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Quality and shelf life of calcium-enriched wholemeal bread stored in a modified atmosphere
Journal of Cereal Science 56 (2012) 418e424
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Quality and shelf life of calcium-enriched wholemeal bread stored in a modified atmosphere M. Fik, K. Surówka*, I. Maciejaszek, M. Macura, M. Michalczyk Department of Refrigeration and Food Concentrates, University of Agriculture in Krakow, Balicka Street 122, PL-30-149 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2012 Received in revised form 11 June 2012 Accepted 12 June 2012
Calcium-enriched wholemeal bread packed in a modified atmosphere (60% CO2, 40% N2), was examined for stability and microbiological changes throughout 32-day storage. The product was still acceptable after 24 days at 20 1 C. At this time no microbiological changes were observed; however, there was a continuous decline in sensory quality, mainly due to deterioration of the texture. Crumb hardness increased, whereas its springiness and cohesiveness decreased. These observations were accompanied by physical and chemical changes characterized by a steady increase in acidity and a sharp decrease in blue value, especially at the beginning of storage. The first microbiological changes occurred only after 27 days of storage and were due to the growth of moulds and amylase-negative Gram-positive cocci, coccibacillus or bacillus. The principal component analysis showed that nearly 86% of the variance in ten considered variables could be represented by two new variables: PC1, defined by eating quality and physicochemical attributes, and PC2 defined mainly by chewiness. Three groups of stored bread were identified on the score plot. The first group, fresh and 3-day stored bread, was characterized by low hardness and sourness; the second, bread stored from six to twenty days, scored lower for overall sensory quality, low springiness, cohesiveness and blue value; and the third group, the product during the final stages of storage, exhibited a clear increase in chewiness and hardness and showed the first indications of microbial deterioration. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Enriched wholemeal bread Bread shelf life Modified atmosphere packaging Textural properties
1. Introduction Bakery products are characterized by short-term stability and limited shelf life due to the rapid course of staling (Fik, 2004). This complex and not fully investigated process is generally believed to stem from manifold changes in the composition of bread, caused by factors other than microbial growth, which result in a loss of freshness and a reduction in quality. Such changes cause a deterioration of both sensory attributes and physicochemical properties, especially in the structure and texture of the bread’s crumb and crust. The reduction observed in starch solubility is also a significant factor, correlating with increasing starch recrystallization and a fall in water holding capacity in the bread crumb during storage
Abbreviations: A, acidity; BV, blue value; C, cohesiveness; CAP, controlled atmosphere packaging; Ch, chewiness; H, hardness; MA, modified atmosphere; MAP, modified atmosphere packaging; MC, moisture content; OSQ, overall sensory quality; PCA, principial component analysis; PC1, principial component 1; PC2, principial component 2; S, springiness; TPA, texture profile analysis; WHC, water holding capacity. * Corresponding author. Tel.: þ48 12 662 47 59; fax: þ48 12 662 47 58. E-mail address: [email protected] (K. Surówka). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2012.06.006
(Hoseney and Miller, 1998). The intensity of such processes increased throughout storage depending on the conditions applied, resulting in the following changes being observed: an increase in the hardness, dryness and crumbliness of the crumb along with a fall in its elasticity; a decrease in the crispness of the crust; and a decline in aroma and other characteristic attributes of bread freshness. All this is evidence of staling, an indicator of the limited shelf life of bread and a phenomenon that may to some extent be explained by moisture migration from the crumb to the crust due to changes in colloid structure (Burrington, 1998) and not necessarily by moisture loss. Thus, retaining the freshness of bread remains an important problem for both consumers and bakers, which explains why bread ageing and the retardation of this process are widely reported in the literature (Fik and Celej, 1993; Fik et al., 2000; Gerrard et al., 1997; Patel et al., 2005). The practice of packaging and storing bread in a modified atmosphere (MAP) is becoming increasing common as a method of extending its shelf life and maintaining good quality. Many authors have confirmed the benefits of this method in the storage of bakery products (Smith, 1993; Kotsianis et al., 2002). Some of them emphasize its role in extending the microbial shelf life of bread (Phillips, 1996). According to Smith (1993), the application of MAP
M. Fik et al. / Journal of Cereal Science 56 (2012) 418e424
extends shelf life in three ways: chemically, it controls biochemical and degradation processes and slows oxidation; microbiologically, MAP may increase shelf life by inhibiting the growth of mould and bacteria; and physically, MAP lengthens a product’s stability by reducing moisture loss. It is similar to controlled atmosphere packaging (CAP) in that a specific gas mixture is applied to the product but differs in the level of control. Bread can be packed in a modified atmosphere in order to delay staling and hinder the growth of moulds using 60% to 80% (v/v) carbon dioxide and 20% to 40% nitrogen. Since bread is one of the most frequently consumed food products, it is reasonable to enrich it with nutritive substances. Commonly supplemented with protein preparations (Begum et al., 2011), bread is also frequently fortified with minerals (Karadzhov and Iserliyska, 2003) and vitamins (Czeizel and Merhala, 1998). Antioxidant substances may be added to bread to achieve healthpromoting effects (Park et al., 1997), while other recipes may enrich the product with dietary fibre (Dalgetty and Byung-Kee, 2006). However, the beneficial nutritional effects of such enrichment do not always correlate positively with the storage stability of bread. Therefore, the aim of this paper was to investigate the effect of a modified atmosphere (60% CO2 and 40% N2) on the quality and shelf life of calcium-enriched wholemeal bread stored at room temperature (20 1 C). In contrast to bread generally available, such bread shows health-promoting properties due to higher levels of dietary fibre and calcium. The use of principal component analysis (PCA) enabled an evaluation of the interrelations between sensory quality, instrumentally measured textural properties and physicochemical indicators important for the consumer and the baker. Microbiological analyses were also carried out. 2. Experimental material 2.1. Bread making and packaging Calcium-enriched wholemeal bread was produced under industrial conditions during one shift in a bakery packing bread in a modified atmosphere. The bread was made according to the following dough formulation: 34.7 parts (weight basis) Graham wheat flour type 1850 (according to ash content) containing 11.4% of protein and characterized by 27% wet gluten yield and a falling number value of 210 s; 6.4 rye flour type 720 (6.8% protein, a falling number value of 170 s); 6.2 wheat flour type 750 (11.9% protein, 33% wet gluten and a falling number value of 320 s); 28.0 leaven; 20.0 water; 1.7 whey; 1.2 salt; 1.6 baker’s yeast and 0.2 calcium carbonate Calcipur 2 OG with granulometry 45 mm (90%). The flours were purchased in ZPZ Szymanów, Polskie M1yny (Teresin, Poland); leaven was obtained due to spontaneous fermentation of the rye flour type 720 with water (1:3); whey was provided by a local dairy; baker’s yeast originated from the Lesaffre S.A. (Wo1czyn, Poland), while calcium carbonate from the Brenntag Sp. z o.o (Ke˛ dzierzyn-Ko zle, Poland). All ingredients were mixed in _ a Kemper SP125 spiral mixer (Sispo, Znin, Poland) for 6 min at low speed (105 rpm) followed by 6 min at higher speed (210 rpm). The resulting dough was left to rise for 90 min at 30 C and then punched down. After shaping, placing in loaf tins and final fermentation for 40 min at 30 C, loaves were baked at 200 C for 45 min in a conventional oven, cooled at room temperature and packed in polyamide/polyethylene bags in a modified atmosphere (60% CO2, 40% N2). The bags used fulfilled the EC 90/128/EEC Regulation on materials intended to come into contact with food and were characterized by total thickness of 80 mm and maximum O2, CO2 and H2O permeability of 100 cm3/(m2 24 h bar), 140 cm3/(m2 24 h bar) and 8 g/(m2 24 h bar), respectively.
