1. Introduction
Sourdough fermentation can impart health-benefiting properties to bread beyond being a source of nutrients. Such functional properties of sourdough may include increased bioavailability of phytonutrients, decreased glycemic response, and an increase in dietary fibre [
1,
2]. Within the cereal matrix, microbial activities of LAB and yeasts can result in the production of nutritionally active compounds, such as peptides, amino acids (and amino acid derivatives, such as γ-amino butyric acid), and exopolysaccharides [
3,
4].
Sourdough fermentation leads to phytase-dependent dephosphorylation of phytic acid [
5]. Phytic acid [myo-inositol hexakis (dihydrogenphosphate)] constitutes 1–4% by weight of cereal grains, being a source of myo-inositol and the major storage form of phosphorus. This molecule is highly charged, with six phosphate groups extending from the central myo-inositol ring.
However, phytic acid chelates several divalent nutritional minerals; hence, it is considered an antinutritional factor. Phytic acid also complexes the basic amino acid group of proteins, thus decreasing the dietary bioavailability of these nutrients [
6,
7]. Therefore, its degradation improves the availability of nutrients and minerals, such as phosphorous, magnesium, iron, calcium, and zinc. Some of the phytase activities are endogenous to cereals but activated by LAB acidification, whereas other phytase activities are directly produced by both yeasts and LAB [
8]. In addition, the LAB in the sourdough produce bioactive compounds, volatile fatty acids, lactic acid, and amino acids during the fermentation process [
9].
Glutamic acid is an amino acid that plays a very important role in taste perception, intermediary metabolism, and excitatory neurotransmission. It plays a pivotal role in the gastrointestinal tract during gastric-phase digestion and enhances gastric exocrine secretion. It is also a precursor for γ-amino butyric acid (GABA), glutathione, arginine, and proline. GABA possesses several well-known physiological functions (i.e., anti-hypertension and anti-diabetic), and glutathione plays a key role in protecting the mucosa from peroxide damage and dietary toxins. A study by Zareian et al. (2013) [
10] reported that
L. plantarum has the potential to synthesize both glutamic acid and GABA in fermented foods. LAB also play a role in increasing the shelf-life and safety of foods, improving food texture, and contributing to the nutritional value of food products. The LAB can impart pleasant sensory properties to sourdough bread. Li and Cao et al. 2010 [
11] have reported that production of L-glutamic acid and GABA (γ-aminobutyric acid) was of utmost importance to producing a functional food product. Komatsuzaki et al. (2005) [
12] have reported that during fermentation, the production of glutamic acid was positively correlated with an increase in GABA concentration. Thus, using LAB capable of producing glutamic acid might facilitate production of functional foods rich in bioactive molecules, such as GABA [
13].
The main metabolic activities of sourdough LAB that influence the nutritional importance of sourdough are the proteolytic activity [
14], the formation of volatile compounds and antibacterial and anti-mold compounds [
15], as well as exopolysaccharide (EPS)-producing characteristics [
16]. Exopolysaccharides (EPS) are microbial polysaccharides produced outside of the cell wall [
17]. They consist of dietary oligosaccharides, which are non-digestible and aid in modulating the activity and composition of the intestinal microflora. EPS may also contain a variety of proteins, glycoproteins, glycolipids, and, in some cases, surprising amounts of extracellular DNA (e-DNA) [
18].
Sourdough LAB synthesize a variety of EPS through the activity of glycosyltransferases. A previous study carried out on EPS suggests that some of the polymers from the fermentation of cereal foods by LAB may be available for food applications and processing [
19,
20]. Synthesis of EPS (glucans and fructans) with prebiotic potential has been reported for sourdough fermentation of sorghum and wheat flours by LAB, such as
L. frumenti,
L. pontis,
L. acidophilus, and
L. reuteri [
21,
22,
23]. Kralj et al. (2002) [
24] reported that by adding 12% sucrose to the dough during fermentation,
L. reuteri strain 121 can form two types of EPS, a 4,6-disubstituted ά-glucan (reuteran) and a levan. Glucansucrase and fructosyltransferase are the enzymes responsible for the synthesis of the abovementioned EPS [
25].
