Effect of Storage on Probiotic Viability, Physicochemical and Sensory Properties of Probiotic-enriched Orange Juice

Emmanuel Lucky Orike^1*, Temidayo Emmanuel Olajugbagbe² and Bridget Okiemute Omafuvbe^3
*Correspondence: Emmanuel Lucky Orike, Department of Microbiology and Biotechnology, First Technical University, Ibadan, Oyo State, Nigeria, Tel: +2348038013110, Email:

Keywords

Probiotic • Fruit • Storage • Functional foods • Lactobacillus plantarum • Lactobacillus bulgaricus • Human health

Introduction

Fruits and vegetables are important in the maintenance of good health. They are good sources of carbohydrates, vitamins, minerals, fiber and numerous bioactive compounds and their regular intake is reported to reduce the risk of chronic diseases in human [1,2]. The World Health Organization (WHO) recently promoted the inclusion of at least 400 g of fruit and vegetables per day for the prevention of cancer, diabetes, obesity and heart disease [3,4]. Modern food technology has introduced the act of transferring the valuable fruit components into juices containing all the essential physical, chemical, organoleptic and nutritional characteristics which are found in the original healthy and ripe fruit [5]. It is believed that with the presence of these nutrients and absence of competing starter cultures, fruit juices would be an ideal medium for the delivery of probiotics [6].

Probiotics are regarded as live microorganisms that, when administered in minimum concentration of 10⁶ CFU/ml or gram, confer a health benefit on the host [7,8]. Traditionally, dairy products including fermented and unfermented milk and cheese have been found to be an ideal carrier for delivering probiotics to the human gastrointestinal tract. However, to increase consumers’ consciousness in probiotic functionalities and overcoming the challenges posed by lactose intolerance, milk allergies, vegetarianism as well as dyslipidemia, it is important to improve the functional product vehicles through other non-dairy products such as fruit juices [9].

The cultures of microorganisms mostly used for producing probiotic foods are Lactic Acid Bacteria (LAB) such as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricusi, Lactobacillus plantarum, Bifidobacterium bifidum, Bifidobacterium infantile and Streptococcus spp [10]. Probiotic strains mainly of the species of Lactobacillusplantarum and Lactobacillus bulgaricus have been used extensively in the development of many probiotic fruit and vegetable juices and their suitability as carrier for probiotic bacteria as well as the sensory acceptability by the consumer has been reported [11,12]. It has been reported that Lactobacillus plantarum and Lactobacillus bulgaricus have potential to modify the phenolic composition fruit juice and enhance its overall antioxidant capacity. This is done by incorporating the probiotic strain(s) directly into the acidic and other unfavorable processing conditions of the juice.

Orange is the fruit of the citrus species Citrus sinensis. It contains high concentration of vitamin C, flavonoids and a good source of hesperidin. Orange juice is highly acidic due to its citric acid content [13]. Probiotic strains involved in the fruit juice probiotication process therefore must remain viable through the harsh conditions via a stable and favourable storage conditions over a reasonable period of time without jeopardizing the organoleptic properties of the juice itself.

Materials and Methods

Probiotic strains (Lactobacillus plantarum (A₂) and Lactobacillus bulgaricus (C₂) used in this study were previously isolated from fermenting cassava, characterized and screened for probiotic potentials [14].

Preparation of pasteurized orange juice

The ripeness index that was considered during the selection of the fruits are skin colour, aroma and texture or firmness, fresh, sizable, mature, ripened and yellow-oranges were purchased from the fruit market in Ile-Ife, Nigeria, washed and squeezed by pressing the pulp through a juice extractor to extract the juice. The extracted orange juice was filtered through a sieve and the filtered orange juice was dispensed in 40 mL portion into several sterile 100 mL capacity conical flasks, covered with foil paper and then pasteurized for 30 seconds in a water bath set at 95 °C [15].

Inoculum preparation

The cell suspension of each of the two probiotic organisms (L. plantarum and L. bulgaricus) was prepared by adding 10 mL of sterile normal saline to 18-24 h old De Man Rogosa and Sharpe (MRS) agar slant cultures of the test strain in McCartney bottle and shaken to wash the cells. The suspension was centrifuged at 8000 rpm for 10 min and pellets were washed with sterile normal saline. The cells were re-suspended in sterile normal saline and standardized to contain 1 × 10⁹ CFU /mL using a spectrophotometer [16].

