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Potential application of four types of tropical fruits in lactic fermentation
LWT - Food Science and Technology 86 (2017) 254e260
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Potential application of four types of tropical fruits in lactic fermentation Rafael Resende Maldonado a, *, Lucas da Costa Araújo a, Letícia Caroline da Silva Dariva a, ~o Pedro Rodrigues Prado a, ssia Nazar Rebac a, Isadora Amalfi de Souza Pinto a, Joa Ke a a Juliana Kazumi Saeki , Thainara Santos Silva , Emerson Kazuhiro Takematsu a, lia Vilela Tiene a, Elizama Aguiar-Oliveira b, Roberto Elias Buosi c, Nata Marcela Aparecida Deziderio d, Eliana Setsuko Kamimura d ~o Paulo, Food Department, Technical College of Campinas (COTUCA), University of Campinas, R. Jorge Figueiredo Corr^ ea, 735, 13087-261, Campinas, Sa Brazil b Exact Sciences and Technology Department, Santa Cruz State University, R. Jorge Amado, km 16, Salobrinho, 45.662-900, Ilh eus, Bahia, Brazil c ~o Paulo, Brazil Integrated College Maria Imaculada, R. Paula Bueno, 240, 13840-040, Mogi Guaçu, Sa d ~o Paulo, Campus Pirassununga, Av. Duque de Caxias Norte, Food Engineering Department, Faculty of Animal Science and Food Engineering, University of Sa ~o Paulo, Brazil 225, Campus da USP, 13635-900, Pirassununga, Sa a
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Article history: Received 30 January 2017 Received in revised form 13 July 2017 Accepted 1 August 2017 Available online 3 August 2017
Fruit juice has been used for the production of lactose-free or low-lactose beverages. This study evaluated the potential of four types of tropical fruit (carambola, guava, mango and pitaya) for the production of fermented lactic beverages with and without whey addition. Pasteurized fruit juices were used at (% v/v): 100% fruit juice, 100% whey and 50:50% of juice: whey. The fermentations were conducted with a lactic culture (L. casei, S. thermophilus, and L. bulgaricus) at 37 C for 72 h. The final characteristics obtained were pH 3.0e4.0, soluble solids concentration (SS) 5.0e7.0 Brix (g/100 g), and acidity 0.2e1.0 %w/v. A validation test obtained 4.0e4.8 (pH), 6.0 Brix (SS), and 0.30e0.50 %w/v (acidity) at 24 h. The lactic bacteria had better growth (107e108 CFU/mL) and the highest sensory acceptance rates (70% for taste, 75% for flavor and 90% for color) utilizing mango or guava. Carambola and pitaya caused a partial inhibition to the cellular growth (103e104 CFU/mL) and resulted in beverages with lower sensory acceptance. In all cases, formulations containing only fruit juice were lactose-free and better sensory acceptance than the ones with of whey. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Carambola Guava Lactobacillus Mango Pitaya
1. Introduction Lactic fermentation of food has several advantages and it is utilized mainly for the production of different dairy products. In this process, the lactose is fermented by acid-lactic bacteria which reduce the concentration of this sugar in the fermentation medium, promote the acidification and the formation of secondary metabolites. The production of lactic acid and other metabolites is responsible for the characteristics of lactic fermented products, such as flavor, taste, increase of shelf life and reduction of the lactose content (Fox & McSweeney, 1998).
* Corresponding author. E-mail address: [email protected] (R.R. Maldonado). http://dx.doi.org/10.1016/j.lwt.2017.08.005 0023-6438/© 2017 Elsevier Ltd. All rights reserved.
The lactic fermentation is usually associated with milk or dairy products; however, it can be applied successfully to other substrates, such as fruit pulp or juice, due to the high fermentable sugar contents. Moreover, fruits and vegetables naturally contain acid lactic or acetic bacteria in their microbiota (DiCagno, Codda, De Angelis, & Gobbetti, 2013; Nyanga et al., 2007; Sagdic, Ozturk, Yapar, & Yetim, 2014). The application of fruit as substrate for lactic fermentation also has the advantage of the incorporation of flavors and nutrients specific to each type of fruit, resulting in products with different sensory and physico-chemical characteristics. Furthermore, the fruit based fermented products are lactose free, which meet the needs of consumers with lactose intolerance. There are reports in the literature of some studies with fruits and vegetables juices applied successfully to the lactic fermentation process, such as: pomegranate (Punica granatum L.) (Filannino et al.,
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2013), cantaloupe melon (Cucumis melo L.) (Fonteles et al., 2013; 2012), cashew apple (Anacardium occidentale) (Pereira, Maciel, & Rodrigues, 2011), carrot, cabbage, beetroot and onion (Gardner, Savard, Obermeier, Caldwell, & Champagne, 2001; Yoon, Woodams, & Hang, 2006, 2005; 2004). Kourkoutas, Xolias, Kallis, Bezirtzoglou, and Kanellaki (2005) utilized fruit as supports for the growth of acid-lactic bacteria and DiCagno, Minervini, Rizzello, De Angelis, & Gobbetti, 2011 obtained fermented fruit smoothies. However, it is known that the survival of lactic bacteria in fruit substrates tends to be more complex than in the dairy ones. This is due mainly to the natural acidity of fruit, the high level of polyphenols and the absence of lactose, which may interfere in the survival of certain sorts of microorganisms as well as in their growth (DiCagno et al., 2011; Fonteles et al., 2013). Based on the promising results reported on the literature for the lactic fermentation of fruit, the aim of this study was to evaluate the potential of four types of tropical fruit (carambola - Averrhoa carambola L.; guava - Psidium guajava; mango - Magifera indica L. var. Tommy Atkins; and red bark pitaya - Hylocereus undatus) for the production of fermented lactic beverages. Additionally, whey, a sub-product largely utilized in dairy products, was also investigated in association with the tropical fruits studied. The fermented lactic beverages were evaluated regarding the concentration of soluble solids (SS), cellular viability, pH and titratable acidity along the fermentation time and the sensory acceptance of the final product. 2. Materials and methods 2.1. Fruit juice preparation For this study, four types of tropical fruits were chosen: carambola (Averrhoa carambola L.), guava (Psidium guajava), mango (Magifera indica L. var. Tommy Atkins), and red bark pitaya with white pulp (Hylocereus undatus). The fruits were purchased in a ~o Paulo, Brazil). They were all ripe, local supermarket (Campinas, Sa undamaged and visually adequate for consumption (good color, odor and texture). For the preparation of the pulps, the fruits were washed, peeled and crushed in a blender. The initial SS of pulps were adjusted by the dilution with distilled water, until the obtainment of juice with 7e8 Brix (g/100 g of sugar). Thereafter, the juice samples were pasteurized in batches at 80 C for 5 min (Nagpal, Kumar, & Kumar, 2012) and bottled hot in glass flasks of 500 mL. After bottling, the flasks were closed hermetically and, after cooled to room temperature, were frozen at 18 C (de Paula Valim, Aguiar-Oliveira, Kamimura, Alves, & Maldonado, 2016). 2.2. Whey preparation Fresh milk (15.0 L) was obtained from a local dairy processor ~o Paulo, Brazil). The coagulation was conducted by (Campinas, Sa the addition of a lactic culture (170 g of industrialized yogurt resulting in an inoculation of 1.106 CFU/mL) and 5.0 mL of liquid rennet (as indicated by the producer). After 1 h of coagulation at room temperature (25 C), the coagulated mass was separated from the whey by filtration. The whey was pasteurized and stored under the same conditions as for the fruit juice. 2.3. Lactic acid fermentation The fermentations were performed with three substrate volume compositions: 100% fruit juice; a mixture of 50:50% fruit juice and whey and 100% whey, totaling nine different formulations. All formulations were evaluated in triplicate utilizing 500 mL Erlenmeyer flasks previously sterilized. To each flask, 300 mL of substrate were added and inoculated with a lyophilized lactic culture
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containing Lactobacillus casei, Lactobacillus bulgaricus and Streptococcus termophillus (Rhodia Foods®) in quantity enough to reach the initial counting of 1.106 CFU/mL. The flasks were kept at 37 C without agitation for a period of 72 h. Samples of each fermentation were collected at 24, 48 and 72 h for the quantification of the concentration of soluble solids (SS), pH, and titratable acidity and to evaluate the microbial growth. The fermented beverages obtained at the end of the fermentation were utilized for the sensory evaluation. 