Suitability of Bifidobacterium spp. and Lactobacillus plantarum as Probiotics Intended for Fruit Juices Containing Citrus Extracts Antonio Bevilacqua, Daniela Campaniello, Maria Rosaria Corbo, Lucia Maddalena, and Milena Sinigaglia
Abstract: A strain of Lactobacillus plantarum and 4 strains of bifidobacteria were inoculated in apple juice and in a commercial beverage labeled as “red-fruit juice,” containing citrus extracts as natural preservatives; the suitability of the probiotics was evaluated in relation to their resistance to 2 kinds of citrus extracts (biocitro and lemon extract), survival in juices at 4 and 37 ◦ C, and inhibition of Zygosaccharomyces bailii. Cell count of L. plantarum and bifidobacteria over time was fitted through the Weibull equation, for the evaluation of the first reduction time (δ), death time, and microbiological shelf life (the break-point was set to 7 log cfu/mL). Bifidobacterium animalis subsp. lactis experienced the highest δ-value (23.21 d) and death time (96.59 d) in the red-fruit juice at 4 ◦ C, whereas L. plantarum was the most promising strain in apple juice at 37 ◦ C. Biocitro and lemon extract did not exert a biocidal effect toward probiotics; moreover, the probiotics controlled the growth of Z. bailii and the combination of L. plantarum with 40 ppm of biocitro reduced the level of the yeast after 18 d by 2 log cfu/mL. Keywords: Bifidobacterium spp., fruit juice, Lactobacillus plantarum, probiotics
M: Food Microbiology & Safety
Probiotics are often added to different products to provide physiological benefits, thus producing “functional food.” B. animalis subsp. lactis and L. plantarum survived in apple juice and in a commercial drink (red-fruit juice), containing a citrus extract as natural preservative, and were able to control the growth of Z. bailii, a spoiling yeast of juices.
Practical Application:
Introduction The term functional food was introduced in Japan in 1980 and Japan was the first Country that stated specific regulatory approval process for functional foods, known as Foods for Specified Health Use (FOSHU) (Sanders 1998). The concept of functional food derives from the realization that specific components of the diets have the capacity to contribute benefits beyond those of basic nutrition; although there is not a general and accepted definition of functional foods, they can be defined as those with functional and health claims made (Binns and Howlett 2009). Foods and beverages containing probiotic microorganisms can be defined “functional.” The viability of probiotics is an important trait for these products: the minimum concentration of live probiotic bacteria should be above 107 cfu/mL or g (Krasaekoopt and others 2003; Homayouni and others 2008), or 106 cfu/mL according to Italian law (Fortina 2007). Fermented milk and other dairy products such as yoghurt, ice cream, cheese, and so on are considered functional foods thanks to their content of calcium and other beneficial components; they are also used as vehicles for probiotic bacteria in humans. Although most of the current probiotic foods are dairy based, there is a growing interest toward nondairy probiotic prodMS 20130700 Submitted 5/24/2013, Accepted 9/8/2013. Authors Bevilacqua Campaniello Corbo and Sinigaglia are with Dept. of the Science of Agriculture, Food and Environment, Univ. of Foggia, Foggia, Italy. Author Maddalena is with Dept. of Economy, Univ. of Foggia, Foggia, Italy. Direct inquiries to author Sinigaglia (E-mail: [email protected]).
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ucts, as a consequence of lactose intolerance, and the unfavorable cholesterol content of fermented dairy products (Ranadheera and others 2010), as well as for the increasing demand for vegetarian probiotic foods (Heenan and others 2004). Fruit juices are appealing, healthy, and “refreshing” foods (Tuorila and Cardello 2002; Luckow and Delahunty 2004a, b; Yoon and others 2004; Sheehan and others 2007), and could be considered a good vehicle for probiotics, due to the high content of sugars and beneficial nutrients (minerals, vitamins, dietary fibers, and antioxidants) (Prado and others 2008). Several authors studied in the past the survival of probiotics in different fruit juices, in relation to cell adaptation (Champagne and Gardner 2008), storage temperature (Saarela and others 2006a), presence of fibers and/or other prebiotics (Saarela and others 2006b), oxygen (Shah 2000), pH and kind of juice (Nualkaekul and Charalampopoulos 2011), organic acids (Nualkaekul and others 2011), vitamins and antioxidant compounds (Shah and others 2010), packaging (Champagne and others 2008). Some limiting elements for the viability of probiotic in juices are the concentration of phenolic compounds (Nualkaekul and others 2011), and the pH (Prado and others 2008); thus, some authors proposed different approaches to enhance microbial viability, like the use of wild strains, able to survive under acidic conditions (Saarela and others 2011), the microencapsulation of probiotics (Sohail and others 2012), and the use of “in situ fermentation” to attain microbial adaptation (Mousavi and others 2011; Pereira and others 2011). Nowadays many probiotic juices are available in Australian and American markets and some products are covered by patents (Holmgren and Lingdren 2012). R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12280 Further reproduction without permission is prohibited
Probiotics in juices . . .
Materials and Methods Microorganisms L. plantarum c19 (Bevilacqua and others 2010a) (Culture Collection of the Dept. of the Science of Agriculture, Food and Environment, Univ. of Foggia), 4 strains of bifidobacteria from Deutsche Sammlung von Mikroorganismem und Zellkulturen’s collection-DSMZ (Braunschweig, Germania) (B. animalis subsp. lactis DSMZ 10140; B. animalis subsp. animalis DSMZ 20104; B. bifidum DSMZ 20456; B. longum subsp. infantis DSMZ 20088), and a yeast (Z. bailii DSMZ 70492) were used throughout the research. L. plantarum and bifidobacteria were grown in MRS broth (Oxoid, Milan) and MRS broth + 0.5% cysteine (cMRS) (Sigma-Aldrich, Milan, Italy) respectively, incubated at 37 ◦ C for 48 h under anaerobic conditions, while the yeast was grown in YPD broth (peptone 20 g/L; yeast extract 10 g/L; glucose 20 g/L) incubated at 25 ◦ C for 48 to 72 h. Thereafter, the cultures were centrifuged at 4000 rpm for 20 min and washed twice with sterile distilled water.
mula of Blaszyk and Holley (1998), modified by Bevilacqua and others (2009): GI =
Abss × 100, Absc
(1)
where, for each time of sampling Abss is the absorbance of the sample containing the extract, and Abcc is the optical density of the control. The minimum concentration of the extracts able to inhibit the growth of the probiotics for 96 h was assumed as the minimum inhibitory concentration (MIC). The viability of the probiotics after 96 h was evaluated through the pour plate method (MRS agar and cMRS agar, incubated at 37 ◦ C for 48 to 72 h).