Inoculation of pasteurized orange juice

The pasteurized orange juice (40 mL) in conical flask was aseptically inoculated with 1% (v/v) of the standardized cell suspension of the selected probiotic lactic acid bacteria as single and mixed culture (1:1). The freshly produced probiotic orange juice was divided into two batches. One batch was stored at 4 °C in the refrigerator and the other half at room temperature (30 °C ± 2 °C) for a period of 20 days. The cold storage (4 °C) temperature was stabilized and maintained by placing a thermometer in the refrigerator powered by two different power sources; government and power generating plants in order to avoid any power fluctuation. During the storage period, samples were withdrawn from the triplicate flasks for analysis at 5 days intervals starting from time zero.

Evaluation of changes in the viable counts of lactic acid bacteria in probiotic orange juice

The viable count of Lactic Acid Bacteria (LAB) in the probiotic orange juice was determined following standard plate count method with De Man Rogosa and Sharpe (MRS) agar. An aliquot (1.0 mL) of the probiotic orange juice sample was serially diluted in sterile maximum recovery diluent (MRD) up to 10^-5 and 0.1 mL of the appropriately diluted sample was spread-plated on De Man Rogosa and Sharpe (MRS) agar and incubated at 35 °C for 24-48 h anaerobically. After incubation, the plates were observed and colonies were enumerated and expressed as log CFU/mL probiotic orange juice.

Determination of changes in pH and titratable acidity of probiotic orange juice

The pH of the probiotic orange juice sample was measured using an electronic digital pH meter (HANNA INSTRUMENT 8021). The juice sample (10 mL) was poured into a clean 100 mL capacity beaker and the calibrated pH electrode was dipped into it and read electronically [17].

Total Titratable Acidity (TTA) of the probiotic orange juice was determined using titration method with phenolphthalein as end point indicator [17]. Exactly 10 mL of the sample (probiotic orange juice) was diluted with equal volume of distilled water and titrated against 0.1N NaOH solution with two drops of phenolphthalein (1% w/v) indicator to give a faint pink colour end point of pH 8.3 (monitored with a pH meter). Each mL of 0.1 NaOH is equivalent to 90.08 mg. 

Titratable Acidity = Volume (mL) of NaOH × Normality (N) of NaOH × Lactic acid Equivalent/volume of sample used.

Organoleptic analysis of the probiotic orange juice

The organoleptic property of the probiotic orange juice stored at refrigeration temperature for 20 days was assessed by a trained panel of 10 regular consumers of orange juice. The probiotic orange juice samples were evaluated for colour, taste, aroma, appearance and general acceptability. Uninoculated orange juice served as control. The parameters were scored using a 5-point Hedonic scale of dislike extremely (1), dislike (2), neither dislike nor like (3), like (4) and like extremely (5) [18].

Statistical analysis

The data obtained in this study were subjected to one-way analysis of variance followed by Student -Newman - Keuls post hoc test (Primer for Biostatistics software package version 3.01) for difference between means [19]. Statistical significance was accepted at P value equal to or less than 0.05.

Results

Changes in the viable counts of LAB in probiotic orange juice duringstorage

There was no significant viable cell count in the control set-up as shown in the Table 1, since there was no inoculation.