2.4. Physico-chemical, microbiological and sensory analyses The concentration of soluble solids (SS) was directly measured with a refractometer (Atago - Master-53M) and the pH was measured with a pHmeter (Hanna - HI 3222-01). The acidity was measured by titration with NaOH 0.1 mol/L and the end point was determined by monitoring the pH within the range 7.8e8.2 using the pHmeter. The microbial growth was measured qualitatively by Gram stain method in order to identify the type of microorganisms as well as their growth in the fermentation medium. The slides were prepared as described by Barile (1983) and analyzed with a microscope (Bioval - L-2000B-PL) with a 1000x oil-immersion lens. The fermented beverages obtained were stored in a refrigerator at 4 C for 24 h and then submitted to the sensory evaluation by 32 non trained panelists, by means of a consumer affective test. A 9 points verbal hedonic scale (1 ¼ dislike it very much and 9 ¼ like it very much) was utilized to evaluate the attributes taste; flavor and color and a 9 points ideality scale (1 ¼ much lower than the ideal and 9 ¼ much higher than the ideal) was utilized to evaluate the attributes acidity and sweetness. From the data obtained, the average and the deviation standard for each attribute were calculated and the analysis of variance (ANOVA) and the Tukey test of average were applied to verify if there were significant differences among samples. The sensory evaluation was performed in two sessions. The nonsugared fermented beverages of mango and pitaya (with and without whey) were evaluated in the first session. The sugared [10% (g/100 mL) of sucrose] fermented beverages of guava and carambola (with and without whey) were evaluated in the second session. In both sessions, the fermented beverage obtained using just whey was included in the sensorial analysis. The addition of sucrose to beverages from guava and carambola was made due to the high acidity verified in the sensory evaluation of the first session. This study was conducted according to the ethical standards established by the Brazilian legislation for research involving human beings and is registered in the Brazilian Ministry of Health in the Brazil Platform (CAAE: 50655215.6.0000.5425). 2.5. Validation experiment A validation experiment was performed with the same formulations of the first experiment, but the concentration of soluble solids (SS) for all formulations was standardized (6 Brix) by dilution with distilled water. The fermentations were conducted using 1.106 CFU/mL of inoculum, at 37 C and without agitation. The fermentation time was limited to 24 h because the major changes in fermentation, observed in the previous experiments, occurred during this period. Samples were collected at the beginning and at the end of fermentation for the physico-chemical analyses. The cellular growth was measured through the colony forming units (CFU). Serial decimal dilutions of each sample were plated in triplicate onto Man, Rugosa and Sharpe (MRS) Agar plates (Kasvi®, Brazil) and incubated at 37 C for 48 h. Plates with number of colonies from 30 to 300 were enumerated and the results were expressed as CFU/mL (counting forming units) (Fonteles, Costa, De
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Jesus, & Rodrigues, 2012). 3. Results and discussion 3.1. Lactic acid fermentation of tropical fruits 3.1.1. Soluble solids concentration (SS) The soluble solids concentration (SS) for this type of beverage may be considered as a balance between the consumption of fermentable sugars and the production of acids (lactic acid, mainly) during the fermentation process. The results obtained for SS varied according to the formulation evaluated (Fig. 1a and b). The formulations containing only fruit juice presented a higher initial SS value (7.0e8.0 Brix) in comparison to the mixtures of fruit and
whey (6.5 Brix) and the formulation with only whey (6.0 Brix). For all formulations there was a decrease of SS in the first 24 h of fermentation, which indicates consumption of sugars for cellular growth and production of lactic acid (Fig. 1a, 1b, 1c and 1d). After this initial period, there was a stabilization or a slight increase of SS and the production of lactic acid until the end of the process, which suggests that, after 24 h, the sugar consumed was utilized only for the production of lactic acid, which is also a soluble solid. The final concentration of SS ranged from 5.0 to 7.0 Brix. However, different initial concentrations of soluble solids may influence the fermentation behavior. Thus, to verify possible interferences of the initial concentration of soluble solids (SS) on the fermentation process, a validation experiment was conducted using a standard initial SS at 6 Brix for all formulations. Fig. 2a and 2b
Fig. 1. a) and b) Soluble solids concentration ( Brix), c) and d) titratable acidity (% w/v, g/100 mL) and e) and f) pH for the lactic acid fermentation of four tropical fruits (carambola, guava, mango and pitaya) pure or in composition with whey (50:50% v/v). Fermentations were carried out in triplicate at 37 C, without stirring for 72 h and were inoculated with 1.106 CFU/mL with a commercial lyophilized lactic culture.
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Fig. 2. a) and b) Soluble solids concentration ( Brix), c) and d) titratable acidity (% w/v, g/100 mL) and e) and f) pH for the lactic acid fermentation of four tropical fruits (carambola, guava, mango and pitaya) pure or in composition with whey (50:50% v/v) in validation experiment. Fermentations were carried out in triplicate at 37 C, without stirring for 24 h and were inoculated with 1.106 CFU/mL with a commercial lyophilized lactic culture.
shows that the behavior of SS for all formulations using standard initial SS followed the same profile as the previous experiment. There were small SS reduction for formulations with mango, guava and whey and there was no change in SS for formulations with pitaya and carambola. 3.2. Acidity and pH profiles The acidity profile (Fig. 1c and 1d) showed an increase of this variable during the fermentative process [0.1e0.6% at 24 h and 0.2e1.0% (g/100 mL) at 72 h], while the pH profile (Fig. 1e and 1f) resulted in a more intense decrease in the first 24 h (from 4.5 to 6.5 for 3.5e4.5), followed by a less accentuated decrease after this
period. Both profiles indicate that the lactic bacteria in general were able to adapt well to the substrates of tropical fruit juice. The final pH values (after 72 h of fermentation) stabilized between 3.0 and 4.0, which is similar to results obtained with milk based fermented lactic beverages. Furthermore, the final pH range is inferior to 4.5, which hinders the proliferation of a great number of pathogenic microorganisms during the storage of the beverages. The formulations containing carambola presented the lowest pH reduction rates and also did not present variation of the SS during the fermentation, which indicates a difficulty of growth of the lactic culture in this substrate. Fig. 2cef shows the acidity and pH profiles for the validation experiment. Acidity increased (from 0.11 to 0.35 g/100 mL at 0 h for
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0.28e0.53 g/100 mL at 24 h) and pH decreased (from 5.2 to 4.1 at 0 h to 4.5e4.1 at 24 h), which indicated the production of acid by the lactic bacteria inoculated in the medium. The initial pH in the validation experiment was lower than in the first experiment; however the drop of pH was also less intense, resulting in beverages with similar pH (after 24 h) in both experiments. On the other hand, it is not possible to compare the initial acidity of both experiments; it can be assumed, however, that due to the lower initial pH, the initial acidity in the validation experiment was higher than in the first experiment. However, after 24 h the acid values of the beverages in both experiments were very close. The comparison of the pH and acidity results of both experiments indicates that there was a good reproducibility of the profiles obtained after 24 h, suggesting that the pH and final acidity of the beverages tend to be very similar despite possible differences in the raw materials used. Studies cited in the literature indicate different pH values for fermented lactic beverages utilizing fruit. Kourkoutas et al. (2005) applied immobilized L. casei in pieces of apple and quince and obtained a final pH of 4.8 after 16 h of fermentation in a substrate with initial pH of 6.0. Filannino et al. (2013) observed a final average pH of 3.20 for fermented lactic beverages of pomegranate. However, the initial substrate already presented a rather low pH (3.52). 3.3. Cellular growth The qualitative evaluation of the cellular growth (Table 1) indicated that there was adaptation of the lactic culture with all substrates assessed. The microbial population observed was formed by Gram-positive bacteria (cocci and bacilli types) in the most of the samples analyzed. There were differences in the formulations with carambola, in which only cocci were identified during the fermentation and in formulations with pitaya in which lactic bacteria were no more detected after 72 h of fermentation. These results can be correlated with the behavior observed for SS, pH and acidity in the formulations using carambola and pitaya juice. For the formulation using carambola, SS was practically constant (Fig. 1a and 2a), there was a lower reduction or better stability in the pH (Fig. 1e and 2e) and the acidity was also practically constant (Fig. 2c). The few variations of these parameters indicate a difficulty of adaptation or survival of the lactic culture in this substrate. The Gram stain in the first experiment identified only cocci in the Table 1 Qualitative evaluation of growth of lactic bacteria using Gram stain. Formulation*
24 h
48 h
72 h
Cocci
Bacilli
Cocci
Bacilli
Cocci
Bacilli
Carambola Guava** Mango** Pitaya
P P P P
A P1 P P2
P P P P2
A P P P2
P P P A
A P P A
Carambola þ whey Guava þ whey Mango þ whey Pitaya þ whey
P P P P
P P P P2
P P P P
P P P P
P P P2 P
P P P P
Whey 1* Whey 2*
P P
P1 P
P P
P P
P P
P P
*All formulations were evaluated in triplicate; P ¼ lactic bacteria present in all replicates; P2 ¼ present in 2 replicates; P1 ¼ present in 1 replicate; A ¼ absence. *There were whey 1 and 2 because the fermentation is divided in two sessions (first session ¼ mango, pitaya and whey 1 and second session ¼ guava, carambola and whey 2). **The quantity of lactic bacteria observed in the slams was much higher in the formulations containing mango and guava.
