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Yogurt is traditionally fermented by a symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. These bacteria exchange metabolites with each other to meet their nutritional demands during protocooperation, resulting in a shorter fermentation time. In this study, we investigated whether fumaric acid functions as a symbiotic agent to promote the growth of Lb. bulgaricus by evaluating 8 strains of Lb. bulgaricus and 7 strains of Strep. thermophilus. All the tested Lb. bulgaricus strains metabolized the added fumaric acid into succinic acid during monoculture in milk, and 6 strains (75%) showed shorter fermentation time compared with the control. The addition of malic acid showed similar trends as that of fumaric acid, indicating that the reverse tricarboxylic acid cycle was functioning in Lb. bulgaricus. All 7 Strep. thermophilus strains tested produced fumaric acid during monoculture in milk. Further, in Lb. bulgaricus 2038, the gene expression of fumarate reductase that converts fumaric acid to succinic acid, was higher in the coculture with Strep. thermophilus 1131 than in the monoculture. These findings indicate that fumaric acid produced by Strep. thermophilus can function as a symbiotic substance during yogurt fermentation to stimulate the growth of Lb. bulgaricus.
Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus are widely used lactic acid bacteria for fermenting yogurt. Although each bacterium can grow in milk individually, they can also protocooperate by exchanging their metabolites (
). Identifying metabolites that influence this symbiosis is important for optimizing fermentation conditions, which can increase the efficiency of industrial manufacture of yogurt.
In addition to the metabolites described above, comprehensive analyses of the symbiosis between Strep. thermophilus and Lb. bulgaricus have revealed candidates functioning in symbiosis (
Postgenomic analysis of Streptococcus thermophilus cocultivated in milk with Lactobacillus delbrueckii ssp. bulgaricus: Involvement of nitrogen, purine, and iron metabolism.
). Some studies demonstrated that Strep. thermophilus produces fumaric acid from arginine and argininosuccinate through the action of argH (argininosuccinate lyase;
Postgenomic analysis of Streptococcus thermophilus cocultivated in milk with Lactobacillus delbrueckii ssp. bulgaricus: Involvement of nitrogen, purine, and iron metabolism.
). These reports suggest that fumaric acid functions as the symbiotic substance between Strep. thermophilus and Lb. bulgaricus. Another study showed that the addition of fumaric acid promoted the growth of Lb. bulgaricus (
Studies on symbiosis in yogurt cultures. II. Isolation and identification of growth factor of Lactobacillus bulgaricus produced by Streptococcus thermophilus..
). However, as the study investigated the effect of fumaric acid using only 1 Lb. bulgaricus strain by measuring acidity, it was not clear whether the growth-stimulating effect of fumaric acid is common to Lb. bulgaricus species. Further, the mechanism of how Lb. bulgaricus utilizes fumaric acid during fermentation was also not described.
Fumaric acid is a component of the tricarboxylic acid (TCA) cycle. Lactic acid bacteria have been reported to have an incomplete TCA cycle, and some lactobacilli were shown to utilize the TCA cycle in the direction of the reductive reactions (
). Lactobacillus bulgaricus also appears to have an incomplete TCA cycle, as all 4 strains of Lb. bulgaricus (ATCC 11842T, ATCC BAA-365, ND02, and 2038) available to the Kyoto Encyclopedia of Genes and Genomes database (https://www.genome.jp/kegg/) contain only 2 genes active in this cycle, both of which are involved in the metabolism of fumaric acid. One is fumarate hydratase (EC 4.2.1.2), which catalyzes the reversible conversion between fumarate and malate; the other is fumarate reductase (FRD; EC 1.3.5.4), which catalyzes the reduction of fumarate to succinate. Therefore, it can be inferred that Lb. bulgaricus is capable of metabolizing fumarate to either malic acid or succinic acid; however, the direction via which Lb. bulgaricus metabolizes fumaric acid needs to be evaluated.
