LWT - Food Science and Technology 163 (2022) 113581

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LWT
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Fermented sheep’s milk enriched in gamma-amino butyric acid (GABA) by

the addition of lactobacilli strains isolated from different
food environments
Inés María Ramos , Justa María Poveda *
Department of Analytical Chemistry and Food Technology, Faculty of Chemical Sciences and Technologies and Regional Institute for Applied Scientific Research (IRICA),
University of Castilla-La Mancha, Avda. Camilo José Cela, s/n, 13071, Ciudad Real, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Lactic acid bacteria, and especially lactobacilli, are the main producers of γ-amino butyric acid (GABA), which
GABA has numerous potential health benefits. In this work, the GABA production capacity in milk of 38 native strains of
Functional fermented milk lactobacilli isolated from different food ecosystems was evaluated, and three of them were selected for the
Lactic acid bacteria
production of fermented sheep’s milk enriched in GABA without the addition of monosodium glutamate.
Health properties
Sheep’s milk
Physico-chemical analyses (pH, titratable acidity, total solids), including water retention capacity, viscosity,
production of GABA, glutamic acid, were carried out, as well as descriptive sensory analysis in fermented milks
for a storage period of 28 days at 4 ◦ C. GABA concentrations around 200 mg/L were found in fermented milks
made with the strains Lacticaseibacillus paracasei Lb41 and Lactiplantibacillus plantarum Lb56. Furthermore, these
fermented milks also presented higher viscosity values than the control, and they had good and typical sensory
characteristics.

1. Introduction Quílez, & Rafecas, 2014).

Functional foods constitute an important sector of the current food
industry in which the dairy industry plays a relevant role (Balthazar
et al., 2016). The bioactive compounds produced by dairy fermentation
could offer health benefits. Among these bioactive compounds,
gamma-aminobutyric acid (GABA) has received significant recent
attention from the food industry.
GABA is a nonprotein four-carbon free amino acid, synthesised by
the irreversible α-decarboxylation reaction of L-glutamic acid or its salts;
the reaction is catalysed by glutamic acid decarboxylase (GAD; EC
4.1.1.15) and its cofactor pyridoxal 5-phosphate (PLP; active vitamin
B6). GABA has potential health benefits, including antidepressant,
sedative, antihypertensive, antidiabetic, anticancer and immune system
enhancement benefits (Abdou, 2006; Adeghate & Ponery, 2002; Hay­
akawa et al., 2004; Huang et al., 2013; Pouliot-Mathieu et al., 2013;
Schuller, Al-Wadei, & Majidi, 2008). In animals, GABA performs
essential activities as an inhibitory neurotransmitter via several path­
ways, namely the central nervous system and peripheral tissue. GABA
also has a positive effect in patients with Huntington’s, Parkinson’s,
Alzheimer’s and stiff-person syndromes and schizophrenia (Diana,
Studies have mainly focused on GABA-producing microorganisms
rather than GABA in isolation. Microbiological production of GABA is
safer and eco-friendlier than chemical methods. Therefore, lactic acid
bacteria (LAB) and yeasts are very important GABA producers, since
they can be used in the manufacture of many fermented foods. Many
investigations have shown the GABA-producing ability of LAB isolated
from fermented foods, with lactobacilli being the major producers
(Quílez & Diana, 2017). High GABA production by LAB is related not
only to its GAD activity but also to the sufficient concentration of glu­
tamic acid in the food matrix. Accordingly, GABA-producing LAB can be
used to develop fermented health-oriented foods. It has been reported
that many foods are a natural source of GABA, and the application of
lactic acid fermentation can increase its content severalfold (Gan, Li,
Gunaratne, Sui, & Corke, 2017). Indeed, this has been verified by recent
studies conducted using lactic acid bacteria strains with high
GABA-producing capacity to produce fermented dairy products (Abd
El-Fattah, Sakr, El-Dieb, & Elkashef, 2018; ChenAlcazar, Yang, Lu, & Lu,
2018; Han, Liao, Wu, Gong, & Bai, 2020; Yu et al., 2020). However, in
the reviewed literature, monosodium glutamate (MSG) is added as
GABA precursor to optimise its production. This has numerous

* Corresponding author.
E-mail addresses: inesmaria.ramos@uclm.es (I.M. Ramos), justamaria.poveda@uclm.es (J.M. Poveda).

