Physicochemical and Microbiological Evaluation of Antioxidant-Rich
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Sharma et al. Bull Natl Res Cent (2021) 45:143 https://doi.org/10.1186/s42269-021-00594-y Bulletin of the National Research Centre RESEARCH Open Access Physicochemical and microbiological evaluation of antioxidant-rich traditional black carrot beverage: Kanji Chetna Sharma*, Param Pal Sahota and Sarabjit Kaur Abstract Background: Daucus carota subsp. sativus (Black carrots) are underutilized in context to their nutritional properties. In this study, Kanji, a naturally fermented probiotic beverage, was prepared using Daucus carota subsp. sativus (var. Punjab Black Beauty). Analysis of the physicochemical and microbiological profle of the naturally fermented Kanji was investigated to boost its utilization for commercial purpose. Results: The physicochemical parameters observed in the fermented Kanji beverage were pH 3.47, total soluble solids 3°B, lactic acid 0.99%, total sugars 36.32 mg/mL, total reducing sugars 27.16 mg/mL, favonoids 38.14 mg/mL, phenols 40.8 mg/mL, antioxidant activity 79.96% and ascorbic acid 110 mg/100 mL. The microbiological analysis revealed an exponential increase in lactic acid bacterial count from 3.96 to 8.33 log CFU/mL. Out of 11 bacterial strains isolated from Kanji, the bacterial strain with high growth potential was genotypically characterized as Pediococcus acidilactici with accession number MK028218. Conclusion: The lactic acid bacterial Kanji beverage was characterized as a potential plant-based probiotic with high antioxidant activity. This functional autochthonous starter from the Kanji can be used for selective fermentation of black carrots for Kanji ascertaining its microbiological safety, unique favor and aroma, and consumption as a commer- cial non-dairy-based probiotic beverage. Keywords: Black carrots, Fermentation, Functional foods, Lactic acid bacteria, Nutraceuticals, Pediococcus Background suggested by health and medical professionals (Perricone Fermentation, from the ages, serves as a food processing et al. 2010). Probiotics, by defnition, are “viable single or method harnessing microorganisms, primarily lactic acid mixed bacterial preparation which, when provided to the bacteria (LAB), and yeast. In contrast with their unfer- host, benefts them by enhancing the properties of the mented counterparts, consumers prefer fermented prod- gut fora” (In’t Veld et al. 1994). Probiotics are required 6 ucts because of their characteristic aroma, texture, taste, to be taken regularly in adequate doses (≥ 10 CFU/mL and color (Rice-Evans et al. 1997). Probiotic food prod- daily) to avoid washout and ensuring the benefts are ucts are in concatenation with such foods and are viable acquired in a sustained manner. selective microbial dietary supplements provided in an Most probiotic foods were constricted to dairy prod- appropriate amount to bestow health benefts apart from ucts but nowadays, plant-based fermented products are the general nutrition. Te impact on human health by being increasingly considered as vectors for probiotic probiotic lactic acid fermented foods has been promptly delivery (Soccol et al. 2010). Signifcant number of lac- tose intolerant people, higher cholesterol levels of dairy *Correspondence: [email protected] foods, and pronounced economic reasons in various Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab 141004, India © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Sharma et al. Bull Natl Res Cent (2021) 45:143 Page 2 of 8 developing countries necessitate the seek out for the Methods alternatives with higher nutritive value along with health- Fermentation process promoting factors, e.g., vegetables, fruits, cereals, and Black carrots (Daucus carota subsp. sativus) of variety legumes, etc. (Perricone et al. 2010) and from products Punjab Black Beauty were procured from the Depart- which have lower cholesterol content nevertheless rich ment of Vegetable Sciences, PAU, Ludhiana, Punjab, in protein, starch, minerals, fbres, vitamins, and antioxi- India. Common salt and rye as adjuvants were purchased dant contents. Besides this, with the popularism of veg- from the local market, Ludhiana, Punjab. Fresh black car- etarian diets in both developed and developing countries, rots (3 kg) were washed with lukewarm water containing the demand for plant-based probiotics has elevated sig- 0.01% potassium metabisulfte (KMS) solution for surface nifcantly. A large number of non-dairy-based tradition- sterilization. Te calyx was removed and carrots were ally fermented products are consumed all over the globe. aseptically lightly peeled. 