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Journal of Food Protection, Vol. 73, No. 4, 2010, Pages 758762

Copyright G, International Association for Food Protection

Research Note

A Ready-to-Use Antifungal Starter Culture Improves the Shelf Life of Packaged Bread
C. L. GEREZ,1 M. I. TORINO,1 M. D. OBREGOZO,1
1Centro

AND

G. FONT

DE

VALDEZ1,2*

de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145 (4000), Tucuman, Argentina; and 2Facultad de Bioqumica, Qumica y Farmacia, Universidad Nacional de Tucuman (UNT), Tucuman, Argentina MS 09-301: Received 14 July 2009/Accepted 17 December 2009

ABSTRACT
Fungal spoilage is the main cause of economic loss in the baking industry. In this study, we developed a ready-to-use biopreserver (slurry [SL]) for nonsliced packed bread by using selected antifungal lactic acid bacteria (LAB) and low-cost ingredients that are compatible with the food matrix. Four LAB strains (Lactobacillus brevis CRL 772, L. brevis CRL 796, L. plantarum CRL 778, and L. reuteri CRL 1100) tested in bread preservation were able to inhibit Penicillium sp. growth and lengthen shelf life twofold with respect to breads prepared using only Saccharomyces cerevisiae (2 days shelf life). The best biopreservation effect (5 days shelf life) was obtained with 40% antifungal slurry SL778 containing L. plantarum CRL 778; this was as effective as 0.2% calcium propionate (PCa). The antifungal effect of SL778 was related to the synthesis of acetic and phenyllactic acid as well as lactic acid, which was produced at a high concentration (31.2 mmol/kg) and lowered the pH of the dough, favoring the undissociated fraction of the organic acids. The combination of the starter SL778 with 0.4% PCa extended the shelf life of packaged bread to 24 days, 2.6-fold longer than breads prepared with only 0.4% PCa.

Fungal spoilage is the leading cause of economic loss in the baking industry and may also cause serious public health problems due to the production of mycotoxins (15). Several factors influence the contamination of baked goods by mold, among them, the type of product (breads or sweet baked goods), ingredients, starter culture, bakery layout, and product packaging. Although fungal spores are killed in bread dough during baking, airborne molds typically contaminate the end product during cooling, slicing, wrapping, and storage. Several methods, including the addition of propionic acid and its salts, modified atmosphere packaging, and irradiation and pasteurization of packaged bread, can be used to minimize microbial spoilage. Most bakeries in Argentina use calcium propionate (PCa) at the maximum concentration (0.4%, wt/wt) allowed by the Argentine Alimentary Code for packaged sliced breads. However, results are not always satisfactory. During the last decades, increasing consumer demand for natural products has renewed food industry attention in biopreservation, especially with lactic acid bacteria (LAB) of particular interest. This microbial group is generally recognized as safe and has been used for centuries in food fermentation. Sourdough (a mixture of wheat or rye flour and water, fermented by LAB with or without yeasts) has been used to enhance bread quality (4, 6). Compared with traditionally processed sourdough, liquid sourdough offers
* Author for correspondence. Tel: (54-381) 431-0465; Fax: (54-381) 4005600; E-mail: gfont@cerela.org.ar.

several technological and analytical advantages, including reproducibility, easier control of the fermentation parameters, and greater suitability under different technologies for the production of various baked goods (2, 9). Most of the beneficial properties ascribed to sourdough result from the acidifying activity of LAB. LAB create an optimum pH for the activity of endogenous proteinases (21) and directly contribute to the loaf volume (5) and the bread flavor, especially through the synthesis of acetic acid (11). However, the need for carefully selected LAB strains as starter cultures and the development of novel technologies for enhancing microbial performance are current challenges for the bakery industry. In previous work, 95 homo- and heterofermentative LAB strains were evaluated for their antifungal activity (10). Only four strains isolated from sourdough (Lactobacillus plantarum CRL 778, L. reuteri CRL 1100, L. brevis CRL 772, and L. brevis CRL 796) were able to inhibit the growth of Aspergillus niger, Penicillium sp., and Fusarium graminearum, the major spoilage molds found in the small bakeries of Tucuman, in northwest Argentina. The antifun gal effect of the selected LAB strains was related to the presence of lactic, acetic, and phenyllactic acids and was dependent on both the pH and the ratio of each acid (10). These results were the starting point of the present study, which addresses the development of a ready-to-use biopreservative starter culture and its evaluation in packaged nonsliced bread.

