Effect of mixed fermentation of Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus on physicochemical attributes, volatile compounds and functional properties of pear juice

Abstract

This study investigated the fermentation of pear juice by a mixed culture of lactic acid bacteria (LAB). Dynamic changes in physicochemical properties, nutritional composition, volatile organic compounds, and functional activities were analyzed. Results showed significant increases (P < 0.05) in total acidity, total phenols, total flavonoids, and DPPH radical scavenging capacity after fermentation. HPLC analysis revealed decreases in citric, tartaric, and malic acids, but increases in lactic and succinic acids. HS-GC-IMS detected 31 volatile compounds, with elevated levels of esters, aldehydes, and acids in the fermented juice. Electronic tongue analysis indicated reduced sweetness and enhanced sourness and richness, while electronic nose confirmed substantial flavor profile changes. LAB fermentation effectively enhanced the nutritional and functional qualities of pear juice, demonstrating its potential as a substrate for novel plant-based fermented beverages.

Highlights

• •Mixed fermentation of pear juice using Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus.
• •Fermentation significantly enhanced antioxidant activity and phenolic content.
• •LAB metabolism altered organic acid profile and sugar/acid balance.
• •GC-IMS revealed increased esters and alcohols, enriching the aroma profile.
• •E-nose and E-tongue confirmed improved overall flavor and acceptability.

1. Introduction

The pear (Pyrus spp.), a prominent member of the Rosaceae family (Gan et al., 2023), is widely cultivated and consumed globally for its desirable flavor, texture, and nutritional value (Hong et al., 2021). It is a valuable source of dietary fiber, phenolic compounds, vitamin C, and potassium, which contribute to health benefits such as antioxidant and anti-inflammatory effects (R et al. Sathya et al., 2024). However, the high moisture content and susceptibility to enzymatic browning of fresh pears accelerate postharvest deterioration, leading to substantial economic losses and a limited shelf life (Yang et al., 2022, Yap et al., 2022). Thus, developing effective postharvest processing techniques is crucial for maintaining quality and reducing waste.

Probiotics, notably lactic acid bacteria (LAB), confer health benefits when consumed adequately. Although the global probiotic market is dominated by dairy-based beverages (Li et al., 2021), concerns regarding their cholesterol, fat content, lactose intolerance, and allergies have spurred interest in non-dairy alternatives (Lee et al., 2025). Fermented fruit beverages are particularly palatable to consumers and fruits serve as ideal media for LAB growth owing to their rich nutrients and fermentable sugars (Saud et al., 2024). LAB fermentation of fruit juices has been shown to enhance functional properties (e.g., antioxidant, antibacterial and improve sensory quality and shelf life, making it a research focus (Žvirdauskienė et al., 2025).

Pears, with their sweet taste and abundance of vitamins and dietary fiber, hold significant potential for valorization. Lactobacillus plantarum (Lp) and L. acidophilus (La) are among the main LAB strains suitable for fruit juice fermentation (Muhialdin et al., 2021; Ruiz Rodríguez et al., 2021). Lp is well-adapted to plant-based substrates (Lan et al., 2023), while La thrives in low-pH environments and utilizes diverse substrates. Both strains have been successfully used in fermenting various fruit juices (Chen et al., 2023). Nonetheless, the fermentation of pear juice using a combination of Lp and La remains unexplored, despite pear juice's apparent suitability as a LAB carrier.

This study aimed to investigate the effects of mixed LAB (Lp + La) fermentation on the physicochemical properties, functional qualities, volatile flavor compounds, and sensory characteristics of pear juice. The goal is to provide a scientific basis and reference for the deep processing of pears into functional fermented beverages.

2. Materials and methods

2.1. Preparation of LAB strains and raw materials

Lactobacillus acidophilus (La, strain JYLA-191) and Lactobacillus plantarum (Lp, strain JYLP-326) powders were obtained from Zhongke Jiayi Biological Engineering (Shandong, China) and stored at 4 °C. Fresh Hongxiang pears (a local Pyrus cultivar) were purchased from a local market (Hebei, China) and stored at 4 °C until processing.

2.2. Preparation and fermentation of pear juice

Fresh pears were washed, peeled, and diced. The pieces were immersed in 0.6 % (w/w) ascorbic acid for 15 min to prevent browning, then juiced using a commercial extractor (Joyoung Z8-V82, China). The crude juice was clarified by filtration through an 80-mesh sieve.

Aliquots (150 mL) of the clarified juice were pasteurized in 250 mL Erlenmeyer flasks at 100 °C for 15 min and cooled. Based on preliminary trials, sterile sucrose was added to increase the sugar concentration by 5 % (w/v), and the pH was adjusted to 5.5 with 1 mol/L NaOH. The La and Lp strains were combined at a 1:1 ratio (v/v), revitalized in sterile saline for 20 min, and inoculated (1.5 mL inoculum per flask) into the juice. Fermentation was carried out at 36 °C for 60 h, with samples taken at 12 h intervals for analysis.

