Research Article

Extraction of Phytochemicals from Beetroot Pomace and Formulation of Phytochemical Enriched Functional Yogurt

Hafsa Tauqir1, Imran Ahmad2 and Muhammad Bilal Sadiq1*

1School of Life Sciences, Forman Christian College (A Chartered University), Lahore, 54600, Pakistan; 2Food Agriculture and Biotechnology Innovation Lab (FABIL), Florida International University, Biscayne Bay Campus, North Miami, Florida USA.

Received | November 21, 2023; Accepted | February 20, 2024; Published | May 27, 2024

*Correspondence | Muhammad Bilal Sadiq, School of Life Sciences, Forman Christian College (A Chartered University), Lahore, 54600, Pakistan; Email: [email protected]

Citation | Tauqir, H., I. Ahmad and M.B. Sadiq. 2024. Extraction of phytochemicals from beetroot pomace and formulation of phytochemical enriched functional yogurt. Sarhad Journal of Agriculture, 40(2): 483-489.

DOI | https://dx.doi.org/10.17582/journal.sja/2024/40.2.483.489

Keywords | Beetroot pomace, Anthocyanins, Antioxidants, Bioactive compounds, Yogurt, Antibacterial

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Fruits and vegetables are rich in bioactive compounds such as high amount of antioxidants, fiber, phytochemicals, betalains, vitamins and minerals. Red beetroot (Beta vulgaris subsp. Vulgaris) is a dicotyledonous biennial plant belonging to Chenopodiaceae family and rich in antioxidants such as flavonoids, carotenoids and biologically active compounds such as vitamins (A, B, C, E and K), betalains, fibers, and inorganic nitrate and folates (Ben Haj Koubaier et al., 2014; Ceclu and Nistor, 2020). Beetroot has various pharmacological activities including antimicrobial, antifungal, antioxidant, anti-inflammatory, expectorant, diuretic, anticancer, antimutagenic and anti-depressant potential (Jasmitha et al., 2018). Due to the medicinal benefits, beetroot as a whole and its parts (its peel, juice and pomace) have been incorporated into various products. Beetroot pomace due to its bioactive potential and color can serve as a functional food ingredient (Janiszewska-Turak et al., 2021). Beetroot has been used in various food products in dairy, bakery and beverage industries due to the presence of colored pigments and associated health benefits (Punia-Bangar et al., 2023). Beetroot pomace is a by-product of beetroot juice processing industry and it is a rich source of antioxidants, betalains and polyphenols (Ceclu and Nistor, 2020; Vulić et al., 2013). Betalains are nitrogen-containing water-soluble pigments that are derived from the amino acid tyrosine. Betalains are abundant in beetroot and can be divided into two groups based on absorption of wavelength; betaxanthins (yellow pigments which show maximum absorbance at the wavelength of 480 nm) and betacyanin (red pigments and their maximum absorbance occurs at 540 nm). They are commercially important as they impart a stronger color than anthocyanin and the intensity of color depends highly on the ratio between the concentrations of the red violet betacyanin to the yellow orange betaxanthins (Nemzer et al., 2011). The aim of this research was to optimize the extraction of betalain pigments from beetroot pomace collected as domestic waste and evaluation of antioxidant and antimicrobial potential of BPE. Moreover, a functional yogurt product was developed by incorporating beetroot betalains. The effective utilization of beetroot pomace can provide a sustainable solution in the management of food waste and formulation of functional food products.

Materials and Methods

Sampling

The fresh beetroot pomace was collected from the local juice shops of the Lahore City of Pakistan. The pomace was dried in a hot air oven (POL-EKU-APARATURA, Poland) at 38 °C till constant weight, followed by grinding into fine powder. The powdered samples were stored in Ziplock bags covered with aluminum foil at 4 °C till further use.

Ultrasonic assisted extraction

UAE was used to extract bioactive compounds from beetroot pomace powder. The frequency (20 kHz) and amplitude (40%) were taken as constant. The independent variables for the experimental runs were taken as the solvent concentration (30%, 50%, 70%), time (15, 30, 45 min) and sample to solvent ratio (1:10, 1:15, 1:20 w/v). The solvent taken for the extraction was a mixture of ethanol and 0.5 % acetic acid (Hidalgo et al., 2018). The sample and solvent were mixed as per the experimental design and the sonication probe (Industrial sonomechanics, USA) was immersed into the solvent mixture. RSM was used for the extraction optimization and response (independent) variables were TPC, betalain concentration and DPPH Inhibition (%).