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MATERIALS AND METHODS

Formulation of the antifungal starter culture (slurry). The strains L. reuteri CRL 1100, L. plantarum CRL 778, L. brevis CRL 796, and L. brevis CRL 772 used in this study were obtained from the Culture Collection (CRL) of the Centro de Referencia para Lactobacilos, (Tucuman, Argentina). Before experimental use, the strains were grown twice in de Man Rogosa Sharpe (MRS) broth (8) without acetate at 37uC for 16 h. The cultures were used as single or mixed (ratio, 1:1) cultures and inoculated (15%, vol/vol) in a mixture of 250 g of wheat flour (000 type: 71.1% carbohydrates, 2.8% fiber, 10% protein), 5 g of sucrose, 12.5 g of skimmed milk, and 0.75 liters of tap water. After homogenization, the pH was adjusted to 5.90 0.15 with Na2HPO4, and fermentations were allowed to proceed at 37uC for 16 h with continuous stirring at 100 rpm. Each resulting semiliquid fermented product was named slurry (SL) and was used as a bioingredient in the dough formula. LAB growth was determined by the plate dilution method using MRS agar; the plates were incubated at 37uC for 48 h, and results were expressed as log CFU per milliliter. The rate of slurry acidification was determined by measuring pH (Altronix-TPX1 pH/ mV-Meter, Argentina) and total titratable acidity (TTA) (potentiometric method with Dornic solution [0.1 N NaOH] using phenolphthalein as an indicator) in 10-ml samples. Results were expressed in milliliters of Dornic solution needed to achieve a pH of 8.3 to 8.6. Organic acid levels were determined by high-performance liquid chromatography (HPLC) and by enzymatic methods. Dough fermentation and bread manufacture. The lactic slurries were used for the production of wheat bread. The doughs were prepared as follows: 1,000 g of commercial wheat flour (000 type), 10 g of NaCl, 20 g of sucrose, 50 g of skim milk, 20 g of fat, 10 g of NaCl, and 0.5 liters of tap water. To incorporate the fermented slurry, tap water was partially replaced (20, 30, 40, or 60%) in the dough preparation by equal amounts of the slurry fermented by each of the selected LAB strains. A commercial strain of Saccharomyces cerevisiae (7 log CFU/g; Calsa, Buenos Aires, Argentina) was used as the leavening agent. Doughs containing yeast only (Y-dough) and doughs with yeast plus 0.1 to 0.4% (wt/wt) PCa (YP-dough) were used as controls. The doughs were individually placed in aluminum pans for fermentation (2 h, 30uC). The pH was determined using a Sartorius portable meter PT-10 model (Sartorius AG, Goettingen, Germany) equipped with a puncture electrode (Hanna Instruments, Woonsocket, RI). Ten-gram portions of dough were homogenized with 90 ml of sterile tap water (homogenizer, VirTis, Gardiner, NY) for analysis of TTA, microbial viability, and organic acid determination. Colony counts were determined by the plate dilution method using MRS agar (8) supplemented with cycloheximide (200 mg/ml) for LAB counts and yeast peptone dextrose agar supplemented with chloramphenicol (500 mg/ml) for yeast counts. The plates were incubated at 37uC for 48 h (LAB) and at 30uC for 72 h (yeasts). Results were expressed as log CFU per gram of dough. The leavening power, determined by fermenting a 50-g portion of dough in a test tube at 30uC for 2 h, was calculated as V (cm3) ~ Vf 2 Vi, where Vf and Vi are the final and initial leaven volumes, respectively. After fermentation, the doughs were baked in a batch oven (160uC for 45 min), and the bread loaves were cooled at room temperature for 90 min. The volume of the baked breads (length by width by height) was expressed in cm3. The bread loaves were surface sprayed (1 ml per 100-g loaf) with a conidial suspension (103 conidia per ml) of Penicillium sp. (strain CH 102); then they were packed into polyethylene bags and stored at 30uC. The bread shelf life was defined as the time (in days) for

molds to become visible on the surface of the packaged loaves. Observations were performed daily. Organic acids determinations. Phenyllactic acid was determined by HPLC (ISCO 2350 model) using an Aminex HPX-87H ion-exclusion column (300 mm | 7.8 mm, Bio-Rad, Hercules, CA) under the following conditions: mobile phase, 2.5 mM H2SO4; flow rate, 0.6 ml/min; column temperature, 45uC. A UV (210 nm) detector (ISCO V 4 model, Teledyne Isco Inc., Lincoln, NE) connected to the software (Peak Simple II, SRI Instruments, Torrance, CA) for data analysis was used. Acetic and lactic acid levels were assessed using commercial enzymatic kits (Boehringer-Mannheim, Mannheim, Germany). Statistical analysis. The results presented are the mean values standard deviation (SD) from three independent experiments. Data were compared by analysis of variance and Dunnett t test, and statistical significance (P , 0.05) was determined (Minitab-12 software trial version, Minitab Inc.; InfoStat v 2008, Cordoba, Argentina).