beverage produced with carambola (Table 1) and in the validation experiment, the quantity of bacteria was estimated in 2.103 CFU/mL (Table 2), less than the quantity inoculated. The macronutrients composition of the fruits assessed is rather similar, which suggests that the carambola may contain certain anti-nutritional factor that hinders the development of the lactic culture. Other studies demonstrated that carambola pulp and peel may present antimicrobial activity against different sorts of microorganisms. Mohamed, Hassan, and Abd Hamid (1994) evaluated different tropical fruit extracts (from ripe and unripe fruit). The unripe carambola extract presented antimicrobial activity against Bacillus subtillis, B. cereus and Proteus vulgaricus, while the ripe carambola extract was also efficient against Staphylococcus aureus, Lactobacillus bulgaricus, Escherichia coli and Pseudomonas aeruginosa. Das et al. (2013) verified that the carambola skin extract, obtained with petroleum ether, presented antimicrobial activity against pathogenic bacteria, especially Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli and B. megaterium. For the formulation using pitaya it was possible to observe that SS, pH and acidity were practically constant (Fig. 1a, 1e, 2a, 2c and 2e). These results suggest a greater difficulty of adaptation for the lactic culture to this particular substrate. Furthermore, the Gram stain in the first experiment showed the presence of cocci, but a few quantities of bacilli (at 24 h); the reducing in the quantity of both (cocci and bacilli) after 48 h; and neither cocci nor bacilli were detected in the beverage after 72 h (Table 1). In addition, in the validation experiment the quantity of lactic bacteria after 24 h was also lower than in the initial time (Table 2). These observations indicate that red pitaya with white pulp is not a bacilli inhibitor such as carambola pulp; however it might be a poor substrate for growth and survival of lactic bacteria according to results of Gram stain. The analyses in MRS plates confirmed this fact because the quantity of bacteria (7.104 CFU/mL) was also lower than the inoculated quantity (Table 2). In other study, Tahera, Feroz, Senjuti, Das, and Noor (2014) verified that aqueous, ethanolic and methanolic red pitaya (Hylocereus polyrhizus) extracts presented certain antimicrobial activity against Bacillus spp.; however there was no antimicrobial activity of these extracts against Staphlycoccus spp. On the other hand, beverages produced with mango and guava showed better results. In the first experiment the Gram stain confirmed the presence of cocci and bacilli in practically all samples using these substrates (Table 1) and in the validation experiment mango and guava presented more than 108 CFU/mL when they applied as the only substrate for lactic fermentation (Table 2). Beverages produced with whey showed both cocci and bacilli in the Gram stain (Table 1); however the quantity of lactic bacteria (Table 2) was lower compared to formulations containing mango
Table 2 Estimated quantity of lactic bacteria in the fermented lactic beverages from tropical fruits after 24 h or fermentation. Formulation*
CFU/mL
Carambola Guava Mango Pitaya
2.103 1.108 6.108 7.104
Carambola þ whey Guava þ whey Mango þ whey Pitaia þ whey
7.104 1.107 4.107 1.104
Whey
1.108
*Formulation ¼ substrates used to production the lactic fermented beverage.
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and guava. Probably, mango and guava contain more specific nutrients for the development of lactic bacteria than the whey. For all formulations, except carambola, the addition of whey reduced the quantity of lactic bacteria after 24 h (Table 2), which indicates that whey had a negative effect when mixed with these fruits. 3.4. Sensorial evaluation The sensory evaluation indicated that among the fermented lactic beverages obtained with the utilization of fruit only, the grades in general rank were: guava > mango > carambola > pitaya. The exception to this behavior was in relation to the attribute color, in which the fermented beverage of mango obtained a superior grade than guava. Moreover, for all attributes assessed, the addition of whey resulted in a worse sensory evaluation than that for the beverages without whey (Table 3). In the comparison between the two sessions, it is noticeable that the addition of sucrose to the beverages before the sensory evaluation improved their results for the attributes taste, flavor and sweetness, but there was no alteration in the attribute acidity. The addition of sucrose enhances the ratio of the beverage (balance between acidity and sweetness) and increases the sensory acceptance of beverages with elevated acidity. Such behavior was already reported for the sensory evaluation of mixed beverages of milk and açaí fermented by kefir (Nogueira, Aguiar-Oliveira, Kamimura, & Maldonado, 2016). The results obtained in this study show that from the tropical fruits assessed, the more commonly consumed in Brazil (mango and guava) presented a higher sensory acceptance than the other two (carambola and pitaya). Furthermore, mango and guava were those in which the lactic bacteria developed better, while carambola and pitaya were less favorable substrates (under the evaluated conditions) for the development of lactic acid bacteria, which probably affected the sensory qualities of the beverages. DiCagno et al. (2011) verified improvement of some sensory attributes such as taste, texture, sweetness and global acceptance in green and red smoothies made of mixed fermented fruit and vegetables. In this present study, it was also possible to notice the improvement of some attributes compared to studies cited in the literature. The lactic fermented beverage of mango had a better-rated taste (6.20) than the mango juice sweetened with 7% of sucrose (4.40) (Cadena et al., 2013). The lactic fermented beverage of guava obtained better-rated flavor and color (7.1 and 7.8, respectively) than the guava juice in the concentration 12 Brix (5.93 and 7.03, respectively) (Thongsombat, Sirichote, & Chanthachum, 2007). The highest sensory acceptance, with the marks 7.7 for appearance, 7.0