This study was conducted to determine the effect of fumaric acid on the growth of Lb. bulgaricus and clarify how fumaric acid is utilized during fermentation in milk. We also examined whether fumaric acid functions as a symbiotic substance during yogurt fermentation by using Lb. bulgaricus 2038 and Strep. thermophilus 1131 as a model starter, as these strains show a strong symbiotic relationship (
Table 1 shows the bacterial strains used in this study. We included strains that exhibited a variety of fermentation properties in skim milk broth. Lactobacillus bulgaricus 2038, ME-810, ME-811, and ME-812, and Strep. thermophilus 1131, ME-813, ME-814, and ME-815 were obtained from Meiji Innovation Center. Lactobacillus bulgaricus ATCC 11842T was obtained from the Japan Collection of Microorganisms (JCM). Lactobacillus bulgaricus NCIMB 702074, NCIMB 701373, NCIMB 701978, and Strep. thermophilus NCIMB 8510T were obtained from the National Collection of Industrial Food and Marine Bacteria. We obtained LMG18311 and LMD-9 from American Type Culture Collection. These strains were anaerobically precultured twice at 37°C for 16 h in autoclaved (at 121°C for 7 min) preculture medium containing 10% (wt/wt) skim milk powder (Meiji Co. Ltd.) and 0.1% (wt/wt) yeast extract (Asahi Food and Healthcare).
Properties of Lactobacillus delbrueckii ssp. bulgaricus were defined by the fermentation time of monoculture in skim milk broth supplemented with 1 mM formic acid. Fast acidifying: <400 min; medium acidifying: 400–600 min; slow acidifying: >600 min. prtS = cell envelope proteinase gene; Strep. thermophilus possessing prtS (prtS+) show fast-acidifying phenotype compared with strain without prtS (prtS−) in monoculture (Fernandez-Espla et al., 2000).
Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus β-galactosidase sequences.
1 Properties of Lactobacillus delbrueckii ssp. bulgaricus were defined by the fermentation time of monoculture in skim milk broth supplemented with 1 mM formic acid. Fast acidifying: <400 min; medium acidifying: 400–600 min; slow acidifying: >600 min. prtS = cell envelope proteinase gene; Strep. thermophilus possessing prtS (prtS+) show fast-acidifying phenotype compared with strain without prtS (prtS−) in monoculture (
2 JCM = Japan Collection of Microorganisms; NCIMB = National Collection of Industrial Food and Marine Bacteria; ATCC = American Type Culture Collection.
For fermentation experiments, skim milk powder broth containing 10% (wt/wt) skim milk was heated at 95°C for 2 min. For monoculture of Lb. bulgaricus, sodium formate (Tokyo Kasei Kogyo) was supplemented at a final concentration of 1 mM. For monoculture of Strep. thermophilus, Hyvital casein CMA500 (Friesland Campina) was supplemented at a final concentration of 0.1% (wt/wt). Fumaric acid, malic acid, and succinic acid (all from Fujifilm Wako) solution (pH 6.5 adjusted with NaOH) was also added after heat treatment. All additives were filtered through a 0.22-µm filter before supplementation. For monoculture of Strep. thermophilus 1131 and all the strains of Lb. bulgaricus, the preculture was inoculated at 2% (wt/wt). For monoculture of Strep. thermophilus, except 1131, the preculture was inoculated at 0.8% (wt/wt). For coculture, the precultures of Lb. bulgaricus 2038 and Strep. thermophilus 1131 were inoculated at 0.2% (wt/wt) and 0.3% (wt/wt), respectively. Fermentation experiments were carried out at 40°C under aerobic conditions. The pH during fermentation was measured using a multiple-electrode measuring device (Horiba) with the pH sensor SE 555 (Knick). The time required for the pH to reach 4.6 was defined as the fermentation time.
