Development and Taste Improvement of Polyamine-Containing Sakekasu Beverages Using Highly Polyamine-Producing Bacteria from Fermented Foods

Abstract

In our previous study, when Levilactobacillus brevis FB215, derived from blue cheese, was cultured in a water extract of Sakekasu, a byproduct of brewing Japanese rice wine, putrescine, a polyamine that has been reported to have health-promoting effects, accumulated. However, the culture supernatant exhibited an undesirable taste. A metabolome analysis revealed that the major metabolites that were increased by the fermentation of Sakekasu extract were lactate, citrulline, and putrescine. Sakekasu extract fermented by FB215 and cultured at 20 °C, 25 °C, 30 °C, and 37 °C contained lactate at concentrations of 35, 49, 58, and 59 mM, respectively, while the putrescine concentrations were approximately 1 mM at all culturing temperatures. Furthermore, 500 mL of Sakekasu extract fermented by FB215 contained 0.02 and 2.2% of the acceptable daily intake of tyramine and histamine, respectively, which are biogenic amines that raise safety concerns regarding their use in fermented foods. Supplementation with sucrose at a final sugar concentration of 16% (w/v) significantly improved the overall palatability of the Sakekasu extract fermented by FB215 to a level statistically equivalent to that of commercially available sugar-sweetened lactic acid bacterial beverages. A daily intake of 500 mL of Sakekasu extract fermented by FB215 provided approximately 28 mg of polyamines, which is equivalent to the increase in blood polyamine concentrations reported in a previous study.

1. Introduction

Polyamines are aliphatic hydrocarbons with two or more amino groups within their molecular structure. In physiological environments, polyamines carry a positive charge, which enables them to interact with negatively charged molecules such as nucleic acids, phosphorylated proteins, phospholipids, and adenosine triphosphate [1]. These interactions have been demonstrated to facilitate various cellular processes, including gene transcription [1,2], translation [1], and cell differentiation [1].

Recent studies have reported that polyamine supplementation can extend lifespan [3,4], improve brain function [5], alleviate colitis [6], alleviate depression [7], and enhance antitumor immunity [8,9]. Although humans are capable of biosynthesizing polyamines [10], endogenous production declines with age [11]. This decline could be compensated by dietary intake or production by the gut microbiota. Indeed, a high-polyamine diet has been shown to increase polyamine concentrations in human blood [12,13], and putrescine, a polyamine produced by the gut microbiota in the colon, has been reported to be absorbed by mice colonic tissue [6].

However, the polyamine content of most foods is comparatively low. Among the foods analyzed, turban shell viscera contain the highest polyamine concentration (approximately 20 mg/g), followed by wheat germ (approximately 0.5–0.6 mg/g [11]). However, the strong flavor of turban shell viscera makes it difficult to consume in large quantities, which limits its practicality as a dietary source of polyamines. An intervention study using spermidine-rich plant extract supplement has been conducted [14]; however, the daily intake in this study was only 1.2 mg, corresponding to merely 4–10% of the typical polyamine intake reported across various countries [15,16,17]. In earlier mouse studies that demonstrated a lifespan extension, the polyamine intake of a high-polyamine group was over five times that of a low-polyamine group [18]. In previous rat studies that showed anti-aging effects, the intake was ten times higher [19]. These findings suggest that obtaining sufficient polyamines from currently available food sources alone may be challenging for achieving health-promoting effects.

The primary focus of this study was Levilactobacillus brevis. L. brevis is a Gram-positive, rod-shaped lactic acid bacterium. It is heterofermentative, producing CO₂, lactic acid, and acetic acid or ethanol during fermentation [20]. There is a long history of this species’ use in various fermented foods [21,22], and several strains of this species have been granted the “Generally Recognized As Safe (GRAS)” status. Some L. brevis strains have been demonstrated to harbor an agmatine deiminase gene [23] that catalyzes the conversion of agmatine to putrescine. Additionally, these strains are characterized by the presence of an antiporter gene that facilitates the import of extracellular agmatine and export of intracellular putrescine [23]. Additionally, L. brevis FB215, the strain used in the present study, has been reported to convert ornithine to putrescine and subsequently export it to the medium [24].

Tyramine, produced through the decarboxylation of tyrosine, may induce symptoms such as elevated blood pressure and headaches when ingested in large amounts. Similarly, histamine, produced through the decarboxylation of histidine, is known to cause food poisoning symptoms such as headaches, nausea, and vomiting when consumed excessively. Since these bioactive amines can be present in fermented foods and potentially affect consumer health, it is important to assess their production to ensure food safety. Strains of L. brevis capable of producing tyramine and histamine have previously been isolated from fermented foods [25,26,27,28], and some lactic acid bacteria isolated from blue cheese, the source of L. brevis FB215 used in this study, have been reported to produce bioamines such as tyramine and histamine, highlighting the need for safety evaluations [29,30,31,32,33].