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for global acceptance and 6.7 for flavor was obtained in the evaluation of fermented soy milk (90%) with sucrose (10%) based fermented beverages with the addition of pineapple and guava flavor (Behrens, Roig, & da Silva, 2004). In counterpart, the lactic fermented beverage of carambola obtained lower marks for color (4.4) and flavor (4.8) in comparison to the carambola juice at 8 Brix and pH 3.52 (5.90 for color and 5.95 for flavor) (Lee & Faridah, 2005) and the lactic fermented beverage of pitaya obtained lower marks for aroma (3.1) and color (3.5) when compared with reconstituted red pitaya juice (5.0 and 6.0, respectively) (Siow & Wong, 2016). In another study, the addition of 10% (v/v) of pitaya to a yogurt formulation containing 10% (w/v) of sucrose and 0.8% (w/v) of gelatin enhanced the attributes taste, texture, color and global acceptance when compared with commercialized yogurt (Jayasinghe, Fernando, Jayamanne, & Hettiarachchi, 2015). In the qualitative answers provided by the panelists in our study, an intense sweet aroma of mango was indicated as the main positive aspect in the beverages produced with this fruit and a viscous and unpleasant consistency in the beverages containing pitaya was indicated as the main negative aspect. Other studies conducted by our research group (unpublished data) presented elevated sensory acceptance rates in non-dairy lactic fermented beverages of mango and guava (using shorter periods of fermentation), confirmed their potential. 4. Conclusion Based on the results obtained in this study, it was concluded that the mango and guava juice presented high potential as substrates for lactic fermentation utilizing a commercial lyophilized lactic culture (L. casei, L. bulgaricus and S. termophillus) at 37 C and without agitation. Under these conditions, it is possible to state that 24 h time is enough for the growth of the lactic culture and for the stabilization of the pH in the fermented beverages. The utilization of carambola demonstrated an inhibitory effect to the development of the lactic culture in regarding the bacilli. The utilization of pitaya juice augmented the lag phase in comparison with the remaining types of fruit juice and reduced the survival rate of the bacteria in longer periods of fermentation. Furthermore, the addition of whey resulted in reduction of the sensory acceptance (for all fruits) and the growth (except with carambola) compared to those for the beverages produced with fruit juice only. Finally, this study was important to investigate the behavior of these types of tropical fruit (carambola, guava, mango and pitaya) as substrates for lactic fermentation, generating relevant data for the development of new lactose free lactic beverages containing sensory and nutritional
Table 3 Sensory evaluation of fermented lactic beverages of tropical fruit and whey (n ¼ 32 panelists). Formulation
Taste
Flavor
Color
Acidity
Sweetness
1.1
0.9
HSD (p ¼ 0.05)**
1.4
Carambola(*) Guava(*) Mango Pitaya
(5.3 (6.3 (6.2 (2.5
± ± ± ±
2.4)a,b,c 1.9)a 1.0)a 1.2)d
(4.8 (7.1 (6.7 (3.1
± ± ± ±
2.0)c 1.7)a 1.3)a 1.5)d,e
(4.4 (7.8 (8.2 (3.5
± ± ± ±
1.8)b 1.2)a 0.8)a 1.6)b,c
(4.4 (4.8 (5.4 (6.8
± ± ± ±
1.1)e 1.5)d,e 1.6)c,d,e 1.5)a,b
(5.4 (5.0 (4.0 (2.6
± ± ± ±
1.3)a 1.5)a,b 1.2)c,d,e 1.1)e,f
Carambola þ whey(*) Guava þ whey(*) Mango þ whey Pitaya þ whey
(4.5 (5.9 (4.4 (2.1
± ± ± ±
2.4)b,c 2.1)a,b 0.7)c 1.1)d
(4.1 (7.0 (6.3 (2.6
± ± ± ±
2.0)c,d 1.6)a 1.1)a,b 1.2)e
(4.4 (7.2 (7.0 (2.5
± ± ± ±
1.6)b 1.5)a 1.5)a 1.6)c
(5.4 (5.9 (6.5 (7.0
± ± ± ±
1.4)b,c,d,e 1.1)a,b,c,d 1.1)a,b,c 1.6)a
(4.3 (4.2 (3.4 (2.4
± ± ± ±
1.3)b,c,d 1.0)b,c,d 1.1)c,d,e 1.2)f
Whey Whey(*)
(4.3 ± 1.5)c (5.6 ± 2.3)a,b,c
1.3
1.2
(4.6 ± 1.3)c (5.1 ± 2.0)b,c
(5.3 ± 1.3)b (5.3 ± 1.8)b
(5.6 ± 1.1)b,c,d (5.7 ± 1.3)b,c,d
*The samples marked with (*) received the addition of 10% w/v (g/100 mL) of sucrose before the sensory evaluation. **For each attribute, formulations marked with different letters present significant difference at 95% of confidence (p < 0.05).
(3.1 ± 1.1)e,f (4.4 ± 1.1)a,b,c
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characteristics of these tropical fruits. Lactose free beverages meet the needs of a growing lactose intolerant public. Acknowledgement ~o de Pais e Mestres” Authors are grateful to the “Associaça ~o Paulo, Brazil) for its financial support. (COTUCA, Campinas, Sa References Barile, M. F. (1983). Gram staining technique. In S. Ranzin, & J. G. Tully (Eds.), Mycoplasma characterization: Vol. 1. Methods in mycoplasmology (pp. 39e41). New York: Academic Press. Behrens, J. H., Roig, S. M., & da Silva, M. A. A. P. (2004). Fermentation of soymilk by commercial lactic cultures: Development of a product with market potential. Acta Alimentaria, 33, 101e109. Cadena, R. S., Cruz, A. G., Netto, R. R., Castro, W. F., Faria, J. D. A. F., & Bolini, H. M. A. (2013). Sensory profile and physicochemical characteristics of mango nectar sweetened with high intensity sweeteners throughout storage time. Food Research International, 54, 1670e1679. Das, J., Datta, Z., Saha, A., Nur, S. M., Barua, P., Rahman, M. M., et al. (2013). A compreenshive study on antioxidant antibacterial, cytotoxic and phytochemical properties of Averrhoa carambola. International Journal of Bioassays, 2, 803e807. http://dx.doi.org/10.21746/ijbio.2013.05.006. DiCagno, R., Codda, R., De Angelis, M., & Gobbetti, M. (2013). Exploitation of vegetables and fruits through lactic acid fermentation. Food Microbiology., 33, 1e10. http://dx.doi.org/10.1016/j.fm.2012.09.003. DiCagno, R., Minervini, G., Rizzello, C. G., De Angelis, M., & Gobbetti, M. (2011). Effect of lactic acid fermentation and antioxidant, texture, color and sensory properties of red and green smoothies. Food Microbiology, 28, 1062e1071. http:// dx.doi.org/10.1016/j.fm.2011.02.011. Filannino, P., Azzi, L., Cavoski, I., Vincentini, O., Rizzello, C. G., Gobbetti, M., et al. (2013). Exploitation of the health-promoting and sensory proprieties of organic pomegranate (Punica granatum L.) juice through lactic acid fermentation. International Journal of Food Microbiology, 163, 184e192. http://dx.doi.org/10.1016/ j.ijfoodmicro.2013.03.002. Fonteles, T. V., Costa, M. G. M., De Jesus, A. L. T., Fontes, C. P. M. L., Fernandes, F. A. N., & Rodrigues, S. (2013). Stability and quality parameters of probiotic cantaloupe melon juice produced with sonicated juice. Food Bioprocess and Technology, 6, 2860e2869. http://dx.doi.org/10.1007/s11947-012-0962-y. Fonteles, T. V., Costa, M. G. M., De Jesus, A. L. T., & Rodrigues, S. (2012). Optimization of the fermentation of cantaloupe juice by Lactobacillus casei NRRL B-442. Food Bioprocess, 5, 2819e2826. http://dx.doi.org/10.1007/s11947-011-0600-0. Fox, P. F., & McSweeney, P. L. H. (1998). Dairy chemistry and biochemistry (1st ed.). London: Blackie Academic & Professional. Gardner, N. J., Savard, T., Obermeier, P., Caldwell, G., & Champagne, C. P. (2001). Selection and characterization of mixed starter cultures for lactic acid fermentation of carrot, cabbage, beet and onion vegetable mixtures. International Journal Food Microbiology, 64, 261e275. http://dx.doi.org/10.1016/S01681605(00)00461-X. Jayasinghe, O., Fernando, S., Jayamanne, V., & Hettiarachchi, D. (2015). Production of a novel fruit-yoghurt using dragon fruit (Hylocereus undatus L.). European