Viability of probiotics in fruit juices Apple juice (pH, 3.7; soluble solids, 11.0 Bx; sugars, 10.5%) and a commercial beverage known as “red-fruit juice” (pH, 3.23; 11.25 Bx; sugars, 11.3%; 20% red orange; 20% blueberry; 10% pomegranate) were purchased from a local market; the juices did not contain any preservative and were thermally treated by producers at 95 ◦ C for 30 to 60 s. Aliquots of 50 mL of each juice were inoculated separately with bifidobacteria or L. plantarum to 8 log cfu/mL and stored at 4 and 37 ◦ C to mimic, respectively, the storage under refrigeration and an accelerated shelf-life test. The duration of the storage was 43 d for the red-fruit juice and 17 d for apple juice. Cell viability was assessed immediately after the inoculation and periodically throughout the storage (every 2 to 4 d) through the pour plate count. The analyses were performed over 2 different batches; for each batch the analyses were repeated twice. The significant differences were pointed out through one-way analysis of variance (ANOVA) using the statistical package Statistica for Windows 10.0 (Statsoft Inc., Tulsa, Okla., U.S.A.) and Tukey’s test (P < 0.05); then, the data were modeled through the Weibull equation, reparameterized by Mafart and others (2002): p N = N0 − t δ ,
(2)
Natural extracts Neroli (citrus flower extract) (Sigma-Aldrich), citrus extract R ) (Quinabra, Probena, Spagna), and lemon extract (Biocitro (Spencer Food Industrial, Amsterdam, the Netherlands) were used as natural compounds. Stock solutions of each chemical (from 1000 to 10000 ppm) were prepared in ethanol/water (1:1), filtered and used immediately.
where, N is the cell concentration over the time (log cfu/mL); N0 , the initial cell concentration; δ, the first reduction time (days), that is, the time to attain a decrease in cell concentration of 1 log cfu/mL; p, the shape parameter. The equation of Weibull, reparameterized by Bevilacqua and others (2008), was used to assess the death time (d.t., days):
Bioactivity of extracts toward probiotics MRS or cMRS containing variable amount of antimicrobials (from 10 to 100 ppm) were inoculated separately with L. plantarum or bifidobacteria to 3 log cfu/mL; the samples were prepared as follows: 19.3 mL of broth, 0.5 mL of the stock solutions of extracts, 0.2 mL of a cell suspension of each probiotic separately (5 log cfu/mL). The controls were prepared by adding to the broth 0.5 mL of the ethanol/water solution, without extracts, and 0.2 mL of probiotics. The samples were stored at 37 ◦ C for 96 h and microbial growth was evaluated periodically through absorbance measurements at 600 nm; the analyses were performed in duplicate over 2 different batches. Data were modeled as growth index (GI), using the for-
p N t N0 = 1 − d .t. .
(3)
The fitting parameters of the Weibull equation were also used for the evaluation of the microbiological shelf life (SL-7) (Krasaekoopt and others 2003; Homayouni and others 2008), defined as the time to attain a cell count of 7 log cfu/mL as follows: SL = δ · (N0 − L c )1/ p ,
(4)
where Lc is the break-point for the cell count of probiotics (7 log cfu/mL). Vol. 78, Nr. 11, 2013 r Journal of Food Science M1765
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An increasing trend in food technology is the “green consumerism,” that is, the use of “friendly compounds” (Burt 2004), like citrus extract, for food preservation. Extracts from citrus were successfully proposed to reduce the contamination of Enterococcus spp. on fruit products (Fisher and Phillips 2008; Fisher and others 2009), and for the inhibition of spoiling yeasts and moulds in juices (Bevilacqua and others 2010b, 2012b, 2013). Red fruit and apple juices are very popular beverages for their health-based functions (Luckow and Delahunty 2004a,b; Yoon and others 2004); thus the main goal of this research was the evaluation of suitability of some bifidobacteria and a strain of Lactobacillus plantarum as probiotics for apple and red-fruit juices, containing citrus extracts as preservatives. This final goal was achieved through some intermediate milestones, as follows: (a) studying the bioactivity of citrus extracts toward L. plantarum and bifidobacteria; (b) evaluating the microbiological shelf life of probiotics in juices; (c) studying the effects of probiotics on the sensory scores of juices; (d) assessing the antagonistic effect of probiotics toward Zygosaccharomyces bailii, a spoiling yeast of juices.
Probiotics in juices . . . Table 1–Coded levels of the factors of the design (L. plantarum Table 2–Combinations of the design. compared with Z. bailii and B. animalis subsp. lactis compared Coded levels with Z. bailii). Coded levels
–1
0
+1
Biocitro (ppm) Thermal abuse (storage at 15 ◦ C, h) Z. bailii (log cfu/mL)
0 0 0
20 24 2
40 48 4
Biocitro
A B C D E F Choosing the most promising probiotics Principal component analysis (PCA) (XLSTAT, Addinsoft, G H Paris) was used to select the most promising probiotics, using I the following input variables: Control 1a Control 2a
1 0 0 –1 –1 1 –1 0 1 –1 1
Values
Thermal Biocitro Thermal Z. bailii abuse Z. bailii (ppm) abuse (h) (log cfu/mL) –1 1 –1 –1 0 0 1 0 1 –1 –1
0 –1 1 –1 1 –1 0 0 1 1 1
40 20 20 0 0 40 0 20 40 0 40
0 48 0 0 24 24 48 24 48 0 0
2 0 4 0 4 4 2 2 4 4 4
M: Food Microbiology & Safety
(1) Cell count of probiotics in apple juice after 17 d of storage a Without probiotics. at 4 and 37 ◦ C; (2) Shelf life (SL-7) at 4 and 37 ◦ C in red-fruit juice; of the design (inoculum of yeast, duration of the thermal abuse (3) MIC of biocitro and lemon extract. and amount of citrus extract); the statistical analysis of the results A sensory test was conducted to evaluate the acceptability of was performed through the package Statistica for Windows. juices inoculated with probiotics, using the approach proposed by Luckow and Delahunty (2004a,b). The test was conducted as Results and Discussion follows: 16 untrained assessors (students and researchers from Univ. The main aim of this paper was the evaluation of apple and of Foggia), analyzed 6 samples, identified by a letter: red-fruit juices as vehicles for probiotics; 4 different strains of bifidobacteria from a Public Collection and a wild strain of L. A, apple juice; plantarum were used as targets. Namely, the wild strain of L. planB, apple juice inoculated with L. plantarum (8 log cfu/mL); tarum was isolated from table olives and studied previously for its C, apple juice inoculated with B. animalis subsp. lactis probiotic traits: in fact, it showed some interesting properties in (8 log cfu/mL); terms of adhesion to model intestinal cell line, antagonistic activity D, red-fruit juice; toward foodborne pathogens (Escherichia coli O157:H7 and StaphyE, red-fruit juice inoculated with L. plantarum; lococcus aureus), survival under simulated gastrointestinal conditions F, red-fruit juice inoculated with B. animalis subsp. lactis. (Bevilacqua and others 2010a). On the other hand, bifidobacteria are generally regarded as probiotics; moreover, the 4 strains used The assessors were requested to give a score for color, odor, and in this research were assessed for their functional traits and B. anoverall acceptability from 0 (bad) to 5 (very good), 2 being the imalis subsp. lactis and B. longum subsp. infantis showed interesting break point. Finally they were requested to answer this question: probiotic traits (survival at pH 2.5 and with bile salts added) and “Would you buy this product?” and choose 3 possible answers: promising technological properties (growth/survival under differ“definitely would buy,” “maybe buy/maybe not buy,” “definitely ent conditions) (Bevilacqua and others 2012a). would not buy” (Bevilacqua and others 2012b).