Sake is a traditional Japanese fermented alcoholic beverage. It is brewed using Aspergillus oryzae and Saccharomyces cerevisiae. A. oryzae is used to convert the starch in rice into sugar, which is then converted into ethanol by S. cerevisiae [34]. After fermentation, the mixture is pressed to separate the liquid sake from the solid residue—this residue is known as Sakekasu. Sakekasu is rich in proteins, peptides, amino acids, carbohydrates, dietary fiber, fat, ash, and vitamins derived from the rice used as a raw material and the microorganisms used in the brewing of the sake [35,36]. Sakekasu has traditionally been consumed as a foodstuff in Japan, often used in soups, marinades, or pickling. Nevertheless, a substantial portion of Sakekasu is currently discarded, and there is growing interest in exploring its potential applications due to its nutritional value and functional properties. Additionally, whether L. brevis FB215 produces histamine or tyramine, which are harmful compounds generally produced by lactic acid bacteria, is important information for evaluating the suitability of this strain as a fermentation bacterium for producing fermented foods.

Fermentation conditions (such as temperature and fermentation time) have been shown to play a crucial role in the quality and sensory characteristics of fermented products and require careful optimization. For example, in kimchi, a traditional fermented vegetable product made using lactic acid bacteria, it is well known that the fermentation temperature significantly influences its flavor [37].

Therefore, in this study, we have optimized the fermentation conditions (especially temperature and fermentation time) of Sakekasu extract using L. brevis FB215 and performed a metabolome analysis of the culture supernatant after fermentation to analyze the contents of various metabolites, including harmful compounds, to improve its flavor. Finally, we evaluated the effect of the addition of sucrose on its flavor.

2. Materials and Methods

2.1. Preparation of Sakekasu Extract

Sakekasu (10 g), stored at −20 °C, was suspended in 40 mL of deionized water. The suspension was centrifuged at 3600× g for 20 min. Sodium hydroxide was added to the supernatant to adjust the pH to 7. The mixture was sterilized by autoclaving at 121 °C for 20 min.

2.2. Cultivation of L. brevis FB215 Using Sakekasu Extract

A portion of the L. brevis FB215 stock culture, stored at −80 °C with 50% glycerol, was streaked onto an MRS agar plate and cultured at 37 °C for 48 h to obtain colonies for the pre-culture. A single colony was inoculated into the MRS medium and pre-cultured at 37 °C for 24 h. The pre-culture was then inoculated into the Sakekasu extract at an initial OD₆₀₀ of 0.03 and cultured aerobically at 20 °C, 25 °C, 30 °C, or 37 °C until it reached the stationary phase.

2.3. High-Performance Liquid Chromatography (HPLC) Analysis of Putrescine and Histamine

Putrescine concentration was measured according to the following protocol [38]. An aliquot of 200 µL of the culture media was treated with 20 µL of 100% trichloroacetic acid to precipitate proteins. After centrifugation (18,700× g, 5 min), the supernatant was filtered through a Cosmonice W filter (Merck, Darmstadt, Germany) and subjected to HPLC analysis. The putrescine concentration was determined using an HPLC system (Chromaster, Hitachi, Tokyo, Japan) equipped with a cation-exchange column (#2619PH, 4.6 × 50 mm; Hitachi) maintained at 67 °C. Putrescine was eluted with mobile phase A (45.2 mM trisodium citrate, 63.3 mM sodium chloride, and 60.9 mM citric acid) and mobile phase B (200 mM trisodium citrate, 2 M sodium chloride, 5% ethanol, and 5% 1-propanol). The concentration of mobile phase B was increased linearly from 50% to 85% over 0–6 min, maintained at 85% for 6–12 min, increased to 100% over 12–18 min, maintained at 100% for 18–45 min, and returned to 50% over 45–60 min. The eluted putrescine was derivatized with o-phthalaldehyde in a post-column reaction. Derivatized putrescine was detected using a fluorescence detector at excitation and emission wavelengths of 340 and 435 nm, respectively. The derivatization reaction was initiated by combining reaction solution 1 (0.4 N NaOH) and reaction solution 2 (234 mM boric acid, 0.05% Brij-35, 5.96 mM o-phthalaldehyde, and 0.2% 2-mercaptoethanol) with the eluate at a temperature of 67 °C. The concentration of putrescine was determined from a standard curve generated using a known standard with a retention time of 15.2 min. Histamine dihydrochloride (FUJIFILM Wako, Osaka, Japan; product no. 081-03351) was used as the standard, and the retention time for histamine was 18 min.