https://doi.org/10.1016/j.lwt.2022.113581
Received 16 February 2022; Received in revised form 21 April 2022; Accepted 17 May 2022
Available online 20 May 2022
0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
I.M. Ramos and J.M. Poveda
drawbacks, first of all, for the health of the consumer, since MSG has
been linked to various forms of toxicity, such as neurotoxic effects and
other adverse effects (Niaz, Zaplatic, & Spoor, 2018). On the other hand,
consumers are increasingly demanding natural products without addi­
tives (Román, Sánchez-Siles, & Siegrist, 2017), so the prospect of pro­
ducing GABA-rich foods without the addition of MSG offers potential
health benefits and commercial possibilities.
Among various fermented dairy products, yoghurt has the largest
share in sales and is massively produced and consumed in Europe
(Serafeimidou, Zlatanos, Kritikos, & Tourianis, 2013). Yoghurt has been
defined by medical professionals, nutritionists and food scientists as one
of the ’’superfoods’’ touted to improve health, challenge aging and
prevent the progression of changes that lead to diabetes, hypertension,
Alzheimer’s disease and cancer (Kok & Hutkins, 2018). Yoghurt has
proven to be an excellent vehicle for the production of such functional
foods, especially when it contains probiotic bacteria (Papadimitriou
et al., 2007).
Among the milks that are suitable for making yoghurt, sheep’s milk is
becoming increasingly attractive to consumers (Balthazar et al., 2017).
Compared to goat and cow’s milk, sheep’s milk has a higher content of
proteins, lipids, minerals and vitamins, conjugated linolenic acid, short-
and medium-chain fatty acids and n-3 fatty acids (Balthazar et al.,
2016), making it very interesting both from a nutritional and techno­
logical point of view. These characteristics lead sheep’s yoghurt to
produce a strong gel with minimal syneresis and superior organoleptic
properties (Domagała, 2009). On the other hand, fermented sheep’s
milk products may be a good option for people allergic to bovine pro­
teins. For these reasons, some authors have claimed that, in the new era
of functional foods, formulations of products fermented with sheep’s
milk will play a fundamental role in providing benefits for human health
(Mohapatra, Shinde, & Singh, 2019). Furthermore, from a
socio-economic point of view, sheep farming for milk production and its
transformation into dairy products plays a crucial role in the Mediter­
ranean region (Murgia, Scano, Cacciabue, Dessì, & Caboni, 2019),
although it should be noted that most of the sheep milk produced is
processed into cheese (Pulina et al., 2018).
Numerous studies have been carried out on the microbial production
of GABA in yoghurt and fermented milks (Abd El-Fattah et al., 2018;
ChenAlcazar et al., 2018; Hagi, Kobayashi, & Nomura, 2016; Han et al.,
2020; Linares, O’Callaghan, O’Connor, Ross, & Stanton, 2016; Shan
et al., 2015; Zhou et al., 2019). In the literature reviewed, however,
there are no studies on the manufacture of GABA-enriched fermented
milk or yoghurt using sheep’s milk as raw material. Therefore, the
objective of this work was to produce GABA-enriched fermented sheep’s
milk using as adjuncts selected strains of lactobacilli from different food
sources based on their ability to produce GABA, without the addition of
glutamate or other additives, while maintaining the characteristics of
the product that make it acceptable from a sensory point of view. All this
will result in the diversification of the available dairy products made
Table 1
Origin of the strains assayed for the GABA production capacity.
Species Origin Strainsa
Lactobacillus acidophilus Almagro eggplants Lb91, Lb104, Lb116
Levilactobacillus brevis Almagro eggplants Lb27, Lb28, Lb61, Lb62, Lb86, Lb99, Lb100, Lb105, Lb111
Wine Lb47
Lactobacillus delbrueckii Almagro eggplants Lb32
Lacticaseibacillus paracasei Goat cheese Lb5, Lb24
Manchego cheese Lb41
Lactiplantibacillus plantarum Winery air Lb53, Lb54, Lb56
Almagro eggplants Lb93, Lb95, Lb65, Lb70, Lb72, Lb75, Lb76, Lb77, Lb79, Lb106,
Lb107, Lb108, Lb112, Lb114
Manchego cheese Lb33, Lb36, Lb37
Wine Lb43, Lb44
a
Strains belong to the LAB-UCLM collection and were maintained frozen at − 80 ◦ C.
2
LWT 163 (2022) 113581
with sheep’s milk.
2. Materials and methods
2.1. Bacterial strains
A total of 38 autochthonous lactic acid bacteria (LAB) strains from
different origins were assayed for their GABA production capacity
(Table 1). They belonged to the species Lactobacillus acidophilus, Levi­
lactobacillus brevis, Lactobacillus delbrueckii, Lacticaseibacillus paracasei
and Lactiplantibacillus plantarum. The strains had been genotyped and
identified in previous studies (Nieto-Arribas, Poveda, Seseña, Palop, &
Cabezas, 2009; Pérez-Martín, Seseña, Fernández-González, Arévalo, &
Palop, 2014; Sánchez, Seseña, & Palop, 2003; Sánchez, Seseña, Poveda,
Cabezas, & Palop, 2005) and had been selected for their safety proper­
ties, which include their non-production of biogenic amines (Rodrí­
guez-Sánchez, Ramos, Seseña, Poveda, & Palop, 2021). Pure cultures
were maintained frozen at − 80 ◦ C, supplemented with 20% (v/v)
glycerol as a cryoprotectant and were twice revitalised by aerobic
cultivation at 30 ◦ C in MRS broth (Condalab, Madrid, Spain) before
being assayed.
The DVI commercial culture Type I (ABIASA, La Coruña, Spain),
comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp.
bulgaricus (1:1), was used to manufacture the fermented milks.
2.2. GABA production capacity of the indigenous LAB strains in milk
To determine the capacity of the LAB strains to produce GABA from
milk, revitalised strains were cultivated in Ultra High Temperature
(UHT) semi-skimmed sheep’s milk (COVAP, Spain) at 30 ◦ C until they
reached a cell population of 7 log Colony Forming Units (CFU)/mL. For
the measurement of the bacterial population, a growth curve was pre­
viously made for each strain, then the strains were incubated for the
appropriate time to obtain the desired population.
Then, the milk was inoculated (1% v/v) with each strain and incu­
bated for 72 h at 30 ◦ C. Next, 3 mL of each sample were added to 3 mL of
12% trichloroacetic acid (TCA) and homogenised in an Ultra-Turrax for
2 min at 14,000 rpm. The samples were then centrifuged at 15,000 × g
for 15 min at 4 ◦ C, and the supernatants were kept at − 20 ◦ C until
analysis by Reversed Phase-High Performance Liquid Chromatography
(RP-HPLC). The experiment was done in triplicate.
The quantification of GABA was carried out by RP-HPLC as in
Poveda, Ruiz, Sesena, and Llanos Palop (2017) with the following
modifications: (i) derivatisation reaction: 400 μL of sample, 900 μL of 1
