Accepted Manuscript

Formation of tyramine in yoghurt during fermentation

Interaction between yoghurt starter bacteria and Lactobacillus
plantarum

Cemile Ylmaz, Vural Gkmen

PII: S0963-9969(17)30163-1
DOI: doi: 10.1016/j.foodres.2017.04.014
Reference: FRIN 6673
To appear in: Food Research International
Received date: 22 February 2017
Revised date: 10 April 2017
Accepted date: 16 April 2017

Please cite this article as: Cemile Ylmaz, Vural Gkmen , Formation of tyramine in
yoghurt during fermentation Interaction between yoghurt starter bacteria and
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 ACCEPTED MANUSCRIPT

Formation of Tyramine in Yoghurt during Fermentation Interaction between

Yoghurt Starter Bacteria and Lactobacillus plantarum

Cemile Ylmaz, Vural Gkmen*

Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering,

Hacettepe University, 06800 Beytepe, Ankara, Turkey

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* Corresponding Author:

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Prof. Dr. Vural Gkmen, e-mail: vgokmen@hacettepe.edu.tr, tel: +90 312 2977108
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Abstract

This study aimed to investigate the formation of tyramine during yoghurt fermentation

with the focus on interaction between Streptococcus thermophilus RSKK 04082,

Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 and Lactobacillus plantarum

RSKK 02030. These microorganisms were used in the yoghurt fermentation as single

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strains or mixed cultures containing double or triple strains. The interactions between

microorganisms have been also revealed by determining total free amino acids and the

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pH of the medium together with the microbial count of the strains. It was observed that

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L. delbrueckii subsp. bulgaricus DSM 20081 did not produce tyramine while S.
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thermophilus RSKK 04082 and L. plantarum RSKK 02030 could produce tyramine

depending on the fermentation conditions. Synergistic interactions between S.

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thermophilus RSKK 04082 and L. delbrueckii subsp. bulgaricus DSM 20081 and,

between L. delbrueckii subsp. bulgaricus DSM 20081 and L. plantarum RSKK 02030
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were found in terms of tyramine production. It was observed in this study that L.

delbrueckii subsp. bulgaricus DSM 20081 had indirect effect on accumulation of

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tyramine in the yoghurts.

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Keywords: Yoghurt, fermentation, tyramine formation, Lactobacillus plantarum,

microbial interaction
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1. Introduction

Probiotics are defined as live microorganisms that, when administered in adequate

amounts, confer a health benefit on the host (Hill et al., 2014). Due to health benefits of

probiotic microorganisms, there is an increasing interest in developing novel foods

containing probiotics (Sidira et al, 2017). Probiotics reduce symptoms of lactose

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intolerance, prevent inflammatory bowel disease and improve balance of intestinal

microflora (Lomer, Parkes, & Sanderson, 2008; Sazawal et al., 2006; Zhang, Li,

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Caicedo, & Neu, 2005).

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The genera Lactobacillus and Bifidobacterium are mostly used probiotic
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microorganisms in food products (Tripathi & Giri, 2014). Dairy products are one of the

most effective carriers for probiotics due to the fact that milk contains all of the growth
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factors required for probiotics (Garcia, de Oliveira, Queiroga, Machado, & de Souza,

2012; Shori, 2016). Probiotics are generally added to dairy products such as cheese,
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yoghurt and ice cream (Brinques & Ayub, 2011; Bezerra, de Oliveira Arcanjo, de

Araujo, de Queiroz, de Oliveira, Gomes, & Madruga, 2017; Cruxen, Hoffmann,

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Zandona, Fiorentini, Rombaldi, & Chaves, 2017).

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Yoghurt is defined as milk fermented with symbiotic starter cultures of Streptococcus

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thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, which shall be in a

viable state, active and still present in the product through the end of shelf life

(CODEX, 2003). Yoghurt is the most famous food matrix for delivery of probiotics

(Linares, Several probiotic bacteria

such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis and

Bifidobacterium bifidum were added into yoghurts (Shoji et al., 2013; Li, Wang, Sun,

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Li, Liu, & Meng, 2016; Settachaimongkon et al., 2015; Vijayendra & Gupta, 2013).

Apart from these bacteria, Lactobacillus plantarum was also used as a probiotic culture

in yoghurts in recent studies (Settachaimongkon et al., 2016; Sidira et al., 2017;

Brinques & Ayub, 2011).

Bioactive amines, low molecular weight organic bases, are mainly formed by

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decarboxylation of related amino acids or by amination/transamination of aldehydes and

ketones (Benkerroum, 2016). At physiological concentrations, they have important roles

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in the body, either in the nervous system as neurotransmitters, or in the vascular system

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as vasoactive substances (Benkerroum, 2016). The most important biogenic amines in
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dairy foods are histamine, tyramine, putrescine, cadaverine, -phenylethylamine and

tryptamine (Linares, Martin, Ladero, Miguel, & Fernandez, 2011). Tyramine, which is
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formed by decarboxylation of tyrosine, has a role in the central nervous system as a

releasing agent of catecholamines including adrenaline, noradrenaline and dopamine

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(Benkerroum, 2016). However, high amount of exogenous tyramine causes adverse

effects on human health such as migraine and hypertensive crisis (EFSA, 2011). The
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formation of these amines, including tyramine, is mainly related with microorganisms

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participating in fermentation process (Lineras et al., 2012). Therefore, interactions

between microorganisms could have a role on the production of tyramine in fermented

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dairy products.

The aim of this study is to reveal the interactions between S. thermophilus, L.

delbrueckii subsp. bulgaricus and L. plantarum in the formation of tyramine during

yoghurt fermentation. For this reason, these microorganisms were used in the yoghurt

fermentation as single strains or mixed cultures containing double or triple strains. Since

their presence as mixed cultures might also affect some physicochemical properties of

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the yoghurt, the interactions between microorganisms have been revealed by also

determining total free amino acids and the pH of the medium together with the

microbial count of the strains.

2. Materials and Methods

2.1. Chemicals and consumables

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Acetonitrile (HPLC grade), serotonin hydrochloride (98%), tryptamine (>98%),

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tyramine (99%), and dopamine hydrochloride were obtained from Sigma-Aldrich

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(Steinheim, Germany). Formic acid (98%), lactic acid (90%) and high-purity (>98%)

alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid
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(Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu),

lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),
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tryptophan (Trp), tyrosine (Tyr), valine (Val), and gamma-aminobutyric acid (GABA)

were purchased from Merck Co. (Darmstadt, Germany). De Man, Rogosa & Sharpe
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(MRS) broth and bacteriological agar were obtained from Lab M (Lancashire, UK).
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M17 agar was obtained from Liofilchem (Roseto Degli Abruzzi, Italy). Vancomycin

was purchased from Koak Pharmaceutical (Turkey).