419
The bread was stored in cardboard boxes at room temperature (20 1 C) for up to 32 days. Samples were examined at regular intervals. 2.2. Sensory evaluation In the sensory evaluation, each attribute of crumb (taste, smell, and elasticity) and crust (taste, smell, and crispness) was assessed using a 5-point scale (Fik et al., 2000). Each evaluation was carried out by nine panellists appropriately trained and tested for sensory sensitivity. Because of the different relative importance of particular attributes to the overall sensory quality of the product, the following contribution coefficients were used: 0.5 for taste and smell of crumb; 0.2 for taste and smell of crust; 0.2 for crumb elasticity; and 0.1 for crust crispness. In the overall sensory quality assessment, scores of 5, 4, 3, 2 and 1 corresponded to evaluations of very good, good, acceptable, unacceptable, and bad respectively. 2.3. Microbiological analyses For the microbiological analysis of bread, 10 g of crumb sample was diluted with 90 ml of 0.1% peptone solution and blended for 2 min in a Stomacher 80 (Seward, England). Serial decimal dilutions were made using 0.1% peptone solution. Total aerobic mesophylic bacteria count was determined on plate count agar medium (Karaoglu et al., 2005) after incubation at 30 C for 72 h. Yeasts and moulds were determined on plates with Sabouraud agar medium, incubated at 25 C for 3e5 days (Odds, 1991). The levels of vegetative and spore-forming amylolytic aerobic bacteria were assayed on agar medium with starch, according to Waksman (Polish Standard PN-A-74134-4, 1998). In addition, the microbial stability of bread was evaluated by means of thermostat trials (at 30 C for 30 days), in which samples of bread packed in a modified atmosphere were analyzed in terms of sensory changes resulting from mould growth (Polish Standard PN-A-74102, 1999). Additionally, samples of fresh bread and bread stored in MAP for 3, 6 and 8 days were thermostated at 37 C for 3 days in Weck jars to investigate changes in the aroma and appearance of the product due to the growth of spore-forming amylolytic aerobic bacteria (Polish Standard PN-A-74102, 1999). 2.4. Textural analysis Measurements of bread texture were carried out using a TA-XT2 Texture Analyser (Stable Micro Systems, England) coupled with a computer equipped with XT.RA Dimension, v.3.7 software, enabling the instrument to be controlled and data to be processed automatically. Crumb samples (in the form of cubes of 60 mm side) of individual leaves baked from the same batch of dough were subjected to texture profile analysis (TPA) (Breene, 1975; Szczesniak, 1963). The samples were compressed twice with an aluminium cylinder-shaped plunger with a diameter of 90 mm to a depth of 36 mm (40% strain). The plunger used moved at a rate of 2 mm/s and the time between strokes was 20 s. The apparatus recorded the force exerted by the plunger as a function of time, from which the following texture parameters were determined: hardness, springiness, cohesiveness and chewiness. Hardness was defined as the peak force [N] during the first compression cycle. Cohesiveness was calculated as a ratio of areas delimited by the curves of the second and first bite. Springiness was determined as a ratio of the time measured between the start of the second area and the second probe reversal divided by the time measured between the start of the first area and the first probe reversal. Chewiness [N] was assessed by multiplying hardness, cohesiveness and springiness.
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2.5. Moisture content and water holding capacity
in terms of millilitres of sodium hydroxide (0.1 M) solution required to neutralize the free acids contained in 100 g of the bread crumb.
Moisture content of the breadcrumb was determined after oven-drying at 105 C until attaining a constant weight. Water holding capacity (WHC) was measured as follows: the crumb (10 g) was gently homogenized with 50 ml of water in a grinder (Heidolph Diax 900, Schwabach, Germany) for 2 min. Then the homogenate was mixed at room temperature for 20 min using a magnetic stirrer. The slurry was centrifuged (1600 g, 10 min) following 20 min of holding. The sediment weight was recorded, whereas the supernatant was used for the determination of blue value. The amount of water in the sediment was calculated by subtracting the dry sample weight. WHC was expressed as grams of water in the sediment per gram of dry sample weight.
2.8. Sorption isotherm The moisture sorption isotherm was plotted for the bread on the basis of data obtained after balancing the dry matter at 20 C in desiccators containing saturated salt solutions of different water activities (Leung, 1986). The monolayer capacity was determined applying the BET equation given by Brunauer, Emmett and Teller and using the data obtained for the low-moisture end of the moisture sorption isotherm (Fennema, 1985). 2.9. Statistical analysis
2.6. Blue value determination The complex of amylose with iodine produces a blue colour. Its intensity can be used to indicate the level of free amylose in a soluble fraction, which informs about starch retrogradation. The blue value was determined by a spectroscopic method. To this end, 20 ml of the supernatant obtained using the method described for WHC, was poured into a centrifuge tube. Next, 2 ml of Carrez I and 2 ml Carrez II solutions were added to precipitate the protein matter. The whole sample was carefully mixed, allowed to stand for 10 min and finally centrifuged (5000 g, 5 min). Afterwards, 4 ml of the supernatant obtained was mixed with 25 ml of iodine solution (0.004%); The blue value was expressed as the absorbance measured at 580 nm using distilled water as a blank. 2.7. pH and acidity For pH determination, 10 g of the crumb was homogenized with 50 ml of distilled water using an RW 11 basic blender (IKA - Werke GMBH & Co. KG, Germany) for 30 s at high speed. The pH of prepared homogenates was determined with a Hanna Instruments pH meter (Model HI 9025, England) equipped with a combined electrode. In order to measure acidity, 25 g of the crumb was shaken for 2 min with distilled water (250 ml) in an Erlenmeyer flask. Then the sample was allowed to stand for 1 h, during which time it was shaken at 15-min intervals. It was then filtered through cotton wool into a dry Erlenmeyer flask. Finally, 50 ml of filtrate was titrated with sodium hydroxide (0.1 M) in the presence of phenolphthalein indicating solution until attaining a constant (not changing within 1 min) light-pink colour. The degree of bread acidity was expressed
Textural measurements were performed nine-fold, whereas physico-chemical analyses were done in triplicate. The results obtained were analyzed statistically using the CSS Statistica v. 9.1, (StatSoft, Inc., Tulsa OK, USA) package. Mean values and standard deviations were calculated for each texture characteristic and physico-chemical measure. ANOVA variance analysis at P 0.05 confidence level for comparing means of variables and principal component analysis (PCA) was used to analyse relationships between variables. 3. Results 3.1. Sensory quality Table 1 shows the changes in overall sensory quality of calciumenriched wholemeal bread during storage in a modified atmosphere. The initial quality of the bread was found to be very good. All sensory attributes scored the maximum value 5. Crumb and crust were characterized by the intensive taste and aroma typically associated with fresh bread. Of the other attributes, crust crispness and crumb elasticity achieved the highest scores. Bread quality was still found to be fairly good after 11 days of storage. However, with continued storage, crumb elasticity was the attribute most prone to rapid deterioration, although this characteristic is not a very important quality factor for this type of bread, which is firm and of low elasticity by nature. The loaf appearance remained almost unchanged during the whole period of storage, making it impossible to evaluate how long the bread had been stored. The product was still acceptable after 24 days of MAP storage; however, after 27 days the panellists found it unacceptable.
Table 1 Effect of storage time in modified atmosphere on overall sensory quality, TPA texture measures for crumb, and some physicochemical changes in calcium-enriched wholemeal bread. Storage time (days)
Overall sensory quality
Hardness (N)
0 3 6 8 11 14 17 20 24 27 32
5.0a 4.9a 4.6 4.2 3.9b 3.8b 3.7bc 3.5c 3.2 2.9d 2.7d
57.48 5.80 64.86 6.71 72.53 7.30 82.03 7.80a 87.50 8.84ab 93.32 9.12bc 95.67 9.68cd 100.61 10.41d 112.32 11.30 118.41 11.50 130.52 12.00
Springiness
0.83 0.05 0.70 0.03a 0.69 0.05a 0.61 0.05b 0.60 0.08b 0.59 0.03b 0.60 0.05b 0.60 0.06b 0.60 0.02b 0.60 0.05b 0.59 0.04b
Cohesiveness
0.30 0.02a 0.28 0.02a 0.23 0.02b 0.22 0.03bc 0.21 0.03bc 0.21 0.01bc 0.21 0.02bc 0.20 0.03cd 0.20 0.02cd 0.20 0.03cd 0.19 0.03d
Chewiness (N)
14.31 1.74a 12.71 2.7bc 11.51 2.61bc 11.01 2.80c 11.02 2.14c 11.56 1.33c 12.05 2.50b 12.07 2.03bc 13.48 2.30ab 14.21 2.10a 14.63 2.37a
Moisture content (%)
Water holding capacity (g water/g dry sample)
Blue value
45.00 1.12 43.52 1.50a 43.83 0.76a 43.77 0.76a 42.80 1.29ab 42.00 0.85b 41.54 1.54b 39.10 0.29c 38.65 0.39c 38.00 0.84c 37.05 0.05
3.17 0.00a 2.88 0.02b 2.96 0.08bc 3.05 0.05c 3.04 0.05c 3.31 0.04d 3.35 0.06d 3.16 0.05a 3.12 0.04a 3.07 0.07c 3.06 0.02c
0.66 0.02 0.37 0.03 0.27 0.02a 0.25 0.01ab 0.24 0.01b 0.23 0.02bc 0.22 0.01c 0.21 0.01c 0.22 0.01c 0.21 0.01c 0.12 0.01
Results are means SD; Means in columns not denoted or denoted with different letters are significantly different (P 0.05).
580nm A 1cm
Acidity (degree)
pH
2.63 0.06a 2.73 0.11a 2.99 0.10b 3.03 0.07b 3.07 0.07b 3.12 0.10b 3.03 0.17b 3.17 0.05 3.30 0.05c 3.35 0.03c 3.36 0.06c
5.67a 5.68a 5.67a 5.65a 5.64a 5.65a 5.64a 5.65a 5.65a 5.64a 5.64a
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3.2. Microbiological analyses
3.6. Acidity and pH
Microbiological analysis of the bread prior to storage showed that the initial total bacteria count as well as yeast and mould levels were below 10 cfu/g. At this stage the bread did not contain any spore-forming amylolytic bacteria or their spores. Throughout storage at 20 1 C, no microbiological changes were observed until the 24th day. Thereafter, especially after 27 and 32 days of storage, the presence of yeasts and moulds 2 101e3 103 cfu/g and bacterial growth 2 102e2 103 cfu/g were detected. The bacteria formed very small white colonies on plate count agar and Waksman medium. Microscopic analysis revealed them to be mainly Gram-positive cocci, cocci-bacillus or bacillus, all of them amylase-negative. Thermostat trials carried out after 0, 3, 6 and 8 days of storage to evaluate sensory changes due to the growth of aerobic amylolytic bacteria were negative for all the breads examined. Breads thermostated at 30 C showed a low incidence of moulds (occurring only in individual samples stored in a modified atmosphere).
