CHANGES IN BITTERNESS, ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC CONTENT OF GRAPEFRUIT JUICE FERMENTED BY LACTOBACILLUS AND BIFIDOBACTERIUM STRAINS

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Nowadays, fruit juice is consumed frequently worldwide because of its freshness and health promoting eff ects.Citrus family fruits such as grapefruit, orange, lemon, tangerine, etc. are rich in bioactive compounds such as minerals, vitamins, fi bres, and antioxidants (Z , 2007).Unfortunately, bitterness makes these fruit juices undesirable and unacceptable by consumers (R R , 2008).Bitterness in citrus fruits is primarily related to two compounds -naringin and limonin, and naringin is the main bitter component.Grapefruit juices contain more than 300-400 μg ml -1 naringin (S H , 1998).Some techniques have been reported for debittering, including adsorption technique (K et al., 2010) or use of β-cyclodextrin (M et al., 2006).These methods have some drawbacks aff ecting juice acidity, fl avour, sweetness, and turbidity as well as low effi ciency (R R , 2008).The application of enzyme in reduction of naringin concentration is a promising technique, because it improves the quality of citrus juices while maintaining health properties.

Grapefruit juice
The 100% grapefruit juice was purchased from the local market.The initial pH of the juice was adjusted to pH 6.3 with 4N NaOH before fermentation.
All strains of Lactobacillus were cultured in MRS medium at 37 °C for 24 h.B. bifi dum B7.5 was cultivated in TPY medium at 37 °C for 24 h under anaerobic conditions (in Bugbox anaerobic chamber, Ruskin Technology, USA).

Fermentation of grapefruit juice
Grapefruit juice in 100 ml fl ask was inoculated with bacteria cultures and kept under aerobic conditions (in the case of lactobacilli) or anaerobic conditions (in the case of B. bifi dum B7.5) at 37 °C for 24 hours.Samples were taken at given time intervals and analysed.TPY and MRS agar media were used to determine the cell number of bifi dobacteria and lactobacilli, respectively (B et al., 2018).

Analysis of carbohydrates and organic acids
The samples of fermented grapefruit juice were centrifuged at 14 000 r.p.m. for 10 min.The cell-free supernatant of grapefruit juice was used to determine the concentration of sugars and organic acids by HPLC method (B et al., 2018).

Analysis of antioxidant capacity
Ferric-reducing power (FRAP) assay was used to measure the total antioxidant capacity of the fermented grapefruit juices (N et al., 2019).

Analysis of total polyphenol content (TPC)
Total phenolic content was determined using the method described previously (N et al., 2019).

Statistical analysis
All data were analysed by one-way ANOVA as well as unpaired and paired t-tests using Statistica v9.0 software package (StatSoft, USA).Generally, P<0.05 was accepted as statistical signifi cance level.The results were presented as mean and standard deviation (SD).

Changes of cell counts, pH, carbohydrates, and organic acids in grapefruit juice
Grapefruit juice was inoculated with diff erent strains of Lactobacillus and Bifi dobacterium (L.plantarum 01, L. rhamnosus B01725, L. fermentum D13, and B. bifi dum B7.5) with about 10 6 CFU ml -1 of initial cell concentration.The results are presented in Figure 1.All investigated lactic acid bacteria grew well in grapefruit juice without nutrition supplementation.After 24 h of fermentation, almost all strains reached cell counts of 10 9 CFU ml -1 except L. rhamnosus B01725, where the population was 5.3×10 8 CFU ml -1 .W and co-workers ( 2009) produced probiotic noni juice with lactic acid bacteria and bifi dobacteria, and they reported that all L. casei, L. plantarum, B. longum strains reached about 1×10 9 CFU ml -1 after 48 h of fermentation at 30 °C.In another study, L. rhamnosus was used to ferment carrot juice (N et al., 2008).The cell count of L. rhamnosus was higher than our data in the case of L. rhamnosus B01725 (about 5×10 9 CFU ml -1 ).However, this cell count was reported after 48 h of fermentation at 37 °C.Our results were signifi cantly higher than those published by B and co-workers (2018), when apricot juice was fermented by mono and mixed cultures of probiotic Lactobacillus and Bifi dobacterium strains; when carrot juice was fermented by Bifi dobacterium strains (K et al., 2008), or in the case of mulberry (Z et al., 2014).During fermentation, short chain fatty acids were produced decreasing the pH values (Fig. 2), and at the end of the process, the fi nal pH ranged from 4.4 to 4.7.The reduction of pH during fermentation of probiotic products is of great importance for deciding the time of fermentation as well as maintaining the quality of product.Lactic acid and acetic acid were produced in the ranges of 9.6-186 mM and 8.9-1074 mM, respectively (Table 1).These results were higher than those conducted by C and co-workers ( 2009), when they cultured mono and mixed starters of probiotic strains in milk and laboratory soy beverage.In the case of grapefruit juice fermented by Lactobacillus spp., greater acetic acid production was observed.The presence of citric acid in the grapefruit juice with the initial concentration of 123 mM can explain the formation of acetic acid by Lactobacillus species through the citric cycle (Krebs cycle) leading to decreasing concentration of citric acid and increasing acetic acid content (T et al., 2005).Citric acid could not be detected in grapefruit juice after fermentation by Lactobacillus, while the amount of citric acid decreased by B. bifi dum B7.5 fermentation (Table 1).Our results are in agreement with data reported by M and co-workers (2010), where the initial concentration of citric acid in pomegranate juice Unauthenticated | Downloaded 09/28/23 11:51 PM UTC decreased signifi cantly during the fermentation by lactic acid bacteria (L.plantarum, L. delbruekii, L. acidophilus, and L. paracasei).The production of organic acids in lactic fermentation in diff erent media including fruit and vegetable juices could depend on the quality of media and strains (Z et al. 2009; H et al., 2014).During the fermentation of grapefruit juice, the concentration of sucrose decreased in all cases, and there was no signifi cant diff erence of sucrose content after fermentation (p < 0.05) among strains (Table 2).The result of sucrose consumption was in agreement with the study of B and co-workers ( 2018), where they carried out lactic fermentation of apricot juice by mono and mixed cultures of probiotic Lactobacillus and Bifi dobacterium strains.The contents of glucose and fructose were utilized at diff erent rates.In general, the orders of sugars consumption were fructose > glucose > sucrose in cases of L. rhamnosus B01725 and B. bifi dum B7.5, and glucose > fructose > sucrose in cases of L. plantarum 01 and L. fermentum D13 strains.The sugars can be fermented by probiotic bacteria via the Embden-Meyerhof pathway (EMP) or the phosphoketolase pathway (PKP), leading to homolactic and heterolactic fermentation profi les, respectively.