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Deionized water (5.6 S/m) was used throughout the experiments (Milli Q-System,
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Millipore, Milford, MA, USA). Syringe filters (nylon, 0.45 m) and Atlantis HILIC

Silica column (2.1 x 150 mm i.d., 3 m) were supplied by Waters Corp. (Milford, MA,

USA).

Ultra high temperature (UHT) milk (3.0% protein, 3.3% fat and 4.7% carbohydrate)

was supplied from a local market in Turkey. Streptococcus thermophilus RSKK 04082

and Lactobacillus plantarum RSKK 02030 were obtained from Refik Saydam National

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Type Culture Collection (Ankara, Turkey), a member of World Federation for Culture

Collections. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 was obtained

from DSMZ-German Collection of Microorganisms and Cell Culture (Braunschweig,

Germany).

2.2. Preparation of yoghurts

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2.2.1. Culture preparation

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Freeze-dried pellets of S. thermophilus, L. delbrueckii subsp. bulgaricus and L.

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plantarum were propagated in MRS broth and stored in a glycerol solution (40%) at -80

C. Before fermentation, frozen bacteria were re-propagated in MRS broth at 37 C for

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24 h.
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2.2.2. Fermentation

Sterile containers were filled with UHT milk (100 mL) aseptically and milk was heated
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to 42 C. 100 L of broth containing related culture (9 log cfu/mL broth) was added into

the milk to perform fermentation. The milk was rapidly inoculated with S. thermophilus
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RSKK 04082 (S), L. delbrueckii subsp. bulgaricus DSM 20081 (B) and L. plantarum
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RSKK 02030 (P), and combinations of these bacteria as S and B (SB), S and P (SP), B

and P (BP), S, B and P (SBP). The initial counts of S were 6.5, 6.2, 6.5 and 6.2 log
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cfu/mL in milks fermented with S, SB, SP and SBP, respectively. The initial counts of P

were 6.2, 6.2, 6.5 and 6.2 log cfu/mL in milks fermented with P, BP, SP and SBP,

respectively. The initial counts of B were 5.6, 5.4, 5.4 and 5.4 log cfu/mL in milks

fermented with B, BP, BS and SBP, respectively. Fermentation was completed in 8

hours and sampling was performed every 2 hours of the fermentation. Fermentation was

carried out in duplicates.

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The pH of yoghurts was measured directly by using a pH meter (PHM210 MeterLab,

France) at room temperature during fermentation. Enumeration of bacteria was carried

out after fermentation periods. For analyses of amino acids and bioactive amines,

yoghurt samples were lyophilized. Samples were kept at -80 C and freeze-drying was

performed for 48 h (Christ Alpha 1-2 LD+, Osterode, Germany).

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2.3. Analysis of bioactive amines and free amino acids

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2.3.1. Extraction

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Extraction of free amino acids was carried out according to the method described by
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Kocadal, zdemir, & Gkmen, (2013). Lyophilized samples (500 mg) were extracted

with water in three stages (5, 2.5, 2.5 mL). After vortexing (10 min) and centrifugation
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(7500xg for 5 min) in each stage, supernatants were collected in a test tube. Combined

supernatants were precipitated with acetonitrile and filtered through a 0.45 m syringe
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filter into an autosampler vial prior to the analyses of tyramine, other bioactive amines

and free amino acids. All samples were extracted in duplicates.

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2.3.2. UPLC-MS/MS analysis of tyramine and other bioactive amines

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Tyramine and other bioactive amines were determined by ultra high performance liquid

chromatography-tandem mass spectrometer (UPLC-MS/MS) (Waters Corp., Milford,

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MA, USA). Chromatographic separation was performed on Atlantis HILIC column (2.1

150 mm, 3 m) by using a isocratic mixture of 0.1 % formic acid in water and 0.1 %

formic acid in acetonitrile (40:60, v/v) at a flow rate of 0.3 mL/min at 40 C. The

injection volume was 3 L. The electrospray source operated in positive ionization

mode, and had the following settings: capillary voltage of 0.80 kV; cone voltage of 20

V; extractor voltage of 4 V; source temperature of 120 C; desolvation temperature of

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370 C; desolvation gas (Nitrogen) flow of 800 L/h and cone gas flow of 100 L/h.

Bioactive amines were identified by multiple reaction monitoring (MRM), using the

conditions in Table 1. Concentrations in the samples were calculated according to the

calibration curves built in the concentration range of 5 and 100 g/L for each

compound. The linearity was evaluated by plotting peak areas against the

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concentrations of bioactive amine standards. Limit of detection (LOD) and limit of

quantification (LOQ) were determined at a signal to noise ratio of 3 and 10,

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respectively. Reproducibility of the method was determined by analyzing three

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replicates in three consecutive days. NU
2.3.3. UPLC-MS/MS analysis free amino acids

Free amino acids were also determined using an UPLC-MS/MS method described
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elsewhere (Kocadal, zdemir, & Gkmen, 2013). Chromatographic separations were

performed on Atlantis HILIC column (2.1 150 mm, 3 m) by using a gradient mixture
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of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) at a flow rate

of 0.4 mL/min. The eluent composition starting with 15 % of A linearly increased to 40

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% in 4 min and held for 3 min. Then, it was linearly decreased to its initial conditions
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(15 % of A) in 1 min. The total chromatographic run was completed in 8 min. The

column equilibrated at 40 C. Electrospray ionization was operated in positive mode.

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The electrospray source had the following settings: capillary voltage of 3.5 kV; cone

voltage of 20 V; extractor voltage of 3 V; source temperature of 120 C; desolvation

temperature of 350 C; and desolvation gas (nitrogen) flow of 900 L/h. Concentration of

amino acids in samples was calculated according to the calibration curve prepared by

using the standard solutions of each amino acid in the concentration range of 0.1 and 2.0

mg/L.

