1 Introduction

A recent review of the prevalence of mental disorders according to United Nations statistics estimates that approximately one billion individuals, constituting 14% of the global teenage population, are affected by various forms of mental illness [1]. Among these, depression and anxiety rank as two of the most prevalent mental health disorders worldwide. These conditions can also serve as symptoms or co-occurring factors with severe mental illnesses like epilepsy, seizures, bipolar disorder, and Huntington's disease [2].

Gamma-aminobutyric acid (GABA), a potent non-protein amino acid neurotransmitter predominantly functions inhibitory across the synapse by reducing the excitability in the Central Nervous System (CNS) [3]. It has been reported that more than 40% of inhibitory synaptic processing in the mammalian brain uses GABA [4]. GABAergic inhibition is thus essential for maintaining a balance between neuronal excitation and inhibition [5]. Disruptions in the GABAergic system have been observed in individuals suffering from depression and anxiety disorders, often resulting in reduced GABA levels [6, 7]. Antidepressant medications alleviate depressive symptoms by stabilizing GABA levels in the brain, thereby promoting a sense of calm and relaxation. These pharmaceutical interventions typically involve synthetic GABA analogues which aim at restoring neurotransmitter equilibrium and enhancing mental stability [8]. However, it is important to acknowledge that certain antidepressants, owing to their chemical composition, may carry disadvantages such as side effects encompassing drowsiness, dizziness, and, in some instances, withdrawal symptoms [9]. To address these concerns, we propose the formulation of a GABA fortified functional beverage as a safe natural alternative to treat low GABA levels in the brain.

In recent years, consumers interest in the consumption of healthier and nutritious functional food and beverage has been increasingly accepted [10]. Among emerging functional foods and beverages in the market, probiotics based functional beverages are more prominent with wider acceptability among consumers [11]. Probiotics, the live microorganisms which when administered in adequate amounts confers various health benefits to human [12]. Such beneficial effects postulated after administration of probiotics through functional food and beverage include; enhancing immune system [13]; lowering the risk of various cancers [14]; balancing the intestinal homeostasis by inhibiting pathogenic microbial flora [15] and aiding lactose intolerance [16]. Dairy based products are the main delivery vehicle for probiotic administration [17]. However, due to the major concerns (lactose intolerance, high cholesterol, allergic milk proteins and saturated fatty acids) associated with the consumption of dairy based functional foods and beverage, demands the need to explore the possibility of novel non-dairy food matrices to deliver probiotics [18]. In this context, the research interests on probiotic functional food and beverage formulation have now been diverted towards non-dairy based products [19]. Therefore, plant-based sources may be viewed as an alternative non-dairy sources for probiotic delivery as they are reported to contain high level of vitamins, minerals, antioxidants, and dietary fibers [20].

Guava (Psidium guajava L.) being the important tropical fruit with significant amount of vitamin C, essential minerals and carbohydrates can be viewed as an alternative raw material for the formulation of a functional beverage. The presence of high amounts of antioxidants such as phenols, flavonoids are reported to prevent the risk of many [21]. Prebiotics are a group of non-digestible oligosaccharides (NDOs) that are consumed by probiotics in the gut to offer potential health benefits to the host [22]. Prebiotic oligosaccharides are defined as the probiotics modulating substances, thereby conferring specific or selective change in the gut to support host health via improvements in metabolic functions [23]. Fructo-oligosaccharides (FOS), Galacto-oligosaccharides (GOS), Isomalto-oligosaccharides (IMO), Manno-oligosaccharides (MO), Pectin-oligosaccharides (POS), Xylo-oligosaccharides (XOS), and chitin-oligosaccharides (COS) are few examples of classical prebiotics. Precisely, FOS have shown potential as supportive complementary therapeutic options against oxidative damage caused mental illness [22]. FOS are known to offer neuroprotective and neuromodulating benefits by modulating brain-derived neurotrophic factors via direct and indirect mechanisms. The rationale for the current study lies in the potential of combining GABA, a known neurotransmitter with calming effects, and a prebiotic like FOS, which promotes the growth of beneficial gut bacteria, in a guava-based beverage. Thus, our objective is to formulate GABA fortified guava based functional beverage supplemented with a prebiotic FOS and to evaluate its neuroprotective effect in a suitable cell line model.

