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LOW WATER ACTYVIlY PACKAGED 0 3 FILE W ei'WHITE BREAD

Contract No. DAAK 60-83-C-0039

Final Report(Jan. 1, 1984 - Dec. 31, 1985)

U.S. Army Natick R & D CenterKansas Street

Natick, MA 01760

Principal Investigator: Dr. Henry K. Leung D T ICCo-investigators: Dr. Charles W. Nagel , ELECTE

Mr. Gordon G. Rubenthaler M 22 mResearch Associates: Dr. Debbie Hsu-- ,n W 2- 2 19 88

Ms. Nancy Larson-Powers IDr. A. Abdelrahman

Technical Assistants: Ms. Dorothy MacEachern HMr. Mike Costello

Department of Food Science and Human Nutrition, Washington StateUniversity, Pullman, WA 99164-6330

Western Wheat Quality Laboratory, USDA, Pullman, WA 99164-4004

APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED

10156:Nattc2:pdh &8 3 21 056

Natick Final ReportPage 2

TABLE OF CONTENTS

PAGE

SUMMARY . . . . . . . . . . . . . . . . . . . . . . .. . . 3INTRODUCTION . . . ................. 4MATERIALS AND METHODS

Baking . . . .... .............. 5Laboratory. . . . ............. .. 5Local Bakery ......................... 5

Physical and Chemical Analysis . ........... 5Moisture and Water Activity .............. 5pH. . . ..................... 8Color . . . . . . ................ 8Firmness. . . . .. .............. 8Thermal Analysis. . ................. 8Headspace Analysis .................. . . 8Humectant Analysis ...... ................ 8Sensory Evaluation . .............. 9

Microbial Studies. ............. . . .. . 10Storage Studies. . . . ... ...... . . . . 10

RESULTS AND DISCUSSION . . ... ...... 110I.) Effects of Humectants on Baking, Sensory and

Physical Properties of Bread ............... 11Dough Mixing Properties ............... 11Baking Properties ..... .............. 11Sensory Properties . ............ 11Staling. . ... ....... . 14Refinement of Formuiation.......... 14pH . . .. . . . . . . . . . . . .. . . 14Water Activity Measurement ........... 14

i1.) Microbial Challenge Studies ............ . 15Molds. . . . . . . . . . . . . . . . .. . . 15Clostridium sporogenes ......... . . 15staphylococcus aureus ............. . 17conclusions . ........... 22

(III.) Storage Stability Studies . ......... 22Microbial Stability. ............... ... 22Moisture and Water Activity. . ....... 22pH ................... 25Firmness . ........... .. 26Color .. . ............ 27Sensory Evalu;tio;............. 29Conclusions ...................... 37

(IV.) Refinement of Formula .. ........ 37Effect of Shortening and Oii ........ 37Sponge vs. Straight Dough.......... 37

CONCLUSIONS .. .. ..... . . . . . . . . . . . 42RECOMMENDATIONS . . . . . . . . . . . . . . 44REFERENCES . . . . . . . . ....... . 45

Natick Final Report

Page 3

SUMMARY

The purpose of this study was to develop a shelf-stable packaged white panbread for military use. Shelf life of bread may be extended to several monthsby proper control of water activity, pH, packaging conditions, and by additionof antimicrobial compounds and dough conditioners. Water activity of bread waseffectively lowered from 0.97 to 0.92-0.93 by addition of 7% sorbitol, 1%propylene glycol and 2% glycerol (flour basis). Incorporation of 0.3% lacticacid powder to the bread reduced its pH to 5.0. Other formulation changesincluded 10% shortening and addition of sodium stearoyl-2-lactylate (anti-staling). The resulting humectant bread was satisfactory for baking and sensoryquality. To prevent dehydration and to exclude oxygen, the bread was packagedin a partial CO, atmosphere in a retort pouch.

The packag d humectant bread was microbiologically stable up to about fourmonths. Growth of Aspergillus flavus, Staphylococcus aureus and other aerobicorganisms were inhibtd. However, pH of the product s be lowered to 4.8to guard against anaerobic organisms.

Storage of the humectant bread resulted in a number of undesirable changes:increases in browning, firmness, dryness, crumbliness, rancid flavor and aroma,and decreases in pH and overall preference. Further improvement will be neededto extend the shelf-life of the bread to one year at 30*C.

Based on this study, a maximum a of 0.92-0.93 and a maximum pH of 4.8 arerecommended for the humectant bread. Partial CO2 packaging using a retort pouchor metal can is preferred for maximum stability.

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INTRODUCTION

Regular United States white pan bread is susceptible to microbial spoilagebecause of its relatively high moisture content (35%). Even with addedpreservatives such as calcium propionate, packaged white bread usually spoilswithin two weeks at room temperature. In the early 1940's, a shelf-stablecanned white bread was developed for military uses. This was accomplished bybaking the bread in a can and sealing the container while it was still hot.When the bread cooled, a vacuum was created inside the can. The combination ofheat treatment, exclusion of oxygen, as well as a reduction in pH (< 4.8) andmoisture content (< 35%) contributed to the microbial stability of the product(Matz, 1972). However, the bread produced by this method is different inquality from the normal white pan bread.

It has been well recognized that water activity is a key factor incontrolling microbial spoilage of food. Water activity of a material is definedas:

a = Pi/PoWere Pi = vapor pressure of water in the atmosphere in

equilibrium with the materialPo = vapor pressure of pure water at the same temperature

In general, most bacteria, yeasts and molds do not grow below a of 0.90,0.87 and 0.80, respectively (Troller, 1979). Bread crumb has a wateW activityof about 0.97, and is therefore susceptible to microbial spoilage.

Water activity of a food material can be effectively lowered by theaddition of water binders or humectants. The common humectants include salt,sugars, gums and polyhydric alcohols (polyols) such as glycerol, sorbitol andpropylene glycol. The polyols are more effective water binders than sugars.Propylene glycol has a bitter-sweet taste; glycerol is slightly sweet andsorbitol is sweet. In addition, sorbitol has a laxative effect if consumed athigh concentration. In baked goods, sorbitol can be used at a concentration upto 30%, and propylene gly col is limited to 2%. Glycerol is in the Generally

* Recognized As Safe (GRAS) list.Besides water activity, other factors such as pH, oxygen, antimicrobial

-aents, heat treatment and packaging are also important in controlling microbialgrowth. The purpose of this project was to develop a shelf-stable white panbread in a flexible pouch by utilizing the above factors for preservation. Thespecific objectives of this project were:-

(1) To investigate the effect of humectants on bread making properties.(2) To determine the effects of different ingredients on water activity, pH,

sensory properties, staling and microbial stability of bread.(3) To develop an acceptable, shelf-stable white pan bread by controlling its

water activity, pH and packaging environment.(4) To conduct a storage stability study of the final products.

Natick Final Report

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MATERIALS AND METHODSBaking

LaboratoryThe wheat flour used in this study was a blend of Montana hard wheat flour

with 12.6% moisture, 12.8% protein and 0.44% ash. The straight dough method wasused throughout the study except where indicated otherwise. The basic formulaincluded the following ingredients unless otherwise specified: 100 g flour (14%moisture basis), 5 g yeast, 6 g sucrose, 3 g shortening (Crisco), 1.5 g salt and0.25 g malt. Sorbitol (Sigma Chemical Co.), glycerol Sigma Chemical Co.),propylene glycol (Baker CHemical Co.), and sodium stearoyl-2-lactylate (SSL)(Patco Products, Kansas City, MO), were added at different concentrations.Baking absorption (g water/1Og flour), mixing time and oxidation were optimizedfor each formula. The fermentation and proofing (35*C) times were adjusted fordifferent formulas. Breads were baked at 204.40C for 26-28 min, and weighedimmediately after removal from the oven. Loaf volume was determined by rapeseeddisplacement.

Local BakeryThe humectant breads were produced in a local bakery using the straight

dough method. The formulas for the bread are summarized in Table 1. The flourwas a blend of all purpose flours containing 12.7% protein (14% m.b.). Lacticacid powder (Purac),.sorbitol (Sigma Chemical Co.), glycerol (Sigma ChemicalCo.), propylene glycol (Baker Chemical Co.) and sodium stearoyl-2-lactylate(SSL) (Patco Products, Kansas City, MO) were added to different concentrations.Baking absorption (g water/100 g flour), mixing time and oxidation wereoptimized for each formulation.

The ingredients, except water, were blended for five minutes. Water wasadded gradually to the mixture while the dough was mixed to optimum consistency.The dough was fermented at room temperature, divided (scaled) and rounded byhand. After molding, the dough was panned and placed inside the proofingcabinet. Proofing was completed when the top of the dough reached 1/2 inchabove the rim of the pan. The breads were baked at 3750 and cooled for 3 hr at,room temperature before packing and delivery to Washington State University.The baking procedures of the humectant breads are summarized in Table 2.

Physical and Chemical Analysis

Moisture and Water ActivityMoisture content of bread crumb and crust was determined using a

modification of AACC method 44-4D. One (1) g of sample was placed in a glasspetri dish, weighed and placed in a vacuum oven at 700C for 24 hours.