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2.4. Enumeration of viable bacteria

Yoghurt sample (1 mL) was diluted in dilution liquid (9 mL) (0.85% NaCl) and diluted

samples were vortexed (Marienfeld Superior, Germany). 100 L of diluted sample was

seeded on the surface of plates. Viable counts of S were determined on M17 agar at 37

C for 24 h (Ashraf & Shah, 2011). Enumeration of B was carried out according to the

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method described by Sule et al. (2014) with some modifications. Viable counts of B

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were determined on MRS agar (pH 6.4) supplemented with 0.05% Cys after anaerobic

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incubation at 40 C for 72 h. Cys was used to provide anaerobic condition and enhance

the growth of L. delbrueckii subsp. bulgaricus. Viable counts of P were determined on

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MRS agar supplemented with 50 mg/L vancomycin after anaerobic incubation at 37 C

for 24 h (Settachaimongkon et al., 2016). Anaerobic incubation was carried out in an

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anaerobic jar with AnaeroPack (MGC, Japan). Enumeration of viable bacteria was

carried out in duplicates.

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2.5. Statistical analysis

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The results were reported as mean standard deviation. Significant differences (p <
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0.05) were evaluated by Duncan test via analysis of variance (ANOVA) by using SPSS

17.0 (Chicago, IL, USA).

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3. Results and Discussion

3.1. pH Changes

pH values of yoghurts might change depending on using single strain or mixed culture

for yoghurt production. Fig. 1 indicates the changes in pH of yoghurts fermented with

single strain and mixed cultures. pH values of milk samples fermented with S, B and P

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decreased from 6.6 to 5.4, 6.1 and 5.6 at the end of 8 hours of fermentation,

respectively. When yoghurts were fermented with SB, SP, BP and SBP, pH values

decreased to 4.8, 5.3, 5.0 and 4.6, respectively. It was obvious that pH values of

yoghurts fermented with double and triple strains were lower than those of fermented

with single strains. Considering the changes in pH values of yoghurts, it was found that

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acid development in B containing yoghurts (SB, BP and SBP) was significantly higher

(p<0.05) than the others at the end of 8 hours of fermentation. Similar to the findings of

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this study, stimulation of acid production in mixed cultures of S. thermophilus and L.

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delbrueckii subsp. bulgaricus compared to their single strain cultures was also reported

by other researchers (Herve-Jimenez et al., 2009; Sieuwerts et al., 2010).

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3.2. Changes in microbial counts
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Changes in viable counts of S, B and P of yoghurt samples fermented with single strain

and mixed cultures are given in Table 2. At the end of 8 hours of fermentation, the
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count of S increased 1.6 log units. When yoghurt samples were fermented with SB and

SBP, the increases in S count were 2.2 log and 2 log units, respectively. Nevertheless,
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1.5 log units increase in S count was observed in yoghurt fermented with SP.
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Therefore, it could be said that B had positive effect on the growth of S. On the other

hand, P did not have positive effect on the growth of S in SP fermented yoghurts. It was
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previously reported that the association between L. delbrueckii subsp. bulgaricus and S.

thermophilus resulted in a higher final population for S. thermophilus in milk (Herve-

Jimenez et al. 2009; Sieuwerts et al. 2010).

When milk was fermented with P, increase in P count was found to be 2 log units at the

end of 8 hours of fermentation. Increases in P count were 2.2 log, 1.5 log, and 1.9 log

units in yoghurts fermented with BP, SP and SBP, respectively. It was obvious that

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growth of P was affected positively by the incorporation of B.

In milk fermented with B, increase in B count was 1.1 log units at the end of 8 hours of

fermentation. When yoghurts were produced with BP, BS and SBP, increases in B

count were 2.3 log, 1.8 log, and 2.4 log units, respectively. As a consequence, S and P

promoted the growth of B in yoghurt samples. It was also reported by Herve-Jimenez et

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al. (2009) that the count of L. delbrueckii subsp. bulgaricus was higher in milk

fermented with mixed culture than single-strain culture.

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3.3. Formation of bioactive amines

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Bioactive amines in yoghurt samples were identified with their retention times and
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specific MRM transitions by means of UPLC-MS/MS (Table 1). There were high linear

correlations between the concentrations of bioactive amine compounds and their peak
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areas (Table S1, see supplementary material). LOD and LOQ values were given in

Table S2 (see supplementary material). The method allowed quantitation of low ppb
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levels in yoghurt samples. Day-to-day reproducibility of the ESI-MS signal intensities

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of the ions was found high with a coefficient of variation of less than 5.0%.

The concentration of bioactive amine in the milk was determined before yoghurt was
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inoculated with selected cultures. GABA was the only bioactive amine with a
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concentration of 0.90 mg/kg d.w (dry weight) while tryptamine, tyramine, dopamine

and serotonin were below the detection limit used. Concentrations of the GABA in

yoghurts did not change significantly at the end of 8 hours of fermentation (p>0.05)

(data was not shown). No other bioactive amines except tyramine were formed at the

end of 8 hours of fermentation.

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The changes in tyramine concentration of yoghurts fermented with single strain and

mixed cultures are given in Table 3. The cultures of S, B and P prepared in MRS broth

and transferred to the milk resulted in the initial tyramine concentration of milk samples

changing from 0.060.01 to 6.270.30 mg/kg d.w. After inoculation of strains to the

milk, tyramine concentrations of milk samples fermented with single strain cultures

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increased significantly during fermentation (p<0.05) except for milk sample fermented

with B (p>0.05). Concentrations of tyramine in milk samples fermented with S and P

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increased significantly from 2.600.02 to 6.750.35 mg/kg d.w and from 2.410.06 to

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4.910.08 mg/kg d.w at the end of 8 hours of fermentation, respectively (p<0.05). In a

previous study, it was reported that only one strain produced tyramine within the eight
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strains of L. delbrueckii subsp. bulgaricus and within the twelve strains of S.
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thermophilus (Bunkova et al., 2009). Moreover, Bover-Cid, Hugas, Izquierdo-Pulido, &

Vidal-Carou, (2001) found that some strains of L. plantarum isolated from fermented
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foods have capability to produce tyramine.

When yoghurt samples were fermented with SB, BP, SP and SBP, concentrations of
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tyramine raised continuously reaching to 20.462.62, 8.750.17, 8.540.10 and

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29.232.19 mg/kg d.w at the end of 8 hours of fermentation, respectively (p<0.05)

(Table 3). Considering the threshold toxic level of tyramine which was 600 mg and
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acceptable level of tyramine in cheese which was given as 100-800 mg/kg

(Benkerroum, 2016), concentrations of tyramine in yoghurt samples were not in the

range of toxic levels.

