Plant related Bioactive compounds are usually used in numerous biomedical applications, but nowadays there is great interest in fermented and fabricated Bio-derived materials in exploring the biomedical applications (Ige et al. 2012; Halibet al. 2017).
For the initiation of fermentation, hybrid and effective bioactive compounds should be achieved from fermented products. Usually fermentation can be done using many micro-organisms, but its effective by using the pro biotic bacteria (Oh et al.
The activity of microbes and yield of new Bio-active compounds can be stimulated with different wavelengths of led lights. It is because the microbial fermentation is found to have less production of bioactive compounds with absence of light. Different wavelengths of lights can yield new Bio-active compounds and can stimulate the activity of microbes (Oh et al. 2017).
Industries had to face many challenges over the consumers requiring for the safer, healthier and nutritional foods [Oliveira, A.
, Amaro, A. L., & Pintado, M. (2018)].Food that contain bioactive compounds like dietary fiber, oligosaccharides,flavonoids, phenolics and probiotics are the functional foods.
Probiotics consists of friendly bacteria. They can promote the equillibrium of intestinal flora. (Plessas, S., Bosnea, L., Alexopoulos, A., & Bezirtzoglou, E. (2012)). Hence food industries have planned for producing high probiotic foods. Dietary habits have been turned to more healthy options over past years while increasing awareness to health benefits of food based products.
Fruit juices beverages are novel substrates that are non-diary probiotic foods ( Ephrem, E.
, Najjar, A., Charcosset, C., & Greige-Gerges, H. (2018)). It is because the diary products produce more allergies and cholestrol diseases and hence the fermented probiotic foods are produced( Perricone, M., Bevilacqua, A., Corbo, M. R., & Sinigaglia, M. (2014)).
Hence, this study is designed to examine the probiotic foods using effects of various color led lights and darkness on the yield of bioactive compounds during fermentation of Guava, Chikoo and Papaya.
Guava (psidium guajava) is a common tropical fruit cultivated in many tropical and subtropical regions. It belongs to Myrtaceae family. Guavas are originated from Mexico or Central America (Morton JF (1987)). Guavas were espoused in tropical and in subtropical asia.
These fruits are usually 4 to 12 centimeters long and round or oval depending on their shapes (Morton JF (1987)). They have typical fragrance and are similar to lemon. World production of guavas were 46.5 million tones in the year 2016 (Tridge. 2016).
The outer skin of guavas can be rough with bitter taste. And pulp of the fruit may be sweet or sour with deep pink (red guavas) or off-white (white guavas). Depending on the species, the central seeds may be harder or softer. Single guava fruit contains four times the vitamin C of an orange.
They are rich in Vitamin C and dietary fibre ( Nutritiondata.com Retrieved August 17, 2010). Phytochemicals like carotenoids and polyphenols produce the skin and flesh color of the fruits. Also the red guava has high content of polyphenols and carotenoids when compared to the white colored guavas (Nutritiondata.com Retrieved August 17, 2010)
Chikoo (Manilkara zapota) is also commonly known as sapota, nispero or sapodilla ( Morton, J. (1987)). It is an evergreen tree native to Central America andd Southern Mexico. The name zapota is derived from Spanish word zapote. The fruit is about 4-8cm in diameter (Harris, Kate (2009).
The flush has pale yellow to earthy brown color. It also has a grainy texture. There will be about one to six seeds in it. The seeds resemble like a beans and are hard, black and glossy. The fruit is extraordinarily sweet and has malty flavor. The unripe fruit is hard and has an firm outer skin(Harris, Kate (2009).
They have an enormous amount of saponin which has astringent properties. They have properties similar to tanin which can easily dry out the mouth. These trees are temperature sensitive. They can survive only in warm condition and in typically tropical environments.
These sapota trees usually take five to eight years to bear fruit. These trees can yield fruits only twice a year. The extracted compounds show anti-diabetic, antioxidant and cholesterol lowering effects ((Fayek NM, Monem AR, Mossa MY, Meselhy MR, Shazly AH (2012). The seed extract shows in-vivo antibacterial effects.( Kothari V, Seshadri S (2010))
Papaya (Carica papaya) is also called as papaw or pawpaw ( Collins Dictionary. n.d. Retrieved 2014-09-25). It is widely cultivated tropical fruit plant. It was originated in northern South America and in Mexico (U.S. National Plant Germplasm System. 9 May 2011).