Measurement of Fumaric, Malic, and Succinic Acid Concentrations
Samples were diluted 2-fold with distilled water. To remove the milk protein, 2.5% (wt/wt) Carrez I and 2.5% (wt/wt) Carrez II were added to the diluted samples (
), which were then centrifuged at 4°C and 20,000 × g for 5 min. The supernatants were analyzed by HPLC (Shimazu) with 2 connected ICSep ICE ORH-801 columns (Concise Separations). The mobile and reaction phases consisted of 7.5 mM p-toluene sulfonic acid monohydrate (Fujifilm Wako) in distilled water and 7.5 mM p-toluene sulfonic acid, 150 µM EDTA-2Na (Dojindo), and 30 mM Bis-Tris (Dojindo) in distilled water, respectively. The injection volume, elution flow, and column temperature were set to 10 µL, 0.5 mL/min, and 55°C, respectively. Detection was carried out using a CCD-10A electrical conductivity detector (Shimazu). Fumaric acid, malic acid, and succinic acid were used as standards to calculate the concentrations in the culture samples.
Measurement of Intracellular NAD+ and NADH Concentrations
Five grams of fermented sample at pH 5.5 was mixed with 1.7 mL of 1 M sodium citrate and 0.7 mL of saline solution consisting of 0.85% (wt/vol) NaCl (Fujifilm Wako), 0.5% (wt/vol) β-glycerophosphate disodium salt (EMD Millipore Corp.), and 0.1% (wt/vol) Tween 80 (Fujifilm Wako, pH 7.0). After vortexing and centrifugation at 4°C and 20,000 × g for 5 min, the cell pellet was resuspended in 10 mL of 50 mM potassium phosphate buffer and centrifuged again at 4°C and 20,000 × g for 5 min. The cell pellet was stored at −80°C until measurement. Intracellular NAD+ and NADH concentration was measured using NAD/NADH Assay Kit-WST (Dojindo) with the addition of the cell-lysing process described below. The cell pellet was resuspended in 1.5 mL of 50 mM potassium phosphate buffer, and the volume containing 5.0 × 106 cfu was centrifuged at 4°C and 20,000 × g for 5 min. The cell pellet was resuspended in 600 µL of NAD-NADH extraction buffer in the kit and transferred to a screw-cap tube with 0.6 g of 0.1-mm zirconium beads. The cell was lysed in FastPrep-24 (MP Biomedicals) twice for 30 s at a speed of 6.5 m/s and centrifuged at 4°C and 20,000 × g for 5 min. The supernatant was deproteinated using a 10K molecular weight cutoff spin filter included in the kit, and NAD+ and NADH concentrations in the filtered sample were measured according to the manufacturer's instruction.
RNA Extraction
Total RNA was extracted from 20 mL (3 h) and 1 mL (4, 5 h) of fermented samples. Briefly, a stop solution consisting of 5% (vol/vol) phenol (Fujifilm Wako) in ethanol (Fujifilm Wako) was added to the sample at one-eighth of the sample volume. Next, one-third of the sample volume of 1 M sodium citrate and one-eighth of the sample volume of the saline solution described in the previous section were added. After vortexing and centrifugation at 4°C and 20,000 × g for 5 min, the cell pellet was resuspended in 1 mL of Tris-EDTA buffer (pH 8.0; Nippon Gene) and centrifuged at 4°C and 20,000 × g for 3 min. The cell pellet was frozen in liquid nitrogen and stored at −80°C until RNA extraction. To extract RNA, the frozen cell pellet was resuspended in 500 µL of extraction buffer consisting of 100 mM LiCl, 10 mM EDTA (pH 7.4), 10 mM Tris-HCl (pH 7.4), and 1% SDS, and the sample was transferred to Lysing Matrix B (MP Biomedicals) supplemented with 500 µL of 25:24:1 phenol-chloroform-isoamyl alcohol (Nippon Gene). The cells were lysed in FastPrep-24 (MP Biomedicals) twice for 45 s at a speed of 6.5 m/s and then centrifuged at 4°C and 4,500 × g for 10 min. Each supernatant was transferred to a MaXtract High-Density tube (QIAGEN) supplemented with 500 µL of 25:24:1 phenol-chloroform-isoamyl alcohol. The tube was gently mixed and centrifuged at 4°C and 20,000 × g for 10 min. The supernatant was transferred to a new tube and mixed with one-tenth volume of 1 M LiCl and 2.5-fold volume of 99.5% ethanol. After vortexing for 20 s, the tube was centrifuged at 4°C and 20,000 × g for 15 min, and the pellet was washed with 200 µL of 70% ethanol. The tube was centrifuged at 4°C and 20,000 × g for 5 min, and RNA was extracted from the pellet by using NucleoSpin RNA Plus (Macherey-Nagel) according to the manufacturer's instructions. The quantity and quality of the extracted RNA were measured with an Agilent 2100 Bioanalyzer (Agilent Technologies), and RNA samples with RNA integrity number >7.1 were used for further analysis.