2.3. Determination of viable cell counts and physicochemical properties

Viable LAB counts were enumerated using the standard pour-plate method on MRS agar after anaerobic incubation at 36 °C for 48 h (Jideani et al., 2021). The pH and total soluble solids (°Brix) were measured with a pH meter (PHS-3E, OLABO, China) and a refractometer, respectively. Total and reducing sugars were quantified by the DNS (3,5-dinitrosalicylic acid) methods, respectively (Saeed et al., 2024). Titratable acidity was determined by titration with 0.1 M NaOH and expressed as g/L citric acid. Color properties of the juice were evaluated using a Minolta CR-400 colorimeter (Konica Minolta, Japan) to obtain CIELAB color values (L*, a*, b*). The total color difference (ΔE) between initial and fermented juices was calculated using the standard formula:

$$∆E=\sqrt{{\left(L^{\ast }-{L_0}^{\ast }\right)}^2+{\left(a^{\ast }-{a_0}^{\ast }\right)}^2+{\left(b^{\ast }-{b_0}^{\ast }\right)}^2}$$ (1)

2.4. Determination of total phenolic and total flavonoid contents

Total phenolic content (TPC) was determined using the Folin-Ciocalteu method (Sip et al., 2024). Briefly, 0.2 mL of diluted juice was mixed with 0.5 mL of 10 % Folin-Ciocalteu reagent and 1.5 mL of 20 % sodium carbonate. The mixture was diluted to 10 mL, incubated in the dark for 60 min, and the absorbance was measured at 765 nm. TPC was expressed as mg gallic acid equivalents (GAE) per 100 mL of juice (R² = 0.9987).

Total flavonoid content (TFC) was measured by the aluminum chloride colorimetric method. Briefly, 4 mL of the diluted sample was reacted with 1 mL of 5 % NaNO₂ and 1 mL of 10 % Al(NO₃)₃ for 5 min, followed by the addition of 10 mL of 10 % NaOH and a 15-min incubation. The absorbance of the resulting mixture was then measured at 510 nm. TFC was expressed as mg rutin equivalents (RE) per 100 mL of juice (R² = 0.9997).

2.5. Determination of antioxidant activities

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of pear juice was determined using the stable DPPH radical method (Wang, Mi, et al., 2023). The DPPH scavenging capacity was calculated as a percentage of radical inhibition relative to a blank control:

$$\begin{array}{c}\text{DPPH}\left(\%\right)=\frac{A_0-A_1}{A_0}\times 100\%\end{array}$$ (2)

The ABTS⁺ scavenging capacity was measured using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) decolorization assay (Cele et al., 2022). The ABTS⁺ scavenging activity (%) was calculated relative to a control:

$$\begin{array}{c}{\text{ABTS}}^{+}\left(\%\right)=\frac{A_{\mathrm{s}}-A_{\mathrm{i}}}{A_{\mathrm{s}}}\times 100\%\end{array}$$ (3)

2.6. Determination of organic acids

Organic acids were analyzed by HPLC following a reported method (Wei et al., 2019). Filtered (0.45 μm) juice samples were separated on an Agilent 1200 system with a C18 column (250 mm × 4.6 mm, 5 μm) at 35 °C. The mobile phase was a gradient of methanol and 0.01 mol/L KH₂PO₄ (pH 2.7) at a flow rate of 0.7 mL/min. Detection was performed at 210 nm. The gradient elution program was as follows: 0–5 min, 5 % methanol; 5–20 min, methanol linearly increased from 5 % to 30 %; 20–25 min, held at 30 % methanol; 25–26 min, returned to 5 % methanol; and 26–30 min, re-equilibration at 5 % methanol. Compounds (e.g., citric, malic, lactic, succinic acids) were identified by comparing retention times with authentic standards and quantified using external calibration curves.

2.7. Identification of volatile components (GC-IMS analysis)

Based on the methodology of Wang et al. (Wang, Qi, et al., 2023), the volatile compounds in pear juice were analyzed using GC-IMS (Agilent 490 GC coupled with FlavourSpec® IMS). Samples (3 mL) were equilibrated at 40 °C for 15 min in 20 mL headspace vials before 500 μL headspace injection (85 °C). Separation was performed on an FS-SE-54-CB capillary column (15 m × 0.53 mm, 0.5 μm) at 60 °C with nitrogen carrier gas at programmed flow: 2 mL/min (2 min), 10 mL/min (8 min), 100 mL/min (10 min), and 130 mL/min (10 min). Ionization used a ³H source (300 MBq) in positive mode, with detection in a 98 mm drift tube (45 °C, 5 kV).

Raw IMS data were acquired and processed using Laboratory Analytical Viewer (LAV, G.A.S., Dortmund, Germany). The retention index (RI) values of the volatiles were calculated by using n-ketones C4-C9 (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China) as external standards. The volatile compounds were identified by comparing the RI values and IMS drift time (DT, the time it takes for ions to reach the collector through a drift tube, in milliseconds) with those of the authentic reference compounds in the GC × IMS Library (Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany). To compare the abundance of individual compounds between samples, the signal intensity of each compound was expressed relative to the total signal intensity of all identified volatiles within the same sample, yielding a relative percentage for comparative purposes.

2.8. Electronic nose analysis

The odor profiles of pear juice were analyzed using an electronic nose (PEN3, AIRSENSE, Germany) equipped with 10 sensors. The sensor array includes: W1C (aromatic compounds), W5S (nitrogen oxides), W3C (ammonia, aromatic molecules), W6S (hydrogen), W5C (alkanes, aliphatic compounds), W1S (methane, broad range), W1W (sulfur compounds), W2S (alcohols, partially aromatic compounds), W2W (aromatic and sulfur-organic compounds), and W3S (alkanes, aliphatic compounds). A 5 mL juice sample was equilibrated in a 50 mL vial at room temperature for 30 min. Measurement was performed via headspace sampling with an injection flow rate of 400 mL/min. The settings were as follows: sample preparation time, 5 s; sampling interval, 1 s; analysis sampling time, 100 s; and sensor cleaning time, 100 s.

2.9. Electronic tongue analysis

The taste profile of pear juice was evaluated using an electronic tongue (SA402B, Insent, Japan) with nine sensors for basic and aftertaste attributes. Samples were diluted 50-fold with distilled water to ensure the sensor response signals fell within their optimal linear detection range, as determined by preliminary tests and according to established methodology (Zhang et al., 2020). The measurement cycle comprised 120 s of sample analysis, 40 s of aftertaste recording, and a 10 s sensor rinse with reference solution.

