Nutritional and phytochemical properties of Pakhoi a traditional fermented beverage from Uttarakhand, India

Abstract

Pakhoi is a traditional fermented beverage consumed by the Jaunsari community of Uttarakhand, India. To ferment Pakhoi a starter culture rich in ethnomedicinal plants named Keem is used. Despite the cultural significance and traditional health claims associated with Pakhoi, scientific evidence regarding its chemical and nutritional composition remains limited. In this study, Pakhoi samples collected from three sites within the Chakrata region were analysed to evaluate their metabolomic profile, nutritional composition, antioxidant capacity, and antimicrobial activity. LC-QTOF-MS analysis revealed variation in bioactive compounds among sampling sites, reflecting differences in local ingredients and fermentation practices. The main metabolites detected could be 6-gingerol, caffeine, capsaicin, camptothecin, caffeyl alcohol, butin and carteolol. The sample with the most metabolites richness was also assessed concerning physicochemical and nutritional parameters. Pakhoi was found to have an acidic pH (3.01 ± 0.02), high carbohydrate and significant crude protein levels. Phytochemical revealed that the total phenolic (7.05 mg GAE mL-1) and flavonoid content (56.85 mg rutin equivalents mL-1) is high and is associated with high antioxidant activity (DPPH: 46.34 ± 0.01%; ABTS: 41.45 ± 0.07; FRAP: 65.38 mM Fe equivalents). Pakhoi is also inhibitory in food-borne pathogens such as Escherichia coli, Salmonella typhi, Shigella sonnei and Vibrio cholerae. In general, the results indicate that Pakhoi is a valuable nutritionally rich phytochemically diverse fermented drink with a strong antioxidant and antimicrobial potential, which proves its traditional use and suggests its potential as a functional fermented beverage.

Introduction

Fermented consumables have known to be a vital part of human society throughout history, with traditional fermentation techniques remaining largely unchanged across generations. Tribal communities worldwide have preserved the expertise in fermenting foods and beverages¹. The rising popularity of plant-based diets has further increased the demand for fermented products^2,3. During the fermentation process the substrates goes through a variety of chemical processes that in turn enhances the sensory qualities of the food or drinks along with providing an extended shelf life, it also increase the amount of bioactive compounds. The resulting products offer improved texture and flavours while containing beneficial probiotic microflora that enhance nutritional value^4–6.

Research has documented numerous health benefits of traditional fermented beverages. Chinese huangjiu contains catechin and syringic acid which have antioxidant properties^7,8. Japanese sake exhibits significant antioxidant and anti-inflammatory effects⁹. Traditional wines and beers contain compounds that may reduce cardiovascular disease, arteriosclerosis, hypertension, and diabetes^10–12.

The high-altitude provinces of India are well recognized for the indigenous fermented beverages which are prepared by utilizing ethnomedicinal plants, which contribute to the distinctive therapeutic properties of these beverages^13,14. Tongba a millet-based traditional drink from Sikkim and Arunachal Pradesh, appreciated for digestive and cardiovascular benefits¹⁵. Chyaang from Sikkim and Darjeeling, used to alleviate respiratory symptoms¹⁶.Chaang from Uttarakhand’s Bhotiya community, traditionally used for joint pain¹⁷and Jaan and Kacchi from Uttarakhand, consumed for digestive and respiratory ailments^18,19. The Jaunsari tribe of Uttarakhand produces a traditional fermented beverage known as Pakhoi. A unique starter culture called Keem, made from over 40 indigenous medicinal plants²⁰ (listed in Supplementary Table 1) is used for the preparation of Pakhoi. Detailed preparation methods and the ethnocultural significance of Keem in Himalayan fermented beverages have been reported previously²¹.

Pakhoi can be fermented using sugar-rich fruits such as apples, peaches, pears, and apricots, or cooked cereals including barley, finger millet, and rice as substrates. Approximately 10 kg of fruit pulp or cooked grain is mixed with jaggery and one-quarter of a powdered Keem cake in an earthen pot. The pot is then sealed with wheat dough and wrapped in woollen cloth to retain warmth. Fermentation proceeds for 7–10 days, continuing until CO₂ generation subsides. Pakhoi is traditionally consumed as a mildly alcoholic fermented beverage. It is a non-distilled drink consumed occasionally during events of cultural and social importance, such as marriage and festivals.

