Journal of Functional Foods 38 (2017) 409–414

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Fermented sweet lemon juice (Citrus limetta) using Lactobacillus

plantarum LS5: Chemical composition, antioxidant and antibacterial
activities
Seyed Mohammad Bagher Hashemi a,⇑, Amin Mousavi Khaneghah b,⇑, Francisco J. Barba c,⇑, Zahra Nemati a,
Samaneh Sohrabi Shokofti a, Fatemeh Alizadeh a
a
Department of Food Science and Technology, College of Agriculture, Fasa University, Fasa, Iran
b
Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
c
Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de
València, Avda. Vicent Andrés Estellés, s/n 46100 Burjassot, València, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Sweet lemon juice was fermented with Lactobacillus plantarum LS5 to produce a probiotic juice. The cell
Received 17 June 2017 counts of the L. plantarum LS5 increased from 7.0 ± 0.1 to 8.63 ± 0.38 log CFU/mL during fermentation
Received in revised form 6 September 2017 (37 °C for 48 h) (p < 0.05) and decreased from 8.63 ± 0.38 to 7.14 ± 0.21 log CFU/mL after storage (4 °C
Accepted 14 September 2017
for 28 d). Antibacterial activity against S. Typhimurium and E. coli O157:H7 was observed in fermented
Available online 22 September 2017
sweet lemon juice. Immediately after fermentation, pH, lactic acid and antioxidant capacity of the sam-
ples increased, while glucose and fructose, total acidity, citric acid and phenolic decreased. During stor-
Keywords:
age, phenolics, and antioxidant decreased in both fermented and non-fermented samples during storage.
Lactobacillus plantarum
Sweet lemon juice
Moreover, the ascorbic acid was more stable in fermented juice compared to non-fermented juice.
Citrus limetta Fermentation of sweet lemon juice could improve the chemical as well as antibacterial properties of
E. coli O157:H7 the juice.
S. Typhimurium Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction the time of consumption should be at least 107 CFU/mL to achieve

Over the last decades, the consumption of probiotics has
attracted considerable attention. According to the Food and Agri-
culture Organization (FAO) and the World Health Organization
(WHO), probiotics are ‘‘live microorganisms that confer health
benefits to the host when administrated in adequate amounts”
(Hashemi et al., 2017; Sanders, 2008). Their consumption could
provide beneficial effects, such as enhancing mucosal barriers, syn-
thesizing different types of vitamin B, decreasing serum choles-
terol, stimulating the immune system, inhibiting intestinal
inflammation, inhibiting the effect of pathogenic microorganisms,
reducing symptoms of diarrhea, and increasing antimutagenic
activity (Hashemi et al., 2017; Hashemi, Shahidi, Mortazavi,
Milani, & Eshaghi, 2014; Guarner & Schaafsma, 1998). Moreover,
it is generally believed that the minimum concentration of living
probiotic microorganism in the product (probiotic contained) at
⇑ Corresponding authors.
E-mail addresses: Hasshemii@yahoo.com, Hashemi@fasau.ac.ir (S.M.B. Hashemi),
mousavi@fea.unicamp.br (A. Mousavi Khaneghah), francisco.barba@uv.es
(F.J. Barba).
the proposed health benefits (Hashemi, Shahidi, Mortazavi, Milani,
& Eshaghi, 2016; Hill et al., 2014).
The crucial roles of probiotic such as lactic acid bacteria (LAB) in
maintaining the human gastrointestinal tract (GIT) microbial
ecosystem by the growth inhibition of ingested pathogens were
well demonstrated (Liévin-Le Moal & Servin, 2014). Lactobacillus
strains also can be used in improving of food products due to their
probiotic properties as well as fermentative activities (Hashemi,
Amininezhad, Shirzadinezhad, Farahani, & Yousefabad, 2016;
Hashemi et al., 2014; Leone et al., 2017; Mota, Lopes, Delgadillo,
& Saraiva, 2015). Antibacterial activity of L. plantarum strains iso-
lated from traditional Kurdish cheese against Staphylococcus aureus
and E. coli O157: H7 was observed by Hashemi et al. (2014). In
another study, Maldonado Galdeano and Perdigón (2006) reported
that Lactobacillus casei stimulated the immune system of the host.
Moreover, Sah, Vasiljevic, McKechnie, and Donkor (2014) found
Lactobacillus strains enhanced antioxidant and antimutagenic
activities of crude peptide extracts from yogurt. Ekıncı (2005)
reported that fermentation increased water-soluble vitamins of
Turkish fermented wheat-flour-yogurt. According to Mousavi
et al. (2013), fermentation of pomegranate juice reduced total