EPS acts as a hydrocolloid to improve the overall textural properties of the sourdough bread produced. Cereal-based LAB species have been associated with the formation of homopolymeric EPS, but recent studies also showed the production of heteropolymeric EPS by sourdough isolates, which were shown to alter the physicochemical properties of sourdough [
26]. Identification of the EPS-producing LAB in sourdough LAB could help improve the quality of sourdough.
Therefore, the objective of this study was to establish the functional properties of the LAB, yeast, and acetic acid bacteria isolated from the coconut-water-kefir-fermented sourdough, CWK, and kefir. This study specifically aims to determine the following properties of the isolates, such as glutamic acid production, phytase enzyme activity, and exopolysaccharide production.
3. Results and Discussion
3.1. Determination of Glutamic Acid Concentration for LAB, AAB, and Yeast Isolates Using the LCMS Analysis Method
To investigate the concentration of glutamic acid produced by each isolate of LAB, ABB, and yeast, LCMS analysis was carried out. From
Table 2, it can be observed that a significantly high concentration of glutamic acid was produced by
Lactiplantibacillus plantarum (264.89 ± 8.57 µMoles/L), followed by
Limosilactobacillus fermentum (260.38 ± 12.09 µMoles/L) and
Lactobacillus reuteri (251.16 ± 10.61 µMoles/L)
p < 0.05. Gobbetti et al. (1994) [
45] have reported that using
Lactiplantibacillus plantarum in sourdough fermentation caused a considerable increase in the total concentration of free amino acids. Zareian et al. (2012) [
13] reported
Lactiplantibacillus plantarum as a glutamic acid producer. A similar study by Pozo-Bayón et al. (2005) [
46] detected the presence of glutamic acid by
Lactiplantibacillus plantarum strains J-39 (19.17 mg/L) and J-51 (16.82 mg/L) in wine after malolactic fermentation by LAB. A study carried out by Coda, Rizzello, and Gobbetti (2010) [
47] reported considerably high glutamic acid concentrations of 107 ± 20 mg/L in sourdoughs made with common wheat flour and fermented with
Lactiplantibacillus plantarum strain C48.
AAB strains (
Acetobacter aceti,
Acetobacter lovaniensis, and
Acetobacter pasteurianus) produced average quantities between 155.28 ± 4.84 µMoles/L and 164.37 ± 4.11 µMoles/L of glutamic acid (as shown in
Table 2). Significantly low concentrations of glutamic acid are produced by all of the individual yeast species, with
Candida guilliermondii significantly producing the lowest (114.29 ± 1.95,
p < 0.05).
Gobbetti et al. (1994) [
48] reported the production of enhanced amounts of glutamic acid by
S. cerevisiae strain 141 during sourdough fermentation.
3.2. Phytate Zone of Hydrolysis
All LAB, AAB, and yeast strains isolated from kefir, CWK, and CWK-fermented sourdough were screened for their phytate-degrading ability using two different types of media: modified MRS agar (MRS-MOPS) in which inorganic phosphate was replaced by sodium phytate (0.65 g/L, Sigma) and 0.2% CaCl2, and MRS-MOPS + AA supplemented with 10 g/L of arginine and glycine.
All of the isolates were incubated for 48 h at 30 °C. As shown in
Table 3, all were positive for phytate hydrolysis by exhibiting a zone of clearance (translucent) around the inoculated area on the agar. A counterstaining method was used by flooding the agar with aqueous cobalt chloride, which helps determine the false positives for phytate-negative microorganisms. The exact mechanism of the counterstaining procedure is not yet known. However, it is estimated that phytate can form complexes with cobalt and metals and that it has a pH dependence on these complexes, with relative binding of different cations with phytate [
49].
After counterstaining, all of the phytase-positive LAB, AAB, and yeast displayed a halo ranging between 2.3 mm and 14.24 mm in diameter.