mol/L borate buffer pH 9.0, 300 μL of methanol and 12 μL of DEEMM
(derivatising agent); (ii) injection volume: 10 μL.
Reference
Sánchez et al. (2003)
Sánchez et al. (2003)
Pérez-Martín et al. (2014)
Sánchez et al. (2003)
Sánchez et al. (2005)
Nieto-Arribas et al. (2009)
Pérez-Martín et al. (2014)
Sánchez et al. (2003)
Nieto-Arribas et al. (2009)
Pérez-Martín et al. (2014)
I.M. Ramos and J.M. Poveda
2.3. Strain activation for fermented milk manufacture
Lacticaseibacillus paracasei Lb24 and Lb41 and Lactiplantibacillus
plantarum Lb56 were activated twice consecutively in MRS broth (37 ◦ C,
24 h), followed by sixth passages in UHT sheep’s milk at 37 ◦ C/24 h until
they reached approximately 8 log CFU/mL. The commercial culture was
pre-cultured in UHT semi-skimmed sheep’s milk (COVAP, Spain)
following the manufacturer’s instructions (5 g/100 L) and incubated for
approximately 16 h at 37 ◦ C until reaching a population of 6 log CFU/
mL.
2.4. Fermented milk manufacture
According to legal regulations, yoghurt must be made only from the
culture composed of Streptococcus thermophilus and Lactobacillus bul­
garicus. In this work, other Lactobacillus strains will be added as an
adjunct culture, therefore the samples produced should be referred to as
fermented milks (BOE-A-2014-4515). Actually, the control samples
produced do correspond to the definition of yoghurt, but it was decided
to call it fermented milk control so that it would not be interpreted as a
different product.
To standardise the production of the different fermented milks using
a raw material with a homogeneous composition and sensory charac­
teristics, UHT semi-skimmed sheep’s milk from the same production
batch (COVAP, Spain) was chosen. The milk composition was: protein:
5.2, fat: 1.7, lactose: 4.8, (expressed in g/100 mL); calcium: 179.5 mg/
100 mL. Due to the high protein content of sheep’s milk, it was not
necessary to enrich it with skimmed milk powder.
Fig. 1 shows the manufacturing process of the fermented milks.
Briefly, two litters of UHT semi-skimmed sheep’s milk were inoculated
with the revitalised LAB strains. To promote the growth of the selected
lactobacilli strains and their GABA production, they were added 24 h
before the addition of the commercial starter and incubated at 37 ±
0.5 ◦ C. Then, the milks were heated to 42 ◦ C, inoculated with a specific
Fig. 1. Flowchart for the fermented milk manufacturing process. CS: com­
mercial starter.
3
LWT 163 (2022) 113581
commercial starter (6 log CFU/mL), dosed into sterile and transparent
plastic 50 mL-cups and incubated at 41.5 ± 0.5 ◦ C in a controlled
temperature incubator. Table 2 shows the cultures and proportions used
in each batch of fermented milk. The coagulation of milk was monitored
for pH during the incubation period until a pH of 4.40 ± 0.05 was
reached, obtaining four different batches of set fermented milks. The
batches were then immediately cooled and stored at 4.0 ± 0.5 ◦ C for 28
days. Fermented milk manufacture was performed in duplicate for each
type of starter combination one week later.
Physico-chemical, microbiological and sensory analyses were con­
ducted on the fermented milks at different times: time 0 (when the
fermented milks have just reached a pH of 4.40 before cooling down)
and after 1, 7, 14, 21 and 28 days of storage at 4 ◦ C. Physico-chemical
and microbiological analyses were performed in triplicate, and sen­
sory analysis was carried out in duplicate.
2.5. Microbiological analysis
In order to know the population of autochthonous LAB present in
fermented milks, appropriately diluted samples were plated on Man,
Rogosa and Sharpe agar (MRS agar; Condalab, Madrid, Spain) with 1
mg/L vancomycin (Fluka, Thermo Fisher Scientific, Madrid, Spain (Bai
et al., 2020), since the indigenous strains Lb24, Lb41 and Lb56 are
vancomycin-resistant (Rodríguez-Sánchez et al., 2021), while the com­
mercial culture species Lb. bulgaricus and S. thermophilus are
vancomycin-sensitive (Coeuret, Gueguen, & Vernoux, 2004). The plates
were incubated at 37 ◦ C for 72 h.
2.6. Physico-chemical analysis
The pH was measured by direct reading, using a digital pH meter
(Crison, Barcelona, Spain); total solids (TS) and titratable acidity (TA)
were determined according to AOAC methods (AOAC, 2010).
2.7. Water-holding capacity
The water-holding capacity (WHC) was defined as the weight per­
centage of concentrated fermented milk in the original sample after
centrifugation at 5,000 × g for 20 min at 4 ◦ C (ChenAlcazar et al., 2018).
For this purpose, 20.0 g of the sample (processed directly in a 50 mL
centrifuge tube) were centrifuged. The WHC was calculated using the
following equation:
WHC = (W/W0) × 100
Where W is the weight of the residue after centrifugation and W0 is the
weight of the sample.
2.8. Viscosity measurement
The apparent viscosity of the fermented milks was measured with a
Brookfield digital rotational viscometer (model DV-II+, Brookfield En­
gineering Laboratories Inc., Middleboro, MA, USA) at 4 ◦ C using a
spindle 63 at a rotational speed of 30 rpm in 150 mL of fermented milk.
Prior to analysis, the yoghurt samples were maintained in an ice water
bath for 30 min until a temperature of 4 ◦ C was achieved. The samples
were manually stirred clockwise for 60 s before taking the measure­
ments (Ahmed et al., 2021). The apparent viscosity reading in centi­
poises (cP) was taken at the point of the 30th second, and torque of
10–100% was maintained at all times.
2.9. GABA, glutamic acid and biogenic amine production in fermented
milks
The quantification of GABA and glutamic acid (Glu) in the fermented
milks was carried out by RP-HPLC using a diethyl
I.M. Ramos and J.M. Poveda
ethoxymethylenemalonate (DEEMM) derivatisation method (Poveda
et al., 2017) with modifications, as described in section 2.3., for GABA
production by autochthonous strains. The biogenic amines were deter­
mined in the same analysis.
2.10. Sensory analysis
Quantitative descriptive analysis (QDA) was run for appearance,
odour, flavour and texture attributes (ISO 13299:2016). The panel
consisted of eight trained assessors (six females and two males between
23 and 60 years of age) who were all members of the university staff.
They had previously been trained following the ISO 8586:2012, and all
were experienced in sensory analysis and familiar with dairy sensory
attributes. Fermented milks were served at 4 ◦ C in plastic 50 mL-cups,
and identified by three-character codes. Samples were presented to the
tasters in a complete block design, in which all panellists tasted all the
samples in different order. The terms describing the attributes of the