Water activity of bread crumb or crust was determined using a SC-10Thermocouple Psychrometer (Duagon Devices, Pullman, WA). The instrumentconsisted of a sample changer, a chromel cnstantan thermocouple, and amicrovoltmeter (100 m V sensitivity). The sample changer can hold up to ninesample and one water cups. Samples were taken from the center portion of theloaf from crumb and the bottom crust near the end for crust. Each sample wasfirmly packed into the sample cup until it was about half full. A sample ofsaturated salt solution of potassium nitrate (KN03) was included for determiningthe cell constant. After the samples were loaded, they were allowed toequilibrate for at least I hour. When a reading was taken, the thermocouple waswetted, then placed above the desired sample and the chamber sealed. Thevoltage output was read at 2 minutes and chamber temperature noted. Wateractivity was computed using an Apple II computer according to the InstructionManual.


Table 1. Formulas of the humectant breads baked in a conmercial bakery

% Flour Weight

Ingredient 7-1-2a 6-1.5-2a

Flour(14% mb; 12.7% protein) 100 100

Water 47 57Yeast (Compressed) 5.25 5.25Shortening 10 10Sugar 3 3Salt 1.5 1.5Ascorbic Acid (40 ppm) (40 ppm)Potassium bromate (10 ppm) (10 ppm)Malt - -Sodium stearoyl lactylate 0.5 0.5Calcium propionate 0.3 0.3Lactic acid (60% powder) 0.3 0.3Sorbitol 7 6Propylene glycol 1 1.5Glycerol 2 2

a% sorbitol - % propylene glycol - % glycerol

Natick Final Report

Page 7

Table 2. Baking procedures and times (min) of the humectant breadsproduced in a commercial bakery

Steps 7-1-2a 6-1.5-2a

Mixing 30 30First Fermentation 38 14Dividing and Rounding 21 27Second Fermentation 5 5Molding and Panning 12 12Proofing (93-F, 70% RH) 83 102Baking (375"F) 30 30Cooling 180 180

a% sorbitol - % propylene glycol - % glycerol


pH The pH of bread crumb was determined according to AACC Method 02-52 (AACC,1984). The bread crumb was disintegrated into particles using a blender. A10 g sample was added to 100 g distilled water in a beaker and mixed thoroughlybefore pH measurement.

ColorA Hunter Color and Difference Meter (Model D-25 D, Hunterlab, Fairfax, VA)

was standardized to the white tile prior to determining the L, a, b values ofbottom crust and crumb.

FirmnessFirmness of bread crumb was determined with a Fudoh Rheometer model

NRM-2002J (Fudoh Kohyo, Co., Tokyo, Japan). A custom-built adapter consistingof a stainless steel rod with a circular lucite plate (32 mm diameter) attachedto the end was used for the test. A 2 cm cube sample was removed from thecenter of the 6 slices taken from the center of the loaf (the seventh slicewhich was cut first was discarded because of width variation). Each sample wascompressed to a height of 0.6 cm using a crosshead speed of 2 cm/min and gramsforce was recorded.

Thermal AnalysisA Perkin-Elmer (Norwalk, CT) Differential Scanning Calorimeter (DSC-4)

equipped with the Data Analysis Station was used to determine the starchcrystallinity of the bread. A No. I Cork Borer was used to remove a plug ofbread crumb from the center of the loaf. The plug was compressed and a 5-6 mgslice was removed using a scalpel. The sample was placed in a volatile samplepan and weighed. Two parts of water (by weight) was added to the sample. Thepan was sealed with a volatile sample sealer and allowed to equilibrate at roomtemperature for at least one hour before the thermal analysis.

The temperature scale of the DSC was calibrated using pure indium (M.P. =156.6'C). An automatic device was built into the instrument for heat flowcalibration. A pan containing 10 mg water was used as the reference. Thesample was scanned from 20-1000C at a rate of 10°C/min. The onset temperature&1d peak area of the curve were determined using the Perkin-Elmer TADS DSC-4Standard Software Program.

Headspace AnalysisCarbon dioxide content of the headspace in the packaged samples was

determined using a Beckman GC-2A Gas Chromatograph (Beckman Instruments, Inc.,Irvine, CA) equipped with a thermal conductivity detector, a Beckman Col SystemSer CS41 Column and a Spectraphysics integrator. Mixtures of CO and air wereused to prepare the standard curves. A 5 ml gas sample was withdrawn from thebag through a silicon system and injected into the GC. The ratio of CO2 to 02was used to determine the percent CO2 present in the pouch.

Humectant AnalysisSorbitol, glycerol and propylene glycol of the humectant breads were

determined using the high performance liquid chromatography (HPLC) methoddeveloped in our laboratory (Nagel et al., 1981). Duplicate bread slice samplesof about 30 g each were removed from the center of two separate loaves of bread.The sample was homogenized with water in a Waring Blender for one minute, andmade up to 250 ml. The mixture was centrifuged at 10,000 x g for 5+min. Twentyfive milliliters of supernate was run through an Amberlite TR 120 H column andthen an Amberlite IR 45 OH" column.. The sample was made up to 100 ml with


distilled water, filtered and analyzed by HPLC (Nagel et al., 1981).

Sensory EvaluationAn untrained panel, consisting of 38 graduate students and staff members at

Washington State University, was used to conduct the preference test of thebread samples. A nine point hedonic scale (Larmond, 1970) was used for overallacceptance. A copy of the score card is given in Appendix A. The bread wassliced and kept in plastic bags before serving. The samples were coded andrandomized in order of presentation. Each judge was presented five samples:control, 5% sorbitol, 20% sorbitol, 8% glycerol, and 10% sorbitol-5% glycerol-2%propylene glycol. The data were analyzed statistically for any significantdifferences.

To evaluate the effect of different humectants on bread quality, asemi-trained panel consisting of ten judges was used. Most of the judges weremembers of the Western Wheat Quality Laboratory. After cooling, six slices weretaken from each loaf of bread, cut into halves, and placed in plastic wrap toprevent dehydration. The panelists were asked to evaluate crumb texture,off-flavor and overall preference (Prentice and D'Appolonia, 1977). A copy ofthe scorecard is given in Appendix B.

To select a descriptive analysis panel, nineteen members of the Departmentof Food Science and Human Nutrition at Washington State University were given aseries of nine triangle tests. Panelists were screened for their ability todifferentiate between various commercial white bread samples. Twelve finalistsand one alternate (10 males and 3 females) were selected to participate infurther training.

Panelists were trained for the first three one-hour training sessions, thejudges evaluated the samples, listing as many texture, aroma and flavordescriptors as possible. Then the group discussed and developed definitions forthese terms. The evaluation was broken down into appearance, aroma, texturedetermined by fingers, mouthfeel textural characteristics, crumb flavor andcrust flavor. The judges were also allowed to express an overall preference;however, these results were different from consumers' since the judges were

,.-ghly trained and sensitive to small differences not detected by consumers.In the fourth through seventh sessions, the judges evaluated samples using

the scorecard developed from the terms derived in previous sessions, anddiscussed how the evaluation was done. In the final three training sessions,the judges evaluated four samples on a scorecard. The sample consisted of aslice of 100 g loaf bread (12 mm thick) placed in a Ziploc bag and labeled witha 3-dig it code. The testing was replicated three times with (I.) one-day old5-2-2 (5% sorbitol - 2% propylene glycol - 2% glycerol) bread, (2.) six-monthold 5-2-2 bread, (3.) one-day old 1-4-4 bread, and (4.) one-day old 8-2-2 bread.The data from these evaluations were analyzed statistically to determine how thejudges performed individually and as a group. Finally, the judges participatedin a one-hour retraining session where the scnrecard (No. 2) was modified(Appendix C).

Scorecard No. 2 was used for the descriptive analysis of breads by thetrained panel. A preference test was conducted when the bread had been storedfive weeks. An untrained panel consisting of 32 people from Washington StateUniversity evaluated preference using an unstructured hedonic scale. A copy ofthe scorecard for the preference test is given in Appendix D.

The bread samples were sliced with a Bosch Kitchen Slicer, cut into halvesand placed in Ziploc bags (Dow chemical) before serving. The samples were codedwith 3-digit numbers and presented in a balanced complete block design. TheJudges were presented with the samples one at a time, with a spit cup, water anda scorecard. The data were analyzed for statistical significance by analysis ofvariance.

Natick Final Report

Page 10

Microbial StudiesStandard Plate Count (SPC) and anaerobic count were conducted according to

the standard procedures (Speck, 1976). Eleven grams of sample were placed in asterile polyethylene bag (18 cm x 30 cm) with 99 ml of dilution water. Thesample was homogenized in a Stomacher Lab Blender Model 400 (Seward Laboratory,London) before microbial analysis. The anaerobic count was determined accordingto the most probable number (MPN) method.

For the challenge studies, Penicillium expansum, Aspergillus niger, andClostridium sporogenes cultures were obtained from the cultL-e col tHon of theDepartment of Food Science & Human Nutrition at Washington State University.The Staphylococcus aureus culture was obtained from the American Type CultureCollection (ATCC 1335)

The molds used for the challenge study were grown on regular bread slicesat 30*C. When the bread slices were overgrown with mold spores, they werefreeze dried and ground into powder using a mortar and pestle. About 2 mg ofthe mold powder was transferred to a one-ml ampule, flushed with carbon dioxideand sealed. The breads were baked as described previously and placed intometallized polyester (2.5 mil) bags (Kapak Corporation, Bloomington, MN). Anampule containing the mold spores was placed inside each bag containing thebread sample. The bags were evacuated and flushed with carbon dioxide twicebefore heat sealing. After sealing, the ampule inside each bag was broken andthe bag was shaken to allow distribution of mold spores on the bread surface.The bags were stored' at 30C and removed periodically for observation of visiblemold growth.

