Food Research International 43 (2010) 1444–1448
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Survival analysis methodology to predict the shelf-life of probiotic ﬂavored yogurt Adriano G. Cruz a,*, Eduardo H.M. Walter a, Rafael Silva Cadena b, José A.F. Faria a, Helena M.A. Bolini a, Hidelte P. Pinheiro b,c, Anderson S. Sant’Ana d a
Department of Food Technology, Faculty of Food Engineering, University of Campinas, Cidade Universitária ‘‘Zeferino Vaz”, CEP 13083-862 Campinas, SP, Brazil Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Cidade Universitária ‘‘Zeferino Vaz”, CEP 13083-862 Campinas, SP, Brazil c Institute of Mathematics, Statistics and Scientiﬁc Computing, University of Campinas, Cidade Universitária ‘‘Zeferino Vaz”, CEP 13083-970 Campinas, SP, Brazil d Department of Food and Experimental Nutrition, Faculty of Pharmaceuthical Sciences, University of São Paulo, Butantã, CEP 05508-900 São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 10 March 2010 Accepted 28 April 2010
Keywords: Survival analysis Probiotic yogurt Biﬁdobacteirum animalis Biﬁdobacteria Shelf-life
a b s t r a c t The feasibility of survival analysis methodology was used to determine the shelf-life of probiotic strawberry ﬂavored yogurt supplemented with Biﬁdobacteirum animalis DN 173010 W was investigated. The quality parameters of probiotic yogurts were related to storage conditions which they are submitted. The consumers were shown sensitive to changes towards sensory characteristics introduced into the products. Using the survival analysis and considering 25% and 50% probability of consumer rejection, the shelf-life of the probiotic yogurt was estimated at 38 and 53 days, respectively. The ﬁndings of this research highlighted the feasibility this technique to determine the shelf-life of foods, in particular, functional foods, as probiotic yogurts. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Probiotics are live microorganisms administered in quantities sufﬁcient to confer health beneﬁts (FAO/WHO, 2001). Of the clinically proven objectives produced by probiotic bacteria, the following can be cited: anti-carcinogenic and anti-mutagenic activities, combating infection by Heliobacter pylori, treatment of the inﬂammatory bowel disease, prevention and treatment of gastrointestinal disorders, increase in activity of the immunological system, antimicrobial, reduction of lactose intolerance, and reduction in blood cholesterol levels (Agrawal, 2009; Shah, 2007). Recently, there have also been recent reports on the potential beneﬁt of probiotics for human skin (Krutman, 2009) and against colds and ﬂu (Leyer, Li, Mubshaer, Reifer, & Ouwehan, 2009). Throughout the World the consumption of probiotic foods has greatly increased in recent years. In Europe this sector involves a total of 1.4 billion euros, led by yogurts and desserts, responding for approximately 72% of the total (Saxelin, 2008). In 2008 a 2.4% growth was registered in Brazil, involving 2.65 billion real (Rocha & Madureira, 2009). Although technological directives for the processing of ice creams and cheeses supplemented with probiotic cultures have been published recently (Cruz, Antunes, Pilleggi, Faria, & Saad, 2009; Cruz, Buriti, Souza, Faria, & Saad, 2009), yogurt is still the main vehicle of probiotic cultures preferred by the consumer * Corresponding author. Tel.: +55 19 35214019. E-mail address: [email protected]
(A.G. Cruz). 0963-9969/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.04.028
(Hailu, Boecker, Henson, & Cranﬁeld, 2009; Siegrist, Stampﬂi, & Kastenholz, 2008). This has resulted in the development of numerous products (Almeida et al., 2008; Antunes, Cazetto, & Bolini, 2005; Arayana, Plauche, & Nia, 2007; Dave & Shah, 1997; Ramasubramanian, Restuccia, & Deeth, 2008) and also in the need to evaluate the interaction and stability of the different probiotic strains (Korbekandi, Jahdi, Maracy, Abedi, & Jalali, 2009; Sacarro, Tamine, Pilleggi, & Oliveira, 2009) and of different operational parameters (Mortazavian et al., 2006, 2007; Sodini, Lucas, Oliveira, Remeuf, & Corrieu, 2002) during their processing. However, independent of the different strategies related during the development and/or processing of these products, it is crucial the viable counts of probiotic bacteria are not decreased along 6 log10 CFU/g, so that we have sufﬁcient numbers of this microbial group able to exert the desired therapeutic effects (Shah, 2000). Survival analysis is an area of statistics extensively used in clinical, epidemiological, biological and product conformity studies. In survival studies, people are accompanied by way of the occurrence of events that can be the diagnosis of a disease, the carrying out of a surgical operation or a birth (Bustamante-Teixeira, Faerstein, & Latorre, 2002). Details about this methodology is published elsewhere (Hough, Langohr, Goméz, & Curia, 2003), as well as its application to estimate the shelf-life of different processed food products (Araneda, Hough, & Pena, 2008; Ares, Giménez, & Gámbaro, 2008; Gambarro, Ares, & Gimenez, 2006; Varela, Salvador, & Fiszman, 2005). This study aimed to use survival analysis methodology to determine the shelf-life of yogurt supplemented with probiotic bacteria.
