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Hurdle technology for shelf stable minimally processed French beans (Phaseolus vulgaris): A response surface methodology approach
LWT - Food Science and Technology 48 (2012) 182e189
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Hurdle technology for shelf stable minimally processed French beans (Phaseolus vulgaris): A response surface methodology approach Sumit Gupta*, Suchandra Chatterjee, Jasraj Vaishnav, Vivekanand Kumar, Prasad S. Variyar, Arun Sharma Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400094, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2011 Received in revised form 23 February 2012 Accepted 5 March 2012
Citric acid treatment in combination with gamma radiation and modified atmosphere packaging was employed as hurdles for control of microorganisms and extending shelf life of minimally processed French beans. Response surface methodology was used to optimize citric acid treatment and g-irradiation dose to obtain product with desired microbial and sensory quality. Optimum processing conditions (citric acid 8.4 g L1; irradiation dose 0.7 kGy; 10 C) resulted in modified atmosphere of 18% O2 and 4% CO2 at end of storage period. Under these conditions shelf life of the product could be extended by one week with acceptable sensory and nutritional quality as evaluated by total antioxidant, phenolics, flavonoids and vitamin C content. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Minimally processed French beans Hurdle technology Citric acid treatment g-Irradiation Modified atmosphere packaging Response surface methodology
1. Introduction The consumption of minimally processed food products including ready-to-eat or readyeto-use has increased worldwide in last decade due to its convenience, freshness and improved quality (Baskaran et al., 2007). The production of fresh cut vegetables usually involve processing such as cleaning, trimming, slicing, washing, drying and packaging (Yildiz, 1994), that causes rapid deterioration by increasing respiration rate, transpiration, enzymatic activity and microbial proliferation (Nguyen-the & Carlin, 1994). Several preservation methods including antioxidant treatment, modified atmosphere packaging (MAP), refrigeration and chlorine wash are currently employed for commercial preparation of fresh cut vegetables (Ahn et al., 2005). Psychrotrophic bacteria, such as Listeria monocytogenes are known to grow at low temperature even under modified atmospheres (Beuchat & Brackett, 1990). Disinfectants used in commercial processing lines, such as chlorine have limited effect (approx. 1 log reduction) on microbial population and can not be relied for eliminating pathogenic microorganisms like L. monocytogenes (Nguyen-the & Carlin, 1994). Thus there is an increasing concern on microbial safety of such produce. Hurdle technology involves the use of several preservation techniques in combination. This reduces the intensive use of one * Corresponding author. Tel.: þ91 22 25590560. E-mail address: [email protected] (S. Gupta). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2012.03.010
preservation technique producing a lower impact on the sensory quality (Ahn et al., 2005). Citric acid treatment, gamma irradiation, low temperature storage and modified atmosphere packaging were employed as hurdles in the present study to develop minimally processed French beans, a widely used vegetable. Citric acid can act synergistically with ionizing radiation in reducing microbial load (Bhide, Paturkar, Sherikar, & Waskar, 2001). French bean is prone to extensive microbial spoilage during storage even under refrigerated temperatures restricting its shelf life. Use of hurdle technology for preservation and extension of shelf life of this vegetable has not been investigated. Objective of the present investigation was to study the effects of irradiation, citric acid, low temperature storage and cling film packaging on sensory and microbial quality of minimally processed French beans. In order to optimize the levels of various hurdles multivariate statistical techniques such as response surface methodology (RSM) have been suggested. When many factors and interactions affect desired response, RSM is an effective tool for optimization of process (Baskaran et al., 2007). Various process parameters employed were optimized using RSM. 2. Materials and methods 2.1. Plant material French beans (Phaseolus vulgaris) grown in outskirts of Mumbai (India) were purchased from a local grower. The vegetable was brought
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to the laboratory within 12 h after harvesting and immediately stored at refrigerated temperatures (10 C) before processing. Beans, washed in running tap water to remove all adhered dust and unwanted plant debris were dried between folds of blotting sheets and subjected to citric acid treatment by dipping in aqueous solutions of citric acid (concentrations chosen according to experimental design) for 5 min. Beans were trimmed from ends, weighed accurately (110 g), and stored in polystyrene trays (inner dimensions: 22 cm 15.7 cm 3 cm) wrapped completely with cling film (Flexo film wraps Ltd., Aurangabad, Maharashtra, India). Film used in the present study had thickness of 8e10 mm and permeability to oxygen and carbon dioxide of 15,000 and 90,000 cm3 m2 day1 atm1 (data as provided by supplier). 2.2. Irradiation and storage Packaged beans were subjected to various irradiation doses up to 2.5 kGy in a cobalt-60 irradiator (GC-5000, BRIT, Vashi, India) having activity of 8.6 kCi and dose rate of 6.5 kGy h1. Irradiator was calibrated using a Fricke dosimeter before the start of experiment and dose uniformity ratio (Dmax/Dmin) was found to be 1.2. The optimum storage temperature for French beans has earlier been reported to be 10 C (Proulx, Yagiz, Cecilia, Nunes, & Emond, 2010). Hence irradiated samples were stored at this temperature. Radiation dose and storage time was chosen according to experimental design. 2.3. Experimental design The Design of experiments and subsequent analysis of results were performed by Design expert 8.0 software (State-ease Inc., U.S.A.). Central composite rotatable design (CCRD) was employed in the present study (Abreu, Beirão-da-Costa, Gonçalves, Beirão-daCosta, & Moldão-Martins, 2003). The variables optimized were citric acid concentration (X1), irradiation dose (X2) and storage time (X3). The independent variables were coded at five levels (a, 1, 0, þ1 and þa). Coded and actual values are shown in Table 1. Complete design consisted of 20 experimental points including six replications of the center points (all variables coded as zero) (Table 2).
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Table 2 Experimental design as central composite rotatable design. Factors Std
Run
X1 (Citric acid) g L1
X2 (Radiation dose) kGy
X3 (Storage time) Days
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1 14 15 3 7 12 20 2 8 6 18 19 17 13 16 11 5 4 9 10
1 1 1 1 1 1 1 1 1.68 1.68 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 1.68 1.68 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 0 0 1.68 1.68 0 0 0 0 0 0
(4) (16) (4) (16) (4) (16) (4) (16) (0.0) (20) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10)
(0.51) (0.51) (1.99) (1.99) (0.51) (0.51) (1.99) (1.99) (1.25) (1.25) (0.00) (2.50) (1.25) (1.25) (1.25) (1.25) (1.25) (1.25) (1.25) (1.25)
(4.00) (4.00) (4.00) (4.00) (16.00) (16.00) (16.00) (16.00) (10.00) (10.00) (10.00) (10.00) (0.00) (20.00) (10.00) (10.00) (10.00) (10.00) (10.00) (10.00)
Values for various factors are shown in their coded form while actual values are given in bracket.
