Comparison of Aroma and Taste Profiles Between Two Fermented Pea Pastes Using Intelligent Sensory Analysis and Gas Chromatography–Mass Spectrometry

Abstract

The traditionally produced pea paste (PP) suffers from suboptimal flavor and inferior quality. Based on the study of single-strain fermentation, we further selected S. cerevisiae, Z. rouxii, and L. paracasei for PP production by dual-strain fermentation (SL, ZL). By combining intelligent sensory technology, gas chromatography–mass spectrometry (GC-MS), and ultra-high-performance liquid chromatography (UPLC) technology, the aroma and taste characteristics of SL- and ZL-fermented PP were compared. The electronic nose and tongue revealed the differences in the aroma and taste characteristics between the two fermentation methods for fermenting PP. In total, 74 volatile compounds (VOCs) in PP were identified through GC-MS analysis. In contrast, the number of VOCs and the concentrations of alcohols and acids compounds in SL were higher than in ZL. Among the 15 VOCs that were common to both and had significant differences, the concentrations of ethanol, 1-pentanol, and ethyl acetate were higher in SL. For taste characteristics, SL demonstrated significantly higher levels of sweet and bitter amino acids, as well as tartaric acid, compared with ZL. These results elucidate the flavor differences of dual-strain fermented PP, providing a theoretical basis for selecting suitable strains for fermenting PP.

1. Introduction

Peas are rich in nutrients and contain essential protein, starch, and vitamins. They are also one of the main edible beans in China and have been widely welcomed by consumers [1,2]. Pea paste (PP), a distinctive sauce from Sichuan, China, is made from peas through the processes of soaking and high-pressure steaming. PP serves as an indispensable ingredient in traditional dishes, including rice with bean soup, noodles with mixed pea sauce, and fish in pea soup. Currently, traditional bean PP is produced in small batches for home use or small-scale operations, leading to inconsistent product quality. A simple steamed and boiled PP lacks flavor, seriously affecting the consumer’s appetite. This has brought challenges for the development and utilization of the food industry. Therefore, improving the flavor of PP is crucial for further enhancing its overall product quality.

To enhance the flavor profile of food, researchers have increasingly investigated the incorporation of microorganisms utilized in fermentation to improve quality [3]. For example, when three strains of lactic acid bacteria were mixed and inoculated during fermentation of prune wine, the concentration of key aromatic compounds such as isoamyl acetate, ethyl heptanoate, and methyl heptanoate in the final product were significantly increased. This suggests that Lactobacillus inoculation positively enhances prune wine’s flavor [4]. Xiao et al. inoculated Pichia spp. strains into chili peppers and found that the yeast accelerated the fermentation process of chili peppers. Amino acids and their derivatives, flavonoid glycosides, carbohydrates, and carbohydrate conjugates were the main differential metabolites in the samples cultured with Pichia spp. starters [5]. Furthermore, the rich protein and starch content in peas provide an energy source for the growth of microorganisms.

It is worth emphasizing that monoculture fermentation has obvious limitations in practical applications and cannot efficiently achieve fermentation with complex substrate compositions and biochemical processes [6]. In contrast, the mixed fermentation approach remarkably augments the capacity to adapt to environmental changes and ameliorates the flavor as well as the nutritional content of fermented products, thereby significantly enhancing the sensory qualities and taste of food [7]. In the study by Li et al., the co-fermentation of two strains, namely P. kluyveri and L. plantarum, had a significant impact on the biochemical transformation and sensory characteristics of plant-based beverages [8]. Consequently, the artificial inoculation of beneficial microorganisms in fermentation not only reduces the fermentation time of the product, but also enhances the flavor quality. However, to date, there have been no reported studies that have employed the dual-strain mixed fermentation method to enhance the quality of PP, particularly regarding flavor.

The flavor of PP is mainly determined by its aroma and taste characteristics and is one of the most important indicators for evaluating its quality [9]. The main methods for aroma detection currently include the E-nose and gas chromatography–mass spectrometry (GC-MS), which was commonly used for volatile components analysis in fermented foods due to its excellent sensitivity, efficiency, and material separation and identification capabilities [10]. Li et al. utilized an electronic nose in conjunction with GC-MS technology to analyze the differences in volatile compounds between two fermented chili pastes, confirming the significant contributions of pyrazine, 2-methoxy-3-(2-methylpropyl), and linalool compounds to the chili paste’s aroma [11]. In addition to volatile compounds, various non-volatile compounds such as organic acids and amino acids also play a significant role in influencing the taste characteristics of PP. However, the complex taste components in PP present challenges in comprehensively capturing its unique taste characteristics by using a single detection method, so it brings great challenges in detecting the taste of PP using a single analysis method. E-tongues, when combined with amino acids and organic acids, have great advantages in detecting the texture of foods. Fortunately, in Cai et al.‘s study, a method combining the E-tongue, amino acids, and organic acids demonstrated great potential for application in taste detection of foods [12].