The C. sporogenes spores used for the challenge study were preparedaccording o Vareltzis et al. (1984). The spore suspension was mixed withsterilized wheat flour and freeze-dried. The spores and vegetative cells wereenumerated using the PE-2 medium (Speck, 1976). To inoculate the bread, thespore-flour mixture was added to the dough before mixing. The inoculum size wasapproximately 1000 spores/g flour. The breads were baked, cooled, sealed inmetallized polyester bags with a carbon dioxide flush, and stored at 300C.Samples were removed periodically for spore and repetitive cell counts.

The Staphyococcus aureus culture was grown overnight on T-soy slant andviwshed of? with 7 ml ste-rie water. A series of cell suspensions was preparedby dilution and the turbidity of the samples was determined using aKlett-Summerson Photoelectric Colorimeter (Klett Mfg. Co., N.Y.). Concentrationof the culture was determined from a standard curve 5relating cell count to Klett(turbidity) units. An inoculum containing about 10 CFU/ml was prepared bydilution and 0.1 ml of the culture was applied onto the bread crust in acircular area of about 4 cm in diameter at one end of the loaf. Afterincubation, the crust portion containing the inoculum was removed and mixed with100 ml sterile saline using a Stomacher. It was enumerated using Staph-110 Agar(Difco, Detroit, MI).

Storage StudiesAfter the bread was delivered, they were repackaged in retort pouches made

of polyester-foil-polyethylene laminate (Kapak Corp., Bloomington, MN) to whicha silicon septum had been added. Except for the samples designated for storagein air,. the bags were evacuated and flushed with CO twice before heat sealingusing a Kenfield Vacuum Sealer (International Kenfild Distributing Co.,Chicago, IL). The breads were stored at -20, 20, 30 and 45*C, and duplicatesamples were.removed for analysis periodically.

When the breads were removed from storage for analysis, one quarter of theloaf was cut off by electric knife (Hamilton Beach, Scovill Division, Waterbury,CT) then the bottom 1/2" of the loaf was removed and stored in a Ziploc bag (Dow


Chemical, Indianapolis, IN) for color evaluation. Seven 2-cm thick slices wereremoved with a Bosch food slicer (Model EAS 55, Robert Bosch, Hausgerate GMBI,Munich, West Germany) for firmness measurement. Moisture, water activity and pHwere determined on the quarter endpleces of the loaf.

RESULTS AND DISCUSSION

(I) Effects of Humectants on Baking, Sensory and Physical Properties of Bread.

Dough Mixing PropertiesIn breadmaking, the first step is mixing of the ingredients into a dough.

The ultimate quality of the bread is closely related to the mixingcharacteristics of the flour dough. Humectants may change the dough propertiesby imbibing water and interacting with other components.

Addition of sorbitol up to 20% had little effect on water absorption, butincreased the mixing time of the eough (Figure 1). Unlike sorbitol, glycerol(20%) did not affect the mixing time, but caused a decrease in water absorptionform 65 to 57%. Propylene glycol was detrimental to dough properties even atlow concentrations (2-5%). The flour dough containing 5% propylene glycolshowed a decrease in dough consistency and an increase in stickiness.

Baking PropertiesLoaf volume and water activity (a ) of bread crumb decreased with

increasing sorbitol and glycerol concentrations in bread (Figure 2). Theeffects were more pronounced with glycerol than with sorbitol. For instance,addition of 15% sorbitol to bread lowered the a from about 0.97 to 0.94 and theloaf volume from 990 c.c. to 960 c.c. By compa ison, 8% glycerol was far moredetrimental to loaf volume than 15% sorbitol although both humectants at thespecified concentration would achieve the same effect in depressing a of the'read crumb.

Additional data showing the humectant effect on baking properties andsensory quality of bread are presented in Table 3. Increased hurnectant

,-concentrations resulted in longer mixing time, longer proof time and decreasedloaf volume. Water activity of bread crumb ranged from 0.907 for the 4-4-4 (41sorbitol-4% glycerol-4% propylene glycol) to 0.939 for the 4-1-1 sample (Table4).

Sensory Properties

The first sensory preference test was conducted using a panel of 38 judges.The samples evaluated were: control, 5% sorbitol, 200 sorbitol, 8% glycerol,and 10% sorbitol-5% glycerol-2% propylene glycol. A nine point hedonic scale (1= dislike extremely, 9 = like extremely) was used. There was no significantdifference in overall acceptability between the control and the breadscontaining humectants. The preference scores were in the range of 6.2 - 6.7,corresponding to "like slightly" or "like moderately". The results are somewhatunexpected since the humectants impart a certain bitter-sweet flavor to foods(Karel, 1973). Some panelists may have preferred the humectant breads becauseof the sweetness.

The ten samples shown in Table 3 were evaluated by a separate panel and theresults are summarized in Table 4. The best flavor scores (4.0 - 4.5) wereobtained with the 1-1-4, 4-1-1, 8-1-2, 1-2-1 and 4-2-1 samples. Taking intoconsideration a, loaf volume and sensory scores, a humectant combinationsimilar to 8-1- seemed to be the best choice. Since the original canned breadcontained only 7% sorbitol, the 7-1-2 combination was selected for the storagestudies.


Table 3. Baking data of bread made with different concentrations of humectants.

Ferment Proof Loaf a Loaf a

Sorb. P.G. Gly. Absorption Mix Time Time Time Wt. Vol. Crumbb

(%) (0) (%) (%) (min:sec) (Min) (Min) (g) (cc) Grain

Control 65 2:40 90 30 141.2 980 6

1 1 4 59 3:45 103 42 156.3 950 5

4 1 1 61 4:10 103 35 159.8 908 6

8 1 2 59 5:00 103 42 160.6 940 6

1 2 1 59 3:50 103 38 155.1 923 5

4 2 1 59 3:55 103 42 158.3 945 6

4 2 2 59 4:20 103 40 157.8 903 5

4 2 4 59 4:10 103 50 159.5 890 5

8 2 4 59 5:10 103 56 163.0 910 5

1 4 2 59 4:00 103 46 158.3 913 4

4 4 4 59 4:30 103 64 161.6 890 2

8 4 1 59 4:30 103 63 164.9 925 3

a Mean of duplicate samples

b = excellent, 1 poor


Table 4. Effect of humectants on water activity and sensory properties of

Crumba Crumbc OveralI d

Sorb. P.G. Gly. Grain Flavorb Texture Preference awe

1 1 4 5 4.0 2.5 3.5 0.934

4 1 1 6 4.4 4.0 3.9 0.939

8 1 2 6 4.2 3.3 3.0 0.912

1 2 1 5 4.5 3.9 4.0 0.936

4 2 1 6 4.2 3.3 3.8 0.927

4 2 2 5 3.0 3.1 2.7 0.932

4 2 4 5 3.1 3.6 3.1 0.920

8 2 4 5 2.7 3.0 2.5 0.919

1 4 2 4 3.2 2.9 2.3 0.932

4 4 4 2 3.1 2.7 2.5 0.907

8 4 1 3 2.5 3.1 2.4 0.909

-aCrumb Quality: 9 excellent; 1 = unsatifactory

b 5 = Absence of off-flavor; 0 = pronounced off-flavor

c Crumb Texture: 5 = smooth and moist; 0 = coarse and dry

d Overall Preference: 5 = like very much; 0 = dislike very much

e aw of crumb was determined 24 hours after baking


Stalin 9read staling was followed by the increase in crumb firmess and the

increase in starch crystallinity as detected by differential thermal analysis(DSC). As bread stales, the starch reverts from the amorphous form to the morestable, crystalline form. Melting of the crystalline amylopectin occurs at50-60*C. The endothermic peak area is proportional to the amount of crystallineamylopectin present in the aged bread. Figure 3 shows the DSC scans of a freshbread and a stale bread stored at 20°C for 14 days. Fresh bread shows no peakat 50*C since the amylopectin is in the amorphous form.

Finning of bread crumb was accelerated by the addition of 10' and 201sorbitol (Figure 4). The DSC data also shows more starch crystallinity ill thebreads containing sorbitol compared to the control during the first week ofstorage at 20°C (Figure 5). However, the control showed more starchcrystallinity than the sorbitol bread at day 14. The discrepancy between thefirmness and the DSC results indicates the complexity of the staling phenomenon.Factors other than starch retrogradation may also contribute to firming of breadcrumb during storage.

Several experiments were conducted to study the effect of sodiumstearoyl-2-lactylate (SSL) and shortening on bread staling. For prolongedstorage (14 days or longer), 3-9% shortening and/or 0.5-1.5% SSL were noteffective in retarding bread staling.

Refinement of FormulationWe observed that browning was a problem with the humectant breads during

storage, especially at elevated temperatures. Therefore, an experiment wasconducted to determine the effect of sugar (0-6%), malt (0-0.3%)and lactic acid(0-0.3%) concentrations on nonenzymatic browning of the humectant bread madewith 8% sorbitol, 2% propylene glycol and 2% glycerol (8-2-2). It was concludedthat 3% sugar was the optimum level for minimizing browning while retaining goodloaf volume of the bread. Also, addition of 0.15-0.30% lactic acid or 0.1-0.3%malt had little effect on gas production or loaf volume of the bread (see theAnnual Report for details). Therefore, malt was completely eliminated and sugarwas reduced from 6 to 3% in all subsequent studies to minimize nonenzymatic,-brwning.

pHTwo different types of lactic acid have been used to lower pH of the

humectant breads in this study. A lactic acid powder (Purac Powder H)containing 60% lactic acid and 40% calcium lactate was obtained from PatcoProducts (Kansas City, MO) and a lactic acid solution (85%) was purchased fromSigma Chemical Co. (St. Louis, MO). Because of the buffering effect of calciumlactate, the powder is not as effective as the lactic acid solution in pHreduction (Figure 6). However, the dough handling properties were much betterwith the lactic acid powder than the solution. Taking into consideration pH andflavor of the bread, 0.3% lactic acid powder was used in the final formulation.