2 Materials and methods

2.1 Microorganism and inoculum preparation

Lactobacillus rhamnosus GG was procured from Aristo Pharmaceuticals Pvt. Ltd. (India). The cells were activated by inoculating onto sterilized de Man, Rogosa, and Sharpe, MRS broth (HiMedia laboratories, India). The procedure was repeated twice for the successful activation of the culture. The cells were collected after 24 h of incubation at 37 °C through centrifugation at 4 °C for 15 min at 8000 rpm. The harvested cells underwent two washes with sterile distilled water [24].

2.2 Preparation of GABA fortified fermented guava beverage (GFGB)

The collection of plant material for this study was conducted in strict accordance with local and national guidelines. All necessary permissions were obtained prior to collection. Necessary considerations were followed to ensure sustainable and responsible use of plant resources. Mature and ripe white guava fruits were carefully selected from local super market (Mathikere, Bangalore, India), washed in tap water, and then trimmed to remove the stalk. The fruits were sliced into pieces and pressed using a fruit mill to obtain crushed pulp. This crushed pulp underwent further processing through a cotton cloth, yielding a clear pulp. The resulting guava pulp, free from seeds, was utilized as the primary ingredient in guava beverage formulation. Guava beverage was formulated to contain 10 g of guava pulp suspended in 90 ml of distilled water, which was further pasteurized using a water bath at 90 °C for 10 min. The below following preparations were then formulated:

  1. 1.

    FE + FOS: 10% Fruit extract (FE) + 1% FOS: Pasteurized guava beverage (100 ml) supplemented with FOS was inoculated using L. rhamnosus GG (~ 106 CFU/ml), subjected to fermentation at 37 °C for 48 h.

  2. 2.

    FE + FOS + GABA: 10% FE + 1% FOS + 0.05% GABA: Pasteurized guava beverage (100 ml) supplemented with FOS was inoculated using L. rhamnosus GG (~ 106 CFU/ml), and incubated at 37 °C. After 48 h of fermentation, fermented guava beverage was fortified using GABA (0.05%) under aseptic conditions.

  3. 3.

    Control: 10% FE + 1% FOS: Un-inoculated sample served as fermentation control.

The pH of the samples was measured using a digital pH meter (VSI-01, VSI Electronics Pvt. Ltd., Chandigarh, India) after proper calibration. Total acidity, expressed as percent total acids, was determined by titrating with 0.1 N NaOH to pH 8.2, using Phenolphthalein as an end point indicator. Viable counts of probiotics in the inoculated beverages were measured using serial dilution technique and by culturing on MRS agar medium [12]. Control and the fermented beverage samples frozen at − 84 °C and later freeze-dried using TFD5503 bench-top freeze-dryer (IlShin BioBase Co. Ltd., Korea).

2.3 Determination of total phenolics and total flavonoids

The total phenolic content was measured using the Folin-Ciocalteu method [25]. To achieve this, 0.5 ml of Folin-Ciocalteu reagent and 1 ml of 20% sodium carbonate were introduced to 0.5 ml (0.1 mg/ml) of the sample. The resulting mixture was then incubated at 75 °C for 10 min, after which the total volume was adjusted to 5 ml with distilled water. The mixture was allowed to stabilize at RT for 30 min. Subsequently, the absorbance of the solution was assessed using a spectrophotometer at a wavelength of 760 nm. A calibration curve was established using a standard solution of gallic acid (GA) with concentrations varying between 0 and 500 µg/ml. The total phenolic content was quantified as µg GAE/g (d.w).

The total flavonoid content was assessed using the aluminum chloride colorimetric method with certain modifications as outlined by [26]. Initially, 0.5 ml (0.1 mg/ml) of the sample was mixed with 0.5 ml of distilled water, followed by the addition of 0.3 ml of 5% (m/v) sodium nitrite. After a 10 min of incubation, 0.3 ml of a 7.5% (w/v) aluminum chloride solution was introduced, and the mixture was allowed to incubate for another 5 min at RT. To terminate the reaction, 2.0 ml of 4% (w/v) sodium hydroxide was added. The absorbance of the resulting solution was measured at 510 nm spectrophotometrically. A calibration curve was prepared using a standard solution of quercetin (QE), with concentrations ranging from 0 to 100 μg/ml. The total flavonoid content was calculated as µg QE/g (d.w).