In this research, building on the findings of previous studies with individual strains, we conducted further fermentation using a combination of Saccharomyces cerevisiae, Zymomonas rouxii, and Lactobacillus paracasei to produce fermented PP (SL, ZL). For this purpose, we employed intelligent sensory technology, gas chromatography–mass spectrometry (GC-MS), and ultra-high-performance liquid chromatography (UPLC) to compare the aroma and taste profiles of SL- and ZL-fermented PP. The objective of this study was to investigate the flavor differences in dual-strain fermented PP and to identify a more suitable dual-strain for PP fermentation, thereby providing valuable references for enhancing the flavor of fermented PP.

2. Materials and Methods

2.1. PP Production

The strains and media were provided by the Key Laboratory of Science of Sichuan Tourism University. Saccharomyces cerevisiae (CICC 1239) and Zygosaccharomyces rouxii (CICC 1239) were activated and cultured in PDA medium (30 °C, 48 h). Lacticaseibacillus paracasei (CICC 20241) was cultured in MRS medium at 37 °C for 24 h. Single colonies were selected and cultured in liquid medium, and the suspension’s concentration was adjusted to 1.0 × 10⁸ CFU/mL. We placed 300 g of double boiled white peas (Beijing, China, Jinhe Luyuan Agricultural Technology Co., Ltd.) in a conical bottle and added distilled water according to the ratio of material to liquid of 1:0.9. The peas were steamed at 121 °C for 15 min, and then cooled to PP. According to previous experiments, Saccharomyces cerevisiae was combined with Lactobacillus paracasei (SL), Zygosaccharomyces rouxii, and Lactobacillus paracasei (ZL) in a composite formulation. The ratio of the composite strain to the individual strain was maintained at 10:1, with an inoculation rate of 2%. The best flavor was obtained after solid fermentation for 5 days at 30 °C in a culture box. After fermentation, the samples were stored at −60 °C.

2.2. Intelligent Sense Analysis of PP

2.2.1. E-Nose Analysis

Eighteen sensors in the E-nose (FOX 4000, A MOS, Toulouse, France) are sensitive to different volatile gases. PA/2, P30/1, P30/2, TA/2, and LY2/AA are sensitive to organic compounds, LY2/LG, P40/1, P40/2, T40/2, and T40/1 are sensitive to high oxidizing gases; LY2/G and LY2/gCTI are sensitive to amines; and LY2/Gh is selective for aniline-type compounds. LY2/gCT is sensitive to alkanes and aromatic components, T30/1 is sensitive to polar compounds, and P10/1 is sensitive to non-polar compounds. P10/2 is sensitive to alkanes, and T70/2 is sensitive to aromatic compounds. Sample analysis was conducted following Wu et al.’s method with slight modifications [13]. Specifically, 2 g of evenly broken PP was placed into a 10 mL headspace bottle for the electronic nose test and covered with a polyethylene cap film. Before detection, it was incubated and balanced at 60 °C for 5 min, then 1.5 mL of gas was manually injected into the electronic nose analyzer. To minimize error, each sample was repeated 10 times, and the last 3 stable measurements were selected for analysis.

2.2.2. E-Tongue Analysis

The E-tongue (α-Astree, Alpha MOS, Toulouse, France) has seven potentiometric sensors that are particularly sensitive to sweetness (ANS), saltiness (CTS), umami (NMS), sourness (AHS), bitterness (SCS), and two reference electrodes (PKS and CPS). The combined information from these seven sensors was used to study the taste characteristics of PP. With reference to Deng et al.’s method and modifying it, PP was mixed with pure water at a ratio of 1:10 before crushing [10]. The clarified solution of PP was obtained for electronic tongue analysis. The measurement conditions of the E-tongue were set as follows: the signal acquisition time was 120 s, the stirring speed was 60 r/min, and the process of analysis was 3 min. Additionally, after each analysis and before measurement, the metal probe attached to the sensor was thoroughly cleaned with deionized water for 1 min to remove residues that may have affected subsequent measurements, thus ensuring accuracy and reliability. To minimize error, each sample was repeated 10 times, and the last 3 stable measurements were selected for analysis [12].

