Asian Journal of Dairy and Food Research, volume 41 issue 4 (december 2022) : 403-409

​Health-promoting Effects of Developed Probiotic Orange Beverage: An in vitro Study

Deeptimayee Mahapatra1,*, Mamoni Das1
1Department of Food Science and Nutrition, Assam Agricultural University, Jorhat-785 013, Assam, India.
Cite article:- Mahapatra Deeptimayee, Das Mamoni (2022). ​Health-promoting Effects of Developed Probiotic Orange Beverage: An in vitro Study . Asian Journal of Dairy and Food Research. 41(4): 403-409. doi: 10.18805/ajdfr.DR-1690.
Background: Probiotic food has evolved as the new trend among the health fanatics because of their proven benefits in preventing many diseases. With change in time the way of consuming probiotics has also changed. Unlike past dairy is not the only option for commercial probiotic production, recently fruit juices have become the popular choice for it. So the current study aimed to assess the feasibility of orange juice (Citrus reticulate) as a potential probiotic carrier for the production of probiotic orange juice with lactic acid bacteria.
Methods: Three test samples (TS) were developed with different combination of lactic acid probiotic bacteria viz. test sample 1 (TS1) (L. bulgaricus and L. casei), TS2 (L. bulgaricus, L. casei and L. gasseri) and TS3 (L. bulgaricus, L. casei, L. gasseri and L. fermentum). The orange juice was pasteurized for 2 min at 90°C and was inoculated at a rate of 10% inoculum. All the test samples were fermented for 4 hrs at 37°C and the physicochemical and nutritional characteristics were evaluated along with their in vitro hypocholesterolemic and in vitro hypoglycemic efficacies.
Result: The probiotic orange test samples did not show inferior properties than the control in terms of physicochemical and nutritional properties. The bacterial count was decreased with time but remained above standard limit (107cfu/100ml) until 28th day of refrigerated storage. All the test samples showed promising antioxidant activity, in vitro hypocholesterolemic activity and in vitro hypoglycemic activities. Hence orange juice could be used as a suitable probiotic carrier for production of novel probiotic beverages.
Now-a-days healthy food means “functional foods”, which provides specific health promoting effects other than their traditional nutritional benefits. In addition to the well established functional ingredients like vitamins, minerals, and micronutrients probiotics have emerged as a new class of active functional ingredients (Jankovic et al., 2010). In Latin the term probiotic means “for life” which means these are essential for life and can be bacteria (Lactobacillus, bifidobacterium etc.) or yeast (Saccharomyces). Literature suggested that the use of probiotics in the form of fermented food was started from Egyptian and Tibetan times as a method to carry milk for long treks (Guo et al., 2016). Since that time dairy has always been exploited for probiotic food production. But recently the introduction of concepts like high cholesterol percentage in milk products, increase of cardiac disease incidence rates, diets like vegan and conditions like lactose intolerance, has accentuated the need for non-dairy functional food options (Granato et al., 2010). Hence fruit and vegetable juices come into existence as an alternative for probiotificated dairy drinks. Besides juices are a good source of both macro (sugars and fiber) and micro (vitamins and minerals) nutrients, which are valuable components to human health. Again the current craze of new generations towards healthier diets in less time and labor has made juices an important natural food alternative.
Different fruit juices are a more agile and effortless way of consuming a required dietary portion of fruits for a day and addition of probiotic to fruit juices would increase the nutritional properties of the juices and would provide additional health benefits (Zuntar et al., 2020). Several researches are present indicating fruit juices like mango, watermelon, grape, pomegranate, peach, noni, mulberry and pineapple etc. as potential probiotic carriers (Okereke et al., 2016; Mousavi et al., 2011; Thakur and Sharma et al., 2017; Pakbin et al., 2014; Chaudhary et al., 2019; Ghafari and Ansari, 2018). But fruits from citrus family are experimented less for probiotification due to their acidic nature. So an attempt was made to develop probiotificated orange juice in the present study because of their vast health effects. Orange juice is one of the most consumed fruit juice around the world (Galaverna and Dall’Asta, 2014). It is a good source of vitamin-C, folate and polyphenols (Ohrvik et al., 2008; Galaverna, et al., 2014). Studies have reported long term consumption of orange juice can reduce total cholesterol, LDL, LDL/HDL ratio and apo B, with significantly increase the folate and Vit-C content of blood (Aptekmann and Cesar, 2013). Evidences also suggested that orange juice consumption can also increase the concentration of HDL cholesterol (Ali et al., 2015; Kurowska et al., 2000) which makes it a healthier choice.
The experiment was conducted at the laboratories of Assam Agricultural University, Assam during the session of 2019-2020. Mature and healthy oranges were selected randomly from a lot of oranges for the study.
Determination of physical parameters of oranges (Citrus reticulate)
Weight of the fruits
Weights were recorded for 7 oranges consecutively and the mean was expressed in gram per fruit.
Peel percentage
Peel percentage was determined by taking the peel weight of 7 oranges consecutively.
Juice content
Juice content was determined by extracting juice of 500 g fruit each for 3 times and expressed as percent juice content (Gupta et al., 2009).
Preparation of starter culture
The strains were revived in 50 ml MRS broth separately and incubated for 45 hrs at 37°C as per the method used by Shukla et al., (2017) with little modification. All the strains were sub-cultured and incubated for 24 hrs before used for fermentation of orange juice.
Fermentation of orange beverage
Freshly pressed orange juice was autoclaved at 90°C for 2 min according to the method outlined by Deshpande et al., (2019). A 10% (v/v) inoculum was used to ferment all the three test samples (Deshpande et al., 2019). Three different combinations of strains were used to ferment orange juice test samples and were named as test sample 1(TS1), test sample 2 (TS2) and test sample 3 (TS3). TS1 contained L. bulgaricus and L. casei (1:1), TS2 contained L. bulgaricus, L. casei and L. gasseri (1:1:1) and TS3 contained L. bulgaricus, L. casei, L. gasseri and L. fermentum (1:1:1:1) respectively. After inoculation the test samples were incubated for 4 hrs at 37°C and were then stored at 4°C till further evaluations.
Enumeration of viable bacteria
The viable cell count of each orange test sample was
evaluated through standard pour plate method using the formula:

