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Romanian Biotechnological Letters Vol. 22, No. 5, 2017 Copyright © 2017 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 22, No. 5, 2017 12915

Synergistic effects of the biological and chemical preservatives

in dough fermentation

Received for publication, November 27, 2015 Accepted, March 24, 2016

VALERIA GAGIU1*, FLORENTINA ISRAEL-ROMING2, DIANA PELINESCU, ALINA ALEXANDRA DOBRE1, MIRELA ELENA CUCU1, IRINA SMEU1, ENUTA IORGA1, CALINA PETRUTA CORNEA2, ILEANA STOICA3, NASTASIA BELC1 1National Research&Development Institute for Food Bioresources–IBA Bucharest, 5th Baneasa Ancuta street, 2nd district, code 020323, Bucharest, Romania 2University of Agronomic Sciences and Veterinary Medicine Bucharest/ Centre for Applied Biochemistry and Biotechnology (CBAB) Biotehnol, 59 Marasti Blvd., postcode 011464 Bucharest, Romania 3University of Bucharest, Faculty of Biology, Department of Genetics, 1-3 Aleea Portocalelor, postcode 060101 Bucharest, Romania *Address for correspondence to: [email protected]

Abstract

Increasing the shelf life of bread and food security can be achieved through biological (sourdough fermented by microbial strains with inhibitory activity), chemical (calcium propionate, essential oils), and physical (packaging system) methods. There were evaluated the synergistic effects of rye sourdough fermented by Lactobacillus plantarum (strains C71, C78 and C81) and calcium propionate or essential oils (onion, garlic, oregano) on the fermentation of the wheat dough. In relation to the flour amount, preservatives were used in percentage of 0.2% (w/w) for the rye sourdough, 0.1% to 0.2% (w/w) for calcium propionate and 0.1% (v/w) for essential oils. Microbiological (lactic acid bacteria and yeasts), biochemical (pH, total titrable acidity, lactic and acetic acids) and molecular genetics of Lactobacillus plantarum strains testing were performed on bread dough. Microbiological analysis of wheat dough with adding of rye sourdough and calcium propionate or essential oils showed that they do not substantially alter the viability of lactic acid bacteria and yeast strains. Genetic and biochemical stability of C71, C78 and C81 strains of Lactobacillus plantarum, as well as their proportion were not affected by calcium propionate or essential oils. Calcium propionate had an antifungal effect stronger than onion, garlic and oregano essential oils.

Keywords: Lactobacillus plantarum, calcium propionate, essential oils, fermentation, rye sourdough, wheat dough, organic acids. 1. Introduction

In baking industry bread occupies a unique position and use compared with other bakery products (P. LIAQAT [1]). Using lactic acid bacteria (LAB) and their metabolites to control fungal growth and mycotoxin accumulation is a biocontrol strategy in food or feed contaminated with toxigenic fungi (I.C. GEREZ & al. [2]; D.K.D. DALIÉ & al. [3]), contributing to the safety, stability, flavor and structure of products (A. LAITILA & al. [4]). Antimicrobial activity of lactic acid bacteria (LAB) may be explained by organic acids (lactic, acetic, propionic etc.) and existence of other compounds (fatty acids, bacteriocins, cyclic dipeptides), especially when acting in synergy (A.C. OUWEHAND [5]; M.L. NIKU-PAAVOLA & al. [6]; A. CORSETTI & al. [7]; U. SCHILLINGER and J.V. VILLARREAL [8]; V. GAGIU & al. [9]). Sourdough (SD) is used due to the consumer demand for food obtained with limited chemical preservatives addition (R. Di CAGNO & al. [10]; V. GAGIU &