In this present study, the Pakhoi samples were collected from three different sites of Chakrata were analysed to find variations in chemical composition by LCMS and further we explored its antimicrobial potential against Vibrio cholerae (MTCC 3906), Escherichia coli (MTCC 687), Shigella sonnei (MTCC 2957) and Salmonella typhi (MTCC 733). We have also examined the physicochemical properties, nutritional composition, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of Pakhoi. Moreover, commercially available red wine, white wine and malt beer were used to conduct a comparative analysis to position Pakhoi in the perspective of other well-known fermented drinks around the world. The research gives the beverage a scientific ground to the health claims which may justify its traditional use.

Materials and methods

Sample collection

Samples were collected from three distinct sites within the Chakrata–Tons Valley region of Jaunsar, Uttarakhand, India. For consistency across analyses and to respect traditional knowledge–associated practices, the sampling sites are referred to as Location 1, Location 2, and Location 3 throughout the manuscript Samples were then transferred into sterile amber bottles and maintained at 7 °C until further analysis. For comparative assessment, red wine (Reisha, Shiraz Reserve 2017), white wine (Sula Dia White Sparkler), and malt beer (Feldschlößchen) were purchased locally in Dehradun, Uttarakhand.

LC-QTOF-MS analysis

Non-targeted metabolomic profiling of the ethyl acetate (EA) extract was performed using an Agilent 1290 Infinity II UPLC coupled to a 6546 QTOF-MS (Agilent Technologies, USA)²². Separation was achieved in negative ion mode on a ZORBAX RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) with mobile phases of 0.1% formic acid in water (A) and methanol (B). The gradient program was 5% B (0–2 min), 5–95% B (2–14 min), held at 95% B (14–16 min), and re-equilibrated to 5% B (18.1–20 min). Flow rate: 0.4 mL min⁻¹; injection volume: 5 µL.

MS parameters: capillary voltage 3500 V; gas temp 320 °C; drying gas 8 L min⁻¹; nebulizer 35 psi; sheath gas 350 °C at 11 L min⁻¹; fragmentor 80 V. Data were acquired from m/z 50–1500 at 3 spectra s⁻¹.

Raw files were processed with MassHunter Workstation (v10.0, Agilent) using molecular feature extraction (MFE). Processed features were aligned and exported to Mass Profiler Professional (v15.1) for statistical evaluation (2720 entities). PCA and ANOVA (p < 0.05) identified significant differences among beverages (201 entities). Data preprocessing and multivariate statistical analysis followed established best-practice guidelines for large-scale LC–MS-based metabolomic studies²³A < 5 ppm mass-accuracy threshold was applied, and tentative identifications (137 entities) were assigned via METLIN metabolite database²⁴.

Physicochemical composition analyses

The physicochemical tests like pH, ash along with energy, sugar, fat, fibre, carbohydrate, and protein were performed using standard protocols suggested by AOAC²⁵.

Preliminary phytochemistry analyses

Presence of phytochemicals flavonoids, alkaloids, steroids, terpenoids, glycosides, saponins, and tannins in Pakhoi, red wine, white wine, and malt beer was unravelled by the use of standard procedures^26,27.

Estimation of total flavonoid content (TFC)

TPC was estimated according to the modified protocol of Anand et al. (2015). Quantification employed a rutin standard curve (R² = 0.9838), and results were expressed as mg rutin equivalents per mL of sample²⁸.

Estimation of total phenol content (TPC)

The Folin–Ciocalteu colorimetric methodwas employed to measure the phenol concentration, and absorbance was measured at 765 nm. The results were expressed as mg gallic acid equivalents (GAE) per gram.)²⁹.