The acidity of the bread showed a slight tendency to increase over the entire storage period (Table 1). However, the degree of change was insufficient to have a perceptible effect on taste and flavour, the attributes examined in sensory evaluation. No significant changes were observed in pH throughout the storage period, remaining more or less constant at around 5.65. Table 1 shows that after 32 days’ storage, the concentration of hydronium ions in highcalcium wholemeal bread packed in MA was almost identical to that in the fresh product even though acidity increased by nearly 28% during this period.
3.3. Texture Individual texture characteristics of crumb as revealed by analyses carried out at regular intervals throughout the storage period are given in Table 1. In the fresh product, the values of all the textural parameters measured were characteristic of a high-quality bread. The highest values were for springiness, cohesiveness and chewiness, while the lowest for hardness. However, after only 3 days of storage, negative changes were recorded for each of these parameters; crumb hardness increased by almost 13% and springiness, cohesiveness and chewiness decreased by about 15, 7 and 11% respectively. During further MA storage, the greatest changes were noted in hardness, which increased steadily so that after 32 days it was 73 N greater than in fresh bread. Springiness and cohesiveness, on the other hand, decreased up to the 8th day of storage; thereafter it remained stable until the end of the experiment. Chewiness, which initially decreased to approximately 11 N after 8 days of storage, then rose steadily, and after 32 days reached a slightly higher value than in the fresh bread. 3.4. Moisture content and water holding capacity Table 1 presents the results of moisture content and water holding capacity (WHC) of bread during MA storage. The moisture content of calcium-enriched wholemeal bread fell gradually during storage corresponding to changes in sensory quality. The fresh product, which obtained the highest scores in the sensory evaluation, had the highest moisture content. Increasing dry matter content had a detrimental effect on the sensory attributes of bread. The data show that 3 days of storage reduced WHC by 9% compared with the initial value. During further storage, the value for this indicator increased gradually to 3.35 g of water per g of dry sample after 17 days. After a subsequent decrease, the value at the end of experiment was close to that in fresh bread. 3.5. Blue value The blue value defines the content of water-soluble starch, so its amount in crumb generally decreases as bread stales. In calciumenriched wholemeal bread packed in MA, this value decreased considerably (by 44% compared to the initial value) after only 3 days (Table 1), and after 6 days a further fall of 15% was recorded. During subsequent storage, the changes observed in the blue value were not so pronounced.
3.7. Sorption isotherm The sorption isotherm established for high-calcium wholemeal bread is sigmoidal in shape and falls into type II (Fig. 1). The shape is typical for food products containing high-molecular substances. In bread these are chiefly starch and gluten, although in the case of wholemeal bread, non-starch polysaccharides such as pentosans should also be considered. The BET monolayer value of the bread examined was 0.0685 g H2O/g d.m., which corresponds to Aw ¼ 0.214. At this value, a product is covered by a monolayer of water, which ensures the best storage stability of the product by protecting non-water substances. At the same time, the amount of water is insufficient to become a reagent or medium for unfavourable processes of a physical, chemical or microbiological nature. Bread distributed in retail outlets contains significantly more water than the monolayer value. In the product examined it was 45%, which is equal to 0.818 g H2O/g of dry matter and 12 times greater than the monolayer. This means that the water activity of the product is high, exceeding 0.95, making it susceptible to unfavourable physicochemical and microbiological changes such as staling or mould development during the storage period. Therefore, protecting the bread by means of a modified atmosphere can play a crucial role. 3.8. Principal component analysis It is generally held that Principal Component Analysis (PCA) is a very convenient method for the multivariate characterization of food products (Baardseth et al., 2000). This method was applied in the present work to identify the most effective variables, point out their mutual relationships, and discriminate groups of samples indicating differences in bread characteristics at various stages of storage. In order to reduce variables for the PCA, correlation coefficients were calculated between the sensory attributes themselves as well as overall sensory quality. Since it was found that all correlations exceeded 0.98 (P < 0.01), overall sensory quality would appear to be a fairly accurate representation of specific sensory attributes in PCA. Table 2 illustrates the correlation coefficients between TPA texture measures, physicochemical variables and overall sensory quality. This table shows that correlations between WHC and the remaining variables are not significant. Similarly, there was no correlation between chewiness and other variables. All other variables correlated mutually, the correlation being very strong in some cases. Principal Component Analysis was carried out using data given in Table 1. The results for the five principal components (PCs) are presented in Table 3. The analysis shows that about 69% of the total variation is explained by the first PC, and that nearly 86% of the variance in the 10 considered variables can be represented by two new variables (PC1 and PC2). Table 4 and the loading plot (Fig. 2)
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Moisture content [g H2O/g d.m.]
0.818 = 45,0% water 0,8 0,7 0,6 0,5 0,4 0,3 0,2 BET=0,0685
0,1
Aw=0,953
Aw=0,214
0 0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
Water activity Fig. 1. Sorption isotherm of a calcium-enriched wholemeal bread.
demonstrate that overall sensory quality, hardness, cohesiveness and springiness, moisture content, blue value, acidity, and pH, were the most important variables for PC1. Thus, the first principal component is defined by eating quality (sensory, textural) and physicochemical attributes. These variables are situated far from the origin of PC1. In particular one textural (hardness) and one chemical parameter (acidity) placed to the left in the loading plot are close together and therefore positively correlated. Similarly, positive correlations exist between the remaining abovementioned variables important for PC1. The second component (PC2) explained about 16% of the variance. The dominant variable for this component is chewiness, although some other variables are also of significance. On the other hand, water holding capacity was the variable that was least represented by the first two PCs. The score plot (Fig. 2) on the axes of the first two PCs shows the distribution of calcium-enriched wholemeal bread at various stages of storage. It allows the identification of three groups: the first comprising fresh bread and bread after three days’ storage (PC1 > 0; PC2 > 0); the second bread stored for 6e20 days (PC2 < 0); and the third comprising the product in the final stages of storage, namely after 24, 27 and 32 days (PC1 < 0; PC2 > 0). 4. Discussion Bread, characterized by its attractive taste, is a basic food product for the majority of consumers all over the world, also satisfying their nutritional needs. Depending on its formulation,
bread may be enriched with ingredients other than standard. The present work provides an analysis of high-fibre, calcium-enriched bread. In order to extent its shelf-life, the bread was packed in a modified atmosphere, which, according to a number of studies, has a beneficial effect on the stability of the stored food (Kotsianis et al., 2002). A significantly higher concentration of carbon dioxide together with the elimination of oxygen provided storage conditions, which allowed the product to maintain acceptable sensory quality for almost 4 weeks. The higher concentration of carbon dioxide in the modified atmosphere may lead to increased perceived acidity in the taste. Wholemeal bread tends to be relatively sour and therefore, in this case, the possible effect of CO2 would not appear to be significant. Indeed, sourness was not perceived in either the sensory evaluation or pH measurements despite a slight increase in titratable acidity. The quality decline of bread is affected by two fundamental processes: staling and the growth of microorganisms, mainly moulds. However, according to Rasmussen and Hansen (2001), who investigated the extension of storage time of wheat bread, a modified atmosphere does not adversely affect starch retrogradation and the staling rate of bread, but rather has a remarkably positive effect on its microbial stability. Our findings showed good microbial stability of calcium-enriched wholemeal bread, proving that a modified atmosphere effectively protects the bread against the growth of moulds and bacteria. Slight proliferation was detected but only after the product had been assessed as unacceptable in the sensory evaluation.
Table 2 Correlation coefficients between calcium-enriched wholemeal bread variables.
OSQ H S C Ch MC WHC BV A
pH
A
BV
WHC
MC
Ch
C
S
H
0.84** 0.79** 0.79** 0.84** ns 0.64* ns 0.68* 0.78**
0.96** 0.95** 0.84** -0.94** ns 0.88** ns 0.86**
0.77** 0.78** 0.95** 0.93** ns 0.68* ns
ns ns ns ns ns ns
0.95** 0.96** 0.65* 0.75** ns
ns ns ns ns
0.88** 0.86** 0.93**
0.78** 0.77**
0.99**
*P < 0.05; **P < 0.01, levels of significance, ns: not significant (P > 0.05). OSQ: overall sensory quality; H: hardness; S: springiness; C: cohesiveness; Ch: chewiness; MC: moisture content; WHC: water holding capacity; BV: blue value; A: acidity.