Changes of antioxidant capacity and total polyphenol content of grapefruit juice
Fermented fruit juice is considered "functional food" because of bioactive compounds such as fi bre, oligosaccharides, and bacteria that promote the equilibrium of intestinal bacterial strains (P et al., 2015).The highest TPC and antioxidant activity were observed in juice fermented by B. bifi dum B7.5 strain (Table 3).Signifi cant reduction of antioxidant activity was observed in all other cases, especially when grapefruit juice was fermented by L. rhamnosus B01725 strain (7.72 mM FeSO 4 ).Due to anaerobic fermentation, fermented Unauthenticated | Downloaded 09/28/23 11:51 PM UTC grapefruit juice did not get exposed to oxygen, leading to minimum decrease in TPC as well as antioxidant activity.An increase in antioxidant capacity was obtained in carrot juice fermented by L. bulgaricus and L. rhamnosus (N et al., 2008), and in noni juice by B. longum, L. casei, and L. plantarum (W et al., 2009).It confi rms that antioxidant activity varies with starter microorganism and cannot be aff ected synergistically (B et al., 2018).

Change of naringin concentration
The initial naringin content in the grapefruit juice was about 2.5 g l -1 .In all cases, the maximum decrease of naringin (about 28%) was obtained after 24 h of fermentation by mono starter L. plantarum 01 strain (Fig. 3).This result can be explained by the plant-related origin of the L. plantarum species.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, it is expected that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds (B et al., 2009).Z and co-workers (2017) purifi ed naringinase from Aspergillus oryzae 1125 and used to debitter orange juice.The naringin concentration decreased to below 30 μg ml -1 , meaning that the bitterness can be effi ciently lowered by naringinase to below the threshold of taste.Naringinase from Cryptococcus albidus can reduce up to 84% naringin at 40 °C and 100% at 60 °C after 60 min of incubation (B et al., 2018).P and co-workers ( 2017) reported that the biotechnological potential of naringinase producing yeast, Clavispora lusitaniae, has been exploited for the processing of kinnow and lemon in the form of lowalcoholic naturally carbonated debittered fermented beverage.After three months of storage at refrigerated temperature, the naringin content in fermented kinnow-lemon beverage decreased from 443.2 ppm to 176.4 ppm.
The effi ciency of naringinase in our study is not as high as of other naringinase sources, however, it is the fi rst report on simultaneous fermentation and reducing of naringin content in grapefruit juice.

Conclusions
Grapefruit juice can be used as substrate for growth of probiotic bacteria without any nutrient supplementation.All investigated Lactobacillus and Bifi dobacterium strains have the ability to reduce naringin concentration, the main component causing the bitterness in grapefruit, while only minimal changes in the levels of antioxidants and TPC were observed.Our results are very promising and can form the basis of technology development for the production of less bitter probiotic citrus juices rich in nutrients.

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This work is supported by the New Széchenyi Plant Project No. EFOP-3.6.3.-VEKOP-16-2017-00005 and by the Higher Education Institutional Excellence Program (20430-3/2018/FEKUTSTRAT) awarded by the Ministry of Human Capacities within the framework of plant breeding and plant protection researches of Szent István University, as well as a Bolyai Research Grant from the Hungarian Academy of Sciences.

Table 1 .
Organic acid concentrations of grapefruit juice after fermentation

Table 2 .
Concentration of carbohydrates in grapefruit juice after fermentation a: Signifi cant at 90% level, b : signifi cant at 95% level, c : signifi cant at 99% level

Table 3 .
Changes of TPC and antioxidant capacity in fermented grapefruit juice