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LWT - Food Science and Technology 75 (2017) 195e201

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LWT - Food Science and Technology


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

Effect of ascorbic acid or oligofructose supplementation on L. paracasei


viability, physicochemical characteristics and acceptance of probiotic
orange juice
Guilherme Mamede da Costa, Jose  Vitor de Carvalho Silva, Je
ssica Dias Mingotti,
~o, Suellen Jensen Klososki, Tatiana Colombo Pimentel*
Carlos Eduardo Bara
 (IFPR) e Campus Paranavaí, Rua Jos
Instituto Federal do Parana , 87703-536, Brazil
~es, Paranavaí, Parana
e Felipe Tequinha, 1400, Jardim das Naço

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

Article history: This study aimed to evaluate the effect of oligofructose (prebiotic) or ascorbic acid supplementation on
Received 17 June 2016 the viability of Lactobacillus paracasei ssp. paracasei probiotic culture, physicochemical characteristics
Received in revised form and acceptance of orange juice during cold storage (4  C for 28 days). Juices with probiotic culture,
3 August 2016
ascorbic acid and/or oligofructose supplementation showed physicochemical characteristics, acceptance
Accepted 23 August 2016
Available online 24 August 2016
and storage stability similar to the pure juices; but lower turbidity (except oligofructose added juice) and
yellow color. Oligofructose or ascorbic acid did not exert a protective effect on probiotic cultures during
storage, but the juices showed viability of probiotic culture higher than 106 CFU/mL during 28 days of
Keywords:
Orange juice
cold storage. The oligofructose content was maintained at appropriated concentrations (1.65e1.68 g/
Probiotic 100 mL) during storage. It is possible to develop synbiotic orange juice added oligofructose (prebiotic)
Prebiotic and probiotic cultures without changing the physicochemical characteristics and acceptance of pure juice
Vitamin C and that may be cold stored for 28 days.
Citrus sinensis © 2016 Published by Elsevier Ltd.

1. Introduction sugars and bioactive compounds (minerals, vitamins, fiber and


antioxidants) which could be utilized by probiotic cultures. In
Probiotics are living microorganisms that provide beneficial addition, they do not contain starter cultures, which compete for
effects to consumers when administered in adequate amounts nutrients with the probiotic cultures (Costa, Fonteles, Jesus, &
(FAO/WHO, 2002). Yoghurts and fermented milks are the main Rodrigues, 2013; Ding & Shah, 2008; Sheehan, Ross, & Fitzgerald,
probiotic products available in the market; however, there is a 2007).
growing interest in the development of non-dairy probiotic prod- However, the maintenance of the viability of probiotic cultures
ucts (Antunes et al., 2013). in fruit juices is difficult, since these products have low pH, high
Non-dairy probiotic products might be useful especially to dissolved oxygen concentration and insufficient amounts of free
consumers who do not appreciate or cannot consume dairy prod- amino acids and peptides. The survival of probiotic cultures in fruit
ucts, including people who are lactose intolerant, allergic to milk juices is dependent on the juice parameters and the probiotic strain
proteins, hypercholesterolemic or strictly vegetarian (Bevilacqua, used. In addition, probiotic cultures may alter the sensory charac-
Campaniello, Corbo, Maddalena, & Sinigaglia, 2013). Furthermore, teristics of the products, especially the aroma and flavor attributes
non-dairy probiotic products would be another way to the general (Antunes et al., 2013; Luckow & Delahunty, 2004). In order to
population to obtain this type of beneficial cultures. improve the survival of probiotic cultures in fruit juices, prebiotic
Fruit juices can be considered suitable products for the addition components or ascorbic acid could be used.
of probiotic cultures as they are considered healthy and refreshing Oligofructose is one of the main prebiotic components available
beverages, are consumed regularly by people of all ages, and have in the market, being capable to provide beneficial health effects to
hosts associated with modulation of their microbiota (FAO/AGNS,
2007). Because they are substrates available for the metabolism
of the probiotic cultures, oligofructoses could increase the stability
* Corresponding author.
of these cultures in fruit juices during cold storage (Donkor,
E-mail address: tatiana.pimentel@ifpr.edu.br (T.C. Pimentel).

http://dx.doi.org/10.1016/j.lwt.2016.08.051
0023-6438/© 2016 Published by Elsevier Ltd.
196 G.M. da Costa et al. / LWT - Food Science and Technology 75 (2017) 195e201