‘b’ values. Instrument was calibrated using a white tile supplied along with the equipment. Firmness was measured using a texture analyzer (TA.XTPLUS, Stable Microsystems Ltd., Surrey, England) with a 100 kg load cell, equipped with a 5 mm diameter flat head cylindrical probe. Maximum force required to compress sample to a target distance of 4 mm was recorded. Test was designed with a trigger force of 15 gm and test speed of 0.5 mm s1. The peak force required to compress samples was referred as a measure of firmness of sample. The data was collected for six replicates and results were expressed as their average. 2.6. Sensory analysis
2.4. Microbial analysis Standard methods were used to enumerate total microbial load present in French beans at each sampling time and treatment conditions according to experimental design (López-Rubira, Conesa, Allende, & Artés, 2005). Enumeration of total aerobic mesophilic bacteria was done by pour plate method on plate count agar (Himedia, Mumbai, India). Incubation was done at 37 C for 24 h. Total yeast and mold count was performed by pour plate method on potato dextrose agar (Himedia, Mumbai, India) supplemented with 0.01 g L1 tartaric acid to lower pH of media to 3.5. Plates were incubated at 37 C for 48 h. Microbial counts were expressed as log10 CFU g1. Each analysis was performed in triplicate. 2.5. Determination of color and texture Surface color values were evaluated using a colorimeter (CM3600d Konica Minolta sensing Inc., Japan) by measuring ‘L’, ‘a’ and Table 1 Original and coded levels of independent variables. Variable
Original
Citric acid Irradiation dose Storage time
X1 (g L1) X2 (kGy) X3 (Days)
Sample for sensory analysis was prepared by cooking French beans in boiling water for 5 min. Beans were then cut into 2 cm long pieces and presented to assessors in white trays. Hedonic testing was carried out by 15 members of experienced sensory panel using a 9 point scale with 1, dislike extremely or not characteristic of product and 9, like extremely or very characteristic of product (López-Rubira et al., 2005). Parameters evaluated were color, aroma, texture, taste, after taste and overall acceptability. The sensory evaluation was done on the same days as microbial and other quality evaluation parameters. For optimization of treatments (irradiation dose and citric acid) hedonic sensory scores were used. For experiment carried out in optimized conditions along with hedonic analysis, sensory evaluation was done by quantitative descriptive analysis (QDA) (Murray, Delahunty, & Baxter, 2001). A trained panel consisting of 12 members, 7 males and 5 females assessed the samples using unstructured 150 mm scale. The 15 sensory attributes assessed were color (green), aroma (French beans like, cooked vegetables, grassy, irradiated and musty), texture (crispy, fibrous and soggy) and taste (sweet, sour, bitter, French beans like, rotten and irradiated). Assessments were repeated twice and sensory data was collected by measuring distance (mm) from origin.
Coded a (1.68)
1
0
þ1
þa (þ1.68)
0.00 0.00 0.00
4.1 0.51 4.00
10 1.25 10.00
16 1.99 16.00
20 2.50 20.00
2.7. Nutritional evaluation 10 g of French beans was extracted twice with 40 ml distilled methanol in an omnimixer (Sorvall, U.S.A). Extract was centrifuged
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at 4000 rpm for 30 min and the supernatant thus collected was made to a final volume of 50 ml. This solution was used for subsequent assays. Total antioxidant activity was evaluated by DPPH radical scavenging assay (Wen, Prasad, Yang, & Ismail, 2010). Total phenolic content was evaluated in accordance with FolineCiocalteu procedure (Han et al., 2011). Total antioxidant activity and total phenolic content was expressed as mg gallic acid equivalent g1 French bean. The AlCl3 method (Luximon-Ramma, Bahorun, Soobrattee, & Aruoma, 2002) was used for determination of total flavonoid content. Flavonoid content was expressed as mg quercetin equivalent g1 of French bean. Total vitamin C content of French beans was estimated in accordance with standard AOAC official microfluorometric method (AOAC, 1990). All analysis was carried with three independent samples each analyzed in triplicate.
Table 3 Significance statistics, p values and signal to noise ratio (S/N) of RSM predicted models.
2.8. Headspace gas composition
According to European standards for minimally processed produce, total microbial count should not increase (>107 cfu g1) during their entire intended storage period (Oms-Oliu, AguiloAguayo, Martin-Belloso, & Soliva-Fortuny, 2010). Thus, the present study aimed at developing minimally processed French beans product having microbial load less than 107 cfu g1 and with excellent sensory qualities during the entire shelf life. Statistical analysis by ANOVA showed that the models generated by RSM were significant (p < 0.05) while lack of fit was insignificant (p > 0.05). Further, signal to noise ratio (s/n) for all models were above 4 indicating sufficient data to navigate designed space (Table 3). Coefficients for predicted regression models and coefficient of determination (R2) are shown in Table 4. High values for R2 also suggest that the models are good fit.
Gaseous composition in package headspace was measured using gas chromatograph (GC-2010 plus, Shimadzu Corporation, Japan) equipped with split/splitless injector, molecular sieve column (30 m length, 0.35 I.D., RT-Msieve 5A, Restek Corporation, U.S.A.) and a TCD detector. Temperature of injection port was kept at 35 C. Initial column temperature was kept at 30 C for 5 min, then raised at the rate of 10 C min1 to 100 C and held at 100 C for 5 min. Sampling was made by piercing a hypodermic needle into package through an adhesive septum, previously stuck to the package. 0.1 ml of sample headspace was taken and injected under a split ratio of 5. From the column used in present study concentrations of only O2 and N2 can be evaluated. Based on observed pO2 and pN2 in package headspace actual concentrations of O2 and CO2 (pO2 and pCO2) were calculated using following equations:
pO2 ¼ ðObserved pO2 =Observed pN2 Þ 78:084
(1)
pCO2 ¼ 100 ½ðObserved pO2 =Observed pN2 Þ 78:084 þ 78:084
(2)
Analysis was done in triplicates and concentrations of gases are expressed as their partial pressures. 2.9. Statistical analysis Model and statistical analysis was performed by Design expert software (Version 8.0, State-ease, USA). Models for individual responses were generated by fitting experimental data into third order polynomial and then removing insignificant terms by backward regression method. Model terms with highest values of partial probability (p-value) are removed first and process is stopped when p-value of next term out satisfies the specified alpha out criterion. Value of alpha out was kept 0.1 which leads to an overall model with terms significant at 0.05 levels. Models were analyzed by analysis of variance (ANOVA) and the predictive capability of the model was quantified by coefficient of determination (R2). Fitted polynomial equations were then used to plot response surface plots in order to visualize the interrelationship of response and experimental levels of each factor. Optimum conditions were then derived using numerical optimization process. Criteria for desired microbial and sensory qualities were given and model equations solved to obtain optimum processing conditions (Table 5). Data for quality evaluation by total antioxidant, total phenolics, total flavonoids and vitamin C was analyzed by two-way ANOVA to determine effect of radiation processing and storage time. Multiple means comparison was done by Tukey’s test (p < 0.05).
Model
Residual
S/N
Response
Sum of squares
df
p-value
Sum of squares
df
p-value
Total plate count Yeast and mold count Texture Color Aroma Taste
26.62
13
0.0047
0.81
5
0.31
16.33
6.26
11
0.0345
1.31
8
0.38
6.4
76.22 348.93 2.90 4.11
11 8 12 11
0.0006 0.024 0.0036 0.0025
4.87 95.50 0.14 0.37
8 9 6 8
0.51 0.73 0.82 0.85
14.71 7.4 9.039 10.99
3. Results and discussions
3.1. Microbial analysis A significant (p < 0.05) increase in microbial load during storage was observed (Fig. 1A). Effect of irradiation and citric acid treatment on microbial load is depicted in Fig.1B. At zero day mesophilic counts reduced with both increasing radiation dose and citric acid treatment (Fig. 1B). A 5 log cycle reduction of mesophilic counts can be observed at a dose of 2 kGy and a 2 log cycle reduction can be observed with citric acid treatment of 16 g L1. Similar results were reported earlier for irradiation of carrots (Chervin & Boisseau, 1994), lettuce (Prakash, Guner, Caporaso, & Foley, 2000), diced celery Table 4 Coefficients of the fitted polynomial representing the relationship between the response and the process variable and R2 values. Yeast and Color mold count ‘a’ (log value cfu g1)
Texture Taste Aroma (sqrt (g)) (Hedonic) (Hedonic)
4.49a 0.11a 0.99a 0.87a 0.15a 0.27a 0.098 0.28a 0.83a 0.58a 0.65a 1.27a 0.52a 0.88a
2.44a 0.43a 0.036a 0.37a 0.13 0.17a 0.32a 0.029a 0.066a 0.14a 0.40a 0.056 0.23 0.58a
8.38a 1.76a 1.65a 3.13a 1.25a 1.76a 1.12 1.98a 3.41a 1.38 2.05 3.47a 3.40 1.00
40.49a 1.91a 1.22a 1.08a 0.76a 0.46a 0.73a 1.09a 0.12 0.42 0.15 0.82a 0.053 1.38a
6.38a 0.18a 0.45a 0.37a 0.017a 0.18a 0.18a 0.12a 0.20a 0.092 0.013 0.33a 0.044 0.34a
0.97
0.82
0.78
0.93
0.91
Coefficients Total plate count (log cfu g1) X0 X1 X2 X3 X11 X22 X33 X12 X13 X23 X123 X112 X113 X122 R2 a
Significant terms at p < 0.05.