Gene Expression Analysis
Extracted RNA (500 ng) was reverse-transcribed using PrimeScript RT Master Mix (Takara). Quantitative reverse transcription-PCR was performed using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific) and a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). The mRNA level of the frd gene from Lb. bulgaricus 2038 was measured using the forward primer frd-F (5′-TGCTGGTCTTGAAGCAACTG-3′) and reverse primer frd-R (5′-TGGGTGAATGTGGATAGCAA-3′). Relative mRNA levels were normalized to that of groEL, which was measured using the forward primer groEL-F (5′-TGAACTGAGCGTGGTTGAAG-3′) and reverse primer groEL-R (5′-GGCAAGATGTCCTGGATGTT-3′). None of the above primers matched any sequence in the Strep. thermophilus 1131 genome sequence, indicating that they were unique to Lb. bulgaricus 2038. The mRNA level of the argininosuccinate lyase gene from Strep. thermophilus 1131 was measured using the forward primer argH-F (5′- CATGCTCAGCCGATTTCTTT-3′) and reverse primer argH-R (5′-CACCAAGTGGGGAAATATCG-3′), reported in a previous study (
Postgenomic analysis of Streptococcus thermophilus cocultivated in milk with Lactobacillus delbrueckii ssp. bulgaricus: Involvement of nitrogen, purine, and iron metabolism.
). Relative mRNA levels were normalized to that of glyS (glycine-tRNA-ligase), which was reported to be suitable for reference gene in Strep. thermophilus (
Short communication: Genome-wide identification of new reference genes for reverse-transcription quantitative PCR in Streptococcus thermophilus based on RNA-sequencing analysis.
). The expression of glyS was measured using the forward primer glyS-F (5′-GACAGGTTCAGCTAGCGACA-3′) and reverse primer glyS-R (5′-AACAGCCACATCATCTGCCA-3′), reported by
Short communication: Genome-wide identification of new reference genes for reverse-transcription quantitative PCR in Streptococcus thermophilus based on RNA-sequencing analysis.
. None of these primers matched any sequence in the Lb. bulgaricus 2038 genome sequence, indicating that they were unique to Strep. thermophilus 1131. A standard curve for each gene was prepared by plotting the threshold cycle values as a function of the log serial dilutions of the cDNA template, and the relative value corresponding to one-fortieth–diluted cDNA was calculated from the standard curve.
Statistical Analysis
All experiments were carried out at least 3 times, and the results are expressed as the mean ± standard deviation. Data were compared by Student's t-test, and P-value <0.05 indicated statistical significance compared with the control.
RESULTS
Effects of Addition of Fumaric, Malic, and Succinic Acids on the Monoculture of Lb. bulgaricus 2038 and Strep. thermophilus 1131
To examine the effect of fumaric acid on fermentation, 1 mM concentrations of fumaric acid, malic acid, or succinic acid was added before fermenting the monoculture of Lb. bulgaricus 2038 and Strep. thermophilus 1131. As shown in Figure 1A, the addition of either fumaric acid or malic acid remarkably shortened the fermentation time of Lb. bulgaricus 2038, whereas succinic acid had no effect. The shortening of fermentation time by fumaric acid and malic acid was observed at a concentration as low as 0.1mM, whereas the most effective concentration was 0.3 mM and 0.5 mM for fumaric acid and malic acid, respectively (Supplemental Figure S1, https://doi.org/10.5281/zenodo.4713062,
). On the contrary, the fermentation time of Strep. thermophilus 1131 did not change following the addition of any of these substances (Figure 1A). Furthermore, the addition of fumaric acid or malic acid, even at a higher concentration (1 mM), did not shorten the fermentation time of the coculture of Lb. bulgaricus 2038 and Strep. thermophilus 1131 (Supplemental Figure S2, https://doi.org/10.5281/zenodo.4713062,
Figure 1Fermentation time and the concentrations of fumaric, malic, and succinic acids in Lactobacillus bulgaricus 2038 and Streptococcus thermophilus 1131 monoculture. (A) Fermentation time until pH 4.6 when 1 mM concentration for each substance was added. Asterisks indicate highly significant differences compared with control (**P < 0.01). (B, C) Concentrations of fumaric, succinic, and malic acids when (B) Lb. bulgaricus 2038 or (C) Strep. thermophilus 1131 was fermented in (i) skim milk broth, and skim milk broth supplemented with (ii) 1 mM fumaric acid, (iii) 1 mM malic acid, and (iv) 1 mM succinic acid. All results are shown as the mean ± SD.