Nilmini, Stolic, Vasiljevic, & Shah, 2007). juices were then supplemented with ascorbic acid (0.24 g/L; ACID
The probiotic strains used in foods are usually aerobic or and PRO-ACID formulations) or oligofructose (20 g/L; PRE and SYNB
microaerophilic and, therefore, the presence of oxygen in the formulations) (Pimentel, Madrona, & Garcia, 2015; Shah et al.,
product may cause toxicity and death of the micro-organism, with 2010).
consequent loss of functionality (Cruz, Faria, & Van Dender, 2007). All formulations were placed in glass flasks, pasteurized at 80  C
The ascorbic acid (vitamin C) could have a protective effect on for 20 min in a water bath (Marconi®, Piracicaba, Brazil) and cooled
probiotic cells during storage, presumably because it is an oxygen in an ice bath until reaching 37  C. Formulations with probiotic
scavenger, thus promoting a more favorable anaerobic environ- cultures (PRO, PRO-ACID and SYNB) were supplemented with
ment (Shah, Ding, Fallourd, & Leyer, 2010). Ascorbic acid is 20 mL/L activated probiotic cultures (3  107 CFU/mL of juice). The
commonly added to fruit juices to prevent browning and to provide orange juices were then stored at 4 ± 2  C for 28 days, which is the
an additional source of vitamin C. The total vitamin C content in shelf life of commercial pasteurized Brazilian orange juice.
200 mL of orange juice (50 mg/100 mL) is higher than the recom-
mended daily intake (RDI) for adults (75 mg/day for women and 2.5. Physicochemical evaluations and probiotic viability
90 mg/day for men), according to the National Institutes of Health
(2011). Measurements of moisture, protein, lipid, ash and carbohy-
Few studies have evaluated the effect of adding probiotic cul- drates were performed according to the Association of Official
tures to orange juice (Ding & Shah, 2008; Luckow & Delahunty, Analytical Chemists guidelines (Aoac, 2004). The oligofructose
2004; Nualkaekul, Salmeron, & Charalampopoulos, 2011; concentration was determined using a Fructan HK enzymatic kit
Rodrigues et al., 2012; Saarela, Virkajarvi, Nohynek, Vaari, & Matto, (Megazyme International Ireland®).
2006; Sheehan et al., 2007), but none evaluated the concomitant The pH was determined using a digital potentiometer (MS
supplementation of the product with oligofructose or ascorbic acid. Technopon®, Piracicaba, Brazil). The titratable acidity was
Therefore, the objective of this study was to evaluate the effect of measured according to Aoac (2004) and expressed as a percentage
oligofructose or ascorbic acid supplementation on the viability of of citric acid. The level of total soluble solids (TSS), as  Brix, was
Lactobacillus paracasei ssp. paracasei probiotic culture, physico- assessed using a digital refractometer (Instruterm®, Sa ~o Paulo,
chemical characteristics and acceptance of orange juice during cold Brazil). Analysis of ascorbic acid was performed by iodometric
storage (4  C for 28 days). titration (IAL, 2008, Chap. XIX).
A colorimeter (Minolta®, model CR400, Osaka, Japan) was used
2. Material and methods for the assessment of color parameters values. Turbidity was
measured using an UV/VIS spectrophotometer at 600 nm (Shah
2.1. Material et al., 2010).
The texture parameters (firmness, consistency, cohesiveness
Pera Orange (Citrus sinensis L. Osbeck, var. Pera), oligofructose and viscosity index) of the orange juices were determined by a
(P95, Orafti®), ascorbic acid (Synth®), probiotic culture Lactobaciilus single compression test using a texture analyzer (TA-XT plus®,
paracasei ssp. paracasei (L. casei-01, Christian Hansen®) and glass Stable Micro System, London, England) equipped with a 5 Kg load
flasks (Farma®) were used in the present study. cell. The formulations (40 mL), in plastic containers of 55 mm
diameter and 60 mm height, were compressed with a 36-mm
2.2. Activation of the probiotic culture diameter cylindrical probe (P36 R) to a depth of 10 mm at a con-
stant speed of 1 mm/s (pre-test and test speeds).
A culture aliquot was inoculated into 5 mL of sterilized orange L. paracasei ssp. paracasei count was performed in Mann Rogosa
juice and incubated at 37  C for 15 h. After this time, 0.05 mL was and Sharpe (MRS) agar (Difco®) and anaerobic incubation (Anae-
again inoculated into 10 mL of sterilized orange juice and incubated robac, Probac®) at 37  C for 72 h (Tharmaraj & Shah, 2003).
at 37  C for 15 h (pre-inoculum) (Pimentel, Madrona, Garcia, &
Prudencio, 2015). To obtain the biomass, 0.1 mL of the pre- 2.6. Sensory evaluation
inoculum was transferred into 300 mL of sterilized orange juice
and re-incubated under the same conditions. The biomass was The sensory panel was composed of 70 untrained individuals
separated by centrifugation in a refrigerated centrifuge (Eppen- (54 women and 26 men), ranging in age from 15 to over 50 years,
dorf®, model 5804R, Piracicaba, Brazil) at 2200g for 10 min at 4  C with majority (51 individuals) ageing 15e25 years.
and washed three times in 0.85 g/100 mL sterile saline solution The acceptance testing of attributes (appearance, aroma, flavor,
(NaCl Dinamica®) to remove the residual fermented juice. The texture and overall acceptance) using a 9-point hedonic scale (1 -
biomass was then resuspended into 50 mL of 0.85 g/100 mL sterile disliked very much and 9 - liked very much) (Stone & Sidel, 2004)
saline solution, thus obtaining the activated probiotic culture. was performed on day 1 of product storage. The judges evaluated
the six formulations in one evaluation session. The formulations
2.3. Formulations (25 mL) were coded with 3-digit numbers and served at a tem-
perature of 4  C in plastic cups, one at a time, in random order. The
Six formulations of orange juice were prepared: PURE (pure tests were performed in individual tables with fluorescent lamps.
juice), PRO (juice added probiotic culture), ACID (juice added acid Drinking water at room temperature and cream crackers were
ascorbic), PRE (juice added oligofructose), PRO-ACID (juice added provided to clean the mouth before and between evaluations of the
probiotic culture and ascorbic acid) and SYNB (juice added pro- formulations.
biotic culture and oligofructose).
2.7. Statistical analysis
2.4. Preparation of orange juices
The complete experiment was replicated two times using a
Oranges were washed in running water, sanitized (6 mL/L Pury completely randomized design. The physicochemical and probiotic
Vitta® fruit disinfectant with 0.96 g/100 mL active chlorine), peeled viability characteristics were performed in triplicates in each
and crushed using a fruit processor (Walita®, Joinville, Brazil). The experiment repetition, every seven days for a 28 day period. The
G.M. da Costa et al. / LWT - Food Science and Technology 75 (2017) 195e201 197