A.G. Cruz et al. / Food Research International 43 (2010) 1444–1448
2. Materials and methods 2.1. Sample preparation A highly consumed, strawberry-ﬂavored whole-milk probiotic yogurt was used in this study, whose label indicated the presence of the strain Biﬁdobacteirum animalis DN 173010 without specifying the yogurt bacteria. Samples from a single batch of probiotic yogurt were acquired from a supermarket in Campinas, Brazil, in the beginning of their shelf-life (1 day after its processing date), and maintained at 10 °C during 0, 14, 28, 42, 56, 70, and 84 days. The choice of the storage conditions was similar to the ones used by Curia, AGuerrido, Langhor, and Hough (2005) to calculate the shelf-life of conventional yogurts using survival analysis methodology. 2.2. Chemical analyses The pH of the yogurts was determined with a pH meter Micronal B-375 (Micronal, São Paulo, Brazil) equipped with a penetration electrode model (Marshall, 1993). The proteolytic activity of the Yoghurt was determined using the OPA Method. Yogurt samples (2.5 mL) were added to 5 mL of 0.75% (w/v) trichloroacetic acid and the mixture was vacuum-ﬁltered using an Advantec # 231 ﬁlter paper (MFS Inc., Dublin, CA, USA). The permeate (150 mL) was added to 3 mL of OPA reagent and the absorbance of the solution was measured spectrophotometrically at 340 nm after 2 min at room temperature (20 °C). The proteolytic activity of the bacterial cultures was expressed as the absorbance of OPA derivatives at 340 nm (Donkor, Henriksson, Vasiljevic, & Shah, 2005). All the analysis was performed in triplicate. Concentrations of lactic and acetic acids were determined by high-performance liquid chromatography (Varian 9010 model, Varian, Inc. Scientiﬁc Instruments, Palo Alto, CA), equipped with an Aminex HPX-87 H, ion exclusion column (Bio-RAD Laboratories, Richmond, CA, USA), ion exchange micro-guard cartridges (BioRAD Labs.); maintained at 65 °C in a column thermostat, model SpH 99 (Spark Holland B.V., Netherlands) was used. Retention times for lactic and acetic acids were 12.2 and 14.4 min, respectively. Refractive index detector models RI 2000 (Chrom Tech Inc., Apple Valley, MN) at 35 °C, and Varian UV at wavelength of 220 nm were applied for sugar and acids detections, respectively. The eluent (mobile phase) was a ﬁltered and degassed solution of H2SO4 prepared with Ultrapure water obtained from Milli-Q Puriﬁcation System (Millipore Corporation, Billerica, MA), at a pH of 2.8, and ﬂow rate of 0.6 mL/min. The standards were solutions of lactic and acetic acids at concentration intervals from 0.125 to 40 kg/m3. The injection volume was held at 25 lL by an automatic sampler. The software Millenium was applied for peaks integration and quantiﬁcation of samples. The preparation of the samples is published elsewhere (Donkor, Nilmini, Stolic, Vasiljevic, & Shah, 2007).
technique was used and plates were incubated at 30 °C/3–5 days. For coliforms, most probable number technique was used. Brilliant green bile 2% and Lauryl sulfate broths (Oxoid, Basingstoke, UK) (37 °C/48 h) were used for enumeration of coliforms. For enumeration of thermotolerant coliforms, EC broth (Oxoid, Basingstoke, UK) was used (44.5–45.5 °C/24 h). All the microbiological analyses were performed in duplicate.