Concentration of tyramine increased about 6 fold in yoghurt fermented with SB whereas

it increased only 2.5 fold in milk fermented with S. On the other hand, tyramine

concentration did not increase in milk fermented with B. It was obvious that there was a

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synergistic relationship between S and B in the conditions of yoghurt fermentation

resulted in more tyramine production. A similar synergistic effect was also observed in

yoghurt fermented with BP although it was not as strong as in case of SB. Tyramine

concentration in yoghurt fermented with BP increased about 3.5 fold. As opposed to the

SB and BP fermented yoghurts, tyramine concentration did not reach to the total

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tyramine concentration of their single strain yoghurts in SP fermented yoghurts.

Therefore, relationship between S and P in terms of tyramine production could be

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explained as antagonism. The highest relative increase in tyramine concentration was

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observed in yoghurt fermented with SBP. In case of triple strain culture, SBP, the

tyramine concentration increased compared to SP.

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Considering the tyramine concentrations of mixed culture yoghurts, it was remarkable
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that whenever B involved in the mixed culture, the tyramine concentration increased

much more than their additive effects during fermentation. Interestingly, B itself had no
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effect on tyramine concentration. The hidden role of B on the tyramine concentration of

mixed cultures could be explained based on the two hypotheses that will be further
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explained and discussed in detail below. One of the hypotheses was when S or P was in
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the mixed culture with B, they reduce the pH of the medium to the optimum pH for B

(Fig. 1). Then, B might begin to produce tyramine. The other hypotheses was if B had
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proteolytic activity resulting in the increase in the concentration of free amino acids, it

would enrich the medium and provide sources for the production of tyramine for S and

P. Under favor of these hypotheses, two modifications in fermentation medium were

carried out to explain the reason of the positive interaction between B and S and, B and

P in mixed cultures.

3.4. Modifications in the growth medium

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3.4.1. Decrease in pH

To reveal the first hypothesis, the pH values of the milks were adjusted to 5.5 and 6,

which were the optimum pH values for the activity of L. delbrueckii subsp. bulgaricus.

It was observed that growth of B in milk samples fermented at pH 5.5 and pH 6.0 was

higher than that of fermented at pH 6.6. However, concentration of tyramine did not

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change significantly during fermentation (p>0.05). These results proved that B could

not synthesize tyramine even if pH of milk was optimum for B (Table 4).

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3.4.2. Increase in the concentrations of free amino acids

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The concentrations of total free amino acids in yoghurts fermented with mixed cultures
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and single strain cultures are given in Table 5. In milk samples fermented with S and P,
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concentrations of total free amino acids decreased significantly from 949.929.2 to

673.748.6 mg/kg d.w and from 1005.422.8 to 918.612.5 mg/kg d.w at the end of 8
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hours of fermentation, respectively. However, when milk was fermented with B,

concentration of total free amino acid increased from 860.226.9 to 1280.211.2 mg/kg
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d.w. Due to having proteolytic activity (Rajagopal & Sandine, 1990; Tamime &

Robinson, 2000), L. delbrueckii subsp. bulgaricus increased the total free amino acid
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content of yoghurt. It was previously demonstrated that the predominant proteolytic

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activity in yoghurt is due to PrtB, a cell-wall protease from L. delbrueckii subsp.

bulgaricus. The first step of proteolysis in yoghurt is carried out by cell-wall proteases,

which hydrolyze the milk casein into peptides, and thereby L. delbrueckii subsp.

bulgaricus has a significant role for enriching the medium with the nitrogen sources.

The second step of proteolysis is the degradation of peptides into smaller peptides and

amino acids via endopeptidase and exopeptidase enzymes from S. thermophilus and L.

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delbrueckii subsp. bulgaricus (Tamime & Robinson, 2000). As considered that only a

few strains of S. thermophilus comprise cell-wall proteases (Tamime & Robinson,

2000), a decrease in the total amino acid content of milk fermented with S was

observed. Approximately 9% reduction in total free amino acid content of milk

fermented with P might be explained with low proteolytic activity of most of the L.

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plantarum strains (Georgieva et al., 2009).

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When milk was fermented with SB, BP and SBP, approximately 28%, 13% and 10%

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increases in total free amino acid content were observed at the end of the fermentation,

respectively. Nevertheless, total free amino acid content of milk fermented with SP did
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not change significantly (p>0.05) (Table 5).

Changes in the concentrations of individual free amino acids of yoghurts fermented

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with single, double and triple strains are also given in Table 6. In milk fermented with

S, concentration of all amino acids decreased except Ala, Asp, Cys, His and Pro. The
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concentrations of Ala, Asp, Cys and His did not change significantly (p>0.05) and the
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concentration of Pro increased during fermentation (p<0.05). Due to the proteolytic

activity, the concentrations of most amino acids increased in yoghurt fermented with B.
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However, concentration of Asp decreased (p<0.05) and concentration of Glu and Cys

did not change at the end of the 8 hours of fermentation (p>0.05). In yoghurt fermented
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with P, the concentrations of most amino acids decreased whereas the concentrations of

Asp, Cys and Pro increased (p<0.05) and the concentration of Glu, Thr and Trp did not

change during fermentation (p>0.05). It was previously reported that total free amino

acid content of milk fermented with S. thermophilus decreased at the end of the

fermentation (Beshkova, Simova, Frengova, Simov & Adilov, 1998). On the other

hand, total free amino acid content of milk fermented with L. delbrueckii subsp.

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bulgaricus increased from 8.67 mg/100g to 90.54 mg/100g at the end of the

fermentation (Beshkova, Simova, Frengova, Simov & Adilov, 1998).

When milk was fermented with complex cultures, concentrations of amino acids

decreased except Asp, Cys, Glu, His, Pro and Trp even though total free amino acid

content increased at the end of the fermentation. Therefore, increase in total free amino

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acid content of yoghurts was due to accumulation of Asp, Cys, Glu, His, Pro and Trp. It

was previously reported that Glu and Pro accumulated in large quantities in yoghurt

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samples, because they were not preferred as nitrogen source by S. thermophilus and L.