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second stage being performed by the consumer at home or by retail outlets. Fik and Surówka (2002) revealed that the optimal time for the initial prebaking is within a wide range of 74e86% of the time required for the complete baking of 0.5 kg unfrozen wheat bread. This study observed that the storage of bread led to an increase in hardness and a decrease in springiness and cohesiveness, the latter two parameters declining sharply during the first week. Similarly, during the early stages of storage, the most pronounced decline was noted in springiness and cohesiveness of bread stored in an unmodified atmosphere (in air) in polyethylene bags at ambient temperature (Fik et al., 2000). Chewiness is defined as the energy required to masticate solid food to make it ready for swallowing. In the view of instrumental texture profile analysis is expressed by multiplying hardness, springiness and cohesiveness together (Szczesniak, 1995). Thus, the decline in chewiness observed here in the first days of storage was affected by decreasing springiness and cohesiveness, whereas its subsequent increase, recorded after the second week of storage, was mainly the result of increasing bread hardness, as the latter two parameters stabilized after the initial period of storage. In this investigation, detrimental changes in bread texture during storage were accompanied by decreasing moisture content in the crumb. According to a number of authors (Zeleznak and Hoseney, 1986; Eliasson and Larsson, 1993), texture deterioration corresponds to changes in water content within a storage period, although, as Czuchajowska and Pomeranz (1989) claimed, these changes are not responsible for bread staling. The above may be explained by the fact that they are accompanied by a hardly noticeable fall in water activity throughout storage. In bakery products water is quite tightly bound by large quantities of hydrophilic groups of starch, gluten and by other biopolymers as well as, in this study, calcium carbonate added as an enriching agent. A sigmoidal plot of the sorption isotherm typical for macromolecules with hydrophilic moieties capable of binding water confirms this phenomenon. On the other hand, intensive amylose retrogradation was recorded throughout the storage of bread, especially during the first week, reflecting a decline in blue value. As the water released in this process has the characteristics of free water, it intensifies the drying process observed in this experiment. However, these
Table 3 Results from the principal component analysis (PCA) for the first five principal components. Principal component
PC1 PC2 PC3 PC4 PC5
Eigenvalue
Explained variance (%)
For PC
Cumulative
By PC
Cumulative
6.944 1.613 1.082 0.209 0.085
6.944 8.557 9.638 9.847 9.932
69.44 16.13 10.82 2.09 0.85
69.44 85.57 96.38 98.47 99.32
Table 4 Principal component loadings for the first two principal components. Variable
PC1
PC2
Overall sensory quality (OSQ) Hardness (H) Springiness (S) Cohesiveness (C) Chewiness (Ch) Moisture content (MC) Water holding capacity (WHC) Blue value (BV) Acidity (A) pH
0.369 0.366 0.341 0.364 0.045 0.335 0.105 0.335 0.369 0.328
0.175 0.203 0.305 0.190 0.776 0.330 0.118 0.245 0.053 0.110
Texture is a crucial quality attribute for bakery goods. Texture profile analysis was therefore conducted to assess the dynamics of changes in this indicator. Our findings revealed that the bread examined is strongly prone to textural changes throughout storage. Patel et al. (2005) claimed that such changes are significantly affected by the heating rate during baking, which, in turn, was responsible for the extent of hydration and swelling of starch granules, disordering of amylopectin crystals, amylose leaching and, as a result, the effective formation of crumb structure. However, the mechanism of bread firming still awaits detailed explanation. Starch-gluten interactions, leading to cross-linkage of gluten by gelatinized starch, play a significant role; however, it has also been found that gluten-free bakery products will firm even faster than bread containing gluten (Gambus, 2005). The problem of staling is commonly solved by a two-stage baking process, the
a
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Fig. 2. PCA loading (a) and score (b) plots in the plane of principal component 1 vs. principal component 2. The labels correspond to variables listed in Table 2 and samples stored throughout the different period of time (in days), respectively.
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phenomena did not result in pronounced changes in the water holding capacity of the crumb. The above observations were confirmed by the results of the PC analysis. There is a regularity between samples situated in a particular direction in relation to the axis origin in the score plot and the high values for variables placed in the same direction in the loading plot. This demonstrates that freshly baked bread and bread in the early stages of storage had good springiness and cohesiveness together with a high blue value (Fig. 2). Such bread scored the highest values in the sensory evaluation as well as having low hardness and sourness. Bread from the second group obtained lower scores for overall sensory evaluation, springiness, cohesiveness and BV. Products in the third group (final stages of storage) exhibited a clear increase in the chewiness and hardness of bread. Thus, in view of the arrangement of samples in relation to variables, MAP storage can be seen to have resulted in substantial differences in the characteristics of calcium-enriched wholemeal bread. However, despite such differences, the modified atmosphere applied (60% CO2 and 40% N2) extends consumer acceptability of the product even up to 24 days. References Baardseth, P., Kvaal, K., Lea, P., Ellekjaer, M.R., Faergestat, E.M., 2000. The effect of bread making process and wheat quality on French baguettes. Journal of Cereal Science 32, 73e87. Begum, R., Rakshit, S.K., Rahman, S.M.M., 2011. Protein fortification and use of cassava flour for bread formulation. International Journal of Food Properties 14, 185e198. Breene, W.M., 1975. Application of texture profile analysis to instrumental food texture evaluation. Journal of Texture Studies 6, 53e82. Burrington, K.J., 1998. Prolonging bakery product life. Food Product Design 7, 12e20. Czeizel, A.E., Merhala, Z., 1998. Bread fortification with folic acid, vitamin B12, and vitamin B6 in Hungary. Lancet 352, 1225. Czuchajowska, Z., Pomeranz, Y., 1989. Differential scanning calorimetry, water activity, and moisture contents in crumb centre and near-crust zones of bread during storage. Cereal Chemistry 66, 305e309. Dalgetty, D.D., Byung-Kee, Baik, 2006. Fortification of bread with hulls and cotyledon fibres isolated from peas, lentils, and chickpeas. Cereal Chemistry 83, 269e274. Eliasson, A.-C., Larsson, K., 1993. Cereals in Breadmaking, vol. 1. Marcel Dekker, New York. 249e370. Fennema, O.R., 1985. Water and ice. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekker Inc., New York, pp. 23e67. _ s Fik, M., 2004. Bread staling and methods of prolonging its freshness. Zywno c. Nauka. Technologia. Jakos c. 2, 5e22.
Fik, M., Celej, A., 1993. Some aspects of bread staling and methods of its restraining. Ch1odnictwo 28, 29e31. Fik, M., Michalczyk, M., Surówka, K., Maciejaszek, I., 2000. Characterisation of the staling process of wholemeal bread. Polish Journal of Food and Nutrition Sciences 9/50, 23e28. Fik, M., Surówka, K., 2002. Effect of prebaking and frozen storage on the sensory quality and instrumental texture of bread. Journal of the Science of Food and Agriculture 82, 1268e1275. Gambus, H., 2005. Linseed (Linum uistatissimum L.) as a source of nutrients in gluten _ s - free bread. Zywno c. Nauka. Technologia. Jakos c. 4 (Suppl.), 61e74. Gerrard, J.A., Every, D., Sutton, K.H., Gilpin, M.J., 1997. The role of maltodextrins in the staling of bread. Journal of Cereal Science 26, 201e209. Hoseney, C., Miller, R., 1998. Current Understanding of Staling of Bread. Technical Bulletin, vol. 20. American Institute of Baking Research Department. 1e6. Karadzhov, G., Iserliyska, D., 2003. Sensory quality of minerally fortified bread. European Food Research and Technology 216, 274e276. Karaoglu, M.M., Kotancilar, H.G., Gurses, M., 2005. Microbiological characteristics of part-baked white pan bread during storage. International Journal of Food Properties 8, 355e365. Kotsianis, I.S., Giannou, V., Tzia, C., 2002. Production and packaging of bakery products using MAP technology. Trends in Food Science & Technology 13, 319e324. Leung, H.K., 1986. Water activity and other coligative properties of food. In: Okos, M.R. (Ed.), Physical and Chemical Properties of Food. American Society of Agricultural Engineers, pp. 138e185. Odds, F.C., 1991. Sabouraud(‘s) agar. Journal of Medical and Veterinary Mycology 29, 355e359. Park, H., Seib, P.A., Chung, O.K., Seitz, L.M., 1997. Fortifying bread with each of three antioxidants. Cereal Chemistry 74, 202e206. Patel, B.K., Waniska, R.D., Seetharaman, K., 2005. Impact of different baking processes on bread firmness and starch properties in breadcrumb. Journal of Cereal Science 42, 173e184. Phillips, C.A., 1996. Modified atmosphere packaging and its effects on the microbiological quality and safety of produce. International Journal of Food Science and Technology 31, 463e479. Polish Standard PN-A-74102, 1999. Bakery Products and Semi-products e Sampling and Microbiological Examination. Polish Committee for Standardization. Polish Standard PN-A-74134-4, 1998. Pastry Products and Semi-products. Microbiological Examination. Determination of Number of Spore Forming Bacteria of Decomposing Starch. Polish Committee for Standardization. Rasmussen, P.H., Hansen, A., 2001. Staling of wheat bread in modified atmosphere. Lebensmittel Wissenshaft und Technologie 34, 487e491. Smith, J.P., 1993. Bakery products. In: Parry, R.T. (Ed.), Principles and Applications of Modified Atmosphere Packaging of Foods. Blackie Academic and Professional, Glasgow, pp. 134e169. Szczesniak, A.S., 1963. Classification of textural characteristics. Journal of Food Science 28, 385e389. Szczesniak, A.S., 1995. Texture profile analysis - methodology interpretation clarified. Journal of Food Science 60 (6), vii. Zeleznak, K.J., Hoseney, R.C., 1986. The role of water in the retrogradation of wheat starch gels and bread crumb. Cereal Chemistry 63, 407e411.