2.3. Free Amino Acid Analysis

The free amino acids of PP were determined by an automatic amino acid analyzer (S-433D, SYKAM, Munich, Germany). Separation was carried out using a strong acidic cation exchange resin column based on sulfonic acid (LCA K07/Li 150 mm × 4.6 mm). The specific determination method was based on the method of Cai et al. with modifications [12]. For this, 3 g of PP was mixed with 15 mL of sulfobromophthalein (7 g/100 mL) under ultrasonic vibration for 30 min to extract the compounds. Then the supernatant was centrifuged at 10,000 rpm for 15 min, and 2 microliters was collected for detection. The retention time and peak area of individual amino acid standards (Sigma-Aldrich, St. Louis, MO, USA) were used for identification and quantification. The measurements were carried out 3 times to ensure accuracy.

2.4. Organic Acid Analysis

The organic acids in PP were determined by high-performance liquid chromatography (1260, Agilent, Santa Clara, CA, USA) on a C18 column (4.6 mm × 250 mm, 5 μm) with the method of determination of organic acids in food (GB 5009.157-2016 [14]). Precisely, 5 g of PP was weighed and added to a 50 mL volumetric bottle with pure water and soaked in a 75 °C water bath for 20 min, and 2 μL of the filtered supernatant was collected for detection. The mobile phase was a 0.1% phosphoric acid–methanol solution, with a flow rate of 1.0 mL/min, a detection wavelength of 210 nm, and a column temperature of 40 °C. The mixed organic acid standard’s retention time and peak area (Shanghai Yuanye Technology Co., Ltd., Shanghai, China) were compared for identification and quantification. The measurements were carried out 5 times to ensure accuracy.

2.5. Analysis of Aroma Components

The aroma components of PP were detected by gas chromatography and mass spectrometry (30 m × 0.25 mm × 0.25, Elite-5MS) (Perkin Elmer Instruments Inc., Waltham, MA, USA). We modified Chen et al.‘s method slightly [15]. Specifically, 4 g of PP was weighed into a headspace bottle and sealed with a polyethylene cap film. Then the SPME fiber coated with DVB/CAR/PDMS was extracted at 70 °C for 30 min with a helium flow rate (99.99%) of 1.5 mL/min. The oven temperature was initially set at 40 °C and then linearly increased to 180 °C at a rate of 4 °C/min. After being kept at 180 °C for 3 min, it was further heated to 230 °C at a speed of 5 °C/min for 5 min. The carrier gas was helium (purity > 99.999%), and the flow rate was 1.0 mL/min. The electron energy of the electron ionization source was set to 70 eV, the ion source temperature was 250 °C, and the temperature of the transmission line was 230 °C. In the full scan mode of the ionization ion source, the mass scan range was set to 45–450 m/z. The retention indices of these compounds relative to standard alkanes (C8–C26) were calculated by comparing the experimental mass spectra with the NIST 11 USA mass spectrometry library. The results were compared with those in the database and could only be determined when the match of the compound was greater than 800 (up to 1000). The measurements were carried out 3 times to ensure accuracy.

Based on the characterization of the compounds’ concentration via gas chromatography and mass spectrometry, the threshold value for each aroma compound in the sample was determined by referring to Xu et al.’s research methodology [16]. The relative odor activity value (ROAV) of each aroma compound was calculated as follows:

$$\mathrm{R}\mathrm{O}\mathrm{A}\mathrm{V}=100\times {\displaystyle \frac{{\mathrm{C}}_{\mathrm{i}}}{{\mathrm{C}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}}\times {\displaystyle \frac{{\mathrm{T}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}{{\mathrm{T}}_{\mathrm{i}}}}$$

In this formula, C_i is the relative content of the aroma compound the in samples (%), T_i is the aroma threshold of the compound in food (μg/kg), and C_max and T_max represent the relative content and aroma threshold of the compound that contributes the most to the overall flavor of the sample, respectively. For all compounds, ROAV ≤ 100; a higher ROAV value indicates a more significant contribution of the component to the overall flavor profile of PP [17,18].