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delbrueckii subsp. bulgaricus (Tamime & Robinson, 2000).
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To reveal the second hypothesis, milk was enriched with amino acids and fermented

with either S or P. For this purpose, the total amino acid content of milk (683.2 mg/kg
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d.w) was increased about 2 fold to the value of 1600 mg/kg d.w prior to fermentation

(Table 6). As it was observed in the count of S and P when they are together with B in
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mixed cultures, the count of S and P increased when the milk was enriched with amino

acids. Concentrations of tyramine in milk samples supplemented with amino acids and
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fermented with S and P increased from 2.790.09 to 21.390.35 mg/kg d.w and from
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3.130.10 to 23.00.04 mg/kg d.w at the end of 8 hours of fermentation, respectively

(Table 4). The second hypothesis was proved as the S and P had capability of
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synthesizing tyramine depending on the amount of nitrogen source found in the food

matrix. Although B did not produce tyramine, it had indirect effect on accumulation of

tyramine in the yoghurts providing nitrogen sources for S and P. It was reported in

several studies that increase in amino acid concentration during cheese ripening is

critical for biogenic amine accumulation in cheeses (Fernandez, Linares, del Rio,

Ladero, & Alvarez, 2007; Ladero, Martinez, Martin, Fernandez, & Alvarez, 2010).

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Biogenic amine formation is an important topic for dairy industry because of its

negative health concerns. Therefore, some strategies (e.g., pasteurization of milk,

irradiation) could be carried out to limit biogenic amine levels in dairy products

(Benkerroum, 2016). One of the strategies to prevent the formation of biogenic amines

is the use of decarboxylase-negative starter cultures. Moreover, antagonistic interaction

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between microorganisms in terms of tyramine production could be another way to limit

the production of tyramine in dairy products.

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The availability of precursor amino acids is a limiting factor for the accumulation of

biogenic amines in food products. The precursor amino acids could be released from
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milk proteins as a result of proteolytic activity of microorganisms in dairy foods

(Benkerroum, 2016). Therefore, reducing proteolytic activity could be suggested to

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control biogenic amine accumulation in dairy foods.

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It was previously reported that some strains of L. delbrueckii subsp. bulgaricus, S.

thermophilus and L. plantarum have capability to produce tyramine (Bunkova et al.,

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2009; Bover-Cid, Hugas, Izquierdo-Pulido, & Vidal-Carou, 2001). In this study,

Streptococcus thermophilus RSKK 04082 and Lactobacillus plantarum RSKK 02030

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produced tyramine in yoghurts. It was found that tyramine formation is related with
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strains used for fermentation.

The results enlightened the possible mechanisms of tyramine formation when a new

strain like L. plantarum RSKK 02030 was incorporated into a dairy product, yoghurt.

Since L. delbrueckii subsp. bulgaricus increased the concentration of amino acids,

formation of tyramine production of L. plantarum RSKK 02030 was triggered in the

yoghurts. Therefore, microbial interactions were found to play a crucial role in the

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production of tyramine. It is suggested that strains that do not synthesize tyramine under

the conditions of fermentation could be potentially used to produce tyramine free dairy

products.

4. Conclusions

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This study demonstrated that L. delbrueckii subsp. bulgaricus DSM 20081 strain did not

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produce tyramine in yoghurt. However, S. thermophilus RSKK 04082 and L. plantarum

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RSKK 02030 produced tyramine depending on the fermentation conditions. Tyramine

formation was not only caused by decarboxylase-positive bacteria but also by microbial
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interactions. Relationship between S. thermophilus RSKK 04082 and L. plantarum

RSKK 02030 in terms of tyramine production could be explained as antagonism.

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Antagonistic interaction between microorganisms could be an effective way to reduce

the amount of tyramine in yoghurt. On the other hand, interaction in tyramine

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production between S. thermophilus RSKK 04082 and L. delbrueckii subsp. bulgaricus

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DSM 20081 and, L. delbrueckii subsp. bulgaricus DSM 20081 and L. plantarum RSKK

02030 was determined as synergism. This interaction was due to the fact that L.
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delbrueckii subsp. bulgaricus DSM 20081 provides nitrogen sources for S.

thermophilus RSKK 04082 and L. plantarum RSKK 02030. Therefore, it should not be
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ignored that microbial interactions could be effective on accumulation of bioactive

amines in foods. The dairy industry must take into account both microbial interactions

and decarboxylase negative strains when it is considered to add a new strain to the

yoghurt. Thus, bioactive amine concentrations are kept under control.

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Figure Captions

Fig. 1. Changes in pH of yoghurts fermented with single strain cultures and mixed

cultures during fermentation. S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P:

L. plantarum

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Figure 1

S B P SB
SP BP SBP
7

6
pH

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5

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4
0 2 4 6 8

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Fermentation time, h
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Table 1. The multiple reaction monitoring (MRM) parameters used to detect individual

bioactive amines by UPLC-MS/MS.

Parent ion Daughter ions Cone voltage Collision energy

Amines
(m/z) (m/z) (V) (V)
Serotonin 177 160* 15 10
Tryptamine 161 144.1* 15 12

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Dopamine 154 137* 20 10
91 20 23

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GABA 104 87* 10 13

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69 10 13
45 10 16
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Tyramine 138 121.1* 15 12
77 15 20
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91 15 20
103 15 20
*Quantitation was performed according to marked daughter ion chromatogram.
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Table 2. Changes in the count of bacteria (log cfu/mL) of yoghurt samples

fermented with single strain cultures and mixed cultures during fermentation.

Fermentation time (h)

0 4 8
S.thermopilus
S 6.50.1a 7.50.0b 8.10.0c

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SB 6.20.3a 8.00.0b 8.40.1b
SP 6.50.2a 7.60.0b 7.90.0c

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SBP 6.20.1a 7.70.0b 8.20.1c
L. plantarum

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P 6.20.2a 7.80.0b 8.20.1b
BP 6.20.2a 8.00.1b 8.40.0b
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SP 6.50.2a 7.60.0b 8.00.1c
SBP 6.20.1a 7.80.0b 8.10.0c
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L. bulgaricus
B 5.60.1a 5.80.2a 6.70.2b
BP 5.40.2a 6.80.1b 7.70.1c
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BS 5.40.4a 6.20.2b 7.20.1c

SBP 5.40.3a 6.60.0b 7.80.0c
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* S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P: L. plantarum

Different letters within the same row indicate statistical significance (p < 0.05).
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P T
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S C
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Table 3. Changes in the concentrations of tyramine (mg/kg d.w) of yoghurt samples fermented with single strain cultures and mixed

cultures during fermentation.