oligofructose content was determined on days 1 and 28 of storage, during the entire shelf life, considering a portion of 200 mL of juice.
in triplicate. A split plot design was used, in which the main
treatment was the formulation and secondary treatment was the 3.2. Physicochemical characteristics
storage duration. The proximate composition was assessed in
triplicates in each experiment repetition on the first day of storage. The physicochemical characteristics of orange juice formula-
For acceptance, the experimental design consisted of randomized tions are shown in Table 2. The orange juices showed pH
complete blocks (the treatments were the formulations, and the (3.88e4.11); titratable acidity (0.53e0.63% citric acid) and TSS
blocks were the judges). Data were submitted to ANOVA and (8.22e10.78  Brix) values typical for orange juice and similar to
Tukey's comparison of the means test (p ¼ 5%). Statistical analyses those reported by other authors (Ding & Shah, 2008; Kelebek et al.,
were performed using the Statistical Analysis System (SAS) 2009; Sohail, Turner, Prabawati, Coombes, & Bhandari, 2012).
software. The pH and titratable acidity of the products were not changed
by the supplementation with oligofructose (PRE), probiotic (PRO) or
3. Results and discussion ascorbic acid (ACID) (p > 0.05) when compared to pure juice (PURE)
during the 28 days of storage (except PRO formulation on day 7).
3.1. Proximate composition The probiotic cultures can metabolize simple sugars present in the
juice, resulting in a production of small amounts of organic acids
In Table 1 is shown the proximate composition of orange juice and decreased pH (Rodrigues et al., 2012). The maintenance of the
formulations. Orange juices supplemented with L. paracasei ssp. pH and titratable acidity of probiotic juices can be related to the
paracasei probiotic culture (PRO, PRO-ACID and SYNB) or ascorbic high buffering capacity of the products and to the fact that some
acid (ACID and PRO-ACID) had similar (p > 0.05) proximate strains, such as L. casei, are able to metabolize citric acid
composition to the pure orange juice (PURE). The addition of oli- (Nualkaekul et al., 2011).
gofructose (PRE and SYNB) resulted in products with lower mois- The maintenance in the acidity of the juices is interesting from a
ture values and higher carbohydrate values (p  0.05) (PURE). sensory point of view, because an increase in acidity could result in
Oligofructoses are soluble oligosaccharides, which causes an in- a decrease in the acceptance of the juices by consumers (Sohail
crease in total solids and carbohydrates contents when added to et al., 2012). Furthermore, high acidity is related to decreased
foods (Pimentel, Madrona, & Garcia, 2015). viability of the probiotic cultures in food products (Pimentel,
The proximate composition (g/100 mL) fell within the following Madrona, & Garcia, 2015).
ranges: moisture (88.7e90.37); protein (1.37e1.85); ash Juices supplemented with probiotic cultures (PRO) or ascorbic
(0.58e0.71), lipid (0.19e0.58) and carbohydrate (6.83e8.44); acid (ACID) presented total soluble solids (TSS) similar (p > 0.05) to
corroborating those reported by other authors (Kelebek, Selli, the pure juice (PURE), while juices supplemented with oligo-
Canbas, & Cabaroglu, 2009; Taco, 2011). fructose (PRE and SYNB) had higher TSS values (p  0.05). The in-
The concentration of oligofructose on day 1 of storage was crease in TSS content is related to the presence of mono- and
1.71e1.86 g/100 mL (PRE and SYNB), lower than the level originally disaccharides in the composition of oligofructose.
added (2 g/100 mL). Oligofructose already has 5e6% of free sugars The orange juice formulations (PURE, PRO, ACID, PRE, PRO-ACID
(fructose, glucose and sucrose) in its formulation (Ro € ßle, Brunton, and SYNB) were stable to cold storage when evaluated for pH,
Gormley, Ross, & Butler, 2010) and the orange juices were sub- titratable acidity and TSS parameters, comparing the products on
jected to a heat treatment (80  C/20 min), which could reduce the the 1st and 28th days of storage (p > 0.05). Stability of the physi-
content of oligofructose in the product (Courtin, Swennen, Verjans, cochemical characteristics of juices during storage is desirable
& Delcour, 2009). because it confirms that the products remain similar to those
During the storage period, the oligofructose content was recently manufactured even after a few weeks of storage (Pimentel,
maintained (p > 0.05) in the initial values, which indicates that the Prudencio, & Rodrigues, 2011).
oligofructose was not decomposed at the low pH (Table 1) and that The formulations ACID and PRO-ACID presented the highest
this component was not used by probiotic culture in its meta- (p  0.05) ascorbic acid content, as expected. In the 1st day of
bolism. The suggested minimum daily effective intake of oligo- storage, probiotic (PRO) and prebiotic (PRE) formulations presented
fructose required to produce a beneficial health effect is 3e8 g of similar ascorbic acid (p > 0.05) content to the pure juice (PURE).
oligofructose/portion of product (Keenan, Brunton, Butler, Wouters, During the storage period all formulations had a decrease in
& Gormley, 2011; Ro € ßle et al., 2010). At the 28th day of storage, the ascorbic acid content by 14e20%, corroborating previous studies
formulations presented 1.65e1.68 g/100 mL of oligofructose, (Del Caro, Piga, Vacca, & Agabbio, 2004; Plaza et al., 2006; Ros-
therefore, the orange juices could be considered prebiotic products  pez, 2007). The methods used in
Chumillas, Belissario, Iguaz, & Lo