Determination of TPC

TPC of the extracts was determined spectrophotometrically using the Folin-Ciocalteu reagent as described by (Hiranrangsee et al., 2016). The results were expressed as mg of GAE/ g of sample (dry basis).

Antioxidant activity

DPPH assay was used to determine the antioxidant activity of the extract (Sadiq et al., 2015). The freshly prepared DPPH solution (5 ml of 40 ppm ethanolic solution) was taken in a test tube and 50 μl extract was added to this test tube, vortexed for 1 min and then kept in the dark for 30 min. The absorbance was read at 517nm and DPPH inhibition was determined by using Equation 1.

Where; AC= absorbance of DPPH solution and AS = absorbance of the sample.

Determination of betalains

The extract was diluted (1:10) with phosphate buffer (pH 6.5) for the determination of betalain pigments (Ravichandran et al., 2013). Betacyanins and betaxanthins were measured at 538 nm and 476 nm respectively and the sum of both yielded the total betalain content. Betalain content (BC) was determined by using the Equation 2.

Where; A= absorbance, DF= dilution factor, l= path of length and ɛ= molar extinction coefficient.

Fourier transform infrared (FTIR) analysis

FTIR analysis of the extract was performed using FTIR (Agilent technologies, USA). The spectra were scanned in the range of 4000-650 cm-1 with a resolution of 4 cm-1 (Sadiq et al., 2015).

Antibacterial activity

Antibacterial activity of the extract was determined against E. coli, S. typhimurium, S. aureus using agar well diffusion method (Khan et al., 2022). Eight different concentrations of the extract were prepared (200, 100, 50, 25, 12.5, 6.5, 3, 1.5 mg/ml) in sterilized distilled water. Nutrient agar (Oxoid, UK) plates were prepared and spread with each bacterial culture (108 CFU/ml) separately. A well of 10 mm was made in each agar plate and 200 µl of each of the extract concentration was added, followed by incubation for 24 h at 37°C. The results were interpreted as the diameter of inhibition zone around the well.

Development and characterization of BPE enriched yogurt

Beetroot extract enriched flavored yogurt was prepared as functional product by using two different concentrations of extract (2% and 4%). One liter of pasteurized full cream milk was inoculated with starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) and incubated in the dark for 5 h at 37°C, followed by refrigeration for another 5 h at 4°C (Oh et al., 2016). The starter culture 4%, v/v was added into the milk at a initial inoculum size of 5 × 10 6 colony forming unit/ml. The yogurt samples were subjected to measurement of pH, titratable acidity and total solid contents. For enumeration of lactic acid bacteria (LAB), yogurt samples were serially diluted and spread onto the surface of de Man, Rogosa and Sharpe agar plates, followed by incubation at 37 °C for 48 h and CFU/ml were counted in each sample.

Sensory evaluation of yogurt

The yogurt samples prepared with BPE (2% and 4% extract) were subjected to sensory evaluation from a panel of 16 individuals. The characteristics to be evaluated were taste, color, odor, texture, acceptance of sourness, aftertaste and overall acceptability. Sensory attributes were measured by a 9-point hedonic scale by using a slightly modified method of Kainat et al. (2023). The yogurt sample without extract was used as a control.

Statistical analysis

The significant differences among mean observations were determined by using One way ANOVA and Tukey’s tests, using SPSS version 23 (Hiranrangsee et al., 2016).