6.88a 0.02a 0.093a 0.18a 0.045a 0.23a 0.39a 0.049a 0.023a 0.26a 0.099a 9.36*103 0.32a 0.13 0.95
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Fig. 1. Response surface curves for microbial counts. A) Variation of total plate counts with radiation dose and storage time at 10 g L1 citric acid B) Variation of total plate counts with radiation dose and citric acid at storage time (0 days) C) Variation of yeasts and molds count with storage time and citric acid at dose of 1.25 kGy D) Variation of yeasts and molds with storage time and radiation dose at 16 g L1 citric acid.
(Prakash, Inthajak, Huibregtse, Caporaso, & Foley, 2000) and other vegetables (Farkas, Seray, Mohacsi-Farkas, Horti, & Andrassy, 1997). It was observed that yeast and mold counts increased significantly (p < 0.05) with storage time. During initial days of storage yeast and mold counts declined with both increasing radiation dose and citric acid treatment (Fig. 1C and D). Surprisingly, with increasing storage time more yeast and mold counts were observed at higher radiation dose and corresponding greater citric acid treatment (Fig. 1D). Low mesophilic bacterial counts leading to diminished competitive flora for yeasts and molds could explain this observation. Further, higher citric acid led to decrease in pH thus creating conditions more favorable for yeasts and molds. 3.2. Texture and color It was observed that during initial storage period firmness of French beans decreased with increasing irradiation dose (Fig. 2A). Radiation induced depolymerization of pectin and other cell wall components such as cellulose and hemicelluloses could lead to
decreased firmness and softening of plant tissues (Prakash, Guner, et al., 2000; Prakash, Inthajak, et al., 2000). At lower radiation doses firmness showed a positive correlation with citric acid treatment while at higher doses (2 kGy) it showed a negative correlation. Protective effect of citric acid on hydrolysis of pectic substances at lower doses of irradiation due to its antioxidant characteristics is suggested. However, higher citric acid concentrations could lead to softening of texture due to acid hydrolysis of pectic substances. Citric acid treatment (10 g L1) has been previously shown to be associated with softening in texture of apple slices (Cocci, Rocculi, Romani, & Rosa, 2006). Surprisingly, at lower citric acid treatments firmness was found to increase with increasing radiation dose (Fig. 2B) on sixteenth day of storage. Irradiation can lead to lower rate of respiration in plant produce which results in lesser loss of water and subsequently better firmness (Rico, Martín-Diana, Barat, & Barry-Ryan, 2007). All three factors analyzed i.e. citric acid, irradiation dose and storage time influenced ‘a’ values significantly (p < 0.05) while effect on ‘L’ and ‘b’ values was not significant (p > 0.05). ‘a’ values
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Fig. 2. Response surface curves for texture and color. A) Effect of radiation dose and citric acid treatment on firmness of French beans at storage time (0 days). B) Effect of radiation dose and citric acid treatment on firmness of French beans after storage time of 16 days. C) Effect of radiation dose and citric acid treatment on ‘a’ values of French beans after storage time of 10 days.
increased with increasing storage period and citric acid treatment indicating a reduction of greenness of samples. Reduction of greenness might be due to degradation of pigments during storage. Interestingly, ‘a’ values demonstrated decreasing trend with increasing radiation dose indicating better greenness of irradiated samples (Fig. 2C). Radiation processing can result in reduction in rate of respiration of plant produce leading to slower rate of degradation of pigments. No significant (p > 0.05) effect of processing conditions was observed on texture and color of French beans as analyzed by hedonic sensory scores. While the physical attributes of color and texture showed significant variance instrumentally, no effect on texture and color values could be observed by sensory panel. 3.3. Aroma and taste Irradiation and storage time significantly (p < 0.05) influenced aroma quality and taste of beans while citric acid treatment had no effect on these parameters. Aroma quality of French beans reduced
with increasing storage period. Samples irradiated at higher doses of radiation demonstrated slightly lesser scores for aroma quality as compared to control samples during initial storage period but demonstrated better aroma scores when analyzed in late storage period (Fig. 3A). Significantly higher aroma scores for irradiated carrots, cilantro, green onions, parsley and red lettuce after 14 days of storage were earlier reported (Fan & Sokorai, 2008). Better aroma scores observed in present study after irradiation and storage as compared to samples stored without irradiation could probably be due to reduced decay in the samples. 3.4. Optimization and verification of results Various processing factors were optimized for achieving maximum shelf life of minimal processed produce with microbial count of <107 cfu gm1 during entire intended storage period and to maximize various quality parameters like texture, aroma and taste. Criteria set for optimization of parameters and solutions obtained are given in Table 5. Solution A was obtained for criteria 1 and solution B
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Fig. 3. Response surface curves for sensory attributes of aroma and taste. A) Effect of radiation dose and storage time on aroma quality of French beans at 10 g L1 citric acid treatment. B) Effect of radiation dose and storage time on taste of French beans at 10 g L1 citric acid treatment.
was obtained for criteria 2. Both solutions were used for validation of models generated. Predicted and actual values of quality parameters for both solutions are shown in Table 5. Close agreement between actual and predicted values indicate suitability of model generated. Values for microbial counts obtained were slightly higher than predicted values. Since, for validation experiments French beans were harvested separately, the variation in microbial counts could be due to inherent variability in biological samples. Solution B demonstrated acceptable microbial load (<107 cfu gm1) with a shelf life of 14 days. Further, it had high values for sensory attributes like texture, aroma and taste. This solution was therefore taken up for further studies regarding nutritional evaluation of developed product, descriptive sensory analysis and gaseous headspace composition. 3.5. Nutritional evaluation As shown in Fig. 4A, antioxidant activity reduced significantly (p < 0.05) during storage but no affect due to irradiation treatment Table 5 Criteria for various factors and responses for process optimization and corresponding optimized solutions obtained. Criteria 1
Criteria 2
Solution A
Solution B
Minimize
Minimize
14
8.4
Minimize
Minimize
1.04
0.7
Maximize (>7 < 15) Should be <5 Minimize
Maximize (>12 15) Should be <6 Minimize
10.41
Maximize
Maximize
Color (a)
Minimize
Minimize
Aroma (Hedonic) Taste (Hedonic)
Maximize
Maximize
Maximize
Maximize
Citric acid (g L1) Irradiation dose (kGy) Storage time (Days) TPC (Log cfu g1) Y & M count (Log cfu g1) Texture (g)
a b
Predicted values for solutions. Actual values.
a
4.76 (5.28 0.29)b 2.17a (2.73 0.5)b 1543a (1463 86)b 8.4a (8.0 0.6)b 6.84a (6.66 0.27)b 6.34a (6.5 0.5)b
14 5.681a (6.01 0.31)b 2.42a (2.93 0.17)b 1646a (1615 112)b 8.8a (8.6 0.4)b 6.62a (6.8 0.35)b 6.2a (6.0 0.25)b
was observed. A 40% decrease in antioxidant activity was observed after 14 days of storage period. Maximum reduction of antioxidant activity was, however, observed during initial storage period of five days. Reduction in antioxidant activity might be due to reduction in content of total phenolics and flavonoids. Total phenolic content reduced significantly (p < 0.05) during storage (Fig. 4B). Slight decrease in phenolic content due to radiation processing was also observed. Contrary to antioxidant results no decrease in phenolics was observed up to a storage period of five days. A decrease of 20% was however, observed on storage up to fourteen days. Flavonoid content showed a similar trend as antioxidant capacity during storage. A significant (p < 0.05) decrease was observed up to a period of five days. No significant decrease was noted beyond this period (Fig. 4C). Fan, Toivonen, Rajkowaski, & Sokorai, 2003 reported that radiation induced phenolic synthesis in fresh-cut iceberg lettuce treated by warm water dipping. Radiation induced increased antioxidant and phenolic content has been previously demonstrated in other vegetables like cabbage (Ahn et al., 2005), carrot and kale juice (Arvanitoyannis, Stratakos, & Tsarouhas, 2009) and fresh cilantro leaves (Fan, Niemira, & Sokorai, 2003). During irradiation the free radicals generated may act as a stress signals and may trigger stress responses in vegetables, resulting in increased antioxidant synthesis (Fan, Toivonen, et al., 2003). Surprisingly, no increased phenolic content was observed due to processing and storage in present study. Observed reduction in total antioxidant and phenolic content could be attributed to the fact that during storage high O2 concentrations were present in packages resulting in oxidation of phenolic compounds. No statistical difference was observed among control and processed samples in total ascorbate content (p > 0.05). Vitamin C content remained similar during entire storage period (Fig. 5). In previous reports on vegetables decrease in ascorbic acid content during storage and irradiation was reported (Arvanitoyannis et al., 2009). Ascorbic acid undergoes oxidation during storage to dehydroascorbic acid (DHAA) which is also bioactive and in a bio available form but to a lesser extent than ascorbic acid (Ogiri et al., 2002). In present study entire vitamin C was oxidized to DHAA and quantified. This might explain the fact that no reduction in vitamin C content was observed.