Water'Activity MeasurementMoisture content and a of bread crumb and bread crust change continuously

after baking due to moisturl transfer from the crumb to the crust. Therefore,widely variable a values may be obtained if the sampling time and location arenot well controll~d. Figure 7 illustrates the variation of aw in crumb andcrust of the.5-2-2 humectant bread with storage time at 200C. At day zero, thea was 0.94-0.96 for bread crumb and 0.71-0.78 for bread crust. As moisturemgrated from the crumb to the crust, aw of crumb decreased with a correspondingincrease in crust a.


After 3 weeks of storage, the a values of crumb and crust were about 0.92and 0.88, respectively. Thus, the aw of the bread would approach an equilibriumvalue of 0.90. Since the a gradualYy leveled off after one week of storage, itwas decided to take the a Measurement of bread seven (7) days after baking.The crumb samples were tagen from the center of the slices that were 1-2 inchesfrom the loaf center. This sampling procedure should help minimize thevariation of a w measurement.

(II) Microbial Challenge Studies

Microbial stability of the bread samples is probably the most importantaspect of this project. It has been shown that the lower a limit for growth ofmost molds is 0.80-0.87 (Beuchat, 1981). Since the humectawt breads had a pH of4.8-5.0 and an a value of about 0.94-0.95 for fresh crumb, they may besusceptible to mcrobial spoilage. Therefore, a series of studies wereconducted to challenge the bread with Penicillium expansum, Aspergillus niger,Clostridium sporogenes and Staphylococcus aureus.

MoldsInitially, the challenge study was conducted with humectant breads

containing 5% sorbitol, 2% propylene glycol and 2% glycerol (5-2-2). No visiblegrowth of A. niger or P. expansum was observed after the breads had been storedfor 1,2,4 and 8 weeks at 30r in retort pouches flushed with CO2 . However,these two molds grew rapidly on regular breads, which served as the positivecontrol.

The minimal aw values of growth are 0.83 - 0.85 for P. expansum (Beuchat,1981) and 0.77 forwA. n (Pitt, 1975). These values ate sbstantially belowthe a of the fresh-b rumb (a = 0.94). The pH of the bread samples wasalso too high (4.8 - 4.9) to be inwibitory against the molds. Therefore, thetwo organisms may be inhibited by the presence of calcium propionate, thehumectants, the carbon dioxide, or a combination of these and other factorsincluding a and pH. Propylene glycol has been shown to be an effectivehumectant-a~timicrobial agent (Acott et a]., 1976). Most likely not any singlefactor alone was solely responsible for the inhibitory action against the twomold species.

A recent report in Cereal Foods World (Brody, 1985) pointed out that someEuropean bakeries have used carbon dioxide packaging to extend shelf-life ofbreads. Research conducted by the British Flour Milling Research Associationdemonstrated that 25% or more carbon dioxide is effective in inhibiting moldgrowth in baked goods. Therefore, the CO2 packaging used in this study may playan important role in preventing microbial spoilage of the humectant breads.

Clostridium sporogenesThe vegetative cell count (21-42) and spore count (10-29) of Clostridium

sporogenes did not increase with storage time. Although the bread dough wasinocuate with about 1000 spores/g of C. sporogenes, only 42 cells and 18spores per g were detected in the fresh crumb sample located at the center ofthe loaf. Thus the vast majority of the spores were destroyed during the bakingprocess. The vegetative cells detected probably resulted from sporegermination.

Additional challenge studies were conducted using the humectant breads madewith the modified formulas (7-1-2 and 6-1.5-?). The results for Clostridiumsporogenes are summarized in Table 5. The vegetative cell and spore countsdecreased from 46-49 and 13-79 to 0-5 and 2-7, respectively. Also, a slightreduction in crust aw was observed. The decrease in aw may indicate a gradual


Table 5. Effect of storage time at 30C on microbial counts of humectantbreads challenged with Clostridium sporogenes.

Time 7-1 -2b 6-1.5-2b

(weeks) Crust pH Veg. Cell Spores Crust pH Veg. Cells Sporesaw (#g) (#19) aw (#/g) (#/g)

0 - - 49 79 46 13

2 - - 11 8 23 13

4 - 4 23 - - 14 2

8 0.923 4.85 7 11 0.926 4.82 2 17

11 0.922 4.75 2 8 0.925 4.77 7 2

16 0.911 4.80 10 4 0.915 4.77 7 2

21 0.913 4.74 0 2 0.913 4.76 5 7

a Initial inoculum - 1400/g

b % sorbitol - % propylene glycol - % glycerol


dehydration due to the permeability of the film and a small leak.For a positive control, regular breads containing no humectants were

challenged with C. sporogenes and stored in a retort pouch flushed with CO2'The initial spore count was 130/g and the vegetative cell count was 46/g.During storage at 30'C, the spore count decreased to 8-13/g after 1-3 weeks.However, the vegetative cells multiplied rapidly upon storage. The cellcounts/g were 2400 (day 7), 14,300 (day 14) and 14,000 (day 21). This clearlydemonstrates the ability of C. sporogenes to grow in bread under favorableconditions.

The minimal a for growth is 0.935-0.965 for C. sporogenes (Sperber, 1983).This range is similar to those for C. botulinum (a = 0.93-0.94) and C.perfrinqens (a = 0.93-0.95). Although these a vglues are close to that of thebread crumb (aw 0.93-0.95) the relatively low pW (4.8-4.9) of the humectantbreads was inh bitory to the growth of C. sporogenes. While pH ( <4.5) or a( <0.93) alone could be used to control-botulism, inhibition of C. botulinumwshould be considered in light of the combination of a , pH and other factors.Further studies to determine the combined pH and a N'mits for growth and toxinproduction of C. botulinum in different food systems are necessary.

Staphylococcus aureusGrowth of Staphylococcus aureus on the crust of humectant breads stored at30C in CO pouches is shown i~F'Te 6. S. aureus was inhibited in some of the

samples. it is not clear why the 6-1.5-2 rea-ds appeared to be more favorablefor S. aureus growth than the 7-1-2 breads. Both formulations included 0.3%lactTc -ac-in powder form. Since the bread samples supported the growth of S.aureus, further studies were conducted to evaluate the pH effects.

Rumectant breads (7-1-2) were prepared using different concentrations oflactic acid in liquid or powder forms. Loaf volume and pH of the breadsdecreased with increasing lactic acid concentrations (Table 7 and Figure 8).Mixing time and fermentation time were also affected by the addition of lacticacid. The lactic acid powder contains 60% lactic acid and 40% calcium lactate.Because of the buffering effect of calcium lactate, the powder is not aseffective as lactic acid solution in pH reduction. However, the dough handlingrproperties were much better with the lactic acid powder than the solution.

Table 8 shows the effect of lactic acid concentration (powder form) on thegrowth of S. aureus in humectant breads after one week of storage at 30'C. Thepreliminary res-ults indicate that 0.25% lactic acid was effective in inhibitingS. aureus when the crust a was 0.90-0.91. However, an extended storage studyndicated that S. aureus g~owth was detected in the humectant breads containing0.3% lactic aciU after four weeks of storage at 30C (Table 9). No growth wasobserved in the 0.4% lactic acid sample. It should be noted that crust aincreased continuously after baking, thus creating a more favorable envirwnmentfor microbial growth. Therefore, adequate storage time should be allowed toevaluate growth of the inoculated organism on the crust. To prevent S. aureusgrowth, the pH should be reduced to 4.80 or less by incorporating 0.4'% or morelactic acid in the humectant breads.

The combined effect of water activity, pH and temperature on growth of S.aureus in brain-heart infusion broth was investigated by Notermans and HeuveTman(1983.. At 30-C, S. aureus was inhibited at a 0.90 and pH 4.9. This findingis similar to what-we observed with the humect~nt breads.

Increasingly more attention is given to the importance of microbial controlby a combination of parameters such as aW, pH, temperature and inhibitors (Foxand Loncin, 1982; Chang et al., 1983; We ster et al., 1985) This studydemonstrates the feasibility of imparting microbial stability to bread by propercontrol of aw, pH, packaging and heat treatment.


Table 6. Effect of storage time at 30*C on microbial gount of humectantbreads challenged with Staphylococcus aureus

Time 7-1-2 b 6-1.5-2b

(weeks) Crust pH Count Crust pH Counta aw w

2- - 1600 - - 5.7x10 5

0 7.8x10 5

4 - - 7.6x103 - - 5.2x10 6

0 8.6x10 6

8 0.917 4.97 400 0.918 4.92 8.8x10 5

0.920 4.96 200 0.922 4.94 400

11 0.914 4.75 0 0.914 4.71 1.32x0 6

0.916 4.75 0 0.918 4.72 8.2x10 5

16 0.919 4.80 6.8x10 6 0.908 4.81 1.5x106

0.917 4.80 1.3x10 5 0.908 4.83 0

a Initial inoculum - 104 CFU on crust surface (1.25 in. diameter circle)

sorbitol - % propylene glycol - % glycerol

o|


Table 7. Effect of liquid and powdered lactic acid on baking properties ofhumectant breads containing 7% sorbitol, 1% propylene glycol and2% glycerol (7-1-2).