DPPH free-radical scavenging assay for the DPPH free-radical scavenging activity

A DPPH stock solution (24 mg in 100 mL methanol) was made and kept at -20 °C. 10 mL of stock was diluted with 45 mL of methanol to create the working solution (absorbance = 1.10 ± 0.02 at 515 nm). 1.9 mL of DPPH solution and 100 µL of sample extract were combined for the experiment, which was then incubated in the dark for two hours. At 515 nm, absorbance was then measured. The standard was ascorbic acid (0.0156–1.0 mg/mL).

Scavenging rate (%) = [(A₀ − A₁)/A₀] × 100 was used to determine the scavenging percentage.

where A1 represented the absorbance while the extract was present and A0 represented the absorbance of the control (without extract)^30,31.

Evaluation of reducing Power–Based antioxidant activity (FRAP)

Solution A (0.30 M acetate buffer, pH 3.6), solution B (0.01 M TPTZ in 0.04 M HCl), and solution C (0.02 M FeCl₃·6 H₂O) were mixed in a 25:2.5:2.5 ratio and incubated at 37 °C for 4 min to create the fresh FRAP reagent. 2.85 mL of FRAP reagent and 0.1 mL of sample were mixed for analysis, and the mixture was left in the dark for 30 min. At 593 nm, absorbance was observed. The results were reported as mmol Trolox equivalents (TE) per L using trolox (10–50 µmol) as a benchmark^32,33.

Trolox equivalent antioxidant capacity (TEAC) using ABTS (2,2’-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid)

By mixing 7 mmol/L ABTS with 2.45 mmol/L potassium persulfate and letting the mixture sit in the dark, the ABTS•⁺ radical was produced. Ethanol was used to dilute the resultant stock until its absorbance at 734 nm was 0.70 ± 0.02. After mixing 4.85 mL of ABTS•⁺ solution with 1 mg of extract in 1 mL of methanol, the absorbance was measured at 734 nm after 6 min. The standard curve was built using trolox (10–50 µmol). The percentage of scavenging was calculated as follows:

% ABTS radical scavenging = [(A0 − A1)/A0] × 100.

where A₀ and A₁ represent the control and sample absorbance, respectively. The values were reported as Trolox equivalents (mM/g) of extract^34,35.

Antimicrobial activity of Pakhoi

Antimicrobial efficacy of sample was evaluated with the standard well diffusion technique, following the methods suggested by Ginovyan and co-workers with slight modification, against the targeted microorganisms³⁶. Additional wells with Different concentrations of ethanol were also accessed for antimicrobial activity. The test organisms used were Vibrio cholerae (MTCC 3906), Escherichia coli (MTCC 687), Shigella sonnei (MTCC 2957), and Salmonella typhi (MTCC 733).

Statistical analyses

The results represent the mean ± standard deviation (SD) of three independent experiments. The calibration equations and data validation were done using Microsoft Excel 2013 (version 15.0.4981.1001; Microsoft Corporation, Redmond, WA, USA). Statistical significance between samples was assessed using two-way ANOVA without replication, with p < 0.05 acting as the significance criteria.

