Introduction

Kanji is an Indian ready-to-serve fermented drink, particularly popular in North India, especially during festivals like Holi. Fermented foods are a category of functional foods that offer health benefits beyond their basic nutritional value. The diverse Indian food culture provides a wealth of functional foods that can meet our nutritional requirements and offer additional health advantages. The fermentation process not only imparts a unique flavor to the food-stuff, but also provides benefits in terms of good gut health, primarily associated with the probiotic potential of Lactobacilli.

Fermented foods and beverages also contain certain compounds that can serve as prebiotics for beneficial host microorganisms (probiotics), such as various strains of Lactobacillus and Bifidobacterium [1]. Prebiotics help to promote the growth and activity of these probiotic microbes, leading to many health benefits, including improved digestive health, nutrient absorption, and potential immune system support. Probiotic lactic acid fermented foods have become popular around the world due to their promotive health effects. Thus, the identification and incorporation of prebiotics into fermented foods and beverages has become an area of interest for researchers and food producers looking to produce food products with potential health-promoting effects.

Kanji is made using a natural lactic acid fermentation process [2]. It is traditionally prepared with grated or diced Anthocyaninrich black carrots (Daucus carota subsp. sativus), black mustard seeds (rye) and various condiments and spices for a unique tangy flavor. It has a cultural and traditional significance in some regions, where it is consumed during specific seasons to combat the effects of extreme heat and promote well-being. This unique beverage, having both prebiotic and probiotic components, has shown good diuretic effect, soothing effect on the digestive tract, and hepatoprotective actions, testifying to its immense beneficial potential.

Recipe of the beverage

The ingredients used in making the fermented black carrot drink are only a few. These include, black carrots, Brassica nigra (called raee in Hindi) seeds, powdered black pepper (Piper nigrum), red chilli (Capsicum annuum) powder, black salt (kala namak) and table salt (Figure 1). The Brassica nigra seeds are coarsely ground. The black carrots are peeled and cut into thin, long slices. These diced carrot pieces are immersed in water, the other ingredients are added and the mixture is stirred. The mixture in then placed in sun or sunlight for few days (3-5 days), preferably in a baked mud pot (matka), after which the beverage is ready for consumption.

Figure 1: The ingredients used in preparing Kanji beverage. All the ingredients are mixed together in a utensil and kept in sunlight for few days. Traditionally, clay pots are used.

Phytochemical Constituents and Therapeutic Benefits of Kanji

Phyto-compounds contained in black carrots and their beneficial effects

Carrots are rich in phytochemicals and nutrients, and provide the necessary minerals to support the growth of several bacterial strains. Phenolic compounds and antioxidants contained in carrot pulp promote anaerobic conditions and multiplication of probiotic microbes [3]. Nutritional analysis has indicated that Kanji can be a promising alternative approach to probiotic dairy products while serving as an effective plant-based vehicle for novel probiotic strains. According to the Food Safety and Standards Authority of India, it is safe to consume Kanji drinks. With 2% (w/v) sucrose, 1% (w/v) glucose, and 0.8% (w/v) fructose, carrot juice provides an excellent growth medium for probiotics by providing a good source of energy and carbon, mainly from glucose and sucrose. L. rhamnosus and L. bulgaricus showed a good growth rate in fermented carrot juice [4].

Being a multi-nutritional food source, black carrots are rich in minerals such as calcium, potassium, magnesium, phosphorus, sodium, zinc, and iron [5]. Black carrots are also a good source of ascorbic acid (vitamin C), which is a vital nutrient for various bodily functions such as collagen synthesis, wound healing, and immune system support. Black carrots contain a higher concentration of flavonoids compared to orange carrots [6]. Furthermore, black carrots are rich in carotenoids, including lutein and beta-carotene [7]. Black carrots have a high content of anthocyanin pigments, a group of phenolic compounds that provide color and are known for their anti-inflammatory, antioxidant, and anti-cancer effects [7]. The major anthocyanins in these carrots are derived from the acylation of cyanidins. Other phenolic compounds including chlorogenic acid, caffeic acid, quercetin, and ferulic acid are also present in black carrots.

Being a cheap source of anthocyanins, which have gained popularity in the food industry as natural alternatives to food colorants, black carrots have received much attention [8,9]. The Anthocyanin pigments have been found to exhibit antioxidant, anticancer, anti-inflammatory, antimicrobial, anti-obesity, and antidiabetic effects [10-12]. Thus, adding black carrot anthocyanins to food products as a coloring agent can promote better health [13,14]. Song et al., 2016 highlighted the antioxidant properties of black carrot sponge cakes. In their study, they observed an increase in total phenolic content and DPPH radical scavenging activity as the proportion of black carrot flour (BCF) was increased in sponge cake. It was concluded that black carrot sponge cake meets consumer preferences while offering several health benefits [15].