https://doi.org/10.1016/j.jff.2017.09.040
1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
410 S.M.B. Hashemi et al. / Journal of Functional Foods 38 (2017) 409–414
anthocyanin content. In this context, the use of probiotic bacteria is
a useful strategy to obtain products with longer shelf life as well as
safer properties due to their ability to delay or preventing the
growth of common contaminant bacteria (Madureira et al., 2015).
Citrus limetta belongs to a type of citrus that is commonly
known as sweet lemon, which can be cultivated in South and
Southeast Asia. Sweet lemon has a flattened apex and a prominent
nipple and has a thin and light yellow rind. Hesperidin, eriocitrin,
and diosmin are the main flavonoids in sweet lemon. Essential
oil of sweet lemon peel contains limonene (49.24%) and c-
terpinene (12.14%) and has antibacterial effect against Pseu-
domonas putida (Oussalah, Caillet, Saucier, & Lacroix, 2006). In folk
medicine, it is used for its antihypertensive effect (Perez, Jimenez-
Ferrer, Alonso, Botello-Amaro, & Zamilpa, 2010; Barreca, Bellocco,
Caristi, Leuzzi, & Gattuso, 2011). Sweet lemon is also a rich source
of nutrients, such as vitamin C and essential minerals (Del Río
et al., 2004). The concentration of these compounds differs accord-
ing to sweet lemon cultivars, cultural practices, maturity stage,
growing field, conditions of storage as well as the extraction pro-
cess and post-processing pasteurization (heat or high-pressure
processing) (Lorente et al., 2014).
The suitability of fruit juices as media for cultivating probiotic
bacteria was recently proposed (Mousavi et al., 2013). The regular
consumption of fruit juice by a variety of age groups make them as
an ideal non-dairy medium based probiotics. Recently, a fermented
pomegranate juice was introduced (Mousavi et al., 2013). How-
ever, some attempts in order to approach of probiotic in other fruit
juices were carried out (Ankolekar, Pinto, Greene, & Shetty, 2012;
Antunes et al., 2013; Pereira, Maciel, & Rodrigues, 2011), citrus
juices due to their popularity, might also be a good target for a fer-
mented probiotic drink (Luckow & Delahunty, 2004; Prado, Parada,
Pandey, & Soccol, 2008).
Therefore, the current study was designed to produce a probi-
otic sweet lemon juice using a L. plantarum LS5 fermentation for
48 h at 37 °C. Furthermore, the viable counts, chemical composi-
tion, antimicrobial activities and antioxidant properties, as well
as sensory properties, were evaluated during fermentation at inter-
vals of 7 days for 28 days.
2. Material and methods
2.1. Raw materials and chemicals
Thirty kg of sweet lemon (a cultivar of Jahrom, at full maturity
stage) were purchased (November 2015) from a local market in
Jahrom City (Fars, Iran). After peeling, the juice was produced using
a mechanical fruit juicer (Pars Khazar, CITRUS model, Gilan, Iran).
The extracted lemon juice was kept at 4 °C to precipitate sus-
pended solids, which were separated using a cotton cloth filter.
The sweet lemon juice was pasteurized at 80 °C for 5 min and
stored frozen ( 20 °C, maximum 2 wk) prior to the day of use.
For thawing, sterile containers containing the juice were stored
at ambient temperature. All chemicals used were supplied by
Merck Co. (Darmstadt, Germany) and Sigma Chemical Co. (St.
Louis. MO, USA).
2.2. Bacterial strains and cultures
Probiotic L. plantarum LS5 (Hashemi et al., 2014), E. coli O157:
H7 ATCC 35150 and Salmonella Typhimurium ATCC 14028 were
obtained from the strain collection of Ferdowsi University of Mash-
had, Mashhad, Iran. L. plantarum was reactivated overnight in MRS
broth (Oxoid, Altrincham, UK) under anaerobic conditions at 37 °C.
E. coli O157:H7 and S. Typhimurium were reactivated in Mueller-
Hinton broth (Oxoid) and incubated at 37 °C overnight. Stock cul-
tures were prepared from the activated cultures and sterile glyc-
erol (1:1 v/v) and were stored frozen ( 20 °C) in sterile screw
caps tubes for a maximum one week.
2.3. Inoculum preparation
Inoculums were prepared by transferring the stock culture of L.
plantarum LS5 to 100 mL of MRS broth. Cells were grown statically
at 37 °C until the cell population reached 9.00 log CFU/mL, which
was then used to initiate the juice fermentation (Hashemi et al.,
2016).
2.4. Fermentation of sweet lemon juice using L. plantarum LS5
Sweet lemon juice (100 mL) was inoculated with 7.00 log CFU/
mL of L. plantarum LS5. Fermentations were carried out in Erlen-
meyer flasks for 48 h at 37 °C. Viable counts, chemical composition,
antioxidant and antibacterial activities of samples were deter-
mined during every hour and at the end of the fermentation
process.
2.5. Probiotic juice storage
Sterile PET bottles (250 mL volume) containing 250 mL of fer-
mented sweet lemon juice were stored in the refrigerator
(4 °C). Viable counts, chemical analyses and antioxidant activity
of juice samples were measured prior to cold storage and at inter-
vals of 7 days for 28 days.
2.6. Determination of viable cells
Serial dilutions (using peptone water) of juice containing L.
plantarum were prepared and aliquots of 0.1 mL of appropriate
dilutions were plated in MRS agar (pH 6.2 ± 0.2) plates (spread
plate method) then incubated at 37 °C for 72 h (Hashemi et al.,
2017; ISO/TS 19036, 2006).
2.7. Chemical tests
2.7.1. pH and acidity
A digital pH meter (MI150, Milwaukee, Italy) was used for the
pH measurements. The acid content was determined by titrating
a sample (5 mL) with 0.1 N NaOH and expressed based on a stan-
dard curve using 0 to 15 mg/mL citric acid.
2.7.2. Bioactive compounds
Ascorbic acid (AA) was measured spectrophotometrically (UV/
visible, Philips, Cambridge, UK) at 500 nm using the method of
(Robinson & Stotz, 1945) and (Burdurlu, Koca, & Karadeniz,
2006). Metaphosphoric acid extraction of the 2,6-dichlorophenol
indophenol dye reduced by ascorbic acid was used. Furthermore,
the ascorbic acid contents were reported as mg/100 g. The concen-
tration of total phenol compounds of sweet lemon juice was mea-
sured spectrophotometrically according to the method of Sudan,
Bhagat, Gupta, Singh, and Koul (2014) at 765 nm. Total phenolics
were reported as gallic acid equivalents (mg/L) from a calibration
curve constructed using gallic acid (100–700 mg/L).
2.7.3. Antioxidant activity
Sweet lemon juice was centrifuged at 4000g for 5 min at 4 °C
and the supernatant was collected for stable free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH) analysis according to the
method of Barreca, Bellocco, Caristi, Leuzzi, and Gattuso (2010). A
volume of 0.3 mL of different concentrations of juice samples was
mixed with 2.7 mL of a methanolized solution of DPPH radical
 S.M.B. Hashemi et al. / Journal of Functional Foods 38 (2017) 409–414 411

