A Comparative study the effect of mango (Mangifera India L.) and pomegranate (Punica granatum L.) juices on probiotic yogurt

The present study aimed to investigate the effects of adding mango and pomegranate juices to probiotic yogurt. Mango ( Mangifera indieaL .) and pomegranate ( Punica granatum L .) juices were added at a ratio of 10 % to yogurt with and without probiotic bacteria over storage periods of 1, 7 and 14 days at 4 o C. The chemical composition, syneresis, viscosity


Introduction
Probiotic bacteria are earning significance in human nutrition due to their multifaceted health benefits and are included in functional foods. Probiotic supplementation is useful in mitigating inflammatory stress andimmune senescence ( Sharma and Padwad, 2020). Also, it is beneficial for improving cognitive and mental health (Kim et al., 2021). It also improves blood glucose profiles in type II diabetes and gestational diabetes patients (Wang et al., 2022). Probiotic/synbiotic supplementation can significantly increase serum total antioxidant capacity, glutathione and nitric oxide levels, as well as reduce malondialdehyde levels in adults. Therefore, probiotic/synbiotic supplementation plays a role in improving antioxidant indices and reducing oxidative stress in the body (Pourrajab et al., 2022). A synbiotic is defined as a "mixture of probiotics and prebiotics that beneficially affects the host by improving the survival and activity of beneficial microorganisms in the gut" (Gourbeyre et al., 2011).
Symbiotic promote the growth of native, beneficial microflora like Bifidobacterium after ingesting prebiotics alone (Rabin et al., 2019). Additionally, probiotic bacteria guard the gut epithelium's mucosal barrier against harmful pathogens (Zommiti et al., 2020). As an illustration, lactic acid bacteria (LAB) produce mucin and decrease intestinal permeability to stop pathogen growth and access to intestinal epithelial cells. Another defense mechanism is the lactic fermentation carried out by LAB, which results in the generation of lactate and acetate as byproducts. This process produces an acidic environment that prevents the growth of infections (Tachedjian et al., 2017).
Probiotics can reduce cholesterol, and their metabolites, known as "postbiotics" such as lactic acid, bacteriocins, and hydrogen peroxide, work as antimicrobials against different pathogenic bacteria ( Silva et al., 2017).Bifidobacterium species are among the most common microorganisms in the natural microflora of the colon. Gram-positive Lactobacillus bacteria are the most prevalent probiotic it functions as probiotic, giving it the advantages of biological therapy. (Muhammad et al., 2019).
Fermented milk and yogurt contain probiotics, especially Lactobacillus and Bifidobacterium, which are commercially available. They are very well suited to various types of food matrixes because of their physiological functions (Fonteles et al., 2011).
Two types of yogurts can be categorized: regular cultured yogurt and probiotic or "bio" yogurt. The activity of L. bulgaricus and S. thermophilus produces standard yogurt. Bio-yogurts are those prepared with probiotic strains of Bifidobacterium and L. acidophilus, which offer several health benefits. These bacteria can promote the friendly microflora found in yogurt, helping to maintain overall The pomegranate (Punica granatum) has numerous nutritional and bioactive compounds such as sugars, proteins, crude fibers, pectin, vitamins, minerals, phenolic compounds, isoflavones, flavonoids, and primarily anthocyanins (Elfalleh et al., 2011;Aruna et al., 2016;Rafraf et al., 2017).Studies have shown the pomegranate's numerous physiological activities, including its antibacterial and anti-inflammatory characteristics, in addition to its antioxidant activity (Kandylis and Kokkinomagoulos, 2020).
Therefore, the main purpose of this study was to investigate the effects of adding mango and pomegranate juices on probiotic yogurt.
Bacterial strains Streptococcus thermophilus (S.thermophiles), Lactobacillus bulgaricus (L.bulgaricus) and Bifidobacterium infantis were obtained from Cairo Microbiological Resources Center in Egypt. These strains were consecutively transferred twice into skim milk (12 % T.S), and they were activated at 42 °C for 24 h before to the yogurt-making process (Jayalalitha et al., 2015 andSaleh et al., 2018) Mango (Mangifera indiea L) and pomegranate (Punica granatum L.) fruits were obtained from the local market in Fayoum governorate, Egypt.
El-GomhoriyaCo. for Trading Drugs, Chemicals and Medical Instruments, Egypt, was used to purchase all of the chemicals.