2.4. Metabolomic Analysis Using Capillary Electrophoresis Time-of-Flight Mass Spectrometry (CE-TOF-MS) of the Culture Supernatant

Metabolome analysis was conducted using CE-TOF-MS by Human Metabolome Technologies, Inc. (Tokyo, Japan). Peaks detected using CE-TOF-MS were then extracted automatically using the MasterHands software (ver. 2.19.0.2, Keio University) with a signal-to-noise ratio threshold of 3 or higher. This yielded data for the mass-to-charge ratio (m/z), peak area, and migration time (MT). The resulting peak areas were converted into relative areas using the following formula:

relative area value = target peak area/internal standard area × sample volume

Given the presence of adduct ions, such as Na⁺ and K^+, along with fragment ions arising from dehydration or deamination, the ions associated with these molecular weights were excluded from the dataset. However, owing to the presence of substance-specific adducts and fragments, complete removal was not feasible. For the peaks that underwent detailed analysis, peak matching and alignment across samples were performed based on m/z and MT values. The detected peaks were compared against all entries in the Human Metabolome Technologies metabolite library using m/z and MT values. The tolerances for matching were set at ±0.5 min for MT and ±10 ppm for m/z, and the mass error (ppm) was calculated as follows:

mass error (ppm) = [(measured value − theoretical value) × 10⁶]/measured value

When a single candidate metabolite matched multiple peaks, a branch number was assigned to each peak. Qualification of the target metabolites was achieved by generating calibration curves using the peak areas that had been corrected by the internal standard. Each metabolite was then quantified using a single-point calibration at 100 µM (internal standard at 200 µM).

2.5. Quantification of Carbohydrates in the Culture Supernatant

Carbohydrate quantification was performed using a Carbohydrate Assay Kit [39] (Sigma, St. Louis, MO, USA; product no. MAK104-1KT). D-glucose standard (Sigma, product no. MAK104C) was diluted in deionized water to achieve concentrations of 0, 4, 8, 12, 16, and 20 µg/tube. A mixture of 200 µL of Assay Buffer (Sigma; product no. MAK104A) and 50 µL of diluted D-glucose standard or the sample was centrifuged at 13,000× g for 5 min. Next, 30 µL of diluted D-glucose standard or the supernatant was combined with 150 µL of concentrated sulfuric acid (Sigma; product no. 258105), followed by incubation at 90 °C for 15 min using a heat block (Takara Bio; product no. DS-TP350). Thirty microliters of developer (Sigma; product no. MAK104B) was then added to the reaction mixture and mixed thoroughly. The absorbance of the mixture was measured at 490 nm. A standard curve was constructed using the D-glucose standard, and the carbohydrate concentrations in the samples were determined accordingly.

2.6. Taste Evaluation Test

The samples tested were composed of Sakekasu extract fermented by L. brevis FB215 at various temperatures, as well as Sakekasu extract that had been fermented by L. brevis FB215, followed by supplementation with sucrose. These samples were evaluated by a sensory panel of 10 participants following a previously published method [40]. To ensure blinding, each sample was labeled with a unique three-digit code, and participants were provided with a glass of water to rinse their mouths between samples. The evaluation questionnaire covered six categories: appearance, odor, acidity, sweetness, aftertaste, and overall impression. Participants were asked to use a 9-point hedonic scale (1 = they strongly dislike it; 9 = they strongly like it) to rate each sample based on a scale developed to assess soldiers’ food preferences [40]. This study was conducted in accordance with the ethical standards of the Ethics Committee of the Faculty of Biology-Oriented Science and Technology at Kindai University (approval no. R6-1-006).

3. Results

3.1. Effects of Culturing Temperature on Bacterial Growth

To analyze the effect of culturing temperature on the growth and putrescine concentration of the culture supernatant, L. brevis FB215 was cultured in Sakekasu extract at 20 °C (Figure 1A), 25 °C (Figure 1B), 30 °C (Figure 1C), and 37 °C (Figure 1D). The maximum OD₆₀₀ values at 20 °C, 25 °C, 30 °C, and 37 °C were 1.8, 2.5, 2.9, and 1.7, respectively, reaching their maximum at 119, 83, 83, and 95 h of cultivation. The highest concentrations of putrescine of the culture supernatant of the Sakekasu extract fermented by L. brevis FB215 (Sakekasu extract fermented by FB215) at 20 °C (Figure 1A), 25 °C (Figure 1B), 30 °C (Figure 1C), and 37 °C (Figure 1D) were approximately 1000, 1300, 1400, and 1000 µM for 119, 71, 71, and 71 h, respectively.