Scientific Journal, 11, 208e215. Kourkoutas, Y., Xolias, V., Kallis, M., Bezirtzoglou, E., & Kanellaki, M. (2005). Lactobacillus casei cell immobilization on fruit pieces for probiotic additive, fermented milk and lactic acid production. Process Biochemistry, 40, 411e416. http://dx.doi.org/10.1016/j.procbio.2004.01.029. Lee, Y. S., & Faridah, M. S. (2005). Physical chemical and quality properties of starfruit juice agglomerate and drink. Journal of Tropical Agriculture and Food Science, 33, 237. Mohamed, S., Hassan, Z., & Abd Hamid, N. (1994). Antimicrobial activity of some tropical fruit wastes (guava, starfruit, papaya, passion fruit, langsat, duku, rambutan and rambai). Pertanika Journal of Tropical Agricultural Science, 17, 291e227. Nagpal, R., Kumar, A., & Kumar, M. (2012). Fortification and fermentation of fruit juices with probiotic lactobacilli. Annals Microbiology, 62, 1573e1578. http:// dx.doi.org/10.1007/s13213-011-0412-5. Nogueira, L. K., Aguiar-Oliveira, E., Kamimura, E. S., & Maldonado, R. R. (2016). Milk and açaí berry juice improve sensorial acceptability of kefir-fermented milk beverage. Acta Amazonica, 46. http://dx.doi.org/10.1590/1809-4392201600173, 471e424. Nyanga, L. K., Nout, M. J. R., Gadaga, T. H., Theelen, B., Boekhout, T., & Zwietering, M. H. (2007). Yeasts and lactic acid bacteria microbiota from masau (Ziziphusm auritiana) fruits and their fermented fruit juice in Zimbabwe. Internationl Journal of. Food Microbiology, 120, 159e166. http://dx.doi.org/ 10.1016/j.ijfoodmicro.2007.06.021. de Paula Valim, F., Aguiar-Oliveira, E., Kamimura, E. S., Alves, V. D., & Maldonado, R. R. (2016). Production of star fruit alcoholic fermented beverage. Indian Journal of Microbiology, 56, 476e481. http://dx.doi.org/10.1007/s12088016-0601-9. Pereira, A. L. F., Maciel, T. C., & Rodrigues, S. (2011). Probiotic beverage from cashew apple juice fermented with Lactobacillus casei. Food Research International, 44, 1276e1283. http://dx.doi.org/10.1016/j.foodres.2010.11.035. Sagdic, O., Ozturk, I., Yapar, N., & Yetim, H. (2014). Diversity and probiotic potentials of lactic acid bacteria isolated from gilaburu, a traditional Turkish fermented European cranberry bush (Viburnum opulus L.) fruit drink. Food Research International, 64, 537e545. http://dx.doi.org/10.1016/j.foodres.2014.07.045. Siow, L. F., & Wong, Y. M. (2016). Effect of juice concentration on storage stability, betacyanin degradation kinectis, and sensory acceptance of red-fleshed dragon fruit (Hylocereus polyrhizus) juice. International Journal of Food Properties, 1e10. http://dx.doi.org/10.1080/10942912.2016.1172086. Tahera, J., Feroz, F., Senjuti, J. D., Das, K. K., & Noor, R. (2014). Demonstration of antibacterial activity of commonly available fruit extracts in Dhaka, Bangladesh. American Journal of Microbiology Research, 2, 68e73. http://dx.doi.org/10.12691/ ajmr-2-2-5. Thongsombat, W., Sirichote, A., & Chanthachum, S. (2007). The production of guava juice fortified with dietary fiber. Songklanakarin Journal Science and Technology, 29, 187e196. Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2004). Probiotication of tomato juice by lactic acid bacteria. Journal of Microbiology, 42, 315e318. Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2005). Fermentation of beet juice by beneficial lactic acid bacteria. LWT Food Science and Technology, 38, 73e75. http://dx.doi.org/10.1016/j.lwt.2004.04.008. Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2006). Production of probiotic cabbage juice by lactic acid bacteria. Bioresource Technology, 97, 1427e1430. http:// dx.doi.org/10.1016/j.biortech.2005.06.018.
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Potential application of four types of tropical fruits in lactic fermentation Rafael Resende Maldonado a, *, Lucas da Costa Araújo a, Letícia Caroline da Silva Dariva a, ~o Pedro Rodrigues Prado a, ssia Nazar Rebac a, Isadora Amalfi de Souza Pinto a, Joa Ke a a Juliana Kazumi Saeki , Thainara Santos Silva , Emerson Kazuhiro Takematsu a, lia Vilela Tiene a, Elizama Aguiar-Oliveira b, Roberto Elias Buosi c, Nata Marcela Aparecida Deziderio d, Eliana Setsuko Kamimura d ~o Paulo, Food Department, Technical College of Campinas (COTUCA), University of Campinas, R. Jorge Figueiredo Corr^ ea, 735, 13087-261, Campinas, Sa Brazil b Exact Sciences and Technology Department, Santa Cruz State University, R. Jorge Amado, km 16, Salobrinho, 45.662-900, Ilh eus, Bahia, Brazil c ~o Paulo, Brazil Integrated College Maria Imaculada, R. Paula Bueno, 240, 13840-040, Mogi Guaçu, Sa d ~o Paulo, Campus Pirassununga, Av. Duque de Caxias Norte, Food Engineering Department, Faculty of Animal Science and Food Engineering, University of Sa ~o Paulo, Brazil 225, Campus da USP, 13635-900, Pirassununga, Sa a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2017 Received in revised form 13 July 2017 Accepted 1 August 2017 Available online 3 August 2017
Fruit juice has been used for the production of lactose-free or low-lactose beverages. This study evaluated the potential of four types of tropical fruit (carambola, guava, mango and pitaya) for the production of fermented lactic beverages with and without whey addition. Pasteurized fruit juices were used at (% v/v): 100% fruit juice, 100% whey and 50:50% of juice: whey. The fermentations were conducted with a lactic culture (L. casei, S. thermophilus, and L. bulgaricus) at 37 C for 72 h. The final characteristics obtained were pH 3.0e4.0, soluble solids concentration (SS) 5.0e7.0 Brix (g/100 g), and acidity 0.2e1.0 %w/v. A validation test obtained 4.0e4.8 (pH), 6.0 Brix (SS), and 0.30e0.50 %w/v (acidity) at 24 h. The lactic bacteria had better growth (107e108 CFU/mL) and the highest sensory acceptance rates (70% for taste, 75% for flavor and 90% for color) utilizing mango or guava. Carambola and pitaya caused a partial inhibition to the cellular growth (103e104 CFU/mL) and resulted in beverages with lower sensory acceptance. In all cases, formulations containing only fruit juice were lactose-free and better sensory acceptance than the ones with of whey. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Carambola Guava Lactobacillus Mango Pitaya
1. Introduction Lactic fermentation of food has several advantages and it is utilized mainly for the production of different dairy products. In this process, the lactose is fermented by acid-lactic bacteria which reduce the concentration of this sugar in the fermentation medium, promote the acidification and the formation of secondary metabolites. The production of lactic acid and other metabolites is responsible for the characteristics of lactic fermented products, such as flavor, taste, increase of shelf life and reduction of the lactose content (Fox & McSweeney, 1998).
* Corresponding author. E-mail address: [email protected] (R.R. Maldonado). http://dx.doi.org/10.1016/j.lwt.2017.08.005 0023-6438/© 2017 Elsevier Ltd. All rights reserved.