The viable counts (Log CFU/mL) of LAB in probiotic orange juice inoculated with single and mixed cultures of L. plantarum (A₂) and L. bulgaricus (C₂) during storage at 4 °C and room temperature (30 °C ± 2 °C) for 20 days is presented in Table 1. The probiotication involving L. plantarum (A₂) and L. bulgaricus (C₂) applied as single culture showed no significant difference (P ≤ 0.05) in the viable cell counts of LAB during storage at 4 °C. There was a slight decrease in the viable count of probiotic LAB strains from 8.16 ± 0.03 to 7.51 ± 0.04 and from 8.23 ± 0.02 to 7.81 ± 0.03 for L. plantarum and L. bulgaricus respectively after 15 days of storage. Also, L. plantarum and L. bulgaricus as mixed cultures maintained a continuous viability ranging between 8.05 ± 0.01 and 8.50 ± 0.03 till the 15^th day followed by a slight drop in viable count to 7.54 ± 0.03 at the end of the storage period. This viability could be attributed to the inactive state and the behaviour of the probiotic strains added to the juice. This corresponds with the report of Boudjou, similarly, a stable pH of the juice at this temperature condition could contribute to the abundance of probiotic strains exceeding the minimum bacteria populations required for probiotic foods to possess health claims as fermentation was limited by keeping the samples in refrigerator. This conforms with the report that refrigeration could promise a more prolonged survival of probiotics while thermal abuse could be detrimental to the viability of probiotics in orange and vegetable juices [9]. It is imperative from the health point of view that probiotic strains selected for probiotication retain their viability and functional activity throughout the shelf-life of the delivery product; an attribute which is strain dependent [20]. Probiotics need to maintain a minimum therapeutic level of 10⁶ - 10⁷ CFU/mL in food product in order to confer health benefit [21]. On the other hand, at room temperature (30 °C ± 2 °C), both L. plantarum and L. bulgaricus maintained a steady growth till the 5th day. The strains gained a significant increase in viable counts at the 10th day of storage after which a significant decrease was observed till the end of the storage period. The viability of L. plantarum and L. bulgaricus as mixed cultures showed a steady growth till the 10^th day followed by a sharp decrease from 9.09 ± 0.03 to 7.53 ± 0.02 at the 15^th day and further decreased to 6.91 ± 0.03 at the end of the storage period. The steady cell count of probiotic strains suggests that orange juice favors a synergistic relationship and enables beneficial bacteria to thrive as mixed probiotic cultures during an extended storage period. This has been effective in the treatment of several gastrointestinal disorders. However, the drop in viable cell count of strains from 10⁹ to 10⁶ CFU/mL (although still within the healthy limit) after the storage period signals the inability of the probiotic strains to tolerate high acidic condition over an extended storage period (above 15 days). This agrees with the report of Ghafari and Ansari [6].

Changes in pH and titratable acidity of probiotic orange juice during storage

The pH changes in orange juice inoculated with single and mixed cultures of probiotic Lactobacillus species are shown in Table 2. Probiotication of orange juice involving L. plantarum and L. bulgaricus as single culture and in combination showed no significant change in pH throughout the course of storage at 4 °C due to low rate of fermentation and production of organic acids. However, at room temperature storage, a sharp increase in acidity was observed for the single and mixed culture probiotic orange juices. The probiotic orange juice inoculated with L. plantarum showed changes in pH from an initial value of 3.80 to 3.10 at the end of the room temperature storage. The probiotication involving L. bulgaricus and a mixture of both lactic acid bacteria also showed the same pattern in pH changes from an initial pH of 3.8 to 3.0 at the 20^th day of storage. It is believed that fermentation was rapid at room temperature compared to the refrigeration condition thus increasing the acidity of the orange juice with time due to the accumulation of organic acid from the fermentation of fermentable polysaccharides by the probiotic strains. Similar result was reported by Gallina DA, et al. [22] in the development and characterization of probiotic fermented smoothie beverage. The viability of probiotics and increase in acidity is of great importance to the quality of the juice as it minimizes the influence of spoilage bacteria stimulate protein digestion and enhance the sensory properties of the juice [23].

The total titratable acidity (expressed as mg lactic acid/mL) of probiotic orange juice involving L. plantarum and L. bulgaricus as single and mixed culture showed a slight reduction initially from 6.52 at day zero to 6.41, 6.46 to 6.42, and 6.51 to 6.42 respectively at the 15^th day of cold storage period (Table 3). The TTA later increased to 6.46, 6.49 and 6.49 respectively at the end of storage at 4 °C. On the other hand, a sharp increase in TTA was observed throughout the room temperature storage of the probiotic orange juice. The L. plantarum and L. bulgaricus as single and mixed culture produced significantly more TTA (from between 6.46 and 6.52 mg lactic acid/mL at the onset to between 16.21 and 16.27 mg lactic acid/mL at the 20^th day).

The titratable acidity of probiotic orange juice produced by L. plantarum and L. bulgaricus at the cold storage (4 °C) period can be linked to the inability of the probiotic strains to metabolize or produce organic acids because fermentation was not possible at refrigeration storage. On the contrary, probiotication involving L. plantarum and L. bulgaricus as single and mixed cultures at room temperature can be linked to the decrease in pH of the probiotic orange juice at room temperature. As the inoculated LAB strains ferment the carbohydrate content of the orange juice, lactic acid is produced which reduces the pH making the juice more acidic and a corresponding increase in the TTA. This result corresponds with the report of Shukla and Kushwaha [13].