Lactic H20 Abs. Mix Time Ferment Loaf LoafSample Acid (%) (min:sec) Time(min) wt.(g) Vol.(c.c) pH

Control - 64.4 2:30 136 152.3 983 5.26

7-1-2 - 58.4 4:00 144 163.1 970 5.49

0.3% Powder 58.4 4:10 168 162.0 945 4.91

0.5% Powder 58.4 4:22 201 162.6 913 4.60

0.6% Powder 58.4 4:46 212 160.8 873 4.51

0.2% Liq. 58.4 4:00 156 163.6 953 4.94

0.3% Liq. 58.4 4:22 170 162.6 895 4.78

0.5% Liq. 58.4 5:02 198 161.5 890 4.51

Nlatick Firdl ReportPage 20

Table 8. Effect of lactic acid (powder form) on survival of Staphylococcusaureus in a CO atmosphere at 30°C in humectant breadscontaining 7% s~rbitol, 1% propylene glycol and 2% glycerol.

Lactic Crust aw Staph. Count

Acid pH Day 2 Day 7 Day 2 Day 7(%)

0a 5.48 0.899 0.918 5.7x10 4

3.1x04 TNTC

0.25 5.04 0.902 0.909 1.2x04

1.8xi04 100

0.30 4.97 0.898 0.914 1.3x10 4

1.6x104 <100

0.35 4.87 0.898 0.910 1.2xi04

1.2xi04 <100

0.425 4.76 0.892 0.914 0.9xi0 4

0.7xlO4 <100

0.50 4.71 0.900 0.915 0.7xi04

0.65x104 <100

a Initial count at day zero - 1.4x104 on crust surface (1.25 in. diametercircle)

Natick Final Report

Page 21

Table 9. Effect of lactic acid (powder form) on survival of Staphylococcusaureus at 30C on hunmectant breads containing 7% sorbitol, 1%propylene glycol and 2% glycerol.

Time 0.3% lactic 0.4% lactic

(weeks) pH aw Count pH aw Count

0a 5.00 0.947 1.28xi0 4 4.87 0.953 1.31xi0 4

5.01 0.954 1.40x10 4 4.87 0.948 1.39104

2 4.84 0.902 0 4.72 0.905 50

4.84 0.896 0 4.73 0.903 0

4 4.81 0.911 0 4.69 0.907 0

4.81 0.929 8.8x0 4 4.67 0.916 0

a pH and crust aw measured at day 5

~ -.- -


Conclusions(1) Penicillium expansum and Aspergillus niger did not grow in the

humectant breads containing 5% sorbitol, 2% propylene glycol and 2%glycerol (5-2-2) when packaged in a CO2 atmosphere.

(2) The 7-1-2 and 6-1.5-2 humectant breads did not support the growth ofClostridium sporogenes during 21-weeks storage at 30'C, although rapidgrowth was observed in regular breads stored under the same conditionsafter 1-2 weeks.

(3) To prevent S. aureus growth on bread crust, pH of the humectant breadsshould be redueTt 4.8 or less with lactic acid.

(III.) Storage stability Studies

An extensive storage stability test was conducted using the 7-1-2 and6-1.5-2 humectant breads. The two formulas were selected based on theinformation obtained from previous experiments regarding acceptability andmicrobial stability of the samples. The breads were packaged in retort pouchesin air or CO and stored at 20,30 or 45°C. Samples were analyzed periodicallyfor mold, aerobic, and anaerobic counts, moisture content, a,, firmness, pH andcolor. Also, a number of sensory attributes were evaluated By a trained tastepanel. A complete set of the data for the physical and sensory properties isgiven in Appendix E-I.

Microbial StabilitNo visible mo d growth was detected in any of the samples throughout the

170 day storage period (Table 10). The aerobic counts of the 7-1-2 samples didnot exceed 1500/g. However, the 6-1.5-2 sample showed excessively high aerobiccounts at days 113 and 170. Anaerobic counts of the breads remained low untilday 170 when they became too numerous to count. Thus the anaerobes in thehumectant breads grew between day 113 and day 170. It is not clear why such along lag period was observed for the growth of the anaerobes. It is possiblethat sufficient oxygen was present to inhibit them and it took this long for theaerobes to deplete it. Based on the microbiological data, the shelf lives were

t-llmited to about 113 days for the 7-1-2 bread, and 85 days for the 6-1.5-2bread. Both formulations included only 0.3% powdered lactic acid. As pointedout in the "Microbial Challenge Study" section, lactic acid concentration shouldbe increased to 0.4% or higher to inhibit S. aureus. Therefore neitherformulation offered adequate protection ag-ainst-microbial spoilage during longterm storage at ambient temperature.

Relatively little information is available in the literature regardingmicrobial stability of bread packaged in carbon dioxide. A storage study ofEnglish style crumpets gackaged in go showed a substantial increase in aerobicplate count from 3 x 10 to 21 x 10 ifter 19 days at 30* C (Smith et al.,1983). The pH of the product was 6.5, considerably higher than that of thehumectant bread. The short shelf life of the CO -packaged crumpets illustratesthe importance of pH and aw reduction in control1tng microbial growth in thehumectant bread.

Moisture and Water ActivityChanges in moisture content of crumb and crust of the two humectant breads

(7-1-2 and 6-1.5-2) during storage are shown in Figures 8-11 and Table 11. Thecrust moisture increased rapidly during the first two weeks of storage andleveled off at about 36% (dry basis) for the 7-1-2 sample and at about 39% (drybasis) for the 6-1.5-2 sample. Conversely, crumb moisture decreased rapidlyduring the first two weeks and leveled off at about 44% (dry basis) for the


Table 10. Microbial counts of humectant breads stored at 30'C

7-1-2 a 6-1.5-2 a

Days Mold Aerobic Anaerobic Mold Aerobic Anaerobic

(CFU/g) (CFU/g)

0 390 5 0 0 0 0

6 0 5 0 0 0 0

32 0 0 0 0 43 0

66 0 35 0 0 1300 0

85 0 1500 2 0 0 0

113 0 260 0 0 104 0170 b 0 85 TNTCc 0 1.5x107 TNTCc

a . sorbitol - % propylene glycol - % glycerol

b Packed in CO,

C Too numerous to count


Table 11. Statistical comparison of the storage data of the twohumectant breads.

Parameters 7-1-2c 6-1.5 -2c

Moisture (% d.b.)

Crumb 46 .0a 49.3 b

Crust 35.5 a 37.0 b

aw

Crumb 0 .927a 0.932a

Crust 0.915 a 0 .921a

pH 4.93a 5 .0 1b

Firmness (g)

200 CO2 1107a 970 b

30c CO2 1023a 880b

300 Air 880a 736b

450 CO2 1015

a,b Values on the same row with different letters are

significantly different (p< 0.05).

c % sorbitol - % propylene glycol - % glycerol.


7-1-2 sample and 48% (dry basis) for the 6-1.5-2 sample. Based on the moisturedata, the retort pouch was an excellent moisture barrier for the packagedbreads.

Water activity changes in crumb and crust of the two humectant breadsduring storage are shown in Figures 12-15 and Table 11. The a values were morescattered than the moisture values. This illustrates the diffIculty inobtaining precise and accurate a, measurements. For the 7-1-2 humectant bread,the crust a varied between 0.903 - 0.925 and the crumb a was 0.920 - 0.930after 10 days of storage. For the 6-1.5-2 bread, the crult a and crumb a were0.910 - 0.930 and 0.920 - 0.935, respectively. Thus, aw of t~e bread sampIesdid not exceed 0.935 after 10 days of storage.

Statistical analysis of the moisture and a data shows no significantdifference in a between the 7-1-2 and the 6-1,-2 humectant breads (Table 11).However, the 6-Y.5-2 breads had significantly higher crumb and crust moisturecontents than those of the 7-1-2 breads. This could be associated with thegrowth of aerobic organisms in the 6-1.5-2 samples but not in the 7-1-2 samplesafter 113 days of storage at 30°C.

PHThe pH of the bread crumb decreased with increasing storage time (Table 12,

Figures 16-19). Similar pH reduction in CO -packaged English style crumpetsduring storage at 20'C was observed by Smit et al. (1983). They ascribed thedecrease in pH to absorption of CO in the package. Products of nonenzymaticbrowning could also contribute to the decrease ii pH during storage. The pHvalues of 4.93 and 5.01 are higher than desired. As indicated int the section onmicrobial challenge studies, the pH should be 4.8 or less in order to improvemicrobial stability.

FirmnessFimness of bread crumb as measured by the Fudoh Rheometer in g force

increased with increasing storage time (Figures 20-22). The 7-1-2 bread hadfirmer crumb than the 6-1.5-2 bread. This is probably due to the highermoisture content of the 6-1.5-2 samples. Firming of bread crumb occurred more

"-rapidly in breads stored at 45'C than those stored at 30'C. This is somewhatsurprising since lower storage temperature is known to accelerate firming ofbread crumb. Perhaps mechanisms other than starch retrogradation contributed tocrumb firming at high temperature during prolonged storage.