fermented milks were selected by consensus from a freely generated list
created by the assessors after evaluating some of the samples. The at­
tributes selected were: two appearance terms (curd firmness and whey
quantity), three odour attributes (yoghurt odour, odour intensity and
odour quality), four flavour attributes (acid flavour, yoghurt flavour,
flavour intensity and flavour quality), one texture-in-mouth attribute
(viscosity, defined as the pressure needed to move the sample between
the tongue and the palate) and a global impression. An unstructured line
scales (from 0 to 10 cm, without divisions) was used for the sensory
evaluation. All samples were tasted in duplicate in two different sessions
in a tasting room that complied with ISO standard 8589:2007.
2.11. Statistical analysis
A one-way analysis of variance (ANOVA) using the Student-
Newman-Keuls (S–N–K) test for comparison of the means (P < 0.05)
and Pearson’s correlation analysis were performed using the IBM SPSS
statistics package version 24.0 (SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. GABA production capacity of the indigenous LAB strains
All the indigenous LAB strains analysed produced GABA after 72 h of
incubation in sheep’s milk (Fig. 2), with concentrations ranging between
3 and 63 mg/L. The amounts of GABA produced in milk were lower than
those produced by the same strains when grown in an optimal medium,
such as MRS supplemented with glutamate and PLP (Rodríguez-Sánchez
et al., 2021). This is a finding worth considering, since the strains are
usually selected based on the analysis of the activity in an optimal me­
dium rather than the medium in which the strains will later be used,
normally a food. Furthermore, there was no supplementation of milk
with sodium glutamate as a GABA precursor (as was performed in most
of the reviewed literature Galli, Venturi, Mari, Guerrini, & Granchi,
2021; Yu et al., 2020). The initial concentration of glutamic acid in the
sheep’s milk used was 25.9 mg/L, higher than that found in the litera­
ture for cow’s and goat’s milk, with 14.9 and 8.6 mg/L, respectively (Gu,
Wang, Yang & Ren, 2020; Kehagias et al., 2008). Therefore, sheep’s milk
could be a suitable raw material for the production of GABA-enriched
products. The amounts of GABA produced by the LAB strains in
Table 2
Cultures and proportions used in fermented milks.
Batch Strain cultures
FMC Commercial starter, CS (8 log CFU/mL)
FM1 Lacticaseibacillus paracasei Lb24 (8 log CFU/mL) + CS (6 log CFU/mL)
FM2 Lacticaseibacillus paracasei Lb41 (8 log CFU/mL) + CS (6 log CFU/mL)
FM3 Lactiplantibacillus plantarum Lb56 (8 log CFU/mL) + CS (6 log CFU/mL)
4
LWT 163 (2022) 113581
sheep’s milk were similar to those reported by Yu et al. (2020) and Galli
et al. (2021) for different strains of lactobacilli in fermented cow’s milk.
The strains Lb24, Lb56 and Lb41 produced the highest (P < 0.05) levels
of GABA and therefore were selected for the production of the fermented
sheep’s milks.
3.2. Microbiological analysis
To ensure the presence of the inoculated selected LAB strains and
determine their viability in the experimental fermented milks, microbial
counts were carried out in specific culture media throughout the storage
period (Table 3). The autochthonous lactobacilli population inoculated
into the experimental fermented milks was around 8 log CFU/mL, which
remained constant until 7 days in batches FM1 and FM3 and until 21
days in FM2. All batches showed a decline in their viable cell counts at
28 days, reaching counts of 7 log CFU/mL for FM2 and 5 log CFU/mL for
fermented milks FM1 and FM3. Decreased viability of the bacteria may
be the result of a decrease in the available lactose they require to keep
growing. During the entire storage period, the count in FM2 was higher
(P < 0.05) than in the other fermented milks. These results are consistent
with those of other authors in fermented cow’s milk, who also found a
reduction in counts of LAB after refrigerated storage (Abd El-Fattah
et al., 2018). According to these results, it can be stated that the indig­
enous LAB strains inoculated into the fermented sheep’s milks remained
viable throughout the entire storage process.
3.3. Physico-chemical analysis
Table 4 shows the results of the pH, titratable acidity and total solids
analysis. The pH of all fermented milks decreased significantly (P <
0.05) during storage due to the activity of the bacteria that remain viable
under refrigeration; the continued growth of the cultures results in the
continuous production of lactic acid and thus a reduction in pH. The pH
of the experimental fermented milks was similar to that of the control,
with the exception of FM2, which showed higher (P < 0.05) pH values.
The TA values were consistent with those of the pH, as was expected.
All batches showed an increase in TA as storage time increased, and, as
expected, FM2 was the batch with the lowest acidity at all times. This is
corroborated by the strong correlation found between pH and TA
(− 0.888, P < 0.01). According to legal regulations, yoghurt must have a
minimum of 0.6% TA, i.e. 6 g lactic acid/L of yoghurt (CODEX STAN
243–2003), so that the fermented milks produced have the acidity
required. These results are consistent with those of similar studies in
cow’s milk yoghurts (Akalın, Unal, Dinkci, & Hayaloglu, 2012; Kar­
iyawasam, Lee, & Paik, 2021).
The results obtained from the TS analysis did not show significant
differences (P < 0.05) either between the batches of fermented milks or
between different storage times.
3.4. Water-holding capacity
Spontaneous syneresis is the spontaneous separation of whey due to
the partial denaturation of milk proteins during heat treatment, result­
ing in the expulsion of whey from the protein network that then becomes
visible as surface whey (Lee & Lucey, 2010). WHC refers to the ability of
yoghurt’s protein gel network to retain water, reflecting the texture and
degree of compactness of the yoghurt (Cui, Chang, & Nannapaneni,
2021). Changes in WHC are a useful method of indirectly assessing
variations in macroscopic changes in the molecular network and
appearance of fermented milk in the mouth.
The fermented milks showed texture and firmness characteristics
similar to those of yoghurt. No syneresis was observed in any of the
fermented milk samples from the different batches at any of the storage
times. This could be attributed to the use of sheep’s milk, which has a
high protein content. The cross-linking of the proteins leads to the for­
mation of a finer network structure, which is related to less syneresis and
I.M. Ramos and J.M. Poveda LWT 163 (2022) 113581