3.2. Metabolites in Sakekasu Extract Fermented by FB215

The Sakekasu extract before inoculation, as well as the Sakekasu extract fermented by FB215 at 20 °C (Figure 2A), 25 °C (Figure 2B), 30 °C (Figure 2C), and 37 °C (Figure 2D) for 95 h (Supplementary Table S1), were subjected to CE-TOF-MS analysis. The concentrations of lactate, putrescine, and citrulline increased in the Sakekasu extract fermented by FB215 compared with the concentrations prior to fermentation at all temperatures. Sakekasu extract fermented by FB215 cultured at 20 °C, 25 °C, 30 °C, and 37 °C contained lactate at concentrations of 35, 49, 58, and 59 mM, respectively. Similarly, Sakekasu extract fermented by FB215 cultured at 20 °C, 25 °C, 30 °C, and 37 °C contained citrulline at concentrations of 0.1, 0.2, 0.4, and 0.6 mM, respectively. In contrast, the concentration of putrescine remained approximately 1 mM, regardless of the culture temperature (Figure 2A–D). The precursors of putrescine, agmatine (Supplementary Figure S1A), and ornithine (Supplementary Figure S1B) were initially present in the Sakekasu extract at concentrations of 90 and 917 µM, respectively. After 95 h of fermentation by L. brevis FB215, these concentrations decreased to a level of less than a few µM. Tyramine and histamine are biogenic amines that raise safety concerns in fermented foods [41]. The concentration of tyramine (Supplementary Figure S1C) increased by 2.6-, 3.1-, 3.0-, and 2.8-fold by fermentation at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, resulting in final concentrations of 1.3, 1.5, 1.5, and 1.4 µM. In contrast, histamine concentrations were analyzed using HPLC (Supplementary Figure S1D) and increased by only 1.0-, 1.0-, 1.1-, and 1.1-fold by fermentation at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, resulting in final concentrations of 16.6, 17.3, 18.5, and 20.3 µM. For the Sakekasu extract fermented by FB215 at 20 °C for 71 h, lactate was detected at concentrations of 2.4 mM (Supplementary Figure S2). This was merely 7% of the concentration reached after 95 h (34.8 mM) of fermentation at the same temperature. In contrast, the putrescine concentration in the Sakekasu extract fermented by FB215 at 20 °C for 71 h remained relatively high at 642 µM (Supplementary Figure S2, Supplementary Table S1), equivalent to 67% of the concentration after 95 h (960 µM).

3.3. Sensory Evaluation

Ten test subjects consumed Sakekasu extract fermented by FB215 at 20 °C, 25 °C, 30 °C, and 37 °C for 95 h. When comparing the overall palatability across all temperature conditions, there were no significant differences among the Sakekasu extracts fermented by FB215 (Figure 3A). Despite the temperature-dependent decrease in lactate concentration, there was no improvement in acidity preference (Supplementary Figure S3A). Furthermore, shifts in culture temperature did not influence the preference for appearance (Supplementary Figure S3B), odor (Supplementary Figure S3C), sweetness (Supplementary Figure S3D), or aftertaste (Supplementary Figure S3E).

To enhance palatability, sucrose was supplemented to Sakekasu extract fermented by FB215 at 20 °C for 29 h, containing 950 mg of putrescine and expected to contain a low concentration of lactic acid based on the results obtained thus far, to attain the same final sugar concentration as in a commercial sugar-sweetened lactic-acid-bacteria-fermented beverage and a commercial black vinegar beverage (Supplementary Figure S4). Consequently, the overall palatability of the Sakekasu extract fermented by FB215 was statistically equivalent to that of a commercial sugar-sweetened lactic-acid-bacteria-fermented beverage (Figure 3B), with an increased preference for sweetness (Supplementary Figure S5A) and aftertaste (Supplementary Figure S5B). The addition of sucrose did not affect preferences for appearance (Supplementary Figure S5C), acidity (Supplementary Figure S5D), or odor (Supplementary Figure S5E). However, the addition of sucrose to the beverage to match the sugar content of the commercial black vinegar beverage did not improve the overall palatability (Figure 3B). A comparison of the aftertaste between the Sakekasu extract fermented by FB215 without sucrose supplementation and a commercial black vinegar beverage revealed a significant difference, which disappeared upon sucrose supplementation (Supplementary Figure S6A). The appearance (Supplementary Figure S6B), odor (Supplementary Figure S6C), sweetness (Supplementary Figure S6D), and acidity (Supplementary Figure S6E) of the Sakekasu extract fermented by FB215 remained consistent when adjusted for the sugar content of the commercial black vinegar beverage, and the overall palatability was comparable to that of the commercial black vinegar beverage (Figure 3B).