Physico-chemical analysis of probiotic orange beverage
Physico-chemical properties like pH, TTA, colour and TSS were determined using standard methods outlined by AOAC (2005).
Nutritional analysis
Test samples were subjected to nutritional evaluations using different suitable methods. The total carbohydrate was evaluated through anthrone method (Hofreiter, 1962). Crude protein, crude fat and crude fiber contents were evaluated by the methods outlined by Ekanem and Ekanem in 2019. The potassium content of the sample was measured through the method described by Nerdy in 2018 and Vitamin-C content was measured through standard AOAC method.
Antioxidant activity
Antioxidant activity/DPPH free radical scavenging activity was determined as per the procedure described by Delgado et al., (2010). 12 ml of juice was blended with 3 ml methanol and water solution (4:1) and mixed well.100 ml of methanol was taken and 0.0024 g DPPH (2, 2-diphenyl-1-picrylhydrazyl) was added to prepare DPPH solution. 300 µL of ethanol blended sample was added to 2.7 mL of DPPH solution. After addition of DPPH solution the test tubes were kept in dark at room temperature for 1 hr. After 1 hr the absorbance of sample, DPPH solution and blank [methanol/water (4:1)] were measured at 517 nm. DPPH radical scavenging activity was calculated from the graph of DPPH radical scavenging activity percentage vs. extract concentration.

A DPPH    =  Absorbance of DPPH.
A Sample  =  Absorbance of sample.
In vitro hypocholesterolemic efficacy
In vitro hypocholesterolemic efficacies of all the samples were performed according to the procedure cited by Liong and Shah (2005) with some modifications. MRS broth was prepared and 0.3% oxgall (bile salt) was added and autoclaved at 121°C for 15 minutes. Cooled MRS media was supplemented with water soluble cholesterol procured from Hi media. 1 ml of orange juice was added to 9 ml of cholesterol mixed broth and incubated for 24 hr at 35°C. After incubation the mixture was centrifuged for 10 min at 10000 rpm. 1 ml of supernatant was transferred to a sterilized test tube. 1 mL of KOH (33%w/v) and 2 mL of absolute ethanol was added to the supernatant, the mixture was vortexed for 1 min to ensure mixing. Then the mixture was incubated for 15 min at 35°C. The mixture was cooled down a little and 2 mL of distilled water and 3 mL of hexane was added to it. The mixture was vortexed for a min and set aside for 1-2 min. Two separate layers were appeared among which hexane formed the upper layer. 1 ml of hexane layer was taken and the contents were evaporated. The residue was dissolved in 2 mL of o-phthalaldehyde solution. Concentrated sulphuric acid (0.5 mL) was added to the mixture and then incubated at room temperature for 10 min.  Absorbance of the mixture was measured at 550 nm.

In vitro hypoglycemic efficacy
10 ml of sample was added with 10 ml of MRS broth containing 20% glucose in it. Mixture of sample and MRS broth was incubated at 37°C and aliquots were collected at 0 and 24 hrs and the remaining glucose in the medium were measured by a Blood glucose meter (Wang et al., 2020).
Physical and physicochemical characteristics
The orange variety Citrus reticulate is used for the production of probiotic orange beverage. The average weight for the orange is 120.41±7.04 g, the average peel percentage is 28.79±1.23% and the average juice content is 47.14±2.54% respectively (Table 1). The TSS and pH of orange juice decreased during the fermentation process in the fermented and non-fermented (Control) orange beverage samples (Table 2). But no significant change was observed among the test samples. The decrease of pH and elevation of total titrable acidity during lactic acid fermentation was due to accumulation of organic acid especially lactic acid in the beverage sample. The results obtained are in agreement with findings by Do et al., 2019, Chaudhary et al., 2019, and Silva et al., 2016. The fermented and non-fermented orange beverage samples showed little difference in their colour. The fermented test samples showed positive values for a* and b* and higher value of b* indicated that the test samples had desirable yellow-orange colour. The L* value of control was significantly lower than the test sample which indicated that the lightness was increased in the test sample and this could be due to the application of thermal processing/pasteurization, which is supported by the findings of Lee et al., (2003).