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Fig. 5. Effect of radiation processing (0.7 kGy), citric acid treatment (8.4 g L1)) and storage on total ascorbate content in French beans. ( ) Control and ( ) processed samples.
attainment of equilibrium between package headspace and atmosphere. Processed samples demonstrated higher CO2 concentration as compared to control till eight days of storage, which could possibly be due to enhanced rate of respiration in plant produce immediately after radiation processing (Fan & Sokorai, 2008). O2 content less than 2% in package headspace can result in anaerobic conditions leading to generation of off-flavors and possible growth of anaerobic pathogens like Clostridium botulinum (Sanchez-Mata, Camara, & Dıez-Marques, 2003). Lowest O2 concentrations observed for the processed samples during entire storage period was 16.48 1.5% thus maintaining aerobic conditions within the package. Processing and packaging conditions chosen in present study are therefore suitable for MAP of French beans. 4. Conclusions Fig. 4. Effect of radiation processing (0.7 kGy), citric acid treatment (8.4 g L1)) and storage on nutritive quality of French beans. A) Total antioxidative capacity. B) Total phenolics content. C) Total flavonoid content. ( ) Control and ( ) processed samples.
3.6. Quantitative descriptive analysis
In summary, RSM approach was successfully employed to optimize process parameters for minimally processed French beans with desired microbial and sensory qualities. Analysis of response surfaces indicates a complex interaction among variables such as irradiation dose, citric acid and storage time. Irradiation and citric
Processed French bean after 14 days of storage was further evaluated by QDA and data obtained is graphically represented by spider diagram (Fig. 6). Untreated sample after storage was not evaluated as it demonstrated visible fungal growth. After 14 days of storage processed product had slightly lesser scores for aroma attributes like green, French beans and grassy. A possible loss of volatile aroma compounds during storage could be suggested. In terms of textural attributes the developed product had less fibrous nature while no difference in taste was observed compared to the control sample. No irradiated off flavor or taste was perceived by assessors (Fig. 6). QDA results established that processed French beans had desired aroma, taste and textural properties even after storage period of fourteen days. 3.7. Headspace composition in packages Significant (p < 0.05) effects of irradiation and storage time were observed for CO2 and O2 during storage (Fig. 7). In control samples an increase in CO2 was observed up to eight days of storage with composition remaining nearly constant thereafter, indicating
Fig. 6. Quantitative descriptive analysis of fresh unprocessed and processed (radiation dose (0.7 kGy) and citric acid treatment (8.4 g L1) after 14 days of storage) samples. ) Fresh unprocessed and ( ) processed sample after 14 day storage. (
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Fig. 7. Headspace gas composition in packages. A) Variation of O2 content during storage in control and processed (radiation processing (0.7 kGy) and citric acid treatment ) (8.4 g L1)) samples. B) Variation of CO2 content during storage in control and processed (radiation processing (0.7 kGy) and citric acid treatment (8.4 g L1)) samples. ( Control and ( ) processed sample.
acid significantly (p < 0.05) reduced microbial contamination. Citric acid was found to reduce radiation induced softening of French beans. Thus, use of citric acid treatments may be useful for maintaining texture of vegetable during radiation processing. Advantage of using RSM also stems from the fact that different quality criteria when employed for optimization resulted in different suitable solutions. This can be useful for commercial production as processing conditions can be suitably calculated based on quality characteristics required, thus reducing severity of treatments. Validation experiments proved that mathematical models generated were suitable for optimizing process parameters for desired product properties. Analysis of developed product by QDA demonstrates that processed French beans had desired aroma, taste and textural attributes. This study demonstrates usefulness of RSM for optimization of treatments for production of minimally processed vegetables.
References Abreu, M., Beirão-da-Costa, S., Gonçalves, E. M., Beirão-da-Costa, M. L., & MoldãoMartins, M. (2003). Use of mild heat pre-treatments for quality retention of freshcut ‘Rocha’ pear. Postharvest Biology and Technology, 30(2), 153e160. Ahn, H.-J., Kim, J.-H., Kim, J.-K., Kim, D.-H., Yook, H.-S., & Byun, M.-W. (2005). Combined effects of irradiation and modified atmosphere packaging on minimally processed Chinese cabbage (Brassica rapa L.). Food Chemistry, 89(4), 589e597. AOAC. (1990). Official methods of analysis (15th ed).. Arlington, Va: Association of Official Analytical Chemists. Arvanitoyannis, J. S., Stratakos, A. Ch., & Tsarouhas, P. (2009). Irradiation applications in vegetables and fruits: a review. CRC Critical Reviews in Food Science and Nutrition, 49(5), 427e462. Baskaran, R., Devi, A. U., Nayak, C. A., Kudachikar, V. B., Prakash, M. N. K., Prakash, M., et al. (2007). Effect of low-dose g-irradiation on the shelf life and quality characteristics of minimally processed potato cubes under modified atmosphere packaging. Radiation Physics and Chemistry, 76(6), 1042e1049. Beuchat, L. R., & Brackett, R. E. (1990). Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment modified atmosphere packaging and temperature. Journal of Food Science, 55(3), 755e758. Bhide, M. R., Paturkar, A. M., Sherikar, A. T., & Waskar, V. S. (2001). Presensitization of microorganisms by acid treatments to low dose gamma irradiation with special reference to Bacillus cereus. Meat Science, 58(3), 253e258. Chervin, C., & Boisseau, P. (1994). Quality maintenance of ready-to-eat shredded carrots by gamma irradiation. Journal of Food Science, 59(2), 359e362. Cocci, E., Rocculi, P., Romani, S., & Rosa, M. D. (2006). Changes in nutritional properties of minimally processed apples during storage. Postharvest Biology and Technology, 39(3), 265e271. Fan, X., Niemira, B. A., & Sokorai, K. J. B. (2003). Sensorial, nutritional and microbiological quality of fresh cilantro leaves as influenced by ionizing radiation and storage. Food Research International, 36(7), 713e719.