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Quality and shelf life of calcium-enriched wholemeal bread stored in a modified atmosphere M. Fik, K. Surówka*, I. Maciejaszek, M. Macura, M. Michalczyk Department of Refrigeration and Food Concentrates, University of Agriculture in Krakow, Balicka Street 122, PL-30-149 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2012 Received in revised form 11 June 2012 Accepted 12 June 2012
Calcium-enriched wholemeal bread packed in a modified atmosphere (60% CO2, 40% N2), was examined for stability and microbiological changes throughout 32-day storage. The product was still acceptable after 24 days at 20 1 C. At this time no microbiological changes were observed; however, there was a continuous decline in sensory quality, mainly due to deterioration of the texture. Crumb hardness increased, whereas its springiness and cohesiveness decreased. These observations were accompanied by physical and chemical changes characterized by a steady increase in acidity and a sharp decrease in blue value, especially at the beginning of storage. The first microbiological changes occurred only after 27 days of storage and were due to the growth of moulds and amylase-negative Gram-positive cocci, coccibacillus or bacillus. The principal component analysis showed that nearly 86% of the variance in ten considered variables could be represented by two new variables: PC1, defined by eating quality and physicochemical attributes, and PC2 defined mainly by chewiness. Three groups of stored bread were identified on the score plot. The first group, fresh and 3-day stored bread, was characterized by low hardness and sourness; the second, bread stored from six to twenty days, scored lower for overall sensory quality, low springiness, cohesiveness and blue value; and the third group, the product during the final stages of storage, exhibited a clear increase in chewiness and hardness and showed the first indications of microbial deterioration. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Enriched wholemeal bread Bread shelf life Modified atmosphere packaging Textural properties
1. Introduction Bakery products are characterized by short-term stability and limited shelf life due to the rapid course of staling (Fik, 2004). This complex and not fully investigated process is generally believed to stem from manifold changes in the composition of bread, caused by factors other than microbial growth, which result in a loss of freshness and a reduction in quality. Such changes cause a deterioration of both sensory attributes and physicochemical properties, especially in the structure and texture of the bread’s crumb and crust. The reduction observed in starch solubility is also a significant factor, correlating with increasing starch recrystallization and a fall in water holding capacity in the bread crumb during storage
Abbreviations: A, acidity; BV, blue value; C, cohesiveness; CAP, controlled atmosphere packaging; Ch, chewiness; H, hardness; MA, modified atmosphere; MAP, modified atmosphere packaging; MC, moisture content; OSQ, overall sensory quality; PCA, principial component analysis; PC1, principial component 1; PC2, principial component 2; S, springiness; TPA, texture profile analysis; WHC, water holding capacity. * Corresponding author. Tel.: þ48 12 662 47 59; fax: þ48 12 662 47 58. E-mail address: [email protected] (K. Surówka). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2012.06.006
(Hoseney and Miller, 1998). The intensity of such processes increased throughout storage depending on the conditions applied, resulting in the following changes being observed: an increase in the hardness, dryness and crumbliness of the crumb along with a fall in its elasticity; a decrease in the crispness of the crust; and a decline in aroma and other characteristic attributes of bread freshness. All this is evidence of staling, an indicator of the limited shelf life of bread and a phenomenon that may to some extent be explained by moisture migration from the crumb to the crust due to changes in colloid structure (Burrington, 1998) and not necessarily by moisture loss. Thus, retaining the freshness of bread remains an important problem for both consumers and bakers, which explains why bread ageing and the retardation of this process are widely reported in the literature (Fik and Celej, 1993; Fik et al., 2000; Gerrard et al., 1997; Patel et al., 2005). The practice of packaging and storing bread in a modified atmosphere (MAP) is becoming increasing common as a method of extending its shelf life and maintaining good quality. Many authors have confirmed the benefits of this method in the storage of bakery products (Smith, 1993; Kotsianis et al., 2002). Some of them emphasize its role in extending the microbial shelf life of bread (Phillips, 1996). According to Smith (1993), the application of MAP
M. Fik et al. / Journal of Cereal Science 56 (2012) 418e424
extends shelf life in three ways: chemically, it controls biochemical and degradation processes and slows oxidation; microbiologically, MAP may increase shelf life by inhibiting the growth of mould and bacteria; and physically, MAP lengthens a product’s stability by reducing moisture loss. It is similar to controlled atmosphere packaging (CAP) in that a specific gas mixture is applied to the product but differs in the level of control. Bread can be packed in a modified atmosphere in order to delay staling and hinder the growth of moulds using 60% to 80% (v/v) carbon dioxide and 20% to 40% nitrogen. Since bread is one of the most frequently consumed food products, it is reasonable to enrich it with nutritive substances. Commonly supplemented with protein preparations (Begum et al., 2011), bread is also frequently fortified with minerals (Karadzhov and Iserliyska, 2003) and vitamins (Czeizel and Merhala, 1998). Antioxidant substances may be added to bread to achieve healthpromoting effects (Park et al., 1997), while other recipes may enrich the product with dietary fibre (Dalgetty and Byung-Kee, 2006). However, the beneficial nutritional effects of such enrichment do not always correlate positively with the storage stability of bread. Therefore, the aim of this paper was to investigate the effect of a modified atmosphere (60% CO2 and 40% N2) on the quality and shelf life of calcium-enriched wholemeal bread stored at room temperature (20 1 C). In contrast to bread generally available, such bread shows health-promoting properties due to higher levels of dietary fibre and calcium. The use of principal component analysis (PCA) enabled an evaluation of the interrelations between sensory quality, instrumentally measured textural properties and physicochemical indicators important for the consumer and the baker. Microbiological analyses were also carried out. 2. Experimental material 2.1. Bread making and packaging Calcium-enriched wholemeal bread was produced under industrial conditions during one shift in a bakery packing bread in a modified atmosphere. The bread was made according to the following dough formulation: 34.7 parts (weight basis) Graham wheat flour type 1850 (according to ash content) containing 11.4% of protein and characterized by 27% wet gluten yield and a falling number value of 210 s; 6.4 rye flour type 720 (6.8% protein, a falling number value of 170 s); 6.2 wheat flour type 750 (11.9% protein, 33% wet gluten and a falling number value of 320 s); 28.0 leaven; 20.0 water; 1.7 whey; 1.2 salt; 1.6 baker’s yeast and 0.2 calcium carbonate Calcipur 2 OG with granulometry 45 mm (90%). The flours were purchased in ZPZ Szymanów, Polskie M1yny (Teresin, Poland); leaven was obtained due to spontaneous fermentation of the rye flour type 720 with water (1:3); whey was provided by a local dairy; baker’s yeast originated from the Lesaffre S.A. (Wo1czyn, Poland), while calcium carbonate from the Brenntag Sp. z o.o (Ke˛ dzierzyn-Ko zle, Poland). All ingredients were mixed in _ a Kemper SP125 spiral mixer (Sispo, Znin, Poland) for 6 min at low speed (105 rpm) followed by 6 min at higher speed (210 rpm). The resulting dough was left to rise for 90 min at 30 C and then punched down. After shaping, placing in loaf tins and final fermentation for 40 min at 30 C, loaves were baked at 200 C for 45 min in a conventional oven, cooled at room temperature and packed in polyamide/polyethylene bags in a modified atmosphere (60% CO2, 40% N2). The bags used fulfilled the EC 90/128/EEC Regulation on materials intended to come into contact with food and were characterized by total thickness of 80 mm and maximum O2, CO2 and H2O permeability of 100 cm3/(m2 24 h bar), 140 cm3/(m2 24 h bar) and 8 g/(m2 24 h bar), respectively.
419
The bread was stored in cardboard boxes at room temperature (20 1 C) for up to 32 days. Samples were examined at regular intervals. 2.2. Sensory evaluation In the sensory evaluation, each attribute of crumb (taste, smell, and elasticity) and crust (taste, smell, and crispness) was assessed using a 5-point scale (Fik et al., 2000). Each evaluation was carried out by nine panellists appropriately trained and tested for sensory sensitivity. Because of the different relative importance of particular attributes to the overall sensory quality of the product, the following contribution coefficients were used: 0.5 for taste and smell of crumb; 0.2 for taste and smell of crust; 0.2 for crumb elasticity; and 0.1 for crust crispness. In the overall sensory quality assessment, scores of 5, 4, 3, 2 and 1 corresponded to evaluations of very good, good, acceptable, unacceptable, and bad respectively. 2.3. Microbiological analyses For the microbiological analysis of bread, 10 g of crumb sample was diluted with 90 ml of 0.1% peptone solution and blended for 2 min in a Stomacher 80 (Seward, England). Serial decimal dilutions were made using 0.1% peptone solution. Total aerobic mesophylic bacteria count was determined on plate count agar medium (Karaoglu et al., 2005) after incubation at 30 C for 72 h. Yeasts and moulds were determined on plates with Sabouraud agar medium, incubated at 25 C for 3e5 days (Odds, 1991). The levels of vegetative and spore-forming amylolytic aerobic bacteria were assayed on agar medium with starch, according to Waksman (Polish Standard PN-A-74134-4, 1998). In addition, the microbial stability of bread was evaluated by means of thermostat trials (at 30 C for 30 days), in which samples of bread packed in a modified atmosphere were analyzed in terms of sensory changes resulting from mould growth (Polish Standard PN-A-74102, 1999). Additionally, samples of fresh bread and bread stored in MAP for 3, 6 and 8 days were thermostated at 37 C for 3 days in Weck jars to investigate changes in the aroma and appearance of the product due to the growth of spore-forming amylolytic aerobic bacteria (Polish Standard PN-A-74102, 1999). 2.4. Textural analysis Measurements of bread texture were carried out using a TA-XT2 Texture Analyser (Stable Micro Systems, England) coupled with a computer equipped with XT.RA Dimension, v.3.7 software, enabling the instrument to be controlled and data to be processed automatically. Crumb samples (in the form of cubes of 60 mm side) of individual leaves baked from the same batch of dough were subjected to texture profile analysis (TPA) (Breene, 1975; Szczesniak, 1963). The samples were compressed twice with an aluminium cylinder-shaped plunger with a diameter of 90 mm to a depth of 36 mm (40% strain). The plunger used moved at a rate of 2 mm/s and the time between strokes was 20 s. The apparatus recorded the force exerted by the plunger as a function of time, from which the following texture parameters were determined: hardness, springiness, cohesiveness and chewiness. Hardness was defined as the peak force [N] during the first compression cycle. Cohesiveness was calculated as a ratio of areas delimited by the curves of the second and first bite. Springiness was determined as a ratio of the time measured between the start of the second area and the second probe reversal divided by the time measured between the start of the first area and the first probe reversal. Chewiness [N] was assessed by multiplying hardness, cohesiveness and springiness.