a-p
Fig. 2. Production of GABA (mg/L; mean values, standard deviation, n = 3) by the LAB strains in UHT semi-skimmed sheep’s milk after 72 h. : Different letters
indicate significant statistical differences (p < 0.05).

Table 3
Viable counts (mean values ± standard deviation; n = 3) of autochthonous LAB (log ufc/g) in the different types of fermented milks during refrigerated storage at 4 ◦ C.
Period of storage (days)

Batch 0 1 7 14 21 28

FMC nd nd nd nd nd nd
FM1 8.2Ac ± 0.1 8.3Ac ± 0.0 8.0Ac ± 0.1 7.3Bb ± 0.3 7.2Bb ± 0.1 5.1Aa ± 0.1
FM2 8.8Bb ± 0.1 8.3A,Bb ± 0.7 9.0Bb ± 0.3 9.0Cb ± 0.0 8.1Cb ± 0.0 7.1Ba ± 0.1
FM3 8.2Ac ± 0.0 8.2Ac ± 0.0 8.2Ac ± 0.0 6.1Ab ± 0.1 5.0Aa ± 0.0 5.0Aa ± 0.0
A-D
: Means in the same column with different letters indicate significant statistical differences for the different batch in the same time of storage (p < 0.05).
a-f
: Means in the same row with different letters indicate significant statistical differences for the same batch in different times of storage (p < 0.05).
nd: not detected, no growth on plates.

higher firmness of the dairy products (Akalın et al., 2012).
The WHC values (Table 4) increased up to 14 days in all fermented
milks, after which point, they remained constant until the end of storage.
In general, experimental fermented milks showed higher values (P <
0.05) of WHC than did the control throughout the storage period. These
results show that the addition of cultures improved the curd tension of
fermented milks, in agreement with those of other authors for cow’s
milk yoghurt (Aryana & McGrew, 2007). On the other hand, Kar­
iyawasam et al. (2021) observed no differences between the WHC of
cow’s milk yoghurt and those of the same yoghurt with probiotic strains.
which is determinant for the viscosity and texture of yoghurt (Shihata &
Shah, 2002). In addition, the use of milks with a higher proportion of
protein in yoghurt production results in higher viscosity (Sodini, Mon­
tella, & Tong, 2005). For this reason, these fermented milks made with
semi-skimmed sheep’s milk, which contains more protein than cow’s
milk, showed viscosity values higher than those reported by other au­
thors for cow’s milk yoghurt (Abd El-Fattah et al., 2018; Kariyawasam
et al., 2021).
3.6. GABA and glutamic acid contents in fermented milks

3.5. Viscosity
Fig. S1 shows the results obtained from the viscosity measurements.
In general, a trend of decreasing viscosity of the fermented milks was
observed during the early stages of the storage, although there were no
significant differences between the batches. This trend was followed by
an increase in viscosity at the end of the storage period. After 28 days of
storage at 4 ◦ C, the fermented milks FM3 and FM1 accounted for the
highest (P < 0.05) values of viscosity, with 3188 and 2497 cP,
respectively.
Both the bacteria used in fermentation and the composition of the
milk are closely related to rheological characteristics, such as viscosity.
LAB can produce different polysaccharides through its metabolism,
Fig. 3 depicts the concentrations of GABA and glutamic acid (Glu) of
the four types of fermented milks during the storage period. In all
batches the concentration of GABA increased progressively during
storage at 4 ◦ C, while the concentration of glutamate decreased. This
was to be expected, since GABA is primarily formed by the irreversible
α-decarboxylation reaction of L-glutamic acid or its salts, catalysed by
glutamic acid decarboxylase enzyme, which is dependent on PLP. This
enzyme has been found in LAB, among other bacteria (Diana et al.,
2014). This is also supported by the strong negative correlation found
between GABA and Glu (− 0.724, P < 0.01).
The initial levels of Glu in the four batches of processed fermented
milks were similar, around 100 mg/L, except for batch FM1, which at
time 0 presented a concentration of 200 mg/L. The presence of Glu in 0-