Coinoculum of probiotics with Z. bailii Aliquots of red-fruit juice (50 mL), diluted with water (1:1) and containing citrus extract, were inoculated with either L. plantarum or B. animalis subsp. lactis (6 log cfu/mL) and Z. bailii; then, the samples were stored at 15 ◦ C (thermal abuse) and 4 ◦ C (refrigerated conditions). The amount of citrus extract (0 to 40 ppm), as well as the concentration of the yeast (0 to 4 log cfu/mL) and the duration of thermal abuse at 15 ◦ C (0 to 48 h) varied according to a 3k-p factorial design. The coded levels of the factors and the combinations of the design are shown in Table 1 and 2, respectively; 2 samples of red-fruit juice, with or without citrus extract, were inoculated with Z. bailii to 4 log cfu/mL and used as controls (control 1 and control 2). Thus, 2 different designs were analyzed L. plantarum compared with Z. bailii and B. animalis compared with Z. bailii. The viability of L. plantarum, B. animalis subsp. lactis (pour plate method, as reported above) and Z. bailii (spread plating on YPD agar, incubated at 25 ◦ C for 4 d) was assessed immediately after the inoculation and throughout the storage (18 d); the experiments were performed in duplicate. The increase of Z. bailii after 18 d was used as input to build a polynomial equation, showing the significant effect of each factors M1766 Journal of Food Science r Vol. 78, Nr. 11, 2013
Bioactivity of extracts Figure 1 shows the bioactivity profile of biocitro and lemon extract toward L. plantarum c19 (GI of the microbial target as a function of the concentration of the extracts); 20 ppm of biocitro strongly affected the target after 24 h by reducing its GI to 32.62%, but the bioactivity of the extract decreased within the running time, as the same extent of inhibition was found after 48 and 96 h with 40 and 50 ppm of the extract. Concerning the effect of lemon extract, after 24 h L. plantarum was inhibited by 10 ppm of the preservative; however, the bioactivity of the extract decreased within the running time and the inhibition of the target was achieved after 96 h by 30 ppm of the extract (Figure 1B). Ten ppm of the biocitro were required to inhibit B. animalis subsp. lactis after 24 h (Figure 2), otherwise at the end of the running time the target was inhibited by 40 ppm of the extract. On the other hand, lemon extract affected strongly the growth of B. animalis subsp. lactis, since 20 ppm of the antimicrobial inhibited this microorganism for the entire running time (data not shown). Concerning the other bifidobacteria, B. animalis subsp. animalis, B. bifidum, and B. longum subsp. infantis were inhibited by 10 ppm of biocitro or lemon extract for the entire running time (data not shown). Although the MIC of neroli was higher than the
Probiotics in juices . . . values recovered for citrus and lemon extracts, it was not used for the following steps of the research due to its strong organoleptic impact. The viable count of bifidobacteria and L. plantarum was 3 log cfu/mL (that is, the inoculation level) in the samples containing the highest amounts of biocitro and lemon extract after 96 h, thus suggesting that the 2 preservatives exerted a biostatic rather than a biocidal effect (data not shown).
Viability of probiotics in juices Data from red-fruit juice were fitted through the Weibull equation, modified by Mafart and others (2002); the fitting parameters of this model are reported in Table 3. At 4 ◦ C B. animalis subsp. lactis experienced the highest first reduction time (δ, 23.21 d), whereas L. plantarum c19 showed the lowest value (2.93 d); the storage at 37 ◦ C caused a strong decrease of this parameter for bifidobacteria.
Figure 1–Growth Index of L. plantarum c19 in MRS broth containing biocitro (A) and lemon extract (B). Mean values ± standard deviation.
140
A
120 Growth Index (%)
100 80 24 h
60
48 h
40
96 h
20 0
10
20
30
40
50
M: Food Microbiology & Safety
0 -10
60
biocitro (ppm)
B
140
Growth Index (%)
120 100 80 24 h
60
48 h
40
96 h
20 0 0
10
20
30
40
lemon extract (ppm)
Figure 2–Effect of biocitro toward B. animalis subsp. lactis in cMRS broth (mean values ± standard deviation).
120
Growth Index (%)
100 80 60
24 h 48 h
40
96 h
20 0 0
10
20
30
40
50
biocitro (ppm)
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Probiotics in juices . . . Table 3–Weibull parameters (±standard error) of Bifidobacterium spp. and L. plantarum c19 in red-fruit juices. δ
N0 ◦C
R2
p
d.t.
Sl-7
4 B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
8.18 8.27 8.27 8.22 7.65
± ± ± ± ±
0.09a 0.22a 0.11a 0.13a 0.35a
23.21 12.93 10.81 13.12 2.93
± ± ± ± ±
2.10a 3.17b 1.38b 1.82b 0.40c
1.47 1.09 1.03 1.14 0.81
± ± ± ± ±
0.19a 0.20a 0.08a 0.12a 0.19b
0.949 0.916 0.984 0.969 0.976
96.59 88.30 82.98 81.88 34.84
± ± ± ± ±
2.40a 3.41b 0.63c 6.05c 4.96d
25.93 16.15 13.64 15.69 1.73
37 ◦ C B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
8.25 8.23 8.27 8.22 7.56
± ± ± ± ±
0.17a 0.07a 0.09a 0.11a 0.09a
3.90 3.96 3.82 3.58 4.15
± ± ± ± ±
0.59c,d 0.24c,d 0.31c,d 0.34c,d 0.33d
2.24 2.36 2.22 2.12 2.62
± ± ± ± ±
0.51c 0.23c 0.26c 0.27c 0.37c
0.987 0.998 0.996 0.996 0.997
9.95 9.60 9.82 9.63 8.93
± ± ± ± ±
0.82e 0.30e 0.39e 0.37e 0.32e
4.30 4.32 4.24 3.94 3.33
Notes: N0 , initial level (log cfu/mL); δ, first reduction time (days); p, shape parameter; Sl-7 (microbiological shelf life), time (days) to observe a decrease of probiotic below 7 log cfu/mL. For each parameter different letters indicate significant differences (one-way ANOVA and Tukey’s test, P < 0.05).
Table 4–Viability (log cfu/mL) (±standard deviation) of Bifidobacterium spp. and L. plantarum c19 in apple juice. Storage (days)
0
1
3
M: Food Microbiology & Safety
4 ◦C B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
6.45 6.45 7.11 6.76 7.06
± ± ± ± ±
0.12a 0.19a,b,c 0.11a 0.15a 0.13a
7.00 6.69 6.94 6.77 7.11
± ± ± ± ±
0.03b 0.01b 0.08a 0.05a 0.03a
37 ◦ C B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
6.45 6.45 7.11 6.76 6.96
± ± ± ± ±
0.26a 0.25a 0.09a 0.38a 0.21a
6.48 6.22 6.83 6.23 6.93
± ± ± ± ±
0.24a 0.34a 0.14a 0.06a 0.07a
6
/a / / / / 6.47 6.57 7.09 6.4 6.99
± ± ± ± ±
0.24a 0.21a 0.34a 0.4a 0.33
6.87 6.70 6.64 6.62 7.04
± ± ± ± ±
0.09a,b 0.03b 0.10a,b 0.12a 0.05a
6.22 6.96 7.02 6.2 6.98
± ± ± ± ±
0.03a 0.012b 0.04a 0.015a 0.31a
5.45 5.65 5.70 5.20 5.67
10
13
/ / / / /
/ / / / /
± ± ± ± ±
0.10b 0.32b 0.13b 0.11b 0.31b
3.2 2.90 2.80 3.20 4.33
± ± ± ± ±
17 6.87 6.70 6.64 6.62 7.04 0.11c 0.20c 0.30c 0.17c 0.11c
± ± ± ± ±
0.09a,b 0.03b 0.10a,b 0.12a 0.05a
–b – – – 3.73 ± 0.34d
Note: Different letters indicate significant differences within the storage time (one-way ANOVA and Tukey’s test, P < 0.05). a Not assessed. b Cell number below the detection limit (1 cfu/mL).
A modified Weibull equation, proposed by Bevilacqua and others (2008), was used to assess the death time, and evaluate the persistence of microbial targets in the system. As expected, under refrigeration B. animalis subsp. lactis showed the highest death time (96.59 d), followed by the other bifidobacteria (82 to 88 d) and
finally by L. plantarum c19 (34.84 d); at 37 ◦ C the death time was ca. 9 to 10 d for all the strains. A prerequisite for probiotics is to enter the intestine at high concentrations (109 to 1010 ); thus, Rosburg and others (2010) recommended to use 7 log cfu/g (or mL) as the minimal level
Figure 3–Principal component analysis. Input variables: biocitro and lemon, MIC of biocitro and lemon extract; apple-4 and apple-37, cell count in apple juice after 17 d at 4 and 37 ◦ C; Sl-7, microbiological shelf life in red-fruit juice (at 4 and 37 ◦ C). Microorganisms: Lp, L. plantarum; B. an. sp. lac., B. animalis subsp. lactis; B. an. sp. an., B. animalis subsp. animalis; B. lon., B. longum; B. bif., B. bifidum.
