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Quality of and Mold Growth on White Enriched Bread for Military Rations Following Directional Microwave Treatment ABSTRACT: Meals ready-to-eat (MRE) are self-contained and flexible packages used by military personnel while in the field to store food for an extended period of time; however, inclusion of white bread is not a common practice because of short shelf life stability and spoilage. The objective of this study was to determine mold inhibition and quality attributes over a 60-d period after applying directional microwaves. Different bread loaves were used for quality and for microbiological experiments. For microbiological analysis, bread was exposed to 0-, 5-, 6-, 7-, 8-, 9-, and 10-s directional microwave treatments after inoculation with a 3 strain cocktail of common bread mold, stored at 25 ◦ C for 60 d, and monitored for mold growth. For quality analysis, bread was exposed to 0- and 10-s treatments, stored at 25 ◦ C, and moisture, water activity (a w ), softness, and sensory analysis were analyzed on 0, 7, 14, 28, 45, and 60 d. There was no quantifiable mold present at day 0 when treated for 10 s (P < 0.05). By day 60, the 10-s treatment had significantly lower counts (< 3 CFU/g) than the remaining treatments. Directional microwave treatment significantly decreased the moisture content of the bread but was not detectable by consumers. There was no difference in a w through day 45 but differences were detected at day 60. There were no differences in softness (mm) of the treated and untreated bread through day 60. No differences were detected by sensory analysis. Directional microwaves can be used to extend the shelf life of white enriched bread up 2 mo with minimal mold growth and without detrimental effect to quality. Keywords: directional microwaves, mold, quality, ready-to-eat, white bread

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Introduction

eals ready-to-eat (MRE) are used by the U.S. military branches to ensure that soldiers are receiving the basic nutrients to sustain operations. The MRE is a totally self-contained and flexibly packaged meal. The MRE ration development program requires many production constraints related to nutrition, personnel acceptance, wholesomeness, reducibility, cost, shelf life, selfheating capability, weight, volume, ease of sanitation, menu fatigue, and performance enhancement. Each meal contains 1200 calories and includes an entr´ee of crackers, cheese, peanut butter or jelly spread, a desert or snack, beverages, an accessory packet, a plastic spoon, and a flameless ration heater. These meals typically use primarily a cracker base for soldiers to prepare sandwiches. The water activity of these items in MRE packages is low and allows for a long shelf life. Most people consuming the MRE would like to include bread instead of crackers; however, the primary problem with using white enriched bread is the potential for mold growth and deterioration of the quality of the bread, primarily staling (Xie and others 2003). Microwaves are part of a broad spectrum of electromagnetic radiation, which also includes radio waves, infrared radiation, visible light, ultraviolet radiation, x-rays, and Gamma rays (Curnutte

MS 20070415 Submitted 5/30/2007, Accepted 1/8/2008. Authors Lakins, Echeverry, Alvarado, Brooks, and M.M. Brashears are with Dept. of Animal and Food Sciences, Texas Tech Univ., P.O. Box 42141, Lubbock, TX 79409, U.S.A. Author M.T. Brashears is with Dept. of Agricultural Education and Communications, Texas Tech Univ., P.O. Box 42131, Lubbock, TX 79409, U.S.A. Direct inquiries to author Alvarado (E-mail: [email protected]).

 C 2008

Institute of Food Technologists doi: 10.1111/j.1750-3841.2008.00677.x

Further reproduction without permission is prohibited

1980). Microwave heating uses electromagnetic waves of frequencies between 300 and 300 GHz to generate heat in a material. Research has been conducted to determine if electromagnetic heating can be used to sterilize or pasteurize food (Ayoub and others 1974; Mudgett 1982; Guan and others 2003). Jeng and others (1987) stated that the electromagnetic energy in the microwave region (223 to 100 GHz and 34500 MHz) has been studied as an alternative energy source for sterilization. Destruction of microorganisms during microwave heating is believed to be due to the thermal effect (Fujikawa and others 1992). Directional microwaves interact with dielectric materials to generate heat by the agitation of molecules in an alternating electromagnetic field. Water, carbon, and matter with high water or carbon content are good microwave absorbers. Other researchers have also concluded that destruction of microorganisms may be caused by nonthermal effects. Four theories have been used to explain the nonthermal inactivation by directional microwaves or “cold pasteurization”: selective heating, electroporation, cell membrane rupture, and magnetic field coupling (Kozempel and others 1998). The selective heating theory states that directional microwaves heat solid microorganisms more effectively than the surrounding medium and this causes a more rapid killing of the organism (Datta and Davidson 2000). Electroporation is caused when an electrical potential crosses the membrane of the microorganism, causing the formation of pores in the membrane that results in leakage of cellular contents. Cell membrane rupture is related to the voltage drop across a membrane resulting in the rupture of the cell membrane. Magnetic field coupling causes a disruption in the internal components of the cell, which leads to cell lysis. This allows bacteria to be destroyed at lower temperatures than using heat alone. Olsen and others (1966) observed Vol. 73, Nr. 3, 2008—JOURNAL OF FOOD SCIENCE