Limosilactobacillus fermentum,
Lactiplantibacillus plantarum,
Lactobacillus fusant,
Lactobacillus reuteri, and
Lactobacillus kunkeei had significantly larger zones of hydrolysis that ranged between 14.24 ± 0.12 mm and 13.73 ± 0.08 mm (
p < 0.05). LAB, AAB, and yeast were tested for their ability to degrade sodium phytate in the presence of calcium chloride. The supplemented calcium aids in the phytase enzyme activity and does not itself take part in the reaction [
49]. The phytate degradation ability of all of these isolates can be attributed to the presence of the phytase enzyme, which degrades the available phytate (sodium phytate) in the agar in the presence of calcium chloride, because no white precipitate was observed around the zone of enzyme-specific phytate hydrolysis [
50].
Acetobacter species had a significantly smaller hydrolysis zone compared to those of LAB, with values between 8.5 ± 0.2 mm and 9.37 ± 0.15 mm (p < 0.05). No previous study has reported phytate hydrolysis by AAB according to the previous literature. The ability to utilize phytate can thus be attributed to the production of the phytase enzyme by AAB.
A significantly smaller zone of hydrolysis was obtained for all yeast species, which ranged between 2.3 ± 0.17 mm and 3.07 ± 0.15 mm (
p < 0.05). Positive yeast strains for phytate hydrolysis have been previously reported by Howson and Davis (1983) for
S. cerevisiae, a GRAS (Generally Recognized as Safe) organism used in food for human consumption [
51]. Degradation of phytate by phytase produced by
S. cerevisiae is valuable in the industrial production of food from plant seed meals [
52]. Tsang (2011) [
53] showed that two out of the three isolates of
C. kefyr and three out of four isolates of
C. guilliermondii tested positive for phytate hydrolysis. Additionally, Caputo, Visconti, and De Angelis (2015) [
54] reported that
C. colliculosa possessed phytate hydrolysis ability. Phytase activity has also been reported to be positive for
Rhodotorula mucilaginosa [
55].
3.3. Phytase Enzyme Activity Assay
Each of the LAB, AAB, and yeast isolates that had tested positive for phytate hydrolysis (
Table 3) were subjected to phytate assay in liquid medium. In this assay, sodium phytate in the presence of calcium was used to modulate the enzymatic activities of phytate-degrading enzymes in microorganisms [
56,
57]. One percent of sodium phytate added to each medium of growth tested did not act as an inhibitor of phytate-degrading enzymes. In fact, the phytase activity of all of the phytate-degrading strains was detected in all three media used, as reported in
Table 4. Among LAB, AAB, and yeast, the highest phytase values were found for
Limosilactobacillus fermentum (4052.53 ± 171.14 U/mL;
p ≤ 0.05) and
L. plantarum (3151.83 ± 383.07 U/min;
p ≤ 0.05) grown on Chalmers broth and Chalmers broth supplemented with either 1% sodium phytate or 2% calcium chloride (
Table 4).
It was reported previously that all tested strains of LAB exhibited the production of phytase between 2.6 and 146 U/mL in terms of inorganic orthophosphate released [
58]. Positive phytase production between 0.53 and 0.74 U/mL has been reported for
L. plantarum T211,
L. plantarum H10, and
L. plantarum H5, and
L. plantarum L3 has been previously reported by Anastasio et al. (2010) [
59] for its growth on all three types of Chalmers broths (Chalmers broth and Chalmers broth supplemented with either 1% sodium phytate or 2% calcium chloride). Phytase production of 110.6 U/mL by
Limosilactobacillus fermentum DC400 incubated for 24 h at 37 °C has been reported by De Angelis et al. (2003) [
50], along with its capability to hydrolyze the available phytate in the medium. The phytase activity in this study is higher when compared to the literature, which could be due to the longer incubation time of 48 h in contrast to 24 h used by De Angelis et al. (2003) [
50]. Yildirim and Arici (2019) [
60] have reported phytase activity for
Lactiplantibacillus plantarum ELB78 of 797.88 U/mL. Sumengen, Dincer, and Kaya (2013) [
61] reported extracellular phytase activity of 984.50 U/mL from
Lactiplantibacillus plantarum isolated from a fermented product known as “shalgam”. This is significantly lower than the value obtained in this study for
L. plantarum, which could be due to the longer fermentation time of the sourdough sets.
When compared with LAB, AAB strains showed significantly lower phytase activity ranging between 2389.02 ± 14.84 U/mL for A. aceti and 2383.86 ± 10.74 U/mL for A. lovaniensis (p ≤ 0.05), followed by yeast strains with phytase activity ranging between 2360.21 ± 6.36 U/mL for R. mucilaginosa and 2364.08 ± 5.82 U/mL for C. colliculosa (p ≤ 0.05).