Table 1
Proximate composition (g/100 mL) of the orange juice formulations.a

Parameter Formulationb

PURE PRO ACID PRE PRO-ACID SYNB

Moisture 90.25 ± 0.08a 90.37 ± 0.04a 90.08 ± 0.12a 88.70 ± 0.18b 90.28 ± 0.09a 88.86 ± 0.89b
Protein 1.85 ± 0.44a 1.84 ± 0.34a 1.83 ± 0.23a 1.69 ± 0.23a 1.78 ± 0.13a 1.37 ± 0.08a
Lipid 0.36 ± 0.23 ab 0.20 ± 0.04b 0.19 ± 0.08b 0.56 ± 0.07a 0.45 ± 0.08 ab 0.58 ± 0.08a
Ash 0.71 ± 0.30a 0.70 ± 0.11a 0.58 ± 0.14a 0.61 ± 0.22a 0.62 ± 0.09a 0.58 ± 0.13a
Carbohydrate 6.83b 6.89b 7.32b 8.44a 6.87b 8.61a
Oligofructose (Day 1) e e e 1.71 ± 0.01aA e 1.86 ± 0.03aA
Oligofructose (Day 28) e e e 1.68 ± 0.41aA e 1.65 ± 0.19aA
a
Means ± standard deviation in the same row followed by different lower case letters indicate statistically significant differences at p  0.05 between formulations of
orange juice (n ¼ 6). Means ± standard deviation in the same column followed by different upper case letters indicate statistically significant differences in oligofructose
content at p  0.05 for each formulation affected by the storage time (n ¼ 6).
b
Formulation: PURE (pure); PRO (probiotic), ACID (ascorbic acid), PRE (oligofructose), PRO-ACID (probiotic þ ascorbic acid), SYNB (probiotic þ oligofructose).
198 G.M. da Costa et al. / LWT - Food Science and Technology 75 (2017) 195e201

Table 2
Physicochemical characteristics of the orange juice formulations.a

Parameter Storage time (Days) Formulationb

PURE PRO ACID PRE PRO-ACID SYNB

pH 1 4.09 ± 0.11Aa 4.10 ± 0.11Aa 4.06 ± 0.09Aab 4.09 ± 0.02Aa 4.02 ± 0.06Ab 4.06 ± 0.05Aab
7 3.96 ± 0.05Ba 3.88 ± 0.07Bb 3.90 ± 0.05Bab 3.99 ± 0.07Ba 3.88 ± 0.01Bb 3.90 ± 0.03Bab
14 4.08 ± 0.06Aa 4.07 ± 0.02Aab 4.03 ± 0.02Aabc 4.04 ± 0.04Aab 3.98 ± 0.03Ac 4.02 ± 0.03Abc
21 4.09 ± 0.03Aa 4.08 ± 0.06Aab 4.05 ± 0.05Aab 4.11 ± 0.04Aa 4.04 ± 0.01Ab 4.06 ± 0.03Aab
28 4.10 ± 0.02Aa 4.10 ± 0.04Aa 4.08 ± 0.03Aa 4.09 ± 0.05Aa 4.07 ± 0.07Aa 4.06 ± 0.02Aa
Titratable acidity (%citric acid) 1 0.55 ± 0.05Ba 0.55 ± 0.06Ba 0.57 ± 0.06Ba 0.53 ± 0.02Aa 0.57 ± 0.05Ba 0.56 ± 0.05Ba
7 0.59 ± 0.04Abc 0.60 ± 0.07Aab 0.63 ± 0.07Aa 0.56 ± 0.02Ac 0.63 ± 0.05Aa 0.61 ± 0.04Aab
14 0.53 ± 0.04Bc 0.54 ± 0.06Bbc 0.56 ± 0.06Bbc 0.54 ± 0.06Abc 0.58 ± 0.06Ba 0.57 ± 0.06Bab
21 0.59 ± 0.06Aa 0.61 ± 0.07Aa 0.61 ± 0.01Aa 0.56 ± 0.04Aa 0.60 ± 0.05ABa 0.58 ± 0.04ABa
18 0.55 ± 0.06Ba 0.54 ± 0.07Ba 0.57 ± 0.03Ba 0.54 ± 0.06Aa 0.58 ± 0.06Ba 0.56 ± 0.06Ba
Total soluble solids ( Brix) 1 9.33 ± 1.33Ab 9.30 ± 1.66Ab 9.25 ± 1.52Ab 10.78 ± 1.73Aa 9.20 ± 1.80Ab 10.50 ± 1.72Aa
7 8.86 ± 0.16ABb 8.78 ± 0.21ABb 9.18 ± 0.20Ab 9.93 ± 0.77Aa 9.25 ± 0.26Ab 9.93 ± 0.61Aa