The lactic fermentation is usually associated with milk or dairy products; however, it can be applied successfully to other substrates, such as fruit pulp or juice, due to the high fermentable sugar contents. Moreover, fruits and vegetables naturally contain acid lactic or acetic bacteria in their microbiota (DiCagno, Codda, De Angelis, & Gobbetti, 2013; Nyanga et al., 2007; Sagdic, Ozturk, Yapar, & Yetim, 2014). The application of fruit as substrate for lactic fermentation also has the advantage of the incorporation of flavors and nutrients specific to each type of fruit, resulting in products with different sensory and physico-chemical characteristics. Furthermore, the fruit based fermented products are lactose free, which meet the needs of consumers with lactose intolerance. There are reports in the literature of some studies with fruits and vegetables juices applied successfully to the lactic fermentation process, such as: pomegranate (Punica granatum L.) (Filannino et al.,
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2013), cantaloupe melon (Cucumis melo L.) (Fonteles et al., 2013; 2012), cashew apple (Anacardium occidentale) (Pereira, Maciel, & Rodrigues, 2011), carrot, cabbage, beetroot and onion (Gardner, Savard, Obermeier, Caldwell, & Champagne, 2001; Yoon, Woodams, & Hang, 2006, 2005; 2004). Kourkoutas, Xolias, Kallis, Bezirtzoglou, and Kanellaki (2005) utilized fruit as supports for the growth of acid-lactic bacteria and DiCagno, Minervini, Rizzello, De Angelis, & Gobbetti, 2011 obtained fermented fruit smoothies. However, it is known that the survival of lactic bacteria in fruit substrates tends to be more complex than in the dairy ones. This is due mainly to the natural acidity of fruit, the high level of polyphenols and the absence of lactose, which may interfere in the survival of certain sorts of microorganisms as well as in their growth (DiCagno et al., 2011; Fonteles et al., 2013). Based on the promising results reported on the literature for the lactic fermentation of fruit, the aim of this study was to evaluate the potential of four types of tropical fruit (carambola - Averrhoa carambola L.; guava - Psidium guajava; mango - Magifera indica L. var. Tommy Atkins; and red bark pitaya - Hylocereus undatus) for the production of fermented lactic beverages. Additionally, whey, a sub-product largely utilized in dairy products, was also investigated in association with the tropical fruits studied. The fermented lactic beverages were evaluated regarding the concentration of soluble solids (SS), cellular viability, pH and titratable acidity along the fermentation time and the sensory acceptance of the final product. 2. Materials and methods 2.1. Fruit juice preparation For this study, four types of tropical fruits were chosen: carambola (Averrhoa carambola L.), guava (Psidium guajava), mango (Magifera indica L. var. Tommy Atkins), and red bark pitaya with white pulp (Hylocereus undatus). The fruits were purchased in a ~o Paulo, Brazil). They were all ripe, local supermarket (Campinas, Sa undamaged and visually adequate for consumption (good color, odor and texture). For the preparation of the pulps, the fruits were washed, peeled and crushed in a blender. The initial SS of pulps were adjusted by the dilution with distilled water, until the obtainment of juice with 7e8 Brix (g/100 g of sugar). Thereafter, the juice samples were pasteurized in batches at 80 C for 5 min (Nagpal, Kumar, & Kumar, 2012) and bottled hot in glass flasks of 500 mL. After bottling, the flasks were closed hermetically and, after cooled to room temperature, were frozen at 18 C (de Paula Valim, Aguiar-Oliveira, Kamimura, Alves, & Maldonado, 2016). 2.2. Whey preparation Fresh milk (15.0 L) was obtained from a local dairy processor ~o Paulo, Brazil). The coagulation was conducted by (Campinas, Sa the addition of a lactic culture (170 g of industrialized yogurt resulting in an inoculation of 1.106 CFU/mL) and 5.0 mL of liquid rennet (as indicated by the producer). After 1 h of coagulation at room temperature (25 C), the coagulated mass was separated from the whey by filtration. The whey was pasteurized and stored under the same conditions as for the fruit juice. 2.3. Lactic acid fermentation The fermentations were performed with three substrate volume compositions: 100% fruit juice; a mixture of 50:50% fruit juice and whey and 100% whey, totaling nine different formulations. All formulations were evaluated in triplicate utilizing 500 mL Erlenmeyer flasks previously sterilized. To each flask, 300 mL of substrate were added and inoculated with a lyophilized lactic culture
255
containing Lactobacillus casei, Lactobacillus bulgaricus and Streptococcus termophillus (Rhodia Foods®) in quantity enough to reach the initial counting of 1.106 CFU/mL. The flasks were kept at 37 C without agitation for a period of 72 h. Samples of each fermentation were collected at 24, 48 and 72 h for the quantification of the concentration of soluble solids (SS), pH, and titratable acidity and to evaluate the microbial growth. The fermented beverages obtained at the end of the fermentation were utilized for the sensory evaluation. 2.4. Physico-chemical, microbiological and sensory analyses The concentration of soluble solids (SS) was directly measured with a refractometer (Atago - Master-53M) and the pH was measured with a pHmeter (Hanna - HI 3222-01). The acidity was measured by titration with NaOH 0.1 mol/L and the end point was determined by monitoring the pH within the range 7.8e8.2 using the pHmeter. The microbial growth was measured qualitatively by Gram stain method in order to identify the type of microorganisms as well as their growth in the fermentation medium. The slides were prepared as described by Barile (1983) and analyzed with a microscope (Bioval - L-2000B-PL) with a 1000x oil-immersion lens. The fermented beverages obtained were stored in a refrigerator at 4 C for 24 h and then submitted to the sensory evaluation by 32 non trained panelists, by means of a consumer affective test. A 9 points verbal hedonic scale (1 ¼ dislike it very much and 9 ¼ like it very much) was utilized to evaluate the attributes taste; flavor and color and a 9 points ideality scale (1 ¼ much lower than the ideal and 9 ¼ much higher than the ideal) was utilized to evaluate the attributes acidity and sweetness. From the data obtained, the average and the deviation standard for each attribute were calculated and the analysis of variance (ANOVA) and the Tukey test of average were applied to verify if there were significant differences among samples. The sensory evaluation was performed in two sessions. The nonsugared fermented beverages of mango and pitaya (with and without whey) were evaluated in the first session. The sugared [10% (g/100 mL) of sucrose] fermented beverages of guava and carambola (with and without whey) were evaluated in the second session. In both sessions, the fermented beverage obtained using just whey was included in the sensorial analysis. The addition of sucrose to beverages from guava and carambola was made due to the high acidity verified in the sensory evaluation of the first session. This study was conducted according to the ethical standards established by the Brazilian legislation for research involving human beings and is registered in the Brazilian Ministry of Health in the Brazil Platform (CAAE: 50655215.6.0000.5425). 2.5. Validation experiment A validation experiment was performed with the same formulations of the first experiment, but the concentration of soluble solids (SS) for all formulations was standardized (6 Brix) by dilution with distilled water. The fermentations were conducted using 1.106 CFU/mL of inoculum, at 37 C and without agitation. The fermentation time was limited to 24 h because the major changes in fermentation, observed in the previous experiments, occurred during this period. Samples were collected at the beginning and at the end of fermentation for the physico-chemical analyses. The cellular growth was measured through the colony forming units (CFU). Serial decimal dilutions of each sample were plated in triplicate onto Man, Rugosa and Sharpe (MRS) Agar plates (Kasvi®, Brazil) and incubated at 37 C for 48 h. Plates with number of colonies from 30 to 300 were enumerated and the results were expressed as CFU/mL (counting forming units) (Fonteles, Costa, De
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Jesus, & Rodrigues, 2012). 3. Results and discussion 3.1. Lactic acid fermentation of tropical fruits 3.1.1. Soluble solids concentration (SS) The soluble solids concentration (SS) for this type of beverage may be considered as a balance between the consumption of fermentable sugars and the production of acids (lactic acid, mainly) during the fermentation process. The results obtained for SS varied according to the formulation evaluated (Fig. 1a and b). The formulations containing only fruit juice presented a higher initial SS value (7.0e8.0 Brix) in comparison to the mixtures of fruit and
whey (6.5 Brix) and the formulation with only whey (6.0 Brix). For all formulations there was a decrease of SS in the first 24 h of fermentation, which indicates consumption of sugars for cellular growth and production of lactic acid (Fig. 1a, 1b, 1c and 1d). After this initial period, there was a stabilization or a slight increase of SS and the production of lactic acid until the end of the process, which suggests that, after 24 h, the sugar consumed was utilized only for the production of lactic acid, which is also a soluble solid. The final concentration of SS ranged from 5.0 to 7.0 Brix. However, different initial concentrations of soluble solids may influence the fermentation behavior. Thus, to verify possible interferences of the initial concentration of soluble solids (SS) on the fermentation process, a validation experiment was conducted using a standard initial SS at 6 Brix for all formulations. Fig. 2a and 2b
Fig. 1. a) and b) Soluble solids concentration ( Brix), c) and d) titratable acidity (% w/v, g/100 mL) and e) and f) pH for the lactic acid fermentation of four tropical fruits (carambola, guava, mango and pitaya) pure or in composition with whey (50:50% v/v). Fermentations were carried out in triplicate at 37 C, without stirring for 72 h and were inoculated with 1.106 CFU/mL with a commercial lyophilized lactic culture.