2.4. Consumer test Fifty consumers (Hough, Calle, Serrat, & Curia, 2007), all habitual yogurt consumers, were recruited at random by placing notices in various places throughout the campus of the State University of Campinas (UNICAMP). They were invited to take part in an acceptance test for seven yogurts submitted to different storage conditions using a 9 cm hybrid hedonic scale, where: 1 = disliked immensely and 9 = liked immensely, each sample containing 30 mL and presented in coded plastic cups, presented monadically in random order. The test was carried out under controlled conditions, mineral water and cream crackers being made available to the consumers (Meilgaard, Civille, & Carr, 1999). In addition, for each sample, the consumers were asked to reply to the following question in a dichotomous way (yes or no): ‘‘Would you normally consume this product?”. These answers (yes or no) were used to calculate the shelf-life of probiotic yogurt by survival analysis methodology.
2.5. Statistical analysis The analysis of variance (ANOVA) was used to evaluate sample acceptance and the microbiological and physical–chemical data with subsequent comparison by Tukey’s test (p < 0.05), using the software STATISTICA 6.0 for Windows. The survival analysis was carried out using the SAS SYSTEM program, the deﬁnition of the following parameters being important in this analysis: T: random variable, representing the yogurt storage time resulting in sample rejection by the consumer, F(T): rejection function; representing the probability of consumer rejection of the yogurt below time t, that is, F(t) = P(T 6 t), S(T): survival function; probability of a consumer accepting the yogurt after time t, that is, S(t) = P(T > t). It is known that F(t) = 1 – S(t). Since the data were discreet, the random variable T was never observed exactly, due to the presence of censure. Discreet statistical distributions (Weibull, Lognormal, Gama, exponential) were ﬁtted to the data obtained in the consumer test and the best ﬁt (obtained by a visual inspection of the curves) used to express F(T) (Fig. 1). For example, if a Weibull distribution is chosen for T, the rejection function is given by:
2.3. Microbiological analysis Enumeration of B. animalis DN 173010 was carried out through pour plate technique in all the samples after the exposition to the different storage times. Control samples were also analyzed using the same method. It was used Columbia agar base (CAB) medium (Becton Dickinson, Cockeysville, MD), being the plates incubated at 37 °C/72 h under anaerobic conditions (Lamoureux, Roy, & Gauthier, 2002). Besides, coliforms, yeast and moulds counts were also carried out to check the hygienic-sanitary status of the samples (Marshal, 1993). For yeasts and moulds counts potato dextrose agar (PDA) plus tartaric acid was used. Pour plate
FðtÞ ¼ 1—Ssev
where Ssev () is the survival function of the smallest extreme value distribution Ssev (w) = exp(ew), and l and r are the model’s parameters. The shelf-life of the probiotic yogurt was obtained by substituting the parameters found in the previous ﬁt and subsequent consideration of the following values: 25% and 50% consumer rejection (Gambarro et al., 2006; Gimenez, Ares, & Gambarro, 2007; Hough et al., 2003).
A.G. Cruz et al. / Food Research International 43 (2010) 1444–1448
Fig. 1. Overall acceptability score of probiotic yogurt with storage time.