Phytase activity has been reported for
S. cerevisiae by Turk et al. (2000) [
62] and for various other Candida species, including
C. kefyr,
C. guilliermondii, and
C. colliculosa. Maximum phytase activity reached 205.4 U/mL for
Rhodotorula mucilaginosa as reported by Yu, Wang, and Liu (2015) [
55].
3.4. Screening for EPS Production
A method reported by Ruas-Madiedo et al. (2008) and Vescovo et al. (1989) for detecting EPS production was used [
41,
42]. EPS was detected through the examination of slimy colonies on the plate until 72 h of incubation at 30 °C on the abovementioned media. The positive colonies were further confirmed phenotypically through the formation of strings upon touching the colonies with a toothpick aseptically.
LAB, AAB, and yeast strains were considered positive for EPS production if they formed slimy colonies on Chalmers agar (CA) in the presence of 5% sucrose and without CaCO
3 (CA + S), CA supplemented with 5% sucrose and no yeast extract (YE), and CA supplemented with 5% sucrose and 0.5% YE (
Table 5).
All of the strains that tested positive for EPS production on CA + S were then grown on CA + S − YE and CA + S + YE for quantification of EPS produced by all of the individual isolates.
In the presence of only CaCO3 without any sucrose supplementation, there was no EPS production. Limosilactobacillus fermentum, L. plantarum, and L. reuteri produced EPS after 24 h of incubation at 30 °C with 5% CO2 on CA + S, CA + S − YE, and CA + S + YE. However, L. kunkeei produced EPS after 48 h with 5% CO2 upon incubation at 30 °C. L. fusant did not produce EPS on any of the three agars.
Chalmers agar supplemented with 5% sucrose and without CaCO
3 is the most suitable medium for the growth of EPS-producing LAB, which can be visualized in the form of slimy colonies. EPS production depends on the medium’s composition (i.e., nitrogen and carbon source and growth factors) and the temperature, pH, incubation time, and available oxygen to which the microorganisms are exposed. All LAB, AAB, and yeast showed growth on CA + S − CaCO
3, CA + S − YE, and CA + S + YE agar. Homopolysaccharide-producing LAB strains, such as
Lactobacillus reuteri, often originate from cereal products, and heteropolysaccharide-producing LAB strains are present in high proportions in dairy products, which are fermented [
63,
64,
65]. Synthesis of homopolysaccharide is correlated to the presence of sucrose in the media, which is the sole substrate for utilization by glycansucrase, while, on the other hand, heteropolysaccharide production is independent of the sucrose source [
66].
Acetobacter aceti and
Acetobacter pasteurianus produced EPS after 48 h of incubation at 30 °C with 5% CO
2 on CA + S − CaCO
3, CA + S − YE, and CA + S + YE agar (
Table 5). No EPS was produced by
A. lovaniensis. AAB are Gram-negative obligate aerobes and belong to the subdivision of α-proteobacteria (well-known vinegar producers). Almost all AAB species can grow in static culture by “floating” as they produce a pellicle on the surface of the culture medium. This pellicle is an aggregate of all of the cells suspended in the liquid–air interface and strongly bound to one another by polysaccharide or other extracellular matrices on the AAB cell surface. The pellicle polysaccharides occur as a homopolysaccharide of cellulose or as heteropolysaccharides, such as a capsular polysaccharide of
Acetobacter aceti strain IFO3284. The
Acetobacter pasteurianus strain produces two different types of colonies on the agar surface: a rough-surfaced colony and a smooth-surfaced colony. The rough-surfaced-colony-producer strain can produce pellicles, which allows it to float on the medium surface in static culture [
67].