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Probiotics in juices . . . juice and the strong viability loss in apple juice, although the literature reports some controversial data. For example, Nualkaekul and others (2011) reported that the phenolic compounds affected significantly the survival of B. longum in pomegranate juice, but not in currant juice.
Selection of the most promising strains and sensory test Figure 3 shows the principal component analysis (PCA) to select the most promising strains; L. plantarum c19 and B. animalis subsp. lactis were significantly different from the other strains: namely, L. plantarum c19 was characterized by a higher viability in apple juice at 37 ◦ C and a higher resistance to biocitro and lemon extract, while B. animalis subsp. lactis showed a higher viability in red-fruit juice. Based on these results, L. plantarum c19 and B. animalis subsp. lactis DSMZ 10140 were selected and inoculated in juices and used for a sensory test, as suggested by Luckow and Delahunty (2004a, b); the results are reported in Figure 4. The probiotics did not affect the sensory traits of juices, as the assessors proposed the same scores for the control samples and the inoculated ones, thus suggesting the suitability of probiotic inoculation in juices.
Figure 4–Sensory scores (colour, odour and overall quality) for apple juice (apple) and red-fruit juice (red fr) inoculated with L. plantarum (Lp) or B. animalis subsp. lactis (Ba). Mean values ± standard deviation.
1.5
increase of Z. baili (log cfu/ml)
1 0.5
control
0 -0.5 -1 -1.5 -2
18 days
biocitro Lp
Figure 5–Increase of Z. bailii (mean values ± standard deviation) in the diluted red fruit after 18 d. Control 1, juice without biocitro and probiotics; control 2, juice not containing probiotic but added with 40 ppm of biocitro; Lp and Ba, prediction of yeast growth in the juice not containing biocitro but inoculated respectively with L. plantarum and B. animalis subsp. lactis; Lp-biocitro and Ba-biocitro, prediction of yeast growth in the samples containing 40 ppm of biocitro and inoculated with probiotics.
Ba Lp-biocitro Ba-biocitro
-2.5 -3
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of probiotics in a food at the time of consumption. This critical break point was used to evaluate the microbial acceptability of juices (SL-7), that was 25.93 d at 4 ◦ C for B. animalis subsp. lactis, 13.64 to 16.15 d for the other bifidobacteria and 1.73 d for L. plantarum. A thermal abuse (storage at 37 ◦ C) caused a significant decrease of the microbiological acceptability of the juice with bifidobacteria. In apple juice neither bifidobacteria nor L. plantarum experienced the classical non linear trend of a death kinetic, therefore the results were reported as cell count throughout the storage (Table 4). Bifidobacterium spp. and L. plantarum remained at ca. 6 log cfu/mL for the entire running time at 4 ◦ C; bifidobacteria were below the detection limit at 37 ◦ C after 17 d, while L. plantarum showed a residual surviving population (3.73 log cfu/mL). The trend experienced by L. plantarum and bifidobacteria in juices relied upon the kind of juice as well as the microorganisms; namely, the strong reduction of L. plantarum in the red-fruit confirmed the data of Nualkaekul and Charalampopoulos (2011), who studied the survival of L. plantarum in lemon, pomegranate, black currant, cranberry, grapefruit, and pineapple juices, and observed a significant viability loss in pomegranate and currant juices, due probably to the high-phenol content. An interesting result was recovered for bifidobacteria, due to their survival in the red-fruit
Probiotics in juices . . .
M: Food Microbiology & Safety
Effect of probiotics and biocitro on Z. bailii Juices could be contaminated by spore-former microorganisms, mould, and yeasts; although yeasts are the less resistant to thermal treatments, they prevail for their acidophilic behavior and quick growth. The genera mostly involved in juice spoilage are Pichia, Candida, Saccharomyces, and Zygosaccharomyces, being P. membranifaciens, C. maltosa, Z. bailii, and S. cerevisiae the species mainly isolated in spoiled juices (Jay and others 2005). Z. bailii is particularly dangerous to product stability due to its high resistance to low pH and low-water activity as well as to its ability to grow in the presence of 2% of acetic acid and of weak acid preservatives such as benzoic and sorbic acids (600 μg/mL) (Makdesi and Beuchat 1996; Sancho and others 2000; Patrignani and others 2010). Thus, it has been identified as a cause of spoilage in fruit juices (Pitt and Hocking 1999) and chosen in this research as a model strain to assess the biocontrol effect exerted by bifidobacteria and L. plantarum as a function of the level of the spoiling yeast, the concentration of biocitro, and the duration of a thermal abuse. The level of L. plantarum and B. animalis subsp. lactis was 6 to 7 log cfu/mL for the entire running time without any significant effect of the factors of the design (data not shown). On the other hand, Z. bailii was significantly affected by the independent variables; in the red-fruit juice containing B. animalis subsp. lactis, the increase of Z. bailii (ZB ) was influenced by the amount of biocitro (bio) and the initial level of the yeast (Z0 ) and could be described by the following polynomial equation: Z B = −0.31 + 0.024 · bio − 0.0012 · bio2 +0.89 · Z0 − 0.12 · Z 02 .
(5)
The most significant terms of the equation were the linear effects of biocitro and initial level of the yeast. In the juice containing L. plantarum, Z. bailii (ZL ) was affected by the amount of biocitro and the duration of thermal abuse (t) as follows: Z L = 0.83 − 0.038 · bio − 0.0075 · bio2 + 0.11 · t − 0.02 · t 2 (6) being the linear term of biocitro and the quadratic effect of the thermal abuse the most significant terms. The models were highly significant with adjusted regression coefficients of 0.922 to 0.967 and a mean standard error of 0.09 to 0.13. These polynomial equations were used to predict yeast growth in 4 different combinations of probiotics with biocitro; these predicted values were compared with the levels of Z. bailii recovered in the controls, that is, juice (control 1) or juice with biocitro (control 2) but not containing the probiotics (Figure 5). Z. bailii without biocitro and probiotics (control 1), increased by 0.8 log cfu/mL after 18 d; biocitro (control 2) and probiotics (Lp and Ba) as single “hurdles” were able to exert a controlling effect, as the level of the spoiling yeast did not increase. When biocitro was combined with L. plantarum, the model predicted a reduction of Z. bailii by more than 2 log cfu/mL, thus suggesting a strong antagonistic effect of the probiotic toward the yeast. It is well known that lactic acid bacteria could interact with yeasts in different ways and experience both positive (Liu and Tsao 2009) or negative interaction (Perricone and others 2010). The antagonistic effect could be the results of different phenomena (competition for the nutrients, production of lactic acid or other antimicrobial compounds) (C ¸ akir 2010; Mendoza and others 2010; Cizeikiene and others 2013); however, it is not M1770 Journal of Food Science r Vol. 78, Nr. 11, 2013
known why the addition of biocitro enhanced the effect of L. plantarum. We could suppose a combination of the bioactivity of lactic acid, competition for the nutrients between L. plantarum and the yeast and the antimicrobial effect of biocitro: this issue should be addressed and the exact mode of action of the interaction elucidated.
Conclusions Fruit juices could be considered as good vehicles for bifidobacteria and L. plantarum c19. Concerning bifidobacteria, the most promising strain was B. animalis subsp. lactis; it showed the best performances in the red-fruit juice with a microbiological shelf life at 4 ◦ C of 26 d, while apple juice could be proposed as a vehicle for L. plantarum c19. Citrus extract (namely biocitro) could be added to fruit juices as a natural preservative, as the probiotics were quite resistant to this extract; moreover, biocitro enhanced the biocontrolling effect of L. plantarum toward Z. bailii. Finally, the probiotics did not affect the sensory scores of juices.