3. Results and discussion 3.1. Quality parameters of the probiotic yogurt All the samples were submitted to previous microbiological analysis (coliforms, yeasts and molds) which showed that the samples were ﬁt for human consumption. Additionally, enumeration of biﬁdobacteria was carried out in all the samples after the exposition to the different storage times being reported values ranged from 7 to 8 log10 CFU/mL (p > 0.05), respectively, indicating the maintenance of the recommended level for exerting potential beneﬁts to the consumer. As expected, the quality parameters of the yogurts were dependent on the exposure time of the samples dependent on the exposure time in which they were subjected to a refrigeration temperature. The pH value of samples ranged from 4.58 for control sample (0 days/10 °C) to 4.12 for the sample that remained for 84 days at 10 °C (p < 0.05) suggesting the occurrence of post-acidiﬁcation, which is a relevant parameter that inﬂuences the viability of the probiotic bacteria in fermented dairy products, like yogurt. ‘Post-production acidiﬁcation’ is deﬁned as a decrease in pH values after fermentation and during storage under refrigerated temperatures, mainly due to the uncontrolled growth of Lactobacillus delbrueckii ssp. bulgaricus under these conditions (Lourens-Hattingh & Viljoen, 2001). Additionally, the results reinforce which the tolerance of Biﬁdobacterium spp. towards pH values is strain-dependent. Indeed, recent research reported B. animalis ssp. lactis showed signiﬁcantly better (p < 0.0001) survival than B. longum in stressed environmental conditions as acidity environments (Jayamanne & Adams, 2009). The results of proteolysis data and organic acid concentrations indicate an direct relationship with the environmental storage conditions of the samples, suggesting an increased metabolism of the microorganisms presents even in refrigerated conditions. In fact, the proteolysis activity ranged from 0.58 for the control sample (10 °C/0 days) to 0.96 for the sample that remained at 10 °C for 84 days (p < 0.05). Towards the organic acid concentrations, the values ranged from 3.6 and 2.1 (control sample) to 12.3 and 10.6 g/L (10 °C/84 days sample) lactic and acetic concentrations, respectively (p < 0.05).
From the operational point of view, application of survival analysis methodology has the advantage that the work to be carried out is really quite simple. Fifty to a hundred individuals are required to express their acceptance or rejection of samples with different storage times or different formulations, sufﬁcient to estimate the shelf-life or deﬁnitive formulation. Another advantage is that these measures are carried out directly on the data obtained in the affective tests, who were the real consumers of these products (Hough, 2006). In survival analysis statistics, there are no statistical tests to compare the goodness-of-ﬁt of the different parametric models used for interval-censored data, and a visual assessment of how the parametric models ﬁt the nonparametric estimation is the common practice used to choose the most adequate model. The distributions gama, Weibull, log-normal and exponential, were ﬁtted to the probiotic yogurt acceptability data. The Weibull distribution showed the most adequate to the data. In fact, it has been using to preview the shelf-life of dairy foods such as cottage cheese (Schmidt & Bouma, 1992), concentrated yogurt (Al-Kadamany et al., 2002), ricotta cheese (Hough, Puglieso, Sanchez, & Silva, 1999) and yayik butter (Arslan, Sert, Ayar, & Ozcan, 2009). Fig. 1 shows the evolution of the overall acceptability scores for the probiotic yogurts with storage time. As expected, a continuous decrease of the acceptability scores was observed with increasing storage time of the yogurts. This ﬁnding shows that the consumers showed sensitive to changes in the sensory characteristics of the product caused by prolonging the storage time to which they were submitted. The samples submitted to during 70 and 84 days at 10 °C showed presented 5.65 and 4.95 for overall acceptability, ranged between the options ‘‘I disliked slightly” and ‘‘I neither liked nor disliked” and ‘‘I neither liked nor disliked” and ‘‘I liked slightly”, respectively, on the affective hedonic scale used in this study. Tables 1 and 2 show the maximum likelihood estimates for the parameters of these models and their respective standard errors, and the estimated shelf-lives for the probiotic yogurts. Fig. 2 shows the estimate for the cumulative distribution function F(t) = 1 – S(t), using these parameters and showing% rejection over time. Considering 25% and 50% probability of rejection of the product, the shelflife was estimated at 38 and 53 days, respectively. The differences found in this study suggest sensitivity of the consumers to the increasing acid taste of the products, which plays the main sensorial change noted in probiotic yogurts, in the result of increased microbial metabolism. Indeed, it have been reported that the sensorial performance of probiotic dairy foods are sensible to storage temperature along their shelf-life, even that the functionality of the probiotic strain remained (Vinderola, Prosello, Molinari, Ghiberto, & Reinheimer, 2009). It is also important to mention the contribution of probiotic bacteria in proteolysis, which sometimes inﬂuence in a relevant way the characteristics of the products, either in a positive or negative approach (Milesi,
Table 1 l and r values of the Weibull distribution. Distribution
l ± standard error
r ± standard error
4.12 ± 0.12
0.39 ± 0.03
3.2. Survival analysis methodology The shelf-life of food products is not exclusively related to deterioration, but to a complex phenomenon dependent on the interaction of the consumer with the actual food (Hough et al., 2003). In this context, the key concept of survival analysis methodology to focus the shelf-life hazard on the consumer rejecting the product rather than on the product deterioration.