2.4 Determination of the in vitro antioxidant activity

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging ability and Ferrous reducing antioxidant capacity (FRAC) of the samples were determined according to the previous method of our research group [27].

2.5 Evaluating the neuroprotective effect in SH-SY5Y cell line model

2.5.1 Cell viability assay and morphological observation

The MTT assay was used to assess the cytotoxicity of our sample to SH-SY5Y human neuroblastoma cell line which was acquired from the National Centre for Cell Science (NCCS) in Pune, India (NCCS, Pune, India). The SH-SY5Y cells cultured in DMEM medium supplemented with 10% FBS, 1% antibiotic–antimycotic solution, and 1% L-glutamine were incubated at 37 °C in 5% CO2. For optimal growth conditions, the cells were sub-cultured every 2–3 days. The experiments utilized cells at passage number 39. To determine cell viability, SH-SY5Y cells were seeded in a 96-well plate at a density of 2 * 104 cells/well, allowed to grow for 24 h. The cells underwent simultaneous treatment with 100 μM of rotenone and different concentrations of either the control, FE + FOS, or FE + FOS + GABA for a period of 24 h. The probiotics in FE + FOS and FE + FOS + GABA were killed by heating after arresting the fermentation process at 121 °C for 15 min, followed by freezing and freeze-drying in order to get a powdered sample. The cell morphology was observed using an inverted biological microscope from Biolink, India. The percentage of cell viability was calculated based on the MTT assay results using the below equation.

$$Cell \,viability \left(\%\right)= \frac{Absorbance \,of \,treated \,wells}{Absorbance \,of \,control \,wells}$$

2.5.2 Evaluation of cellular anti-oxidant ability

SH-SY5Y cells were seeded at a density of 0.5 * 106 cells/ml in a 12-well plate and incubated for 48 h to allow for cell attachment and to achieve the desired cell density. The cells were then exposed to 100 μM Rotenone for 2 h, followed by treatment with 100 μg/ml of either the control, FE + FOS, or FE + FOS + GABA in 1 ml of culture medium, and incubated for an additional 24 h. The superoxide dismutase (SOD) activity was quantified via a colorimetric SOD assay kit (R & D Systems INC, USA) and catalase (CAT) activity was assessed following Aebi's [28] protocol, using a GENLISA Human Catalase ELISA Kit (KrishGen Biosystems, India).

2.5.3 Quantification of intracellular ROS using confocal microscopy

ROS production was measured by fluorogenic dye 2’, 7’-dichlorodihydrofluorescein diacetate (H2-DCFDA), which is oxidized by intracellular ROS [29]. Cells were cultured in a 96-well glass bottom plate at a density of 10,000 cells/200 μl and incubated at 37 °C for 24 h. Subsequently, cells were treated with the required concentrations of experimental samples and control, followed by another 24 h incubation. Post-treatment, the medium was removed, and the cells were washed with PBS. Furthermore, the cells were treated with 10 μM H2-DCFDA solution and incubated at 37 °C for 30 min, shielded from light. The DCF accumulation is measured by the increase in the fluorescence at 535 nm when the sample is excited at 488 nm. The measured intensity is proportional to the concentration of ROS inside the cells [30]. Observation was conducted using a ZEISS LSM 880 Fluorescence live cell imaging system, employing a 488 nm laser for excitation and detecting at 535 nm for green fluorescence and bright field. Images were analyzed with ZEN Blue Software, and DCF intensity was quantified using Image J software.

2.6 Statistical analysis

All experiments were conducted in triplicate. Data were reported as the mean ± standard deviation (SD). To determine statistical significance, we used one-way ANOVA followed by Dunnett's post-hoc test. The statistical analyses were conducted using InStat3 software, version 3.36.