Results

LC-QTOF-MS

The Venn diagram in Fig. 1 shows a total of 2720 entities, out of which 252 are identified as markers, with 137 entities identified based on accurate HRMS mass by METLIN. The heatmap in Fig. 2 depicts the concentration and distribution of several chemical entities present in pakhoi collected from three different places. The heatmap represents a wide range of chemical substances. The variance in the identified markers can be seen in Fig. 3. The heatmap shows that Location 1 contains much higher quantities of 6-gingerol, caffeine, and capsaicin. Figure 3 shows a comparative level data of compound identified from the analysis. Caffeine and Capsaicin are predominantly higher in Location 1, indicating that this environment or sample condition might favor the synthesis or stability of these compounds. Camptothecin and Caffeyl alcohol are more concentrated in Location 2, Butin and Colneleic acid are significantly present in Location 3. ThePA compounds found only in Location 1 are 3-O-Methylgallate, Cucurbitacin O, Kakuol, Linifolin A, and Triacanthine. The compounds found only in Location 2 are Erythrinasinate A, Gravolenic acid, Lusitanicoside, Montanol, Palmatoside G, Stearolic acid, and Vernodalol. The compounds found only in Location 3 are (-)-Medicocarpin, 5-O-Feruloylquinic acid, Glucofrangulin B, Myristicanol A, and Paeonolide. The compounds found in both Locations 1 and 2 are 7-beta-D-Glucopyranosyloxybutylidenephthalide, Butin, Diferuloylputrescine, Protobassic acid, and Tyromycic acid. The compounds found in both Locations 1 and 3 are 2-Hydroxy-5-methylquinone, 3,3’-Dihydroxy-4’,5,7-trimethoxyflavone, 2-Hydroxy-5-methylquinone, Camellianin A, Dihydrogranaticin, and Ethyl Aconitate. The compounds found in both Locations 2 and 3 are Salvinorin A, Caffeyl alcohol, 6-beta-D-Glucopyranosyl-4’,5-dihydroxy-3’,7-dimethoxyflavone, Genipin, and Depsipeptide. The compounds found in all three locations (Locations 1, 2, and 3) are Location, Aurantio-obtusin, Kaempferol 7-O-glucoside, Dihydrodaidzein 7-O-glucuronide, Multifidol, Menisdaurilide, Nortrachelogenin, Paeonilactone B, Paeonilactone C, Palmitic Acid, Nortrachelogenin, Phloionolic acid, and Tectoridin.

Fig. 1.

The Venn diagram shows a total of 2720 entities, out of which 252 are identified as markers, with 137 entities identified based on accurate HRMS mass by METLIN.

Fig. 2.

The Heatmap displays 137 identified compounds based on the METLIN database search by HRMS accurate mass and illustrates the relative differences between Location 1, Location 2, and Location 3.

Fig. 3.

Venn diagram showing the distribution of LC–QTOF–MS marker compounds in Pakhoi collected from three sampling locations (L1, L2, L3). Numbers in each region indicate the count of compounds unique to or shared among locations; callouts list the compound names for that region.

The metabolite profiles of Pakhoi samples taken from three distinct locations were compared using Principal Component Analysis (PCA) (Fig. 4). Principal component analysis (PCA) performed using 252 biomarkers showed clear separation among the Pakhoi samples, with PC1 and PC2 accounting for 63.33% and 36.45% of the total variance, respectively. The PCA plot made it evident that Location 1’s metabolite composition differs from that of Locations 2 and 3. Despite the fact that the compound richness was similar at all three sites, Location 1’s distinct PCA positioning indicates that it has a distinct chemical profile. This observation led to the selection of Location 1 for more in-depth examinations in order to investigate its phytochemical and biological potential.

Fig. 4.

Principal component analysis (PCA) based on 252 biomarkers detected across Pakhoi samples collected from three locations. Principal Component 1 (PC1) explains 63.33% of the variance, while Principal Component 2 (PC2) explains 36.45%, highlighting differences in overall biomarker profiles among samples.

Physicochemical composition analyses

The traditional beverage, pakhoi, was pre-tested on the basis of pH, ash, calories, sugar, fat, fibre, carbohydrate and protein. A detailed comparison of the nutritional content of the drinks, i.e., Pakhoi, red wine, white wine and malt beer is given in Table 1. The result revealed that Pakhoi (pH 3.01 ± 0.02) is more acidic in nature followed by commercially prepared white wine (pH 3.4 ± 0.1), red wine, or malt beer (pH 4.3 ± 0.01, 4.3 ± 0.1 respectively). Moreover, the carbohydrate content is also higher in Pakhoi (8.26 ± 0.36%) while commercially available red wine, white wine, and malt beer have comparatively low carbohydrate (0.424 ± 0.20%, 0.41 ± 0.03%, 0.362 ± 0.04% respectively) The highest, calorie content per 100mL ranges from 136.61 ± 0.11 Kcal/100 mL in Pakhoi, while the least calorie content of 6.926 ± 0.12 kcal/100mL was reported in white wine. The sugar content in Pakhoi was depicted highest 1.367 ± 0.11 mg/mL, while red wine contains the lowest sugar content at 0.197 ± 0.03 mg/mL. The red wine has the highest total dietary fibre content at 0.833 ± 0.41%, whereas malt beer has the lowest fibre at 0.33%. Malt beer has the highest percentage of total fat at 0.38 ± 0.08. The sodium content varies from 6.05 ± 0.13% in malt beer, the highest. Pakhoi also has the highest percentage of crude protein at 25.15 ± 0.26%, while white wine has the lowest at 0.70 ± 0.46%. Pakhoi has an ash content of 0.48%, while red wine has a relatively low ash content of 0.138%, while both white wine and malt beer have higher ash contents of 0.374% and 0.490% respectively.