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I.M. Ramos and J.M. Poveda LWT 163 (2022) 113581

Table 4
Values (mean ± standard deviation; n = 3) of pH, titratable acidity, total solids and WHC of the different types of fermented milks during refrigerated storage at 4 ◦ C.
Batch Period of storage (days)

0 1 7 14 21 28

pH FMC 4.4a ± 0.0 4.1Ab ± 0.0 3.9Ac ± 0.0 3.9A,Bd ± 0.0 3.9B,Ce ± 0.0 3.8B,Cf ± 0.0
FM1 4.4a ± 0.0 4.2Bb ± 0.0 3.9Ac ± 0.0 3.7Ad ± 0.0 3.7Ae ± 0.0 3.7Af ± 0.0
FM2 4.4a ± 0.0 4.3Cb ± 0.0 4.3Bc ± 0.0 4.0Bd ± 0.0 4.0Ce ± 0.0 3.9Cf ± 0.0
FM3 4.4a ± 0.0 4.1Bb ± 0.0 3.9Ac ± 0.0 3.8Ad ± 0.0 3.8Be ± 0.0 3.8Bf ± 0.0
TA FMC 14.2Ba ± 3.2 14.4Bb ± 2.8 16.6Bc ± 0.9 17.0Bd ± 1.2 17.0Bd ± 16.0 17.0d ± 3.5
(g lactic acid/L) FM1 13.1Ba ± 1.9 13.2Ba ± 1.3 17.1Bb ± 6.0 17.2Bb ± 3.1 17.3Bb ± 0.7 17.3c ± 0.3
FM2 11.8Aa ± 3.2 12.4Ab ± 0.3 13.4Ac ± 2.5 13.6Ad ± 8.6 15.3Ae ± 3.2 17.3e ± 2.9
FM3 13.7Ba ± 0.0 14.0Bb ± 1.3 17.0Bc ± 7.5 17.9Bd ± 1.2 18.3Be ± 1.6 18.3e ± 0.5
TS (%) FMC 12.5 ± 0.5 12.9 ± 0.2 12.5 ± 0.3 12.3 ± 0.1 12.7 ± 0.1 12.6 ± 0.2
FM1 12.4 ± 0.1 12.7 ± 0.1 12.4 ± 0.0 12.7 ± 0.5 12.8 ± 0.1 12.8 ± 0.1
FM2 12.2 ± 0.0 13.3 ± 0.1 12.8 ± 0.1 12.8 ± 0.3 12.7 ± 0.1 12.8 ± 0.1
FM3 12.7 ± 0.1 12.5 ± 0.3 12.3 ± 0.1 12.2 ± 0.1 12.0 ± 0.0 12.9 ± 0.1
WHC FMC 42.1Ba ± 0.1 42.3Aa ± 0.2 42.3Aa ± 0.3 45.5Ab ± 1.4 44.0Ab ± 0.1 43.3Ab ± 1.5
FM1 41.7Aa ± 0.2 51.5Cb ± 0.4 50.4Cb ± 1.6 54.1Ac ± 1.0 53.5Cc ± 0.9 52.6Bc ± 0.0
FM2 46.9Da ± 0.1 47.8Ba ± 0.4 48.7Ba ± 1.8 54.7Ab ± 3.6 54.8Cb ± 0.9 53.3Bb ± 1.7
FM3 45.9Ca ± 0.0 53.6Dc ± 0.2 52.42Dc ± 0.0 56.1Bd ± 2.2 55.8Bd ± 2.3 55.6Bd ± 1.9
A-D
Means in the same column with different letters indicate significant statistical differences for each parameter (p < 0.05).
a-f
Means in the same row with different letters indicate significant statistical differences (p < 0.05).

day fermented milks may be due in part to the intrinsic content of this
amino acid in milk (25.9 mg/L, results not shown), as well as to its
synthesis through the proteolytic activity of bacteria at the beginning of
fermentation. On the other hand, the initial levels of GABA in the fer­
mented milks were similar for all four batches with values lower than 40
mg/L, and, since no GABA was found in the milk (results not shown),
they were necessarily due to the GAD activity of the LAB strains used in
fermented milks manufacture.
After 24 h of storage, the fermented milk control reached a GABA
concentration of 112.3 mg/L and continued to increase slightly,
although the GABA concentrations remained lower for the FMC (P <
0.05) than for the experimental fermented milks. On the other hand, in
the experimental fermented milks the highest increase occurred be­
tween 7 and 14 days of storage. The highest (P < 0.05) concentrations of
GABA were determined at 28 days of storage for batches FM2 and FM3,
with values of 191.9 and 197.9 mg/L, respectively. This means that
GABA production continues during the storage of the fermented milk, so
it would be beneficial for one’s health to consume fermented milks after
a certain storage time. These results are in accordance with those re­
ported by Abd El-Fattah et al. (2018) and ChenAlcazar et al. (2018) in
which the addition of LAB increased the GABA content of fermented
cow’s milk.
Pearson’s correlation analysis showed a strong correlation of GABA
with pH (− 0.920, P < 0.01). This was to be expected, since pH has been
shown to influence the activation pathway of the GAD enzyme, which is
responsible for maintaining cellular homeostasis (Sanchart, Rattana­
porn, Haltrich, Phukpattaranont, & Maneerat, 2017). In response to a
decrease in pH, the activity of the GAD enzyme, and therefore the
accumulation of GABA, increases to mitigate cytosolic acidification
(Rashmi, Zanan, John, Khandagale, & Nadaf, 2018).
Some studies have shown that probiotic dairy products with GABA
and GAD activity can be sustained through the digestive system and
have health benefits. GABA ingestion may have a beneficial effect as an
antihypertensive, insomnia-relaxant and antidepressant agent at certain

Fig. 3. Concentrations (mg/L) of GABA and glutamic acid in the different types of fermented milk during storage at 4 ◦ C. For identification of the fermented milk
batches see Table 2.