(6  10 5 mol/L). The inhibition against DPPH radical was calcu- 2.9. Sensory evaluation
lated at 517 nm (UV/visible, Philips, Cambridge, UK).
The ferric-reducing antioxidant power (FRAP) was measured Sensory evaluation of fermented sweet lemon juice was done
using the procedure described by Benzie and Strain (1996) using using a 12 member panel (six men and six women, 20–25 years
FRAP reagent. For each assay, 20 lL of sample and 2.98 mL of FRAP old). The 9-point hedonic scale was used according to the method
reagent were mixed and then the absorbance was measured at of Hashemi et al. (2016).
593 nm.
2.10. Statistical analyses
2.7.4. HPLC measurement of sugars and organic acids
Quantitative determinations of sugars (fructose and glucose) One way ANOVA and Duncan’s multiple range test using SPSS
and organic acids (lactic and citric acids) were performed accord- software (SPSS 20.0 for Windows; SPSS Inc., Chicago, IL, USA) were
ing to the method of Mousavi et al. (2013) using an HPLC (Knauer, used for determination of differences between groups for all times
Azura, Germany) equipped with a K-2600UV–visible and K-2310 at P < 0.05.
refractive index (RI) detector. The separation columns for sugars
and organic acids were a Eurokat H (250  30 mm, 5 mm, Knauer
3. Results and discussion
column) and an Ultrasep ES-FS special (250  30 mm, 5 mm,
Knauer column), respectively. The ChromGate Software A1493
3.1. Fermentation of sweet lemon juice by probiotic L. Plantarum LS5
was used as software to determine targeted compounds.
According to Fig. 1A, a slow growth of L. plantarum was
2.8. Antibacterial activity assay observed during the initial period of fermentation while after
12 h, a rapid growth was found. Furthermore, after fermentation
Culture supernatant (cell-free) was obtained by centrifugation for 36 h, cell counts of 8.52 log ± 0.34 log CFU/mL were recorded.
(8000g, 4 °C, 20 min, Tuttlingen, Germany) of fermented juice. Fil- According to Mousavi, Mousavi, Razavi, Emam-Djomeh, and Kiani
tration of the supernatant was done using a nominal 0.22 mm filter (2011); after 72 h fermentation, the bacterial population of L. del-
to remove residual cells. The well diffusion method was used to bruekii, L. paracasei, L. acidophilus and L. plantarum increased in
detect growth inhibition of E. coli O157:H7 and S. Typhimurium. pomegranate juice. It was found that L. plantarum and L. delbruekii
The inoculum was spread on Mueller-Hinton Agar (Oxoid) plates had a good growth rate. Additionally, Hashemi et al. (2017) found
resulting in 106 CFU/plate. The plates were incubated at 37 °C for population of L. plantarum strains increased during fermentation of
24 h. Measurement of the diameter of the inhibition zone (with a sarshir.
caliper) was considered an inhibitory effect (Kong, Wang, & The acidity and pH of sweet lemon juice are shown in Fig. 1B.
Xiong, 2007). The measured values for acidity (%) and pH were 1.9 and 3.4,