This particular fruit plant can grow in three different sexes. They are male, female and hermaphrodite. The male fruit produces only pollens, female fruit produces small inedible fruits. And the hermaphrodite can self-pollinate itself. It is highly frost-sensitive. This plant grows rapidly fruiting within three years. Commonly two different kinds of papayas are grown. They are Red papaya and Yellow papaya.
Red papaya is red or orange flesh with sweet flesh. Yellow papaya has yellow flesh (Papaya Australia. 2015). Carica papaya is the first transgenic fruit tree. Genome sequencing was first performed to this fruit tree ( Ronald, Pamela and McWilliams, James (14 May 2010)).
In 2010, eighty percent of Hawaiian papaya was genetically modified. In 2016, the global production of Carica papaya was about 13.05 million tonnes. And it was led by India with 44% . Usually the pulp of raw papaya contains 88% water, 11% carbohydrates, proteins and fats. Papaya provides 44 kilocalories of in 100 gram amount of flesh. It has high source of Vitamin C ( Rivera-Pastrana DM, Yahia EM, Gonz?lez-Aguilar GA (2010)).
Lactobacillus casei is a probiotic bacteria. It is a species of genus lactobacillus belonging to the Lactobacillaceae family. And is usually found in the human urinary tract and in mouth. They have wide ph and temperature range. They have high application in diary productions.
Dominant species of nonstarter lactic acid bacteria is Lactobacillus casei. They are most widely studied and are present in the functional fruits. In the food preparation, they are also commonly used for natural fermentation (Kazuyoshi Takeda; Ko Okumura (2007)).
Hai et al. (2000) and Yan et al.(2013) had already reported that LED’s have high potential to be used in the bioreactors. LED lights are usually electroluminescent. And the color change of the light is identified by the band gap of the semiconductor.
Main objective is LED’s can emit high intensity using low energy. In this study, antioxidant activities and anti-diabetic activities are analyzed. These activities are done using the photo-fermented samples. Different colored led lights are used for their different wavelength reaction to the fruit samples along with Lactobacillus casei.
Different colored LED lights like red (~700-635 nm), blue (~ 490-450 nm), green (~560-520 nm), and yellow (~ 590-560 nm). Additionally, functional groups are also identified using Fourier Transform Infrared (FTIR) Analysis.
Lactobacillus casei colonies were used from the department of Genetic Engineering, SRM. Different fruits like Psidium guajava(Guava), Manilkara sapota(Chikoo), Carica papaya(Papaya) were purchased from the Pazhamudhir cholai (fruits and vegetable store) in South India.
All materials for the DPPH assay and alpha amylase inhibition test were purchased from the Hi-Media and SRL chemicals. The instruments involved were UV Spectrophotometer and FTIR spectrometer. Media and all other chemicals were purchased from Hi-Media.
Inoculum preperationDe Man, Rogosa and Sharpe Media (MRS Media) was dissolved completely. And they were Sterilized by autoclaving at 120°C and at 1-1.5atm for 15 min prior to use. Probiotic Bacteria, Lactobacillus casei was added into it. The media was incubated at 37°C for 24 hours.
The fruit sample extracts were prepared for fermentation . The fruits were purchased from the Fruits and Vegetable store (Pazhamudhir cholai) in South India. The purchased fruits were washed with autoclaved water and they were peeled off.
The fruit extracts were smashed using autoclaved motor and pistil individually. Furtherly, distilled water was added to the fruit extracts and are dissolved. This extract sample was sterilized by autoclaving at 120°C and at 1-1.5atm for 15 min.
Commercially available LED (Light Emitting Diode ) lights of illumination strips of different colors and 9V HI-WAAT battery was purchased. Red, Green, Blue and Yellow light strips were connected to the battery. They were assembled around the Erlenmeyer flasks accordingly.
The flask containing probiotic bacteria and the fruit extracts were placed on a shaking incubator at 120rpm.A flask with no LED light was also setup in the same incubator. Erlenmeyer flask was covered with black colored chart for the dark and to avoid the distraction of different wavelength of LED. It was setup with appropriate temperature and left in the incubator for about 48hours.