6
I.M. Ramos and J.M. Poveda LWT 163 (2022) 113581

Table 5
Olfactive, gustative and viscosity in mouth analysis scores of the fermented milks during cold storage. The intensity of each attribute was rated by the assessors using
the scale of 7 cm.
Atributes Batch Period of storage (days)

1 7 14 21

Olfactory phase Yogur FMC 3.6a ± 1.1 5.6b ± 1.0 5.6b ± 0.6 5.3b ± 1.1
FM1 3.4a ± 1.1 5.6b ± 1.1 5.7b ± 0.9 5.1b ± 1.0
FM2 3.5a ± 0.6 5.4b ± 1.1 5.8b ± 1.2 5.2b ± 0.5
FM3 3.7a ± 0.9 5.6b ± 0.5 5.0b ± 1.0 5.6b ± 1.2

Cheese FMC 1.3A ± 0.3 nd nd nd

FM1 2.1Bb ± 0.5 1.1a ± 1.1 nd 1.6Aa ± 0.6
FM2 2.7Bc ± 1.0 1.4a ± 1.0 1.5Aa ± 0.6 2.0Bb ± 0.4
FM3 3.3Cc ± 0.1 1.0a ± 0.9 2.0Bb ± 1.1 2.1Bb ± 1.1

Sour FMC 0.4Aa ± 0.6 1.7Bc ± 0.4 2.3Bd ± 0.5 1.0b ± 2.3
FM1 0.5Aa ± 0.7 0.5Aa ± 0.4 1.7Ab ± 0.2 1.2b ± 1.8
FM2 0.4Aa ± 0.2 1.5Bb ± 0.5 2.1Bc ± 1.8 1.6b ± 1.7
FM3 1.7Ba ± 0.1 1.9Ba,b ± 0.3 2.2Bb ± 1.0 2.0b ± 1.6

Intensity FMC 4.3Bc ± 1.0 3.7Ab ± 1.6 2.5Aa ± 0.1 2.7Aa ± 1.9
FM1 1.9Aa ± 1.3 3.2Ab ± 1.1 4.8Bc ± 0.1 5.0Bd ± 1.7
FM2 3.5Ba ± 2.3 4.0Aa
± 0.2 5.1Cb ± 0.1 4.9Bb ± 0.3
FM3 4.9Ba ± 1.8 4.6Ba ± 0.1 5.4Cb ± 1.2 5.3Bb ± 1.2

Quality FMC 5.2Cc ± 0.2 5.9Ad ± 1.7 4.0Ab ± 0.0 3.7Aa ± 1.7
FM1 4.7A,Bb ± 1.0 5.6Ac ± 0.5 4.3Ba ± 0.0 4.1Aa ± 0.3
5.0B,Cc
FM2 ± 0.4 6.2Db ± 1.5 3.9Aa ± 0.2 4.5Bb ± 1.2
FM3 4.4Aa ± 0.7 6.2Bc ± 1.9 4.5Ba ± 0.2 5.0Cb ± 0.6
Gustative phase Yogur FMC 5.8Bb ± 1.0 7.0Cd ± 0.6 6.5Bc ± 1.1 4.2Aa ± 0.1
FM1 5.5Ba ± 1.1 5.6Ba ± 0.9 5.3Aa ± 1.0 7.0Cb ± 0.3
FM2 2.9Aa ± 1.1 4.2Ab ± 1.2 5.5Ac ± 0.5 5.6Bc ± 0.1
FM3 6.2Cc ± 0.5 4.2Aa ± 1.0 5.9Ab ± 1.2 6.2Cc ± 0.1

Cheese FMC 1.0A ± 0.1 nd nd nd

FM1 2.6Ca ± 0.5 nd 3.0Cb ± 0.1 2.5Ba ± 1.1
FM2 1.7Bb ± 0.2 2.8 c
± 0.8 1.2Aa ± 1.5 2.0Ab ± 1.0
FM3 1.7Ba ± 1.8 nd 2.0Bb ± 0.1 2.6Bc ± 0.1

Acid FMC 4.4Ca ± 1.6 5.0Cb ± 2.0 5.1Cb ± 0.2 4.9Aa,b ± 0.5
FM1 3.5Ba ± 1.4 4.3Bb ± 0.9 4.3Bb ± 0.4 5.0Ac ± 1.0
FM2 1.9Aa ± 1.5 2.8Ab ± 0.7 3.0Ab ± 0.0 5.1Ac ± 1.3
FM3 4.6Cb ± 1.4 4.6Bb ± 0.5 4.4Ba ± 0.2 5.4Bc ± 1.1

Intensity FMC 4.1Bc ± 0.1 3.8Ab ± 1.6 4.0Ab

± 2.0 3.5Aa ± 0.0
FM1 4.3Bb ± 1.2 3.2Aa ± 1.4 3.3Aa ± 0.9 4.5Bc ± 0.4
FM2 2.6Aa ± 1.2 4.6Bb ± 1.5 4.6Bb ± 0.7 5.0Cc ± 0.0
FM3 5.0Ca ± 1.9 5.1Ca ± 1.4 5.1Ca ± 0.0 5.6Db ± 0.2

