involved samples,
in theirthree aromatic
formation.
BAs (2-phenylethylamine, tyramine, and tryptamine), for FCLOs (sourced
Indeed, the present study found that such patterns comprised cadaverine only for cheese samples, from fermented cod livers),
and those from the sequential metabolic pathway which transforms
three aromatic BAs (2-phenylethylamine, tyramine, and tryptamine), for FCLOs (sourced fromthe amino acid substrates ornithine
or argininecod
fermented to spermine
livers), and (i.e., putrescine,
those from thespermidine, and spermine
sequential metabolic pathwayitself) for wine/vinegar
which transforms the products.
amino
Such idiosyncratic, fermented food product-dependent signatures for CS-normalized
acid substrates ornithine or arginine to spermine (i.e., putrescine, spermidine, and spermine itself) fermented food
BAwine/vinegar
for concentrations may serve
products. Such toidiosyncratic,
provide valuable information
fermented regarding the fermentative
food product-dependent signaturesbacterial
for CS-
sources, routes involved in fermentation, and product manufacturing
normalized fermented food BA concentrations may serve to provide valuable information regardingconditions employed for them.
the fermentative bacterial sources, routes involved in fermentation, and product manufacturing
conditions employed for them.
Foods 2020, 9, 1807 23 of 28

For the putrescine → spermidine → spermine metabolic pathway, which was identified as
representing a wine/vinegar-specific one from analysis of the CS-normalized dataset, and which
accounted for >70% of total BAs in this fermented food class (Table 2), both positive or negative
correlations could arise between a BA catabolite and its immediate upstream precursor, but not
necessarily between the terminal spermine metabolite and that upstream of its spermidine substrate
(i.e., putrescine).
With regard to toxic concentrations and health risk recommendations available in [25], it should be
noted that all mean histamine levels determined in the fermented food samples tested here lie markedly
below the recommended 100 ppm limit for it (with no single product exceeding this value—the
highest level observed was 57 ppm in one of the fish sauce products assessed). Furthermore, with the
exception of the cheese products evaluated, the mean total BA values all food groups were <200 ppm,
the wine/vinegar classification substantially so (Table 2). However, although three of the cheese
products tested had total BA contents of <200 ppm, two of them had levels ranging from 600–800 ppm,
and therefore their dietary consumption may present a health risk for susceptible individuals.
Mean BA concentrations for the FCLO product examined ranged from 0 (histamine) to only
34 ppm (2-phenylethylamine), with the highest levels observed for the most predominant species,
2-phenylethylamine and tyramine, being 103 and 88 ppm. Since the United States of America’s
recommended dietary intake of health-friendly, highly unsaturated omega-3 (O-3) fatty acids (FAs) is
a maximum of 1.0 g/day [41], and the oil explored here contains a mean of 29% (w/w) total O-3 FAs
(predominantly the sum of eicosapentaenoic and docosahexaenoic acids) [6], then daily consumption
of 100/29% × 1.0 g = 3.45 g of this FCLO product would provide estimated absolute maximal daily
intake levels of 3.45 × 103 µg = 355 µg, and 3.45 × 88 µg = 304 µg of 2-phenylethylamine and tyramine,
respectively. Based on the 10 samples of this product analyzed, estimated mean daily intakes of
these BAs will be 111 and 95 µg only. Therefore, it appears that daily consumption of this product
at the recommended U.S.-recommended dosage levels will certainly not provide any health risks to
consumers, even if they are susceptible to the adverse effects experienced by their excessive intake
(e.g., migraines induced by 2-phenylethylamine).
As noted above, one potentially important health benefit offered by the ingestion of dietary BAs is
their novel antioxidant properties, both for the prevention of food spoilage during storage or transport
episodes, but also in vivo following their ingestion. Indeed, our laboratory recently explored the
powerful antioxidant capacities of BA-containing natural FCLO products, and their resistivities to
thermally-mediated oxidative damage to unsaturated FAs therein, particularly O-3 PUFAs [6]. These
marine oil products, which arise from the pre-fermentation of cod livers (Section 2), were indeed found
to display a very high level of antioxidant activity, and PUFAs therein were also more resistant towards
thermally-mediated peroxidation than other natural cod liver oil products evaluated. Resonances
assignable to aromatic BAs, specifically those arising from 2-phenylethylamine and tyramine, were
directly observable in the 1 H NMR profiles of ca. 1/3 (v/v) diluted solutions of these products with
C2 HCl3 . Additionally, corresponding spectra acquired on both 2 H2 O and C2 H3 O2 H extracts of these
oils confirmed the presence of both these BAs, together with a series of others, both aromatic and
aliphatic. In the present study, mean concentrations of 2-phenylethylamine and tyramine detectable in
these products were found to be 34 and 24 ppm, respectively. Antioxidant actions of the phenolic BA
antioxidant tyramine found in this FCLO product may be explicable by its chain-breaking antioxidant
effects, and this may offer contributions towards the potent resistance of PUFAs, particularly O-3
FAs, present therein. However, in view of the absence of a phenolic function in 2-phenylethylamine,
its antioxidant potential is likely to involve an alternative radical-scavenging mechanism, presumably
that involving O2 -consuming carbon-centered pentadienyl radical species, as found in [22,23].
The TBARS method employed here to determine the lipid peroxidation status of fermented
foods, which involved an extended low temperature equilibration process [28], successfully
avoids the artefactual generation of TBA-reactive aldehydes, including malondialdehyde (MDA),
during commonly-employed alternative protocols for this assay system, which generally involve a
Foods 2020, 9, 1807 24 of 28