14 8.55 ± 1.00ABb 8.45 ± 1.09ABb 8.41 ± 1.15Ab 9.87 ± 1.57Aa 8.22 ± 1.45Bb 9.70 ± 1.62Aba
21 8.30 ± 0.11Bb 8.28 ± 0.12Bb 8.50 ± 0.33Ab 9.88 ± 0.32Aa 8.52 ± 0.26ABb 9.60 ± 0.20Aa
28 9.33 ± 1.68Ab 9.11 ± 1.82Ab 9.37 ± 1.99Ab 10.58 ± 1.70Aa 9.10 ± 1.98ABb 10.47 ± 2.01Aa
Ascorbic acid (mg/100 g) 1 45.00 ± 5.25cA 51.20 ± 1.50bcA 70.25 ± 4.34aA 49.50 ± 0.58bcA 68.50 ± 2.89aA 56.50 ± 1.73bA
7 40.00 ± 4.61dAB 47.80 ± 0.96cB 66.50 ± 2.89aB 48.90 ± 1.27cA 64.50 ± 1.91aB 54.30 ± 2.66bA
14 38.00 ± 4.69eBC 45.50 ± 0.58cBC 62.25 ± 1.25aC 42.55 ± 3.43dB 61.00 ± 1.15aC 50.75 ± 3.84bB
21 36.00 ± 2.31eC 44.50 ± 1.73cC 60.00 ± 0.00aC 39.70 ± 3.90dB 59.13 ± 1.75aC 48.30 ± 5.89bB
28 35.90 ± 2.55eC 43.90 ± 1.89cC 59.90 ± 1.25aC 39.70 ± 3.25dB 58.70 ± 1.25aC 47.90 ± 5.25bB
Turbidity at 600 nm 1 2.41 ± 0.11Bab 2.30 ± 0.14Ac 2.32 ± 0.14Bbc 2.41 ± 0.13Bab 2.41 ± 0.09Aab 2.43 ± 0.11Ba
7 2.51 ± 0.13ABa 2.32 ± 0.07Ab 2.45 ± 0.11Aa 2.52 ± 0.08Aa 2.44 ± 0.05Aab 2.56 ± 0.08Aa
14 2.51 ± 0.13ABa 2.34 ± 0.18Ab 2.35 ± 0.16ABb 2.41 ± 0.16Bab 2.44 ± 0.08Aab 2.46 ± 0.11ABa
21 2.59 ± 0.15Aa 2.35 ± 0.07Ab 2.39 ± 0.09ABb 2.55 ± 0.07Aab 2.49 ± 0.05Aab 2.45 ± 0.09ABab
28 2.58 ± 0.22Aa 2.39 ± 0.13Ab 2.39 ± 0.14ABb 2.48 ± 0.09ABab 2.45 ± 0.08Ab 2.47 ± 0.08ABab
L* 1 37.24 ± 0.14Cbc 36.94 ± 0.50Ccd 37.74 ± 0.48Cab 36.99 ± 0.27Ccd 37.30 ± 0.76Ba 36.44 ± 0.19Bd
7 39.98 ± 0.14Ba 37.07 ± 0.24Cc 37.89 ± 0.19Cb 36.62 ± 0.09Cc 37.30 ± 0.15BCb 36.41 ± 0.17Bd
14 39.58 ± 0.14Ba 38.33 ± 0.27Bb 39.19 ± 0.24Ba 37.79 ± 0.25Bb 38.58 ± 0.36Bb 37.17 ± 0.34Ac
21 43.55 ± 0.14Aa 41.54 ± 0.15Ab 41.61 ± 1.79Ab 38.79 ± 0.20Ac 38.75 ± 0.17Ac 37.11 ± 0.35Ad
28 44.00 ± 0.14Aa 42.00 ± 0.15Ab 42.01 ± 1.75Ab 39.15 ± 0.25Ac 38.99 ± 0.23Ac 37.71 ± 0.40Ad
a* 1 0.89 ± 0.33Cb 1.03 ± 0.27Cb 1.31 ± 0.07Ca 1.22 ± 0.14Ca 1.22 ± 0.03Ca 1.00 ± 0.15Cb
7 1.44 ± 0.64Ba 1.28 ± 0.06Bab 1.43 ± 0.06Ba 1.35 ± 0.03Cab 1.40 ± 0.04Bab 1.26 ± 0.04Bb
14 1.74 ± 0.14Aa 1.55 ± 0.08Ab 1.81 ± 0.11Aa 1.74 ± 0.08Ba 1.80 ± 0.12Aa 1.62 ± 0.09Ab
21 1.75 ± 0.13Ab 1.45 ± 0.09Ac 1.55 ± 0.11Bc 1.94 ± 0.06Aa 1.64 ± 0.11Abc 1.52 ± 0.11Ac
28 1.79 ± 0.18Ab 1.50 ± 0.11Ac 1.59 ± 0.15Bc 2.04 ± 0.10Aa 1.69 ± 0.21Abc 1.62 ± 0.11Ac
b* 1 17.99 ± 0.35 20.49 ± 1.70 21.39 ± 0.55 21.26 ± 0.69 21.86 ± 0.22 19.94 ± 0.44
7 21.11 ± 2.03Ba 19.95 ± 0.28Bc 20.11 ± 0.21Bb 18.83 ± 0.09Bd 19.85 ± 0.20Bc 19.20 ± 0.13Bc
14 22.26 ± 1.36ABa 20.09 ± 0.44ABc 20.55 ± 0.13ABc 20.10 ± 0.43ABc 21.72 ± 0.44ABb 19.99 ± 0.29BAc
21 23.37 ± 0.78Aa 21.18 ± 0.31Abc 22.30 ± 0.98Ab 22.02 ± 0.28Ab 21.86 ± 0.2AB1b 19.94 ± 0.44BAc
28 24.00 ± 0.84Aa 22.18 ± 0.41Ab 22.40 ± 0.98Ab 22.32 ± 0.38Ab 22.85 ± 0.45Ab 20.92 ± 0.47Ac