2.6. Statistical Analysis

Statistical analysis used the R language. In addition, prior to univariate analysis, the data distribution was transformed to achieve normality according to the methods of Box and Cox [19]. The T-test was used to determine the significant differences between the groups, and the significance level was p < 0.05. To obtain a comprehensive understanding of the data, the robust principal component analysis (rPCA) model was established, based on the molecular concentration. For each rPCA model, the loading score map and Pearson correlation map were calculated to highlight the structure of the data and identify the relationships between variables and the model’s constituents.

3. Results

3.1. Intelligent Sensory Analysis of PP

To analyze the overall aroma and taste characteristics of fermented PP from SL and ZL, we utilized response values from intelligent sensory devices, including E-nose and E-tongue sensors, to construct an rPCA model. In Figure 1a of the rPCA model of the E-nose, PC 1 accounted for 91.5% of the sample variance. In the E-tongue’s rPCA model, PC 1 accounted for 93.8% of the sample variance. The rPCA model effectively summarized the overall features of the sample. The results of the E-nose indicated that the SL and ZL samples exhibited distinct positional characteristics in PC 1. Furthermore, in the Pearson correlation plot (Figure 1b) of PC 1, the response values of SL for LY2/gCT, LY2/LG, P30/1, TA/2, P30/2, and T40/2 were found to be higher. The response values of ZL for LY2/Gh and T30/1 were also higher. It is worth noting that in the map of rPCA results for the E-tongue (Figure 1c), the SL and ZL samples also showed a significant degree of separation. In the PC 1 correlation graph (Figure 1d), the ANS, CPS, NMS, and PKS responses of the SL samples were higher. In contrast, the response values of SCS and CTS in the ZL samples were higher.

3.2. Analysis of the Aroma Profile in PP

In order to obtain the aroma components of the SL and ZL samples, GC-MS was used to further analyze the volatile compounds of PP. In total, 84 molecules were identified, and the specific molecular information is shown in Table S1. These included 28 alcohols, 5 aldehydes, 16 acids, 15 esters, 5 ketones, 8 heterocycles, and 7 others.

As shown in Figure 2a, 47 and 44 volatile compounds were found in SL and ZL, respectively, and 17 compounds were common to them. Among the shared compounds’ categories, alcohols were the most numerous, with a total of seven types, followed by esters, aldehydes and acids. Specifically, isopropyl alcohol and 1-pentanol had the highest content and were therefore the main source of the aroma in PP. Figure 2b shows a significant difference in the volatile compounds between SL and ZL in terms of categories (p < 0.05), including alcohols, aldehydes, esters, and ketone compounds. Specifically, compared with ZL, SL exhibited higher abundance in terms of the number and concentration of alcohols. Interestingly, the esters and ketones of ZL were significantly higher than those of SL (p < 0.05). As for the acids, only three more acid compounds were found in ZL than in SL. To comprehensively and visually summarize the differences between the important shared volatile compounds in SL- and ZL-fermented PP, an unsupervised rPCA model was used to calculate the concentrations of the 14 volatile organic compounds in a Venn diagram (Figure 2a). As shown in Figure 3a, PC 1 accounted for 98.2% of the total PCA, with the ZL and SL samples occupying the positive and negative sides of the number axis, effectively distinguishing the overall information of the samples. In the loading map (Figure 3b), higher concentrations of ethanol, 1-pentanol, ethyl acetate, and hexanal were observed in the SL sample compared with the ZL sample. In contrast, the concentrations of r-1,2-propanediol, dimethyl ether, 1,2-dimethoxyethane, ethylboronic acid, ethyl lactate, 3-chlorobutan-2-ol, methyl formate, 4-penten-2-ol, 2-methoxy-acetaldehyd, 1-methoxy-2,3-epoxypropane, and 5-methyl-5-hexen-2-ol were higher in the ZL samples. Among them, ethyl acetate and ethyl lactate had relative odor activity values (ROAVs) higher than 1, so they contributed significantly to the fragrance characteristics of PP. Meanwhile, hexanal, ethanol, and 1-pentanol exhibited ROAVs less than 1 but greater than 0.01, which were of great importance in modulating the aroma characteristics of PP, as shown in

Table 1. ROAVs of VOCs in SL and ZL.