3 Results and discussion

3.1 Preparation of fermented guava beverage

Lactic acid bacteria (LAB) play a pivotal role in the food industry, primarily due to their ability to ferment, enhancing both the sensory qualities and the health benefits of food products. The process of fermentation by LAB not only transforms the taste, aroma, and texture of the food but also significantly boosts its bioactive components and antioxidant capacity [31]. The surge in demand for non-dairy options has been fueled by the growing number of vegetarians and individuals with lactose intolerance. A notable trend in this area is the development of non-dairy fermented products using LAB, focusing on fruit and vegetable juices as the base. This innovative approach caters to the dietary preferences and needs of a wider audience, offering them beneficial alternatives that are rich in probiotics and nutrients while being lactose-free [32]. The natural sugars present in fruit juices act as fuel for the growth of cultures, opening up possibilities for creating novel flavor profiles in fermented products [33]. Research by Kwaw et al. [34], Liao et al. [35], and Mousavi et al. [36] has shown successful fermentation of fruit juices using Lactobacillus strains, leading to the production of appealing non-dairy fermented beverages. Given the recognized health benefits of fermented goods and the limited exploration into fermenting guava juice, a decision was made to utilize L. rhamnosus GG for formulating a new non-dairy fermented drink. This innovative approach not only diversifies the range of fermented beverages available but also taps into the nutritional and probiotic potential of guava juice, offering a unique and healthful option to consumers.

The preparations viz., FE + FOS and FE + FOS + GABA were inoculated with probiotic bacteria (L. rhamnosus GG) at an initial cell density of about ~ 106 CFU/ml. The cell count, cell productivity, pH and titratable acidity after 48 h of fermentation are summarized in Table 1. According to the table, the population of L. rhamnosus GG significantly (*p < 0.05) increased to 8.7*108 and 8.3*108 CFU/ml in FE + FOS and FE + FOS + GABA samples with the elapse of fermentation time. Our findings align with those of De Oliveira et al. [37] who observed a comparable rise in viable probiotic cells during the spontaneous fermentation of guava by-products. The success of fermentation processes heavily depends on the growth capability of probiotic bacteria, as their viability is essential for ensuring the quality and stability of organic fermented products. According to international standards, for fermented products to claim health benefits, they must contain at least 107 viable probiotic bacteria per gram of the product at the time of purchase [38]. The rate at which cells are produced in a given volume in FE + FOS and FE + FOS + GABA were reported to be 1.81*107 and 1.72*107 CFU/ml*h, respectively. Our research into the guava-based fermented beverage demonstrates that the cell yield of L. rhamnosus GG reaches and surpasses this standard, ensuring that the product is both compliant with these guidelines and potentially beneficial for consumer health. This achievement underscores the efficacy of our fermentation process and the quality of the resulting beverage, making it a viable candidate for health-conscious markets.

Table 1 Cell count, productivity, change in pH and titratable acidity in Control, FE + FOS & FE + FOS + GABA
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The production of short-chain fatty acids (SCFA) during the fermentation process leads to a decrease in pH levels and increase in titratable acidity (% total acids) [39]. In case of FE + FOS, the pH dropped from an initial value of 5.8 ± 0.05 to 5.16 ± 0.03 and while in case of FE + FOS + GABA pH was found to be 4.93 ± 0.02. Fortification of GABA to the FE + FOS + GABA at a concentration of 0.05% resulted in a modification of the pH value. This illustrates the impact of GABA fortification on the acidity level of the sample. Titratable acidity, indicating the total acid content, shows the control sample at 1.48 ± 0.03%, reflecting lower acidity. The FE + FOS sample shows a significant increase in acidity to 3.38 ± 0.11%, suggesting that the supplementation of FOS stimulated more production of SCFA. The FE + FOS + GABA showed the acidity (3.35 ± 0.09%) which is slightly lower than FE + FOS but higher than the control, indicating GABA slightly moderates the acidity increase caused by FOS.