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Vol. 78, Nr. 11, 2013 r Journal of Food Science M1771
Abstract: A strain of Lactobacillus plantarum and 4 strains of bifidobacteria were inoculated in apple juice and in a commercial beverage labeled as “red-fruit juice,” containing citrus extracts as natural preservatives; the suitability of the probiotics was evaluated in relation to their resistance to 2 kinds of citrus extracts (biocitro and lemon extract), survival in juices at 4 and 37 ◦ C, and inhibition of Zygosaccharomyces bailii. Cell count of L. plantarum and bifidobacteria over time was fitted through the Weibull equation, for the evaluation of the first reduction time (δ), death time, and microbiological shelf life (the break-point was set to 7 log cfu/mL). Bifidobacterium animalis subsp. lactis experienced the highest δ-value (23.21 d) and death time (96.59 d) in the red-fruit juice at 4 ◦ C, whereas L. plantarum was the most promising strain in apple juice at 37 ◦ C. Biocitro and lemon extract did not exert a biocidal effect toward probiotics; moreover, the probiotics controlled the growth of Z. bailii and the combination of L. plantarum with 40 ppm of biocitro reduced the level of the yeast after 18 d by 2 log cfu/mL. Keywords: Bifidobacterium spp., fruit juice, Lactobacillus plantarum, probiotics
M: Food Microbiology & Safety
Probiotics are often added to different products to provide physiological benefits, thus producing “functional food.” B. animalis subsp. lactis and L. plantarum survived in apple juice and in a commercial drink (red-fruit juice), containing a citrus extract as natural preservative, and were able to control the growth of Z. bailii, a spoiling yeast of juices.
Practical Application:
Introduction The term functional food was introduced in Japan in 1980 and Japan was the first Country that stated specific regulatory approval process for functional foods, known as Foods for Specified Health Use (FOSHU) (Sanders 1998). The concept of functional food derives from the realization that specific components of the diets have the capacity to contribute benefits beyond those of basic nutrition; although there is not a general and accepted definition of functional foods, they can be defined as those with functional and health claims made (Binns and Howlett 2009). Foods and beverages containing probiotic microorganisms can be defined “functional.” The viability of probiotics is an important trait for these products: the minimum concentration of live probiotic bacteria should be above 107 cfu/mL or g (Krasaekoopt and others 2003; Homayouni and others 2008), or 106 cfu/mL according to Italian law (Fortina 2007). Fermented milk and other dairy products such as yoghurt, ice cream, cheese, and so on are considered functional foods thanks to their content of calcium and other beneficial components; they are also used as vehicles for probiotic bacteria in humans. Although most of the current probiotic foods are dairy based, there is a growing interest toward nondairy probiotic prodMS 20130700 Submitted 5/24/2013, Accepted 9/8/2013. Authors Bevilacqua Campaniello Corbo and Sinigaglia are with Dept. of the Science of Agriculture, Food and Environment, Univ. of Foggia, Foggia, Italy. Author Maddalena is with Dept. of Economy, Univ. of Foggia, Foggia, Italy. Direct inquiries to author Sinigaglia (E-mail: [email protected]).
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ucts, as a consequence of lactose intolerance, and the unfavorable cholesterol content of fermented dairy products (Ranadheera and others 2010), as well as for the increasing demand for vegetarian probiotic foods (Heenan and others 2004). Fruit juices are appealing, healthy, and “refreshing” foods (Tuorila and Cardello 2002; Luckow and Delahunty 2004a, b; Yoon and others 2004; Sheehan and others 2007), and could be considered a good vehicle for probiotics, due to the high content of sugars and beneficial nutrients (minerals, vitamins, dietary fibers, and antioxidants) (Prado and others 2008). Several authors studied in the past the survival of probiotics in different fruit juices, in relation to cell adaptation (Champagne and Gardner 2008), storage temperature (Saarela and others 2006a), presence of fibers and/or other prebiotics (Saarela and others 2006b), oxygen (Shah 2000), pH and kind of juice (Nualkaekul and Charalampopoulos 2011), organic acids (Nualkaekul and others 2011), vitamins and antioxidant compounds (Shah and others 2010), packaging (Champagne and others 2008). Some limiting elements for the viability of probiotic in juices are the concentration of phenolic compounds (Nualkaekul and others 2011), and the pH (Prado and others 2008); thus, some authors proposed different approaches to enhance microbial viability, like the use of wild strains, able to survive under acidic conditions (Saarela and others 2011), the microencapsulation of probiotics (Sohail and others 2012), and the use of “in situ fermentation” to attain microbial adaptation (Mousavi and others 2011; Pereira and others 2011). Nowadays many probiotic juices are available in Australian and American markets and some products are covered by patents (Holmgren and Lingdren 2012). R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12280 Further reproduction without permission is prohibited
Probiotics in juices . . .
Materials and Methods Microorganisms L. plantarum c19 (Bevilacqua and others 2010a) (Culture Collection of the Dept. of the Science of Agriculture, Food and Environment, Univ. of Foggia), 4 strains of bifidobacteria from Deutsche Sammlung von Mikroorganismem und Zellkulturen’s collection-DSMZ (Braunschweig, Germania) (B. animalis subsp. lactis DSMZ 10140; B. animalis subsp. animalis DSMZ 20104; B. bifidum DSMZ 20456; B. longum subsp. infantis DSMZ 20088), and a yeast (Z. bailii DSMZ 70492) were used throughout the research. L. plantarum and bifidobacteria were grown in MRS broth (Oxoid, Milan) and MRS broth + 0.5% cysteine (cMRS) (Sigma-Aldrich, Milan, Italy) respectively, incubated at 37 ◦ C for 48 h under anaerobic conditions, while the yeast was grown in YPD broth (peptone 20 g/L; yeast extract 10 g/L; glucose 20 g/L) incubated at 25 ◦ C for 48 to 72 h. Thereafter, the cultures were centrifuged at 4000 rpm for 20 min and washed twice with sterile distilled water.
mula of Blaszyk and Holley (1998), modified by Bevilacqua and others (2009): GI =
Abss × 100, Absc
(1)
where, for each time of sampling Abss is the absorbance of the sample containing the extract, and Abcc is the optical density of the control. The minimum concentration of the extracts able to inhibit the growth of the probiotics for 96 h was assumed as the minimum inhibitory concentration (MIC). The viability of the probiotics after 96 h was evaluated through the pour plate method (MRS agar and cMRS agar, incubated at 37 ◦ C for 48 to 72 h).
Viability of probiotics in fruit juices Apple juice (pH, 3.7; soluble solids, 11.0 Bx; sugars, 10.5%) and a commercial beverage known as “red-fruit juice” (pH, 3.23; 11.25 Bx; sugars, 11.3%; 20% red orange; 20% blueberry; 10% pomegranate) were purchased from a local market; the juices did not contain any preservative and were thermally treated by producers at 95 ◦ C for 30 to 60 s. Aliquots of 50 mL of each juice were inoculated separately with bifidobacteria or L. plantarum to 8 log cfu/mL and stored at 4 and 37 ◦ C to mimic, respectively, the storage under refrigeration and an accelerated shelf-life test. The duration of the storage was 43 d for the red-fruit juice and 17 d for apple juice. Cell viability was assessed immediately after the inoculation and periodically throughout the storage (every 2 to 4 d) through the pour plate count. The analyses were performed over 2 different batches; for each batch the analyses were repeated twice. The significant differences were pointed out through one-way analysis of variance (ANOVA) using the statistical package Statistica for Windows 10.0 (Statsoft Inc., Tulsa, Okla., U.S.A.) and Tukey’s test (P < 0.05); then, the data were modeled through the Weibull equation, reparameterized by Mafart and others (2002): p N = N0 − t δ ,
(2)
Natural extracts Neroli (citrus flower extract) (Sigma-Aldrich), citrus extract R ) (Quinabra, Probena, Spagna), and lemon extract (Biocitro (Spencer Food Industrial, Amsterdam, the Netherlands) were used as natural compounds. Stock solutions of each chemical (from 1000 to 10000 ppm) were prepared in ethanol/water (1:1), filtered and used immediately.