Compound #	CAS	Threshold Value (mg/kg)	Descriptor	ROAV
				ZL	SL
Ethyl isobutyrate	97-62-1	0.00011	Apple, floral, cream	43.05	<0.01
Isoamyl acetate	123-92-2	0.13	Apple, banana	0.1	<0.01
Isobutyl acetate	110-19-0	0.038	Apple, banana, floral	0.03	<0.01
Ethyl acetate	141-78-6	0.005	Fruit, grape	22.72	17.29
Ethyl lactate	97-64-3	0.014	Rum, creamy	0.03	11.27
Methyl formate	107-31-3	325	Fruit, wine	<0.01	<0.01
Ethyl butyrate	105-54-4	0.000053	Anise, pineapple	19.49	<0.01
Ethyl propionate	105-37-3	1.1	Rum, pineapple	<0.01	<0.01
Ethyl 3-methylvalerate	5870-68-8	0.000008	Pineapple	100	<0.01
Acetone	67-64-1	59	Ether, hay	<0.01	0.04
Acetoin	513-86-0	0.014	Butter, cream	0.02	<0.01
2-Pentanone	107-87-9	0.35	Paraffin, orange peel	<0.01	0.29
2-Heptanone	110-43-0	0.023	Sweet pepper, cinnamon	<0.01	3.07
Styrene	100-42-5	0.068	Balsam	0.01	<0.01
Glutaric acid	110-94-1	24	/	<0.01	<0.01
Formic acid	64-18-6	0.98	Spicy	1.08	6.04
Glycine	56-40-6	4630	/	<0.01	<0.01
Propionic acid	29102	0.05	Raspberry	<0.01	<0.01
Benzoic acid	65-85-0	1	Pungent, sour odor	<0.01	<0.01
3-Hydroxybutyric acid	625-71-8	42	Butter	<0.01	<0.01
Hexanal	66-25-1	0.005	Grassy, apple flavor	0.06	0.22
Ethylbenzene	100-41-4	0.026	Aromatic	<0.01	<0.01
Dimethyl ether	115-10-6	430	Sweet, pungent odor	<0.01	<0.01
3-Methyl-1-butanol	123-51-3	0.01	Banana, cheese	<0.01	<0.01
2-Methyl-1-propanol	78-83-1	0.033	Alcohol, cocoa, malt	1.86	<0.01
Isopropyl alcohol	67-63-0	0.065	Fruit, grape	20.5	100
Ethanol	64-17-5	100	Wine, floral	0.01	<0.01
Propylene glycol	57-55-6	16	Orange	<0.01	<0.01
3-Methyl-2-butanol	598-75-4	0.41	/	<0.01	<0.01
2-Butanol	78-92-2	0.66	Wine aroma	0.02	<0.01
2,4-Pentanediol	625-69-4	9.6	/	<0.01	<0.01
2,3-Butanediol	513-85-9	0.02	Creamy, floral	0.03	<0.01
1-Pentanol	71-41-0	0.36	Almonds, balsamic	0.68	0.33
Amino-2-propanol	78-96-6	28	/	<0.01	<0.01

The “/” symbol in the table indicates that no odor description information was found for this compound. ^# ROAV > 1 indicates that the compound contributes significantly to the aroma profile of the sample. 1 > ROAV > 0.01 indicates that the compound had a modulating effect on the aroma profile of the sample.

.

3.3. Analysis of Free Amino Acids in PP

According to the evaluation of the electronic tongue for the taste of PP, the 17 important amino acids in SL and ZL were analyzed separately using an amino acid analyzer. The free amino acid content of PP is shown in

Table 2. The content of free amino acids in PP was characterized by an amino acid analyzer (mean ± SD).

