INTRODUCTION:

Red beetroot is a vegetable, enthusiastically expended in Europe. Its foundations can be eaten crude or utilized as a significant crude material in nourishment industry for creation of dried of solidified jelly, drinking what's more, concentrated juices, and regular colors (betacyanins), which are utilized as sustenance added substances. Beetroots contain naturally dynamic betalains: Betacyanins and betaxanthins^1-2. Betacyanins are red-violet shades: betanin, isobetanin, neobetanin, betanidin, and isobetanidin, while betaxanthins (red colors) are spoken to by vulgaxanthin I and vulgaxanthin II. Rich wellsprings of betacyanins are such plants as beetroot (Beta vulgaris L. ssp. vulgaris), desert plants of genera Hylocereus and Opuntia, and many blooming plant types of the family Amaranthaceae³ The importance of functional foods is increasing worldwide.

This category of food has numerous natural nutrients present in the food that have positive effect on human health. Functional food comprises products which help in reducing bp, probiotics, prebiotics and food enriched with macro and micro elements. In this paper Beta vulgaris, commonly known as red beet or beetroot is used for its medicinal properties, i.e rich in source of antioxidants, Vit C, sodium, betanin, Vit B6, B12. Researches are now being done on functional beverages made from food or vegetables fermented with probiotic bacteria in a controlled manner since they are present in abundant number in human gut in abundant number. Besides the medicinal properties of functional beverages, they are beneficial for people having lactose intolerance.

Our work aimed in the production of functional beverage by fermenting Beta vulgaris with culture of Pediococcus pentosaseus to determine it’s antibacterial activity against food borne pathogens , and also to check it’s anti-inflammatory and anti-oxidant property.

MATERIALS AND METHODS:

Sample processing and fermentation:

A pre-characterized culture of Pediococcus pentoseus was acquired from Molecular and microbiology lab, VIT University Vellore. A loop full of culture was then inoculated in MRS broth and was then incubated for 24 hours. Then the culture was centrifuged at 6000rpm for 15 mins at 4°C. The supernatant was discarded and the pellet was washed with 0.9% Sodium Chloride solution. The freshly prepared beetroot juice was pasteurized for 15mins at 80ºC to remove the undesirable microorganisms. After pasteurization, the juice was cooled to room temperature and inoculated with culture pellet. The pasteurized Beetroot juice with culture was fermented at 37°C for 24 and 48 hrs. Following fermentation, after 24 and 48hours, sample was tested for change in color and pH.

Biochemical test – Gram staining and Catalse test:

The isolated strain of Pediococcus pentoseus (VITMS07) were chracterized for Gram staining and catalase test.

Determination of pH and color:

After 48hrs of fermentation, the fermented juice samples were analyzed for pH using pH meter. The changes in pH were observed at regular interval of 24h⁴.

Determination of anti-microbial activity by well diffusion method:

The bacterial strains are tested for their antimicrobial activity against three different pathogens. The culture with fermented beet root juice were centrifuged at 10,000 rpm for 20min at 4˚C. The cell free supernatant analysed for anti-microbial activity^5-6.

Determination of antioxidant activity by DPPH method:

Antioxidant property of the fermented beetroot juice samples were analyzed by the DPPH method. DPPH solution was dissolved in methanol for the assay. Ascorbic acid were kept as control. The OD was observd at 517 nm an calculated by following formula⁷.

Percentage (%) =

volume of control - volume of test sample X 100

Volume of control

Determination of Anti-inflammatory activity:

Using HRBC method, the anti-inflammatory activity of the fermented beetroot samples were observed⁸.

RESULTS AND DISCUSSION:

Gram’s test – Gram positive cocci were observed under 100X oil immersion in bright field microscope (Fig 1).

Figure 1: Gram staining test

Catalase test:

There is no appearance of gas bubbles was observed when the organism was placed over the hydrogen peroxide indicates that the organism is cant produce catalase enzyme (Fig 2).

Figure 2: Catalase test

Determination of pH:

The pH of the raw beetroot juice was measured as 4.51 before inoculation and fermentation. After fermentation for 48 hours , the pH was recorded to be 4.32. For the next 6 days the pH of the fermented beetroot juice was monitored and decrease in ph was noted which indicates the presence of organic acids. The pH were tested for another 8 days which again shows the increase in pH. The final pH observe was 3.81 (Fig 3).

Figure 3: Change in pH and color

Determination of antibacterial activity:

The fermented beetroot juice with culture inoculum for 48hrs, showed a clear zone of inhibition of 22mm was observed against Escherichia coli. But no significant anti-bacterial action sere observed against other common food borne microbes such as Salmonella typhimurium, Staphylococcus aureus or Listeria monocytogenis . The antibacterial activity of the 48hrs fermented beetroot juice was may be due to the presence of organic acids such as lactic acid or citric acid. It may be also due to the presence of antibacterial proteins such as bacteriocins which can inhibit E.coli(Fig 4).

Figure 4: Anti-microbial activty (Zone of inhibition)

Determination of anti-oxidant activity:

The anti-oxidant property of the beetroot juice, i.e the property of inhibiting the formation of free radicals was checked. Ascorbic acid was taken as a reference to check the percentage of anti-oxidant property of our sample.The reduction capability of DPPH radical is determined by the decrease in its absorbance at 517 nm, induced by antioxidants. The decrease in absorbance of DPPH radical is caused by antioxidants, because of the reaction between antioxidant molecules and radicals, progresses, which results in the scavenging of the radical by hydrogen donation. It is visually noticeable as a change in colour from purple to yellow. Hence, DPPH is usually used as a substrate to evaluate the antioxidative activity.

Figure 5: Anti-Oxidant by DPPH method (Change in purple to yellow color and graph)

Determination of anti-inflammatory activity:

The 48hrs fermented beetroot juice also showed anti-inflammatory activity. For different concentrations of sample, the percentage of protection also varied. Three different concentrations (50µl,25µl and 15µl) were made mixing the HRBC solution and the fermented beetroot juice. The highest Percentage of protection was recorded as 77 for 50µl sample concentration (Fig 6).

Figure 6: Anti-inflammatory activity

High performance Liquid chromatography (HPLC)

High performance liquid chromatography was done to check the presence of organic acids that were formed after 48hrs of fermentation.. HPLC analysis confirmed the presence of organic acids like citric acid and lactic acid in a greater quantity in 48 hours fermented sample, on the basis of retention time, where the respective peaks were observed to be 3.58 and 5.48. The presence of more amount of lactic acid in the 48 hour fermented sample implied greater lactic acid fermentation. Also, in the 48 hour fermented sample, the presence of kaempferol rutinoside (Retention time of 9.72) has been revealed. The presence of kaempferol rutinoside, which is known to be an effective antioxidant, anti-carcinogenic and anti-mutagenic compound can be due to acid hydrolysis in the fermented sample (Fig 7a and 7b).

CONCLUSION:

The fermented beetroot juice can be used as a functional food because apart from it’s anti-bacterial and anti-inflammatory activities it is evident from the HPLC results that it contains kaemoferol rutinoside which is a potent anti-cancer compound. Thus it can be used for treating many diseases in a natural way without any side effects.

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Received on 28.06.2017 Modified on 18.07.2017

Research J. Pharm. and Tech 2018; 11(5): 1757-1760.

DOI: 10.5958/0974-360X.2018.00326.8