Anthocyanins present in black carrots have been found useful in the prevention of metabolic syndrome through nine biomechanisms [16,17]. Poudyal et al. 2010 found that anthocyaninrich black carrot juice was beneficial for glucose tolerance, liver function, plasma lipid profiles, abdominal obesity, and cardiac fibrosis with anti-inflammatory effects in a rat model [18]. Furthermore, anthocyanins from black carrots have shown good anti-cancerous effects [17,19]. Netzel et al found that Anthocyanin pigments from ethanolic extracts of black carrots exhibited substantial antiproliferative activity against cancer cells [20].

An experiment was conducted by Cho and Chung, 2019 where black carrot cookies were prepared with varying concentrations of black carrot powder (0%, 5%, 10%, 15%). The ingredients, including flour, baking powder, and black carrot powder, were mixed with eggs. In a separate bowl, butter and sugar were creamed and then added to the mixture. Finally, this dough was shaped into cookies and baked. Though there were no significant differences in texture, odor, and taste, however, the phenolic content was found to be increased as the concentration of black carrot powder increased, ranging from 24.0 to 103.5 mg GAE/100g. Furthermore, the addition of black carrot powder improved the cookies’ DPPH radical scavenging ability, highlighting their antioxidant potential. It was recommended that 15% black carrot powder be added to the cookie formulation to meet consumer preferences [21].

The phenolic content present in black carrot roots is higher than in other colorful root vegetables [22]. Black carrots contain a substantial quantity of phenolic acids like hydroxycinnamates and caffeic acid [9]. These phenolic compounds aid in amelioration of inflammation by inhibiting inflammatory pathways [23]. The polyacetylene compounds present in black carrots can decrease Nitric oxide production (macrophage-derived NO) by 65%. Polyacetylenes content helps in enhancing the anti-inflammatory effects of black carrots [24]. The phenolic compounds in black carrots also prevent the risk of diabetes, and fermented black carrots are effective in diabetes management and prevention [25]. The phytochemicals found in black carrots have also been associated with reduced risk of cardiovascular diseases (CVDs).

Effect of fermentation in augmenting the therapeutic effects of black carrot

During the fermentation process of this unique beverage, microflora mainly Lactobacilli, play a crucial role in breaking down the sugars in the carrots and producing lactic acid. Fermentation not only imparts a unique flavor but also provides benefits in terms of good gut health and digestion, primarily associated with the probiotic potential of Lactic Acid Bacilli (LAB). Sowami and Thorat in 2012 carried out a study to analyze the antimicrobial effect of bacteriocin derived from the Lactobacillus growing in fermented carrot kanji [26]. Bacteriocin shows potent bacteriocidal activity against Staphylococcus aureus, which is a major cause of food poisoning through toxin-mediated virulence [27]. This inhibitory activity suggests potential application of black carrot kanji as a probiotic. Singh et al. in 2021 compared the therapeutic potency of homemade Kanji and Marketed Probiotic Drink. The study results proved that traditional homemade Kanji possesses a higher probiotic, antioxidant, and antimicrobial profile as compared to the bottled probiotic drink [28].

A study by Park et al. in 2015 demonstrated that carrying out the fermentation process using Lactiplantibacillus plantarum or Aspergillus oryzae can substantially increase the total carotenoid content of black carrots while also altering the anthocyanin composition [29]. It may be noted that Lactiplantibacillus plantarum (formerly called Lactobacillus plantarum) is commonly found in many fermented food products as well as anaerobic plant matter. L. plantarum was first isolated from saliva. It is a hardy microbe and demonstrates the ability to temporarily persist in plants, and in the intestinal tract of insects and vertebrate animals. Aspergillus oryzae, is a filamentous fungus (a mold) used in East Asia, especially Japan, to saccharify rice, sweet potato, and barley in the making of alcoholic beverages. It is also used to ferment soybeans for making soy sauce and miso. The Park study confirmed that fermented carrot beverage has higher bioavailability and bio accessibility of important phyto-constituents, as compared to carrot juice.

In an experimental attempt to explore an alternative way to produce a functional bread type for healthy diet, black carrot fiber (BCF) was used to fortify bread. The quality and antioxidant properties of the Fiber Fortified Flat Bread were evaluated. It was found that there was a regular increase in phenolic content and anti-oxidant activity of both the outer and inner parts of the bread due to BCF level used. The process of making the fortified bread involved collecting black carrot pomace from blended carrots, drying it to 12% moisture, pounding it, and storing it at 4°C. Wheat flour was then mixed with carrot fiber to prepare dough, which was fermented and baked at 250°C for 5 minutes. The experiment’s results showed that a black carrot fiber level of up to 2.5 percent is suitable for formulating black carrot fiber-fortified bread, considering its physical qualities [30]. Thus, adding black carrot fiber to flatbread was recommended for boosting its antioxidant content and physical appearance. This approach presents a potential method for producing a functional bread variety for a healthy lifestyle.