RESULTS The chemical and microbiological characteristics of the slurries obtained with each LAB strain (SL772, SL796, SL778, and SL1100) are summarized in Table 1. Cell counts (9.2 to 9.4 log CFU/ml) and pH values (pH ~ 3.7) were similar for all slurries after 16 h of fermentation. SL778 yielded the highest values for TTA (8.7 ml of 0.1 N NaOH per 10-ml sample) and lactic acid (219 mM), while SL1100 produced the highest level of acetic acid (24 mM). Only slurries produced with SL778 and SL1100 yielded phenyllactic acid (PLA), at 0.34 and 0.29 mM, respectively. Each fermented slurry was subsequently used in different proportions (20 to 40%, vol/vol) to produce doughs (SL-doughs) that were identified with the number of the LAB strain. Regardless of the LAB strain, use of 60% slurry did not yield a suitable dough or bread; the leavening power was low (,50 cm3), yielding a bread with low volume (,250 cm3) and a sharp acidic flavor. In contrast, no negative effects were obtained with slurry at 20 to 40% (vol/vol). Results are presented in Table 2. The SL-doughs attained a significantly (P , 0.05) lower pH (5.0 to 5.4) and higher TTA (4.7 to 7.7 ml of 0.1 N NaOH per g) than Y- and YP-doughs due to the high concentration of lactic acid (9.0 to 34.2 mmol/kg) and acetic acid (5.4 to 11.2 mmol/kg). The best FQ values (1.3 to 2.8) corresponded to the SL-doughs prepared with SL778, SL796, and SL772. In contrast, Y- and YP-doughs gave FQ values .5.0. Differences in shelf life were also seen for breads prepared with SL, Y, and YP. The addition of slurry to doughs extended the shelf life to 4 to 5 days compared with Y-breads, an effect that was dependent on both the lactobacilli involved and the percentage of slurry used; the antifungal effect was lost after slurry neutralization (data not shown). Best results (5 days shelf life) were obtained using SL778 at 40% (vol/vol). The antifungal effectiveness of SL778 at 40% was similar to that of YP-bread containing 0.2% PCa, while Y-breads were spoiled within 2 days of ambient storage. The shelf life of breads prepared from doughs containing SL778 (40%, vol/vol) and PCa (0.1 to

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TABLE 1. Characteristics of the slurries fermented with lactic acid bacteria

Organic acids (mM) Slurriesa Colony countsb pH TTAc Acetic Lactic Phenyllactic

SL772 SL778 SL796 SL1100

a

9.4 9.4 9.3 9.2

0.1 0.3 0.6 0.2

3.7 3.6 3.7 3.7

0.0 0.0 0.0 0.0

7.6 8.7 5.7 6.2

0.2 0.7 0.2 0.5

11 16 10 24

2.0 3.0 5.0 0.4

167 219 150 138

11 18 15 2.0

0.0 0.34 0.0 0.29

0.0 0.03 0.0 0.02

The slurries (SLs) were fermented with L. brevis CRL 772 (SL772), L. plantarum CRL 778 (SL778), L. brevis CRL 796 (SL796), and L. reuteri CRL 1100 (SL1100) at 37uC for 16 h. Semiliquid slurries contained an inoculated mixture: wheat flour, 250 g; sucrose, 5 g; skimmed milk, 12.5 g; and tap water, 0.75 liters. b Colony counts of LAB expressed as log CFU per milliliter of slurry. c TTA, total titratable acidity, reported as milliliters of 0.1 N NaOH needed to achieve a pH of 8.3 to 8.6 in 10-ml samples.

TABLE 2. Characteristics of doughs prepared with fermented slurries a

Organic acids (mmol/kg) Preservative (%)b pH TTAc Lactic Acetic Phenyllactic FQd

SL772 20 30 40 SL796 20 30 40 SL778 20 30 40 SL1100 20 30 40 PCa 0.0f 0.1 0.2 0.3 0.4
a