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D.G. LAKINS, A. ECHEVERRY, C.Z. ALVARADO, J.C. BROOKS, M.T. BRASHEARS, AND M.M. BRASHEARS

Effect of directional microwaves on bread quality . . .

M: Food Microbiology & Safety

that the nonthermal effect of directional microwaves played a vital role in the inactivation of the microorganism in suspension causing the formation of hydrogen peroxide and other chemical transformations of small molecules, which resulted in cleavage of chemical bonds. The directional microwave technique used at Texas Tech Univ. is very different from a commercial or a household microwave and was developed by an Italian company (ITACA New Tech, Brescia, Italy). In general, this technology developed in Italy differs from traditional/home directional microwaves technology because of the following factors: r This equipment uses a horizontal and rotary movement. Traditional directional microwaves ovens only have a rotary movement. In this way, food exposure to directional microwaves is more uniform. r This equipment has several sources of directional microwaves: horizontal and vertical. With this procedure, you can vary the power over a wider range of values and provide a more homogeneous distribution of power within the chamber. Traditional directional microwaves ovens have only 1 source. r In addition to heating, this equipment utilizes fast cooling using CO 2. The ability to cool product quickly following directional microwaves is important in preserving quality of the product since heat can denature proteins. Based on these differences, this directional microwave technology available at Texas Tech Univ. is able to destroy pathogens at a lower temperature and without destroying the quality of the food. Research with this new technology has been conducted with eggs, cheese, and spices. Results in eggs indicated that there was a 2 log reduction of Salmonella Enteritidis in both the high (105 CFU/g) and low (102 CFU/g) inoculum and there were limited quality changes (Ferroni and others 2003; Lakins 2006). However, this new technology has not ever been researched in bread. Enriched white bread has an approximate water activity of 0.92 and research has indicated that an a w > 0.80 level will allow mold growth. Because of this, using enriched white bread in an MRE poses some potential problems with mold growth; however, the application of directional microwaves on foods has indicated that molds and spores are receiving detrimental treatment. Therefore, the objectives of this study were to determine mold inhibition and quality attributes after applying directional microwaves on enriched white bread over a 60-d period.

Materials and Methods Pasteurization unit The microwave unit is made of stainless steel. The cavity of the unit measures approximately 130 cm long, 170 cm high, and 100 cm deep and weighs 130 kg. Inside the cavity, a polycarbon cylinder is used to house the food product during pasteurization. A sleeve that protrudes from the left side of the unit is used to insert the axis of rotation. This rotation unit consists of a pneumatic piston that moves the rotating structure horizontally to ensure that the product receives equivalent dosages of electromagnetic rotation. A computer is connected to the unit that allows strict operator control of time, percentage power, and speed of movement of the cylinder.

Preparation of bread Loaves of white enriched bread were purchased from a local grocery store from the same processing day. Two treatments were used in this study: a control 0-s directional microwaves and treatment 10-s directional microwaves. Loaves were either assigned to qualM100

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ity testing or microbiological testing. For quality testing, 2 pieces of the white enriched bread were placed in a 3-mil bag (Cryovac, S.C., U.S.A.) and sealed. These bags were then placed into the directional microwave unit and treated for 10 s (2.45 GHz; 12.2-cm wavelength). Four replications of 8 samples each for the quality and 4 replications were conducted for the microbiology.