Table 1.

Physicochemical and proximate analysis of traditional beverage pakhoi, and commercially available red wine, white wine and malt beer.

S. No.	Parameter	Pakhoi	Red wine	White wine	Malt beer
1	Calories(kcal/100mL)	136.61 ± 0.11	6.926 ± 0.12	12.67 ± 0.28	18.10 ± 0.13
2	Sugar(mg/mL)	1.367 ± 0.11	0.241 ± 0.025	0.937 ± 0.03	1.597 ± 0.03
3	Total Dietary Fiber (%)	0.436 ± 0.56	0.833 ± 0.41	1.53 ± 0.37	0.33 ± 0.11
4	Total Fat (%)	0.33 ± 0.01	0.27 ± 0.23	0.27 ± 0.07	0.38 ± 0.08
5	Sodium (%)	0.41 ± 0.41	10.62 ± 0.37	4.74 ± 0.15	6.05 ± 0.13
6	Total Carbohydrate (%)	8.26 ± 0.36	0.424 ± 0.20	0.41 ± 0.03	0.362 ± 0.04
7	Crude Protein (%)	25.15 ± 0.26	0.70 ± 0.46	2.15 ± 0.5	3.91 ± 0.44
8	pH	3.01 ± 0.02	4.3 ± 0.01	3.4 ± 0.1	4.3 ± 0.1
9	Ash (%)	0.48 ± 0.09	0.138 ± 0.00	0.374 ± 0.012	0.490 ± 0.009

Preliminary qualitative phytochemical analysis

The beverages were analysed for the presence of the phytochemicals like phenols, terpenoids, alkaloids, flavonoids, and carbohydrates, saponins and glycosides was also investigated. The outcomes of the qualitative test conducted on Pakhoi, red wine, white wine, and malt beer were shown in Table 2. Glycosides were present in all the beverages, Pakhoi, and Malt beer have a moderate concentration while both red wine and white wine demonstrating a high presence. Phenols were detected in Pakhoi, Red wine, White wine, and Malt beer. Among these beverages, Pakhoi and Red wine showed a high presence of phenols, while White wine have a moderate presence. White wine showed a high presence of steroids while Pakhoi, Malt beer and Red wine exhibited a moderate presence. Similarly, flavonoids were detected in all the drinks with Pakhoi and Red wine showing a high presence of flavonoids and White wine and malt beer displaying a moderate presence. Terpenoids were found only in Pakhoi and Red wine however, the presence of terpenoids was moderate in Pakhoi and low in red wine. Alkaloids were also present in Pakhoi and Red wine, with Pakhoi displaying a moderate presence and red wine showing a low presence. Saponins were only detected in Malt Beer, where they displayed a moderate presence. The overall results suggest that the presence and quantity of phytochemicals in the tested beverages vary widely.

Table 2.

Qualitative Estimation of phytochemicals in the traditional drink pakhoi.

S.No.	Qualitative Test	Pakhoi	Red wine	White wine	Malt Beer
1.	Glycosides	+	++	++	+
2.	Phenols	++	++	+	+
3.	Steroids	+	+	++	+
4.	Flavonoid	++	++	+	+
5.	Terpenoids	+	+	-	-
6.	Alkaloid	+	+	-	-
7.	Saponins	-	-	-	+

Where “+” indicates the presences of phytochemicals and “-“indicates absence of the phytochemical.