Fan, X., & Sokorai, K. G. B. (2008). Retention of quality and nutritional value of 13 fresh-cut vegetables treated with low dose radiation. Journal of Food Science, 73(7), S367eS372. Fan, X., Toivonen, P. M. A., Rajkowski, K. T., & Sokorai, K. J. B. (2003). Warm water treatment in combination with modified atmosphere packaging reduces undesirable effects of irradiation on quality of fresh-cut iceberg lettuce. Journal of Agricultural and Food Chemistry, 51(5), 1231e1236. Farkas, J., Saray, T., Mohacsi-Farkas, C., Horti, K., & Andrassy, E. (1997). Effect of low dose gamma radiation on shelf-life and microbiological safety of pre-cut/ prepared vegetables. Advances in Food Science, 19(3/4), 111e119. Han, J., Britten, M., St-Gelais, D., Champagne, C. P., Fustier, P., Salmeri, S., et al. (2011). Polyphenolic compounds as functional ingredients in cheese. Food Chemistry, 124(4), 1589e1594. López-Rubira, V., Conesa, A., Allende, A., & Artés, F. (2005). Shelf life and overall quality of minimally processed pomegranate arils modified atmosphere packaged and treated with UV-C. Postharvest Biology and Technology, 37(2), 174e185. Luximon-Ramma, A., Bahorun, T., Soobrattee, M. A., & Aruoma, O. I. (2002). Antioxidant activities of phenolic, proanthocyanidin, and flavonoid components in extracts of Cassia fistula. Journal of Agricultural Food Chemistry, 50(18), 5042e5047. Murray, J. M., Delahunty, C. M., & Baxter, I. A. (2001). Descriptive sensory analysis: past, present and future. Food Research International, 34(6), 461e471. Nguyen-the, C., & Carlin, F. (1994). The microbiology of minimally processed fresh fruits and vegetables. CRC Critical Reviews in Food Science and Nutrition., 34(4), 371e401. Ogiri, Y., Sun, F., Hayami, S., Fujimura, A., Yamamoto, K., Yaita, M., et al. (2002). Very low vitamin C activity of orally administered L-dehydroascorbic acid. Journal of Agricultural and Food Chemistry, 50(1), 227e229. Oms-Oliu, G., Aguilo-Aguayo, I., Martin-Belloso, O., & Soliva-Fortuny, R. (2010). Effects of pulsed light treatments on quality and antioxidant properties of fresh cut mushrooms (Agaricus bisporus). Postharvest Biology and Technology, 56(3), 216e222. Prakash, A., Guner, A. R., Caporaso, F., & Foley, D. M. (2000). Effects of low-dose gamma irradiation on the shelf life and quality characteristics of cut Romaine lettuce packaged under modified atmosphere. Journal of Food Science, 65(3), 549e553. Prakash, A., Inthajak, P., Huibregtse, H., Caporaso, E., & Foley, D. M. (2000). Effects of low dose gamma irradiation and conventional treatments on shelf life and quality characteristics of diced celery. Journal of Food Science, 65(6), 1070e1075. Proulx, E., Yagiz, Y., Cecilia, M., Nunes, N., & Emond, J. (2010). Quality attributes limiting snap bean (phaseolus vulgaris l.) Postharvest life at chilling and nonchilling temperatures. HortScience, 45(8), 1238e1249. Rico, D., Martín-Diana, A. B., Barat, J. M., & Barry-Ryan, C. (2007). Extending and measuring the quality of fresh-cut fruits and vegetables: a review. Trends in Food Science and Technology, 18(7), 373e386. Sanchez-Mata, M. C., Camara, M., & Diez-Marques, C. (2003). Extending shelf-life and nutritive value of green beans (Phaseolus vulgaris L.), by controlled atmosphere storage: macronutrients. Food Chemistry, 80(3), 309e315. Wen, T. N., Prasad, K. N., Yang, B., & Ismail, A. (2010). Bioactive substance contents and antioxidant capacity of raw and blanched vegetables. Innovative Food Science and Emerging Technologies, 11(3), 464e469. Yildiz, F. (1994). Initial preparation, handling and distribution of minimally processed refrigerated fruits and vegetable. In R. C. Wiley (Ed.), Minimally processed refrigerated fruit & vegetables (pp. 15e65). New York: Chapman and Hall.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Hurdle technology for shelf stable minimally processed French beans (Phaseolus vulgaris): A response surface methodology approach Sumit Gupta*, Suchandra Chatterjee, Jasraj Vaishnav, Vivekanand Kumar, Prasad S. Variyar, Arun Sharma Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400094, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2011 Received in revised form 23 February 2012 Accepted 5 March 2012
Citric acid treatment in combination with gamma radiation and modified atmosphere packaging was employed as hurdles for control of microorganisms and extending shelf life of minimally processed French beans. Response surface methodology was used to optimize citric acid treatment and g-irradiation dose to obtain product with desired microbial and sensory quality. Optimum processing conditions (citric acid 8.4 g L1; irradiation dose 0.7 kGy; 10 C) resulted in modified atmosphere of 18% O2 and 4% CO2 at end of storage period. Under these conditions shelf life of the product could be extended by one week with acceptable sensory and nutritional quality as evaluated by total antioxidant, phenolics, flavonoids and vitamin C content. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Minimally processed French beans Hurdle technology Citric acid treatment g-Irradiation Modified atmosphere packaging Response surface methodology
1. Introduction The consumption of minimally processed food products including ready-to-eat or readyeto-use has increased worldwide in last decade due to its convenience, freshness and improved quality (Baskaran et al., 2007). The production of fresh cut vegetables usually involve processing such as cleaning, trimming, slicing, washing, drying and packaging (Yildiz, 1994), that causes rapid deterioration by increasing respiration rate, transpiration, enzymatic activity and microbial proliferation (Nguyen-the & Carlin, 1994). Several preservation methods including antioxidant treatment, modified atmosphere packaging (MAP), refrigeration and chlorine wash are currently employed for commercial preparation of fresh cut vegetables (Ahn et al., 2005). Psychrotrophic bacteria, such as Listeria monocytogenes are known to grow at low temperature even under modified atmospheres (Beuchat & Brackett, 1990). Disinfectants used in commercial processing lines, such as chlorine have limited effect (approx. 1 log reduction) on microbial population and can not be relied for eliminating pathogenic microorganisms like L. monocytogenes (Nguyen-the & Carlin, 1994). Thus there is an increasing concern on microbial safety of such produce. Hurdle technology involves the use of several preservation techniques in combination. This reduces the intensive use of one * Corresponding author. Tel.: þ91 22 25590560. E-mail address: [email protected] (S. Gupta). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2012.03.010
preservation technique producing a lower impact on the sensory quality (Ahn et al., 2005). Citric acid treatment, gamma irradiation, low temperature storage and modified atmosphere packaging were employed as hurdles in the present study to develop minimally processed French beans, a widely used vegetable. Citric acid can act synergistically with ionizing radiation in reducing microbial load (Bhide, Paturkar, Sherikar, & Waskar, 2001). French bean is prone to extensive microbial spoilage during storage even under refrigerated temperatures restricting its shelf life. Use of hurdle technology for preservation and extension of shelf life of this vegetable has not been investigated. Objective of the present investigation was to study the effects of irradiation, citric acid, low temperature storage and cling film packaging on sensory and microbial quality of minimally processed French beans. In order to optimize the levels of various hurdles multivariate statistical techniques such as response surface methodology (RSM) have been suggested. When many factors and interactions affect desired response, RSM is an effective tool for optimization of process (Baskaran et al., 2007). Various process parameters employed were optimized using RSM. 2. Materials and methods 2.1. Plant material French beans (Phaseolus vulgaris) grown in outskirts of Mumbai (India) were purchased from a local grower. The vegetable was brought
S. Gupta et al. / LWT - Food Science and Technology 48 (2012) 182e189
to the laboratory within 12 h after harvesting and immediately stored at refrigerated temperatures (10 C) before processing. Beans, washed in running tap water to remove all adhered dust and unwanted plant debris were dried between folds of blotting sheets and subjected to citric acid treatment by dipping in aqueous solutions of citric acid (concentrations chosen according to experimental design) for 5 min. Beans were trimmed from ends, weighed accurately (110 g), and stored in polystyrene trays (inner dimensions: 22 cm 15.7 cm 3 cm) wrapped completely with cling film (Flexo film wraps Ltd., Aurangabad, Maharashtra, India). Film used in the present study had thickness of 8e10 mm and permeability to oxygen and carbon dioxide of 15,000 and 90,000 cm3 m2 day1 atm1 (data as provided by supplier). 2.2. Irradiation and storage Packaged beans were subjected to various irradiation doses up to 2.5 kGy in a cobalt-60 irradiator (GC-5000, BRIT, Vashi, India) having activity of 8.6 kCi and dose rate of 6.5 kGy h1. Irradiator was calibrated using a Fricke dosimeter before the start of experiment and dose uniformity ratio (Dmax/Dmin) was found to be 1.2. The optimum storage temperature for French beans has earlier been reported to be 10 C (Proulx, Yagiz, Cecilia, Nunes, & Emond, 2010). Hence irradiated samples were stored at this temperature. Radiation dose and storage time was chosen according to experimental design. 2.3. Experimental design The Design of experiments and subsequent analysis of results were performed by Design expert 8.0 software (State-ease Inc., U.S.A.). Central composite rotatable design (CCRD) was employed in the present study (Abreu, Beirão-da-Costa, Gonçalves, Beirão-daCosta, & Moldão-Martins, 2003). The variables optimized were citric acid concentration (X1), irradiation dose (X2) and storage time (X3). The independent variables were coded at five levels (a, 1, 0, þ1 and þa). Coded and actual values are shown in Table 1. Complete design consisted of 20 experimental points including six replications of the center points (all variables coded as zero) (Table 2).