Microbiology For microbiological testing, the bread was inoculated with a 3 × 103 CFU/g of a cocktail mixture of 3 separate mold strains obtained from the American Type Culture Collection (ATCC) (ATCC cultures Penicillium crustosum 90174, Rhizopus microsporus 52807, and Aspergillus niger 58133). The inoculated bread was allowed to equilibrate for 10 min under a hood, then was placed into MRE packaging (Cryovac bags). After packaging, it was subjected to directional microwave treatment from 5 to 10 s (2.45 GHz; 12.2-cm wavelength). After treatment, all bags were stored at 25 ◦ C for the 60-d period and observed on a daily basis for mold growth. A total of 4 replications were used for the analysis for statistical soundness. Duplicates of the treated loaves were used to conduct an analysis of the inhibitory effect of the directional microwaves treatment. Mold populations were determined on days 0 and 60, respectively. Bread samples (11 g) were aseptically collected from each of the MRE packs and placed inside sterile, filter stomacher bags. A total of 99 mL of buffered peptone water (BPW, Difco) was added in the bag to make a 1:10 dilution of the sample after which it was homogenized for 2 min in a laboratory blend stomacher (Model 400, Stomacher Lab System, Seward Medical, London, U.K.). Serial dilutions were performed and 0.1 mL of each dilution was plated on plate count agar (PCA, Difco) containing 100 mg chloramphenicol per liter and placed at 25 ◦ C for 3 to 5 d. In addition, noninoculated untreated samples (control) were also subjected to enumeration on PCA to be able to compare naturally present levels of mold on the bread.

Quality Moisture. Bread moisture was determined over the 60-d period (days 0, 7, 14, 28, 45, and 60) according to the AOAC (1995) official method 950.46 (Moisture in Meat) using a drying oven. Water activity. Water activity of the bread was measured over the 60-d period (days 0, 7, 14, 28, 45, and 60) using an Aqua-Lab CX-2. For each analysis, 3 g of bread were placed in a plastic cup and inserted into the machine for analysis. The Aqua-Lab CX-2 was calibrated using a saturated NaCl solution obtaining a w of 0.754 Rahman (1995). Softness/ Texture. Softness of the bread was measured over the 60-d period (days 0, 7, 14, 28, 45, and 60) according to the AACC (2000) method 74-10A using an Instron (Norwood, Mass., U.S.A.) with a compression head. Sensory analysis. Sensory of the bread was performed on 7 d following treatment using an untrained panel of 50 people. Untrained panelists were recruited on the campus of Texas Tech Univ., included both males and females, and ranged in age from 18 to 60 years old. The panelists were administered a triangle test and asked to choose the different sample. They were asked to rank the samples for texture on a 1 to 8 hedonic scale. Before the sensory analysis, the bread slices were manually cut into 3 × 3 cm in pieces for serving. A separate random 3-digit number was used to identify the individual samples. Each panelist was asked to rinse their mouth with tap water and eat a cracker in between samples. They were served at individual booths under red lights to keep results from becoming biased. Differences were determined using a critical number table of correct responses in a triangle test.

Effect of directional microwaves on bread quality . . . Statistical analyses The experimental design was a completely randomized design. Data were analyzed using proc GLM in SAS (SAS 1996). All statistically significant comparisons were at P < 0.05. For the microbiological part, least significant difference (LSD) of the mold counts (log transformed) was obtained to compare differences between the treatments. Replication was treated as a random effect.

Quality analysis Quality analysis was performed on the bread over a 60-d period. Bread has a tendency to lose moisture and have a decreased water activity over time as a result of natural dehydration and staling. Therefore, total moisture (%) was measured to determine the amount of moisture in the bread samples (Table 1). As expected, there was an overall decrease in moisture (%) over time. There was no difference between control and treated samples on days 0, 7, 28, and 45 in moisture (%). However, on days 14 and 60, the control bread was slightly higher in moisture (35.11; 31.33) and (27.83; 25.00), respectively, compared to the directional microwave treated bread. Water activity is another measurement of moisture and is correlated to water available for microbiological growth. This measurement is especially important in determining the amount of water available for mold growth (> 0.80 required). Table 2 indicates the water activity results over a 60-d period in control and treated bread. On days 0, 7, 28, and 45, there was no difference in the water activity of the control compared to the directional microwave treatment. However, on days 14 and 60, the water activity of the control was slightly higher compared to the treated samples (0.90, 0.89, and 0.87, 0.85 respectively). These differences in moisture and water activity could be due to natural differences occurring during the loss of moisture over time during storage. However, these differences, even though significant, may not be noticeable to the average consumer. Bread staling is one of the most common problems during bread storage. Bread softness is negatively correlated with staleness. Therefore, softness was measured in this study to determine the amount of hardening (staling) of the treated bread during storTable 1 --- Total moisture (%) of white enriched bread treated with directional microwaves over 60-d storage period. Day 0