3.2 Total phenolics, total flavonoids and in-vitro antioxidant activity

Bioactive properties, namely total phenolics, total flavonoids, DPPH radical scavenging activity, ferrous reducing antioxidant capacity (FRAC) of the samples were evaluated. The Fig. 1 demonstrates a significant increase in total phenolics in the samples after fermentation (*p < 0.05). Specifically, the total phenolics in the FE + FOS and FE + FOS + GABA samples were measured at 33.45 ± 0.19 and 33.21 ± 0.11 µg GAE/g (d.w), respectively, marking a notable difference from the control (29.63 ± 0.08 µg GAE/g (d.w)). This increase in total phenolics is likely due to the enzymatic degradation of polyphenol complexes by the microorganisms involved in fermentation [40]. Fermentation facilitates the hydrolysis of glycosylated phenolic compounds into free phenolic compounds, with enzymes like β-glucosidase, produced by fermenting bacteria, playing a crucial role in these complex transformations [37]. This is supported by prior studies that have noted an uptick in TPC in fruit juices fermented with Lactobacillus strains [34, 41].

Fig. 1
figure 1

Change in Total Phenolics in FE + FOS and FE + FOS + GABA. All values are expressed as Mean ± SD (n = 3). Significant difference (*p < 0.05) was observed between control versus FE + FOS/FE + FOS + GABA. Un-inoculated 10% FE + 1% FOS served as control

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Flavonoids are secondary metabolites known for their antioxidant, antibacterial, and antimicrobial properties [42]. Figure 2 illustrates the variation in total flavonoids as a result of fermentation. This enhancement is attributed due to the metabolic activities of the fermenting bacteria. Lactobacillus strains are capable of producing specific enzymes which aids in breaking down complex compounds, including flavonoid glycosides, into simpler, more absorbable forms. This enzymatic breakdown releases bound flavonoids, increasing the total flavonoid content available in the fermented beverage. Moreover, the acidic environment created during fermentation might further facilitate the release of these compounds. The process not only potentially increases the bioavailability of flavonoids but also contributes to the nutritional value of the fermented beverage by enhancing its antioxidant properties [43].

Fig. 2
figure 2

Change in Total Flavonoids in FE + FOS and FE + FOS + GABA. All values are expressed as Mean ± SD (n = 3). Significant difference (*p < 0.05) was observed between control versus FE + FOS/FE + FOS + GABA. Un-inoculated 10% FE + 1% FOS served as control

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Dietary antioxidants that are enhanced through probiotic fermentation in beverages have the remarkable ability to support and stimulate the body's cellular defense mechanisms. These natural compounds, including vitamins, polyphenols, and flavonoids, play a crucial role in safeguarding cells against oxidative stress and damage [44]. DPPH and FRAC assays were performed to examine the antioxidant potential of fermented beverage. The FE + FOS and FE + FOS + GABA exhibited significant (*p < 0.05) increase in their ability to scavenge DPPH radicals (Table 2). The capability to neutralize DPPH radicals can be assessed through the IC50 value, which indicates the necessary concentration of a sample to neutralize 50% of the DPPH radicals in the mixture. This study compared the DPPH scavenging abilities of FE + FOS and FE + FOS + GABA against a control sample. It was observed that the scavenging efficiency improved as the concentration of the samples increased. Notably, FE + FOS (0.41 ± 0.09 mg/ml) and FE + FOS + GABA (0.43 ± 0.05 mg/ml) exhibited IC50 values that were almost identical and significantly lower than that of the control (0.67 ± 0.03 mg/ml), indicating a stronger antioxidant activity. The enhanced antioxidant performance of FE + FOS and FE + FOS + GABA samples is likely attributed to the increased levels of total phenolics and flavonoids resulting from the fermentation process [43].

Table 2 IC50 values of control, FE + FOS & FE + FOS + GABA
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The FRAC of FE + FOS and FE + FOS + GABA demonstrated a positive correlation with its concentration, as indicated by the rising absorbance at 700 nm when compared to the control (Fig. 3). This relationship highlights the critical role of both the fermentation process and the integration of prebiotics like FOS and bioactive compounds such as GABA in amplifying the beverage’s ability to counter oxidative stress.