Results and Discussion

Optimized extraction

Response surface methodology (RSM) was used to determine the optimized conditions for the extraction of bioactive compounds from beetroot pomace through ultrasonic assisted extraction (UAE). The total 17 runs were proposed by RSM for the extraction process and highest responses for 2, 2-diphenyl-1-picryl hydrazyl (DPPH) inhibition (66.6%), betaxanthins (174 mg/L), betacyanins (265 mg/L) and total phenolic content (TPC) of 2.78 mg gallic acid equivalent (GAE) per gram (dry basis) were observed during optimization (Table 1). Linear model was used to assess the influence of extraction parameters on TPC and the model was significantly fit to the experimental data (p < 0.05). All the extraction parameters influence the TPC, however the nature of influence was only significant (p < 0.0001) for sample to solvent ratio. Lack of fit was insignificant, which indicated that the data was well fitted. The coefficient of correlation (R2) was 0.8. The other response variables such as DPPH and betalain content were not significantly influenced by the extraction parameter. UAE relies on the rupturing the cells of the vegetable/fruit by sound waves and hence ensures the increases surface contact between the solvent itself and the plant material. This ultimately leads to a higher extraction yield of the bioactive compounds (Kainat et al., 2023). In this study at the optimized extraction condition (48% solvent concentration, 28 min extraction time and, 1:17 sample to solvent ratio), obtained extract was dried into powder and TPC of 37.05 mg GAE/g extract was obtained, which was significantly higher than the previous result for the TPC quantified (3.67 mg GAE/g) in beetroot juice (Vasconcellos et al., 2016). UAE was reported to be an effective technique for the high yield extraction of pigments and phenolic compounds from beetroot byproducts (Fernando et al., 2021).

FTIR analysis

FTIR analysis was used to detect the various functional groups present in the beetroot pomace extract (BPE) by detecting of the peaks in the infrared region. These functional groups were correlated to the various active compounds found within the extract (Figure 1).

 

Table 1: Optimization of beetroot pomace extraction using response surface methodology.

Exp no.	Time (min)	Solvent (%)	Sample to solvent ratio	TPC (mg GAE/g)	Betacyanin (mg/L)	Betaxanthins (mg/L)	DPPH inhibition (%)
1	45	70	1/15	1.94	127	43	37.6
2	30	50	1/15	2.26	108	101	66.6
3	30	30	1/20	2.77	101	56	36.5
4	45	50	1/10	1.68	96	99	39
5	30	50	1/15	2.36	265	140	40
6	30	70	1/20	2.78	28	23	60
7	15	50	1/20	2.39	83	53	38
8	30	50	1/15	1.81	108	101	18
9	30	30	1/10	1.46	81	65	10
10	45	50	1/20	2.54	37	20	43
11	15	70	1/15	2.36	30	37	50
12	15	50	1/10	1.55	65	45	43.54
13	30	50	1/15	2.06	152	92	35
14	30	70	1/10	1.53	58	40	13
15	45	30	1/15	2.24	38	27	13
16	30	50	1/15	2.11	162	174	35
17	15	30	1/15	2.54	96	52.5	25.45

 

Table 2: FTIR analysis of beetroot pomace extract.

Range (cm-1)	Functional groups and class of compounds	Beetroot pomace extract (cm-1)
3450-3250	-OH in phenols and alcohols	3293.8
2936-2913	-CH3, -CH2 stretch in aliphatic compounds	2927.9
1650-1580 1618-1498	N-H, 1° Amines Benzene ring in aromatic compounds	1619.4
1360-1150 1410-1310	-CH2X representing alkyl halides. -OH in tertiary alcohols or phenols	1367.7
1055-1020 1150-1000	Si-O-Si in silicone or organic siloxane C-F stretch in aliphatic fluoro compounds	1026.5
700-610	Alkyne bend	667.07

 

 

The peaks identified by FTIR were assigned to the corresponding functional groups (Table 2). The most intense peak was observed at 3293.8 cm-1 indicating the –OH group in alcohols and phenols (Sadiq et al., 2015). The bands observed at 2936-2913 cm-1 referred to the presence of carboxylic acid in BPE (Kushwaha et al., 2018). The second intense peak was observed at 1026.5 cm-1 which corresponds to the Si-O-Si group in silicones or organic siloxanes or the C-F stretch in aliphatic fluoro compounds (Nandiyanto et al., 2019). The third intense peak was observed at 1619.4 cm-1 corresponding to the range 1650-1500 cm-1 indicating primary amines or benzene rings in aromatic compounds. Kushwaha et al. (2018) also reported that the absorption peaks in the range of 1000-1300 cm-1 corresponded to the presence of phenols and alcohols in BPE.

Antibacterial activity

All the test pathogens were found susceptible to BPE. At highest test concentration (200 mg/ml), inhibition zones of 29.67, 31 and 30 mm were observed against E. coli, Salmonella typhimurium and S. aureus, respectively (Table 3). Previously, Vulić et al. (2013), reported antibacterial activity of BPE against both Gram +ve and -ve bacteria, with predominant activity against S. typhimurium. Salamatullah et al. (2021) also reported that beetroot pomace extract exhibited predominant antibacterial activity against Gram negative bacteria which might be due to the difference in the cell wall composition of Gram positive and negative bacteria. The phytochemicals present in the beetroot have been reported to exhibit antibacterial and antifungal properties (Salamatullah et al., 2021).