It should be pointed out that in the packaging of the bread totaldisplacement of the air atmosphere by CO2 was not obtained. Analysis of the CO2content indicated an average value of 63.7% with a range of 44.8-77%.Comparison of the firmness readings for breads stored in CO2 atmosphere versusair (Figure 20) indicates that firming is more rapid in the presence of COThis is diametrically opposed to the observations of Knorr and Tomlins (1995)who showed that compressibility of breads stored for 1-2 weeks increased muchmore slowly in the presence of CO atmospheres as opposed to air or nitrogen.This apparent discrepancy may be ue to the differences in storage time in thetwo studies (2 weeks vs. 6 months).

Although SSL and additional shortening were added to the humectant bread,their.effect on bread firmness was probably minimal after the first two weeks ofstorage.

ColorThe Hunter L values for the crumb and crust of the stored breads are

presented in Tables 13 and 14. Very little change in the lightness value was

0LL

a) I

4-u LA ( ' a' 00-0 LA U-).

CCUv

4- .0 C2 L

4-' 4

-0 0o c4 I'D-~l LA ( pI LA L

4- .- ' ' 0

4- )I M \

E-nv~ -Qk 4--

4) (u *Q a u ' 02 LEA u C)-4

4J4~

a) a* a' m'0~

LA * -u

"0 C! 0m -4 " "

= to

.4- 0

q- * CD' ' LO LO (U

41 a, ' 0 rCA C) -

oo 4.)

4-) C

4.) U .

4j. .0 02 0 -

( 6. LACj 4nU) ~ ~ = ('4 4-3a' C 0.

k'0 0 0 LA 0L. E-

0 QLL 9(a 4-CL U


lable 13. Hunter L values ot the crumb of stored bredd.

Time (weeks)

Sample 0.14 1 2 3 6 9 12 16 20

7:1:2200 CO2 73.2 - - 71.4 73.9 74.7 71.0 73.2

6:1.5:2200 CO2 74.9 - - 74.1 75.1 75.4 68.9 74.3

7:1:2300 CO2 - 73.4 72.3 73.5 72.5 72.0 71.8 66.0 71.6

6:1.5:2300 CO2 - 74.3 73.7 74.5 74.6 73.3 74.0 66.2 73.6

7:1:2300 Air - 73.4 73.6 74.5 74.4 73.8 72.7 71.5 71.4

6:1.5:2300 Air - 74.9 74.0 74.7 74.4 74.5 74.5 72.7 73.5

7:1:2450 CO2 - 72.0 71.9 69.8 67.7 60.2 - - -

6:1.5:2450 CO2 - 73.5 73.4 73.9 71.0 67.8 -

Natick Final Report

Page 28

Table 14. Hunter L values of the crust. of stored bread.

Time (weeks)

Sample 0.14 1 2 3 6 9 12 16 20 24

7:1:2200 CO2 39.6 - - 31.2 29.2 - 36.4 31.6 27.9 30.4

6:1.5:2200 CO2 34.5 - - 33.3 34.5 - 34.2 27.7 27.8 32.3

7:1:2300 CO2 - 32.6 27.5 30.8 29.5 29.1 29.3 29.9 29.2 32.6

6:1.5:2300 CO2 - 33.5 29.7 29.8 31.9 34.7 29.1 25.2 34.0 28.6

7:1:2300 Air - 30.6 31.2 31.9 39.8 31.6 28.3 30.5 30.4 32.1

6:1.5:230° Air 31.8 30.7 29.4 29.6 29.4 29.9 24.8 26.8 29.4

7:1:2450 CO2 - 27.3 28.0 26.3 26.0 26.8 -

6:1.5:2450 CO2 - 30.3 25.7 30.0 28.9 35.8


observed for the bread stored at 20 and 30C. However, at 45°C there was adramatic decrease in the lightness value for the crumb indicating a rapid stateof browning. Not as dramatic a trend was observed in the crust stored at 450C(Table 14).

Sensory EvaluationA total of 25 sensory attributes of the humectant breads were evaluated as

a function of storage time (Tables 15-17, Figures 23-72, Appendix F). Asexpected, crumb and crust color increased during storage (Figures 23, 24, 48,49, Table 15). Browning was accelerated at 45'C. The control stored at -20Cshowed no significant changes in crumb or crust color during storage. Carbondioxide has no significant effect on browning.

In general, the aroma values showed significant increases during storageand this was reflected in the overall aroma intensity (Tables 15-17, Figures25-29, 50-54). The only aroma that did not significantly change during storagewas the sour aroma. Of interest is the observation that the caramel characterdid not begin to appear until 36 days of storage and then it increaseddramatically by the end of 78 days. Among all of the aroma attributes, rancidaroma showed the most pronounced changes (Figures 29, 54). The bread stored at45C became highly rancid after two weeks. The overall aroma intensity of thesamples stored in CO, was lower than those stored in air at 30*C during thefirst five weeks (Ta le 16). It should be noted that yeasty aroma, anindication of freshness of bread, decreased with storage time (Figures 26, 50).

Textural changes in bread during storage are shown in Tables 15-17, andFigures 30-33 and 50-58. Firmness, dryness, crumbliness and springinessincreased with storage time. These changes generally occurred more rapidly at45C than at 20 and 300C. This is contrary to the general belief that rate ofbread staling decreases with increasing temperature. Perhaps mechanisms otherthan starch retrogradation are involved in bread staling at high storagetemperature. Similar changes occurred in mouthfeel (Figures 34-37, 59-62),which can also be considered textural in nature. Graininess and dryness showedthe most dramatic changes during storage. Gumminess and rate of hydration did

-show a tendency to decrease in values but not as dramatically as the perceived-increase in graininess. Carbon dioxide had no significant effect on texturalchanges in bread during storage at 30C (Tables 16 and 17).

Among all of the flavor attributes, rancid and caramel flavors showed themost dramatic changes during storage (Tables 15-17, Figures 38-46, 63-71).Sourness and bitterness increased with storage time, while yeasty flavor, anindicator of freshness, decreased. Sweetness showed little change duringstorage. As expected, there was a significant interaction between temperatureand sensory properties.

All of the changes in sensory properties in bread during storage arereflected in the overall preference (Figures 47 and 72). The control (-20°C)remained unchanged during the storage period, while preference of the 45Csample decreased markedly with storage time. No beneficial effect of CO 2storage was observed regarding sensory preference'of the bread.

In addition to the trained taste panel tests, a preference test was alsoconducted after five weeks storage of the bread (Table 18) using an untrainedpanel.. No significant difference was observed between the 7-1-2 and the 6-1.5-2samples. Preference score decreased with increasing temperature. The humectantbreads stored at 30C were rated as "neither like nor dislike", while the 45Csamples were unacceptable. It should be pointed out that bread as a foodnormally would not generate a high preference score since bread is usually usedas a carrier.

0

9- 4-)

M0 C'j C\1 UlA C\j \

.- 0 in .- ) N LA re).6 ) a cli m~

.0 E %y

4-) 0 UlA U-) U- CV) A

(a -4 O'E c=;.4-) ko Z: L C\J m~

E

0 u3OC O 0 CO Ol 14:~ A U

4) co qd C\J C0 CC) -4 co N. cr) LAL.0 C.Jt ~ -4* C~J m~ C'

4-0

0 0 .0 .0 to .0 .0 u .0 ucm (\j r- ON N.l 00 0I CO C

S.. c-4 0 -N. CO N. C4) C0 - enJ r) - -4 C\j (VI) \

4-)in V

N.~ U-) PL . ~ L a% -

co 0; CO CO LA O' 0 N.E -N. C%j ' ' C.

E'' >1 .0 .0 U)4-1 to to (a fu EU to.0 . .0EL u CD Csj qr CO ON N. co co C*.j

CO LA O CO LA L L A; CJ _ 0C=C-I) m 4 C\j c\J m) C

S-o0-0in C .0CEU (a (a E O eU E .0 .0a) as a) (\i 00 -4 CO

W3 C) C.J (VI -

4~- -C-)Ut o ( or

00 CO LA LAO~ Cl) 0 N

o CO)

t-

to 0uCl)

EUEIA ~~4-3 o0 E A i

010 0 o -4) atc) CD L. 0 to

0U m- 0 u E. S- V )

0 03 5 . x 5>L. U E U 0 5 .5.. 0cUc U 5 .. 0 i

-

oLL

-44

I~ C cn O c L O -

C ) * * CO.

C* . LC * -l

LA 4- A L .4 C N

>1 m U 0EU (a EU c0 . 0. .

(N .; clA r.: 1,: C LA mm(Ni C\)i l-4 -

.0 -0 to

* . U U

In '4-J

(1

co a

Cn %D C) C'J ,:: m (Ni C)

9A . .a

=s 0A N- C- LA(L U0'0

-. 4-1

c 01

0. OL N7 - IA 00 Q . 41

4--

C --

0 4 c)fe.to

4) 3


Table 16. Statistical analysis of the sensory data for two humectantbreads stored for five weeks at -20, 30 and 45°C.