short (ca. 10–15 min) heating stage at 95–98 ◦ C in order to develop the monitored pink/red chromophore
rapidly. However, from an analysis of TBARS and (TBARS):(total lipid) ratios determined on the
preliminary FCLO-excluded fermented food samples, there appears to be only little evidence for the
ability of BAs to offer any protection against lipid peroxidation in such products. Although Table 3
shows that the above ratio is significantly greater in the wine/vinegar group, this observation is
perceived to be derived from their very low lipid contents, and the presence of a range of non-MDA
TBA-reactive aldehydes present therein, including acetaldehyde and acrolein, for example, although
these are also lipid oxidation products. Moreover, despite taking steps to avoid the artefact-generating
heating stage of this assay, this test still remains poorly specific in view of the reactions of a variety of
non-aldehydic substrates to react with it to form interfering chromophores, which also absorb at a
monitoring wavelength of 532 nm. Nevertheless, TBARS level appeared to be positively correlated
with fermented food spermidine concentration (Figure 2), and both this lipid peroxidation index and
its lipid-normalized value appeared to be positively correlated with fermented food histamine content
(loading on PC2, Table 4). However, in view of the many complications associated with this TBARS
lipid peroxidation index, which offers only a very limited and still often erroneous viewpoint on the
highly complex lipid peroxidation process [42], such observations cannot be rationally considered at
this stage. As expected, the lipid-normalized TBARS value was negatively correlated with total lipid
content (Figure 3b). The latter variable also appeared to be negatively correlated with histamine and
putrescine levels (loading on PC5, Table 4). Unfortunately, results from unspecific TBARS assays are
still widely employed as important quality indices throughout the food industry.
One quite surprising observation made in the current study was the detection of lipids, albeit at
low levels, in wine and vinegar samples. Notwithstanding, as noted above, FAs have been detected in
Zhenjiang aromatic vinegar products at similar contents to those found here [32]. Furthermore, Yunoki
et al. [43] explored the FA constituents of some commercially-available red wine products, and found
that lipid constituent concentrations varied from 27 to 96 mg/100 mL for n = 6 domestic (Japanese)
wines, and 31 to 56 mg/100 mL for n = 6 foreign products, and that a total of 12 different FAs were
detectable, mainly saturated ones. Although the extraction method described in the latter report was a
2:1 chloroform:methanol (Folch) one that targets non-polar triacylglycerols (TAGs) and more polar
phospholipids, it is likely that the FAs detectable in the wine/vinegar products explored here, and also
those present in Zhenjiang aromatic vinegars [32], are present as free non-glycerol-esterified species
and their corresponding anions, and this would account for their higher levels detectable in these
studies than those reported in [43]. Indeed, fermentation processes readily induce the hydrolysis of
TAGs to free FAs, together with mono- and diacylglycerol adducts, and free glycerol [44]; such FAs will
be expected to contribute towards the food pH values determined here. Similarly, Phan et al. [45] found
a broad spectrum of lipidic species, specifically TAGs, polar lipids, free FAs, sterols, and cholesterol
esters present in pinot noir wines.
The official AOAC gravimetric method for lipid determination employed in the current study
involves an acid hydrolysis step involving HCl in any case, followed by extraction with mixed ethers,
i.e., both diethyl and petroleum ethers. Hence, the HCl added will be sufficient to hydrolyze any
residual TAGs present to free FAs and glycerol, and also fully protonate the former so that they are
extractable as such into ether solvents. Indeed, it has been demonstrated that such free FAs are readily
soluble and extractable into these ether solvent systems [46,47]. Hence, the passage of lipidic species
from grapes and/or micro-organisms to finalized bottled wine and vinegar products has been confirmed
in further investigations.
Interestingly, 1 H NMR analysis of 2 H2 O extracts of the FCLO product investigated found
proportionately high concentrations of free FAs and free glycerol therein (data not shown). These FAs
were mainly present as PUFAs, as would be expected from the overall lipid composition of this
product which contains high levels of omega-3 FAs as TAG species prior to fermentation induction.
This observation is fully consistent with the ability of lactobacilli-mediated fermentation processes to
partially hydrolyze TAGs in such a product. High levels of the short-chain organic acids propionic
Foods 2020, 9, 1807 25 of 28