where, N is the cell concentration over the time (log cfu/mL); N0 , the initial cell concentration; δ, the first reduction time (days), that is, the time to attain a decrease in cell concentration of 1 log cfu/mL; p, the shape parameter. The equation of Weibull, reparameterized by Bevilacqua and others (2008), was used to assess the death time (d.t., days):
Bioactivity of extracts toward probiotics MRS or cMRS containing variable amount of antimicrobials (from 10 to 100 ppm) were inoculated separately with L. plantarum or bifidobacteria to 3 log cfu/mL; the samples were prepared as follows: 19.3 mL of broth, 0.5 mL of the stock solutions of extracts, 0.2 mL of a cell suspension of each probiotic separately (5 log cfu/mL). The controls were prepared by adding to the broth 0.5 mL of the ethanol/water solution, without extracts, and 0.2 mL of probiotics. The samples were stored at 37 ◦ C for 96 h and microbial growth was evaluated periodically through absorbance measurements at 600 nm; the analyses were performed in duplicate over 2 different batches. Data were modeled as growth index (GI), using the for-
p N t N0 = 1 − d .t. .
(3)
The fitting parameters of the Weibull equation were also used for the evaluation of the microbiological shelf life (SL-7) (Krasaekoopt and others 2003; Homayouni and others 2008), defined as the time to attain a cell count of 7 log cfu/mL as follows: SL = δ · (N0 − L c )1/ p ,
(4)
where Lc is the break-point for the cell count of probiotics (7 log cfu/mL). Vol. 78, Nr. 11, 2013 r Journal of Food Science M1765
M: Food Microbiology & Safety
An increasing trend in food technology is the “green consumerism,” that is, the use of “friendly compounds” (Burt 2004), like citrus extract, for food preservation. Extracts from citrus were successfully proposed to reduce the contamination of Enterococcus spp. on fruit products (Fisher and Phillips 2008; Fisher and others 2009), and for the inhibition of spoiling yeasts and moulds in juices (Bevilacqua and others 2010b, 2012b, 2013). Red fruit and apple juices are very popular beverages for their health-based functions (Luckow and Delahunty 2004a,b; Yoon and others 2004); thus the main goal of this research was the evaluation of suitability of some bifidobacteria and a strain of Lactobacillus plantarum as probiotics for apple and red-fruit juices, containing citrus extracts as preservatives. This final goal was achieved through some intermediate milestones, as follows: (a) studying the bioactivity of citrus extracts toward L. plantarum and bifidobacteria; (b) evaluating the microbiological shelf life of probiotics in juices; (c) studying the effects of probiotics on the sensory scores of juices; (d) assessing the antagonistic effect of probiotics toward Zygosaccharomyces bailii, a spoiling yeast of juices.
Probiotics in juices . . . Table 1–Coded levels of the factors of the design (L. plantarum Table 2–Combinations of the design. compared with Z. bailii and B. animalis subsp. lactis compared Coded levels with Z. bailii). Coded levels
–1
0
+1
Biocitro (ppm) Thermal abuse (storage at 15 ◦ C, h) Z. bailii (log cfu/mL)
0 0 0
20 24 2
40 48 4
Biocitro
A B C D E F Choosing the most promising probiotics Principal component analysis (PCA) (XLSTAT, Addinsoft, G H Paris) was used to select the most promising probiotics, using I the following input variables: Control 1a Control 2a
1 0 0 –1 –1 1 –1 0 1 –1 1
Values
Thermal Biocitro Thermal Z. bailii abuse Z. bailii (ppm) abuse (h) (log cfu/mL) –1 1 –1 –1 0 0 1 0 1 –1 –1
0 –1 1 –1 1 –1 0 0 1 1 1
40 20 20 0 0 40 0 20 40 0 40
0 48 0 0 24 24 48 24 48 0 0
2 0 4 0 4 4 2 2 4 4 4
M: Food Microbiology & Safety
(1) Cell count of probiotics in apple juice after 17 d of storage a Without probiotics. at 4 and 37 ◦ C; (2) Shelf life (SL-7) at 4 and 37 ◦ C in red-fruit juice; of the design (inoculum of yeast, duration of the thermal abuse (3) MIC of biocitro and lemon extract. and amount of citrus extract); the statistical analysis of the results A sensory test was conducted to evaluate the acceptability of was performed through the package Statistica for Windows. juices inoculated with probiotics, using the approach proposed by Luckow and Delahunty (2004a,b). The test was conducted as Results and Discussion follows: 16 untrained assessors (students and researchers from Univ. The main aim of this paper was the evaluation of apple and of Foggia), analyzed 6 samples, identified by a letter: red-fruit juices as vehicles for probiotics; 4 different strains of bifidobacteria from a Public Collection and a wild strain of L. A, apple juice; plantarum were used as targets. Namely, the wild strain of L. planB, apple juice inoculated with L. plantarum (8 log cfu/mL); tarum was isolated from table olives and studied previously for its C, apple juice inoculated with B. animalis subsp. lactis probiotic traits: in fact, it showed some interesting properties in (8 log cfu/mL); terms of adhesion to model intestinal cell line, antagonistic activity D, red-fruit juice; toward foodborne pathogens (Escherichia coli O157:H7 and StaphyE, red-fruit juice inoculated with L. plantarum; lococcus aureus), survival under simulated gastrointestinal conditions F, red-fruit juice inoculated with B. animalis subsp. lactis. (Bevilacqua and others 2010a). On the other hand, bifidobacteria are generally regarded as probiotics; moreover, the 4 strains used The assessors were requested to give a score for color, odor, and in this research were assessed for their functional traits and B. anoverall acceptability from 0 (bad) to 5 (very good), 2 being the imalis subsp. lactis and B. longum subsp. infantis showed interesting break point. Finally they were requested to answer this question: probiotic traits (survival at pH 2.5 and with bile salts added) and “Would you buy this product?” and choose 3 possible answers: promising technological properties (growth/survival under differ“definitely would buy,” “maybe buy/maybe not buy,” “definitely ent conditions) (Bevilacqua and others 2012a). would not buy” (Bevilacqua and others 2012b).