Preliminary quantitative phytochemical analysis

The following tests were conducted to determine the TPC, TFC, DPPH, ABTS, and FRAP levels of each drink. Table 3 provides the results of different tests conducted on four different drinks: Pakhoi, Red Wine, White Wine, and Malt Beer. There were significant level of difference (P < 0.05) in the TPC, TFC, antioxidant potential of the drinks. TPC is a measure of the quantity of phenolic compounds in a sample. It was found that Red Wine had the highest TPC which is 12.477 mg GAE/mL, followed by Pakhoi 7.05 mg GAE/mL, and White Wine 3.2334 mg GAE/mL. Malt beer had the lowest TPC at 3.6801 mg GAE/mL. Red Wine presented the highest level of TFC 192.70 mg of rutin equivalent/mL, followed by Pakhoi 56.85 mg of rutin equivalent/mL and Malt Beer 15.17 mg of rutin equivalent/mL while white wine had the lowest TFC at 12.28 mg of rutin equivalent/mL. The DPPH test results showed that Red Wine had the highest antioxidant activity with a % scavenging of 68.89 ± 0.01, followed by pakhoi 46.34 ± 0.01, White Wine with a value of 45.05 ± 0.1, and Malt Beer with the value of 11.07 ± 0.29. ABTS assay results showed that Red Wine 43.50 ± 0.01 have the highest antioxidant activity, followed by pakhoi 41.45 ± 0.07, White Wine with a value of 40.04 ± 0.01, and Malt Beer with 37.23 ± 0.0. The higher FRAP value, implies higher the antioxidant activity. It was found that Red Wine had the highest FRAP value at 87.44 ± 0.01, followed by Pakhoi at 65.38, White Wine at 64.10 ± 0.01, and Malt Beer at 54.62 ± 0.02. The results of the tests indicate that Red Wine and Pakhoi have higher total phenolic and flavonoid content, as well as higher antioxidant activity compared to White Wine and Malt Beer.

Table 3.

Quantitative Estimation of total phenolic content (TPC), total flavonoid content (TFC), and DPPH free radical scavenging activity for various beverages.

Drink	Total Phenolic content (mg of GAE/mL)	Total Flavonoid Content (mg of rutin equivalent/mL)	DPPH (%)	ABTS (%)	FRAP (µM Fe(II)/L)
Pakhoi	7.08 ± 0.009a	56.86 ± 0.00b	46.34 ± 0.01c	41.45 ± 0.07d	65.38 ± 0.04e
Red wine	12.477 ± 0.05a	192.71 ± 0.04 b	68.89 ± 0.01 c	43.50 ± 0.01 d	87.44 ± 0.01 e
White wine	3.2334 ± 0.01a	12.29 ± 0.00 b	45.05 ± 0.1 c	40.04 ± 0.01 d	64.10 ± 0.01 e
Malt beer	3.6801 ± 0.009a	15.17 ± 0.01 b	11.07 ± 0.29 c	37.23 ± 0.0 d	54.62 ± 0.02 e

*Values are expressed as mean SD (n = 2). Means in the same column with same letters are significantly different (P < 0.05). Row (degree of freedom = 3), column (degree of freedom = 4).

Antimicrobial activity of Pakhoi

The antimicrobial activity tests result of the sample against selected pathogenic bacteria is provided in Table 4; Fig. 5. In the case of Vibrio cholerae, the positive control (Ciprofloxacin 200ppm) demonstrated a zone of inhibition of 22.83 ± 0.64 mm, while negative control (NC) showed no zone of inhibition. In comparison, Pakhoi resulted in a ZOI of 6.33 ± 0.57 mm, which was higher than the zones observed with 5% ethanol (9.50 ± 0.44 mm) and 10% ethanol (10.57 ± 1.91 mm). For Escherichia coli, the positive control exhibited a zone of inhibition of 30.90 ± 1.90 mm, with the negative control showing no inhibition zone. Pakhoi demonstrated an inhibitory zone of 22.80 ± 0.69 mm, which is better than inhibitory effect of 5% ethanol (11.10 ± 0.85 mm) and 10% ethanol (12.97 ± 0.96 mm). In a similar manner, the positive control contained 28.43 ± 1.56 mm zone of inhibition in the case of Shigella sonnei and the zone of inhibition of 21.23 ± 1.66 mm in Pakhoi as compared to 8.37 ± 0.64 mm in the case of 5% ethanol and 9.13 ± 0.60 mm in the case of 10% ethanol. Lastly, in case of Salmonella typhi, positive control gave a zone of inhibition of 28.40 ± 0.53 mm and negative control did not inhibit. Pakhoi had a zone of inhibition of 21.53 ± 0.59 mm, which was stronger than that of 5% ethanol (5.13 ± 4.46 mm) and 10% ethanol (8.03 ± 0.85 mm).