183
Table 2 Experimental design as central composite rotatable design. Factors Std
Run
X1 (Citric acid) g L1
X2 (Radiation dose) kGy
X3 (Storage time) Days
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1 14 15 3 7 12 20 2 8 6 18 19 17 13 16 11 5 4 9 10
1 1 1 1 1 1 1 1 1.68 1.68 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 1.68 1.68 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 0 0 1.68 1.68 0 0 0 0 0 0
(4) (16) (4) (16) (4) (16) (4) (16) (0.0) (20) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10)
(0.51) (0.51) (1.99) (1.99) (0.51) (0.51) (1.99) (1.99) (1.25) (1.25) (0.00) (2.50) (1.25) (1.25) (1.25) (1.25) (1.25) (1.25) (1.25) (1.25)
(4.00) (4.00) (4.00) (4.00) (16.00) (16.00) (16.00) (16.00) (10.00) (10.00) (10.00) (10.00) (0.00) (20.00) (10.00) (10.00) (10.00) (10.00) (10.00) (10.00)
Values for various factors are shown in their coded form while actual values are given in bracket.
‘b’ values. Instrument was calibrated using a white tile supplied along with the equipment. Firmness was measured using a texture analyzer (TA.XTPLUS, Stable Microsystems Ltd., Surrey, England) with a 100 kg load cell, equipped with a 5 mm diameter flat head cylindrical probe. Maximum force required to compress sample to a target distance of 4 mm was recorded. Test was designed with a trigger force of 15 gm and test speed of 0.5 mm s1. The peak force required to compress samples was referred as a measure of firmness of sample. The data was collected for six replicates and results were expressed as their average. 2.6. Sensory analysis
2.4. Microbial analysis Standard methods were used to enumerate total microbial load present in French beans at each sampling time and treatment conditions according to experimental design (López-Rubira, Conesa, Allende, & Artés, 2005). Enumeration of total aerobic mesophilic bacteria was done by pour plate method on plate count agar (Himedia, Mumbai, India). Incubation was done at 37 C for 24 h. Total yeast and mold count was performed by pour plate method on potato dextrose agar (Himedia, Mumbai, India) supplemented with 0.01 g L1 tartaric acid to lower pH of media to 3.5. Plates were incubated at 37 C for 48 h. Microbial counts were expressed as log10 CFU g1. Each analysis was performed in triplicate. 2.5. Determination of color and texture Surface color values were evaluated using a colorimeter (CM3600d Konica Minolta sensing Inc., Japan) by measuring ‘L’, ‘a’ and Table 1 Original and coded levels of independent variables. Variable
Original
Citric acid Irradiation dose Storage time
X1 (g L1) X2 (kGy) X3 (Days)
Sample for sensory analysis was prepared by cooking French beans in boiling water for 5 min. Beans were then cut into 2 cm long pieces and presented to assessors in white trays. Hedonic testing was carried out by 15 members of experienced sensory panel using a 9 point scale with 1, dislike extremely or not characteristic of product and 9, like extremely or very characteristic of product (López-Rubira et al., 2005). Parameters evaluated were color, aroma, texture, taste, after taste and overall acceptability. The sensory evaluation was done on the same days as microbial and other quality evaluation parameters. For optimization of treatments (irradiation dose and citric acid) hedonic sensory scores were used. For experiment carried out in optimized conditions along with hedonic analysis, sensory evaluation was done by quantitative descriptive analysis (QDA) (Murray, Delahunty, & Baxter, 2001). A trained panel consisting of 12 members, 7 males and 5 females assessed the samples using unstructured 150 mm scale. The 15 sensory attributes assessed were color (green), aroma (French beans like, cooked vegetables, grassy, irradiated and musty), texture (crispy, fibrous and soggy) and taste (sweet, sour, bitter, French beans like, rotten and irradiated). Assessments were repeated twice and sensory data was collected by measuring distance (mm) from origin.
Coded a (1.68)
1
0
þ1
þa (þ1.68)
0.00 0.00 0.00
4.1 0.51 4.00
10 1.25 10.00
16 1.99 16.00
20 2.50 20.00
2.7. Nutritional evaluation 10 g of French beans was extracted twice with 40 ml distilled methanol in an omnimixer (Sorvall, U.S.A). Extract was centrifuged
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at 4000 rpm for 30 min and the supernatant thus collected was made to a final volume of 50 ml. This solution was used for subsequent assays. Total antioxidant activity was evaluated by DPPH radical scavenging assay (Wen, Prasad, Yang, & Ismail, 2010). Total phenolic content was evaluated in accordance with FolineCiocalteu procedure (Han et al., 2011). Total antioxidant activity and total phenolic content was expressed as mg gallic acid equivalent g1 French bean. The AlCl3 method (Luximon-Ramma, Bahorun, Soobrattee, & Aruoma, 2002) was used for determination of total flavonoid content. Flavonoid content was expressed as mg quercetin equivalent g1 of French bean. Total vitamin C content of French beans was estimated in accordance with standard AOAC official microfluorometric method (AOAC, 1990). All analysis was carried with three independent samples each analyzed in triplicate.
Table 3 Significance statistics, p values and signal to noise ratio (S/N) of RSM predicted models.
2.8. Headspace gas composition
According to European standards for minimally processed produce, total microbial count should not increase (>107 cfu g1) during their entire intended storage period (Oms-Oliu, AguiloAguayo, Martin-Belloso, & Soliva-Fortuny, 2010). Thus, the present study aimed at developing minimally processed French beans product having microbial load less than 107 cfu g1 and with excellent sensory qualities during the entire shelf life. Statistical analysis by ANOVA showed that the models generated by RSM were significant (p < 0.05) while lack of fit was insignificant (p > 0.05). Further, signal to noise ratio (s/n) for all models were above 4 indicating sufficient data to navigate designed space (Table 3). Coefficients for predicted regression models and coefficient of determination (R2) are shown in Table 4. High values for R2 also suggest that the models are good fit.
Gaseous composition in package headspace was measured using gas chromatograph (GC-2010 plus, Shimadzu Corporation, Japan) equipped with split/splitless injector, molecular sieve column (30 m length, 0.35 I.D., RT-Msieve 5A, Restek Corporation, U.S.A.) and a TCD detector. Temperature of injection port was kept at 35 C. Initial column temperature was kept at 30 C for 5 min, then raised at the rate of 10 C min1 to 100 C and held at 100 C for 5 min. Sampling was made by piercing a hypodermic needle into package through an adhesive septum, previously stuck to the package. 0.1 ml of sample headspace was taken and injected under a split ratio of 5. From the column used in present study concentrations of only O2 and N2 can be evaluated. Based on observed pO2 and pN2 in package headspace actual concentrations of O2 and CO2 (pO2 and pCO2) were calculated using following equations:
pO2 ¼ ðObserved pO2 =Observed pN2 Þ 78:084
(1)
pCO2 ¼ 100 ½ðObserved pO2 =Observed pN2 Þ 78:084 þ 78:084
(2)
Analysis was done in triplicates and concentrations of gases are expressed as their partial pressures. 2.9. Statistical analysis Model and statistical analysis was performed by Design expert software (Version 8.0, State-ease, USA). Models for individual responses were generated by fitting experimental data into third order polynomial and then removing insignificant terms by backward regression method. Model terms with highest values of partial probability (p-value) are removed first and process is stopped when p-value of next term out satisfies the specified alpha out criterion. Value of alpha out was kept 0.1 which leads to an overall model with terms significant at 0.05 levels. Models were analyzed by analysis of variance (ANOVA) and the predictive capability of the model was quantified by coefficient of determination (R2). Fitted polynomial equations were then used to plot response surface plots in order to visualize the interrelationship of response and experimental levels of each factor. Optimum conditions were then derived using numerical optimization process. Criteria for desired microbial and sensory qualities were given and model equations solved to obtain optimum processing conditions (Table 5). Data for quality evaluation by total antioxidant, total phenolics, total flavonoids and vitamin C was analyzed by two-way ANOVA to determine effect of radiation processing and storage time. Multiple means comparison was done by Tukey’s test (p < 0.05).