Fig. 3
figure 3

Ferrous reducing antioxidant capacity (FRAC) assay of FE + FOS and FE + FOS + GABA. All values are expressed as Mean ± SD (n = 3). Un-inoculated 10% FE + 1% FOS served as control

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Therefore, in case of FE + FOS, the fermentation process leverages the synergistic effects of FOS to nourish probiotic bacteria, thereby elevating the levels of antioxidants produced during fermentation. This enhancement is attributed to the probiotics metabolism, which, in the presence of FOS, generated a wider range of phenolic compounds known for their antioxidant activities. These compounds are capable of scavenging harmful free radicals, thus contributing to the beverage's overall antioxidant capacity. With the inclusion of GABA in the FE + FOS + GABA, the fermentation matrix becomes enriched not only with the prebiotic benefits of FOS but also with the neuroactive and health-promoting properties of GABA. The presence of GABA could also directly contribute to the antioxidant activity, considering its potential in reducing oxidative stress in biological systems.

3.3 Neuroprotective effect in SH-SY5Y cell line model

3.3.1 Cell viability assay and morphological observation

The protective effect of FE + FOS and FE + FOS + GABA against rotenone-induced toxicity was evaluated using SH-SY5Y cell lines. The percentage of cell viability treated with different concentrations of samples, subjected to rotenone-induced stress is illustrated in the Fig. 4. The dose-dependent protective effect against the cytotoxicity of rotenone was observed for the FE + FOS and FE + FOS + GABA. Exposure of SH-SY5Y cells to rotenone (100 µM), caused a significant ($$p < 0.001) decline in cell viability (viability reduced by 43.46% in comparison to untreated). Treatment of control, FE + FOS and FE + FOS + GABA (6.25–100 µg/ml) to rotenone exposed SH-SY5Y cells, caused alterations in cell viability. A significant increase (*p < 0.05) in cell viability was observed in rotenone exposed SH-SY5Y cells treated with control only at a concentration ≥ 50 μg/ml. As the concentration of the FE + FOS and FE + FOS + GABA increased from 6.25 to 100 µg/ml, there is a general trend of increasing cell viability, suggesting that the samples are providing protective effect to the SH-SY5Y cells. The FE + FOS + GABA tends to have a higher cell viability (89.34%, ***P < 0.001) relative to the FE + FOS (77.21%, **p < 0.001) at the highest concentration evaluated, suggesting that GABA's presence augments the protective response. In line with the MTT assay findings, morphological examinations indicated that the cellular damage induced by rotenone was substantially mitigated by the treatments with both FE + FOS and FE + FOS + GABA (Fig. 5). Exposure to SH-SY5Y cells to rotenone changed its morphology into an irregular form showing neurites retraction [45]. The cells that remain seem rounded and do not exhibit the elongated processes typical of healthy neuronal cells, indicative of rotenone-induced toxicity. The cells treated with FE + FOS and FE + FOS + GABA exhibit a much healthier morphology, more akin to that of untreated cells. The neurites are more pronounced and spread out, and there's a higher cell density, indicating a significant protective effect from the combined treatment of FE + FOS and GABA against rotenone toxicity.

Fig. 4
figure 4

Effect of FE + FOS and FE + FOS + GABA on cell viability of rotenone exposed SH-SY5Y cells. All values are expressed as Mean ± SD (n = 3). Significant difference ($$p < 0.01) was observed between untreated versus rotenone exposed SH-SY5Y cells; (**p < 0.01) was observed between rotenone exposed SH-SY5Y cells and control treated cells; (***p < 0.001) was observed between rotenone exposed SH-SY5Y cells and FE + FOS/FE + FOS + GABA treated cells. Un-inoculated 10% FE + 1% FOS served as control

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Fig. 5
figure 5

SH-SY5Y Cell Morphology: a Untreated SH-SY5Y cells b SH-SY5Y cells exposed to rotenone (100 µM) c Rotenone (100 µM) exposed cells treated with Control (100 µg/ml) d Rotenone (100 µM) exposed cells treated with FE + FOS (100 µg/ml) e Rotenone (100 µM) exposed cells treated with FE + FOS + GABA (100 µg/ml)

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3.3.2 Cellular anti-oxidant ability