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257
Fig. 2. a) and b) Soluble solids concentration ( Brix), c) and d) titratable acidity (% w/v, g/100 mL) and e) and f) pH for the lactic acid fermentation of four tropical fruits (carambola, guava, mango and pitaya) pure or in composition with whey (50:50% v/v) in validation experiment. Fermentations were carried out in triplicate at 37 C, without stirring for 24 h and were inoculated with 1.106 CFU/mL with a commercial lyophilized lactic culture.
shows that the behavior of SS for all formulations using standard initial SS followed the same profile as the previous experiment. There were small SS reduction for formulations with mango, guava and whey and there was no change in SS for formulations with pitaya and carambola. 3.2. Acidity and pH profiles The acidity profile (Fig. 1c and 1d) showed an increase of this variable during the fermentative process [0.1e0.6% at 24 h and 0.2e1.0% (g/100 mL) at 72 h], while the pH profile (Fig. 1e and 1f) resulted in a more intense decrease in the first 24 h (from 4.5 to 6.5 for 3.5e4.5), followed by a less accentuated decrease after this
period. Both profiles indicate that the lactic bacteria in general were able to adapt well to the substrates of tropical fruit juice. The final pH values (after 72 h of fermentation) stabilized between 3.0 and 4.0, which is similar to results obtained with milk based fermented lactic beverages. Furthermore, the final pH range is inferior to 4.5, which hinders the proliferation of a great number of pathogenic microorganisms during the storage of the beverages. The formulations containing carambola presented the lowest pH reduction rates and also did not present variation of the SS during the fermentation, which indicates a difficulty of growth of the lactic culture in this substrate. Fig. 2cef shows the acidity and pH profiles for the validation experiment. Acidity increased (from 0.11 to 0.35 g/100 mL at 0 h for
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0.28e0.53 g/100 mL at 24 h) and pH decreased (from 5.2 to 4.1 at 0 h to 4.5e4.1 at 24 h), which indicated the production of acid by the lactic bacteria inoculated in the medium. The initial pH in the validation experiment was lower than in the first experiment; however the drop of pH was also less intense, resulting in beverages with similar pH (after 24 h) in both experiments. On the other hand, it is not possible to compare the initial acidity of both experiments; it can be assumed, however, that due to the lower initial pH, the initial acidity in the validation experiment was higher than in the first experiment. However, after 24 h the acid values of the beverages in both experiments were very close. The comparison of the pH and acidity results of both experiments indicates that there was a good reproducibility of the profiles obtained after 24 h, suggesting that the pH and final acidity of the beverages tend to be very similar despite possible differences in the raw materials used. Studies cited in the literature indicate different pH values for fermented lactic beverages utilizing fruit. Kourkoutas et al. (2005) applied immobilized L. casei in pieces of apple and quince and obtained a final pH of 4.8 after 16 h of fermentation in a substrate with initial pH of 6.0. Filannino et al. (2013) observed a final average pH of 3.20 for fermented lactic beverages of pomegranate. However, the initial substrate already presented a rather low pH (3.52). 3.3. Cellular growth The qualitative evaluation of the cellular growth (Table 1) indicated that there was adaptation of the lactic culture with all substrates assessed. The microbial population observed was formed by Gram-positive bacteria (cocci and bacilli types) in the most of the samples analyzed. There were differences in the formulations with carambola, in which only cocci were identified during the fermentation and in formulations with pitaya in which lactic bacteria were no more detected after 72 h of fermentation. These results can be correlated with the behavior observed for SS, pH and acidity in the formulations using carambola and pitaya juice. For the formulation using carambola, SS was practically constant (Fig. 1a and 2a), there was a lower reduction or better stability in the pH (Fig. 1e and 2e) and the acidity was also practically constant (Fig. 2c). The few variations of these parameters indicate a difficulty of adaptation or survival of the lactic culture in this substrate. The Gram stain in the first experiment identified only cocci in the Table 1 Qualitative evaluation of growth of lactic bacteria using Gram stain. Formulation*
24 h
48 h
72 h
Cocci
Bacilli
Cocci
Bacilli
Cocci
Bacilli
Carambola Guava** Mango** Pitaya
P P P P
A P1 P P2
P P P P2
A P P P2
P P P A
A P P A
Carambola þ whey Guava þ whey Mango þ whey Pitaya þ whey
P P P P
P P P P2
P P P P
P P P P
P P P2 P
P P P P
Whey 1* Whey 2*
P P
P1 P
P P
P P
P P
P P
*All formulations were evaluated in triplicate; P ¼ lactic bacteria present in all replicates; P2 ¼ present in 2 replicates; P1 ¼ present in 1 replicate; A ¼ absence. *There were whey 1 and 2 because the fermentation is divided in two sessions (first session ¼ mango, pitaya and whey 1 and second session ¼ guava, carambola and whey 2). **The quantity of lactic bacteria observed in the slams was much higher in the formulations containing mango and guava.
beverage produced with carambola (Table 1) and in the validation experiment, the quantity of bacteria was estimated in 2.103 CFU/mL (Table 2), less than the quantity inoculated. The macronutrients composition of the fruits assessed is rather similar, which suggests that the carambola may contain certain anti-nutritional factor that hinders the development of the lactic culture. Other studies demonstrated that carambola pulp and peel may present antimicrobial activity against different sorts of microorganisms. Mohamed, Hassan, and Abd Hamid (1994) evaluated different tropical fruit extracts (from ripe and unripe fruit). The unripe carambola extract presented antimicrobial activity against Bacillus subtillis, B. cereus and Proteus vulgaricus, while the ripe carambola extract was also efficient against Staphylococcus aureus, Lactobacillus bulgaricus, Escherichia coli and Pseudomonas aeruginosa. Das et al. (2013) verified that the carambola skin extract, obtained with petroleum ether, presented antimicrobial activity against pathogenic bacteria, especially Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli and B. megaterium. For the formulation using pitaya it was possible to observe that SS, pH and acidity were practically constant (Fig. 1a, 1e, 2a, 2c and 2e). These results suggest a greater difficulty of adaptation for the lactic culture to this particular substrate. Furthermore, the Gram stain in the first experiment showed the presence of cocci, but a few quantities of bacilli (at 24 h); the reducing in the quantity of both (cocci and bacilli) after 48 h; and neither cocci nor bacilli were detected in the beverage after 72 h (Table 1). In addition, in the validation experiment the quantity of lactic bacteria after 24 h was also lower than in the initial time (Table 2). These observations indicate that red pitaya with white pulp is not a bacilli inhibitor such as carambola pulp; however it might be a poor substrate for growth and survival of lactic bacteria according to results of Gram stain. The analyses in MRS plates confirmed this fact because the quantity of bacteria (7.104 CFU/mL) was also lower than the inoculated quantity (Table 2). In other study, Tahera, Feroz, Senjuti, Das, and Noor (2014) verified that aqueous, ethanolic and methanolic red pitaya (Hylocereus polyrhizus) extracts presented certain antimicrobial activity against Bacillus spp.; however there was no antimicrobial activity of these extracts against Staphlycoccus spp. On the other hand, beverages produced with mango and guava showed better results. In the first experiment the Gram stain confirmed the presence of cocci and bacilli in practically all samples using these substrates (Table 1) and in the validation experiment mango and guava presented more than 108 CFU/mL when they applied as the only substrate for lactic fermentation (Table 2). Beverages produced with whey showed both cocci and bacilli in the Gram stain (Table 1); however the quantity of lactic bacteria (Table 2) was lower compared to formulations containing mango
Table 2 Estimated quantity of lactic bacteria in the fermented lactic beverages from tropical fruits after 24 h or fermentation. Formulation*
CFU/mL
Carambola Guava Mango Pitaya
2.103 1.108 6.108 7.104
Carambola þ whey Guava þ whey Mango þ whey Pitaia þ whey
7.104 1.107 4.107 1.104
Whey
1.108
*Formulation ¼ substrates used to production the lactic fermented beverage.