Day 7

a

a

Control 36.93 Directional 37.26a microwaves (10 s)

34.82 33.52a

Day 14 Day 28 Day 45 Day 60 35.11b 31.33a

27.74a 27.36a

25.78a 26.09a

27.83b 25.00a

n = 16; 2 replications. a–b Means within column with different subscripts are different (P < 0.05).

Figure 1 (day 0) illustrates the decline in the mold population from an initial 3.3 log10 CFU/g mold count in mold inoculated, nontreated bread samples to no detectable mold spores detected after treatment with the directional microwaves for a period of 10 s. The control bread that was not inoculated had a background population of mold at almost 1.5 log 10 CFU/g. These data suggest that treatment of the bread with the directional microwaves for 10 s was a very effective method to inactivate the mold spores in the product. There was a statistically significant decline in the total mold spores of bread treated for 7 s or less and bread treated for 10 s (P < 0.05). The difference between a 3.3 log 10 CFU/g count and no detectable spores represents a 99.9% reduction in the mold populations. Figure 2 illustrates the microbial analysis performed on the bread after the 60-d storage period. All samples had higher mold counts than those obtained at day 0, as expected. Samples treated for 10 s showed an increase to approximately 1.5 logs of mold when compared to those results obtained at day 0. However, the counts at day 60 on the samples treated for 10 s were very low and showed no visible mold growth after the 60 d of storage. A 1.5 log 10 CFU/g amount of mold in 1 slice of bread is similar to the amount of mold found in control bread at the day 0 sampling period. At day 60, the control bread and samples treated for 7 s or less had mold counts around 6.0 logs 10 CFU/g, which were significantly higher than the counts on the bread treated for 10 s. Visual results. Duplicate samples of the bread were prepared to perform daily observations on the bread over a period of 60 d to determine if there was any visible mold growth. In general, for all 4 replications, all samples that were treated with the directional microwaves for 7 s or less began to show mold growth between days 6 and 16 after treatment. For those samples treated for 10 s, no surface mold growth was observed during the 60-d period except for 1 sample at day 17 after treatment. These results confirmed that the 10-s treatment was long enough to inhibit the mold on the bread over a 60-d period and were consistent with the microbiological testing. There have been limited studies using this new directional microwave technology on foods, and no studies in bread. Therefore, no comparisons can be made to other studies. However, it should be noted that pasteurization studies involving commercial microwaves have indicated that they are problematic and have varying results (Heddleson and Doores 1994). The lack of standardization and nonuniform heating within commercial microwaves

Table 2 --- Water activities of white enriched bread treated Table 3 --- Softness (mm) of white enriched bread treated with directional microwaves over 60-d storage period. with directional microwaves over 60-d storage period. Treatment Control Directional microwaves (10 s)

Day 0 a

0.92 0.92a

Day 7 a

0.91 0.90a

Day 14 b

0.90 0.89a

Day 28 a

0.88 0.88a

Day 45 a

0.86 0.86a

Day 60 b

0.87 0.85a

n = 8. a–b Means within column with different subscripts are different (P < 0.05).

Treatment Control Directional microwaves (10 s)

Day 0 a

7.88 7.84a

Day 7 a

7.91 8.10a

Day 14 a

7.28 6.67a

Day 28 a

4.36 4.94a

Day 45 a

3.63 3.72a

Day 60 1.50a 2.16a

n = 16. a–b Means within column with different subscripts are different (P < 0.05).

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Microbiological study

Results and Discussion

Treatment

age (Table 3). There were no significant differences between the control and directional microwave 10-s treatment. However, as expected, both the control and the directional microwave 10-s treatment had decreasing softness over the 60-d period due to the moisture loss over time. Sensory analysis was performed on the enriched white bread on day 7 using a triangle test in which no differences were indicated between treated and control bread (data not shown).