3 L of boiled and then cooled However, these products have not been exploited com- bacteriologically safe water was added to 1 L of black mercially as probiotic beverages due to a lack of required carrot juice. Condiments salt and rye (60 g each) were scientifc research. Plant-based fermented products are added to the fermenter after roasting them in the micro- preferred on account of their bioactive properties along wave oven at 800 watts for 1 min. No starter culture with their wholesome nutrient profle and unique taste was added as natural autochthonous fora was allowed and favor. for the spontaneous fermentation of the Kanji bever- Anthocyanin-rich black carrots (Daucus carota subsp. age. Fermentation of black carrot beverage was allowed sativus) cultivated mainly in Northern India are utilized at room temperature (RT) for fve days which ensures in the preparation of the lactic acid fermented probiotic the establishment of lactic acid bacteria in the beverage beverage ‘Kanji’ by spontaneous fermentation of uni- and reduces the risk of contamination. Te naturally fer- formly grated black carrots with requisite condiments mented Kanji beverage was stored at RT for six months concentration. Black carrots are in the focus due to their in sterilized glass bottles. Te bottles were sterilized by high anthocyanin content and extraordinary quality autoclaving. Microbiological and physicochemical analy- parameters (Kirca et al. 2006). Kanji beverage exhibits ses were performed at an interval of 20 days. diuretic, digestive tract soothing, hepatoprotective, and uterine stimulating potential. Te major anthocyanins in Microbiological analysis of naturally fermented Kanji these carrots are derived from the acylation of cyanidins Te traditionally fermented Kanji samples were diluted (Rice-Evans et al. 1997) and have greater color stability serially in 0.9% autoclaved saline and inoculated onto at food pH in contrast to the other anthocyanin sources de Man Rogosa and Sharpe (MRS) agar plates and were of plant origin. Studies also suggest that LAB strains incubated at 37 °C for 24–48 h for total lactic acid bacte- from Kanji showed their potential as probiotics and rial counts. For the count of total mesophilic aerobic bac- showed acid-bile salt tolerance, cholesterol assimilation, teria (TMAB), plate count agar was used with incubation and antimicrobial activity against food-borne pathogens at 37 °C for 24–48 h. Enumeration of total yeast count (Lamba et al. 2019). Te identifed strains were K1a: Lac- was performed using potato dextrose agar and plates tobacillus curvatus, Lactobacillus delbrueckii, and K23c: were incubated at 28 °C for three days. Te total coliform Lactobacillus coryniformis (Reddy et al. 2007). Fur- count was performed by sample enumeration on crystal thermore, Lactobacillus plantarum isolated from Kan- violet neutral red bile lactose agar (VRBL agar) at 37 °C jika was evaluated as a potential source of Vitamin B12 for 24–48 h. (Madhu et al. 2010). Te autochthonous LAB varies as a function of the Biochemical and genotypic characterization of bacterial quality of the raw material, harvesting conditions, and strains temperature. It was shown that the use of a starter cul- Te biochemical and phenotypic characteristics of the ture helps to standardize the fermentation by control- lactic acid strains were compared with the previously ling the microbial fora (Font de Valdez et al. 1990). Tis published data for the identifcation of the cultures. study was undertaken to study the nutritional profle of Bacterial isolate with high growth potential was further Kanji beverage and the isolation and characterization of analyzed by molecular characterization. After isolating the lactic acid cultures (LAB) present during fermenta- Genomic DNA using the Schubert method (Schubert tion from their natural environment. Tis study can be et al. 2008), the forward and reverse primers 27F (5′AGA the basis for the development of potential starter cultures GTT TGATCMTGG CTC AG3′) and 1492R (5′TAC GGY with predictable characteristics which can be used for TAC CTT GTT ACG ACTT3′) were used in the amplifca- commercial production of Kanji with consistent quality tion of the 16S gene. Te PCR reaction was carried out and good nutritive profle. in 100 μL volume (10 μL 10 × PCR bufer, 2.5 μL dNTPs Sharma et al. Bull Natl Res Cent (2021) 45:143 Page 3 of 8 mix, 2.5 μL each primer, 1 μL Taq DNA polymerase) Antioxidant activity (%) by DPPH assay with 3 μL DNA template. Te PCR cycle (Peltier Ter- % Antioxidant activity was measured by DPPH method mal Cycler, BIORAD) was conducted with the follow- (Brand-Williams et al. 1995). For estimation, a 100 µL ing program: 95 °C for 5 min, 30 cycles of 1 min at 95 °C, sample was taken and 3.9 mL DPPH solution (1 μM 1 min at 55 °C, 1 min at 72° and 5 min at 72 °C.