Table 1: Physical parameters of oranges (Citrus reticulate).


Table 2: Physico-chemical properties of probiotic orange beverage.

Nutritional characteristics of probiotic orange beverages
The experiment was conducted to examine the effect of fermentation by lactic acid bacteria on the proximate composition of orange juice and also for evaluating the effect of different combination of probiotic strains on the proximate composition of the same. Moisture content of the fermented test samples (TS1, TS2 and TS3) was recorded to be significantly decreased than non fermented orange juice but no such significant difference was noticed among the test samples. The decrease could be explained by the fact that use of thermal processing reduces the moisture content of juice, which was previously documented in several other thermally processed probiotic beverages like pine-apple juice, sweet lime juice and blueberry and carrot blended juice (Ghafari and Ansari, 2018, Khatoon et al., 2015, Mauro et al., 2016). The carbohydrate content of non fermented juice (control) was 9.20% which was significantly higher than the fermented test samples. The reduced values of carbohydrate showed the utilization of available sugar by the probiotic strains for their growth and multiplication. Similar trend was recorded by several other researchers (Mauro et al., 2016, Wang et al., 2009). No significant difference was seen among the test samples and this could be due to shorter time of fermentation. Fermentation showed no significant effect on the crude protein content of fermented and non fermented orange beverage test samples. The results were supported by the findings of Mauro et al., (2016). Similarly no significant difference was observed in the crude fat content between the non fermented and fermented test samples, which was previously reported in a probiotic pine-apple juice (Ghafari and Ansari, 2018). Such a non significant difference was also recorded for the crude fiber and potassium content of the non fermented and fermented test samples (Table 3).

Table 3: Nutritional characteristics of multi-strain probiotic orange beverages.

Ascorbic acid/Vitamin-C the major vitamin available in oranges was evaluated to compare the effect of fermentation on the Vit-C content of the test samples. A non significant relationship was recorded among the test samples but a significant decrease was recorded in the fermented sample compared to the non fermented sample (Table 3). Many studies revealed that application of thermal processing and pasteurization resulted in decreased Vitamin C content of the orange juice (Igwemmar et al., 2013; Martýnez et al., 2006) which could be a possible explanation for the findings of the present study.
Health promoting effects of probiotic orange beverage
Antioxidant properties
The percentage free radical scavenging activity of the test samples was lower than the control (Fig 1) and this could be explained with the fact that application of thermal processing and pasteurization decreases the free radical scavenging activity as it affects the vitamin C content (Igwemmar et al., 2013; Meena et al., 2017; Martýnezet_al2006). Among the test samples TS3 showed highest DPPH free radical scavenging activity which could be due to the presence of several specific strains. Literature documented that lactic acid bacteria namely L. casei, L. fermentum and L. gasseri posses some antioxidant activity (Lin and Yen, 1999; Lee et al., 2005; Wang et al., 2009; Chooruk, et al., 2017; Rastogi et al., 2020).

Fig 1: Antioxidant activity of test samples.

Hypocholesterolemic activity
The in vitro hypocholesterolemic activity of all the developed orange test samples ranged from 25%-28% and among all TS3 had the highest hypocholesterolemic activity (Fig 2). The hypocholesterolemic properties for the probiotic lactic acid bacteria used for the study were previously described by several other researchers with different mechanisms like, incorporation of cholesterol to cell surface and conversion of cholesterol to coprostanol etc (Lye et al., 2010).

Fig 2: In vitro Hypocholesterolemic activity of test samples.

Hypoglycemic activity
The hypoglycemic efficacy of developed probiotic orange test samples TS1, TS2 and TS3 were 50.85%, 54% and 55.78% respectively (Fig 3). All the test samples showed promising results that could be described with the fact that glucose is the primary substrate for growth of probiotic bacteria although the glucose utilization capacity is different from strain to strain (Merkel and Perry, 1977).

Fig 3: In vitro hypoglycemic activity of test samples.

The research demonstrated that all the three probiotic orange beverage test samples maintained viability till 4th week of storage which proves that orange juice can be a potential substrate for probiotic lactic acid bacteria without any nutrient supplementation. The promising antioxidant activity of all the test samples by virtue of both orange juice components (Vit-C and phenols) and probiotic mixtures requires further exploration in terms of their potential use for managing several oxidative stress associated diseases. The results of in vitro hypo-cholesterolemic and hypo-glycemic properties that are recorded in the present study needed to be investigated more with proper animal models. Overall, orange juice with probiotics can be used as healthy non-dairy probiotic drink in the future.
The authors acknowledge Assam Agricultural University, Jorhat, Assam, India, for facilitating the research work.
Ms Deeptimayee Mahapatra conducted the experiments, analyzed the data and drafted this paper. Dr. Mamoni Das designed the experiments, revised the manuscript and approved the final version for publication.
The authors declare no conflict of interest.

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