Model
Residual
S/N
Response
Sum of squares
df
p-value
Sum of squares
df
p-value
Total plate count Yeast and mold count Texture Color Aroma Taste
26.62
13
0.0047
0.81
5
0.31
16.33
6.26
11
0.0345
1.31
8
0.38
6.4
76.22 348.93 2.90 4.11
11 8 12 11
0.0006 0.024 0.0036 0.0025
4.87 95.50 0.14 0.37
8 9 6 8
0.51 0.73 0.82 0.85
14.71 7.4 9.039 10.99
3. Results and discussions
3.1. Microbial analysis A significant (p < 0.05) increase in microbial load during storage was observed (Fig. 1A). Effect of irradiation and citric acid treatment on microbial load is depicted in Fig.1B. At zero day mesophilic counts reduced with both increasing radiation dose and citric acid treatment (Fig. 1B). A 5 log cycle reduction of mesophilic counts can be observed at a dose of 2 kGy and a 2 log cycle reduction can be observed with citric acid treatment of 16 g L1. Similar results were reported earlier for irradiation of carrots (Chervin & Boisseau, 1994), lettuce (Prakash, Guner, Caporaso, & Foley, 2000), diced celery Table 4 Coefficients of the fitted polynomial representing the relationship between the response and the process variable and R2 values. Yeast and Color mold count ‘a’ (log value cfu g1)
Texture Taste Aroma (sqrt (g)) (Hedonic) (Hedonic)
4.49a 0.11a 0.99a 0.87a 0.15a 0.27a 0.098 0.28a 0.83a 0.58a 0.65a 1.27a 0.52a 0.88a
2.44a 0.43a 0.036a 0.37a 0.13 0.17a 0.32a 0.029a 0.066a 0.14a 0.40a 0.056 0.23 0.58a
8.38a 1.76a 1.65a 3.13a 1.25a 1.76a 1.12 1.98a 3.41a 1.38 2.05 3.47a 3.40 1.00
40.49a 1.91a 1.22a 1.08a 0.76a 0.46a 0.73a 1.09a 0.12 0.42 0.15 0.82a 0.053 1.38a
6.38a 0.18a 0.45a 0.37a 0.017a 0.18a 0.18a 0.12a 0.20a 0.092 0.013 0.33a 0.044 0.34a
0.97
0.82
0.78
0.93
0.91
Coefficients Total plate count (log cfu g1) X0 X1 X2 X3 X11 X22 X33 X12 X13 X23 X123 X112 X113 X122 R2 a
Significant terms at p < 0.05.
6.88a 0.02a 0.093a 0.18a 0.045a 0.23a 0.39a 0.049a 0.023a 0.26a 0.099a 9.36*103 0.32a 0.13 0.95
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Fig. 1. Response surface curves for microbial counts. A) Variation of total plate counts with radiation dose and storage time at 10 g L1 citric acid B) Variation of total plate counts with radiation dose and citric acid at storage time (0 days) C) Variation of yeasts and molds count with storage time and citric acid at dose of 1.25 kGy D) Variation of yeasts and molds with storage time and radiation dose at 16 g L1 citric acid.
(Prakash, Inthajak, Huibregtse, Caporaso, & Foley, 2000) and other vegetables (Farkas, Seray, Mohacsi-Farkas, Horti, & Andrassy, 1997). It was observed that yeast and mold counts increased significantly (p < 0.05) with storage time. During initial days of storage yeast and mold counts declined with both increasing radiation dose and citric acid treatment (Fig. 1C and D). Surprisingly, with increasing storage time more yeast and mold counts were observed at higher radiation dose and corresponding greater citric acid treatment (Fig. 1D). Low mesophilic bacterial counts leading to diminished competitive flora for yeasts and molds could explain this observation. Further, higher citric acid led to decrease in pH thus creating conditions more favorable for yeasts and molds. 3.2. Texture and color It was observed that during initial storage period firmness of French beans decreased with increasing irradiation dose (Fig. 2A). Radiation induced depolymerization of pectin and other cell wall components such as cellulose and hemicelluloses could lead to
decreased firmness and softening of plant tissues (Prakash, Guner, et al., 2000; Prakash, Inthajak, et al., 2000). At lower radiation doses firmness showed a positive correlation with citric acid treatment while at higher doses (2 kGy) it showed a negative correlation. Protective effect of citric acid on hydrolysis of pectic substances at lower doses of irradiation due to its antioxidant characteristics is suggested. However, higher citric acid concentrations could lead to softening of texture due to acid hydrolysis of pectic substances. Citric acid treatment (10 g L1) has been previously shown to be associated with softening in texture of apple slices (Cocci, Rocculi, Romani, & Rosa, 2006). Surprisingly, at lower citric acid treatments firmness was found to increase with increasing radiation dose (Fig. 2B) on sixteenth day of storage. Irradiation can lead to lower rate of respiration in plant produce which results in lesser loss of water and subsequently better firmness (Rico, Martín-Diana, Barat, & Barry-Ryan, 2007). All three factors analyzed i.e. citric acid, irradiation dose and storage time influenced ‘a’ values significantly (p < 0.05) while effect on ‘L’ and ‘b’ values was not significant (p > 0.05). ‘a’ values
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Fig. 2. Response surface curves for texture and color. A) Effect of radiation dose and citric acid treatment on firmness of French beans at storage time (0 days). B) Effect of radiation dose and citric acid treatment on firmness of French beans after storage time of 16 days. C) Effect of radiation dose and citric acid treatment on ‘a’ values of French beans after storage time of 10 days.
increased with increasing storage period and citric acid treatment indicating a reduction of greenness of samples. Reduction of greenness might be due to degradation of pigments during storage. Interestingly, ‘a’ values demonstrated decreasing trend with increasing radiation dose indicating better greenness of irradiated samples (Fig. 2C). Radiation processing can result in reduction in rate of respiration of plant produce leading to slower rate of degradation of pigments. No significant (p > 0.05) effect of processing conditions was observed on texture and color of French beans as analyzed by hedonic sensory scores. While the physical attributes of color and texture showed significant variance instrumentally, no effect on texture and color values could be observed by sensory panel. 3.3. Aroma and taste Irradiation and storage time significantly (p < 0.05) influenced aroma quality and taste of beans while citric acid treatment had no effect on these parameters. Aroma quality of French beans reduced
with increasing storage period. Samples irradiated at higher doses of radiation demonstrated slightly lesser scores for aroma quality as compared to control samples during initial storage period but demonstrated better aroma scores when analyzed in late storage period (Fig. 3A). Significantly higher aroma scores for irradiated carrots, cilantro, green onions, parsley and red lettuce after 14 days of storage were earlier reported (Fan & Sokorai, 2008). Better aroma scores observed in present study after irradiation and storage as compared to samples stored without irradiation could probably be due to reduced decay in the samples. 3.4. Optimization and verification of results Various processing factors were optimized for achieving maximum shelf life of minimal processed produce with microbial count of <107 cfu gm1 during entire intended storage period and to maximize various quality parameters like texture, aroma and taste. Criteria set for optimization of parameters and solutions obtained are given in Table 5. Solution A was obtained for criteria 1 and solution B
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187
Fig. 3. Response surface curves for sensory attributes of aroma and taste. A) Effect of radiation dose and storage time on aroma quality of French beans at 10 g L1 citric acid treatment. B) Effect of radiation dose and storage time on taste of French beans at 10 g L1 citric acid treatment.