L* ranging from 0 (black) to 100 (white), a* ranging from red (þa*) to green (a*), b* ranging from yellow (þb*) to blue (b*).
a
Means ± standard deviation in the same row followed by different lower case letters indicate statistically significant differences at p  0.05 between formulations of
orange juice for the same storage time (n ¼ 6). Means ± standard deviation in the same column followed by different upper case letters indicate statistically significant
differences at p  0.05 for each formulation affected by the storage time (n ¼ 6).
b
Formulation: PURE (pure); PRO (probiotic), ACID (ascorbic acid), PRE (oligofructose), PRO-ACID (probiotic þ ascorbic acid), SYNB (probiotic þ oligofructose).

this work, including the type of packaging (glass), the low tem- Vijayanand, & Prapulla, 2009). Therefore, it is necessary to eval-
perature during storage (4  C) and the reduced space left in the uate whether the changes in turbidity and the discoloration of the
glass flasks helped to maintain the ascorbic acid content in products with the addition of the proposed ingredients result in a
adequate levels. Therefore, to obtain the recommended daily intake decrease in product acceptance by consumers.
of ascorbic acid (75 mg/day for women and 90 mg/day for men), the The probiotic cultures were added as an activated probiotic
consumers should drink 150e250 mL of the orange juice, as the culture in a saline solution, what contributed to the decrease in the
formulations had 35.9e59.9 mg/100 g of ascorbic acid in the end of turbidity and darkening of the color. Choi, Kim, and Lee (2002)
the cold storage. report that ascorbic acid can interact with pigments of the juices;
There was no effect (p > 0.05) of oligofructose supplementation resulting in the degradation of both components and decrease in
(PRE and SYNB) in the turbidity of the orange juices (PURE), while a the color intensity and nutritional quality of the products. Quintero,
decrease in this parameter was noted (p  0.05) with the addition Giraldo, and Cortes (2011) obtained a darker mango pulp with the
of probiotic cultures (PRO) and ascorbic acid (ACID), observed from supplementation with oligofructose and suggested that the fiber
the 1st and 14th days of storage, respectively. Considering the color could trap in its interior solids in suspension or low weight mole-
parameters, the addition of probiotic culture (PRO), oligofructose cules, changing the color of the products.
(PRE) or ascorbic acid (ACID) resulted in darker (lower L* values) During storage, it was observed an increase in turbidity of the
and less yellow (lower b* values) products. pure juice (PURE), while the other products (PRO, ACID, PRE, PRO-
The orange juice is characterized as a cloudy product, unlike ACID and SYNB) showed similar values (p > 0.05) on the 1st and
other juices that are submitted to a clarification process, such as 28th days of storage. Considering the color parameters, all juices
apple juice or grape juice. The color of the juices is a primary quality became darker and more yellow, i.e., a reduction in L* values and
attribute for the acceptance of the products by consumers increased values of a* (p  0.05) were observed.
(Pimentel, Madrona, & Prudencio, 2015; Renuka, Kulkarni, The accumulation of lysed or dead bacterial cells can increase
G.M. da Costa et al. / LWT - Food Science and Technology 75 (2017) 195e201 199

the turbidity of the fruit juices (Shah et al., 2010), therefore, the
maintenance of this parameter in the probiotic juices (PRO, PRO-
ACID and SYN) is an indication that a large part of the probiotic
culture survived during refrigerated storage of the orange juice. The
intensification of the color may have been caused by oxidative and
non-oxidative reactions of polyphenols, resulting in colored
condensation products; or by Maillard Reaction or melanoidins
formation (Tajchakavit, Boye, Be langer, & Couture, 2001).
The texture characteristics (firmness, cohesiveness, consistency
and viscosity index) of orange juice formulations are shown in
Table 3. The orange juice formulations showed no differences in the
texture parameters (p > 0.05) during the 28 days of storage. The
texture maintenance of juices with the addition of ingredients and
during the cold storage is important as it indicates that the addi-
tions do not modify the conventional products and that the prod-
Fig. 1. Viability (log CFU/mL) of the Lactobacillus paracasei ssp. paracasei in formula-
ucts are similar to newly manufactured even after 28 days of tions of orange juices (PRO, PRO-ACID e SYNB) during cold storage (4  C). The error
storage. bars represent the standard deviation (n ¼ 6). Formulation: PRO (probiotic), PRO-ACID
The addition of probiotic cultures may result in increased (probiotic þ ascorbic acid), SYNB (probiotic þ oligofructose).
firmness, consistency and adhesiveness in some products, due to
the production of exopolysaccharides (EPS). EPS can act as textur-
izing agents and stabilizers, increasing the viscosity of the final the counts of the probiotic culture also were between 107 and
product and interacting with other constituents of the products, 108 CFU/mL (data not shown).
such as proteins (Duboc & Mollet, 2001). Oligofructose is incorpo- Activation of the freeze-dried probiotic cultures is usually per-
rated into the product matrix and can confer or enforce already formed by propagating the culture in MRS broth followed by the
existent bonding between different components of the product separation of the biomass using a refrigerated centrifuge
(Cruz et al., 2010). These changes were not observed in this study. (Bevilacqua et al., 2013; Costa et al., 2013; Ding & Shah, 2008;
Pimentel, Madrona, & Garcia, 2015; Pimentel, Madrona, &
Prudencio, 2015; Rodrigues et al., 2012). MRS broth is a culture
3.3. Probiotic culture viability medium with higher cost (US$ 191.00/500 g) (Lojalab, 2016) than
the orange juice (US$ 0.15/500 g), increasing the cost of production
The survival of probiotic culture in orange juice formulations is by US$ 2.615/L of juice, which precludes the production of probiotic
shown in Fig. 1. In the first day of storage, the formulations sup- juices by small and medium companies. As far as the authors know,
plemented with probiotic culture (PRO, ACID and SYNB), presented no other study used fruit juices to propagate the probiotic cultures.
similar (p > 0.05) counts of Lactobacillus paracasei ssp. paracasei, To be considered probiotic, the food products must have counts
indicating that the probiotic culture was in the same amount in all above 106 CFU/mL (Donkor et al., 2007) or 108e109 UFC in the daily
formulations. portion of the product (200 mL for juice) (Anvisa, 2008), so all the
The initial counts higher than 107 CFU/mL in the present study juices supplemented with probiotics (PRO, PRO-ACID and SYNB) in
indicate that orange juice is suitable substrate for Lactobaciilus this study could be considered probiotic products during the 28
paracasei propagation. When using MRS as the propagation media,