Table 2 Estimated shelf-life times for probiotic ﬂavored yogurt. Rejection (%)
Shelf-life time (days)
A.G. Cruz et al. / Food Research International 43 (2010) 1444–1448
Fig. 2. Probability of rejecting the product compared with time.
Vinderola, Sabbag, Meinardi, & Hynes, 2009; Yadav, Jain, & Sinha, 2007). These ﬁnding could result in lower notes at the sensorial attributes by the consumers, who reject the product and therefore, do not purchase it. It is crucial that labels of probiotic dairy foods contain clear information about the conservation conditions of the product, as the temperature storage. Different results by using survival analysis methodology to estimate the shelf-life of whole strawberry conventional yogurts in Argentina and Spain, using the same storage conditions of this study (Curia et al., 2005; Salvador, Fiszman, CURIA, & Hough, 2005). Values of 41 and 60 days for the former country and 68 and 103 days for the second one, considering 25% and 50% rejection, respectively, were observed. However, it is worth to note, that, even the presence of intrinsic difference among the people of three countries, like Brazil, Argentine and Spain, the results obtained by using the survival analysis methodology are understanding. The additional presence of probiotic bacteria in the yogurt formulation can contribute to decrease the shelf-life of the product, due to higher production of organic acid levels, which are more perceived by the consumers. Consequently, there are an increased rejection of the product. A sour taste is the aspect of ﬂavor most commonly associated with acids and its perception is a complex event from both chemical and physiological standpoints. The recent hypothesis that its intensity is directly related to the total molar concentration of all the organic acid species present that have one or more protonated carboxyl groups, plus the concentration of free hydrogen ions, may provide a basis for predicting the sour taste in food formulations (Neta, Johanningsmeier, & McFeeters, 2007). Yogurt is a fermented milk product that has traditionally been prepared by allowing milk to sour at 40–45 °C. During its manufacture, changes in the milk constituents are attributed to fermentation and to ingredients added during the manufacturing process. Additionally, the heat treatment induces relevant changes which affect the physical properties of the product such as the whey protein denaturation and also creates conditions for a better development of starter cultures (Tamine & Robinson, 2007). Supplementation with probiotic microorganisms such as B. animalis potentially increases the amount of acid compounds in the yogurt since bacteria of the genus Biﬁdobacterium have an optimum growth temperature range between 37 and 41 °C. In addition they produce acetic and lactic acids in a ratio of 3:2 during the fermentation step, in an ideal synthetic medium. Therefore excessive growth may yield products with a vinegar-like taste and aroma (Gomes & Malcata, 1999). It has been reported that the supplementation of probiotic yogurts with bacteria of the genus Biﬁdobacterium can result in
a greater amount of organic acids during storage (Adhikari, Mustapha, Grün, & Fernando, 2000; Baron, Roy, & Vuillemard, 2000), as well as it can also lead to emergence of vinegar-like off ﬂavor under inappropriate fermentation and/or storage conditions. However, the acid production depends on the strain employed (Samona, Robinson, & Marakis, 1996). Thus their presence could contribute, potentially, to rejection of the product, consequently leading to a shorter shelf-life. As it mentioned earlier, all the probiotic yogurts used in this study, independent of the conditions which they were submitted, presented 7–8 log10 CFU/mL which suggest a possible inﬂuence on the overall sensorial performance of the product. It is also interesting to note that not even the addition of fruit pulp, which constitutes a commonly used technological option to hide the acid nature of yogurts processed in industrial plants where the fermentative and cooling processes are not adequately controlled, exerted any effect on product acceptability. As reported earlier, even yogurts submitted to shorter times at a temperature of 45 °C were rejected by the consumers. 