VALERIA GAGIU, FLORENTINA ISRAEL-ROMING, DIANA PELINESCU, ALINA ALEXANDRA DOBRE, MIRELA ELENA CUCU, IRINA SMEU, ENUTA IORGA, CALINA PETRUTA CORNEA, ILEANA STOICA, NASTASIA BELC


al. [9]; P. SKANDAMIS & al. [11]). Sourdough is a complex food ecosystem in which lactobacilli are highly adapted to the environmental conditions (temperature, pH, acidity, maltose as the most abundant fermentable carbohydrate and fructose as a potential electron acceptor), producing antimicrobials (A. VERA & al. [12]). Baking industry and sourdough preparation require LAB strains capable to produce rapid acid (Ö. SIMSEK & al. [13]) and reduce the pH to about 3.3 in order to create a stable environment (M. KOCKOVÁ & al. [14]), the optimal pH being 5.5...6.2 (A. CORSETTI & al. [15]). Acidification and antimicrobial compounds produced by sourdough microbiota contributes to the product stability and play an important role in regulating complex interactions between starter and contaminant microbiota (T. ZOTTA & al. [16]). Dough bread obtained with the addition of sourdoughs (with low pH and high proportions of acetic and lactic acid) have a high volume and a longer shelf life (Ö. SIMSEK & al. [13]). Lactobacillus plantarum (homofermentative, optional heterofermentative) can be used as a biocontrol agent of Fusarium sp. (R. CODA & al. [17]), being dominant in the ecosystem of spontaneous rye sourdoughs (S. WECKX & al. [18]). In mature sourdoughs, Lactobacillus plantarum represents approximately 90% of the bacterial population, the largest producer of lactic acid (P. DAMIANI & al. [19]) and lead to better growth of Saccharomyces cerevisiae strains (A. HANSEN & al. [20]; A. GOBBETTI & al. [21]).

Calcium propionate (CaP, E282) is an antimicrobial agent without activity against yeasts and limited activity against bacteria, but effective against fungi. Preservative efficacy is dependent on the product’s pH as the antimicrobial effect of undissociated acid is much stronger than the dissociated acid (E. LÜCK and M. JAGER [22]). At pH 4.9, calcium propionate breaks down into equal amounts of propionic and calcium propionate ions, the optimum pH is below 5.0, the efficiency decreasing at pH above 6.0. Like other salts of propionic acid, calcium propionate work by lowering water activity, with pKa 4.5 to 5.0 (M.B. LIEWEN and E.H. MARTH [23]; E. LÜCK and M. JAGER [22]). European Community Directive 95/2/EC allows a maximum amount of 3 mg/ml calcium propionate (for rye bread and packaged sliced bread) and recommends a concentration of 2 mg/ml potassium sorbate, which is rarely used, because of negative side effects on loaf volume (P. LAVERMICOCCA & al. [24]). Reducing the preservatives amount at subinhibitory levels led in some cases to stimulation of spoilage fungi growth (S. MARIN & al. [25]; K.I. SUHR and P.V NIELSEN [26]) and stimulating the production of mycotoxins (L.B. BULLERMAN [27]). The inhibitory agent is propionic acid, while ionized forms or propionate are not inhibitory. A change in pH of 0.3 units results in a doubling of the concentration of inhibitor propionic acid (M. TZATZAKIS & al. [28]). To ensure shelf life and for safety reasons in the bakery, calcium propionate must be used simultaneously with sourdough, imparting a broader antifungal spectrum due to a synergistic effect (L.A.M. RYAN & al. [29]; P. LAVERMICOCCA & al. [24]; V. GAGIU & al. [9]). There has been no correlation between shelf-life and pH of bread, but the type and amount of acid present may have effects on other microstate agents (A. SADEGHI [30]).

Essential oils (EOs) are complex volatile compounds with antimicrobial activity on bacteria and fungi (L.L. BARRERA-NECHA & al. [31]). The compounds efficacy in a food system is influenced by pH, lipids (lowering activity of hydrophobic compounds), proteins (can bind and reduce the activity of certain components) (R.S. FARAG & al. [32]; M. OMYDBEYGI & al. [33]). The antimicrobial activity of essential oils on yeast may be the result of disruption of several enzyme systems involved in energy production and synthesis of structural components (D.E. CONNER and L.R. BEAUCHAT [34]; P.M. DAVIDSON [35]). Some essential oils or compounds proved a good efficacy, tolerance to low pH (2.0) and selectivity against bacterial pathogens, in swine intestinal tract (W. SI & al. [36]).