To understand the anti-oxidative effect of FE + FOS and FE + FOS + GABA, we investigated the activity of antioxidant enzymes viz., SOD, and CAT in SH-SY5Y cells under rotenone-induced oxidative stress. As shown in Fig. 6a, b, the untreated SH-SY5Y cells demonstrated the SOD and CAT activity of 3.75 ± 0.10 IU/ml and 4.36 ± 0.15 ng/ml, respectively. The exposure of cells to rotenone (100 µM) resulted in a significant ($$$P < 0.001) decrease in the activities of both SOD (0.28 ± 0.05 IU/ml) and CAT (0.20 ± 0.03 ng/ml), underpinning the induction of oxidative stress. Subsequent treatment with a control (100 µg/ml) alongside rotenone modestly elevated SOD and CAT levels to 0.41 ± 0.06 IU/ml and 1.11 ± 0.12 ng/ml, respectively, which may reflect a slight attenuation of rotenone's oxidative impact. However, a significant (**p < 0.001) enhancement in the activity of these antioxidant enzymes was observed with FE + FOS (100 µg/ml) to the rotenone exposed cells, achieving SOD and CAT concentrations of 1.17 ± 0.06 IU/ml and 2.44 ± 0.07 ng/ml, respectively. Notably, FE + FOS with GABA at the same concentration (100 µg/ml) under rotenone challenge yielded the most substantial upregulation (***P < 0.001) in SOD and CAT levels, with recorded activities of 2.34 ± 0.13 IU/ml and 4.37 ± 0.11 ng/ml, respectively. These levels are comparable to those of the untreated control, indicating a significant mitigation of oxidative stress. These results suggest that the combined treatment of FE + FOS and GABA may confer a synergistic protective effect against rotenone-induced cellular damage, potentially through the upregulation of endogenous antioxidant defenses. It is in line with evidences from the previous studies wherein fermented Cornus officinalis [46], fermented Mentha arvensis extract [47] enhanced the activities of antioxidant enzymes. By increasing the activities of antioxidant enzymes, fermented plant extracts offer a promising natural strategy for combating oxidative stress and promoting health. Thus, the interest in fermented plant products aligns with the broader quest for natural and effective health solutions, highlighting fermentation as a valuable tool in the development of functional foods and nutraceuticals with antioxidant properties.

Fig. 6
figure 6

a and b Effect of FE + FOS and FE + FOS + GABA on activities of SOD and CAT. All values are expressed as Mean ± SD (n = 3). Significant difference ($$$p < 0.01) was observed between untreated versus rotenone (100 µM) exposed SH-SY5Y cells; (**p < 0.01) was observed between rotenone exposed SH-SY5Y cells and FE + FOS treated rotenone exposed SH-SY5Y cells; (***p < 0.001) was observed between rotenone exposed SH-SY5Y cells and FE + FOS + GABA treated rotenone exposed SH-SY5Y treated cells

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3.3.3 Intracellular ROS

ROS production was measured in the SH-SY5Y cells (Figs. 7 and 8). Figure 7 depicts DCF expression in untreated, rotenone alone and rotenone with either control, FE + FOS or FE + FOS + GABA treated SH-SY5Y cells observed using fluorescence microscopy. Our findings revealed that untreated cells exhibited minimal DCF fluorescence (Fig. 8), with a mean intensity value of 0.45 ± 0.02, serving as a baseline for comparison. Upon exposure to rotenone, a significant increase in oxidative stress was observed, as indicated by a dramatic increase in DCF fluorescence to 14.72 ± 4.73. This substantial rise underscores rotenone's capacity to induce oxidative stress, leading to elevated ROS production within the cellular environment. Rotenone exposed cells treated with control (100 µg/ml) resulted in a slight reduction in DCF intensity to 14.01 ± 4.04. However, this decrement was not statistically significant, suggesting that the control does not mitigate the oxidative stress elicited by rotenone. The FE + FOS (100 µg/ml) alongside rotenone treatment markedly reduced the DCF fluorescence to 7.81 ± 0.63. This observation indicates that FE + FOS possesses antioxidative properties capable of significantly (*p < 0.05) diminishing the oxidative stress induced by rotenone. Furthermore, FE + FOS + GABA at 100 µg/ml, in the presence of rotenone, further significantly (**p < 0.001) lowered the DCF fluorescence intensity to 5.39 ± 0.35. This reduction was the most pronounced among the rotenone-treated groups, suggesting a synergistic effect between FE + FOS and GABA in combating oxidative stress, thereby leading to a more substantial decrease in ROS production. Collectively, these results elucidate the protective effects of GABA fortified fermented guava beverage against rotenone-induced oxidative stress, highlighting their potential as antioxidative agents in cellular models exposed to mitochondrial toxins. These findings are consistent with prior research indicating the potent antioxidative properties of natural compounds in mitigating oxidative stress within neuronal environments [46, 48]. Betarbet et al. [48] demonstrated that systemic exposure to pesticides like rotenone reproduces neurodegenerative disease features by inducing oxidative stress, underscoring the importance of identifying effective antioxidative strategies. Similarly, Tian et al. [49] reported that fermented Cornus officinalis extracts exhibit protective effects against high glucose-induced oxidative stress, highlighting the potential of natural products in neuroprotective interventions.