A) B)
9,5 4,5 2,2
9,0 2,0
4,0
8,5
1,8
8,0

Acidity (%)
Log (cfu/ml)

3,5 1,6
7,5
pH

7,0 3,0 1,4

6,5 1,2
6,0 2,5
1,0
5,5
2,0 0,8
5,0
0 1 2 3 4 5 6
0 12 24 36 48
Time (hours) Time (hours)

C) D)
8 60 7,5
glucose Fructose
7
50 7,0
Citric acid concentration (g/l)

concentration of sugars (g/l)

Lactic acid concentration (g/l)

6
6,5
40
5
6,0
4 30
5,5
3
20
5,0
2
10 4,5
1
4,0
0 0
0 12 24 36 48
0 12 24 36 48
Time (hours) Time (hours)

Fig. 1. Effect of fermentation on microbial cells (Lactobacillus plantarum) (A), pH, acidity (B), organic acids (C) and sugars (D) of sweet lemon juice. Dashed lines indicate pH
and lactic acid changes. Some points are significantly different (P < 0.05).
412 S.M.B. Hashemi et al. / Journal of Functional Foods 38 (2017) 409–414

respectively. The total acidity of fermented sweet lemon juice study showed that the fermentation process had no significant
declined to 1.2, which can be attributed to a decrease in citric acid. effect on the concentration of AA in juices (Fig. 2A). However, a sig-
In this line, Hashemi et al. (2016) reported that L. plantarum LS5 nificant decrease in total phenolic compounds was observed in fer-
increased the acidity and reduces the pH of Doogh. mented juice, which was mainly attributed to phenolic compounds
Moreover, in order to evaluate the changes of organic acid con- metabolism enhanced by L. plantarum (Fig. 2A). Similarly, Othman,
centrations during the fermentation, HPLC analysis was used. Citric Roblain, Chammen, Thonart, and Hamdi (2009) reported that both
acid decreased while lactic acid increased throughout the fermen- spontaneous and controlled fermentations of olives resulted in a
tation (Fig. 1C). Citric acid content (53 g/L) was reduced signifi- large decrease (32–58%) in total phenolic compounds. They found
cantly during the fermentation by L. plantarum, being 13.2 g/L these compounds were metabolized by starter strains.
within 48 h of fermentation. This fact can be attributed to the In addition, the antibacterial effect of fermented and non-
use of citric acid during fermentation as the main available carbon fermented juice was evaluated (Fig. 2B). Results indicated that
source in sweet lemon juice to produce lactic acid. Similar behavior the antibacterial properties increased during fermentation. Fer-
of Lactobacillus species, such as L. casei, incorporated in the growth mented juice showed an acceptable antibacterial activity against
media was reported previously (Mousavi et al., 2011; Palles, E. coli O157: H7 and S. Typhimurium. Results also showed that
Beresford, Condon, & Cogan, 1998). As result of sweet lemon juice the fermented juice had higher antibacterial properties against S.
fermentation, the concentration of lactic acid increased (Hashemi Typhimurium in comparison to E. coli O157: H7 (P < 0.05). These
et al., 2016). The amount of lactic acid produced was not enough findings are in agreement with results of Pereira et al. (2011),
to compensate the observed decrease in citric acid concentration who claimed that L. casei could control the growth of other bacteria
and, consequently, the acidity was reduced. as well as juice spoilage during fermentation of cashew apple juice.
The metabolism of carbohydrate during the fermentation is Indeed, the interaction of lactic acid on the external membrane of
shown in Fig. 1D. The major detected sugars in sweet lemon juice Gram-negative pathogenic bacteria could increase permeabiliza-
were glucose and fructose, which were utilized mainly between 12 tion and consequently allows other antibacterial compounds to
and 36 h of the fermentation during the exponential microbial enter into the bacteria (Alakomi et al., 2000). However, the analyt-
growth rate. The fermentation process of L. plantarum caused a ical methods for detecting pathogens inside probiotics or products
reduction from 6.1 and 6.9 to 5.3 and 5.1 g/L in the concentration with probiotics are not fully validated and accurate internationally.
of fructose and glucose, respectively, after 48 h. Fermentation of Regarding antioxidant capacity, Fig. 2C shows a significant
sugars by microorganisms could be correlated to the strain, fer- increase in the DPPH scavenging and FRAP (P < 0.05) as a result
mentation time and substrate (Mousavi et al., 2011; Palles, of lactic acid fermentation. The antioxidant activity of sweet lemon
Beresford, Condon, & Cogan, 1998). For instance, Mousavi et al. juice significantly was improved. These findings are in good agree-
(2011) reported that glucose and fructose could be considered as ment with findings of Mousavi et al. (2013), who showed that dur-
suitable energy and carbon sources for Lactobacillus strains. ing fermentation of pomegranate juice, the concentration of
On the other hand, the impact of fermentation on bioactive phenolic compounds decreased, whereas the antioxidant activity
compounds was also evaluated. As it is well known, juices are rich of fermented pomegranate juice increased. Dueñas, Fernández,
in AA but it is an unstable compound and under storage conditions Hernández, Estrella, and Muñoz (2005) reported that fermentation
can be decomposed rapidly (Burdurlu et al., 2006). Results of this of cowpea flours by L. plantarum increased antioxidant activity.