Coinoculum of probiotics with Z. bailii Aliquots of red-fruit juice (50 mL), diluted with water (1:1) and containing citrus extract, were inoculated with either L. plantarum or B. animalis subsp. lactis (6 log cfu/mL) and Z. bailii; then, the samples were stored at 15 ◦ C (thermal abuse) and 4 ◦ C (refrigerated conditions). The amount of citrus extract (0 to 40 ppm), as well as the concentration of the yeast (0 to 4 log cfu/mL) and the duration of thermal abuse at 15 ◦ C (0 to 48 h) varied according to a 3k-p factorial design. The coded levels of the factors and the combinations of the design are shown in Table 1 and 2, respectively; 2 samples of red-fruit juice, with or without citrus extract, were inoculated with Z. bailii to 4 log cfu/mL and used as controls (control 1 and control 2). Thus, 2 different designs were analyzed L. plantarum compared with Z. bailii and B. animalis compared with Z. bailii. The viability of L. plantarum, B. animalis subsp. lactis (pour plate method, as reported above) and Z. bailii (spread plating on YPD agar, incubated at 25 ◦ C for 4 d) was assessed immediately after the inoculation and throughout the storage (18 d); the experiments were performed in duplicate. The increase of Z. bailii after 18 d was used as input to build a polynomial equation, showing the significant effect of each factors M1766 Journal of Food Science r Vol. 78, Nr. 11, 2013
Bioactivity of extracts Figure 1 shows the bioactivity profile of biocitro and lemon extract toward L. plantarum c19 (GI of the microbial target as a function of the concentration of the extracts); 20 ppm of biocitro strongly affected the target after 24 h by reducing its GI to 32.62%, but the bioactivity of the extract decreased within the running time, as the same extent of inhibition was found after 48 and 96 h with 40 and 50 ppm of the extract. Concerning the effect of lemon extract, after 24 h L. plantarum was inhibited by 10 ppm of the preservative; however, the bioactivity of the extract decreased within the running time and the inhibition of the target was achieved after 96 h by 30 ppm of the extract (Figure 1B). Ten ppm of the biocitro were required to inhibit B. animalis subsp. lactis after 24 h (Figure 2), otherwise at the end of the running time the target was inhibited by 40 ppm of the extract. On the other hand, lemon extract affected strongly the growth of B. animalis subsp. lactis, since 20 ppm of the antimicrobial inhibited this microorganism for the entire running time (data not shown). Concerning the other bifidobacteria, B. animalis subsp. animalis, B. bifidum, and B. longum subsp. infantis were inhibited by 10 ppm of biocitro or lemon extract for the entire running time (data not shown). Although the MIC of neroli was higher than the
Probiotics in juices . . . values recovered for citrus and lemon extracts, it was not used for the following steps of the research due to its strong organoleptic impact. The viable count of bifidobacteria and L. plantarum was 3 log cfu/mL (that is, the inoculation level) in the samples containing the highest amounts of biocitro and lemon extract after 96 h, thus suggesting that the 2 preservatives exerted a biostatic rather than a biocidal effect (data not shown).
Viability of probiotics in juices Data from red-fruit juice were fitted through the Weibull equation, modified by Mafart and others (2002); the fitting parameters of this model are reported in Table 3. At 4 ◦ C B. animalis subsp. lactis experienced the highest first reduction time (δ, 23.21 d), whereas L. plantarum c19 showed the lowest value (2.93 d); the storage at 37 ◦ C caused a strong decrease of this parameter for bifidobacteria.
Figure 1–Growth Index of L. plantarum c19 in MRS broth containing biocitro (A) and lemon extract (B). Mean values ± standard deviation.
140
A
120 Growth Index (%)
100 80 24 h
60
48 h
40
96 h
20 0
10
20
30
40
50
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0 -10
60
biocitro (ppm)
B
140
Growth Index (%)
120 100 80 24 h
60
48 h
40
96 h
20 0 0
10
20
30
40
lemon extract (ppm)
Figure 2–Effect of biocitro toward B. animalis subsp. lactis in cMRS broth (mean values ± standard deviation).
120
Growth Index (%)
100 80 60
24 h 48 h
40
96 h
20 0 0
10
20
30
40
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biocitro (ppm)
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Probiotics in juices . . . Table 3–Weibull parameters (±standard error) of Bifidobacterium spp. and L. plantarum c19 in red-fruit juices. δ
N0 ◦C
R2
p
d.t.
Sl-7
4 B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
8.18 8.27 8.27 8.22 7.65
± ± ± ± ±
0.09a 0.22a 0.11a 0.13a 0.35a
23.21 12.93 10.81 13.12 2.93
± ± ± ± ±
2.10a 3.17b 1.38b 1.82b 0.40c
1.47 1.09 1.03 1.14 0.81
± ± ± ± ±
0.19a 0.20a 0.08a 0.12a 0.19b
0.949 0.916 0.984 0.969 0.976
96.59 88.30 82.98 81.88 34.84
± ± ± ± ±
2.40a 3.41b 0.63c 6.05c 4.96d
25.93 16.15 13.64 15.69 1.73
37 ◦ C B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
8.25 8.23 8.27 8.22 7.56
± ± ± ± ±
0.17a 0.07a 0.09a 0.11a 0.09a
3.90 3.96 3.82 3.58 4.15
± ± ± ± ±
0.59c,d 0.24c,d 0.31c,d 0.34c,d 0.33d
2.24 2.36 2.22 2.12 2.62
± ± ± ± ±
0.51c 0.23c 0.26c 0.27c 0.37c
0.987 0.998 0.996 0.996 0.997
9.95 9.60 9.82 9.63 8.93
± ± ± ± ±
0.82e 0.30e 0.39e 0.37e 0.32e
4.30 4.32 4.24 3.94 3.33
Notes: N0 , initial level (log cfu/mL); δ, first reduction time (days); p, shape parameter; Sl-7 (microbiological shelf life), time (days) to observe a decrease of probiotic below 7 log cfu/mL. For each parameter different letters indicate significant differences (one-way ANOVA and Tukey’s test, P < 0.05).
Table 4–Viability (log cfu/mL) (±standard deviation) of Bifidobacterium spp. and L. plantarum c19 in apple juice. Storage (days)
0
1
3
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4 ◦C B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
6.45 6.45 7.11 6.76 7.06
± ± ± ± ±
0.12a 0.19a,b,c 0.11a 0.15a 0.13a
7.00 6.69 6.94 6.77 7.11
± ± ± ± ±
0.03b 0.01b 0.08a 0.05a 0.03a
37 ◦ C B. animalis subsp. lactis B. longum subsp. infantis B. animalis subsp. animalis B. bifidum L. plantarum
6.45 6.45 7.11 6.76 6.96
± ± ± ± ±
0.26a 0.25a 0.09a 0.38a 0.21a
6.48 6.22 6.83 6.23 6.93
± ± ± ± ±
0.24a 0.34a 0.14a 0.06a 0.07a
6
/a / / / / 6.47 6.57 7.09 6.4 6.99
± ± ± ± ±
0.24a 0.21a 0.34a 0.4a 0.33
6.87 6.70 6.64 6.62 7.04
± ± ± ± ±
0.09a,b 0.03b 0.10a,b 0.12a 0.05a
6.22 6.96 7.02 6.2 6.98
± ± ± ± ±
0.03a 0.012b 0.04a 0.015a 0.31a
5.45 5.65 5.70 5.20 5.67
10
13
/ / / / /
/ / / / /
± ± ± ± ±
0.10b 0.32b 0.13b 0.11b 0.31b
3.2 2.90 2.80 3.20 4.33
± ± ± ± ±
17 6.87 6.70 6.64 6.62 7.04 0.11c 0.20c 0.30c 0.17c 0.11c
± ± ± ± ±
0.09a,b 0.03b 0.10a,b 0.12a 0.05a
–b – – – 3.73 ± 0.34d
Note: Different letters indicate significant differences within the storage time (one-way ANOVA and Tukey’s test, P < 0.05). a Not assessed. b Cell number below the detection limit (1 cfu/mL).
A modified Weibull equation, proposed by Bevilacqua and others (2008), was used to assess the death time, and evaluate the persistence of microbial targets in the system. As expected, under refrigeration B. animalis subsp. lactis showed the highest death time (96.59 d), followed by the other bifidobacteria (82 to 88 d) and
finally by L. plantarum c19 (34.84 d); at 37 ◦ C the death time was ca. 9 to 10 d for all the strains. A prerequisite for probiotics is to enter the intestine at high concentrations (109 to 1010 ); thus, Rosburg and others (2010) recommended to use 7 log cfu/g (or mL) as the minimal level
Figure 3–Principal component analysis. Input variables: biocitro and lemon, MIC of biocitro and lemon extract; apple-4 and apple-37, cell count in apple juice after 17 d at 4 and 37 ◦ C; Sl-7, microbiological shelf life in red-fruit juice (at 4 and 37 ◦ C). Microorganisms: Lp, L. plantarum; B. an. sp. lac., B. animalis subsp. lactis; B. an. sp. an., B. animalis subsp. animalis; B. lon., B. longum; B. bif., B. bifidum.