Compounds	CAS	Content (mg/kg)		Taste Characteristics
		SL	ZL
Asp	6899-03-2	166.96 ± 8.46 a	83.40 ± 3.40 b	Umami
Glu	56-86-0	511.04 ± 10.56 b	577.17 ± 6.14 a	Umami
Pro	609-36-9	73.09 ± 4.62 a	87.15 ± 4.10 a	Sweet
Gly	56-40-6	115.48 ± 4.40 a	100.28 ± 1.81 b	Sweet
Thr	72-19-5	68.72 ± 4.34 a	39.27 ± 1.55 b	Sweet
Ser	302-84-1	187.98 ± 7.18 a	58.89 ± 1.71 b	Sweet
Asn	132388-64-8	55.01 ± 2.75 b	123.04 ± 3.16 a	Sweet
Ala	338-69-2	223.06 ± 4.83 a	111.81 ± 2.26 b	Sweet
Val	7004-03-7	128.86 ± 7.42 a	118.38 ± 2.98 a	Sweet
Met	63-68-3	29.61 ± 1.49 a	8.96 ± 0.85 b	Bitter
Ile	73-32-5	36.68 ± 2.95 a	10.09 ± 0.38 b	Bitter
Leu	3588-60-1	89.54 ± 4.55 a	29.42 ± 2.61 b	Bitter
Tyr	70642-86-3	49.30 ± 4.47 a	22.36 ± 0.57 b	Bitter
Phe	673-31-4	68.55 ± 4.74 a	46.47 ± 1.42 b	Bitter
His	71-00-1	19.11 ± 1.11 b	25.75 ± 2.32 a	Bitter
Lys	56-87-1	87.01 ± 3.80 a	76.13 ± 1.29 b	Bitter
Arg	74-79-3	963.99 ± 25.52 a	1008.03 ± 5.41 a	Bitter

Different lowercase letters indicate statistically significant differences between compounds according to Tukey’s HSD test (p < 0.05).

. To show the differences in the free amino acid composition between SL and ZL, an unsupervised rPCA model was established based on their content. In the PCA score plot shown in Figure 4a, PC 1 accounts for 97.1% of the total sample variance and explains most of the differences between the two samples, with the two samples clearly located on opposite sides of the number axis.

In addition, the differences in the amino acid composition between SL and ZL are successfully depicted in the load map in Figure 4b. Compared with SL, the samples of ZL had higher contents of Asn, Glu, His, Lys, and Gly. In contrast, the highest concentrations of Thr, Tyr, Asp, Lle, Leu, and Ala were detected in the SL sample. Free amino acids are both nutrient components and taste components in PP. The main free amino acid tastes of SL and ZL were classified, mainly including umami, sweet and bitter flavors. Interestingly, there were significant differences in the sweet and bitter flavors of PP after inoculation (p < 0.05). Compared with ZL, SL had a higher content of sweet and bitter amino acids (Figure 4c).

3.4. Analysis of Organic Acids in PP

The concentrations of six organic acids in PP were analyzed using HPLC, including tartaric acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid. Table 2 provides specific information on the organic acids in PP. An rPCA model was established based on the organic acid information detected by HPLC, as shown in Figure 5, to effectively demonstrate the differences between ZL and SL in terms of organic acids. According to the information captured by PC 1 in PCA, PC 1 accounted for 93.8% of the total variance (Figure 5a). Specifically, ZL and SL were effectively separated and successfully distributed on the positive and negative half-axes of PC 1. The differences observed are shown in Figure 5b, where the contents of lactic acid, tartaric acid, and citric acid were higher in SL, while the contents of malic acid, succinic acid, and acetic acid were higher in ZL. There was a significant difference in the content of succinic acid, tartaric acid, acetic acid, and citric acid (Figure 5c).

3.5. Correlation Analysis of Flavor and Taste

The E-nose and GC-MS instrument can analyze PP from the perspective of volatile components. The E-nose can provide the overall odor profile properties, while GC-MS can provide a complete analysis of the molecular characteristics of aroma compounds. Furthermore, the E-tongue can comprehensively evaluate the sensory characteristics of PP and highlight the amino acids that had a direct impact on the taste attributes by combining information on amino acids and organic acids. As shown in Figure 6a, the E-nose sensors T30/1, P10/1, and P40/1 exhibited positive correlations with methyl formate, 4-penten-2-ol, 2-methoxy-acetaldehyd, ethylboronic acid, dimethyl ether, ethyl lactate, 1,2-dimethoxyethane, 3-chlorobutan-2-ol, r-1,2-propanediol, and 1-methoxy-2,3-epoxypropane. In addition, these compounds were negatively correlated with the LY2/LG, P30/1, P40/2, P30/2, T40/2, T40/1, and TA/2 sensors, but ethanol, 1-pentanol and ethyl acetate were positively correlated with these sensors. As seen in Figure 6b, there was a correlation between amino acids and organic acids and the response of the E-tongue sensor. Specifically, lactic acid, succinic acid, Asn, and His were positively correlated with the E-tongue sensors PKS, CTS, NMS, CPS, ANS, and SCS, while Leu, Met, Ser, Asp, Ile, Tyr, and Thr were negatively correlated.