Preparation of mango and pomegranate juices
Mango and pomegranate fruits were washed and the juice was obtained according to the method described byChavan et al., (2013). The Juice was pasteurized at 90 °C for 15 sec then immediately cooled in an ice bath to below 10 °C and kept in sterilized glass bottles in the refrigerator (4°C) until use.

Yogurt producing
The IDF (1988) was used for yogurt production as follows: heating the milk at 95 °C for 5 minutes, then cooling it to 42 °C and dividing it into the following sex formulas (three replicates each). After that 10 % of pasteurized mango and pomegranate juices were added, stirred quickly after the immediate addition of the 2 % (v/v, S. thermophilus and L.bulgaricus in a1:1 ratio starter culture) with or without Bifidobacterium infantis of 24 h old culture. Samples were incubated in a plastic cup (100 ml) at 37 °C for 6 h until the pH reached 4.3 or 0.8 % acidity. The samples were then kept at 4 °C until they were analyzed.

Determination of the total phenolic and flavonoid contents of different yogurt
The total polyphenols contents of different yogurt were measured using the "colorimetric method" of Folin-Ciocalteau according to Singleton and Rossi, (1965)using A UV/Vis spectrophotometer (HITACHI U900, Japan) as gallic acid mg/100g at 765 nm. Also, the total flavonoids of the samples were determined calorimetrically using the aluminum chloride method according to Ordonez et al., (2006) using UV/Vis spectrophotometer (HITACHI U900, Japan), as quercetin (mg/100g sample).

Determination of the antioxidant activityof different yogurt samples
The antioxidant capacity of yogurt samples using the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) radical, samples' capacity to scavenge radicals was assessed byAkowuah et al., (2005). UV-spectrophotometer was used to detect the absorbance at 515 nm.

Chemical analysis of different yogurt samples
The chemical analyses include the following: total solids, moisture, fat, protein, ash, pH and acidity as total titratable acid (TTA) were analyzed at an interval of 1, 7 and 14 days according to A.O.A.C. (1995). The pH measurement for all samples was done using a digital pH meter (Model pH-Kent EIL 7020). The TTA was determined according to A.O.A.C. (2000) using sodium hydroxide 0.1mol/L. Then, the TTA of the yogurt samples was expressed as the lactic acid percentage.

Rheological Properties
Thesyneresis of the different yogurt samples was determined according to the method described byLawrence, (1959). Viscosity of the different yogurt samples was determined by Arana, (2003).

HPLC analysis of yogurts and juices treatments
An Agilent1260 series, Infinity, II LC, Waldron, Germany, was used for the HPLC analysis of identification of phenolic compounds according to Maraud et al., (2012).

Microbiological analysis:
The bacteria in the yogurt samples were cultured and counted according to Terzaghi and Sandrine (1975); Shah (1996), andMarshall, (1992) for Lactobacillus Bulgaricus, Streptococcus thermophilus and Bifidobacterium infants, in triplicate at 1, 7 and 14 days of the storage. The enumeration condition of each strain is indicated as follows: Lactobacillus Bulgaricus, MRS agar at 37 °C for 72 h in anaerobic condition, Streptococcus thermophilus, M17 agar at 37°C for 48 h in aerobic condition and Bifidobacterium infants count was done using M17 agar at 30 °C for 72 h in anaerobic condition. The counting of these bacteria is expressed as colony-forming units/ gram (log CFU/g) of each sample.

Statistical analysis:
Statistical analysis of the data was conducted using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test using the SPSS statistical software program version 19 (SPSS Inc., USA). Results were indicated by mean ± SD. The significant differences among treatments' means were explored at a ≤ 0.05 probability level using a statistical software package (Info State) according to Casanovas' et al., (2012).

Chemical analysis of mango and pomegranate juices
The chemical properties of mango and pomegranate juices are indicated in Table ( ,found that moisture content and total carbohydrate (78.9-82.8% and 16.20-17.18%) were major contents mango, while minor contents were total fat, total protein and ash (0.30-0.53%, 0.36-0.40% and 0.34-0.52% respectively).
The results of T.S, fat and protein of pomegranate juice in the present study were higher than that mentioned by Hallim et al., 2019, who found that the total solid%, total protein%, fat% and ash% of pomegranate juice were (14.06%, 1.34%, 0.49 % and 0.07%) respectively.