The aims of this study were (i) to analyze characteristics of rye sourdoughs fermented by Lactobacillus plantarum, (ii) to analyze synergistic effect of rye sourdough and calcium

Synergistic effects of the biological and chemical preservatives in dough fermentation


propionate or essential oils on microorganism’s viability in wheat doughs, (iii) to analyze the influence of calcium propionate and essential oils on the microorganisms metabolism in wheat doughs, (iv) to quantify the cells number for each LAB strains from bread doughs based on biochemical and genetic profile. 2. Reagents and methods

2.1. Microorganisms and culture conditions Lactobacillus plantarum (C71, C78, and C81) strains were provided from the culture

collection of University of Bucharest, Faculty of Biology (Center for Research in Microbiology, Genetics and Biotechnology). Lactobacillus plantarum (C71, C78, and C81) strains were isolated from rye sourdough without sugars or salt addition, fermented at 320C and daily refreshment for 7 days, having a strong ability to increase total titrable acidity (TTA) and to decrease pH of sourdough. Based on previous results for SD inoculums, the three Lactobacillus plantarum strains C71, C78 and C81 were selected due to their inhibitory activity against the fungi Fusarium graminearum 96, Fusarium culmorum 46 and Fusarium verticillioides (NIRD Fundulea) and Aspergillus flavus 1038L (CBAB Biotehnol). The highest antifungal activity in MRS broth was recorded for Lactobacillus plantarum C71 strain, while in SDB broth, Lactobacillus plantarum C78 strain inhibited Fusarium graminearum 96 growth more than Lactobacillus plantarum C71 and C81 strains (V. GAGIU & al. [9]).

Pure Lactobacillus plantarum cultures were maintained at -700C in De Man Rogosa and Sharpe (MRS) broth (Biokar Diagnostics, France) with 20% glycerol. Before each experiment strains were reactivated in MRS broth (1% inoculum) by incubation at 320C for 24 hours. Lactobacillus plantarum (C71, C78, and C81) pure strains were combined to obtain the mixed inocula (C71:C78:C81) in a proportion of (3:1:2) for P9.A inoculum and (2:3:1) for P18.A inoculum.

2.2. Rye sourdoughs preparation P9.A and P18.A rye sourdoughs (A - without yeasts) are obtained by fermentation of

rye flour with P9 or P18 mixed inoculum of Lactobacillus plantarum C71, C78 and C81 strains. Rye sourdough (DY 160) was obtained using 250 g rye flour (1% ash), 3.7 g sodium chloride, 150 ml water, 5 ml P9 or P18 mixed inoculum of Lactobacillus plantarum, followed by mixing for 5 min and fermentation at 320C for 24 h. Fermented rye SD was used as 2% to wheat flour in the direct technology of wheat bread dough. Rye sourdough samples were taken after the mixing and 24 h of fermentation, and stored in sterile Falcon tubes at -180C until the analytical testing.

2.3. Chemical preservatives with antifungal activity Calcium propionate (CaP, E282) is an antibacterial and antifungal preservative in food

(Sigma - Aldrich, Germany). Calcium propionate was used in a proportion of 0.1% and 0.2% to flour (w/w) in the direct technology of wheat bread dough. Calcium propionate (0.1%, 0.2% and 0.3%) did not show ,,in vitro’’ inhibitory activity on malt extract agar (MEA) against Fusarium graminearum 96, Fusarium culmorum 46, Fusarium verticillioides and Aspergillus flavus 1038L even when it was used at legal limit (0.3 %). Essential oils were garlic oil (Allium sativum, GAR), onion oil (Allium cepa, ONI), and origanum oil (Thymus capitatus, ORI), obtained by steam distillation and provided by Sigma - Aldrich, Germany. EOs were dissolved in DMSO (Dimethyl sulfoxide, Sigma Aldrich, Germany) 1:2 (v/v) to give stock solutions which were mixed at 180 rpm for 10 min and sterilized by filtration using sterile membrane filters (Millex–GP, pore size 0.22 μm). Working solution (400 ppm) was prepared in sterile distilled water and stored at 40C. Stock solution and working solution of essential oils were stored in a refrigerator at +40C. In experiments, EOs were used at a concentration of 400 ppm, in a proportion of 0.1% to flour (v/w).