Quality FMC 4.5Bb ± 1.1 3.7Aa ± 1.3 3.7Ba ± 1.0 3.3Aa ± 1.1
FM1 5.1Cc ± 0.2 4.2Bb ± 1.1 3.0Aa ± 0.6 5.3Cd ± 1.1
FM2 2.3Aa ± 1.0 5.2Dc ± 1.1 4.5Cb ± 0.8 4.6Bb ± 1.1
FM3 4.4Ba ± 0.5 4.8Ca ± 1.1 5.0Db ± 1.3 6.4Dc ± 0.9
Texture in mouth Viscosity FMC 2.8Ab ± 0.2 2.2Aa ± 1.6 3.5c ± 0.0 2.8Ab ± 0.5
FM1 3.6Bb ± 1.1 3.5Bb ± 0.1 3.6b ± 0.2 2.8Aa ± 1.5
FM2 2.8Aa ± 0.8 2.8Aa ± 0.1 3.5b ± 1.3 3.5Bb ± 0.3
FM3 3.5Ba ± 0.2 3.5Ba ± 1.2 3.9a ± 1.2 4.2Cb ± 0.2
A-D
: Means in the same column for each attribute with different letters indicate significant statistical differences between batches in the same times of storage
(p < 0.05).
a-f
: Means in the same row for each attribute with different letters indicate significant statistical differences for the same batch in different times of storage (p < 0.05).
nd: not detected.

doses for rats and humans (Sahab, Subroto, Balia, & Utama, 2020).
Inoue et al. (2003) reported that a daily intake of 100 mL of fermented
milk containing 10–12 mg of GABA can help control blood pressure in
mildly hypertensive human patients. Likewise, Nakamura, Takishima,
Kometani, and Yokogoshi (2009) observed that consumption of choco­
late enriched with 28 mg of GABA produced a reduction in stress in
subjects. Other authors have reported antihypertensive effects, stress
reduction and a reduction in sleep latency of GABA with doses between
2 and 100 mg (Hepsomali, Groeger, Nishihira, & Scholey, 2020).
Considering the dietary recommendations (USDA, 2015) for dairy
consumption (3 servings per day, that is, a daily intake of 3 × 125 g for
yoghurt and other fermented milks), the fermented milks in our study
would provide between 86 and 100 mg of GABA, which, based on the
studies discussed above, could have some beneficial functional value for
consumers.
3.7. Biogenic amines
The production of biogenic amines was null or practically negligible
in all the samples analysed. This supports the idea that the selection of
the strains based on their non-production of BAs in vitro is an adequate
criterion, since they did not produce BAs in the fermented milks either.

7
I.M. Ramos and J.M. Poveda
3.8. Sensory analysis
The taste, smell and consistency of a fermented milk have an
important influence on the final quality of the product and are largely
determined by the culture that carries out the fermentation. The main
objective of the present work was to produce fermented sheep’s milks
enriched with GABA without altering the typical organoleptic charac­
teristics of this product. A sensory evaluation of the four types of fer­
mented milks produced was performed, and the results obtained are
presented in Table 5.
All samples tested presented a characteristic firm texture, and none
showed the presence of whey. The fermented milks were characterised
by a moderate yoghurt odour on day 1, which increased significantly
from day 7 onwards. In general, the samples presented an intense
yoghurt flavour, with some differences among batches, with the highest
scores for samples of FM1 at 21 d and FMC at 7 d. All the fermented
milks showed moderate levels of acid flavour. The odour and flavour
quality were moderate to high, increasing at the end of the storage
period, and were significantly higher (P < 0.05) for the experimental
fermented milks. There were few significant differences among the
fermented milks in terms of their viscosity in the mouth, although FM3
showed higher values for this quality. This agrees with the instrumental
viscosity of FM3, which was higher than that of the other fermented
milks.
Finally, regarding the overall impression, the tasters gave higher
scores to the samples from the FM3 batch, followed by FM1 and FMC
(results not shown).
These results show that the experimental fermented milks were rated
higher than the control for several attributes and did not present any
defects or atypical characteristics for the product in question.
4. Conclusions
This work demonstrates the GABA enrichment of fermented sheep’s
milks using native LAB strains isolated from different food environ­
ments, without additives and with physico-chemical properties and
sensory characteristics typical of the product.
Semi-skimmed sheep’s milk has proven to be an excellent raw ma­
terial for the production of fermented milk, since it does not require the
addition of skimmed milk powder; in fact, no syneresis was observed in
the fermented milks during the entire refrigerated storage period.
Among the 38 strains tested for their GABA production, Lb24, Lb41
and Lb56 were selected for the production of fermented milks. The
fermented milks made with the strains Lb41 (Lacticaseibacillus paracasei)
and Lb56 (Lactiplantibacillus plantarum) had the highest GABA concen­
trations, around 200 mg/L. These fermented milks also presented higher
values of viscosity and WHC. In addition, they were better evaluated by
tasters than the control.
Future work is warranted to study the production of other bioactive
compounds in fermented milks with these strains, as well as to search for
new strains of LAB to produce health-promoting dairy foods.
Funding
This work was supported by the Fondo Europeo de Desarrollo
Regional (FEDER), Junta de Comunidades de Castilla La-Mancha, Spain
(Project no. SBPLY/17/180,501/000528). Inés Ramos is supported by a
contract (Ref.: 2020-COB-9880) associated with the same project.
Declaration of interest form
Authors declare that there are no conflicts of interest whatsoever.
CRediT authorship contribution statement
Inés María Ramos: Data curation, Formal analysis, Methodology,
8
LWT 163 (2022) 113581
Resources, Investigation, Writing – original draft. Justa María Poveda:
Conceptualization, Data curation, Funding acquisition, Investigation,
Methodology, Formal analysis, Visualization, Writing – original draft,
Writing – review & editing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2022.113581.
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