2.10. Sensory evaluation

A Sensory evaluation was conducted by a panel of 20 trained assessors using a method adapted from literature (Sun et al., 2022). Juice samples (20 mL) were served at room temperature in coded cups. Assessors rated color, aroma, taste, and overall acceptability on a 5-point hedonic scale (1 = extremely dislike, 5 = extremely like), with palate cleansing between samples. The scores for each attribute were averaged.

Ethical approval for this sensory evaluation was exempted by the Ethics Review Committee of Hebei Agricultural University (HAU) as the study involved only routine taste testing of safe food samples with minimal risk. All participants provided written informed consent after being fully informed of the study's purpose and procedures. Their participation was voluntary, their responses were processed anonymously, and no personally identifiable information was collected or disclosed.

2.11. Statistical analysis

All experiments were conducted in triplicate, and results are reported as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Duncan's multiple range test was used to determine significant differences between means at a 95 % confidence level (P < 0.05). Statistical analyses were performed using SPSS software (v22.0, IBM, USA). Graphs were plotted using Origin 2021 (OriginLab, USA).

3. Results

3.1. Viable cell counts and physicochemical properties during fermentation

Pears provide a nutrient-rich medium and can serve as an excellent carrier for LAB growth (Ruiz Rodríguez et al., 2021). The number of viable bacteria is a critical parameter to evaluate the fermentation characteristics of LAB (Wang et al., 2024). A higher LAB count often correlates with better fermentation efficacy and more consistent product characteristics (Peng et al., 2021). As shown in Fig. 1A, at the end of fermentation (60 h), the viable cell counts increased from an initial 6.75 to 8.90 log CFU/mL, illustrating that the two LAB strains adapted well to the pear juice environment. The rapid increase in LAB viability during the first 36 h was likely due to efficient utilization of the abundant sugars in the pear juice matrix (Liu et al., 2022). From 36 h to 60 h, the growth rate of LAB slowed significantly, possibly due to the accumulation of organic acids, which created an inhibitory low-pH environment, a trend consistent with the observations of Lan et al. (Lan et al., 2023).

Fig. 1.

Effect of fermentation on viable counts (A); pH and total acids (B); total and reducing sugars(C). Different letters above the bars indicate significant differences (P < 0.05).

The pH and total acidity served as crucial fermentation parameters, influencing both sensory quality and microbial safety of fermented products. As depicted in Fig. 1B, a marked pH reduction from 5.50 to 3.99 (P < 0.05) accompanied by a corresponding increase in total acidity from 2.33 to 8.65 g/L was observed during LAB fermentation. These pronounced acidification patterns substantiate the robust metabolic activity of the LAB culture, particularly their capacity to ferment sugars into organic acids (Quan et al., 2022). The observed biochemical transformation aligns with established LAB metabolic pathways, wherein fermentable sugars (notably glucose and fructose) are converted into various organic acids and other flavor-active metabolites (Wang et al., 2022). These findings confirm the metabolic adaptability of the LAB consortium in the pear juice matrix and underscore that pear juice provides a nutritionally adequate substrate to support vigorous LAB growth and metabolism.

As shown in Fig. 1C, the contents of total sugars were declined dramatically (P < 0.05) during fermentation, especially within the first 36 h, corresponding to the period of most rapid bacterial growth. This rapid sugar depletion is associated with the active metabolism of LAB, which catabolize sugars to produce energy and metabolic by-products (including organic acids) (Yang et al., 2023). In contrast, the concentration of reducing sugars (monosaccharides such as glucose and fructose) showed an initial increase during the first 24 h of fermentation, followed by a decrease from 24 to 60 h. The transient increase in reducing sugars can be attributed to the hydrolysis of sucrose and other di-saccharides present in pear juice into their monosaccharide components. This inference is strongly supported by the known composition of pear juice and the enzymatic profile of LAB. Many LAB strains, including those commonly used in food fermentation, possess intrinsic or inducible sucrase (invertase) activity that efficiently hydrolyzes sucrose into glucose and fructose (Selvaraj & Gurumurthy, 2024). Therefore, the observed initial rise in reducing sugars is a direct and expected consequence of microbial sucrase activity outpacing the subsequent uptake and catabolism of the resulting monosaccharides. Once accumulated, these reducing sugars were subsequently consumed by LAB to produce organic acids and other metabolites (Devanthi et al., 2021). Interestingly, this trend of reducing sugar variation differs from some reports in other fermented fruits. Li et al. observed a continuous decline in reducing sugars during LAB fermentation of jujube juice (Li et al., 2021). Such differences suggest that the specific carbohydrate composition of the substrate and the metabolic characteristics of the microbial culture can influence sugar utilization patterns.