Phytochemicals contained in B. nigra seeds and their health benefits

Brassica nigra (black mustard) seeds, caller rae in north India, contain a variety of phytochemicals, including alkaloids, saponins, tannins, and a range of polyphenols such as flavonoids (flavonols, flavones, flavan-3-ols, anthocyanidins, flavanones, and isoflavones) and non-flavonoids (phenolic acids, hydroxycinnamates, stilbenes, etc. [31]. The oil extracted from the seeds is rich in major fatty acids such as palmitic, stearic, oleic, linoleic, linolenic, eicosanoic, and erucic acids. The essential oil of B. nigra seeds contains components like di-(9-octadecenoyl)-glycerol, octadecadienoyl chloride, and hexadecanoic acid, 1-(hydroxymethyl)-1,2ethanediyl ester. Additionally, glucosinolates such as Sinigrin and mustard oil glucosides are significant biomolecules in B. nigra seeds [32,33]. The above phytochemicals contained in black mustard seeds have a range of very useful health effects as given below (Figure 2).

Anticancer Potential of Brassica nigra Seed Extract

Brassica nigra seed extract has demonstrated significant anticancer activity, especially against human non-small cell lung cancer cell lines. The growth-inhibitory effects include reducing the viability and clonogenic survival of cancer cells in a concentrationdependent manner. The extract also induces apoptosis, evidenced by increased caspase-3 activity, and causes DNA double-strand breaks. The cell cycle analysis revealed that the extract arrests cancer cells in the S and G2/M phases. Moreover, it suppresses the migratory and invasive properties of the cancer cells, by modulating the expression of key proteins involved in these processes. In liver tissue, the presence of isothiocyanate in the seed extract plays a key role in anti proliferative effects, suggesting protective benefits against the development or progression of liver cancer [34,35]. Furthermore, studies have shown that B nigra extract, with sinigrin as a predominant glucosinolate, exhibits notable antiproliferative activity on both non-tumor and tumor cell lines [36]. The addition of myrosinase enhances these effects, correlated with the modulation of Mitogen-Activated Protein Kinases.

Antimicrobial effects of Brassica nigra Seed Extract

The aqueous extract of Brassica nigra seeds demonstrates notable antimicrobial activity. Utilizing the agar well diffusion method, Danlami et al. found that the extract effectively inhibited several harmful bacteria, including Escherichia coli, Klebsiella pneumonia, Salmonella para-typhi, Pseudomonas aeruginosa, and Staphylococcus aureus, with inhibition zones ranging from 7mm to 23mm [37].

Antimalarial Activity

Brassica nigra seed extract exhibited significant antimalarial properties against Plasmodium berghei infection in mice. Administering different doses of the extract (100, 200, and 400 mg/kg) showed notable chemosuppressive and chemoprophylactic activities, with higher doses enhancing survival time and weight gain in Plasmodium infected mice [38]. This indicates the presence of active compounds in the seeds that could be effective for malaria treatment and prevention. This unique effect could prove to be beneficial in tropical areas like north-west India, where mosquitoes start breeding with the onset of summers around Holi (in March), and proliferate all through the summers. Co-incidentally, the dark maroon coloured Kanji drink is prepared in north India at the time of Holi, since the drink is traditionally associated with this festival of colours.

Antiviral Properties against SARS-CoV-2

Guijarro-Real et al. 2021 found that the methanolic extract of Brassica nigra seeds inhibited the SARS-CoV-2 chymotrypsinlike protease (3CLPro), crucial for the virus’s replication [39].

Protective Effects against Cadmium Chloride Toxicity on lungs

An experimental study on Brassica nigra seeds revealed their protective potential against the toxic effects of cadmium chloride (CdCl2) in female albino rats [40]. Treatment with the aqueous extract of the seeds restored normal blood parameters and mitigated lung tissue damage induced by cadmium chloride, suggesting protective effects against oxidative stress and tissue damage caused by toxic substances.

Anticonvulsant Effects

The anticonvulsant effects of Brassica nigra seed extract were observed in a study using the kindling method in mice. Doses of 75, 150, and 300 mg/Kg significantly reduced the intensity, frequency, and duration of seizures. Additionally, the extract positively affected brain tissue biochemistry, indicating antioxidant properties and suggesting its potential as a treatment for grand mal seizures [41].

Figure 2: Showsa summary of the health advantages accruing from drinking the fermented beverage made from black carrots. Black carrots contain some very useful phytochemicals that are not present in red carrots. Brassica nigra is the other important component of Kanji, that helps in fermentation, and imparts a sour, tangy taste to the beverage.