Time (h) S B P SB BP
P TSP SBP

0 2.600.02a 0.060.01a 2.410.06a 3.410.05a 2.500.16a

R I
5.220.15a 6.270.30a

4
4.330.11b

4.130.24b
0.050.00b

0.050.00b
3.600.14b

3.940.03c
4.930.30ab

9.341.17b U SC
2.920.02a

6.530.07b
6.840.20b

6.540.18b
7.890.38a

15.310.27b

6 5.270.29c 0.050.00b 4.620.22d

A
21.763.64c
N 11.010.77c 9.290.35c 23.702.11c

8 6.750.35 d
0.060.01 a
4.910.08 d
M
20.462.62d 8.750.17d 8.540.10d 29.232.19d

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* S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P: L. plantarum

P T
Different letters within the same column indicate statistical significance (p < 0.05).

C E
A C

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Table 4. Changes in pH, viable counts of related bacteria and tyramine concentrations of

modified yoghurt samples during fermentation.

Time pH Count of bacteria Tyramine

(h) (log cfu/mL) (mg/kg d.w)
Saa 0 6.50.0a 6.20.3a 2.790.09a

4 5.60.1b 8.70.0b 11.42040b

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8 5.10.1c 9.00.0b 21.390.35c

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Paa 0 6.50.0a 6.10.2a 3.130.10a

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4 5.80.1b 8.80.0b 13.000.48b

8 5.20.1c 8.70.2b 23.050.04c

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BpH6 0 5.90.1a 5.00.0a 0.030.02a

4 5.70.0a 5.40.2b 0.030.00a

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8 5.50.1b 6.40.0c 0.060.01a

BpH5.5 0 5.50.1a 5.10.3a 0.030.02a

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4 5.40.0ab 5.70.0a 0.040.00a

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8 5.20.1b 6.60.3b 0.050.01a

* Saa: Yoghurt supplemented with amino acids and fermented with S. thermophilus,
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Paa: Yoghurt supplemented with amino acids and fermented with L. plantarum,
BpH6: Yoghurt fermented with L. delbrueckii subsp. bulgaricus at pH 6,
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BpH5.5: Yoghurt fermented with L. delbrueckii subsp. bulgaricus at pH 5.5

Different letters within the same column in every panel indicate statistical significance
(p < 0.05).

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Table 5. Changes in the total free amino acid concentrations (mg/kg d.w) of yoghurt samples fermented with single strain cultures and mixed

cultures during fermentation.

Time (h) S B P SB BP SP SBP

0 949.929.2c 860.226.9b 1005.422.8d 1050.315.9a 959.215.4a
T
1107.91.1bc

P
1390.533.5b

2 985.573.8 c
694.538.3 a
715.218.8 a
1104.549.7 a
904.429.4 a

R I
1102.159.1bc 1255.146.3a

4 776.022.5ab 826.938.6b 813.020.7b 1167.254.8a

C
1153.446.5bc
S
936.263.0a 1215.33.7a

6 821.510.0 b
1051.450.1 c
826.427.5 b
1360.131.3

N
b

U
1214.97.0c 1082.031.1b 1448.439.9bc

8 673.748.6a 1280.211.2d 918.612.5c

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* S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P: L. plantarum
A
1344.766.4b 1085.824.7b 1222.653.7c 1526.278.7c

E D
Different letters within the same column indicate statistical significance (p < 0.05).

P T
C E
A C

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Table 6. Changes in the individual free amino acid concentrations (mg/kg d.w) of yoghurt samples

fermented with single strain cultures and mixed cultures during fermentation.

Time, h Ala Arg Asn Asp Cys Gln Glu

S 0 31.25.9a 26.93.1a 21.22.3a 44.37.2a 243.177.6a 6.00.1a 308.350.6a
2 45.31.7b 6.70.1b 29.21.6b 93.816.7bc 220.510.6a n.d 265.418.8ab
4 30.74.7a n.d 5.41.2c 64.25.9ab 221.711.4a 0.60.2b 270.74.6ab
6 45.26.2b n.d 2.91.0c 99.021.2c 239.270.9a 1.60.1c 220.50.5b

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8 39.01.4ab n.d 1.80.4c 57.53.5a 210.035.4a 0.90.1b 207.5.53.5b

B 0 50.24.2ab 48.62.9a 25.90.2a 56.20.6a 20.50.7a 5.70.1a 325.610.3b

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2 43.74.2a 34.02.6a 25.42.0a 40.05.0b 28.54.2a 7.00.6ab 249.717.0a
4 48.39.8a 43.814.1a 37.58.6ab 30.22.8a 28.13.3a 7.61.1b 24514.1a
6 63.44.6ab 72.311.1b 45.55.6bc 38.25.5ab 30.27.7a 9.60.9c 291.640.2ab

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8 83.10.8c 90.22.5b 53.81.7c 42.10.9b 26.85.7a 9.50.4c 331.55.0c

P 0 53.43.3a 36.00.3a 25.60.1a 65.39.3ab 148.40.9a 4.30.3a 322.91.1bc

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2 31.80.4b 5.80.8b 15.80.2b 56.28.5a 157.43.4ab 0.40.0b 245.014.1a
4 32.81.2b n.d 10.50.5c 80.50.8b 161.95.1b 0.40.2b 321.82.6bc
6 28.81.1b n.d 2.60.6d 104.06.2c 186.84.9c 0.20.0b 316.714.6b
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8 31.91.4b n.d 2.80.3d 123.00.2c 213.15.2d 0.20.1b 343.62.5c

SB 0 54.11.8a 37.13.3a 36.71.2a 59.41.3a 194.86.3a 3.90.1a 295.15.4ab

2 54.04.3a 6.20.5a 35.44.5a 86.110.9ab 191.80.4a n.d 323.145.3ab
46.84.4a 16.34.0b 88.820.2ab 276.66.0a 0.10.0b 364.853.7b
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4 n.d
6 53.64.2a n.d 7.92.2b 78.714.1ab 700.673b n.d 240.650.4a
8 53.34.8a n.d 9.03.2b 121.915.4c 566.06.5c 0.90.0b 290.826.7ab
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BP 0 49.12.3a 41.53.3a 34.10.6a 54.02.6a 152.01.6a 2.90.6a 284.40.0a