Fermented juice non-fermented juice

Total phenolic compounds (mg/l)
Ascorbic acid content (mg/ 100g)

A) 300 700 B) 25
690 a
250
a
Inhibition zone (mm)

680 20
200 670
15
150
660 b
650 b
10
100 640
630
50 5
620
0 610 0
0 12 24 36 48 E.coli O157:H7 S. Typhimurium
Time (hours)

C) 100 30
Inhibition against DPPH (I%)

90
25
FRAP (mmol Fe/mass)

80
70 20
60
50 15
40
30 10
20 5
10
0 0
0 12 24 36 48
Time (hours)

Fig. 2. Effect of fermentation on ascorbic acid, total phenolic compounds (A), antibacterial activity (B), and antioxidant activity (C) of sweet lemon juice. Dashed lines indicate
ascorbic acid and DPPH radical scavenging changes.
 S.M.B. Hashemi et al. / Journal of Functional Foods 38 (2017) 409–414 413

10 Fermented juice non-fermented juice

A) 9
B) 270

Ascorbic acid (mg/100g)

8
Log (cfu/mL)
250
7
6 230
5
210
4
3 190
2
170
1
0 150
0 7 14 21 28 0 7 14 21 28
Day Day

C) non-fermented juice Fermented juice D) Fermented juice non-fermented juice

750 100
Total phenolic compounds

Inhibition DPPH (I%)

700 80
650 60
(mg/l)

600 40

550 20

500 0
0 7 14 21 28 0 7 14 21 28
Day Day

E) F) Taste Overall aceptability

Fermented juice non-fermented juice 10
30 a a
FRAP( mmol Fe/mass)

25 8 b
b
20
6
Score

15
10 4
5
2
0
0 7 14 21 28 0
Fermented juice non-fermented juice
Day

Fig. 3. Viable cells (Lactobacillus plantarum) (A), ascorbic acid content (B) total phenolic compounds (C), DPPH assay (D), FRAP assay (E) and sensory evaluation (F) of sweet
lemon juice samples during storage at 4 °C. Each column with same small letter is not significantly different.