In addition to chemical changes, LAB fermentation also affected the color attributes of the pear juice. The chromatic properties of the juice underwent significant changes throughout fermentation, as evidenced by shifts in CIELAB color values (Table 1). A consistent decrease in luminosity (L* value) was observed throughout fermentation, principally attributable to increased turbidity resulting from LAB cellular proliferation (Liu et al., 2022). Concurrently, the chromaticity coordinates (a* and b* values) demonstrated progressive augmentation, reflecting complex pigment modifications. These chromatic alterations yielded statistically significant ΔE values (P < 0.05), confirming the substantial visual impact of fermentation. The observed color changes are likely driven by multiple concurrent processes. The primary factors include: (i) the physical light-scattering effect due to increased microbial biomass and turbidity; and (ii) chemical modifications of endogenous pear juice pigments, possibly through pH shifts induced by organic acid production or enzymatic (e.g., polyphenol oxidase) activity that affects phenolic compound stability. While the direct contribution of microbial pigments in this specific system requires further validation, it is noted that certain LAB strains have been reported to produce pigment-like metabolites, such as carotenoids, which could theoretically influence color in some fermented foods (Xie et al., 2023). Notably, previous studies on fermented fruit juices have reported varied color outcomes, with some showing color stabilization or lightening (Wu et al., 2020). The differences observed in our study highlight the importance of specific strain-substrate interactions in determining fermentation-induced color changes, especially in phenolic-rich juices like pear juice. Overall, our results demonstrate that LAB fermentation can induce pronounced modifications in the optical (visual) properties of pear juice through multiple concurrent biochemical processes.

Table 1.

Effects of LAB fermentation on color values of pear juice.

Fermentation	L⁎	a⁎	b⁎	∆E
0 h	47.28 ± 0.12a	0.29 ± 0.01e	6.80 ± 0.05e	/
12 h	46.78 ± 0.09b	0.39 ± 0.01d	6.91 ± 0.02d	0.52 ± 0.06e
24 h	46.35 ± 0.05c	0.41 ± 0.01d	7.19 ± 0.08c	0.96 ± 0.05d
36 h	45.85 ± 0.08d	0.48 ± 0.01c	7.54 ± 0.13b	1.54 ± 0.07c
48 h	45.32 ± 0.31e	0.54 ± 0.01b	7.63 ± 0.08b	2.14 ± 0.28b
60 h	44.52 ± 0.11f	0.61 ± 0.02a	7.73 ± 0.09a	2.93 ± 0.11a

Values are mean ± standard deviation. Different letters in the same column indicate a significant difference between different groups (P < 0.05).

3.2. Effects of fermentation on total phenol and flavonoids contents

Phenolic compounds in fruits exist in both free and bound states, possessing good physiological activities and being of great significance for regulating human health. As shown in Fig. 2A, TPC and TFC increased significantly during fermentation. Previous studies have demonstrated that the polyphenol content in fruit and vegetable juices increases significantly after fermentation by LAB (Li et al., 2021). LAB fermentation has been shown to increase phenolic content in jujube juice and blueberry juice, enhancing their antioxidant activity.

Fig. 2.

Effect of fermentation on the content of total phenols and total flavonoids (A); DPPH and ABTS⁺ radical clearance (B). Different letters above the bars indicate significant differences (P < 0.05).

It is worth noting, however, that not all reports agree on this trend. In some cases, fermentation can decrease phenolic levels depending on the strain or substrate. Li et al. (Li et al., 2019) found that fermenting apple juice with Lp ATCC 14917 led to a significant decrease in TPC. Prada et al. suggested that the increase in TPC observed in many fermentations may be due to the hydrolysis of complex bound polyphenols into simpler free phenolics by LAB-produced enzymes (such as esterases and glycosidases) (Parada et al., 2023). The release of these previously bound phenolics would register as an increase in measurable TPC. In our study, a similar mechanism could explain the rise in TPC. The mixed LAB culture might produce enzymes that break down polyphenol conjugates or release phenolics from the pear pulp matrix. Specifically, lactic acid bacteria are known to produce glycosidases (e.g., β-glucosidase) and esterases, which can hydrolyze glycosidic and ester bonds in polyphenol conjugates, thereby increasing the pool of free phenolic compounds (Xiang et al., 2019). The action of these enzymes likely contributed to the conversion of bound phenolic forms into free, extractable compounds in our fermented pear juice.

Likewise, the TFC of the pear juice showed a significant increase after fermentation. This result contrasts with some literature reports, such as a study on hawthorn juice where LAB fermentation led to a decrease in flavonoids (Zhao et al., 2025). The increase in TFC in our fermented pear juice could be attributed to the breakdown of cellular structures and cell walls during fermentation, which releases flavonoid compounds that were initially sequestered in the fruit tissue (Isas et al., 2020). LAB metabolism (and mild fermentation acidity) can soften or partially degrade plant cell walls, thereby freeing flavonoids and other phytochemicals. Thus, the fermentation process likely enhanced the extractability or liberation of flavonoids in the pear juice, resulting in a higher measured TFC. The production of organic acids by LAB lowers the pH, which can weaken and solubilize plant cell wall polysaccharides (Tangyu et al., 2019). Furthermore, certain LAB strains possess pectinolytic and cellulolytic activities that can degrade structural components of the cell wall, facilitating the liberation of intracellular flavonoids into the juice matrix (Wang et al., 2021). It should be noted that the specific activities of these enzymes (e.g., glycosidases, esterases, pectinases) were not quantified in the present study. Therefore, while the observed increases in TPC and TFC are consistent with, and strongly supported by, the well-documented enzymatic capabilities of LAB reported in the literature, the precise contribution of each enzymatic pathway warrants further investigation through targeted enzyme assays in future work.