2 48.17.5a 12.71.7b 38.31.4b 59.01.6a 131.910.5a n.d 245.510.1a
4 45.82.4a 4.90.2c 27.52.0c 74.54.0b 158.01.0a n.d 391.528.9b
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6 59.12.1b n.d 19.01.6d 83.12.6c 310.916.9b n.d 373.923.2b

8 24.30.4c n.d 0.40.0e 96.71.3d 513.912.3c n.d 255.710.8a
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SP 0 51.52.0a 29.51.9a 34.60.3a 54.41.8a 317.81.6a 1.80.3a 286.43.4a

2 40.57.3b 6.31.4b 33.76.4a 71.712.4a 347.57.7a n.d 264.140.7a
4 63.51.9c 4.80.1b 16.61.9b 65.93.8a 165.50.1b n.d 294.732.2a
6 24.90.8d n.d 1.80.2c 97.311.9b 474.425.0c n.d 285.337.7a
8 27.11.7d n.d 1.80.8c 112.90.4b 509.416.2c n.d 341.28.1a

SBP 0 66.50.7a 39.43.8a 52.72.1a 74.81.8a 336.719.2a 2.90.8a 301.334.8a

2 53.88.4ab 7.20.7b 39.78.2b 81.510.7ab 344.325.8a n.d 295.632.7a
4 46.81.8b n.d 11.30.0c 85.52.0ab 416.518.6b n.d 307.35.5a
6 52.93.7b n.d 9.70.3c 103.06.7b 654.629.2c n.d 278.921.8a
8 47.76.1b n.d 4.91.2c 105.318.0b 867.029.0d n.d 228.350.5a
Milk 41.35.3 33.55.2 7.00.8 52.310.9 n.d 1.00.4 336.878.7

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Milkaa 108.013.9 73.07.9 67.34.5 133.918.6 4.10.4 4.50.8 428.420

* S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P: L. plantarum, Milkaa: Milk supplemented with amino
acids
Different letters within the same column belonged to related yoghurt sample indicate statistical significance (p <
0.05).
n.d: not detected

Continued

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Time, h Gly His Ile Leu Lys Met Phe

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a a a a ab a
S 0 60.73.2 13.70.0 0.90.2 16.62.6 65.27.4 7.60.8 8.11.4a
2 51.44.5ab 15.50.8a 1.20.0b 22.41.6b 75.76.1a 9.21.3a 19.14.0b

SC
4 40.56.0bc 14.11.3a 0.10.0c 4.90.6c 33.95.6c 0.70.3b 1.60.0c
6 45.17.1bc 15.22.8a 0.10.0c 3.10.6c 44.718.0bc 0.60.1b 1.10.0c
8 33.52.1c 13.60.3a n.d 1.90.2c 27.53.5c 0.10.0b 0.40.1c
NU
B 0 64.22.2ab 15.10.4a 1.20.0a 21.40.5a 83.80.9ab 4.50.1a 18.22.4ab
2 53.01.8a 11.90.6a 1.20.0a 20.41.2a 60.30.7a 4.20.8a 15.12.3a
MA

4 64.710.7ab 13.82.0a 1.80.4b 29.75.7b 72.624.2ab 12.14.0b 26.66.6bc

6 78.67.1bc 18.71.1b 2.10.2bc 35.53.7bc 96.416.1b 17.84.9bc 29.74.9c
8 89.02.2c 19.01.4b 2.50.0c 41.90.5c 141.90.2c 24.80.3c 42.81.3d

P 0 66.36.5a 26.70.2c 1.20.0a 20.80.6a 80.00.8a 9.40.2a 15.70.9a

ED

2 36.63.5b 15.82.3b 0.60.0b 11.12.9b 41.01.9b 4.81.1b 7.51.8b

4 42.21.7b 11.30.9a 0.30.0c 8.81.5b 41.23.2b 0.70.1c 6.60.8b
6 42.02.3b 12.70.2ab 0.10.0d 3.80.4c 32.33.7c 0.70.0c 7.20.3b
PT

8 45.91.4b 15.30.8b 0.10.0d 3.70.2c 39.72.2b 0.80.1c 7.50.4b

SB 0 58.00.9a 13.00.4a 1.60.1a 29.00.6a 92.54.4a 13.80.4a 26.81.9a

CE

2 51.73.9a 17.31.4ab 1.40.1b 28.33.7a 90.56.4a 12.31.8a 27.15.7a

4 35.71.9b 29.75.5c 0.20.0c 14.82.9b 59.514.1b 5.31.7b 19.05.6ab
6 34.10.9b 25.76.3bc 0.20.0c 8.52.1b 49.96.3bc 3.51.4b 10.24.5bc
AC

8 24.36.3b 18.34.4abc 0.20.0c 9.32.0b 31.30.9c 2.10.9b 5.13.7c

BP 0 49.21.9a 13.50.5a 1.50.0a 27.60.5a 85.42.1a 12.20.1a 26.11.1a

2 49.58.1a 14.50.2a 1.60.0b 30.41.3a 80.91.9a 14.01.7a 31.03.7a
4 34.07.0ab 34.34.0b 0.50.0c 24.02.0b 84.65.3a 13.11.3a 26.51.7a
6 25.26.9b 25.83.4c 0.30.0d 16.70.5c 56.22.4b 6.40.1b 19.80.3b
8 32.05.9b 18.60.8a 0.10.0e 2.40.2d 30.80.3c 0.60.1c 1.30.5c

SP 0 44.64.5ab 13.20.0a 1.50.0a 28.10.1a 82.18.1a 12.40.3a 24.90.2a

2 31.30.6a 14.72.3a 1.30.2a 28.23.5a 80.117.0a 11.80.7a 31.04.6b
4 56.76.6b 20.32.9ab 0.50.1b 15.61.2b 67.05.5a 8.60.9b 17.71.6c
6 35.64.8a 15.30.5a 0.10.0c 3.30.1c 24.51.8b 0.50.0c 1.60.3d

36
 ACCEPTED MANUSCRIPT

8 41.57.1a 24.45.9b 0.10.1c 3.80.9c 31.210.1b 1.30.5c 2.01.0d

SBP 0 59.03.9a 19.81.0a 2.20.1a 40.11.5a 127.514.6a 20.10.9a 49.02.8a

2 51.04.8b 18.74.3a 1.60.3b 32.96.8a 94.528.4ab 14.42.7b 35.16.3b
4 38.71.7c 28.00.4b 0.20.0c 11.50.2b 71.71.3bc 2.50.1c 17.80.6c
6 30.81.7c 28.41.4b 0.20.0c 10.20.6b 62.10.5bc 2.00.3c 15.20.3cd
8 32.20.3c 21.11.9a 0.10.0c 6.30.8b 47.75.1c 1.30.3c 8.82.4d
Milk 66.56.4 14.91.1 0.60.0 14.01.9 53.50.6 6.41.3 4.81.3
Milkaa 126.615.6 61.75.7 3.60.5 54.78.9 116.71.1 46.42.9 36.711.3
* S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P: L. plantarum, Milkaa: Milk supplemented with amino