5.3 5.1 5.1 5.3 5.3 5.2 5.4 5.3 5.0 5.4 5.2 5.2 5.7 5.6 5.5 5.5 5.5

5.0 5.2 5.2 4.7 5.5 6.6 5.2 7.0 7.5 7.2 7.5 7.7 4.5 4.5 4.6 4.7 4.7

9.1 11.6 16.3 9.0 9.2 13.8 10.2 15.6 31.2 24.4 27.6 34.2 2.5 2.4 2.3 2.6 2.3

6.8 7.7 8.3 5.4 5.6 6.2 7.9 9.2 11.2 6.8 7.7 8.0 0.4 0.4 0.3 0.4 0.3

NDe ND ND ND ND ND 0.4 0.6 0.7 0.7 0.9 1.1 ND ND ND ND ND

1.3 1.5 1.9 1.6 1.6 2.2 1.3 1.7 2.8 3.6 3.6 4.3 6.2 6.0 7.7 5.2 7.7

0.4%, wt/wt) (SLP-doughs) was also evaluated with Y- and YP-breads used as negative and positive controls, respectively. As shown in Table 3, the SLP-breads obtained by combining SL778 and 0.4% PCa reached a shelf life of 24 days. As for organic acids, similar concentrations of lactic, acetic, and phenyllactic acids were found in SL- and SLPdoughs at similar concentrations (Tables 2 and 3). Nevertheless, differences in preservation were obtained in SL- and SLP-breads. Since the inhibitory effect of weak acids depends greatly on their undissociated fraction, this parameter was calculated for lactic acid, acetic acid, PLA, and PCa in SLP- and YP-doughs using the HendersonHasselbach equation (Table 3), which considers the pH of the environment and the dissociation constant (pKa value of the particular acid) (10). SL- and SLP-doughs prepared with SL778 40% (vol/vol) had a pH of 5.0; with only 7% of the lactic acid being undissociated (pKa, 3.83), it was scarcely active despite its high concentration (31.1 to 31.4 mmol/kg dough). Only 3% of the PLA (pKa, 3.46) was undissociated; however, the antifungal effectiveness of PLA is much greater than that of lactic acid (10). In contrast, 37% of the acetic (pKa, 4.76) and 42% of the PCa (pKa, 4.87) acids were undissociated. Undissociated PCa accounted for the major difference between SL- and SLP-doughs and between SLP- and YP-doughs. YP-doughs had a pH of 5.5 with only 18% of the PCa undissociated. DISCUSSION The flavor, taste, and shelf life of wheat bread are improved by the addition of sourdough starter, in which LAB play an important role (3, 14). This kind of starter is widely used in Sudan, India, Mexico, and Europe to make bread from sorghum, rice, maize, and rye flour, but it is not typically used in Argentina and most other South American countries. During the last decade, the main technological advancements in the baking industry have been shortened leavening time and the availability of ready-to-use starter cultures. Although bakers yeast (S. cerevisiae) reduces the time of bread making, the finished bread is less flavorful than bread prepared using specific strains of LAB (1). The ready-to-use antifungal slurry (SL778) developed in this study contains the desired LAB strain L. plantarum CRL 778 along with low-cost ingredients that are compatible

All doughs were prepared with commercial yeast. b SLs, slurries (biopreservatives): SL772 (L. brevis CRL 772), SL796 (L. brevis CRL 796), SL778 (L. plantarum CRL 778), and SL1100 (L. reuteri CRL 1100) were incorporated by partially replacing (20, 30, and 40%) tap water in the dough preparation by equal amounts of the slurry fermented by each of the selected LAB strains. PCa, calcium propionate, chemical preservative added to the dough (YP-doughs) at 0.1 to 0.4% (wt/wt). c TTA, total titratable acidity, reported as milliliters of 0.1 N NaOH per g of fermented dough. d FQ, fermentation quotient, calculated as lactate/acetate molar ratio. e ND, not detected. f When no PCa was used (0% PCa), the resulting control dough was designated as Y-dough.

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TABLE 3. Combined effects of SL778 and calcium propionate on dough characteristics and bread shelf life
Dough Organic acidsb Preservativea pH Lactic Acetic Phenyllactic Propionic FQc Bread Shelf life (days)d

PCa YP0.1 YP0.2 YP0.3 YP0.4 SL778 z PCa SLP0.1 SLP0.2 SLP0.3 SLP0.4
a

5.5 5.5 5.5 5.5 5.0 5.0 5.0 5.0

2.3 2.4 2.3 2.3 31.4 31.2 31.1 31.2

(0.05) (0.05) (0.05) (0.05) (2.4) (2.3) (2.3) (2.3)

0.31 0.42 0.31 0.31 11.0 11.2 11.5 11.2

(0.04) (0.06) (0.04) (0.04) (4.1) (4.1) (4.2) (4.1) 0.7 0.7 0.7 0.7

ND ND ND ND (0.02) (0.02) (0.02) (0.02)