Storage Conditions

Sensory Attributes -20(C0 2) 30(Air) 30(C0 2) 45(C0 2)

Crust browness significant interaction

Crumb color 16.4a 19.8 b 20.1 b 28.1 c

Aroma

overall intensity 24.2 a 28 .0b 26.6 a 31.3 c

yeasty aroma 13.3 a 10.0b 10.7 b 9.5b

acrid aroma 4 .8a 7 .9b 6.3ab 8.0b

sour aroma' 4 .2a 7 .4b 6.8ab 8 .1b

caramel aroma not tested these weeks

rancid aroma 2 .5a 5 .9b 5 .3ab 9.6 c

Texture

dryness 11.1a 22.7 b 2 4 .3 b 29.3 c

firmness significant interaction

springiness 21.1 a 34 .5bc 33.0 b 38.2 c

crumbliness 10.1 a 18.7 b 20.2b 22. b

Mouthfeel

dryness 12.3 a 26.0b 26.0b 32.7 c

gumminess 28.8a 22.7b 23.7b 20.9b

graininess 6 .2a 9.4ab 10 .9b 12.1 b

rate of hydration 36 .9b 27.2 a 26.1a 24.9 a

Natick Final Report

Page 33

Table 16 (cont.)

Storage Conditions

Sensory Attributes -20(C02) 30(Air) 30(C02 ) 45(C02 )

Flavor

sweet N.D

sour 3 .6a 5 .5ab 4 .2ab 6.4b

bitter N.D

yeasty 11 .1b 7 .1a 8 .4a 7 . 1 a

caramel 5.7 a 6.3 a b 6.1 a b 8.8 b

cardboard/rancid 3 .1a 7 .7b 8 .5b 12.6 c

Crust flavor

burnt significant interaction

bitter N.D

sweet N.D

Va1ues in the same row with different letters are significantly different

at 5% level.

N.D.= Not determined


Table 17. Statistical analysis of the sensory data for two humectantbreads stored for 24 weeks at -20 and 30*C

Storage Conditions

Sensory Attributes -20(C02 ) 30(AIR) 30(C0 2 )

Crust browness significant interaction

Crumb color 15 .6a 22.8b 24 .1b

Aroma

overall intensity 24 .6a 31.0b 30 .0b

yeasty aroma 14 .2b 9.3a 9 .6a

acrid aroma 4.3 10.4b 9 .5b

sour aroma 3 .ga 8 .7b 1.7 b

caramel aroma 3.8a 9 .8b 1 1 . 1b

rancid aroma 2 .5 a 9 1 b 8.4 b

Texture

dryness 17.7 a 27.9 b 30 . 5b

firmness significant interaction

springiness 22.1 a 35.7b 37 .5b

crumbliness 10.4 a 23.5b 24 .8b

Mouthfeel

dryness significant interaction

gumminess 31.5b 23.6 a 22.4 a

graininess 6 .3a i4.2b 14.6 b

rate of hydration 37.5b 24.8 a 24.3 a


Table 17 (cont.)

Storage Conditions

Sensory Attributes -20(C0 2) 30(AIR) 30(C0 2 )

Flavorsweet N.D

sour 4.5a 7.2b 7.0b

bitter 2 .9a 5 .9b 5 .2b

yeasty 11 .6b 6 .9b 7.3b

caramel 5.7a 10.6b 10.5 b

rancid 3.6a 1 1.7b 10.9 b

Crust flavor

burnt 10 .6b 9.0a 10.1 a b

bitter N.D

Values in the same row with different letters are significantly different(p<O.O5).N.D. = Not determined

Natick Final Report

Page 36

Table 18. Sensory preference of the humectant breads after fiveweeks of storage

Storage Preference Nine-PointSample Conditions Score Equivalent

6-1.5-2 450C 21.3a 3.2

7-1-2 450C 22.3 a 3.3

7-1-2 300C 29.7b 4.5

6-1.5-2 300C 30 .0b 4.5

6-1.5-2 -200C 37.5 c 5.6

7-1-2 -20C 40 .5c 6.1

Means in the same column:with different letters are significantlydifferent at 5% level.


Conclusions1.No visible mold growth was detected in the 7-1-2 or 6-1.5-2 samples

throughout the 170-day storage period.2. Anaerobic growth occurred between 113 and 170 days at 30'C. Aerobic

count also increased markedly in the 6-1.5-2 sample by 113 days.3. Lactic acid (powder) concentration should be increased from 0.3 to

0.4%.4. The retort pouch provided an excellent barrier for the packaged bread.

No moisture loss occurred during the storage period. Water activityof the bread crumb and crust stabilized at about 0.92-0.93 afterextended storage.

5. Storage of the humectant bread resulted in an increase in crumbfirmness, an increase in browning and a decrease in pH. These changeswere generally accelerated at 45'C compared to 20 and 30'C.

6. Most aroma values of the humectant breads increased with storage time.Rancid aroma showed the most dramatic change. Yeasty aroma, anindicator of freshness, decreased with storage time.

7. Firmness, dryness, crumbliness and springiness of the humectant breadsincreased with storage time. These changes generally occurred morerapidly at 45C than at 20 and 30*C.

8. Rancid and caramel flavors of the humectant bread increased markedlyduring storage.

9. The control (-20'C) showed no significant changes in physical orsensory properties throughout the storage period.

10. Carbon dioxide storage did not improve the sensory acceptability ofhumectant bread during prolonged storage.

(IV.) Refinement of Formula

Effect of Shortening and Oil

Since rancidity was a major problem in bread staling during storage, anexperiment was conducted to evaluate the effect of fat content on bread bakingrroperties. A reduction in fat content was accompanied by an increase in water

absorption in bread dough (Table 19). From the standpoint of microbialstability, a high fat content (e.g. 10%) is preferred.

In an attempt to alleviate the rancidity problem, an oil that is highlystable against lipid oxidation (Durkex 500) was used in place of the Criscoshortening (Table 20). Compared to the humectant bread (7-1-2) containing 10%shortening, the oil sample had a higher specific volume and a higher crumb wateractivity. After 10 days of storage at 45*C, a of the crumb and crust of thetwo samples was about 0.92, indicating an equiyibration between the crumb andthe crust.

In general, there were no significant differences between the oil and theshortening samples stored for 10 days at 45*C (Table 21). The only exceptionwas that sourness was more pronounced in the shortening sample. The changes insensory properties were similar to those reported in Tables 15-17.

Sponge vs. Straight DoughThe humectant breads (7-1-2) prepared by the straight dough and the sponge

dough methods were similar in loaf volume, crust color, crumb moisture and wateractivity (Table 22). However, pH of the sponge dough bread (5.0) was lower thanthat of the straight dough bread (5.2). Thus the sponge dough method ispreferred for the production of the humectant breads.


Table 19. Effect of fat content on baking properties of humectant breedscontaining 7% sorbitol, 1% propylene glycol, 2% glycerol and 0.4%lactic acid.

Formula H20 Abs. Mix Time Ferm. Proof Loaf Wt. Loaf Vol. Crumba

(% Fat) (%) (min:sec) Time(min) Time(min) (g) (cc) Grain

Control 64.5 2:30 94 46 150.7 985 7

3 60.4 3:50 100 82 158.2 896 7

6 59.4 4:05 100 88 159.8 912 7

10 58.4 4:00 100 98 162.6 921 7

a Excellent = 9, Poor 1

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Table 21. Sensory properties of humectant breads (7-1-2) containing 10%shortening or a high stability oil (Durkex 500) after ten days ofstorage.

Senscry Score

Attributes Oil (-20'C) Shortening (-20'C) Oil (45'C) Shortening (45'C)

Crumb Color 9 .7a 10 .0a 18 .7b 18.7b

Overall AromaIntensity 25 .3ab 23 .1a 31.7bc 34 2b

Yeasty Aroma 15.7 b 15.7b 9 .7a 9 .0 a

Rancid Aroma 1 .7a 2 .4a 13. 2b 16.1 b

Dryness 8.9a 8.2a 3 1.0b 33 .4b

Firmness 7.5a S.Oa 36 .1b 36 .2b

Crumbliness 7 .5a 7 .5a 15 .7b 25 .5b

Sour Flavor 3.2a 6 .4ab 8 .3b 14 .2c

Caramel Flavor 4.2a 40 a 14 .3b 8 .9ab

Rancid Flavor 4 .7a 5,0a 17 .6b 15 .1b

iPreference 30 .9b 33 1 b 189 17 5a

Values in the same row with different letters are significantly different at 5%level.

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CONCLUSIONS

1. Dough mixing properties were affected by the addition of sorbitol,glycerol and propylene glycol. Propylene glycol was detrimental todough properties even at low concentrations (2-5%).

2. Loaf volume and water activity (a ) of bread decreased with increasinghumectant concentrations. Compar~d to sorbitol at the same aw 1glycerol was more detrimental to bread quality.

3. The a of crust was lower than the crumb and required time to reachequillbri um.

4. Staling seemed to be accelerated by increased concentration ofhumectants, although at 14 days no difference was observed between 10*%sorbitol and the control. Shortening (3-9%) and sodiumstearyl-2-lactylate (0.5-1.5%) did not retard staling.

5. Removing malt from the formula and reducing the sucrose content to 3%markedly reduced the tendency to browning.

6. Lactic acid powder (Purac Powder H) gave better dough handlingproperties.than lactic acid solution although more was needed toobtain the same decrease in pH.

7. Based on a , loaf volume and sensory evaluation data, a humectantbread contlining 7% sorbitol, 1% propylene glycol and 2% glycerol(7-1-2) was selected for further study.

8. Mold growth in bread can be inhibited by the addition of humectantsand lactic acid, and by packaging in a partial CO2 atmosphere.

9. The 7-1-2 and 6-1.5-2 humectant breads did not support the growth ofC. sporogenes at 300C.

10. S. aureus grew readily in the 6-1.5-2 bread, while results wereVarTa Tewith the 7-1-2 bread. -To prevent S. aureus growth, pH of thehumectant bread should be decreased to 4.8 6r less.