and acetic acids (as their propionate and acetate anions in neutral solution media), both lactobacilli
fermentation catabolites, the former arising from the metabolic reduction of lactate [48], were also
detectable in these extracts. These results will be reported in detail elsewhere.

4. Limitations of the Study

One important limitation of this study is the limited sample sizes of some of the fermented food
sampling classes incorporated into our primary experimental design. This was largely a consequence
of only small numbers of differing fermented food products being available for purchase locally,
for example vegetable sauce and fish products. However, it should be noted that the cheese and
wine/vinegar classifications had BA contents and patterns which markedly contrasted with those
of the other fermented food groups evaluated. These differences, along with those for other food
quality markers observed (Table 3, Figures 1 and 3–5), were found to be very highly statistically
significant, even with these limited sample sizes. Hence, this did not present a major constraining issue.
Moreover, the performance of additional MV analyses on a revised model including a combined fish,
fish sauce/paste, and vegetable sauce classification (on the basis of only a limited level of significant
differences between them) with n = 17 overall served to overcome this problem (Figure 6), and this
incentive did not distract from the main objectives and focus of the investigation in view of their
predominant MV similarities in BA contents. However, univariate analysis found that the mean
spermidine concentration was significantly higher in fermented vegetable sauces than it was in
corresponding fish products (Table 3), and vice-versa for mean 2-phenylethylamine levels (Table 7).
Further evidentiary support was provided by data analysis strategies applied, which were highly
rigorous, and included the preliminary tracking of sample outliers. Furthermore, rigorous Welch tests
were implemented for the ANOVA models employed, and either Bonferroni or FDR corrections were
applied for post-hoc “between-fermented food classification” tests in order to circumvent potential
problems with false positives (type I errors).
Another limitation of the current study was the unavailability of differing manufacturing sources
of FCLO products, and therefore unlike other fermented food products assessed here, statistical
evaluations involved an investigation of 10 separate, randomly-selected batches of a single product,
both separately (Table 5) and jointly with all other classes involved in the primary statistical analysis
conducted (Table 7, and Figures 5 and 6). However, the very wide between-batch variance of all FCLO
samples explored facilitated this approach.
Finally, one further limitation is the poor specificity and interpretability of the TBARS method
employed for the quality assessment of fermented food products here, specifically for assessments of
their degrees of lipid peroxidation. However, one major precautionary step was taken in this study to
minimize problems and potential interferences in this assay system, and this involved the avoidance
of an aldehydic artefact-forming heating stage. Future investigations of the lipid oxidation status
of fermented foods should therefore employ more reliable and specific methodologies such as those
involving high-resolution 1 H NMR analysis for the direct, simultaneous, multicomponent analysis of a
series of both primary and secondary lipid oxidation products, e.g., conjugated hydroperoxydienes
and their aldehydic fragmentation products, respectively. This protocol may be applied directly to
solution-state products, or indirectly to either aqueous or lipid/deuterochloroform extracts of fermented
food products.