6.6 13.2 19.8 26.4 6.6 13.2 19.8 26.4

(1.5) (2.4) (3.6) (4.8) (2.7) (5.5) (8.2) (10.9)

7.4 5.7 7.4 7.4 2.8 2.8 2.7 2.8

4 5 8 9 10 12 18 24

PCa, calcium propionate; YP, bread with PCa at increasing amounts (subscripts) corresponding to 0.1 to 0.4% (wt/wt); SLP, dough prepared with the slurry fermented by L. plantarum CRL 778 (SL778) at 40% plus PCa at concentrations of 0.1 to 0.4% (wt/wt). b The total amount of the organic acid is expressed as millimoles per kilogram of dough fermented at 30uC for 2 h; figures in parentheses indicate the concentration of the undissociated fraction, calculated with the Henderson-Hasselbach equation and expressed as millimoles per kilogram of dough. The amounts of total and undissociated propionic acid were stoichiometrically estimated from the PCa added for bread making and by using the Henderson-Hasselbach equation. ND, not detected. c FQ, fermentation quotient of the dough, calculated as lactate/acetate molar ratio. Total species (undissociated and dissociated forms) of acids were used for the calculations. d Shelf life is the time during which no visible mold growth in the packaged loaves was observed.

with the food matrix (wheat dough). Since the slurry was developed to partially replace tap water in the dough, no major changes in the conventional process of bread making are introduced. All the lactobacilli strains tested (L. brevis CRL 772, L. brevis CRL 796, L. plantarum CRL 778, and L. reuteri CRL 1100) were active against Aspergillus, Fusarium, and Penicillium (10). In the present study, Penicillium sp. was used for dough contamination because of its high incidence (.90%) in wheat bread spoilage (16). The slurries (SL772, SL796, SL778, and SL1100) prepared from each lactobacilli strain also inhibited mold growth, thus allowing a 4- to 5day shelf life, which was twofold higher than Y-breads prepared without preservatives. Use of SL778 produced the best biopreservative effect (5-day shelf life), which is similar to that obtained using 0.2% PCa. The antifungal effect of SL778 was mainly related to acetic acid and PLA, while lactic acid (produced at a high concentration) was essential for lowering the dough pH, thus favoring the undissociated active fraction of the acids, mainly PCa (18). The undissociated acid molecules are lipophilic and diffuse through the plasma membrane into the cell. The higher-pH environment of the cytosol promotes the rapid dissociation of the acid molecules into charged protons and anions, which cannot subsequently diffuse back outside (12). Acidification of the cell cytoplasm resulting from the accumulation of protons inhibits key metabolic activities involved in glycolysis (13) and hence reduces the ATP yield. The above helps explain the lengthy shelf life of bread prepared with 40% (vol/vol) SL778; after fermentation, the dough attained a low pH (pH 5.0), which favored the

undissociated fraction of the organic acids and the antifungal effect of the slurry (Table 3). The antifungal activity of L. plantarum FST1.7, a strain that produces PLA and two antifungal cyclic dipeptides (leucine-proline and phenylalanine-proline) was also confirmed in wheat bread (7). In contrast, other authors were unable to reproduce the antifungal effect of certain LAB strains in wheat bread (19), probably due to a loss in activity of unidentified compounds during baking (17). Similarly, L. sanfranciscensis, which in vitro produces a broad range of organic acids effective against Aspergillus, Fusarium, and Penicillium (20), was not effective in bakery goods (5). The longest shelf life (24 days) was achieved for packaged bread using a combination of 0.4% PCa and 40% of the bio-antifungal culture SL778. A longer shelf life (10 days) for wheat bread inoculated with Penicillium expansum was also reported using a mixture of 0.3% PCa and sourdough fermented with L. plantarum FST1.7. The authors attributed these results to a potential synergistic effect between the LAB strain and PCa. In our study, supplementing traditional doughs (prepared with commercial yeast and PCa) with SL778 significantly (P , 0.05) improved the shelf life of packaged bread. According to our results, combining SL778 with 0.4% PCa yields bread with an extended shelf life of 24 days and using SL778 with 0.2% PCa yields bread with a 12-day shelf life. Both products appear promising for artisanal bakeries in Argentina. ACKNOWLEDGMENTS
The authors acknowledge the financial support of Consejo Nacional de Investigaciones Cientficas y Tecnicas (CONICET), Agencia Nacional de Promocion Cientfica y Tecnologica (ANPCyT), and Consejo de

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Investigacion de la Universidad Nacional de Tucuman (CIUNT) from Argentina.

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