Regarding acetic acid bacteria, conflicting findings have emerged regarding the impact of carbon sources on EPS (extracellular polysaccharides) production. Studies indicate that fructose, sucrose, and glucose are associated with the highest yields of bacterial cellulose [
68,
69,
70,
71,
72]. Yet, there remains a lack of data concerning how carbon sources affect the chemical composition of heteropolysaccharides like acetan or gluconacetan, which are synthesized by Acetobacter strains [
73].
Candida guilliermondii and
Rhodotorula mucilaginosa were positive for EPS production after 48 h of incubation at 28 °C for all CA + S, CA + S − YE, and CA + S + YE, whereas
C. kefyr,
S. cerevisae, and
C. colliculosa were negative for phenotypic EPS production (
Table 5).
Most of the Candida species can form surface-attached microbial communities by producing extracellular polymeric substances [
74]. Gientka et al. (2016) [
75] have reported the production of EPS by
Candida guilliermondii isolated from kefir. A study carried out by Garza et al. (2016) [
76] has reported that EPS is produced by
Rhodotorula mucilaginosa. In most cases, EPS is produced when microorganisms are under conditions of stress and create a shell-like structure that prevents toxic reagents from reaching the cell [
76,
77,
78,
79].
3.5. EPS Quantification
All of the isolates that tested positive for EPS production on CA + S − YE and CA + S + YE agar (
Table 5) were further quantified for EPS production as polymer dry mass in mg/kg. All of the LAB, AAB, and yeast strains show higher EPS production on Chalmers agar supplemented with 5% sucrose and 0.5% YE compared to CA + 5% sucrose without YE (
Table 6).
Three out of the four LAB strains showed the significantly highest EPS production on CA + S + YE, between 85.21 ± 2.35 mg/kg and 94.01 ± 1.65 mg/kg polymer dry mass (PDM) (p ≤ 0.05), when compared to EPS production by AAB and yeast.
The assessment of EPS (extracellular polysaccharides) in LAB (lactic acid bacteria) is commonly conducted using broth medium, which may be modified with various simple sugars, along with complex media. Hence, this study adopted a straightforward and beneficial approach. Another rationale behind employing a solid medium was to minimize the risk of contamination by polysaccharides, including certain proteins or other large molecules found in liquid culture mediums. Additionally, the solid surfaces of agar culture media encourage attached bacteria to produce EPS compared to planktonic cells in broth media [
44].
Acetobacter aceti and
Acetobacter pasteurianus produced higher EPS on CA + S + YE of 24.55 ± 1.99 and 32.27 ± 0.98 mg/kg PDM, respectively (
p ≤ 0.05). The genus Acetobacter is known for its capacity to produce cellulose (a type of EPS), which is used for some of the fermented food products [
80,
81,
82,
83]. A study carried out by Matsushita, Ebisuya, Ameyama, and Adachi (1992) provides evidence that
A. aceti (R-strain or rough-surfaced-colony) produces EPS; however, no quantitative data were presented [
84].
Candida guilliermondii and
Rhodotorula mucilaginosa produced 29.66 ± 1.44 and 34.73 ± 1.58 mg/kg PDM, respectively (
p ≤ 0.05). Vazquez-Rodriguez et al. (2018) have reported that
Rhodotorula mucilaginosa strain UANL-001 L produced 19 mg/mL of EPS after 96 h of incubation at 28 °C [
80]. A study carried out by Gientka et al. (2016) shows that
Candida guilliermondii was able to synthesize 2.9 g/L of EPS after 72 h of incubation and with maltose as the carbon source [
75].
4. Conclusions
The biological and enzymatic phytase assays have shown that Limosilactobacillus fermentum and L. plantarum exhibited the highest phytase production and phytate-hydrolyzing properties.
Similarly, these two isolates exhibited the highest EPS-producing capability.
Except for Lactobacillus fusant, Lactobacillus reuteri, and Lactobacillus kunkeei, all of the LAB produced high glutamic acid concentration. Production of glutamic acid was observed for AAB and yeast isolates, but it was significantly lower when compared with those of LAB isolates. Interestingly, among all of the yeast isolates, Candida guilliermondii had the significantly lowest glutamic-acid-producing capability.
This paper has demonstrated that the microorganisms associated with sourdough fermentation in this study possess functional properties that could improve the sourdough.