3.3. Effects of fermentation on antioxidant activities of pear juice

As illustrated in Fig. 2B, LAB fermentation markedly enhanced the antioxidant capacity of pear juice, with DPPH radical scavenging activity showing a significant increase (reaching 94.74 %) during the first 48 h of fermentation. Notably, the antioxidant activity plateaued between 48 and 60 h, suggesting stabilization of bioactive compounds. This plateau could be attributed to a combination of microbial and biochemical factors. By 48 h, the LAB population may have approached stationary phase, leading to a deceleration in metabolic activities responsible for both the release of bound phenolics (e.g., via glycosidase activity) and the bioconversion of simple phenolic precursors (Yaqoob et al., 2025). Concurrently, a dynamic equilibrium might have been reached between the ongoing microbial production or release of antioxidants and their possible degradation through enzymatic oxidation or polymerization reactions, which can be influenced by the prolonged exposure to fermentation metabolites and the altered redox environment (Filannino et al., 2018). Thus, the observed stabilization in DPPH activity reflects a balance point in the complex interaction between microbial metabolism and the stability of the antioxidant compounds present. This observed enhancement correlates strongly with the concurrent accumulation of phenolic and flavonoid compounds, consistent with established structure-activity relationships between polyphenolic content and free radical scavenging capacity reported in previous studies (Isas et al., 2020). ABTS⁺ scavenging capacity changed smoothly from 0 to 36 h and significantly decreased from 36 to 60 h, which showed a different trend from DPPH radical scavenging capacity. The results of DPPH and ABTS⁺ were in agreement with Kaprasob et al., who reported a similar pattern where LAB fermentation increased DPPH activity but had a variable effect on ABTS activity in cashew apple juice (Kaprasob et al., 2018). The reason, as noted, likely lies in the distinct reaction chemistries: certain phenolic compounds or degradation products may scavenge DPPH radicals efficiently but not ABTS⁺ radicals, or vice versa. The observed divergence likely stems from the distinct chemical nature of the two assays. DPPH is a stable, nitrogen-centered radical soluble in organic solvents, measuring hydrogen-donating capacity. ABTS⁺ is a pre-generated, water-soluble radical cation capable of both electron and hydrogen transfer (Munteanu & Apetrei, 2021). The complex mixture of antioxidants and metabolites produced during LAB fermentation may interact differently with these radical systems, leading to the differential responses. This underscores the value of employing multiple assays to capture the comprehensive antioxidant profile of fermented foods.

Overall, the fermentation led to an enhancement of antioxidant potential (especially in the DPPH assay), suggesting an improvement in the functional quality of pear juice. The improved antioxidant activity, particularly the pronounced increase in DPPH scavenging capacity, signifies that LAB fermentation not only preserves but actively enhances the juice's content of bioactive compounds capable of neutralizing free radicals. This enhancement is directly relevant to the research objective of developing a novel functional beverage, as antioxidant capacity is a critical attribute linked to potential health-promoting properties and consumer appeal. The slight decline in ABTS activity at later stages could also indicate that prolonged fermentation might degrade some antioxidant compounds or produce intermediates that interfere with the ABTS assay. Nonetheless, the substantial increase in DPPH scavenging capacity demonstrates a clear boost in antioxidant constituents due to LAB fermentation.

3.4. Effects of fermentation on organic acid in pear juice

Organic acids are highly effective in suppressing the growth and proliferation of diverse spoilage and pathogenic microorganisms (Punia Bangar et al., 2022). Moreover, they represent crucial constituents of the flavor matrix in LAB fermented products, endowing the final products with unique sour notes and characteristic aromas (Zapaśnik et al., 2022). The results of organic acids in pear juice during the fermentation process were shown in Fig. 3. The total organic acid content, as well as the profile of individual acids, changed significantly as a result of LAB fermentation.

Fig. 3.

The oxalic acid content (A); succinic acid content (B); tartaric acid content(C); citriic acid content (D); lactic acid content (E); malic acid content (F) of pear juice during fermentation detected by HPLC technology. Different letters above the bars indicate significant differences (P < 0.05).

Unfermented pear juice mainly contains malic acid and citric acid. After fermentation, the content of citric acid, tartaric acid and malic acid decreased, while the content of lactic acid and succinic acid increased significantly (P < 0.05). The content of oxalic acid irregularly changed. The content of oxalic acid irregularly changed. This pattern is consistent with oxalate not being a core metabolic product of LAB, whose primary fermentation outputs are typically lactic and acetic acids. Its fluctuation is therefore more likely attributable to pH-dependent physicochemical release from the fruit matrix rather than directed microbial synthesis (Filannino et al., 2018). The decrease in malic acid may be due to its participation in the malolactic pathway (Li et al., 2021). Citric acid is an intermediate product in the tricarboxylic acid metabolic cycle and is consumed during the metabolism of LAB and producing some intermediate products, such as succinic acid, which is the reason for the increase of succinic acid (Peng et al., 2021).

Lactic acid, being the primary fermentation end-product of LAB carbohydrate metabolism, showed the most dramatic increase. Lactic acid concentration rose from a negligible initial level (around 0.09 g/L, likely present from natural pear microflora or minimal initial content) to about 1.60 g/L after fermentation (P < 0.05). This substantial accumulation of lactic acid confirms that the fermentation was predominantly lactic acid fermentation. Lp and La are facultatively heterofermentative and homofermentative LAB, respectively, and their metabolism of sugars (homofermentative pathway for La and flexible metabolism for Lp) largely produces lactic acid as a major end-produc (Xie et al., 2023). Additionally, Lp is capable of malolactic conversion (malic to lactic), further contributing to lactic acid levels. The net effect is a high lactic acid yield, which is desirable for imparting a pleasant tartness and for preservation, as lactic acid acidifies the juice environment.

The content of succinic acid increased modestly but significantly, as noted above, likely linked to citrate metabolism. Succinic acid can contribute a mild acidity and umami-like taste in trace amounts. The irregular changes in oxalic acid content are of interest. Oxalic acid is a diacid that may precipitate or decompose under certain conditions. The fermentation conditions (rising acidity and fluctuations in redox potential) may affect oxalic acid solubility or lead to chemical transformation (e.g., precipitation as calcium oxalate or breakdown). The fluctuations observed suggest that oxalic acid did not follow a clear consumption or production pattern. During active LAB fermentation, the pH drops, and temperature was kept moderate (36 °C), which might lead to some oxalic acid precipitating out of solution or oscillating due to equilibrium shifts. Thus, the inconsistent trend for oxalic acid could be due to its inherent chemical instability in the changing fermentation matrix.