11. An extended storage stability test (170 days) showed no visible moldgrowth in the 7-1-2 and 6-1.5-2 breads. However, anaerobic growthoccurred between days 113 and 170 at 30*C. It is recommended that thelactic acid concentration be increased from 0.3 to 0.4%.

12. Major changes in texture, color, pH and sensory properties of thehumectant breads were observed during storage. The breads showed anincrease in browning, firmness, drynessi crumbliness, rancid flavorand rancid aroma. These changes were accompanied by a decrease in pHand overall preference score. There was a significant interactionbetween these changes and temperature.

13. Carbon dioxide storage did not improve the overall sensoryacceptability of the breads.


14. Replacing the shortening in the formula with a high stability oil(Durkex) had no significant effect on sensory properties of thehumectant bread stored at 45°C for ten days.

15. Humectant bread made with the sponge dough method had a lower pH thanthat made with the straight dough method.


RECOMMENDATIONS

1. The bread formula should include 7% sorbitol, 1% propylene glycol and2% glycerol, or a combination of humectants that results in a final awof 0.92-0.93 or less in the crumb.

2. The pH of the bread should be reducecd to 4.8 or less by addition ofmore lactic acid powder.

3. The bread should be packaged in a hermetically sealed container suchas a retort pouch or a metal can with a partial CO2 atmosphere.

4. The bread should be prepared by the sponge dough method.

5. To avoid post-contamination, the bread may be baked inside thecontainer and sealed after baking.


REFERENCES

AACC. 1983. American Association of Cereal Chemists Approved Method, Method74-10, AACC, St. Paul, MN.

Acott, K., A.E. Sloan and T.P. Labuza. 1976. Evaluation of antimicrobialagents in a microbial challenge study for an intermediate moisture petfood. J. Food Sci. 41:541.

Bashford, L.L. and T.E. Hartung. 1976. Rheologlcal properties related to breadfreshness. J. Food Sci. 41:446.

Beuchat, L.R. 1981. Microbial stability as affected by water activity. CerealFoods World 26:345.

Brody, A.L. 1985. Packaging: Controlled atmosphere bakery product packaging.Cereal Foods World 30:352.

Chang, C.W., T.G. Sebranek, H.W. Walker and D.E. Galloway. 1983. Packagingfilm permeability in conjunction with sodium nitrite, potassium sorbate orlactic acid starter culture for control of Clostridium sporogenes (PA 3679)growth in sliced bologna. J. Food Sci. 48:Z.

D'Appolonia, B.L., K.A. Gilles, E.M. Osman and Y. Pomeranz. 1971.Carbohydrates. In: "Wheat Chemistry and Technology", p. 301. Y. Pomeranz(ed.), Amer. Assoc. Cereal Chemists, St. Paul, MN.

Fox, M. and M. Loncin. 1982. Investigations into the microbiological stabilityof water-rich foods associated by a combination of methods. Lebensm. u.Technol. 15:321;

Frazier, W.C. 1967. Food Microbiology. 2nd edition, McGraw-Hill Book Co., NewYork, NY.

Giovanni, M.G. 1983. Response surface methodology and product optimization.Food Technol. (11):41.

Karel, M. 1973. Recent research and development in the field of low-moistureand intermediate-moisture foods. CRC Critical Rev. Food Technol. 4:329.

Knorr, D. and R.I. Tomlins. 1985. Effect of carbon dioxide modified atmosphereon the compressibility of stored baked goods. J. Food Sci. 50:1172.

Labuza, T.P. and M. Saltmarch. 1981. The nonenzymatic reaction as affected bywater in foods. In: "Water Activity: Influences on Food Quality", p. 605.L.B. Rockland and G.F. Stewart (eds.), Academic Press, New York, NY.

Larmond, E. 1970. Methods for sensory evaluation of food. Canada Dept. ofAgriculture, Ottowa, Ontario, Canada.

Larsen, R.A., R.B. Koch and J.J. McMullen; 1954. The browning of canned breadcrumb. Food Technol. 8:355.

Liewen, M.B. and E.H. Marth. 1985. Growth and inhibition of microorganisms inthe presence of sorbic acid: a review. J. Food Protect. 48:364.

Maga, J.A. 1975. Bread staling.' CRC Critical Rev. Food Technol. 5:443.Marth, E.H. 1977. Chemicals in food preservation. In: "Elements of Food

Technology*, N.W. Desrosler (ed.), p. 82. AVI Publishing Co., Westport,CT.

Mauron, J. 1981. The Maillard Reaction in food; a critical review from thenutritional standpoint. Prog. Fd. Nutr. Sci. 5:5.

Nagel; C.W., C.J. Brekke and H.K. Leung. 1981. Humectant analysis ofintermediate meat by high performance liquid chromatography. J. Food Sci.47:342.

Notermans, S. and C.J. Heuvelman. 1983. Combined effect of water activity, pHand suboptimal temperature on growth and enterotoxin production ofStaphylpcoccus aureus. J. Food Sci. 48:1832.

Pitt, J.1. 1975. Xeroh lic fungi and the spoilage of foods of plant origin.In: "Water Relations of Foods", R.B. Duckworth (ed.), Academic Press,London.

Natick Final Report

Page 46

Pomeranz, Y. and K.F. Finney. 1975. Sugars in breadmdking. Baker's Digest49:20..

Ponte, J.G., Jr. 1981. Staling of white pan bread: fundamental causes. CRCCritical Rev. Food Sci. & Nutr. 15:1.

Prentice, N. and B.L. D'Appolonia. 1971. High-fiber bread containing brewer'sspent grain. Cereal Chem. 54:1084.

Rubenthaler, G.L., P.L. Finney, D.E. Demaray and K.F. Finney. 1980. Gasograph:Design, construction, and reproducibility of a sensitive 12-channel gasrecording instrument. Cereal Chem. 57:212.

Smith, J.P., E.D. Jackson and B. Doraikul. 1983. Storage study of agas-packaged bakery product. J. Food Sci. 48:1370.

Speck, M.L. 1976. Compendium of Methods for the Microbiological Examination ofFoods. American Public Health Association, Washington, D.C.

Sperber, W.H. 1983. Influence of water activity on food borne bacteria - areview. J. Food protect. 46:142.

Vareltzis, K., E.M. Buck and R.G. Labbe. 1984. Effectiveness of a betalains/potassium sorbate system versus sodium nitrite for color development andcontrol of total aerobes, Clostridium perfringens and Clostridiumsporogenes in chicken frankfurter. J. Food Protect. 47:=*2.

Walker, C.E. and A.M. Parkhurst. 1984. Response surface analysis of bake-labdata with a personal computer. Cereal Foods World 29:662.

Webster, S.N., D.R. Fowler and R.D. Cooke. 1985. Control of a range of foodrelated organisms by a multi-parameter preservation system. J. FoodTechnol. 20:311.

CONTROL7 -7SORBITOL

500 10. % -

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Appendix B

Name

Date

Please evaluate these samples on a scale of 0 to 5 for crumb texture,

off-flavor, and overall preference.

Crumb texture: 0 - smooth and moist5 - coarse and dry

Off-flavor: 0 - imperceptible5 - pronounced

Overall preference: 0 - dislike very much1 - dislike moderately2 - dislike slightly3 - like slightly

4 - like moderately5 -like very much

OverallCode Crumb texture Off-flavor preference

Comments:

Appendix C

SAMPLE BREAD EVALUATION NAME

DATE

Directions: Indicate the intensity of each characteristic by placing avertical line through the -orizontal scale.

APPEARANCE

crustbrowness light dbrk

crumb color IIwhite It. yellow blown

AROMA

overallintensity sli ht vkry

yeasty ___

acrid I

sour ___

rancid 1__

other(specify) '

TEXTURE

drynessslight vtry

firmness ___

springiness ___

crumbliness I

Appendix C

SAMPLE NAME

MOUTHFEEL

dryness v ry

gumminess 1 t

graininess Irate ofhydration slow fdst

FLAVOR OF CRUMB

sweet _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

sli ht varysour t__

bitter .

yeasty Icaramel ___

cardboard/ ___rancid

other 1(specify)

FLAVOr OF CRUST

burntslight vdry

bitter ___

sweet

OVERALLPREFERENCE disliked neither ltke

extremely dislike extremelyor like

Appendix C

BREAD EVALUATION

DEFINITIONS

TEXTURE

Dryness - lack of surface moisture

Firmness - resistance to squeezing between thumb and forefinger

Springiness - how fast sample springs back to original shape after deformation

Toughness - how much sample resists being torn apart

Crumbliness - how sample falls apart when apply shearing force

MOUTHFEEL

Dryness - Lack of surface moisture on tongue, abrasive

Toughness - resistance to bite

Stickiness - how much sample sticks to teeth and palate (adhesive like peanutbutter)

Gumminess - how much the sample sticks to itself (like chewing gum)

Rate of hydration - how easily the sample took up moisture while chewing

Graininess - feel particles on-the palate while masticating

Appendix D

SCORECARD FOR PREFEREN CE TEST

NAME

DATE

CODE

SAMPLE #

Directions: Please taste the sample and rate how much you like it by placinga vertical line on the scale below.

DisIlike Neither LikeExtremely Dislike Extremely

or Like

Comments:

NAME

DATE

CODE

SAMPLE #

Directions: Please taste the sample and rate how much you like it by placinga vertical line on the scale below.

isl'ike Neither LikeExtremely Dislike Extremely

or Like

Comments:

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