4. Discussion

The concentration of putrescine in the culture supernatants of L. brevis FB215 cultured in Sakekasu extract increased markedly before the bacterial growth reached the growth phase (Figure 1A–D). Previous research has indicated that culturing human fecal samples at low pH levels results in elevated putrescine concentrations in the culture supernatant [42], suggesting that bacteria may export putrescine to the culture as a mechanism to counteract environmental acidification. However, in this study, despite variations in lactate concentration with changing culturing temperatures, the putrescine concentrations increased to approximately 1 mM at all culturing temperatures (Figure 2A–D), and concentrations of agmatine and ornithine, precursors of putrescine, decreased to below the detection limits or down to a few µM (Supplementary Figure S1A,B). These findings suggest that L. brevis FB215 may activate acid tolerance mechanisms, releasing putrescine at relatively low lactate concentrations or that a novel induction mechanism for putrescine export may be involved.

A daily intake of 500 mL of Sakekasu extract fermented by FB215 provided approximately 28 mg of polyamines, the same concentration as in a previous human intervention study [12], which reported an increase in blood polyamine concentrations. The amount of polyamines that can be extracted from 500 mL of Sakekasu extract fermented by FB215 is 66% of the daily polyamine intake of Europeans [43], 175% of that of Turks [17], 89% of that of Americans [16], and 107% of that of the Japanese [15].

The acceptable daily intake for tyramine and histamine, which are biogenic amines associated with safety concerns in fermented foods, is 800 and 50 mg, respectively [44]. In the event of the daily consumption of 500 mL of Sakekasu extract fermented by FB215, the intake of tyramine and histamine would be approximately 103 µg and 1.1 mg, respectively (Figure 2A–D, Supplementary Figure S1C,D). This intake represented only 0.02% and 2.2% of the acceptable daily intakes for tyramine and histamine, respectively.

The findings of the experiment evaluating the impact of the culture temperature on beverage palatability demonstrated that the taste and other attributes of Sakekasu extract fermented by FB215 above the optimum temperature were only marginally influenced by the culture temperature (Figure 3A, Supplementary Figure S3A–E). Despite a decline in lactate concentration with reduced culture temperatures (Figure 2A–D), there was no enhancement in acidity preference (Supplementary Figure S3A). This suggests that participants may have favored the pronounced acidity level present in each of the culture supernatants tested. The sugar concentrations of commercially available sugar-sweetened lactic-acid-bacteria-fermented and unsweetened acetic acid beverages were measured (Supplementary Figure S4), and sucrose was added to the Sakekasu extract fermented by FB215 at 20 °C to match the sugar concentration of the sugar-sweetened commercial beverage, thereby enhancing its sweetness. This adjustment resulted in a significant enhancement in overall palatability (Figure 3B, Supplementary Figure S5A–E), approaching the level of appeal exhibited by commercial products. The most significant enhancement was observed in preferences for sweetness and aftertaste (Supplementary Figure S5A,B), underscoring the pivotal role these attributes play in the comprehensive assessment of the beverage. Conversely, the addition of a sugar level analogous to that of the unsweetened acetic acid beverage with a low sugar concentration did not enhance overall palatability or sweetness preference, although it did improve aftertaste preference (Figure 3B, Supplementary Figure S5A,D). This finding indicates that the masking effect on aftertaste can be achieved with a lower sugar addition (43 g/L), but a relatively higher sugar level (160 g/L) is necessary to enhance sweetness.

5. Conclusions

In this study, we developed Sakekasu extract fermented by FB215, which contains 1 mM of polyamines. L. brevis FB215 accumulated high concentrations of lactic acid and citrulline in the Sakekasu extract fermented by FB215 in a temperature-dependent manner. Conversely, putrescine was accumulated to approximately 1 mM under all culture temperature conditions, accompanied by the nearly complete consumption of its precursors, agmatine and ornithine. Tyramine and histamine, which are recognized as potentially compromising the safety of fermented foods, were detected in negligible amounts in the Sakekasu extract fermented by FB215, with concentrations below levels deemed to be harmful to human health. Furthermore, the addition of sucrose to the Sakekasu extract fermented by FB215 cultured at 20 °C for 29 h—adjusted to match the sugar content of a commercial sweetened lactic-acid-bacteria-fermented beverage—significantly improved its palatability.