3.5. Characterization of the volatile flavor components by HS-GC-IMS

To gain a more detailed understanding of the aroma changes, the volatile compounds in pear juice before and after fermentation were analyzed using headspace GC-IMS (Fig. 5). Table 2 summarizes the volatile compounds identified in PJ and FPJ, along with their relative contents. In total, 31 volatile substances were detected across both samples by GC-IMS, including 9 alcohols, 4 ketones, 4 aldehydes, 8 esters, 4 acids, and 2 other compounds (such as terpenes or miscellaneous volatiles). In the PJ, the predominant volatile compounds contributing to its aroma were identified as 1-pentanol, isobutyl methyl carbinol (isoamyl alcohol), 3-pentanone, and a hexyl ester (likely hexyl acetate). These compounds impart fruity and floral notes characteristic of fresh pear aroma. After fermentation, the profile shifted. The main volatile components in FPJ were isobutyl methyl carbinol (also present in PJ), benzyl alcohol, 3-hexen-1-ol (a green, leafy-smelling alcohol), hexyl acetate, and ethyl butyrate. The presence of ethyl butyrate is notable as it provides a strong fruity (pineapple-like) aroma, which was low in the unfermented juice but increased with fermentation. Comparing the overall classes of volatiles, alcohols were the most abundant category in both PJ and FPJ, accounting for about 29.04 % of the total volatiles in the fresh juice and approximately 34.48 % in the fermented juice. Alcohols such as various amyl alcohols and benzyl alcohol can impart pleasant aromatic notes and also act as solvents for other aroma compounds, thereby influencing the overall aroma matrix (Chen et al., 2019). Among the alcohols detected, 4-methyl-2-pentanol (an isoamyl alcohol isomer) and n-pentanol had relatively high intensities. These two alcohols have distinctly aromatic and fruity odors, contributing significantly to the aroma character of the juices. The content of these alcohols remained high in the fermented juice, although some alcohols (e.g., n-pentanol as discussed below) showed a decrease after fermentation.

Fig. 5.

The fingerprints of volatile compounds in PJ and FPJ detected by HS-GC-IMS technology. PJ means Pear juice before fermentation; FPJ means Pear juice after fermentation.

Table 2.

Effects of LAB fermentation on volatile compounds of pear juice.

Number	classify	Compounds	CAS	Formula	Odor description	PJ	FPJ
						Concentration (μg/kg)	Concentration (μg/kg)
1	Alcohol	1-Hexanol	111–27-3	C6H14O	Aroma, wine, light fat aroma	0.77 ± 0.09a	0.77 ± 0.02a
2		(E)-hept-2-enal	928–95-0	C6H12O	Unripe fruit smell	0.57 ± 0.05a	0.26 ± 0.03b
3		1-Pentanol	71–41-0	C5H12O	fruity	1.23 ± 0.21a	0.31 ± 0.07b
4		2-Ethylhexanol	104–76-7	C8H18O	Floral scent	0.02 ± 0.01b	1.13 ± 0.03a
5		Benzyl alcohol	100–51-6	C7H8O	fruity	0.12 ± 0.06b	2.35 ± 0.08a
6		2,3-Butanediol	513–85-9	C4H10O2	fruity	0.24 ± 0.05a	0.15 ± 0.03b
7		2-Methyl-1-butanol	137–32-6	C5H12O	Fresh fruity and floral	0.19 ± 0.05b	0.26 ± 0.08a
8		Isobutyl methyl carbinol;	108–11-2	C6H14O	Aromatic smell	1.25 ± 0.11b	3.37 ± 0.12a
9		3-Hexen-1-ol	544–12-7	C6H12O	Fruity, sweet	1.07 ± 0.03b	1.34 ± 0.04a
10	Ketone	2-Nonanone	821–55-6	C9H18O	Fruity flavor	0.02 ± 0.01b	0.13 ± 0.06a
11		4-Methyl-2-pentanone	108–10-1	C6H12O	Pleasant ketoid fragrance	0.15 ± 0.01a	0.05 ± 0.03b
12		3-Pentanone	96–22-0	C5H10O	Distinctive smell	1.61 ± 0.15a	0.77 ± 0.50b
13		3-Heptanone	106–35-4	C7H14O	Fruit and fat aromas	0.18 ± 0.04a	0.23 ± 0.04a
14	Aldehyde	benzaldehyde	100–52-7	C7H6O	Cherry nut aroma	0.36 ± 0.01a	0.33 ± 0.01a
15		2-methylpropanal	78–84-2	C4H8O	Convallaria aroma	0.39 ± 0.09b	0.81 ± 0.12a
16		Pentana	110–62-3	C5H10O	The sweet smell of fruit	0.50 ± 0.03a	0.43 ± 0.06a
17		(E)-Pent-2-en-1-al	1576-87-0	C5H8O	Pungent fruit smell	0.11 ± 0.04b	0.21 ± 0.05a
18	Ester	butyl acetate	123–86-4	C6H12O2	Similar to apple banana aroma	0.55 ± 0.05b	0.72 ± 0.07a
19		Ethyl valerate	539–82-2	C7H14O2	fruity	0.42 ± 0.02b	1.05 ± 0.03a
20		Butyl acrylate	141–32-2	C7H12O2	Fruity aroma	0.10 ± 0.07b	0.36 ± 0.05a
21		Ethyl pyruvate	617–35-6	C5H8O3	Fruit aroma	0.97 ± 0.04a	0.08 ± 0.01b
22		Hexylester	629–33-4	C7H14O2	Fruity aroma	2.08 ± 0.03a	2.10 ± 0.02a
23		Ethyl butyrate	105–54-4	C6H12O2	Pineapple and banana aromas	0.09 ± 0.02b	2.45 ± 0.37a
24		Hexyl ester	13,562–84-0	C8H15O2	Fruity aroma	1.16 ± 0.38a	1.20 ± 0.28a
25		Heptylester	112–23-2	C8H16O2	fruity	0.28 ± 0.04a	0.31 ± 0.14a
26	Acid	Octanoic acid	124–07-2	C8H16O2	Rancid smell, weak aroma	0.41 ± 0.04b	0.83 ± 0.03a
27		Allylacetic acid	591–80-0	C5H8O2	Caramel flavor	0.36 ± 0.04b	0.88 ± 0.13a
28		butanoic acid	107–92-6	C4H8O2	Fruity, fermented	0.30 ± 0.04b	0.59 ± 0.09a
29		hexanoic acid	142–62-1	C6H12O2	Daqu wine smell	0.05 ± 0.01b	0.73 ± 0.24a
30	Hydrocarbon	Ethylbenzene	100–41-4	C8H10	Strong aroma	1.21 ± 0.18b	2.46 ± 0.66a
31		2,5-Dimethylfuran	625–86-5	C6H8O	Sweet fruit aromas	0.10 ± 0.01b	0.39 ± 0.02a