4. Discussion

As one of the top two edible legumes in our country, peas play an important role in daily diets and are also an important cereal crop [1]. Consumers often process peas into pea paste and, in culinary applications, they frequently combine peas with other ingredients to enhance both the taste and nutritional value of various dishes. However, PP made by simple boiling or stewing has the problems of monotonous taste and inferior flavor, which significantly hamper the development and utilization of the food industry. Therefore, improving the flavor of PP is of great importance for promoting its processing and utilization. Currently, leveraging the interactions among microorganisms to enrich the complexity of food flavors has emerged as a popular strategy for improving food quality [20].

Yeast and lactic acid bacteria are common microorganisms that play an important role in the formation of the flavor of fermented foods [7,21]. Concurrently, dual-strain fermentation has been shown to effectively facilitate fermentation processes involving complex substrate compositions and intricate biochemical pathways [22]. Previous research has demonstrated the efficacy of dual fermentation involving edible Saccharomycetes and probiotics to markedly enhance the flavor of Rosa roxburghii Tratt and coix seed beverages, thus meeting the demand for high-quality food [6]. To this end, the incorporation of lactic acid bacteria can establish a mixed fermentation system, where the synergistic interaction between lactic acid bacteria and pure yeast fermentation yields a more diverse array of metabolic products and enhanced efficiency compared with pure yeast fermentation alone [23,24]. Building on previous studies on the fermentation of PP with a single strain, this study further utilized a combination fermentation method with Saccharomycetes, Zygosaccharomyces rouxii, and Lactobacillus paracasei to produce PP (SL, ZL) [25]. The objective was to deepen the theoretical understanding of the differences in PP production through dual-strain fermentation and to identify a more suitable dual-strain combination that enhances the economic value of PP fermentation, thereby providing a solid theoretical basis.

In examining the flavor profile of PP, we initially utilized advanced sensory systems that incorporated electronic nose and electronic tongue technologies. These systems demonstrated remarkable discriminatory capabilities in identifying the aroma and taste characteristics of two fermented PP samples. Fermentation had the most substantial impact on the volatile aroma of PP, and the type of aroma compounds described by the E-nose sensor showed that the type of aroma compounds that had the greatest impact on SL were aromatic compounds. For ZL, the main type of volatile odors were highly oxidizing, polar compounds. Free amino acids, derived from protein degradation, not only exerted a significant impact on flavor but also served as precursors for key volatile flavor compounds that were closely associated with product quality [26,27]. With regard to amino acids, 17 important free amino acids were identified, all of which were found in legume plants [28]. In terms of the taste profile, SL exhibited higher sweetness levels while ZL demonstrated more pronounced bitterness and saltiness, which can influence the perceived quality of the PP. Fermented pea paste was found to contain abundant bitter amino acids, whereas umami and sweet amino acid contents were comparatively lower, consistent with the findings reported by An et al. [29]. Research has reported that Saccharomyces cerevisiae can secrete proteases during fermentation, breaking down the proteins in legumes into amino acids and small peptides, which are important sources of umami flavors in foods [30]. The umami taste in PP primarily originates from the amino acids Asp and Glu, while the sweet-tasting amino acids Gly, Ser, Ala, and Val play a crucial role in determining complexity and flavor balance [31]. Additionally, Lys and Arg influence bitterness. Among these, glutamic acid is recognized as the predominant free amino acid in legume plants, making legume plants an ideal raw material for fermented food products [32]. Through the fermentation process, the substances that enhance freshness in PP can be further released and intensified.

On the other hand, organic acids are also important taste components in PP [9]. The organic acids present in fermented legumes are mainly generated through microbial metabolism, enzymatic degradation of raw materials, and enzymatic reactions, and the fermentation strain has a significant impact on these organic acids. This is because different strains of bacteria differ in their utilization of carbon sources, which, in turn, affects the metabolism of organic acids [33]. In the mixed-fermentation PP, succinic acid was the main organic acid, with a characteristic sour and savory flavor, playing an important role in PP [34]. Acetic acid is produced by the oxidation of ethanol and has a mildly stimulating effect, which can regulate the flavor of fermented foods [35]. Tartaric acid is naturally present in many plants and is one of the main acids found in wine. In fermented zha chili pepper, tartaric acid is also one of the main organic acids [31]. In the PP fermented by two different mixed strains, the content of tartaric acid and citric acid in SL was significantly higher than that in ZL (p < 0.05). During the co-fermentation process, S. cerevisiae and L. paracasei may promote each other’s metabolic activities, especially in the production of citric acid and tartaric acid. Additionally, Z. rouxii may be more inclined to generate other types of organic acids through its metabolic pathway under similar metabolic conditions. This also partially explains the higher concentration of succinic acid in ZL. In addition to amino acids and organic acids, other secondary metabolites such as flavonoids, phenolic compounds, terpenoids, and alkaloids also make important contributions to the taste of plant-based foods [36]. Some compounds are present in flavonoids, which give plant-based foods a bitter taste and thus enhance their flavor [37]. Consequently, future research should also focus on the characteristics of the non-volatile metabolic compounds in PP.