LyophilizationIncubated fruit extracts were centrifuged at 4000rpm for 5min at 4°C. Supernatants were freeze dried using lyophilizer. These powdered samples were stored at -20°C.
Antioxidant potentials for the fermented and unfermented fruit extracts were assessed using 1,1-diphenyl 2-picrylhydrazyl (DPPH) radical scavenging activity according to xxxx.
DPPH free radical scavenging activity for unfermented fruit extract(control) and fermented fruit extract was assessed by suspending 0.5 ml of fruit extract samples in 3 ml of ethanol. To which 0.3ml of freshly prepared DPPH ethanol solution was added, vortexed and kept in room temperature for about 30 minutes for DPPH radical scavenging activity to occur. Control and Auto zero was also freshly prepared for measuring absorbance.
Control was prepared by adding 3.5 ml of ethanol and 0.3ml of DPPH ethanol solution similarly auto-zero was prepared by adding 3.3ml of ethanol and 0.5ml of fruit sample extract. The absorbance of the solutions was then measured using UV-V Spectrophotometer at 490 nm.490 nm was chosen because of purple color DPPH. Percentage Inhibition of free radical DPPH was calculated as follows :
Inhibition (%) = 100 – [Ab (sample) – Ab(blank) / Ab(control)] * 100
where Ab(sample) is the absorbance of the test compound, Ab(blank) is the absorbance of the compound containing ethanol and sample and Ab(control) is the absorbance of the compound containing ethanol and DPPH.
FT-IR Spectra of unfermented and fermented fruit extract samples were obtained after incubation under various light conditions by L.casei and were recorded using Perkin-Elmer FTIR spectrophotometer in diffuse reflectance mode at 400 – 4000 wavelength at a resolution of 4 cm?1 in KBr pellets.
amylase inhibitory activity of the fruit extract samples was evaluated according to the standard procedure of xxxx. Two hundred micro liter of starch (0.4mg/ml) and hundred micro liter of fruit extracts (1mg/ml), were dissolved in phosphate buffer (20mM NaH2PO4 and 6.7mM NaCl, pH 6.9). Followed by 50 micro liter of ? – amylase solution (1U/ ml dissolved in phosphate buffer) added to the fruit extract mixture .
To which phosphate buffer is added to make up for 500 micro liter. The reaction mixture was incubated at room temperature for 5 minutes for the enzymatic reaction to occur. Finally, the reaction was concluded by adding 0.1% HCl acid.
Eventually, iodine reagent was added to the mixture. Starch concentration was absorbed to be decreased due to the activity of ? – amylase . It was measured at 660 nm using multi – well ELISA plate reader.
Results and Conclusion
The Fruit extract samples are photo fermented and the color changes was observed for each individual sample at different LED light wavelengths.
DPPH Radical Scavenging Activity
Fermented fruit extract samples using probiotic bacteria under various light wavelengths were compared to the control and dark samples. DPPH radical scavenging activity has been observed by the color change using DPPH assay.
And furtherly the O.D was observed using spectrophotometer. Anti-Oxidant activity has been observed by the color change from Violet to light Yellow color. lowest O.D shows high concentration.
DPPH free-radical scavenging activity of extract obtained from L.casei fermentation under green, red, blue and yellow LED lights increased in a dose-dependent manner.
Among different Light wavelengths samples fermented with blue, red and yellow shows high radical scavenging activity.
Fourier Transform Infrared Spectrophotometer (FT-IR)
The results of three different fruit extracts of Psidium guajava (Guava), Manilkara sapota (Chikoo), Carica Papaya (Papaya) were analyzed. Their corresponding functional groups were also illustrated.
amylase inhibitory activity of the fermented fruit extract samples were evaluated. The inhibitory percentage of ?-amylase of the different fermented fruit extracts were shown in fig. 8. Among the different samples the light with low wavelengths has shown high inhibition percentage.
Yellow, Blue, Green and Red LED lights has shown high alpha-amylase inhibition than dark. Among which fermented papaya and guava had shown high inhibition with yellow colored led light. And chikoo had shown high inhibition with blue colored led light.
Fig :8 MTT Assay