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2.5. Moisture content and water holding capacity
in terms of millilitres of sodium hydroxide (0.1 M) solution required to neutralize the free acids contained in 100 g of the bread crumb.
Moisture content of the breadcrumb was determined after oven-drying at 105 C until attaining a constant weight. Water holding capacity (WHC) was measured as follows: the crumb (10 g) was gently homogenized with 50 ml of water in a grinder (Heidolph Diax 900, Schwabach, Germany) for 2 min. Then the homogenate was mixed at room temperature for 20 min using a magnetic stirrer. The slurry was centrifuged (1600 g, 10 min) following 20 min of holding. The sediment weight was recorded, whereas the supernatant was used for the determination of blue value. The amount of water in the sediment was calculated by subtracting the dry sample weight. WHC was expressed as grams of water in the sediment per gram of dry sample weight.
2.8. Sorption isotherm The moisture sorption isotherm was plotted for the bread on the basis of data obtained after balancing the dry matter at 20 C in desiccators containing saturated salt solutions of different water activities (Leung, 1986). The monolayer capacity was determined applying the BET equation given by Brunauer, Emmett and Teller and using the data obtained for the low-moisture end of the moisture sorption isotherm (Fennema, 1985). 2.9. Statistical analysis
2.6. Blue value determination The complex of amylose with iodine produces a blue colour. Its intensity can be used to indicate the level of free amylose in a soluble fraction, which informs about starch retrogradation. The blue value was determined by a spectroscopic method. To this end, 20 ml of the supernatant obtained using the method described for WHC, was poured into a centrifuge tube. Next, 2 ml of Carrez I and 2 ml Carrez II solutions were added to precipitate the protein matter. The whole sample was carefully mixed, allowed to stand for 10 min and finally centrifuged (5000 g, 5 min). Afterwards, 4 ml of the supernatant obtained was mixed with 25 ml of iodine solution (0.004%); The blue value was expressed as the absorbance measured at 580 nm using distilled water as a blank. 2.7. pH and acidity For pH determination, 10 g of the crumb was homogenized with 50 ml of distilled water using an RW 11 basic blender (IKA - Werke GMBH & Co. KG, Germany) for 30 s at high speed. The pH of prepared homogenates was determined with a Hanna Instruments pH meter (Model HI 9025, England) equipped with a combined electrode. In order to measure acidity, 25 g of the crumb was shaken for 2 min with distilled water (250 ml) in an Erlenmeyer flask. Then the sample was allowed to stand for 1 h, during which time it was shaken at 15-min intervals. It was then filtered through cotton wool into a dry Erlenmeyer flask. Finally, 50 ml of filtrate was titrated with sodium hydroxide (0.1 M) in the presence of phenolphthalein indicating solution until attaining a constant (not changing within 1 min) light-pink colour. The degree of bread acidity was expressed
Textural measurements were performed nine-fold, whereas physico-chemical analyses were done in triplicate. The results obtained were analyzed statistically using the CSS Statistica v. 9.1, (StatSoft, Inc., Tulsa OK, USA) package. Mean values and standard deviations were calculated for each texture characteristic and physico-chemical measure. ANOVA variance analysis at P 0.05 confidence level for comparing means of variables and principal component analysis (PCA) was used to analyse relationships between variables. 3. Results 3.1. Sensory quality Table 1 shows the changes in overall sensory quality of calciumenriched wholemeal bread during storage in a modified atmosphere. The initial quality of the bread was found to be very good. All sensory attributes scored the maximum value 5. Crumb and crust were characterized by the intensive taste and aroma typically associated with fresh bread. Of the other attributes, crust crispness and crumb elasticity achieved the highest scores. Bread quality was still found to be fairly good after 11 days of storage. However, with continued storage, crumb elasticity was the attribute most prone to rapid deterioration, although this characteristic is not a very important quality factor for this type of bread, which is firm and of low elasticity by nature. The loaf appearance remained almost unchanged during the whole period of storage, making it impossible to evaluate how long the bread had been stored. The product was still acceptable after 24 days of MAP storage; however, after 27 days the panellists found it unacceptable.
Table 1 Effect of storage time in modified atmosphere on overall sensory quality, TPA texture measures for crumb, and some physicochemical changes in calcium-enriched wholemeal bread. Storage time (days)
Overall sensory quality
Hardness (N)
0 3 6 8 11 14 17 20 24 27 32
5.0a 4.9a 4.6 4.2 3.9b 3.8b 3.7bc 3.5c 3.2 2.9d 2.7d
57.48 5.80 64.86 6.71 72.53 7.30 82.03 7.80a 87.50 8.84ab 93.32 9.12bc 95.67 9.68cd 100.61 10.41d 112.32 11.30 118.41 11.50 130.52 12.00
Springiness
0.83 0.05 0.70 0.03a 0.69 0.05a 0.61 0.05b 0.60 0.08b 0.59 0.03b 0.60 0.05b 0.60 0.06b 0.60 0.02b 0.60 0.05b 0.59 0.04b
Cohesiveness
0.30 0.02a 0.28 0.02a 0.23 0.02b 0.22 0.03bc 0.21 0.03bc 0.21 0.01bc 0.21 0.02bc 0.20 0.03cd 0.20 0.02cd 0.20 0.03cd 0.19 0.03d
Chewiness (N)
14.31 1.74a 12.71 2.7bc 11.51 2.61bc 11.01 2.80c 11.02 2.14c 11.56 1.33c 12.05 2.50b 12.07 2.03bc 13.48 2.30ab 14.21 2.10a 14.63 2.37a
Moisture content (%)
Water holding capacity (g water/g dry sample)
Blue value
45.00 1.12 43.52 1.50a 43.83 0.76a 43.77 0.76a 42.80 1.29ab 42.00 0.85b 41.54 1.54b 39.10 0.29c 38.65 0.39c 38.00 0.84c 37.05 0.05
3.17 0.00a 2.88 0.02b 2.96 0.08bc 3.05 0.05c 3.04 0.05c 3.31 0.04d 3.35 0.06d 3.16 0.05a 3.12 0.04a 3.07 0.07c 3.06 0.02c
0.66 0.02 0.37 0.03 0.27 0.02a 0.25 0.01ab 0.24 0.01b 0.23 0.02bc 0.22 0.01c 0.21 0.01c 0.22 0.01c 0.21 0.01c 0.12 0.01
Results are means SD; Means in columns not denoted or denoted with different letters are significantly different (P 0.05).
580nm A 1cm
Acidity (degree)
pH
2.63 0.06a 2.73 0.11a 2.99 0.10b 3.03 0.07b 3.07 0.07b 3.12 0.10b 3.03 0.17b 3.17 0.05 3.30 0.05c 3.35 0.03c 3.36 0.06c
5.67a 5.68a 5.67a 5.65a 5.64a 5.65a 5.64a 5.65a 5.65a 5.64a 5.64a
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3.2. Microbiological analyses
3.6. Acidity and pH
Microbiological analysis of the bread prior to storage showed that the initial total bacteria count as well as yeast and mould levels were below 10 cfu/g. At this stage the bread did not contain any spore-forming amylolytic bacteria or their spores. Throughout storage at 20 1 C, no microbiological changes were observed until the 24th day. Thereafter, especially after 27 and 32 days of storage, the presence of yeasts and moulds 2 101e3 103 cfu/g and bacterial growth 2 102e2 103 cfu/g were detected. The bacteria formed very small white colonies on plate count agar and Waksman medium. Microscopic analysis revealed them to be mainly Gram-positive cocci, cocci-bacillus or bacillus, all of them amylase-negative. Thermostat trials carried out after 0, 3, 6 and 8 days of storage to evaluate sensory changes due to the growth of aerobic amylolytic bacteria were negative for all the breads examined. Breads thermostated at 30 C showed a low incidence of moulds (occurring only in individual samples stored in a modified atmosphere).