The primary volatile compounds identified in this study encompass alcohols, esters, aldehydes, and acids. The alcohol content in PP mainly comes from the fermentation process, primarily facilitated by lactic acid bacteria and yeast during the fermentation process of sugar and starch [9]. These microorganisms break down the sugars (such as glucose) in peas through the glycolytic pathway, thereby increasing the variety and content of alcohols [38]. This process adds a rich taste and flavor to PP. Notably, isopropyl alcohol, ethanol, and 1-pentanol were the alcohol aroma compounds with the highest concentration in PP. Isopropyl alcohol imparts a fruity sweet fragrance, such as grapefruit fragrance [39]. Ethanol is an essential class of volatile organic compounds in fermented products, not only because they can bring pleasant aromas by themselves, but also because they help enhance the effects of other aroma components [40]. 1-Pentanol has been described as having a vinegar-like flavor and a fusel oil taste, which are produced through the degradation of amino acids via the Strecker reaction to generate the corresponding aldehydes, followed by reduction [41]. It has been reported that this compound balances the flavor profile of wine and enhances the overall sensory experience, contributing to both taste and aroma [42]. The content of isopropyl alcohol and 1-pentanol in SL was higher than that in ZL, so the fruit aroma and vinegar taste were more pronounced in SL than in ZL. The esters produced by fermented PP mainly include ethyl acetate, ethyl lactate, and methyl formate. Esters are compounds formed by the esterification of low-level saturated fatty acids and saturated fatty alcohols, derived from the esterification of acid with alcohol [43]. The threshold of esters was found to be lower [44]. The majority of esters possess a fruity fragrance, thereby imparting a pleasant fruity taste to the fermentation products [45]. Aldehydes are the degradation products of lipid oxidation during fermentation and are related to the metabolic products of saccharomycetes [46]. The aldehyde substances in fermented PP include hexanal and 2-methoxy-acetaldehyde. It is worth noting that the combined use of multiple analytical instruments can significantly enhance the evaluation of the aroma and taste characteristics of PP fermented by dual strains. Some electronic nose sensors, namely LY2/LG, T30/1, P10/1, P40/1, P30/1, P40/2, P30/2, T40/2, T40/1, and TA/2, demonstrated significant positive correlations with the aroma compounds. Meanwhile, electronic tongue sensors, including PKS, CTS, NMS, CPS, ANS, and SCS, exhibited significant positive correlations between amino acids and organic acids in terms of taste. These findings may serve as potential candidates for the development of smart sensors (electronic nose and electronic tongue) for the comprehensive analysis of PP samples.

5. Conclusions

To the best of our knowledge, this study aimed to utilize a diverse array of techniques to elucidate the effects of dual-strain fermentation on the flavor profile of PP. The application of mixed fermentation has been found to effectively enhance the richness of PP flavor profile. The E-nose and E-tongue exhibited high recognition capabilities in identifying and distinguishing among fermented PP samples. The primary aromatic compounds identified in fermented PP included alcohols, aldehydes, acids, and esters. Compared with ZL, SL more effectively imparted beneficial aromas to PP, such as floral and vinegar-like aromas. In terms of taste, SL exhibited higher sweetness levels, whereas ZL demonstrated more pronounced bitterness and saltiness. The umami flavor in PP primarily derives from the amino acids aspartic acid and glutamic acid, whereas the sweet flavor predominantly originates from glycine, serine, alanine, and valine. Overall, SL fermentation proved to be more effective than ZL fermentation in enhancing the richness and complexity of PP’s flavor profile. This study provides valuable insights for improving the enhancement of quality and flavor of PP.