M1768 Journal of Food Science r Vol. 78, Nr. 11, 2013
Probiotics in juices . . . juice and the strong viability loss in apple juice, although the literature reports some controversial data. For example, Nualkaekul and others (2011) reported that the phenolic compounds affected significantly the survival of B. longum in pomegranate juice, but not in currant juice.
Selection of the most promising strains and sensory test Figure 3 shows the principal component analysis (PCA) to select the most promising strains; L. plantarum c19 and B. animalis subsp. lactis were significantly different from the other strains: namely, L. plantarum c19 was characterized by a higher viability in apple juice at 37 ◦ C and a higher resistance to biocitro and lemon extract, while B. animalis subsp. lactis showed a higher viability in red-fruit juice. Based on these results, L. plantarum c19 and B. animalis subsp. lactis DSMZ 10140 were selected and inoculated in juices and used for a sensory test, as suggested by Luckow and Delahunty (2004a, b); the results are reported in Figure 4. The probiotics did not affect the sensory traits of juices, as the assessors proposed the same scores for the control samples and the inoculated ones, thus suggesting the suitability of probiotic inoculation in juices.
Figure 4–Sensory scores (colour, odour and overall quality) for apple juice (apple) and red-fruit juice (red fr) inoculated with L. plantarum (Lp) or B. animalis subsp. lactis (Ba). Mean values ± standard deviation.
1.5
increase of Z. baili (log cfu/ml)
1 0.5
control
0 -0.5 -1 -1.5 -2
18 days
biocitro Lp
Figure 5–Increase of Z. bailii (mean values ± standard deviation) in the diluted red fruit after 18 d. Control 1, juice without biocitro and probiotics; control 2, juice not containing probiotic but added with 40 ppm of biocitro; Lp and Ba, prediction of yeast growth in the juice not containing biocitro but inoculated respectively with L. plantarum and B. animalis subsp. lactis; Lp-biocitro and Ba-biocitro, prediction of yeast growth in the samples containing 40 ppm of biocitro and inoculated with probiotics.
Ba Lp-biocitro Ba-biocitro
-2.5 -3
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of probiotics in a food at the time of consumption. This critical break point was used to evaluate the microbial acceptability of juices (SL-7), that was 25.93 d at 4 ◦ C for B. animalis subsp. lactis, 13.64 to 16.15 d for the other bifidobacteria and 1.73 d for L. plantarum. A thermal abuse (storage at 37 ◦ C) caused a significant decrease of the microbiological acceptability of the juice with bifidobacteria. In apple juice neither bifidobacteria nor L. plantarum experienced the classical non linear trend of a death kinetic, therefore the results were reported as cell count throughout the storage (Table 4). Bifidobacterium spp. and L. plantarum remained at ca. 6 log cfu/mL for the entire running time at 4 ◦ C; bifidobacteria were below the detection limit at 37 ◦ C after 17 d, while L. plantarum showed a residual surviving population (3.73 log cfu/mL). The trend experienced by L. plantarum and bifidobacteria in juices relied upon the kind of juice as well as the microorganisms; namely, the strong reduction of L. plantarum in the red-fruit confirmed the data of Nualkaekul and Charalampopoulos (2011), who studied the survival of L. plantarum in lemon, pomegranate, black currant, cranberry, grapefruit, and pineapple juices, and observed a significant viability loss in pomegranate and currant juices, due probably to the high-phenol content. An interesting result was recovered for bifidobacteria, due to their survival in the red-fruit
Probiotics in juices . . .
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Effect of probiotics and biocitro on Z. bailii Juices could be contaminated by spore-former microorganisms, mould, and yeasts; although yeasts are the less resistant to thermal treatments, they prevail for their acidophilic behavior and quick growth. The genera mostly involved in juice spoilage are Pichia, Candida, Saccharomyces, and Zygosaccharomyces, being P. membranifaciens, C. maltosa, Z. bailii, and S. cerevisiae the species mainly isolated in spoiled juices (Jay and others 2005). Z. bailii is particularly dangerous to product stability due to its high resistance to low pH and low-water activity as well as to its ability to grow in the presence of 2% of acetic acid and of weak acid preservatives such as benzoic and sorbic acids (600 μg/mL) (Makdesi and Beuchat 1996; Sancho and others 2000; Patrignani and others 2010). Thus, it has been identified as a cause of spoilage in fruit juices (Pitt and Hocking 1999) and chosen in this research as a model strain to assess the biocontrol effect exerted by bifidobacteria and L. plantarum as a function of the level of the spoiling yeast, the concentration of biocitro, and the duration of a thermal abuse. The level of L. plantarum and B. animalis subsp. lactis was 6 to 7 log cfu/mL for the entire running time without any significant effect of the factors of the design (data not shown). On the other hand, Z. bailii was significantly affected by the independent variables; in the red-fruit juice containing B. animalis subsp. lactis, the increase of Z. bailii (ZB ) was influenced by the amount of biocitro (bio) and the initial level of the yeast (Z0 ) and could be described by the following polynomial equation: Z B = −0.31 + 0.024 · bio − 0.0012 · bio2 +0.89 · Z0 − 0.12 · Z 02 .
(5)
The most significant terms of the equation were the linear effects of biocitro and initial level of the yeast. In the juice containing L. plantarum, Z. bailii (ZL ) was affected by the amount of biocitro and the duration of thermal abuse (t) as follows: Z L = 0.83 − 0.038 · bio − 0.0075 · bio2 + 0.11 · t − 0.02 · t 2 (6) being the linear term of biocitro and the quadratic effect of the thermal abuse the most significant terms. The models were highly significant with adjusted regression coefficients of 0.922 to 0.967 and a mean standard error of 0.09 to 0.13. These polynomial equations were used to predict yeast growth in 4 different combinations of probiotics with biocitro; these predicted values were compared with the levels of Z. bailii recovered in the controls, that is, juice (control 1) or juice with biocitro (control 2) but not containing the probiotics (Figure 5). Z. bailii without biocitro and probiotics (control 1), increased by 0.8 log cfu/mL after 18 d; biocitro (control 2) and probiotics (Lp and Ba) as single “hurdles” were able to exert a controlling effect, as the level of the spoiling yeast did not increase. When biocitro was combined with L. plantarum, the model predicted a reduction of Z. bailii by more than 2 log cfu/mL, thus suggesting a strong antagonistic effect of the probiotic toward the yeast. It is well known that lactic acid bacteria could interact with yeasts in different ways and experience both positive (Liu and Tsao 2009) or negative interaction (Perricone and others 2010). The antagonistic effect could be the results of different phenomena (competition for the nutrients, production of lactic acid or other antimicrobial compounds) (C ¸ akir 2010; Mendoza and others 2010; Cizeikiene and others 2013); however, it is not M1770 Journal of Food Science r Vol. 78, Nr. 11, 2013
known why the addition of biocitro enhanced the effect of L. plantarum. We could suppose a combination of the bioactivity of lactic acid, competition for the nutrients between L. plantarum and the yeast and the antimicrobial effect of biocitro: this issue should be addressed and the exact mode of action of the interaction elucidated.
Conclusions Fruit juices could be considered as good vehicles for bifidobacteria and L. plantarum c19. Concerning bifidobacteria, the most promising strain was B. animalis subsp. lactis; it showed the best performances in the red-fruit juice with a microbiological shelf life at 4 ◦ C of 26 d, while apple juice could be proposed as a vehicle for L. plantarum c19. Citrus extract (namely biocitro) could be added to fruit juices as a natural preservative, as the probiotics were quite resistant to this extract; moreover, biocitro enhanced the biocontrolling effect of L. plantarum toward Z. bailii. Finally, the probiotics did not affect the sensory scores of juices.
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