Kim et al. (2011) concluded that metabolites produced by the Total phenolics of sweet lemon juice decreased in both fer-
selected strains through fermentation may also have a significant mented and non-fermented juice during storage (Fig. 3C). It
impact on increasing antioxidant activity in fermented Cheongguk- was reported that phenolic compounds were affected by both
jang juice. Furthermore, in a previous study, fermentation of Doogh duration and temperature of storage (Klimczak, Malecka,
by L. plantarum LS5 showed radical scavenging activities and Szlachta, & Gliszczyńska-Świgło, 2007). They found the phenolic
increased oxidation stability of Doogh (Hashemi et al., 2016). content of orange juice significantly declined during the storage.
It was reported phenolic compounds can degrade by enzymes
3.2. Storage of the probiotic sweet lemon juice and chemical reactions.
Fig. 3D and E shows the antioxidant capacity of fermented
The viable cell counts of L. plantarum in the fermented juice and non-fermented juice decreased during storage. Both total
were 7.14 log ± 0.21 log CFU/mL after 28 d of storage at 4 °C phenol contents and AA concentration strongly correlated with
(Fig. 3A). For probiotics to survive the unfavorable conditions the antioxidant activity of citrus juices. Additionally, AA
inside gastrointestinal tract and reach the intestine in adequate accounted for 65–100% of the total antioxidant activities of
numbers they should be available initial at 107 CFU/mL in food at citrus juices (Gardner, White, McPhail, & Duthie, 2000).
the end of the shelf-life period; thus, in the case of fermented Klimczak et al. (2007) found the decrease in the content of phe-
sweet lemon juice, it can be used as a probiotic juice for up four nolics and AA during storage was correlated with the reduction
week. It was previously reported that various fruit juices could in the antioxidant capacity of orange juice. However, the pH,
serve as effective media for growth of probiotics, to provide the acidity, lactic acid metabolism and sugar consumption did not
desired viable counts. The observed cell growth profile in this change during storage (P < 0.05).
study corresponded to previous investigations (Sheehan, Ross, & The observed variation in scores during sensory evaluation for
Fitzgerald, 2007). fermented sweet lemon juice during 28 days storage at 4 °C are
Although the AA content of the juice significantly decreased presented in Fig. 3F. Fermented juice resulted in attribute scores
during storage of both fermented and non-fermented juices, AA greater than the rejection limit (6) during the storage. The overall
reduction in fermented juices was lower in comparison to non- acceptability and taste of fermented sweet lemon juice were eval-
fermented juice (Fig. 3B). Several factors such as the pH, sugars, uated by the panelists with scores significantly higher than non-
and minerals concentration in the juice, temperature, oxygen fermented juice, during the storage. This suggests that with this
levels and presence of enzymes can affect the stability of AA during limited panel experience that the fermented sweet lemon juice
storage and processing (Burdurlu et al., 2006). was organoleptically acceptable.
414 S.M.B. Hashemi et al. / Journal of Functional Foods 38 (2017) 409–414
4. Conclusions
From the results obtained in this study, it can be concluded that
citric acid, total phenolic compounds, and sugar contents were sig-
nificantly reduced in fermented sweet lemon juice, thus showing
the capability of L. plantarum to consume high amounts of the sub-
strate compared to other strains measured. However, ascorbic acid
content was not modified significantly immediately after fermen-
tation of sweet lemon juice and lactic acid was increased. After
36 h of fermentation L. plantarum cell counts were 8.52 log ± 0.34
log CFU/mL, while after 28 d of storage at 4 °C, they were reduced
to 7.14 log ± 0.21 log CFU/mL, which was acceptable. Fermentation
of sweet lemon juice could improve the chemical properties such
as antioxidant activity as well as antibacterial properties and the
fermented juice could be used as a nondairy functional food
product.
Acknowledgments
Amin Mousavi Khaneghah gratefully acknowledges the support
of CNPq-TWAS Postgraduate Fellowship (Grant #3240274290).
This study was supported by Fasa University, Fasa, Iran.
Conflict of interest
Authors declare that they have no conflict of interest.
References
Alakomi, H. L., Skyttä, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., &