PT
acids
Different letters within the same column belonged to related yoghurt sample indicate statistical significance (p <

RI
0.05).
n.d: not detected

SC
NU
Continued
MA

Time, h Pro Ser Thr Trp Tyr Val

S 0 27.95.1a 17.74.0a 13.62.3a 7.10.1a 5.80.5a 22.24.3a
2 38.02.2a 23.80.7b 26.00.9c 3.71.0b 4.60.5b 33.52.3b
4 57.910.1ab 5.21.6c 15.24.4a 4.20.7b 0.60.2c 3.31.9c
ED

6 83.321.4b 1.90.5c 10.52.0ab 1.40.2c 1.00.2c 4.70.5c

8 75.07.1b 1.20.3c 6.01.4b n.d 0.40.1c 2.70.5c

37.41.4a 21.52.0a 16.20.8ab 3.50.1ab 8.50.0a 31.52.7ab

PT

B 0
2 27.91.8b 21.00.6a 13.31.6a 2.70.2a 7.31.4a 27.21.5a
4 37.04.2a 32.67.1a 19.35.8ab 5.51.2b 15.54.2a 54.713.3bc
49.44.2c 45.82.0b 23.11.4bc 8.01.2c 27.65.5b 67.28.1cd
CE

6
8 62.41.1d 54.20.5b 28.61.3c 12.00.3d 40.50.1c 83.00.1d

P 0 35.21.4a 31.75.7a 19.02.3a 3.30.6ab 9.21.5a 30.90.8a

AC

2 34.35.7a 10.25.6b 18.04.2a 2.40.6a 2.91.7b 17.63.1b

4 42.33.2a 4.91.5b 20.44.9a 3.10.1ab 1.20.3b 21.70.4b
6 59.12.9b 6.73.1b 15.81.8a 3.90.4b 1.10.2b 1.50.4c
8 63.01.6b 2.60.2b 18.41.0a 4.30.7b 0.80.1b 1.10.1c

SB 0 35.10.8a 22.00.0a 20.80.8a 6.30.7a 6.20.1a 43.30.4ab

2 51.84.2a 30.41.1b 29.83.1b 8.12.0a 4.00.9ab 54.55.8b
4 102.512.5b 5.31.2c 22.14.7ab 10.12.4a 4.01.2ab 64.915.8b
6 90.84.2b 10.03.7c 7.12.1c 9.43.7a 3.50.7b 25.36.6a
8 129.89.1c 8.71.6c 12.13.2c 10.53.2a 2.60.9b 47.87.9ab

BP 0 29.80.0a 22.93.7a 20.72.4a 5.50.5a 5.20.1ab 40.91.4a

37
 ACCEPTED MANUSCRIPT

2 34.30.7a 31.95.7b 22.50.8a 7.20.4b 4.60.6a 45.61.7a

4 89.61.7b 9.12.3c 27.51.8b 10.20.9c 13.30.7c 83.87.7b
6 95.711.2b 5.90.3c 19.70.9a 11.40.4c 6.91.6b 78.14.6b
8 85.81.4b 2.90.5c 6.90.5c 8.30.7b 0.50.2d 4.40.5c

SP 0 29.70.1a 19.50.8a 21.10.2b 6.10.0a 6.80.3a 41.00.7a

2 35.05.5a 21.05.1a 26.74.4c 7.21.6ab 4.60.1b 44.68.7a
4 62.33.0b 9.53.0b 12.80.1a 9.10.1b 5.90.2ab 38.54.5a
6 86.16.5c 4.60.1bc 11.21.2a 7.10.5ab 1.00.3c 6.90.8b
8 92.62.8c 0.410.1c 13.61.3a 8.70.5b 1.30.4c 8.72.4b

PT
SBP 0 35.80.4a 47.25.2a 33.10.0a 11.50.2ab 8.40.4a 61.54.5a

RI
a
2 54.89.8 26.10.5b 29.86.6 a
10.23.8 a
3.40.8 b
58.89.4a
4 100.61.2b 5.60.4c 13.60.7b 12.50.6ab 6.60.0a 38.00.6b
b
6 120.06.8 7.82.3c 10.91.8 b
16.50.2 b
6.31.6 a
38.14.4b

SC
8 100.911.5b 6.60.3c 6.31.0b 13.01.7ab 1.80.8b 26.15.7b
Milk 25.756.6 5.61.7 12.00.9 2.03.8 2.61.8 2.73.6
Milkaa 75.06.7 62.99.9 78.44.7 43.09.6 29.44.2 46.19.1
NU
* S: S. thermophilus, B: L. delbrueckii subsp. bulgaricus, P: L. plantarum, Milkaa: Milk supplemented with amino
acids
Different letters within the same column belonged to related yoghurt sample indicate statistical significance (p <
MA

0.05).
n.d: not detected
ED
PT
CE
AC

38
 ACCEPTED MANUSCRIPT

Graphical abstract

30
yoghurt fermented with SB
25 yoghurt fermented with SBP

yoghurt fermented with S

Tyramine, mg/kg

20
yoghurt fermented with B
15
yoghurt fermented with P

PT
10
S: S.thermophilus RSKK 04082
B: L. delbrueckii subsp. bulgaricus DSM 20081
5 P: L. plantarum RSKK 02030

RI
0
0 2 4 6 8

SC
Fermentation time, h
NU
MA
ED
PT
CE
AC

39
 ACCEPTED MANUSCRIPT

Highlights

L. delbrueckii subsp. bulgaricus DSM 20081 did not synthesize tyramine.

There was a synergistic interaction in tyramine production between S.

thermophilus and L. bulgaricus.

L. delbrueckii subsp. bulgaricus DSM 20081 had indirect effect on tyramine

PT
formation.

RI
GABA, serotonin, dopamine and tryptamine were not formed during yoghurt

fermentation.

SC
NU
MA
ED
PT
CE
AC

40