Determination of total phenolic, flavonoids contents and antioxidant activity of yogurt fortified with mango and pomegranate juices samples
The data in Table (2) indicated that the probiotic yogurt showed higher values of antioxidant activity, total phenolic and flavonoid contents than the control yogurt. Furthermore, probiotic yogurt fortified with mango juice showed higher values for these parameters than yogurt fortified with mango juice only.
The same manner was observed for yogurt fortified with pomegranate juice and probiotic yogurt fortified with pomegranate juice. Probiotic yogurt fortified with pomegranate juice, showed the highest total phenolic, flavonoid contents and antioxidant activity overall treatments. In addition, this treatment during storage times of 1, 7, and 14 days showed antioxidant activity as DPPH inhibition of 93.15±0.66, 88.51±1.05 and 82.67±0.59, respectively. Considerably, as mentioned before in Table (1) that pomegranate juice has higher values of antioxidant activity, total phenolic and flavonoid contents than mango juice.Arjmand et al., (2013),referred to the higher antioxidant activity of pomegranate juice to its high content of anthocyanins, hydrolyzable tannins, ellagic acid derivatives along with other flavonoid compounds. Table (2) showed that total phenolic, flavonoid contents and antioxidant activity values gradually decreased along with the storage for all treatments. This could be due to the oxidation of phenolic compounds or their reaction with casein of yogurt. Also, the presence of lactic acid which is secreted by the cultural bacteria causes phenolic compounds to be decomposed as well (Cho et al., 2017;Han et al., 2019).

Chemical analysis of yogurt fortified with mango and pomegranate juices samples
The chemical composition of yogurt samples fortified with mango and pomegranate juices is shown in Table (3). Data in Table  (3) revealed that control yogurt has the lowest T.S. (13.53±0.01) and highest fat (3.29±0.02). In contrast, adding Bifidobacterium to control yogurt as probiotic yogurt resulted in an increased T.S. percentage, but there was no significant difference in fat or protein percentages. Also, fortifying fortification yogurt with mango juice raised the percentage of the total solids and lowered both the fat and protein percentages when compared with control yogurt. In addition, probiotic yogurt fortified with mango juice showed the highest T.S. percent compared to all treatments.
The elevation of the total solids in mango yogurt may be due to the higher solid content of the fruit juice itself than the used milk (Ronak et al., 2016). However, yogurt fortified with pomegranate juice has more T.S. percent than control yogurt, but it was lower than yogurt fortified with mango. This may be a normal result since mango juice has more T.S. percent than pomegranate juice.
In general, all the chemical composition parameters of T.S., fat, protein and ash percent are gradually increasing with time of storage.Furthermore, during yogurt fermentation with a long storage time of two weeks, the pH is decreasing gradually and the syneresis is elevated, thus causing of losing the moisture of yogurt and causing all the previous parameters to increase over time Previous results by Hassanein et al., (2014), found that fortifying yogurt with fruit juice may lower its chemical composition parameters due to the high moisture and low fat and protein contents of the fruit itself, and these treatments are causing the syneresis to elevate by storage and losing the moisture by evaporation, resulting in increasing the % of the yogurts' chemical parameter. The pH and acidity of yogurt fortified with mango and pomegranate juices showed in Table (4). Yogurt fortified with pomegranate juice had a significantly lower pH (4.29±0.2) and a significantly higher acidity (0.93±0.01) than yogurt fortified with mango juice (4.41±0.01 and 0.91±0.02) compared to the control group (4.69±0.2 and 0.84±0.01) of pH and acidity respectively. The decrease in pH and increase in acidity may be due to the lower content of pomegranate juice pH and higher acidity than mango juice. Similar results were obtained by Ismail et al., (2014) found that the pH of two types of pomegranates ranged (from 4.28 -3.15) and the acidity ranged (from 0.74-1.52) of juice extracted by blending of seed.
The same results were observed in probiotic yogurt when compared to control yogurt. Probiotic yogurt showed a significant decrease in pH p≤0.05 (4.52±0.01) and a significant increase in acidity (0.89±0.01) compared to control yogurt that using regular starter culture (4.69±0.02 and 0.84±0.01respectively). The effect Bifidobacterium on both pH and acidity is contributed by the acidity caused by the regular starter culture, which is a critical factor for Bifidobacterium growth Bifidobacterium produces metabolites, especially acetic acid that lowers the pH and increases the acidity as well (Sarvari et al., 2014 andMeenakshi et al., 2018).
Also, data in the same table revealed that there is significantly different between probiotic yogurt fortified with pomegranate juice and probiotic yogurt fortified with mango juice during the storage period. The lowest pH values of probiotic yogurt fortified with pomegranate juice showed a significant decrease during the storage period.