Table 3
Texture characteristics of the orange juice formulations.a

Parameter Storage time (Days) Formulationb

PURE PRO ACID PRE PRO-ACID SYNB

Firmness (g) 1 26.39 ± 0.41Aa 26.91 ± 1.02Aa 26.68 ± 0.71Aa 26.20 ± 0.70Aa 27.14 ± 0.85Aa 26.49 ± 0.51Aa
7 26.50 ± 0.50Aa 26.91 ± 0.63Aa 27.06 ± 0.63Aa 26.85 ± 0.84Aa 26.11 ± 0.63Aa 25.76 ± 0.64Aa
14 26.49 ± 0.44Aa 26.79 ± 0.70Aa 26.19 ± 0.47Aa 26.41 ± 0.65Aa 26.73 ± 0.48Aa 26.55 ± 0.71Aa
21 26.75 ± 0.76Aa 25.91 ± 0.57Aa 27.04 ± 0.74Aa 26.45 ± 0.75Aa 30.33 ± 3.45Aa 27.44 ± 1.66Aa
28 27.00 ± 1.19Aa 26.68 ± 0.74Aa 26.52 ± 0.67Aa 25.90 ± 1.20Aa 26.95 ± 1.08Aa 26.66 ± 0.47Aa
Viscosity index (g s) 1 0.75 ± 0.10Aa 0.66 ± 0.10Ba 0.78 ± 0.13Aa 0.77 ± 0.11Aa 0.73 ± 0.09Aa 0.73 ± 0.10Aa
7 0.78 ± 0.08Aa 0.84 ± 0.08ABa 0.70 ± 0.11Aa 0.77 ± 0.12Aa 0.82 ± 0.07Aa 0.83 ± 0.06Aa
14 0.76 ± 0.15Aa 0.75 ± 0.07ABa 0.68 ± 0.07Aa 0.67 ± 0.12Aa 0.75 ± 0.08Aa 0.74 ± 0.06Aa
21 0.71 ± 0.09Aa 0.69 ± 0.07ABa 0.78 ± 0.09Aa 0.75 ± 0.08Aa 0.74 ± 0.04Aa 0.80 ± 0.08Aa
28 0.76 ± 0.09Aa 0.62 ± 0.14Ba 0.76 ± 0.08Aa 0.79 ± 0.09Aa 0.74 ± 0.13Aa 0.84 ± 0.16Aa
Cohesiveness 1 5.92 ± 0.39Aa 5.77 ± 0.14Aa 5.83 ± 0.16Ba 6.01 ± 0.39Aa 5.96 ± 0.24Aa 5.96 ± 0.18Aa
7 5.86 ± 0.31Ab 5.88 ± 0.20Ab 6.26 ± 0.49Aa 5.94 ± 0.23Aab 5.94 ± 0.20Aab 5.90 ± 0.26Ab
14 5.84 ± 0.20Aa 5.98 ± 0.21Aa 5.84 ± 0.21Ba 5.92 ± 0.13Aa 5.93 ± 0.18Aa 5.92 ± 0.18Aa
21 5.94 ± 0.33Aa 5.990.15Aa 6.00 ± 0.10ABa 6.01 ± 0.24Aa 5.96 ± 0.18Aa 5.95 ± 0.20Aa
28 5.94 ± 0.19Aa 5.83 ± 0.31Aa 5.86 ± 0.28Ba 5.92 ± 0.25Aa 5.77 ± 0.34Aa 5.94 ± 0.43Aa
Consistency (g s) 1 152.80 ± 2.96Aa 154.36 ± 6.28Aa 155.86 ± 3.22Aa 149.64 ± 4.11Ba 152.90 ± 5.80Ba 153.63 ± 3.26Aa
7 150.37 ± 3.77Aa 153.71 ± 3.17Aa 151.89 ± 3.54Aa 154.82 ± 5.30Aa 147.36 ± 3.95Ca 150.90 ± 5.12Aa
14 152.73 ± 2.04Aa 155.15 ± 3.63Aa 152.38 ± 2.93Aa 149.70 ± 4.53Ba 152.29 ± 3.06BCa 150.73 ± 3.76Aa
21 151.29 ± 3.06Ab 149.86 ± 2.64Ab 150.54 ± 5.34Ab 150.38 ± 3.64Bab 159.35 ± 13.42Aa 151.91 ± 5.94Ab
28 152.84 ± 7.39Aa 151.74 ± 3.49Aa 152.89 ± 3.85Aa 151.97 ± 6.19ABa 154.46 ± 6.36Ba 154.54 ± 2.98Aa
a
Means ± standard deviation in the same row followed by different lower case letters indicate statistically significant differences at p  0.05 between formulations of
orange juice for the same storage time (n ¼ 6). Means ± standard deviation in the same column followed by different upper case letters indicate statistically significant
differences at p  0.05 for each formulation affected by the storage time (n ¼ 6).
b
Formulation: PURE (pure), PRO (probiotic), ACID (ascorbic acid), PRE (oligofructose), PRO-ACID (probiotic þ ascorbic acid), SYNB (probiotic þ oligofructose).
200 G.M. da Costa et al. / LWT - Food Science and Technology 75 (2017) 195e201

Table 4
Acceptance of the orange juice formulations.a

Parameter Formulationb

PURE PRO ACID PRE PRO-ACID SYNB

Appearance 7.03 ± 1.85a 7.35 ± 1.37a 7.26 ± 1.40a 7.37 ± 1.51a 7.62 ± 1.45a 7.03 ± 1.68a
Aroma 6.64 ± 1.81a 6.72 ± 1.43a 6.44 ± 1.77a 6.91 ± 1.83a 7.05 ± 1.47a 6.48 ± 1.72a
Flavor 5.36 ± 2.34a 5.94 ± 1.81a 5.11 ± 2.06a 5.91 ± 2.21a 5.97 ± 2.19a 5.44 ± 1.86a
Texture 6.01 ± 1.92a 6.55 ± 1.85a 6.08 ± 1.99a 6.85 ± 1.72a 6.91 ± 1.70a 6.66 ± 1.64a
Overall impression 5.64 ± 2.33a 6.16 ± 1.96a 5.55 ± 1.99a 6.22 ± 1.95a 6.28 ± 2.06a 5.93 ± 1.88a

Hedonic values (appearance, aroma, flavor, texture e overall impression): 1 e disliked very much; 9 e liked very much.
a
Means ± standard deviation in the same row followed by different lower case letters indicate statistically significant differences at p  0.05 between formulations of
orange juice (n ¼ 6).
b
Formulation: PURE (pure), PRO (probiotic), ACID (ascorbic acid), PRE (oligofructose), PRO-ACID (probiotic þ ascorbic acid), SYNB (probiotic þ oligofructose).