Organoleptic properties of the probiotic orange juice

The probiotic orange juice produced by L. bulgaricus as a single culture (sample B) and in combination with L. plantarum (sample C) showed no significant difference (P ≤ 0.05) in colour, taste, aroma, appearance and general acceptability and were most preferred to the other probiotic orange juice involving L. plantarum as a single culture (sample A) (Table 4). This is an indication that L. bulgaricus plays a vital role in contributing to the development of these sensory attributes. Sample A (orange juice inoculated with L. plantarum) was rated low for taste and general acceptability and was significantly different from the other probiotic orange drinks. It is worthy of note that the probiotic orange drink inoculated with L. bulgaricus and the mixed culture of the two lactic acid bacteria were not significantly different from the uninoculated pasteurized orange juice in all the organoleptic attributes scored by the taste panel. The type of microorganism, the juice, storage conditions and the addition of other compounds may influence the sensory properties of the finished product [24]. The results showed that Sample B (inoculated with L. bulgaricus) and sample C (inoculated with mixture of L. plantarum and L. bulgaricus) were more preferred and acceptable by consumer than the sample involving L. plantarum as a single culture (sample A). This is an indication that L. bulgaricus may have played a vital role in the development of these sensory attributes. Similar result was reported by Maldonado RR, et al. [25] in the potential application of four types of tropical fruits in lactic fermentation.

Conclusion

In conclusion, the combination of L. plantarum and L. bulgaricus as probiotics in orange juice is a suitable in the development of functional foods. The combination of the LAB strains presented a favourable synergy from their metabolism without jeopardizing the integrity of the juice. Orange juice enriched with L. plantarum and L. bulgaricus singly and in combination may provide a new asset in the production of healthy functional drink which may solve the problem associated with probiotic dairy products especially for lactose intolerant individuals.

Declaration

Ethical statement

This study was conducted in line with the required guideline involving informed consent of all participants. All the participants were pre-informed of the nature of the study and they willingly volunteered to participate without any cohesion.

Ethics approval

Not Applicable.

Consent for publication

Not Applicable.

Availability of data materials

All data generated or analyzed during this study are included in this article.

Funding

This Research did not receive any specific grant from funding agencies in the public, commercial or non-for-profit sectors.

Authors Contribution

BOO conceived and supervised the work, ELO performed the methodology, TEO performed the data curation and wrote the original manuscript, ELO AND TEO performed the data analysis and initial report writing, ELO and BOO performed the Reviewing and Editing while all Authors gave the Resources, read and approved the manuscript.

Acknowledgement

The authors wish to appreciate the staff of the General Laboratory, Department of Microbiology, Obafemi Awolowo University, Ile-Ife for the assistance rendered during the study.

Conflict of Interest

The authors have no conflict of interests.

References

1. Volpe, Stella L. "Fruit and vegetable intake and prevention of chronic disease." ACSMs Health Fit J 23 (2019): 30-31.

Google Scholar, Crossref, Indexed at

2. Mostafidi, Mahdieh, Mohammad Reza Sanjabi, Faezeh Shirkhan and Maryam Tamaskani Zahedi. "A review of recent trends in the development of the microbial safety of fruits and vegetables." Trends Food Sci Technol 103 (2020): 321-332.

Google Scholar, Crossref, Indexed at

3. Fruit, W. H. O. "Vegetable Promotion Initiative–report of the meeting." Geneva: World Health Organization (2003).
4. Swain, Manas Ranjan, Marimuthu Anandharaj, Ramesh Chandra Ray and Rizwana Parveen Rani. "Fermented fruits and vegetables of Asia: A potential source of probiotics." Biotechnol Res Int 2014 (2014): 250424.

Google Scholar, Crossref, Indexed at

5. Nicklas, Theresa A., Carol E. O'Neil and Victor L. Fulgoni III. "Consumption of various forms of apples is associated with a better nutrient intake and improved nutrient adequacy in diets of children: National Health and Nutrition Examination Survey 2003–2010." Food Nutr Res 59 (2015): 25948.

Google Scholar, Crossref, Indexed at

6. Ghafari, Sedigheh and Sara Ansari. "Microbial viability, physico-chemical properties and sensory evaluation of pineapple juice enriched with Lactobacillus casei, Lactobacillus rhamnosus and inulin during refrigerated storage." J Food Meas Charact 12 (2018): 2927-2935.

Google Scholar, Crossref, Indexed at

7. Hill, Colin, Francisco Guarner, Gregor Reid and Glenn R. Gibson, et al. "Expert consensus document: The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic." Nat Rev Gastroenterol Hepatol (2014).