The acidity of the bread showed a slight tendency to increase over the entire storage period (Table 1). However, the degree of change was insufficient to have a perceptible effect on taste and flavour, the attributes examined in sensory evaluation. No significant changes were observed in pH throughout the storage period, remaining more or less constant at around 5.65. Table 1 shows that after 32 days’ storage, the concentration of hydronium ions in highcalcium wholemeal bread packed in MA was almost identical to that in the fresh product even though acidity increased by nearly 28% during this period.
3.3. Texture Individual texture characteristics of crumb as revealed by analyses carried out at regular intervals throughout the storage period are given in Table 1. In the fresh product, the values of all the textural parameters measured were characteristic of a high-quality bread. The highest values were for springiness, cohesiveness and chewiness, while the lowest for hardness. However, after only 3 days of storage, negative changes were recorded for each of these parameters; crumb hardness increased by almost 13% and springiness, cohesiveness and chewiness decreased by about 15, 7 and 11% respectively. During further MA storage, the greatest changes were noted in hardness, which increased steadily so that after 32 days it was 73 N greater than in fresh bread. Springiness and cohesiveness, on the other hand, decreased up to the 8th day of storage; thereafter it remained stable until the end of the experiment. Chewiness, which initially decreased to approximately 11 N after 8 days of storage, then rose steadily, and after 32 days reached a slightly higher value than in the fresh bread. 3.4. Moisture content and water holding capacity Table 1 presents the results of moisture content and water holding capacity (WHC) of bread during MA storage. The moisture content of calcium-enriched wholemeal bread fell gradually during storage corresponding to changes in sensory quality. The fresh product, which obtained the highest scores in the sensory evaluation, had the highest moisture content. Increasing dry matter content had a detrimental effect on the sensory attributes of bread. The data show that 3 days of storage reduced WHC by 9% compared with the initial value. During further storage, the value for this indicator increased gradually to 3.35 g of water per g of dry sample after 17 days. After a subsequent decrease, the value at the end of experiment was close to that in fresh bread. 3.5. Blue value The blue value defines the content of water-soluble starch, so its amount in crumb generally decreases as bread stales. In calciumenriched wholemeal bread packed in MA, this value decreased considerably (by 44% compared to the initial value) after only 3 days (Table 1), and after 6 days a further fall of 15% was recorded. During subsequent storage, the changes observed in the blue value were not so pronounced.
3.7. Sorption isotherm The sorption isotherm established for high-calcium wholemeal bread is sigmoidal in shape and falls into type II (Fig. 1). The shape is typical for food products containing high-molecular substances. In bread these are chiefly starch and gluten, although in the case of wholemeal bread, non-starch polysaccharides such as pentosans should also be considered. The BET monolayer value of the bread examined was 0.0685 g H2O/g d.m., which corresponds to Aw ¼ 0.214. At this value, a product is covered by a monolayer of water, which ensures the best storage stability of the product by protecting non-water substances. At the same time, the amount of water is insufficient to become a reagent or medium for unfavourable processes of a physical, chemical or microbiological nature. Bread distributed in retail outlets contains significantly more water than the monolayer value. In the product examined it was 45%, which is equal to 0.818 g H2O/g of dry matter and 12 times greater than the monolayer. This means that the water activity of the product is high, exceeding 0.95, making it susceptible to unfavourable physicochemical and microbiological changes such as staling or mould development during the storage period. Therefore, protecting the bread by means of a modified atmosphere can play a crucial role. 3.8. Principal component analysis It is generally held that Principal Component Analysis (PCA) is a very convenient method for the multivariate characterization of food products (Baardseth et al., 2000). This method was applied in the present work to identify the most effective variables, point out their mutual relationships, and discriminate groups of samples indicating differences in bread characteristics at various stages of storage. In order to reduce variables for the PCA, correlation coefficients were calculated between the sensory attributes themselves as well as overall sensory quality. Since it was found that all correlations exceeded 0.98 (P < 0.01), overall sensory quality would appear to be a fairly accurate representation of specific sensory attributes in PCA. Table 2 illustrates the correlation coefficients between TPA texture measures, physicochemical variables and overall sensory quality. This table shows that correlations between WHC and the remaining variables are not significant. Similarly, there was no correlation between chewiness and other variables. All other variables correlated mutually, the correlation being very strong in some cases. Principal Component Analysis was carried out using data given in Table 1. The results for the five principal components (PCs) are presented in Table 3. The analysis shows that about 69% of the total variation is explained by the first PC, and that nearly 86% of the variance in the 10 considered variables can be represented by two new variables (PC1 and PC2). Table 4 and the loading plot (Fig. 2)
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Moisture content [g H2O/g d.m.]
0.818 = 45,0% water 0,8 0,7 0,6 0,5 0,4 0,3 0,2 BET=0,0685
0,1
Aw=0,953
Aw=0,214
0 0
0,1
0,2
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0,4
0,5
0,6
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1
Water activity Fig. 1. Sorption isotherm of a calcium-enriched wholemeal bread.
demonstrate that overall sensory quality, hardness, cohesiveness and springiness, moisture content, blue value, acidity, and pH, were the most important variables for PC1. Thus, the first principal component is defined by eating quality (sensory, textural) and physicochemical attributes. These variables are situated far from the origin of PC1. In particular one textural (hardness) and one chemical parameter (acidity) placed to the left in the loading plot are close together and therefore positively correlated. Similarly, positive correlations exist between the remaining abovementioned variables important for PC1. The second component (PC2) explained about 16% of the variance. The dominant variable for this component is chewiness, although some other variables are also of significance. On the other hand, water holding capacity was the variable that was least represented by the first two PCs. The score plot (Fig. 2) on the axes of the first two PCs shows the distribution of calcium-enriched wholemeal bread at various stages of storage. It allows the identification of three groups: the first comprising fresh bread and bread after three days’ storage (PC1 > 0; PC2 > 0); the second bread stored for 6e20 days (PC2 < 0); and the third comprising the product in the final stages of storage, namely after 24, 27 and 32 days (PC1 < 0; PC2 > 0). 4. Discussion Bread, characterized by its attractive taste, is a basic food product for the majority of consumers all over the world, also satisfying their nutritional needs. Depending on its formulation,
bread may be enriched with ingredients other than standard. The present work provides an analysis of high-fibre, calcium-enriched bread. In order to extent its shelf-life, the bread was packed in a modified atmosphere, which, according to a number of studies, has a beneficial effect on the stability of the stored food (Kotsianis et al., 2002). A significantly higher concentration of carbon dioxide together with the elimination of oxygen provided storage conditions, which allowed the product to maintain acceptable sensory quality for almost 4 weeks. The higher concentration of carbon dioxide in the modified atmosphere may lead to increased perceived acidity in the taste. Wholemeal bread tends to be relatively sour and therefore, in this case, the possible effect of CO2 would not appear to be significant. Indeed, sourness was not perceived in either the sensory evaluation or pH measurements despite a slight increase in titratable acidity. The quality decline of bread is affected by two fundamental processes: staling and the growth of microorganisms, mainly moulds. However, according to Rasmussen and Hansen (2001), who investigated the extension of storage time of wheat bread, a modified atmosphere does not adversely affect starch retrogradation and the staling rate of bread, but rather has a remarkably positive effect on its microbial stability. Our findings showed good microbial stability of calcium-enriched wholemeal bread, proving that a modified atmosphere effectively protects the bread against the growth of moulds and bacteria. Slight proliferation was detected but only after the product had been assessed as unacceptable in the sensory evaluation.
Table 2 Correlation coefficients between calcium-enriched wholemeal bread variables.
OSQ H S C Ch MC WHC BV A
pH
A
BV
WHC
MC
Ch
C
S
H
0.84** 0.79** 0.79** 0.84** ns 0.64* ns 0.68* 0.78**
0.96** 0.95** 0.84** -0.94** ns 0.88** ns 0.86**
0.77** 0.78** 0.95** 0.93** ns 0.68* ns
ns ns ns ns ns ns
0.95** 0.96** 0.65* 0.75** ns
ns ns ns ns
0.88** 0.86** 0.93**
0.78** 0.77**
0.99**
*P < 0.05; **P < 0.01, levels of significance, ns: not significant (P > 0.05). OSQ: overall sensory quality; H: hardness; S: springiness; C: cohesiveness; Ch: chewiness; MC: moisture content; WHC: water holding capacity; BV: blue value; A: acidity.