Fig. 7
figure 7

Fluorescence microscope images depicting DCF expression in Untreated, Rotenone alone and Rotenone with either control, FE + FOS or FE + FOS + GABA treated SH-SY5Y cells at the magnification of 40 X at bright field and FL1 channels

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Fig. 8
figure 8

Effect of FE + FOS and FE + FOS + GABA on the levels of intracellular ROS. All values are expressed as Mean ± SD (n = 3). Significant difference ($$$p < 0.01) was observed between untreated versus rotenone (100 µM) exposed SH-SY5Y cells; (*p < 0.05) was observed between rotenone exposed SH-SY5Y cells and FE + FOS treated rotenone exposed SH-SY5Y cells; (**p < 0.01) was observed between rotenone exposed SH-SY5Y cells and FE + FOS + GABA treated rotenone exposed SH-SY5Y treated cells

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Moreover, our study's focus on the combined application of FE + FOS and GABA introduces a novel perspective on synergistic antioxidative responses. The significant decrease in ROS production with combined treatments suggests a potential enhancement of cellular defenses against oxidative damage. This aligns with recent studies emphasizing the neuroprotective activities of natural products and their components against Alzheimer’s disease and related neurodegenerative conditions [50]. Martinez et al. [51] further elaborate on the role of antioxidants in modulating glutamate receptors, which are critical in the pathogenesis of neurodegeneration, thus providing a mechanistic insight into how compounds like FE + FOS and GABA might confer their protective effects.

These collective findings underscore the critical role of natural and fermented products in developing therapeutic strategies against oxidative stress-induced neurodegeneration. Our study contributes to this growing body of evidence, suggesting that the combination of FE + FOS and GABA could offer a promising approach to enhancing antioxidant defense mechanisms in the context of rotenone exposure.

4 Conclusion

In conclusion, our comprehensive investigation into the fermentation of guava juice utilizing LAB, particularly L. rhamnosus GG, and its subsequent enhancement with prebiotics and bioactive compounds, has yielded a novel fermented beverage with significant health-promoting potentials. The fermentation process not only resulted in a notable increase in the population of viable probiotic cells, surpassing international health standards, but also significantly enhanced the biochemical properties of the guava beverage, including its acidity and bioactive component content. Furthermore, the introduction of FOS and GABA into the fermentation matrix significantly impacted the phenolic and flavonoid content, as well as the overall antioxidant capacity of the beverage, underscoring the synergistic effects of these additions on enhancing the beverage’s antioxidative properties.

Most importantly, the neuroprotective efficacy of the fermented guava beverage against rotenone-induced toxicity in SH-SY5Y cell lines was demonstrated. The fortified beverages, particularly FE + FOS + GABA, showed a substantial protective effect, as indicated by improved cell viability and morphology, alongside a significant reduction in intracellular ROS levels. These outcomes highlight the potential of fermented guava beverages, enhanced with FOS and GABA, as promising dietary supplements for mitigating oxidative stress and supporting neuronal health. This study not only underscores the importance of fermentation in developing functional foods with enhanced health benefits but also opens new avenues for the utilization of guava juice as a base for creating innovative, health-promoting fermented beverages.