 

Table 3: Antibacterial activity of beetroot pomace extract.

Concentration (mg/ml)	Diameter of inhibition zone (mm)
	E. coli	S. typhimurium	S. aureus
200	29.67 ±0.6a	31 ±1a	30 ± 3a
100	25.5 ±0.5b	27 ±0.6a	27 ±1a
50	21.7 ±1.5a	19.7 ±1.5a	19.7 ±2a
25	17.67 ±1.5a	16 ±1ab	13.7 ±1.5b
12.5	16 ±0.6a	15.3 ±1.1a	15.7 ±0.5a
6.25	15 ±1a	14.5 ±0.7a	14.7 ±1.5a

Different superscript letters within a row indicate means which are significantly different (p < 0.05).

 

Beetroot extract enriched yogurt

Two different yogurt samples were prepared with varying extract concentrations: i.e., 2% and 4% and a control yogurt without extract was also prepared. pH was not significantly different among the samples but there was an increase in the total slid content with the addition of extract (Table 4). Moreover, LAB count and titratable acidity were significantly increased (p < 0.05) with the increasing concentration of the BPE.

 

Table 4: Total solid content, pH, titratable acidity and LAB count for beetroot pomace enriched yogurt.

Yogurt samples enriched with extract	pH	Total solid content (%)	Titratable acidity	Lactic acid bacteria count (Log CFU/ml)
Control	5.1±0.1a	50±1.1b	0.36±0.01b	4.73±0.5b
2% extract	5.2±0.3a	55±1.6a	1±0.1a	6.92± 0.3a
4% extract	5.3±0.25a	57±1.8a	1.08±0.04a	7.5± 0.2a

Different superscript letters within a column indicate means which are significantly different (p < 0.05).

 

Sensory evaluation

After the addition of BPE in yogurt, taste score was significantly decreased in comparison to control sample, however, color and odor scores were significantly high after the extract addition (Table 5). There was no significant difference in the overall acceptability score of control and extract enriched yogurt samples. Jovanović et al. (2021) used 3% beetroot pomace flour to develop drinking yogurt samples. However, in this study the addition of 2% BPE revealed the relatively high acceptable sensory scores. Beetroot peel powder was also reported in the formulation of functional mayonnaise with improved physical, sensory and textural attributes (Lazăr et al., 2022).

 

Table 5: Sensory evaluation of beetroot extract enriched yogurt.

Sensory attributes	Yogurt with 2% extract	Yogurt with 4% extract	Control yogurt
Taste	5.6±1.9b	6.1±1.9b	7.94±0.25a
Color	8.6±0.5a	8.2±1.1a	7±1.1b
Odor	7.3±1.4a	7.3±1.3a	5.6±2.2b
Texture	7.4±1.4a	6.6±1.5a	7.5±1.4a
Aftertaste	6±1.8a	6.6±1.8a	6.37±0.96a
Overall acceptability	6.6±1.3a	6.4±1.7a	6.4±1.2a

Different superscript letters within a row indicate means which are significantly different (p < 0.05).

 

Conclusions and Recommendations

Beetroot pomace is a rich source of phenolic compounds and anthocyanins which are associated with antioxidant activities. The extract showed antibacterial activity against gram +ve and gram -ve bacteria. Based on the bioactive potential BPE can be used in the formulation of functional food products. Functional yogurt prepared by using the pomace extract received significantly high color and odor scores from the panelists due to the color of anthocyanins. Hence, BPE along with its color attributes and bioactive potential can be used in formulation of food and pharmaceutical products.

Novelty Statement

Beetroot pomace is a byproduct and usually exposed as a waste which can pose environmental hazards. Optimized extraction of TPC and anthocyanins can ensure the effective utilization of pomace in the formulation of food products as a coloring agent, preservative and functional component due to its antioxidant and antibacterial potential.

Author’s Contribution

Hafsa Tauqir: Performed all the experiments and analyzed the data.

Muhammad Bilal Sadiq: Designed the study, interpreted the data and write the manuscript.

Imran Ahmad: Did data analysis and manuscript editing.

All authors read and approved the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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