Table 4.

Measures of zone of Inhibition of sample against vibrio cholerae (MTCC 3906), Escherichia coli (MTCC 687), Shigella sonnei (MTCC 2957), and Salmonella Typhi (MTCC 733).

S.No.	Test organism	Well code	Sample	Zone of Inhibition (mm)			Mean ± SD
				R1	R2	R3
1	Vibrio cholerae	S	Pakhoi	6	7	6	6.33 ± 0.57
2		PC	Positive control	23.2	22.1	23.2	22.83 ± 0.64
3		5%	5% Ethanol	9.3	10	9.2	9.50 ± 0.44
4		10%	10% Ethanol	12	11.3	8.4	10.57 ± 1.91
5		NC	Negative control	0	0	0	0.00 ± 0.00
1	Escherichia coli	S	Pakhoi	23.2	22	23.2	22.80 ± 0.69
2		PC	Positive control	32.5	28.8	31.4	30.90 ± 1.90
3		15%	5% Ethanol	10.3	12	11	11.10 ± 0.85
4		20%	10% Ethanol	12.1	14	12.8	12.97 ± 0.96
5		NC	Negative control	0	0	0	0.00 ± 0.00
1	Shigella sonnei	S	Pakhoi	23	21	19.7	21.23 ± 1.66
2		PC	Positive control	27	30.1	28.2	28.43 ± 1.56
3		5%	5% Ethanol	8	9.1	8	8.37 ± 0.64
4		10%	10% Ethanol	8.5	9.7	9.2	9.13 ± 0.60
5		NC	Negative control	0	0	0	0.00 ± 0.00
1	Salmonella typhi	S	Pakhoi	21.1	22.2	21.3	21.53 ± 0.59
2		PC	Positive control	28	29	28.2	28.40 ± 0.53
3		5%	5% Ethanol	8	0	7.4	5.13 ± 4.46
4		10%	10% Ethanol	8.9	7.2	8	8.03 ± 0.85
5		NC	Negative control	0	0	0	0.00 ± 0.00

*All the experiments were conducted in triplicates and zones expressed as mean with their standard deviations. The zone of inhibition expressed includes the size of well ≈6 mm. Ciprofloxacin (200ppm) was used as positive control and distilled water was used as negative control.

Fig. 5.

Antimicrobial activity of Pakhoi against [A] Vibrio cholerae (MTCC 3906) [B] E.coli (MTCC 687) [C] Shigella sonnei (MTCC 2957) [D] Salmonella typhi (MTCC 733).

Discussion

This paper provides a combined evaluation of Pakhoi, a traditional fermented drink in the Jaunsar area of India, in terms of its nutritional parts, phytochemical properties, bioactive compounds diversity, and antimicrobial activity. The findings show that Pakhoi is a fermented drink that is chemically complex and its characteristics are directly related to the traditional preparation, ingredients and long fermentation.

Along with traditional starter culture Keem, cereal grains are the primary substrate in the preparation of Pakhoi. In the earlier studies, the microbial consortium of Keem has been described and it was demonstrated that it harbours a highly diverse population of bacteria and yeasts³⁷. The microorganisms that are in Keem have enzymatic capabilities that aid in degradation of carbohydrates. Fermentation is also known to increase bioactivity by releasing bound phytochemicals that would otherwise not be accessible in the raw substrate and by the formation of secondary metabolites during fermentation by microbes³⁸.

