RESEARCH ARTICLE

Fermentation of Tomato Juice with S. cerevisiae has enhanced nutrition and shelf-life

Shristi Muraka¹, Bharti Sahu², Andrea Kolla²*

¹Parul University, Varodara

²Department of Biotechnology, Seth Phoolchand Mahavidhyalaya, Rajim

shristimurarka7@gmail.com; bharti15august@gmail.com; annpereira@gmail.com

*Corresponding Author Email- annpereira@gmail.com

Introduction

Botanically, tomato (Solanum lycopersicum) is a berry, grown widely for its consumable fruits.  It belongs to the nightshade family Solanaceae, commonly grown in the tropical and warm temperate zone of the world. In terms of significance, it ranks second to sweet potatoes and potatoes.

There has been a noticeable global trend in recent years toward the use of phytochemicals as antioxidants and functional ingredients that are derived from natural resources like fruits, vegetables, oilseeds, and herbs. Considering the vast nutritional and health benefits of tomatoes, it is listed as a power food. Hence, the inclusion of tomatoes in the daily diet in the most natural form must be encouraged. The year round tomatoes are produced but the changing environmental conditions, due to global warming tends to hamper its production resulting in the rise in price. Tomato has high water content and is perishable hence easily susceptible to microbial spoilage (Abdullahi 2016). Over time, a number of techniques for preserving tomatoes have been developed. These include canning, sun-drying, and the use of chemical preservatives like sodium benzoate and sodium metabisulfite to preserve tomato paste, juice, and other products (Guil‒Guerrero et al. 2009; Martinez 2002; Ramos-Bueno et al. 2017). The addition of chemical preservatives is harmful effects to health, especially due to their ability to transform into carcinogenic agents and for their teratogenic effect (Sambu et al. 2022).

This research study therefore investigates methods to prepare nutrition rich tomato juice with a longer shelf-life, in the most natural way possible. The drink was fortified with cinnamon, cardamom, cloves, stevia and tulsi. These ingredients are known to add taste, havehealth benefits and some of themare well known natural preservatives as well (Pinar Kuru 2014; Bina et al. 2018; Bhowmik 2010). Fermentation with Saccharomyces cerevisiae was used, as a method of non-thermal preservation for the tomato juice to further enhance its nutritional value and increase its shelf-life. Fermentation with S. cerevisiae is a naturally occurring process known to improve the physical properties of tomato juice, organoleptic properties, and the in vitro bio-accessibility of lycopene. The digestive enzymes released by S. cerevisiae during fermentation breaks down the tomato cells, and release the intracellular lycopene thus increasing its bioaccessibility. Fermentation also reduces the size of the tomato pulp particles, which improves its stability during storage. Further, S. cerevisiae metabolites are helpful in treating many health problems like cardiovascular disorders, immune-compromised diseases and many more. S. cerevisiae has been traditionally used as a neuro-protective, antioxidant, anti-diabetic, immune booster and antioxidant (Farid et al 2019). Hence, yeast fermentation is a potential bioprocess for not only enhancing the physical properties of tomato juice, but also enriching their nutritional value and health benefits.

Materials and Methods

Preparation of Plant Material

Organic tomatoes were washed with tap water, and rinsed thrice to remove all dirt and impurities. Tomatoes were then boiled in water for 10 minutes, peeled, ground with a kitchen blender and filtered using a cheese cloth. The extract was concentrated by heating at a temperature<50^oC and stored at room temperature.

Processing of Non-fermented juice

1.       Sorting, Washing, Chopping, grinding, filtration

2.       Homogenization and pre-heating to 60^o C

3.       Fortification of natural preservatives (Table 1)

4.       Vacuum de-aeration Pasteurization at 98 ^o C and holding at this temperature for about 2 minutes

5.       Cooling from 90-20 ^o C

6.       Filling under vacuum at 92^o C in glass bottles

7.       Capping /closing of containers

8.       Holding of full containers at 90^o C for about 6-8 minutes in a pasteurization tunnel

9.       Cooling of the full containers is done at 40^o C in a tunnel

Processing of Fermented juice

1.       Sorting, Washing, Chopping, grinding, filtration

2.       Homogenization Pre-heating at 60^o C

3.       Fortification of natural preservatives (Table 1)

4.       Vacuum de-aeration Pasteurization at 98^o C and holding at this temperature for about 2 minutes

5.       Cooling from 90-20^o C

6.       Filling under vacuum at 92^o C glass bottles

7.       Capping /closing of containers

8.       Holding of full containers at 90^o C for about 6-8 minutes in a pasteurization tunnel

9.       Cooling of full containers is done at 40 ^o Cin a tunnel

10.    Fermentation by adding 1gm yeast (first dissolved it in 2ml lukewarm water), mixed well and stored at room temperature.