Google Scholar, Crossref, Indexed at

8. Olajugbagbe, Temidayo Emmanuel, Oluwatosin Esther Elugbadebo and Bridget Okiemute Omafuvbe. "Probiotic potentials of Pediococuss acidilactici isolated from wara; A Nigerian unripened soft cheese." Heliyon 6 (2020).

Google Scholar, Crossref, Indexed at

9. Patel, A. R. "Probiotic fruit and vegetable juices-recent advances and future perspective." Int Food Res J 24 (2017).

Google Scholar, Indexed at

10. James, Armachius and Yousheng Wang. "Characterization, health benefits and applications of fruits and vegetable probiotics." CYTA J  Food 17 (2019): 770-780.

Google Scholar, Indexed at

11. Gao, Yifeng, Nazimah Hamid, Noemi Gutierrez-Maddox and Kevin Kanton, et al. "Development of a probiotic beverage using breadfruit flour as a substrate." Foods 8 (2019): 214.

Google Scholar, Crossref, Indexed at

12. Santos Monteiro, Shênia, Yolanda Albertina Silva Beserra, Hugo Miguel Lisboa Oliveira and Matheus Augusto de Bittencourt Pasquali. "Production of probiotic passion fruit (Passiflora edulis Sims f. flavicarpa Deg.) drink using Lactobacillus reuteri and microencapsulation via spray drying." Foods 9 (2020): 335.

Google Scholar, Crossref, Indexed at

13. Priyanka Shukla, Priyanka Shukla and Anjali Kushwaha Anjali Kushwaha. "Development of probiotic beverage from whey and orange juice." (2017): 629.

Google Scholar, Crossref, Indexed at

14. Orike, E. L., S. M. Adeyemo and B. O. Omafuvbe. "Probiotic potentials of lactic acid bacteria isolated from fermenting cassava." Int J Probiotics Prebiotics 13 (2018).

Google Scholar

15. Petruzzi, Leonardo, Daniela Campaniello, Barbara Speranza and Maria Rosaria Corbo, et al. "Thermal treatments for fruit and vegetable juices and beverages: A literature overview." Compr Rev Food Sci Food Saf 16 (2017): 668-691.

Google Scholar, Crossref, Indexed at

16. Oyewole, O. B. "Optimization of cassava fermentation for fufu production: Effects of single starter cultures." J Appl Bacteriol 68 (1990): 49-54.

Google Scholar, Crossref, Indexed at

17. https://law.resource.org/pub/us/cfr/ibr/002/aoac.methods.1.1990.pdf
18. Pourabedin, M., A. Aarabi and S. Rahbaran. "Effect of flaxseed flour on rheological properties, staling and total phenol of Iranian toast." J Cereal Sci 76 (2017): 173-178.

Google Scholar, Crossref, Indexed at

19. Glantz, S. A. "Premier of biostatistics: The program. San Francisco, EUA." (1992).
20. Shori, Amal Bakr. "Influence of food matrix on the viability of probiotic bacteria: A review based on dairy and non-dairy beverages." Food Biosci 13 (2016): 1-8.

Google Scholar

21. Ryan, Jessica, Scott C. Hutchings, Zhongxiang Fang and Nandika Bandara, et al. "Microbial, physico‐chemical and sensory characteristics of mango juice‐enriched probiotic dairy drinks." Int J Dairy Technol 73 (2020): 182-190.

Google Scholar, Crossref, Indexed at

22. Gallina, Darlila Aparecida, Paula de Paula Menezes Barbosa, Rita de Cássia Salvucci Celeste Ormenese and Aline de Oliveira Garcia. "Development and characterization of probiotic fermented smoothie beverage." Rev Cienc Agron 50 (2019): 378-386.

Google Scholar, Crossref, Indexed at

23. Huang, Zhihai, Lu Huang, Guangliang Xing and Xiao Xu, et al. "Effect of co-fermentation with lactic acid bacteria and K. marxianus on physicochemical and sensory properties of goat milk." Foods 9 (2020): 299.

Google Scholar, Crossref, Indexed at

24. Chaudhary, Aditya. "Probiotic fruit and vegetable juices: Approach towards a healthy gut." Int J Curr Microbiol Appl Sci 8 (2019): 1265-1279.

Google Scholar, Crossref, Indexed at

25. Maldonado, Rafael Resende, Lucas da Costa Araújo, Letícia Caroline da Silva Dariva and Késsia Nazar Rebac, et al. "Potential application of four types of tropical fruits in lactic fermentation." Lwt 86 (2017): 254-260.

Google Scholar