days of cold storage. formulations. The presence of oligofructose (PRE and SYNB), pro-
During the storage period (comparing the products at days 1 biotic culture (PRO, PRO-ACID and SYNB) or ascorbic acid (ACID and
and 28 of storage), the viability of the probiotic cultures was PRO-ACID) did not affect (p > 0.05) the acceptance (appearance,
maintained (p > 0.05), despite the acidic conditions and the pres- aroma, flavor, texture and overall impression) of the orange juice
ence of oxygen in the medium. The results indicate that the pro- (PURE), indicating that although these additions have changed the
biotic used (Lactobacillus paracasei ssp. paracasei) is a resistant color and turbidity of the products, they did not affect how con-
strain and that the orange juice was a suitable carrier for probiotic sumers liked the juices and the desire in consuming or buy them.
supplementation. The results of the acceptance corroborate those observed in phys-
The high survival of the probiotic cultures in orange juice could icochemical evaluations (Tables 2 and 3), in which the additions of
be related to the high acid concentration (11 g/L of malic oligofructose, probiotic culture and ascorbic acid did not change the
acid þ citric acid) and fibers (2.8 g/L) of this product (Nualkaekul pH, titratable acidity and texture parameters (firmness, cohesive-
et al., 2011). Some probiotic cultures have the ability to metabo- ness, consistency and viscosity index) of the juices.
lize citric acid (Nualkaekul et al., 2011), while fibers could physically Maintenance of the acceptability of the products with the sup-
protect the probiotic cells from damage caused by the environment plementation with probiotics and/or prebiotics is important
by the adhesion of the bacterial cells to the oligosaccharide because consumers are not interested in consuming functional
(Nualkaekul, Deepika, & Charalampopoulos, 2012). Furthermore, beverages if the added ingredients occasioning strange aromas or
orange juice already contains vitamin C (ascorbic acid), which may unpleasant flavors in products, even taking into account the ben-
have contributed to the reduction of the dissolved oxygen in the efits to health (Pimentel, Madrona, & Garcia, 2015).
medium, increasing the survival of the probiotic culture Acceptance scores for appearance, flavor and texture were be-
(Champagne, Raymond, & Gagnon, 2008). tween 6 and 7 on a hedonic scale of 9 points, indicating that con-
The addition of oligofructose (SYNB) or ascorbic acid (PRO-ACID) sumers liked slightly to moderate the products. Considering the
had no effect (p > 0.05) on the viability of the probiotic cultures. flavor attribute and the overall impression, the scores were be-
The results of probiotic culture survival corroborate those obtained tween 5 and 6, indicating that consumers were indifferent or liked
for oligofructose content, i.e., oligofructose was not metabolized by slightly the products. The low acceptance of products in taste and
the probiotic culture (Table 1), therefore, this component was not overall impression can be justified by the technological process
able to increase the survival of the probiotic culture during storage. used in this study, i.e., the juices were subjected to a heat treatment
Oligofructose possibly has a protective capacity in the survival of in a water bath (80  C/20 min), impairing the flavor of the products.
probiotic cultures in products where the proximate composition is Furthermore, in Brazil, consumers are accustomed to consuming
not suitable for their survival. The orange juice had a protein con- natural orange juice, and, therefore, the heat treatment should be
tent of 1.8 g/100 mL (Table 1) and glucose content of 3.23 g/100 g subtle in order to not compromise the sensory characteristics of the
(Kelebek et al., 2009). Concentrations of 0.3% protein are suggested product. The technological problems could be solved easily on an
for probiotic products (Nualkaekul et al., 2012), and glucose as the industrial scale, as the industries have heat exchangers for the
main sugar source, because it is easily assimilated. In fact, Pimentel, thermal treatment, which use higher temperatures and lower
Madrona, and Garcia, (2015) observed an increase in the survival of retention time (seconds) than the batch method, maintaining the
probiotic cultures in clarified apple juice with the supplementation sensory properties of the juices.
with oligofructose. The apple juice had lower amounts of proteins Therefore, it is possible to formulate probiotic orange juice using
(z0.04%) and its main sugar was fructose (5.69 g/100 g), a disac- oligofructose as a prebiotic compound without changing the
charide, which possibly contributed to the protective effect of oli- acceptance of the products. The formulations containing oligo-
gofructose on the survival of the probiotic culture. fructose (PRE and SYNB) could exert beneficial health effects, such
Nualkaekul et al. (2011) also found no effect of ascorbic acid on as inhibition of growth of pathogens in the gut; increase in calcium
the viability of Lactobacillus plantarum NCIMB 8826 in model so- absorption from the diet and relief of constipation (Cruz et al.,
lutions. The authors suggest that the lack of effect was related to the 2010).
decrease in ascorbic acid content during storage of the solutions. In
this study, the fact that orange juice already contains ascorbic acid 4. Conclusion
and the degradation of this compound may have contributed to the
absence of effect. The degradation of ascorbic acid was 14e20% Orange juice is a suitable food for the incorporation of ascorbic
during the 28 days of storage (Table 2). acid, Lactobacillus paracasei ssp. paracasei as a probiotic culture
and/or oligofructose as prebiotic, resulting in products with phys-
3.4. Sensory analysis icochemical (excepted color and turbidity), texture, acceptance and
storage stability similar to the pure products. There was no pro-
In Table 4 is presented the acceptance of orange juice tective effect of oligofructose or ascorbic acid on the survival of
G.M. da Costa et al. / LWT - Food Science and Technology 75 (2017) 195e201 201

probiotic cultures, but the products could use the claim of pro- Instituto Adolf Lutz. Sa ~o Paulo: Instituto Adolf Lutz.
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