Effect of directional microwaves on bread quality . . . Figure 1 --- Effects of directional microwave treatment on mold population in inoculated white bread (day 0). Each bar represents the mean of 4 replications. Means with the same letter are not significantly different (P < 0.05).

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Figure 2 --- Effects of directional microwaves treatment on mold population in inoculated white bread (day 60). Each bar represents the mean of 4 replications. Means with the same letter are not significantly different (P < 0.05).

makes pasteurization of foods variable and potentially dangerous. Acknowledgments However, the technology developed by ITACA and used at Texas We would like to acknowledge ITACA New Tech (Brescia, Italy) and Tech Univ. have shown that uniform heating, destruction of mi- all the research scientists associated with this Italian company for crobes, and quality retention can be possible due to the differences their support and knowledge in the completion of this project. in the apparatus compared to the commercial microwave (Ferroni and others 2003; Lakins 2006).

References

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Conclusions

he use of directional microwaves can be applied to white enriched bread to inhibit mold growth without causing detrimental effects on the quality or sensory attributes. With the application of directional microwaves, white enriched bread could be added to the MRE and have an increased shelf life of up to 60 d.

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[AACC] American Assn. of Cereal Chemists. 2000. Approved methods of the AACC. 10th ed. St. Paul, Minn.: AACC. 74 p. [AOAC] Assn. of Official Analytical Chemists. 1995. Official methods of analysis of AOAC Intl. 16th ed. Va.: AOAC Int. Ayoub JA, Berkowitz D, Kenyon EM, Wadworth CK. 1974. Continuous electromagnetic pasteurization sterilization of meat in flexible pouches. J Food Sci 39: 309–13. Curnutte B. 1980. Principles of electromagnetic pasteurization. J Food Prot 43(8):618– 24. Datta AK, Davidson PM. 2000. Microwave and radio frequency processing. J Food Sci 65 (Suppl.): 32–41.

Effect of directional microwaves on bread quality . . . Kozempel MB, Annous A, Cook R, Scullen OJ, Whiting R. 1998. Innactivation of microorganisms with microwaves at reduced temperatures. J Food Prot 61:582–5. Lakins DG. 2006. Salmonella Enteritidis reduction, quality attributes, and nutritional profile in both white and brown shell eggs subjected to an innovative microwave technology [dissertation]. Texas Tech Univ, Lubbock, Tex. Mudgett RE. 1982. Electromagnetic pasteurization properties of foods in electromagnetic pasteurization processing. Food Technol 36:109–12. Olsen CM, Drake CL, Bunch SL. 1966. Some biological effects of electromagnetic pasteurization energy. J Directional Microwaves Power 1:45–6. Rahman S. 1995. Food properties handbook. Boca Raton, Fla.: CRC Press. 500 p. SAS Inst. 1996. SAS users guide. SAS Inst. Inc., Cary, N.C., U.S.A. Xie F, Dowell FE, Sun XS. 2003. Comparison of near-infrared reflectance spectroscopy and texture analyzer for measuring wheat bread changes in storage. Cereal Chem 80(1):25–9.

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Ferroni C, Coccoli G, Baronio G, Piazza S, Paterlini F. 2003. Evaluation of new treatments for pasteurization-sterilization of shell eggs. XLII Convegno Annuale della Societa Italiana Patologia Aviare. October 2–3, 2003. Forli, Italy. Fujikawa H, Ushioda H, Kudo Y. 1992. Kinetics of Escherichia coli destruction by electromagnetic pasteurization. Appl Environ Microbiol 58(3):920–4. Guan D, Gray P, Kang DH, Tang J, Shafer B, Ito K, Younce F, Yang TCS. 2003. Microbiological validation of electromagnetic pasteurization-circulated water combination heating technology by inoculated pack studies. J Food Sci 68(4): 1428–32. Heddleson RA, Doores S. 1994. Factors affecting microwave heating of foods and microwave induced destruction of foodborne pathogens—a review. J Food Prot 57:1025–37. Jeng DK, Kaczmarek KA, Woodworth AG, Balasky G. 1987. Mechanism of electromagnetic pasteurization sterilization in the dry state. Appl Environ Micro 53(9): 2133–7.

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