5. Conclusions
This study demonstrated that almost all fermented foods tested had total BA levels which
lay below the maximum recommended values for them. A composite application of univariate
and MV chemometrics techniques clearly demonstrated that the MV approach applied was
valuable for discriminating between fermented wine/vinegar products and cheeses, and the
distinction between these two fermented food classes and a combination of fish, fish sauce/paste,
and vegetable sauce products. Further MV analysis performed on CS-normalized BA profiles revealed
Foods 2020, 9, 1807 26 of 28

distinctive patterns for cheese (cadaverine only), FCLOs (the aromatic BAs 2-phenylethylamine,
tyramine, and tryptamine), and wine/vinegar products (pathway-associated putrescine, spermidine,
and spermine). Such distinctive signatures for fermented food BA contents may offer useful information
regarding the nature of, and regulatory conditions employed for, fermentation processes utilized
during their commercial production.
The simultaneous untargeted analysis of eight or more BAs using the LC-MS/MS analysis strategy
employed here offers major advantages which are unachievable by alternative, more targeted techniques
with the ability to determine only single or very small numbers of chemometrically-important analytes.
Notably, the diagnostic potential of a series of n (for example, five or more) BA content analyte variables
in a MV chemometrics investigation offers major advantages over the analytical acquisition of only a
single possible marker. Indeed, food sample patterns of BAs and related food quality indices, which are
characteristic of a particular fermented food product classification, will be expected to provide a much
higher level of statistical power, reliability, and confidence concerning the accurate distinction between
these classifications, and their accurate and selective assignment to one of them, than that discernable
from a single BA analyte level only. Secondly, the patterns of BAs and associated food quality criteria
determined, together with their correlations to particular factors or components (predominantly
linear, but occasionally quadratic or higher combinations of predictor BA and supporting variables),
may potentially serve to supply extensive information regarding the sources of such BAs, bacterial,
commercial, or otherwise.

Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/12/1807/s1,

S1: Summary of Antioxidant Activities of BAs, S2: Potential Adverse Health Effects of Dietary Bas, S3: Outline of
Analytical Techniques Available for BA Determinations and the Screening of BA-Generating Bacteria in Foods.
Author Contributions: J.Z. was responsible for the manufacture of FCLO samples and the random distribution
of these samples from different batches for analysis; he was also responsible for surveys of the availabilities,
and purchases of all fermented food products from US retail outlets, together with their distribution for analysis.
M.G. and B.C.P. monitored and validated all chemical analysis methods for fermented food products, involving
those for BAs, TA and pH values, total lipid contents, and TBARS levels. M.G. was responsible for study
experimental design, and also performed the univariate and MV chemometrics analyses of analytical datasets
acquired, with assistance from B.C.P., M.G. also prepared, drafted, and finalized the manuscript for submission
purposes. J.Z., B.C.P., and M.G. reviewed and edited manuscript drafts, and also contributed towards the
interpretation of experimental results obtained. M.G. also fully supervised the complete study. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was part-funded by The Weston-Price Foundation, grant number WP1-MG3.
Acknowledgments: All authors are very grateful to the Weston A. Price Foundation (DC, USA) for part-funding
the study, and to Midwest Laboratories (13611 B Street, Omaha, NE 68144-3693, USA) for performing the
laboratory analysis of BAs. We are also grateful to Dave Wetzel of Green Pastures Products Inc. (NE, USA) for
valuable discussions.
Conflicts of Interest: J.Z. is an employee of Green Pasture Products, 416 E. Fremont Street, O’Neill, NE 68763,
USA. None of the other authors declare any conflicts of interest. The sponsoring body had no role in the design,
execution, interpretation, or writing of the study.

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