Rheological properties of yogurt fortified with mango and pomegranate juices' treatments:
Syneresis is the loss of liquid from yogurt during the storage period and is used as an indicator or a key to the quality of yogurt itself (Vital et al., 2015;Dabija et al., 2018). Results indicated in Table (5) showed that the syneresis amount increased gradually during the storage period. The lowest whey syneresis was for control yogurt which ranged from 6.6±0.07 ml on day 1 up to 11.9±0.07 ml on day 14.
However, the addition of pomegranate juice to yogurt caused the highest syneresis amount of all treatments, which varied from 7.50±0.6 ml on day 1 up to 13.80±1.2 ml on day 14. Also, the yogurt cultured with Bifidobacterium along with regular culture as probiotic yogurt, probiotic yogurt fortified with mango, and probiotic yogurt fortified with pomegranate treatments showed higher syneresis amounts when compared to control yogurt. Furthermore, results in Tables (1 and 2), indicated that mango juice and yogurt fortified with it have lower acidity and a higher total solid content than pomegranate juice and its fortified yogurt.Both acidity and total solid contents affect the syneresis and viscosity parameters of yogurt (Hassanein et al. 2014; Shahbandari et al., 2016).
Also, many studies have indicated that syneresis amount is increasing with storage progress due to yogurt acidity development, which causes the water holding capacity to be reduced during storage progress (Silva et al., 2017; Atwaa et al., 2020; Blassy et  al., 2020; Ismail et al., 2020).
According to the data in the same table, there was no significant difference in viscosity (p > 0.05) between yogurt fortified with mango juice and probiotic yogurt fortified with mango juice. Also, there is no significant difference between yogurt fortified with pomegranate juice and probiotic yogurt fortified with pomegranate juice. However, the viscosity of mango-fortified yogurt was higher (159±10) than that of pomegranate-fortified yogurt (155±11). The water-soluble fiber present in mango may be absorbed by water and increases the viscosity (Mahmoud et al., 2008).
The viscosity of probiotic yogurt fortified with pomegranate juice was the lowest (150±12) while, control yogurt had a higher viscosity (163±11). Fortifying yogurt with fruit juices led to a significant decrease p≤0.05 in viscosity. The decrease in viscosity may be due to fruit juice's declining water-holding capacity of protein, which lowers the viscosity (Celik et al., 2017).

Sensory evaluation of yogurt fortified with mango and pomegranate juices' treatments:
From the data illustrated in Table (6) it is noticeable that, control yogurt was negatively affected by the addition of pomegranate juice. However, control yogurt almost has the same sensory evaluation when yogurt is fortified with mango juice. Data from Table (3) indicated that pomegranate yogurt has more moisture percent and less total solids percent than yogurt fortified with mango juice. This was attributed to the texture scores of these yogurts and these scores were improved by the addition of probiotics to the culture. The addition of Bifidobacterium to yogurt's regular starter culture improved many sensory properties (Vitheejongjaroen et al.,  2021; Ma et al., 2022).
Also, results showed that all the sensory properties indicators decreased gradually with storage for up to 14 days. As shown in Table ( However, fortifying yogurt with mango juice affects its overall acceptability and proved to be the most favorable yogurt, with nearly equal overall acceptability to the control yogurt. Kebary et al., (2020)indicated that mango was the most popular and favorable fruit tested in Egypt when used in the fortification of ice cream.

HPLC analyses of yogurt fortified with mango and pomegranate juices' treatments:
Table (7) showed that both gallic and chlorogenic acids were the predominant phenolic compounds found in all yogurt treatments. The main phenolic component in control yogurt is gallic acid (0.87±0.01µg/ml). The major phenolic compound found in probiotic yogurt and yogurt fortified with mango juice treatments is chlorogenic acid (1.55 and 1.10 µg/ml respectively). Also, gallic acid was found to be the principal phenolic compound of the following treatment: probiotic yogurt fortified with mango juice, yogurt fortified with pomegranate juice and probiotic yogurt fortified with pomegranate juice with an amount of (1.73±0.01, 2.27±0.01 and 2.36±0.01 µg/ml), respectively.
Phenolic compounds such as gallic acid, chlorogenic acid, syringic acid, ferulic acid and quercetin were found to be abundant compounds in yogurt fortified with both probiotics and mango juice. Caffeic acid, catechin, methyl gallate, rutin, coumaric acid, daidzein, pyrocatechol, and naringenin were not detected in this treatment.