was obtained for criteria 2. Both solutions were used for validation of models generated. Predicted and actual values of quality parameters for both solutions are shown in Table 5. Close agreement between actual and predicted values indicate suitability of model generated. Values for microbial counts obtained were slightly higher than predicted values. Since, for validation experiments French beans were harvested separately, the variation in microbial counts could be due to inherent variability in biological samples. Solution B demonstrated acceptable microbial load (<107 cfu gm1) with a shelf life of 14 days. Further, it had high values for sensory attributes like texture, aroma and taste. This solution was therefore taken up for further studies regarding nutritional evaluation of developed product, descriptive sensory analysis and gaseous headspace composition. 3.5. Nutritional evaluation As shown in Fig. 4A, antioxidant activity reduced significantly (p < 0.05) during storage but no affect due to irradiation treatment Table 5 Criteria for various factors and responses for process optimization and corresponding optimized solutions obtained. Criteria 1
Criteria 2
Solution A
Solution B
Minimize
Minimize
14
8.4
Minimize
Minimize
1.04
0.7
Maximize (>7 < 15) Should be <5 Minimize
Maximize (>12 15) Should be <6 Minimize
10.41
Maximize
Maximize
Color (a)
Minimize
Minimize
Aroma (Hedonic) Taste (Hedonic)
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Citric acid (g L1) Irradiation dose (kGy) Storage time (Days) TPC (Log cfu g1) Y & M count (Log cfu g1) Texture (g)
a b
Predicted values for solutions. Actual values.
a
4.76 (5.28 0.29)b 2.17a (2.73 0.5)b 1543a (1463 86)b 8.4a (8.0 0.6)b 6.84a (6.66 0.27)b 6.34a (6.5 0.5)b
14 5.681a (6.01 0.31)b 2.42a (2.93 0.17)b 1646a (1615 112)b 8.8a (8.6 0.4)b 6.62a (6.8 0.35)b 6.2a (6.0 0.25)b
was observed. A 40% decrease in antioxidant activity was observed after 14 days of storage period. Maximum reduction of antioxidant activity was, however, observed during initial storage period of five days. Reduction in antioxidant activity might be due to reduction in content of total phenolics and flavonoids. Total phenolic content reduced significantly (p < 0.05) during storage (Fig. 4B). Slight decrease in phenolic content due to radiation processing was also observed. Contrary to antioxidant results no decrease in phenolics was observed up to a storage period of five days. A decrease of 20% was however, observed on storage up to fourteen days. Flavonoid content showed a similar trend as antioxidant capacity during storage. A significant (p < 0.05) decrease was observed up to a period of five days. No significant decrease was noted beyond this period (Fig. 4C). Fan, Toivonen, Rajkowaski, & Sokorai, 2003 reported that radiation induced phenolic synthesis in fresh-cut iceberg lettuce treated by warm water dipping. Radiation induced increased antioxidant and phenolic content has been previously demonstrated in other vegetables like cabbage (Ahn et al., 2005), carrot and kale juice (Arvanitoyannis, Stratakos, & Tsarouhas, 2009) and fresh cilantro leaves (Fan, Niemira, & Sokorai, 2003). During irradiation the free radicals generated may act as a stress signals and may trigger stress responses in vegetables, resulting in increased antioxidant synthesis (Fan, Toivonen, et al., 2003). Surprisingly, no increased phenolic content was observed due to processing and storage in present study. Observed reduction in total antioxidant and phenolic content could be attributed to the fact that during storage high O2 concentrations were present in packages resulting in oxidation of phenolic compounds. No statistical difference was observed among control and processed samples in total ascorbate content (p > 0.05). Vitamin C content remained similar during entire storage period (Fig. 5). In previous reports on vegetables decrease in ascorbic acid content during storage and irradiation was reported (Arvanitoyannis et al., 2009). Ascorbic acid undergoes oxidation during storage to dehydroascorbic acid (DHAA) which is also bioactive and in a bio available form but to a lesser extent than ascorbic acid (Ogiri et al., 2002). In present study entire vitamin C was oxidized to DHAA and quantified. This might explain the fact that no reduction in vitamin C content was observed.
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Fig. 5. Effect of radiation processing (0.7 kGy), citric acid treatment (8.4 g L1)) and storage on total ascorbate content in French beans. ( ) Control and ( ) processed samples.
attainment of equilibrium between package headspace and atmosphere. Processed samples demonstrated higher CO2 concentration as compared to control till eight days of storage, which could possibly be due to enhanced rate of respiration in plant produce immediately after radiation processing (Fan & Sokorai, 2008). O2 content less than 2% in package headspace can result in anaerobic conditions leading to generation of off-flavors and possible growth of anaerobic pathogens like Clostridium botulinum (Sanchez-Mata, Camara, & Dıez-Marques, 2003). Lowest O2 concentrations observed for the processed samples during entire storage period was 16.48 1.5% thus maintaining aerobic conditions within the package. Processing and packaging conditions chosen in present study are therefore suitable for MAP of French beans. 4. Conclusions Fig. 4. Effect of radiation processing (0.7 kGy), citric acid treatment (8.4 g L1)) and storage on nutritive quality of French beans. A) Total antioxidative capacity. B) Total phenolics content. C) Total flavonoid content. ( ) Control and ( ) processed samples.
3.6. Quantitative descriptive analysis
In summary, RSM approach was successfully employed to optimize process parameters for minimally processed French beans with desired microbial and sensory qualities. Analysis of response surfaces indicates a complex interaction among variables such as irradiation dose, citric acid and storage time. Irradiation and citric
Processed French bean after 14 days of storage was further evaluated by QDA and data obtained is graphically represented by spider diagram (Fig. 6). Untreated sample after storage was not evaluated as it demonstrated visible fungal growth. After 14 days of storage processed product had slightly lesser scores for aroma attributes like green, French beans and grassy. A possible loss of volatile aroma compounds during storage could be suggested. In terms of textural attributes the developed product had less fibrous nature while no difference in taste was observed compared to the control sample. No irradiated off flavor or taste was perceived by assessors (Fig. 6). QDA results established that processed French beans had desired aroma, taste and textural properties even after storage period of fourteen days. 3.7. Headspace composition in packages Significant (p < 0.05) effects of irradiation and storage time were observed for CO2 and O2 during storage (Fig. 7). In control samples an increase in CO2 was observed up to eight days of storage with composition remaining nearly constant thereafter, indicating
Fig. 6. Quantitative descriptive analysis of fresh unprocessed and processed (radiation dose (0.7 kGy) and citric acid treatment (8.4 g L1) after 14 days of storage) samples. ) Fresh unprocessed and ( ) processed sample after 14 day storage. (
S. Gupta et al. / LWT - Food Science and Technology 48 (2012) 182e189
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Fig. 7. Headspace gas composition in packages. A) Variation of O2 content during storage in control and processed (radiation processing (0.7 kGy) and citric acid treatment ) (8.4 g L1)) samples. B) Variation of CO2 content during storage in control and processed (radiation processing (0.7 kGy) and citric acid treatment (8.4 g L1)) samples. ( Control and ( ) processed sample.
acid significantly (p < 0.05) reduced microbial contamination. Citric acid was found to reduce radiation induced softening of French beans. Thus, use of citric acid treatments may be useful for maintaining texture of vegetable during radiation processing. Advantage of using RSM also stems from the fact that different quality criteria when employed for optimization resulted in different suitable solutions. This can be useful for commercial production as processing conditions can be suitably calculated based on quality characteristics required, thus reducing severity of treatments. Validation experiments proved that mathematical models generated were suitable for optimizing process parameters for desired product properties. Analysis of developed product by QDA demonstrates that processed French beans had desired aroma, taste and textural attributes. This study demonstrates usefulness of RSM for optimization of treatments for production of minimally processed vegetables.
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