4. Conclusions Survival analysis was shown to be an adequate methodology to determine the shelf-life of probiotic yogurt. The consumers showed sensitive to changes towards sensory characteristics introduced into the products which can relate to the metabolism of the probiotic bacteria present in the yogurt formulation as expressed to pH values and organic acid concentrations. Considering 25% and 50% probability of consumer rejection, the shelf-life of the probiotic yogurt was estimated at 38 and 53 days, respectively. The ﬁndings of this research highlighted the feasibility of using survival analysis to determine the shelf-life of foods, in particular, functional foods, as probiotic yogurts. References Adhikari, K., Mustapha, A., Grün, I. U., & Fernando, L. (2000). Viability of microencapsulated biﬁdobacteria in set yogurt during refrigerated storage. Journal of Dairy Science, 83(9), 1946–1951. Agrawal, R. (2009). Probiotics: An emerging food supplement with health beneﬁts. Food Biotechnology, 19(3), 227–246. Al-Kadamany, I., Toufeili, M., Khattar, Y., Abou-Jawdeh, S., Harakeh & Haddad, T. (2002). Determination of shelf-life of concentrated yogurt (Labneh) produced by in-bag straining of set yogurt using hazard analysis. Journal of Dairy Science, 85(5), 1023–1030. Almeida, M. H. B., Zoellner, S. S., Cruz, A. G., Moura, M. R. L., Carvalho, L. M. J., & Sant’Ana, A. S. (2008). Potentially probiotic açaí yogurt. International Journal of Dairy Technology, 61(2), 178–182. Antunes, A. E. C., Cazetto, T. F., & Bolini, H. M. A. B. (2005). Viability of probiotic microorganism during storage, post-acidiﬁcation and sensory analysis of fatfree yogurts with added whey protein concentrate. International Journal of Dairy Technology, 58(3), 169–173. Araneda, M., Hough, G., & Pena, E. W. (2008). Current status of survival analysis methodology to estimating sensory shelf-life of ready to eat lettuce (Lactuca Sativa). Journal of Sensory Studies, 23(2), 162–170. Arayana, K. J., Plauche, S., & Nia, T. (2007). Prebiotic and probiotic fat free yogurt. Milchwissenschaft, 62(3), 295–298. Ares, G., Giménez, A., & Gámbaro, A. (2008). Sensory shelf life estimation of minimally processed lettuce considering two stages of consumers’ decisionmaking process. Appetite, 50(2–3), 529–535. Arslan, D., Sert, D., Ayar, A., & Ozcan, M. H. (2009). Shelf life determination of Yayik butter fortiﬁed with spice extracts. International Journal of Dairy Technology, 62(2), 189–194. Baron, M., Roy, D., & Vuillemard, J.-C. (2000). Biochemical characteristics of fermented milk produced by mixed-cultures of lactic starters and biﬁdobacteria. Dairy Science and Technology, 80(5), 465–478. Bustamante-Teixeira, M. T., Faerstein, E., & Latorre, M. R. (2002). Técnicas de análise de sobrevida. Cadernos de Saúde Pública, 18(3), 579–594. Cruz, A. G., Antunes, A. E. C., Pilleggi, A. L. O. P. S., Faria, J. A. F., & Saad, S. M. I. (2009). Ice cream as probiotic food carrier. Food Research International, 42(9), 1233–1239. Cruz, A. G., Buriti, F. C. A., Souza, C. H. B., Faria, J. A. F., & Saad, S. M. I. (2009). Probiotic cheese: Health beneﬁts, technological and stability aspects. Trends in Food Science and Technology, 20(8), 344–354.
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