Next, to determine how these substances were utilized or produced, we measured the concentrations of fumaric acid, malic acid, and succinic acid before and during fermentation. For Lb. bulgaricus 2038, more than half of the added fumaric acid and malic acid were consumed at pH 5.5 and had been depleted when the culture reached pH 4.6 (Figure 1B ii, iii). Notably, a high concentration of succinic acid was observed at pH 4.6 with the addition of both fumaric acid (Figure 1B ii) and malic acid (Figure 1B iii). The production of succinic acid was also confirmed both in control (Figure 1B i) and culture containing added succinic acid (Figure 1B iv). In addition, the intracellular NAD+-NADH ratio of Lb. bulgaricus 2038 at pH 5.5 was higher with the addition 1 mM fumaric acid compared with that of the control (Table 2). In contrast, although Strep. thermophilus 1131 did not consume any added substance, they produced the highest amount of fumaric acid among the substances measured under all conditions (Figure 1C). These results suggest that only Lb. bulgaricus metabolized fumaric acid and malic acid to succinic acid during fermentation, promoting the growth of Lb. bulgaricus.
Effects of Addition of Fumaric and Malic Acids on 7 Lb. bulgaricus Strains
To examine whether shortening of the fermentation time by adding fumaric acid and malic acid was common to Lb. bulgaricus strains, the fermentation time required to reach pH 4.6 was measured for the type strain and 6 strains from the Meiji and National Collection of Industrial Food and Marine Bacteria culture collections. As shown in Figure 2, the fermentation times of 5 strains were shortened by the addition of fumaric acid. All strains, except the type strain, showed shorter fermentation time by the addition of malic acid. Further, succinic acid was produced in all strains tested following supplementation with fumaric acid or malic acid (Table 3). These results indicate that Lb. bulgaricus strains tested here have a common ability to metabolize fumaric acid and malic acid to succinic acid during fermentation, and 5 strains of them resulted in a shortened fermentation time by utilizing fumaric acid.
Figure 2Fermentation time of 7 Lactobacillus bulgaricus strains to reach pH 4.6 in control and after addition of 1 mM fumaric acid or 1 mM malic acid. Results are shown as the mean ± SD. Asterisks indicate highly significant differences compared with control. **P < 0.01, *P = 0.035 (NCIMB 702074), P = 0.024 (NCIMB 701978 fumaric acid), P = 0.014 (NCIMB 701978 malic acid).
Production of Fumaric Acid by 6 Strep. thermophilus Strains
To examine whether the production of fumaric acid was common to multiple Strep. thermophilus strains, fumaric acid concentrations at pH 4.6 or pH 5.0 were measured for the type strain and 5 strains from the Meiji and American Type Culture Collection culture collections. As shown in Table 4, all tested strains produced fumaric acid during fermentation, as observed for Strep. thermophilus 1131 (Figure 1C), regardless of the source or fermentation properties. This result indicates that during fermentation, Strep. thermophilus commonly produce fumaric acid, which can be utilized by Lb. bulgaricus during coculture.