Helander, I. M. (2000). Lactic acid permeabilizes gram-negative bacteria by
disrupting the outer membrane. Applied and Environmental Microbiology, 66(5),
2001–2005.
Ankolekar, C., Pinto, M., Greene, D., & Shetty, K. (2012). In vitro bioassay based
screening of antihyperglycemia and antihypertensive activities of Lactobacillus
acidophilus fermented pear juice. Innovative Food Science and Emerging
Technologies, 13, 221–230.
Antunes, A. E. C., Liserre, A. M., Coelho, A. L. A., Menezes, C. R., Moreno, I.,
Yotsuyanagi, K., & Azambuja, N. C. (2013). Acerola nectar with added
microencapsulated probiotic. LWT – Food Science and Technology, 54(1),
125–131.
Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2010). Flavonoid
composition and antioxidant activity of juices from Chinotto (Citrus 
myrtifolia Raf.) fruits at different ripening stages. Journal of Agricultural and
Food Chemistry, 58(5), 3031–3036.
Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011). Flavonoid profile
and radical-scavenging activity of Mediterranean sweet lemon (Citrus limetta
Risso) juice. Food Chemistry, 129(2), 417–422.
Benzie, I. F. F., & Strain, J. J. (1996). The Ferric Reducing Ability of Plasma (FRAP) as a
measure of Antioxidant Power: The FRAP Assay. Analytical Biochemistry, 239(1),
70–76.
Burdurlu, H. S., Koca, N., & Karadeniz, F. (2006). Degradation of vitamin C in citrus
juice concentrates during storage. Journal of Food Engineering, 74(2), 211–216.
Del Río, J. A., Fuster, M. D., Gómez, P., Porras, I., García-Lidón, A., & Ortuño, A. (2004).
Citrus limon: A source of flavonoids of pharmaceutical interest. Food Chemistry,
84(3), 457–461.
Dueñas, M., Fernández, D., Hernández, T., Estrella, I., & Muñoz, R. (2005). Bioactive
phenolic compounds of cowpeas (Vigna sinensis L). Modifications by
fermentation with natural microflora and with Lactobacillus plantarum ATCC
14917. Journal of the Science of Food and Agriculture, 85(2), 297–304.
Ekıncı, R. (2005). The effect of fermentation and drying on the water-soluble
vitamin content of tarhana, a traditional Turkish cereal food. Food Chemistry, 90
(1), 127–132.
Gardner, P. T., White, T. A. C., McPhail, D. B., & Duthie, G. G. (2000). The relative
contributions of vitamin C, carotenoids and phenolics to the antioxidant
potential of fruit juices. Food Chemistry, 68(4), 471–474.
Guarner, F., & Schaafsma, G. J. (1998). Probiotics. International Journal of Food
Microbiology, 39, 237–238.
Hashemi, S. M. B., Amininezhad, R., Shirzadinezhad, E., Farahani, M., & Yousefabad,
S. H. A. (2016). The antimicrobial and antioxidant effects of Citrus aurantium L.
flowers (Bahar Narang) extract in traditional yoghurt stew during refrigerated
storage. Journal of Food Safety, 36(2), 153–161.
Hashemi, S. M. B., Mousavi Khaneghah, A., Kontominas, M. G., Esß, I., Sant’Ana, A. S.,
Martinez, R. R., & Drider, D. (2017). Fermentation of sarshir (kaymak) by lactic
acid bacteria: antibacterial activity, antioxidant properties, lipid and protein
oxidation and fatty acid profile. Journal of the Science of Food and Agriculture.
https://doi.org/10.1002/jsfa.8329 (in press).
Hashemi, S. M. B., Shahidi, F., Mortazavi, S. A., Milani, E., & Eshaghi, Z. (2014).
Potentially probiotic Lactobacillus strains from traditional Kurdish cheese.
Probiotics and Antimicrobial Proteins, 6(1), 22–31.
Hashemi, S. M. B., Shahidi, F., Mortazavi, S. A., Milani, E., & Eshaghi, Z. (2016). Effect
of Lactobacillus plantarum LS5 on oxidative stability and lipid modifications of
Doogh. International Journal of Dairy Technology, 69(4), 550–558.
Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., & Sanders, M. E.
(2014). Expert consensus document: The international scientific association for
probiotics and prebiotics consensus statement on the scope and appropriate use
of the term probiotic. Nature Reviews Gastroenterology and Hepatology, 11(8),
506–514.
ISO/TS 19036 (2006). Microbiology of food and animal feeding stuffs—Guidelines
for the estimation of measurement uncertainty for quantitative determinations.
Switzerland.
Kim, J., Choi, J. N., Kang, D., Son, G. H., Kim, Y. S., Choi, H. K., & Lee, C. H. (2011).
Correlation between antioxidative activities and metabolite changes during
Cheonggukjang fermentation. Bioscience, Biotechnology, and Biochemistry, 75(4),
732–739.
Klimczak, I., Malecka, M., Szlachta, M., & Gliszczyńska-Świgło, A. (2007). Effect of
storage on the content of polyphenols, vitamin C and the antioxidant activity of