The reason behind the strong antioxidant activity of Pakhoi could be the high amount of phenolic and flavonoid found in it. The same has been observed with other fermented drinks such as the Chinese rice wine where compounds such as catechin and syringic acid play a role in antioxidant capacity and the Japanese sake which has been linked with the antioxidant and anti-inflammatory activity^39,40. The similarities in fermentation-related fertilities in Pakhoi suggest that the functional properties of phytochemical can be changed due to alterations in the fermentation levels of Pakhoi. In line with this observation, the antimicrobial activity observed with the chosen microorganisms indicates that fermentation is one of the factors contributing to the overall bioactivity of Pakhoi. The study of Lee and co-workers proves this as they stated that fermentation enhanced the extraction yield and bioactivity⁴¹. This is probably possible because organic acids and phenols, as well as other secondary metabolites formed during fermentation, coexist. Instead of active single compound, it seems that the antimicrobial response is the result of the general chemical environment generated by the fermentation process.

Pakhoi also has an agreement with the previously reported antimicrobial activity in other fermented products. Vibrio cholerae has been shown to be attacked by Coffee by-product extracts⁴². Kombucha has been reported to inhibit Salmonella sp., Listeria monocytogenes, Staphylococcus spp., and Candida albicans⁴³, while fermented milk permeates reduced Escherichia coli growth⁴⁴. These findings in the current paper thus append Pakhoi to the list of natural fermentation products that have proven antimicrobial activity as well as antioxidant activity.

The analysis of biomarker and principal component analysis showed that there was apparent compositional change between Pakhoi samples across various sites. This variability is typical of traditional fermented drinks, in which the variability of raw materials, the length of fermentation, and local practices affect the microbial activity and metabolic production. Besides its phytochemical and antimicrobial properties, Pakhoi has a nutritional profile. The nutrient availability can be further improved by fermentation by partially breaking down complex carbohydrates and altering macromolecules. Both the cereal-based nutrients and metabolites formed after fermentation make Pakhoi one of the fermented beverages with nutritional and bioactive potential.

Pakhoi is considered to be a weakly alcoholic, non-distilled drink and is seldom drunk except during cultural and social occasions like marriage ceremonies and local festivals. Consumption does not take place as a normal dietary beverage. In the context of this culture, Pakhoi is similar to few other traditional fermented drinks across the world that were formerly restricted to certain geographical areas only to acquire scientific fame. Although its local use is rather restricted, the outcomes of the current research allow regarding Pakhoi as a functional food item that has the same properties as other accepted fermented drinks, which can be regarded as the need to conduct systematic scientific research on traditional beverages.

Conclusion

The primary aim of the research was to investigate the physicochemical properties, nutritional value, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity of the traditional fermented beverages referred to as pakhoi besides providing information to give an idea about the chemical profile. Though the industry of fermented drinks like wines and beers had always dominated the drink industry this research was meant to illuminate on the qualities of the traditional beverages and their potential as an alternative to the commercial production of drinks. The results show that pakhoi and red wine have better phenolic and flavonoid content and antioxidant activity in comparison to white wine and malt beer. According to laboratory results, pakhoi and red wine have health potential since they have high amounts of phytochemical compounds. An extensive evaluation of these drinks forms the basic knowledge in future studies. The determination of the quantity of compounds can be used to enhance the understanding of beverage chemistry by scientists. These discoveries serve as a basic instrument to the scientists and corporate specialists, who must have the chemical knowledge of drink ingredients to inform further studies in food science. Improving knowledge of bioactive compounds requires additional research to validate their health benefits.

Author contributions

ST- writing, data curation and investigation; JA- validated and analysed the data; KB data curation; KP- conceptualization and validation of the study, review and editing; DM and AA -review and editing.

Funding

This work was supported by a grant from Uttarakhand Council of Science and Technology for the project id: UCS&T/R&D-01/19/20/17530. We are highly thankful to the council.

Data availability

All data generated or analyzed during this study are included in this article.

Declarations

Competing interests

The authors declare no competing interests.