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and guava. Probably, mango and guava contain more specific nutrients for the development of lactic bacteria than the whey. For all formulations, except carambola, the addition of whey reduced the quantity of lactic bacteria after 24 h (Table 2), which indicates that whey had a negative effect when mixed with these fruits. 3.4. Sensorial evaluation The sensory evaluation indicated that among the fermented lactic beverages obtained with the utilization of fruit only, the grades in general rank were: guava > mango > carambola > pitaya. The exception to this behavior was in relation to the attribute color, in which the fermented beverage of mango obtained a superior grade than guava. Moreover, for all attributes assessed, the addition of whey resulted in a worse sensory evaluation than that for the beverages without whey (Table 3). In the comparison between the two sessions, it is noticeable that the addition of sucrose to the beverages before the sensory evaluation improved their results for the attributes taste, flavor and sweetness, but there was no alteration in the attribute acidity. The addition of sucrose enhances the ratio of the beverage (balance between acidity and sweetness) and increases the sensory acceptance of beverages with elevated acidity. Such behavior was already reported for the sensory evaluation of mixed beverages of milk and açaí fermented by kefir (Nogueira, Aguiar-Oliveira, Kamimura, & Maldonado, 2016). The results obtained in this study show that from the tropical fruits assessed, the more commonly consumed in Brazil (mango and guava) presented a higher sensory acceptance than the other two (carambola and pitaya). Furthermore, mango and guava were those in which the lactic bacteria developed better, while carambola and pitaya were less favorable substrates (under the evaluated conditions) for the development of lactic acid bacteria, which probably affected the sensory qualities of the beverages. DiCagno et al. (2011) verified improvement of some sensory attributes such as taste, texture, sweetness and global acceptance in green and red smoothies made of mixed fermented fruit and vegetables. In this present study, it was also possible to notice the improvement of some attributes compared to studies cited in the literature. The lactic fermented beverage of mango had a better-rated taste (6.20) than the mango juice sweetened with 7% of sucrose (4.40) (Cadena et al., 2013). The lactic fermented beverage of guava obtained better-rated flavor and color (7.1 and 7.8, respectively) than the guava juice in the concentration 12 Brix (5.93 and 7.03, respectively) (Thongsombat, Sirichote, & Chanthachum, 2007). The highest sensory acceptance, with the marks 7.7 for appearance, 7.0
259
for global acceptance and 6.7 for flavor was obtained in the evaluation of fermented soy milk (90%) with sucrose (10%) based fermented beverages with the addition of pineapple and guava flavor (Behrens, Roig, & da Silva, 2004). In counterpart, the lactic fermented beverage of carambola obtained lower marks for color (4.4) and flavor (4.8) in comparison to the carambola juice at 8 Brix and pH 3.52 (5.90 for color and 5.95 for flavor) (Lee & Faridah, 2005) and the lactic fermented beverage of pitaya obtained lower marks for aroma (3.1) and color (3.5) when compared with reconstituted red pitaya juice (5.0 and 6.0, respectively) (Siow & Wong, 2016). In another study, the addition of 10% (v/v) of pitaya to a yogurt formulation containing 10% (w/v) of sucrose and 0.8% (w/v) of gelatin enhanced the attributes taste, texture, color and global acceptance when compared with commercialized yogurt (Jayasinghe, Fernando, Jayamanne, & Hettiarachchi, 2015). In the qualitative answers provided by the panelists in our study, an intense sweet aroma of mango was indicated as the main positive aspect in the beverages produced with this fruit and a viscous and unpleasant consistency in the beverages containing pitaya was indicated as the main negative aspect. Other studies conducted by our research group (unpublished data) presented elevated sensory acceptance rates in non-dairy lactic fermented beverages of mango and guava (using shorter periods of fermentation), confirmed their potential. 4. Conclusion Based on the results obtained in this study, it was concluded that the mango and guava juice presented high potential as substrates for lactic fermentation utilizing a commercial lyophilized lactic culture (L. casei, L. bulgaricus and S. termophillus) at 37 C and without agitation. Under these conditions, it is possible to state that 24 h time is enough for the growth of the lactic culture and for the stabilization of the pH in the fermented beverages. The utilization of carambola demonstrated an inhibitory effect to the development of the lactic culture in regarding the bacilli. The utilization of pitaya juice augmented the lag phase in comparison with the remaining types of fruit juice and reduced the survival rate of the bacteria in longer periods of fermentation. Furthermore, the addition of whey resulted in reduction of the sensory acceptance (for all fruits) and the growth (except with carambola) compared to those for the beverages produced with fruit juice only. Finally, this study was important to investigate the behavior of these types of tropical fruit (carambola, guava, mango and pitaya) as substrates for lactic fermentation, generating relevant data for the development of new lactose free lactic beverages containing sensory and nutritional
Table 3 Sensory evaluation of fermented lactic beverages of tropical fruit and whey (n ¼ 32 panelists). Formulation
Taste
Flavor
Color
Acidity
Sweetness
1.1
0.9
HSD (p ¼ 0.05)**
1.4
Carambola(*) Guava(*) Mango Pitaya
(5.3 (6.3 (6.2 (2.5
± ± ± ±
2.4)a,b,c 1.9)a 1.0)a 1.2)d
(4.8 (7.1 (6.7 (3.1
± ± ± ±
2.0)c 1.7)a 1.3)a 1.5)d,e
(4.4 (7.8 (8.2 (3.5
± ± ± ±
1.8)b 1.2)a 0.8)a 1.6)b,c
(4.4 (4.8 (5.4 (6.8
± ± ± ±
1.1)e 1.5)d,e 1.6)c,d,e 1.5)a,b
(5.4 (5.0 (4.0 (2.6
± ± ± ±
1.3)a 1.5)a,b 1.2)c,d,e 1.1)e,f
Carambola þ whey(*) Guava þ whey(*) Mango þ whey Pitaya þ whey
(4.5 (5.9 (4.4 (2.1
± ± ± ±
2.4)b,c 2.1)a,b 0.7)c 1.1)d
(4.1 (7.0 (6.3 (2.6
± ± ± ±
2.0)c,d 1.6)a 1.1)a,b 1.2)e
(4.4 (7.2 (7.0 (2.5
± ± ± ±
1.6)b 1.5)a 1.5)a 1.6)c
(5.4 (5.9 (6.5 (7.0
± ± ± ±
1.4)b,c,d,e 1.1)a,b,c,d 1.1)a,b,c 1.6)a
(4.3 (4.2 (3.4 (2.4
± ± ± ±
1.3)b,c,d 1.0)b,c,d 1.1)c,d,e 1.2)f
Whey Whey(*)
(4.3 ± 1.5)c (5.6 ± 2.3)a,b,c
1.3
1.2
(4.6 ± 1.3)c (5.1 ± 2.0)b,c
(5.3 ± 1.3)b (5.3 ± 1.8)b
(5.6 ± 1.1)b,c,d (5.7 ± 1.3)b,c,d
*The samples marked with (*) received the addition of 10% w/v (g/100 mL) of sucrose before the sensory evaluation. **For each attribute, formulations marked with different letters present significant difference at 95% of confidence (p < 0.05).
(3.1 ± 1.1)e,f (4.4 ± 1.1)a,b,c
260
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