orange juices. Journal of Food Composition and Analysis, 20(3–4), 313–322.
Kong, B., Wang, J., & Xiong, Y. L. (2007). Antimicrobial activity of several herb and
spice extracts in culture medium and in vacuum-packaged pork. Journal of Food
Protection, 70(3), 641–647.
Leone, R. D. S., de Andrade, E. F., Ellendersen, L. N., da Cunha, A., Chupel Martins, A.
M., Granato, D., & Masson, M. L. (2017). Evaluation of dried yacon (Smallanthus
sonchifolius) as an efficient probiotic carrier of Lactobacillus casei LC-01. LWT –
Food Science and Technology, 75, 220–226.
Liévin-Le Moal, V., & Servin, A. L. (2014). Anti-infective activities of Lactobacillus
strains in the human intestinal microbiota: From probiotics to gastrointestinal
anti-infectious biotherapeutic agents. Clinical Microbiology Reviews, 27(2),
167–199.
Lorente, J., Vegara, S., Mart, N., Ibarz, A., Coll, L., & Hernandez, J. (2014). Chemical
guide parameters for Spanish lemon (Citrus limon (L.) Burm.) juices. Food
Chemistry, 162, 186–191.
Luckow, T., & Delahunty, C. (2004). Which juice is ‘‘healthier”? A consumer study of
probiotic non-dairy juice drinks. Food Quality and Preference, 15(7), 751–759.
Madureira, A. R., Soares, J. C., Pintado, M. E., Gomes, A. M. P., Freitas, A. C., & Xavier
Malcata, F. (2015). Effect of the incorporation of salted additives on probiotic
whey cheeses. Food Bioscience, 10, 8–17.
Maldonado Galdeano, C., & Perdigón, G. (2006). The probiotic bacterium
Lactobacillus casei induces activation of the gut mucosal immune system
through innate immunity. Clinical and Vaccine Immunology, 13(2), 219–226.
Mota, M. J., Lopes, R. P., Delgadillo, I., & Saraiva, J. A. (2015). Probiotic yogurt
production under high pressure and the possible use of pressure as an on/off
switch to stop/start fermentation. Process Biochemistry, 50(6), 906–911.
Mousavi, Z. E., Mousavi, S. M., Razavi, S. H., Emam-Djomeh, Z., & Kiani, H. (2011).
Fermentation of pomegranate juice by probiotic lactic acid bacteria. World
Journal of Microbiology and Biotechnology, 27(1), 123–128.
Mousavi, Z. E., Mousavi, S. M., Razavi, S. H., Hadinejad, M., Emam-Djomeh, Z., &
Mirzapour, M. (2013). Effect of fermentation of pomegranate juice by
Lactobacillus plantarum and Lactobacillus acidophilus on the antioxidant
activity and metabolism of sugars, organic acids and phenolic compounds.
Food Biotechnology, 27(1), 1–13.
Othman, N. B., Roblain, D., Chammen, N., Thonart, P., & Hamdi, M. (2009).
Antioxidant phenolic compounds loss during the fermentation of Chétoui
olives. Food Chemistry, 116(3), 662–669.
Oussalah, M., Caillet, S., Saucier, L., & Lacroix, M. (2006). Antimicrobial effects of
selected plant essential oils on the growth of a Pseudomonas putida strain
isolated from meat. Meat Science, 73(2), 236–244.
Palles, T., Beresford, T., Condon, S., & Cogan, T. M. (1998). Citrate metabolism in
Lactobacillus casei and Lactobacillus plantarum. Journal of Applied Microbiology, 85
(1), 147–154.
Pereira, A. L. F., Maciel, T. C., & Rodrigues, S. (2011). Probiotic beverage from cashew
apple juice fermented with Lactobacillus casei. Food Research International, 44(5),
1276–1283.
Perez, Y. Y., Jimenez-Ferrer, E., Alonso, D., Botello-Amaro, C. A., & Zamilpa, A. (2010).
Citrus limetta leaves extract antagonizes the hypertensive effect of angiotensin
II. Journal of Ethnopharmacology, 128(3), 611–614.
Prado, F. C., Parada, J. L., Pandey, A., & Soccol, C. R. (2008). Trends in non-dairy
probiotic beverages. Food Research International, 41(2), 111–123.
Robinson, W. B., & Stotz, E. (1945). The indophenol-xylene extraction method for
ascorbic acid and modifications for interfering substances. Journal of Biological
Chemistry, 160, 217–225.
Sah, B. N. P., Vasiljevic, T., McKechnie, S., & Donkor, O. N. (2014). Effect of probiotics
on antioxidant and antimutagenic activities of crude peptide extract from
yogurt. Food Chemistry, 156, 264–270.
Sanders, M. E. (2008). Probiotics: Definition, sources, selection, and uses. Clinical
Infectious Diseases, 46(Suppl. 2), S58–S61.
Sheehan, V. M., Ross, P., & Fitzgerald, G. F. (2007). Assessing the acid tolerance and
the technological robustness of probiotic cultures for fortification in fruit juices.
Innovative Food Science and Emerging Technologies, 8(2), 279–284.
Sudan, R., Bhagat, M., Gupta, S., Singh, J., & Koul, A. (2014). Iron (FeII) chelation,
ferric reducing antioxidant power, and immune modulating potential of
arisaema jacquemontii (himalayan cobra lily). BioMed Research International.
https://doi.org/10.1155/2014/179865. 2014.