11.    Filtering of the juice was done after fermentation.

Qualitative test for carbohydrates

Molisch’s test In 2ml of sample, 2 drops of Molisch’s reagent were added and mixed. To this mixture 2ml of conc. Sulphuric acid was added and colour development is observed. Formation of purple condensation or purple ring indicates presence of carbohydrate (Mishra 2019).

Iodine test was done for starch. In 2ml of sample 2 drops of iodine solution is added and observed for colour change. Development of blue colour indicates presence of starch in a sample (Elzagheid 2018).

Benedict’s test was performed for identification of reducing sugars. To 5ml Benedict’s reagent 8 drops sample was added and mixed. The mixture was boiled and precipitate colour was observed. The colour of the precipitate ranges through green, greenish yellow, yellow, orange or brick red precipitate depending on the quantity of sugar present (Mishra 2019).

In Anthrone test carbohydrate is dehydrated in presence of conc. sulphuric acid and forms furfural and its derivatives. This furfural and its derivatives react with anthrone (10-keto, 9, 10-dihydro anthracene) to give a blue green complex. In 2ml of solution, 2 drops of anthrone reagent and colour changed was observed (Sadasivam S. 1996).

Qualitative test for proteins

Ninhydrin Test In this reaction α-amino group reacts with ninhydrin to give blue - purple product. Amino acids with secondary amino groups react with ninhydrin to give yellow products. In 1ml sample, 0.2 ml 1% ninhydrin solution is added, boiled for 1 minute and the colour is observed (Moore Stanford 1968).

Biuret Test is the test used to detect the presence of protein. In 2ml sample equal amount of 10% NaOH is added and mixed, to this mixture 0.5% copper sulphate is added drop by drop and colour change is observed (Sapan Christine V. 1999).

Xanthoproteic Test was performed to detect the aromatic amino acid in a sample. In 2ml sample 1ml conc. Nitric acid was added and heated, yellow precipitate was observed and cooled and few drops of NaOH were added (Subroto Edy.2020).

Lead Sulphide Test is a test to check sulphur containing amino acid in a sample based on the degradation of sulphur in S-S or S-H group of amino acid under strong alkaline condition at high temperature. In 2ml sample 40% NaOH is added and boiled for 3 mins. To this 5 drops of Lead Acetate was added and mixed and observed for colour change.

Neumann’s Test is a test to detect casein in a sample. In 5ml of sample, 0.5ml 40% NaOH is added and heated for 2-3 minutes and cool under tap water. To this 1ml of concentrated sodium nitrate and a pinch of ammonium molybdate is added and warm gently colour changed is observed.

Phytochemical test

Lead acetate test is a test to detect tannin. In 5ml sample few drops of 10% solution of lead acetate was added and precipitate formation was observed.

Keller Killiani is a test that detects cardiac glycosides in plants. In 2ml acetic anhydride 0.5 ml sample was added. To this we added few drops of ferric chloride and conc. sulphuric acid colour change was observed.

Test for steroids was assayed by Lieberman-Burchard method. 0.5 ml of sample was mixed and solubilized in 3ml chloroform and filtered. To this conc. sulphuric acid was added and colour change was observed.

Test for phytosterol and terpenoid was done using Salkowski method. In 2ml sample, 2ml of chloroform and 2ml of conc. sulphuric acid was added to formation of a coloured layer was observed

Test for Quinone1ml of concentrated sulphuric acid in 1ml of sample formation of red colour indicate the presence of quinone.

Test for Phenol: In 2ml of distil water dilute 10% of ferric chloride and 1ml of sample was mixed and colour change was observed.

Test for Anthocyanin colour change depends on pH of the solution. In 1ml sample, 1ml 2N hydrochloric acid and 1ml ammonia was added and heated at 100ºC for 5 minutes. Development of blue - green colour solution indicates presence of anthocyanin.

Test for Flavonoid in 2ml sample 1ml 2N sodium hydroxide was mixed. Development of yellow colour indicates positive test for flavonoid.

Test for Leuco-anthocyanin in 2ml sample 2ml isoamyl alcohol was added and mixed well. Red colour is positive for leuco-anthocyanin.

Test for Saponin In 2ml sample 2ml distil water and mixed well. The formation bubbles or foam indicate the presence of saponin in the sample (Obadoni & Ochuko 2001).

Test for Alcohol

Reaction of alcohol with Ceric Ammonium Nitrate reagent changes the colour from yellow to red. In 1ml ceric ammonium nitrate reagent few drops of sample was added and mixed well. The colour change was observed.