M. Fik et al. / Journal of Cereal Science 56 (2012) 418e424
second stage being performed by the consumer at home or by retail outlets. Fik and Surówka (2002) revealed that the optimal time for the initial prebaking is within a wide range of 74e86% of the time required for the complete baking of 0.5 kg unfrozen wheat bread. This study observed that the storage of bread led to an increase in hardness and a decrease in springiness and cohesiveness, the latter two parameters declining sharply during the first week. Similarly, during the early stages of storage, the most pronounced decline was noted in springiness and cohesiveness of bread stored in an unmodified atmosphere (in air) in polyethylene bags at ambient temperature (Fik et al., 2000). Chewiness is defined as the energy required to masticate solid food to make it ready for swallowing. In the view of instrumental texture profile analysis is expressed by multiplying hardness, springiness and cohesiveness together (Szczesniak, 1995). Thus, the decline in chewiness observed here in the first days of storage was affected by decreasing springiness and cohesiveness, whereas its subsequent increase, recorded after the second week of storage, was mainly the result of increasing bread hardness, as the latter two parameters stabilized after the initial period of storage. In this investigation, detrimental changes in bread texture during storage were accompanied by decreasing moisture content in the crumb. According to a number of authors (Zeleznak and Hoseney, 1986; Eliasson and Larsson, 1993), texture deterioration corresponds to changes in water content within a storage period, although, as Czuchajowska and Pomeranz (1989) claimed, these changes are not responsible for bread staling. The above may be explained by the fact that they are accompanied by a hardly noticeable fall in water activity throughout storage. In bakery products water is quite tightly bound by large quantities of hydrophilic groups of starch, gluten and by other biopolymers as well as, in this study, calcium carbonate added as an enriching agent. A sigmoidal plot of the sorption isotherm typical for macromolecules with hydrophilic moieties capable of binding water confirms this phenomenon. On the other hand, intensive amylose retrogradation was recorded throughout the storage of bread, especially during the first week, reflecting a decline in blue value. As the water released in this process has the characteristics of free water, it intensifies the drying process observed in this experiment. However, these
Table 3 Results from the principal component analysis (PCA) for the first five principal components. Principal component
PC1 PC2 PC3 PC4 PC5
Eigenvalue
Explained variance (%)
For PC
Cumulative
By PC
Cumulative
6.944 1.613 1.082 0.209 0.085
6.944 8.557 9.638 9.847 9.932
69.44 16.13 10.82 2.09 0.85
69.44 85.57 96.38 98.47 99.32
Table 4 Principal component loadings for the first two principal components. Variable
PC1
PC2
Overall sensory quality (OSQ) Hardness (H) Springiness (S) Cohesiveness (C) Chewiness (Ch) Moisture content (MC) Water holding capacity (WHC) Blue value (BV) Acidity (A) pH
0.369 0.366 0.341 0.364 0.045 0.335 0.105 0.335 0.369 0.328
0.175 0.203 0.305 0.190 0.776 0.330 0.118 0.245 0.053 0.110
Texture is a crucial quality attribute for bakery goods. Texture profile analysis was therefore conducted to assess the dynamics of changes in this indicator. Our findings revealed that the bread examined is strongly prone to textural changes throughout storage. Patel et al. (2005) claimed that such changes are significantly affected by the heating rate during baking, which, in turn, was responsible for the extent of hydration and swelling of starch granules, disordering of amylopectin crystals, amylose leaching and, as a result, the effective formation of crumb structure. However, the mechanism of bread firming still awaits detailed explanation. Starch-gluten interactions, leading to cross-linkage of gluten by gelatinized starch, play a significant role; however, it has also been found that gluten-free bakery products will firm even faster than bread containing gluten (Gambus, 2005). The problem of staling is commonly solved by a two-stage baking process, the
a
b
Ch
1,0
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H A 0,0
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Principal component 2
2,0
0
32 27
1,5 1,0
24 3
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20
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6
17 14 11
-1,0 -1,5
8
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-0,5
0,0
0,5
Principal component 1
1,0
-2,5
-6
-4
-2
0
2
4
6
Principal component 1
Fig. 2. PCA loading (a) and score (b) plots in the plane of principal component 1 vs. principal component 2. The labels correspond to variables listed in Table 2 and samples stored throughout the different period of time (in days), respectively.
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phenomena did not result in pronounced changes in the water holding capacity of the crumb. The above observations were confirmed by the results of the PC analysis. There is a regularity between samples situated in a particular direction in relation to the axis origin in the score plot and the high values for variables placed in the same direction in the loading plot. This demonstrates that freshly baked bread and bread in the early stages of storage had good springiness and cohesiveness together with a high blue value (Fig. 2). Such bread scored the highest values in the sensory evaluation as well as having low hardness and sourness. Bread from the second group obtained lower scores for overall sensory evaluation, springiness, cohesiveness and BV. Products in the third group (final stages of storage) exhibited a clear increase in the chewiness and hardness of bread. Thus, in view of the arrangement of samples in relation to variables, MAP storage can be seen to have resulted in substantial differences in the characteristics of calcium-enriched wholemeal bread. However, despite such differences, the modified atmosphere applied (60% CO2 and 40% N2) extends consumer acceptability of the product even up to 24 days. References Baardseth, P., Kvaal, K., Lea, P., Ellekjaer, M.R., Faergestat, E.M., 2000. The effect of bread making process and wheat quality on French baguettes. Journal of Cereal Science 32, 73e87. Begum, R., Rakshit, S.K., Rahman, S.M.M., 2011. Protein fortification and use of cassava flour for bread formulation. International Journal of Food Properties 14, 185e198. Breene, W.M., 1975. Application of texture profile analysis to instrumental food texture evaluation. Journal of Texture Studies 6, 53e82. Burrington, K.J., 1998. Prolonging bakery product life. Food Product Design 7, 12e20. Czeizel, A.E., Merhala, Z., 1998. Bread fortification with folic acid, vitamin B12, and vitamin B6 in Hungary. Lancet 352, 1225. Czuchajowska, Z., Pomeranz, Y., 1989. Differential scanning calorimetry, water activity, and moisture contents in crumb centre and near-crust zones of bread during storage. Cereal Chemistry 66, 305e309. Dalgetty, D.D., Byung-Kee, Baik, 2006. Fortification of bread with hulls and cotyledon fibres isolated from peas, lentils, and chickpeas. Cereal Chemistry 83, 269e274. Eliasson, A.-C., Larsson, K., 1993. Cereals in Breadmaking, vol. 1. Marcel Dekker, New York. 249e370. Fennema, O.R., 1985. Water and ice. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekker Inc., New York, pp. 23e67. _ s Fik, M., 2004. Bread staling and methods of prolonging its freshness. Zywno c. Nauka. Technologia. Jakos c. 2, 5e22.
Fik, M., Celej, A., 1993. Some aspects of bread staling and methods of its restraining. Ch1odnictwo 28, 29e31. Fik, M., Michalczyk, M., Surówka, K., Maciejaszek, I., 2000. Characterisation of the staling process of wholemeal bread. Polish Journal of Food and Nutrition Sciences 9/50, 23e28. Fik, M., Surówka, K., 2002. Effect of prebaking and frozen storage on the sensory quality and instrumental texture of bread. Journal of the Science of Food and Agriculture 82, 1268e1275. Gambus, H., 2005. Linseed (Linum uistatissimum L.) as a source of nutrients in gluten _ s - free bread. Zywno c. Nauka. Technologia. Jakos c. 4 (Suppl.), 61e74. Gerrard, J.A., Every, D., Sutton, K.H., Gilpin, M.J., 1997. The role of maltodextrins in the staling of bread. Journal of Cereal Science 26, 201e209. Hoseney, C., Miller, R., 1998. Current Understanding of Staling of Bread. Technical Bulletin, vol. 20. American Institute of Baking Research Department. 1e6. Karadzhov, G., Iserliyska, D., 2003. Sensory quality of minerally fortified bread. European Food Research and Technology 216, 274e276. Karaoglu, M.M., Kotancilar, H.G., Gurses, M., 2005. Microbiological characteristics of part-baked white pan bread during storage. International Journal of Food Properties 8, 355e365. Kotsianis, I.S., Giannou, V., Tzia, C., 2002. Production and packaging of bakery products using MAP technology. Trends in Food Science & Technology 13, 319e324. Leung, H.K., 1986. Water activity and other coligative properties of food. In: Okos, M.R. (Ed.), Physical and Chemical Properties of Food. American Society of Agricultural Engineers, pp. 138e185. Odds, F.C., 1991. Sabouraud(‘s) agar. Journal of Medical and Veterinary Mycology 29, 355e359. Park, H., Seib, P.A., Chung, O.K., Seitz, L.M., 1997. Fortifying bread with each of three antioxidants. Cereal Chemistry 74, 202e206. Patel, B.K., Waniska, R.D., Seetharaman, K., 2005. Impact of different baking processes on bread firmness and starch properties in breadcrumb. Journal of Cereal Science 42, 173e184. Phillips, C.A., 1996. Modified atmosphere packaging and its effects on the microbiological quality and safety of produce. International Journal of Food Science and Technology 31, 463e479. Polish Standard PN-A-74102, 1999. Bakery Products and Semi-products e Sampling and Microbiological Examination. Polish Committee for Standardization. Polish Standard PN-A-74134-4, 1998. Pastry Products and Semi-products. Microbiological Examination. Determination of Number of Spore Forming Bacteria of Decomposing Starch. Polish Committee for Standardization. Rasmussen, P.H., Hansen, A., 2001. Staling of wheat bread in modified atmosphere. Lebensmittel Wissenshaft und Technologie 34, 487e491. Smith, J.P., 1993. Bakery products. In: Parry, R.T. (Ed.), Principles and Applications of Modified Atmosphere Packaging of Foods. Blackie Academic and Professional, Glasgow, pp. 134e169. Szczesniak, A.S., 1963. Classification of textural characteristics. Journal of Food Science 28, 385e389. Szczesniak, A.S., 1995. Texture profile analysis - methodology interpretation clarified. Journal of Food Science 60 (6), vii. Zeleznak, K.J., Hoseney, R.C., 1986. The role of water in the retrogradation of wheat starch gels and bread crumb. Cereal Chemistry 63, 407e411.