(Palafox-Carlos et al., 2012; Peng et al., 2019),reported
thatthe most prominent phenolic constituents of mango were gallic acid, chlorogenic acid, quercetin, syringic acid and catechin. Also, gallic acid and chlorogenic acid were found to be the highly contributed compounds in mango. These findings led to the observation that the addition of Bifidobacterium (probiotic) to yogurt fortified with mango juice resulted in increasing its phenolic compounds contents. So, the addition of fruit juices to yogurt led to increases in the total phenolic and flavonoid contents and antioxidant activity of these yogurts fortified with fruit juices While the yogurt fortified with probiotic and pomegranate juice was found to have more varieties of phenolic components such as: gallic acid, chlorogenic acid, catechin, methyl gallate, coumaric acid, ferulic acid and quercetin. Caffeic acid, syringic acid, pyro catechol, rutin and ellagic acid were not detected in this treatment. Hmid et al., (2017),illustrated that pomegranate has an abundant amount of the following polyphenols: quercetin, gallic acid, chlorogenic acid, ferulic acid, caffeic acid, ellagic acids, rutin and phloridzin.

Microbiological examination of yogurt fortified with mango and pomegranate juices.
Results in Tables (8 and 9) showed the viable count of Lactobacillus,Streptococcus and Bifidobacterium (CFU×10 6 /g) in yogurt fortified with mango and pomegranate juices. The addition of mango juice elevated the total viable counts of bacteria in all treatments. Yogurt fortified with mango juice showed a viable count of Lactobacillus during storage times of 1, 7 and 14 days of 8.53±0.02, 8.67±0.04 and 8.82±0.02, respectively. Also, yogurt fortified with mango juice showed a viable count of Streptococcus during storage times of 1, 7 and 14 days of 8.61±0.01, 8.68±0.02 and 8.74±0.02, respectively. These viable counts of bacteria were higher than the count for the control yogurt. The fortification of yogurt with fruits is causing the total bacterial count to elevate as a result of the presence of fruits' metabolites, which are used by the bacteria and enhance their growth (Jin et al.,  2018; Atwaa et al., 2020; Blassy et al., 2020).
However, the addition of pomegranate juice to regular yogurt decreased the total viable counts of bacteria in all treatments. Yogurt fortified with pomegranate juice showed a viable count of Also, the indicated results showed that yogurt culture with Bifidobacterium causes the total viable count of all bacteria to elevate, and the total count of all bacteria is decreased by storage. This may refer to the anaerobic preference nature of these bacteria, with a long storage period there is a reduction in the oxygen percent in yogurt (Tripathi and Giri, 2014). However, it was stated that acidity is a main factor for Bifidobacterium growth, and the acidity of the fruit and starter culture metabolism causes a reduction in the viable count of Bifidobacterium during long storage ( Dave and Shah 1998;Meenakshi et al., 2018).

Conclusion
Control yogurt has lower T.S., acidity, syneresis, total phenol, total flavonoids, antioxidant activity, the number of phenolic compounds detected, and a viable count of lactobacillus and streptococcus. Adding Bifidobacterium to control yogurt resulted in higher T.S., total phenolic, total antioxidant activity, pH, viscosity, and lower overall acceptability compared to control yogurt.
Probiotic yogurt fortified with pomegranate juice increased total phenol, total flavonoid, antioxidant activity, acidity and syneresis compared to probiotic yogurt fortified with mango juice, which revealed an increase in T.S., fat, protein, PH, viscosity, overall acceptability, number of phenolic compounds detected by HPLC and viable count of Bifidobacterium,Lactobacillus and Streptococcus compared to probiotic yogurt fortified with pomegranate juice. In general, the fortification of yogurt with probiotic bacteria with pomegranate juice is better than fortification with mongo juice because the higher antioxidant activity, total phenolic compounds, and flavonoids, which have a protective effect against many diseases.