Table 4Concentration of fumaric acid produced by Streptococcus thermophilus
Expression of Fumarate Reductase in Lb. bulgaricus 2038 and Argininosuccinate Lyase in Strep. thermophilus 1131 in Monoculture and Coculture
To further examine whether fumaric acid functions as the symbiotic substance from Strep. thermophilus to Lb. bulgaricus in coculture, expression of frd gene in Lb. bulgaricus 2038, which metabolizes fumarate to succinate, was measured at 3, 4, and 5 h of fermentation, corresponding to early, mid, and late exponential phase. If Lb. bulgaricus 2038 utilized fumaric acid produced by Strep. thermophilus 1131 in the coculture, the expression of frd was predicted to be upregulated in coculture compared with in monoculture. In support of this hypothesis, frd expression was remarkably higher in coculture than in monoculture at 3 and 4 h of fermentation (Figure 3). At 5 h, it decreased in coculture to approximately the same value as in monoculture. In Strep. thermophilus 1131, the expression of argH, which produces fumaric acid from arginine and argininosuccinate, was upregulated at 4 h of fermentation in coculture as compared with monoculture (Supplemental Figure S3, https://doi.org/10.5281/zenodo.4713062,
). This result suggested that Lb. bulgaricus in coculture upregulated the expression levels of frd in the early to mid-stage of fermentation to utilize fumaric acid produced by Strep. thermophilus.
Figure 3The frd gene expression of Lactobacillus bulgaricus 2038 in monoculture or coculture with Streptococcus thermophilus 1131. Relative expression values of frd were normalized to those of groEL. Results are shown as mean ± SD. Asterisks indicate highly significant differences compared with control; **P < 0.01.
We examined the influence of fumaric acid on the growth of Lb. bulgaricus and clarified how fumaric acid was utilized during fermentation. All 8 Lb. bulgaricus strains tested here metabolized fumaric acid and malic acid to succinic acid, with 6 strains (75%) showing shorter fermentation times. Additionally, all 7 Strep. thermophilus strains tested produced fumaric acid during fermentation, and frd expression by Lb. bulgaricus 2038 in coculture with Strep. thermophilus 1131 was remarkably higher than in monoculture. These findings indicate that fumaric acid produced by Strep. thermophilus can function as a symbiotic substance in coculture to stimulate the growth of Lb. bulgaricus.
We confirmed that fumaric acid and malic acid were metabolized into succinic acid in all Lb. bulgaricus strains tested (Figure 1B and Table 3); these strains were from different sources and exhibited different fermentation properties such as fast and slow acidification and a ropy phenotype (Table 1). Furthermore, fumaric acid stimulated the growth of Lb. bulgaricus strains tested here except for ATCC 11842T and NCIMB 702074 (Figure 2). These results suggest that the ability to reduce fumaric acid to succinic acid is common to Lb. bulgaricus species; however, this utilization does not always stimulate the growth of Lb. bulgaricus. To explain this phenomenon, studies are needed to determine the mechanism by which fumaric acid promotes the growth of Lb. bulgaricus. One probable mechanism involves control of the redox balance in the cell. We confirmed that the addition of fumaric acid to Lb. bulgaricus 2038 increased the intracellular NAD+ concentration and NAD+-to-NADH ratio at pH 5.5 (Table 2). It has been reported that the balance of NAD+ and NADH is closely related to carbon utilization and control of the growth rate in bacteria (
). It has also been shown that the enzyme FRD covalently links to coenzyme FAD to reduce fumaric acid to succinic acid by transferring electrons from FADH (
). Thus, the reduction of intracellular H+ levels by FRD may increase the NAD+-NADH ratio in the cell, promoting the glycolysis and growth of Lb. bulgaricus. Different effects of fumaric acid for each strain could be attributed to several factors affecting the NAD+-NADH balance in the cell; therefore, a comprehensive research is necessary to determine the detailed mechanism of how fumaric acid influences the fermentation properties of Lb. bulgaricus.