Test for Antioxidant

0.2mM DPPH solution was prepared in methanol and 50µl/ml, tomato juice sample was mixed to produce a final DPPH concentration of 0.1mM. The mixture was vigorously shaken and left to stand for 30 minutes in dark and observed for colour change (Ruth et al 2018).

Product Shelf-Life Studies

50 ml of sample was placed in transparent glass bottles and covered with aluminum foil, and left on the laboratory shelf at ambient conditions (temperature 24±2.20 c; humidity 65±3.40C; 12 light/dark cycle) for shelf life studies. Colour change, oxidative rancidity, including spoilage (growth of yeast); objectionable odour and taste were monitored.

Table 1: Fortification was done for both fermented and non-fermented samples

Results and Discussion

Tomato juice was prepared and fortified with natural ingredients like cinnamon, tulsi, cardamom, cloves and stevia to increase its nutritional value, organoleptic properties and shelf-life. The drink was prepared as 5 different samples (S1, S2, S3, S4, S5) containing different fortificants in different concentrations (Table 1).Replicates of S1 -S5 were prepared and fermented by adding 1gm pre-activated yeast (Saccharomyces cerevisieae). These samples were labelled as SF1, SF2, SF3, SF4 and SF5. All the samples were then analysed or proteins, phytochemicals, pH, alcohol, phenols and carbohydrates.

Non-fermented tomato juice: The colour of the finished product was deep red for all samples. The pH ranged from 4-5 (Table 2). Carbohydrate, protein, antioxidants, casein and alcohol was present, while iodine and starch was absent (Table 3 and table 4). Moderate amount of reducing sugar was detected by Benedict’s test. Phytochemicals like tannin, cardiac glycoside, trepenoid, quinone, phenol, anthocyanin, flavonoid, Leuco-anthocyanin were present and saponin was absent in all samples (Table 5). These samples had a shelf-life 15 days at room temperature.

Fermented tomato juice: The colour of the finished product was deep red for all samples. Carbohydrate, protein, antioxidants, casein and alcohol was present, while iodine and starch were absent (Table 3 and table 4). Phytochemicals like tannin, cardiac glycoside, trepenoid, quinone, phenol, anthocyanin, flavonoid, Leuco-anthocyanin were present (Table 5), in all similar to the non-fermented samples. However in fermented samples, the pH ranged from 4-6 (Table 2), sugar was present only in traceable amounts compared to the non-fermented samples and finally saponin was detected in all the fermented samples only. These samples had a shelf life of two months at room temperature.

A global trend in the increase and demand for healthy, natural foods and beverages has been observed. The change in social habits and dietary intake has urged people to prefer and consume Ready-to-serve (RTS) foods and beverages. However, due to the growing quotient of health awareness people now opt for the most natural and organic ready to serve foods and drinks.  Generally, RTS beverages are preserved with synthetic/ chemical preservatives and packed with refined sugars.  This, in the recent years has been a reason for the rising trend in diseases and obesity.  The main aim of this study is to replace the role of chemical preservatives in a nutritious tomato juice preparation with natural preservatives and preservation by fermentation method. The natural preservatives and taste enhancers used in the shelf life extension were cinnamon, cardamom, tulsi, tamarind, and cloves (Keith Singletary 2014). These additives are known for their health benefits as well as play a role in food preservation by inhibiting bacterial growth. These natural preservatives were added to increase the shelf-life of the tomato juice. Stevia was added as a natural sweetener. The low-calorie count of Stevia qualifies it to be a healthy alternative for diabetes control or weight loss (Hannah Nichols 2018).Further, long term preservation was attempted by fermentation with S. cerevisiae (yeast) known for its immunity boosting capability and health benefits of its metabolites. The tomato juice preparation kept well at room temperature / without refrigeration for 15 days in non-fermented samples and for 2 months in fermented samples with no physiochemical or biological changes. The fermentation process increased the nutritional quotient of the tomato juice by the presence of saponins and lower levels of sugar. Saponins help boost immunity, decrease blood lipid levels, lower risks of cancer etc. Hence, a combination of natural preservatives and non-thermal methods like fermentation could be new trend to improve the shelf-life and nutritional value fruit juices. More practically, given their track record of successful microorganism inhibition, prolonged shelf life, and improved nutritional quality, fermentation and natural preservatives would be the way of the future for the preservation of fruit juice and beverages.

Table 2: pH value of samples

Table 3: Test for carbohydrates: All samples were positive for carbohydrate. The content of reducing sugar was lower in fermented samples.

Table 4: Test for proteins: All samples were positive for protein and showed the presence of casein.

Table 5: Test for phytochemicals: All samples showed the presence of protein and casein.