Values are mean ± standard deviation. Different letters in the same line indicate a significant difference between different groups (P < 0.05).

Esters constituted the second major group of volatile compounds. Esters are well-known to provide sweet and fruity aromas in fruit-based products (Wang et al., 2022). In our analysis, the overall ester content increased in the fermented juice compared to the original juice. Key esters included ethyl butyrate and n-hexyl acetate, both of which were present at higher levels in FPJ. Ethyl butyrate has a strong sweet, pineapple-like fragrance, and hexyl acetate offers a characteristic pear-like, sweet aroma; their elevated presence in FPJ suggests that fermentation enhanced certain fruity aromatic notes. Esters are typically formed by the enzymatic esterification of alcohols and acids or by yeast/bacterial metabolism. Here, LAB might contribute to ester formation indirectly via metabolism that produces ethanol or through releasing enzymes from fruit tissues.

These aroma transformations can be explained by microbial activity: LAB can metabolize certain aroma compounds (breaking down some esters or alcohols) and can also release bound aroma precursors from pear (such as glycosidically bound volatiles) or generate new volatiles through pathways like amino acid catabolism (producing higher alcohols or short-chain acids). Additionally, fermentation conditions (low pH, presence of ethanol or CO₂ might cause some volatile loss or chemical equilibrium shifts. The result is a unique flavor profile in the fermented juice. While some of the original fruity notes are diminished, the fermented juice gains a richer aroma complexity that includes mild buttery, floral, and caramel-like characteristics. These changes, as indicated by the E-nose PCA, were substantial enough to differentiate the juices clearly.

3.6. E-nose and E-tongue analysis

Aroma and taste are critical determinants of consumer acceptance for fruit juices. LAB fermentation can substantially modify the aroma profile of juices, owing to microbial metabolism of aroma precursors as well as non-metabolic factors such as enzymatic activities and cell autolysis that release aromatic compounds (Carpena et al., 2021). In this study, an electronic nose was used to compare the headspace aroma of unfermented pear juice (PJ) and fermented pear juice (FPJ). The E-nose sensor array produces a “radar” plot of responses, reflecting the relative intensity of different volatile groups.

Fig. 4A shows the radar plot of the E-nose sensor responses for PJ and FPJ. The response patterns for the two samples were very similar on most sensors, with the plots nearly overlapping for 7 out of 10 sensors. Notable differences were observed for three sensors: W1W, W1S, and W2S. The W1W sensor (sensitive mainly to certain organic compounds, especially long-chain alkanes) showed a similar response for PJ and FPJ, indicating that long-chain alkanes were present at comparable levels in both. In contrast, sensors W1S (sensitive to terpenes and some sulfur compounds) and W2S (sensitive to alcohols, sulfur, and some aromatics) showed divergent responses between the two samples. This suggests that the contents of terpenoid compounds, some alcohols, sulfur-containing volatiles, and other aromatic substances differed considerably between the unfermented and fermented pear juice. In other words, LAB fermentation led to qualitative and/or quantitative changes in certain volatile groups (likely increasing some sulfurous or terpenoid notes while perhaps reducing others). To further interpret the complex sensor data, principal component analysis (PCA) was applied to the E-nose sensor responses (Fig. 4B). The PCA results revealed that the first principal component (PC1) accounted for about 92.2 % of the total variance in the aroma data, and the second principal component (PC2) accounted for an additional 4.4 %, for a cumulative variance of 96.6 %. The score plot shows two well-separated clusters corresponding to PJ and FPJ, with no overlap. The distinct clustering indicates that the electronic nose could clearly discriminate between unfermented and fermented pear juice based on their volatile profiles. The fact that over 96 % of the variance is captured by PC1 and PC2 means the difference between PJ and FPJ aroma is very pronounced and dominated by a few principal factors (likely the differences picked up by W1S and W2S sensors). In summary, the E-nose analysis suggests that fermentation introduced significant changes in the volatile makeup of pear juice, enough to be easily detected and differentiated in multivariate aroma space.

Fig. 4.