We demonstrated that Lb. bulgaricus uses an incomplete TCA cycle reductively. It has been reported that the genome of Lb. bulgaricus has evolved in a reductive manner through the adaptation to milk and protocooperation with Strep. thermophilus (
). Lactobacillus bulgaricus possess only frd and the fumarate hydratase gene from the TCA cycle, suggesting that these genes are important for the growth of Lb. bulgaricus. The metabolism of fumaric acid to succinic acid has also been confirmed in Lactobacillus reuteri (
Metabolic engineering of Lactobacillus plantarum for succinic acid production through activation of the reductive branch of the tricarboxylic acid cycle.
), indicating that this type of metabolism is a common trait among Lactobacillus species. However, one study showed contradictory results, demonstrating that Lb. bulgaricus had the greatest ability to produce malic acid from fumaric acid among the 6 genera of lactic acid bacteria (
). As the authors measured the production of malic acid in buffer solution rather than in milk broth, the medium may have influenced the use of fumaric acid by Lb. bulgaricus.
We confirmed in Table 4 that all 6 Strep. thermophilus strains tested produced concentrations of approximately 0.2 to 0.4 mM fumaric acid regardless of whether the cell wall protease PrtS (
) or ropy phenotype was present, which is consistent with a previous report describing that Strep. thermophilus produced fumaric acid via argH during fermentation (
Postgenomic analysis of Streptococcus thermophilus cocultivated in milk with Lactobacillus delbrueckii ssp. bulgaricus: Involvement of nitrogen, purine, and iron metabolism.
Metabolic profiles of cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine, alanine and glutathione in Streptococcus thermophilus during pH-controlled batch fermentations.
). In addition, we observed high expression of argH in Strep. thermophilus 1131 at 4 h in coculture (Supplemental Figure S3, https://doi.org/10.5281/zenodo.4713062,
), suggesting that this strain produce fumaric acid during coculture with Lb. bulgaricus 2038. We demonstrated that addition of as low as 0.1 mM fumaric acid stimulated the growth of Lb. bulgaricus 2038 and that 0.3 mM fumaric acid shortened the fermentation time most effectively (Supplemental Figure S1, https://doi.org/10.5281/zenodo.4713062,
), indicating that the concentration of 0.2 to 0.4 mM fumaric acid produced by Strep. thermophilus was sufficient to promote the growth of Lb. bulgaricus. Because we could not confirm a fumarate transporter gene, we hypothesize that the release and uptake of fumaric acid by Strep. thermophilus and Lb. bulgaricus, respectively, could have occurred because of diffusion of the undissociated molecules.
We observed that frd showed higher expression in Lb. bulgaricus 2038 when in coculture with Strep. thermophilus 1131 than in monoculture (Figure 3). A previous study demonstrated that fumaric acid produced by Strep. thermophilus was consumed during coculture with Lb. bulgaricus (
), supporting that fumaric acid functions as a symbiotic substance between these species. We also confirmed that the addition of fumaric acid to the coculture of Lb. bulgaricus 2038 and Strep. thermophilus 1131 did not shorten the fermentation time (Supplemental Figure S2, https://doi.org/10.5281/zenodo.4713062,
), suggesting that the concentration of fumaric acid produced by Strep. thermophilus 1131 in coculture met the demand of Lb. bulgaricus.
In conclusion, 6 out of 8 Lb. bulgaricus strains fermented rapidly by metabolizing fumaric acid and malic acid to succinic acid, and all 7 Strep. thermophilus strains produced fumaric acid during fermentation. Though we need further experiments to examine the underlying reason for the differences in the reaction toward fumaric acid among Lb. bulgaricus strains, our results revealed that fumaric acid can be an important symbiotic agent between Lb. bulgaricus and Strep. thermophilus. These findings provide insights into the complex system of symbiosis during yogurt fermentation.
ACKNOWLEDGMENTS
All authors are employees of Meiji Co. Ltd. (Tokyo, Japan). The authors are grateful to Yasuko Sasaki from Meiji University (Kanagawa, Japan) and Seiya Makino from Meiji Co. Ltd. for valuable discussions. The authors also thank Yoshiko Honme from Meiji Co. Ltd. for technical assistance with the experiments. The authors declare no conflicts of interest.
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