(A) Results of electronic nose detection of PJ and FPJ; (B) Principal component analysis (PCA) of aroma components analyzed by electronic nose; (C) Results of electronic tongue detection of PJ and FPJ; (D) Results of sensory evaluation of PJ and FPJ. PJ means Pear juice before fermentation; FPJ means Pear juice after fermentation.

In addition to aroma, the electronic tongue was used to evaluate the taste profile changes due to fermentation. Fig. 4C presents the E-tongue taste intensity radar for PJ and FPJ, focusing on key taste attributes. Compared to the fresh pear juice, the fermented pear juice showed a significant increase in acidity (sourness) and a significant decrease in sweetness (P < 0.05) in the E-tongue readings. This result is consistent with the chemical analysis: the rise in organic acids (especially lactic acid) and the consumption of sugars (Fig. 3 and Fig. 1C) explain the more sour and less sweet taste of FPJ. The conversion of sugars into organic acids during LAB fermentation directly translates to heightened sourness and reduced sweetness in the final product. Notably, the E-tongue also indicated an increase in the taste attribute described as “richness” or “umami/fullness” for the fermented juice. This enhancement in richness could be due to the production of various amino acids and small peptides, or other flavor-active metabolites, by the fermenting bacteria. LAB are known to release amino acids through proteolysis and produce compounds like diacetyl or certain nucleotides that can contribute to a fuller taste. Additionally, the presence of more glycerol or oligosaccharides could impart a richer mouthfeel. The increased richness in FPJ may stem from secondary metabolites produced by LAB as well as the transformation of some free amino acids during fermentation (Sevindik et al., 2022). It is important to note that proteolytic activity and free amino acid profiles were not quantified in this study. Therefore, while the observed increase in “richness” is consistent with the well-documented ability of LAB to generate savory metabolites through proteolysis and amino acid metabolism, as reported in the literature, this specific mechanistic link remains a plausible interpretation based on collective evidence rather than a directly measured outcome in the current experiment.

3.7. Sensory analysis

Sensory evaluation provides the ultimate assessment of the acceptability of fermentation-induced changes. As shown in Fig. 4D, panelists clearly preferred the fermented pear juice (FPJ) over the unfermented juice (PJ) in flavor and overall acceptance.

The improved aroma complexity and preference for FPJ are clearly supported by the volatile compound analysis using HS-GC-IMS (Fig. 5, Table 2). The volatile profile shifted significantly after fermentation. While fresh PJ aroma was characterized by compounds like 1-pentanol and 3-pentanone, FPJ showed a marked increase in key esters such as ethyl butyrate (imparting a strong fruity, pineapple-like note) and hexyl acetate (pear-like aroma). The overall increase in ester content, along with the presence of alcohols like benzyl alcohol and 3-hexen-1-ol, created the unique, more pronounced fruity/fragrant aroma profile described by the panel. These new aroma compounds, such as esters and alcohols, are generated through LAB metabolism, primarily through the activity of enzymes like β-glucosidase and esterases, which cleave glycosidically bound aroma precursors and synthesize esters from acids and alcohols.

The enhanced taste profile of FPJ, as perceived by the sensory panel, is strongly corroborated by the electronic tongue (E-tongue) data (Fig. 4C). Panelists described FPJ as having a more balanced and pleasant sweet-sour taste, which directly aligns with the E-tongue readings showing a significant decrease in sweetness and an increase in sourness (P < 0.05). This shift is chemically driven by the microbial conversion of sugars into organic acids, predominantly lactic acid, during fermentation. Furthermore, the E-tongue detected a notable increase in “richness” or “umami” in FPJ. This attribute can be attributed to the metabolic activities of LAB, which are known to release amino acids, peptides, and nucleotides during fermentation, compounds that contribute to a savory, mouth-filling sensation and enhance flavor complexity (Iosca et al., 2022).

In terms of color, no dramatic difference was noted by the panel, consistent with the subtle changes often observed in LAB-fermented fruit juices. The overall acceptability score of FPJ was significantly higher than that of PJ.

It should be acknowledged that the sensory evaluation in this study was conducted with a trained panel of 20 assessors. While this sample size is sufficient to detect clear trends and is supported by strong instrumental correlations, future consumer studies with larger and more diverse panels would be valuable to confirm and generalize these sensory preferences.

In conclusion, the sensory analysis confirms that LAB fermentation positively transformed the organoleptic properties of pear juice. The clear preference for FPJ is directly linked to the instrumentally-measured improvements in its taste profile (balanced sweetness/sourness and enhanced richness via E-tongue) and aroma profile (increased fruity esters and complex alcohols via HS-GC-IMS). Therefore, fermentation not only enhances the functional quality of pear juice but also optimizes its critical sensory attributes for greater consumer appeal.

4. Conclusion

This study systematically evaluated the effects of LAB fermentation on the physicochemical properties, bioactive compounds, and sensory profile of pear juice. The fermentation process efficiently converted pear sugars into organic acids and substantially increased the TPC, TFC, and antioxidant capacity. Integrated analysis with an electronic nose, electronic tongue, and GC-IMS demonstrated notable changes in the flavor profile of the fermented pear juice. The overall flavor was transformed, exhibiting greater aromatic complexity, a more balanced taste, and a stronger overall fragrance. Collectively, these findings indicate that LAB fermentation is a promising strategy for the development of functional pear beverages, providing the dual benefits of valorizing pear resources and expanding product diversity in the functional beverage market.

CRediT authorship contribution statement

Dongxia Li: Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Yinuo Tian: Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Zhizhou Chen: Supervision, Resources, Investigation. Jianfeng Sun: Supervision, Investigation. Yaqiong Liu: Resources. Huixia Zhu: Writing – review & editing, Supervision, Resources. Jianlou Mu: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The authors do not have permission to share data.