2.4. Bread doughs preparation Direct method to obtain bread doughs with added rye SD (P9 or P18), essential oils (ORI,

GAR or ONI) or calcium propionate (CaP) was: 500 g wheat flour - type 650, 15 g Pakmaya yeast, 7.5 g NaCl, 306 ml tape water, the rye sourdough (2%, w/w), essential oil, ml (0.1%, v/w)



or calcium propionate (0.1% or 0.2% w/w), kneading for 5 min, fermentation at 290C for 90 min. After fermentation, bread doughs with rye sourdough and preservatives (CaP or EOs) were compared to control doughs (CD), without bacterial inoculum, obtained with the same amount of flours (DY 160) and incubated in the same conditions. Dough samples were taken after the kneading and after 90 min of fermentation. Wheat dough samples and CD were stored at -180C in sterile Falcon tubes until analytical testing (LAB and yeasts populations; total titrable acidity, lactic and acetic acids, pH).

2.5. Enumeration and isolation of lactic acid bacteria and yeasts in rye sourdough and wheat doughs

Lactic acid bacteria cell number was determined after homogenization (260 rpm, Stomacher 400 Circulator) of 10 g rye sourdough or bread dough with 90 ml of sterile peptone water (1%, w/v), followed by serial dilutions, inoculation in Man-Rogosa-Sharpe agar (MRS agar) (Biokar Diagnostics, France) and incubation at 370C for 72 h. Yeast cell number from the bread dough was quantified after homogenization (at 260 rpm, Stomacher 400 Circulator) of 10 g rye sourdough or bread dough with 90 ml of sterile peptone water (1%, w/v), followed by serial dilutions, inoculation on glucose chloramphenicol agar (AGC) broth (Biokar Diagnostics, France), followed by incubation at 250C for five days.

2.6. Total titrable acidity, pH and organic acids produced in rye sourdough and wheat doughs Total titrable acidity was determined after homogenization of 10 g of rye sourdough or

wheat doughs with 90 ml of distilled water, and expressed as the amount (ml) of 0.1 M NaOH needed to reach the value of pH of 8.5.

pH was determined by a pH meter (INOLab PH730 WTW, Germany). Detection and quantification of carboxylic acids were performed by high-performance

liquid chromatography in reverse phase (HPLC-RP) with UV detection. Samples (5 g) were homogenized (1:1) with ultrapure water (Millipore, Simplicity 185) for 3 min, and centrifuged at 4,000 rpm for 10 min. Before injection, 1 ml supernatant was filtered using 0.22 μm PTFE syringe filter. Processed samples were placed in the autosampler of Waters chromatographic system (model Alliance 2695-2487); data acquisition and processing were realized with Empower software. Supelcogel H column 4.6 mm x 25 cm and Supelcogel H guard column 4.6 mm x 5 cm were used for separation. The mobile phase was constituted by 0.1% phosphoric acid solution, eluting in isocratic mode. Separation was done at 30°C, injection duration was 30 min, with another 5 min equilibration time between samples. Detection of the two carboxylic acids was carried out at 210 nm, with 15.5 min retention time for the lactic acid and 18.2 min retention time for acetic acid. Calibration was done using a standard solution of lactic acid and acetic acid with 2.5 mg/ml concentration for each. From this solution five standards were made in triplicate by injection of appropriate amounts of the analyte. The calibration curve had a linearity coefficient of 0.9985 and correlation coefficient of 0.9993.

2.7. Biochemical and molecular genetic characterization of LAB Isolation and purification of genomic DNA were performed by a derived technical

protocol of JOHNSON [37]. Electrophoretic analysis of DNA was performed by gel agarose in the horizontal plate and

submerged system: 1X TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 8.0-8.5), agarose 0.8%. Migration was carried out for 3-4 hours at an intensity of electric field of 2.5 V/cm.

Genotypic characterization by Repetitive Element Palindromic-Polymerase Chain Reaction (rep-PCR) analysis. Repetitive sequences of prokaryotes genome may be used as sites for oligonucleotide primers. This method allows amplification of DNA fragments of different size, represented by sequences located between these repetitive elements. Amplicons with different size, may show electrophoretic migration, resulting in a specific pattern of each bacterial species (A.M. RIDLEY [38]). For reaction were used: 10 μl 2x Promega Master Mix,



1 μl DNA (50 ng/ μl), 0.1 μM rep-PCR-primer and MQW, up to a final volume of 20 μl (A.D. MAY and L.H. SHARON [39]). It was used a single primer (5'-GTGGTGGTGGTGGTG-3') and the PCR program of thermocycler M.J. Research includes: initial denaturation (950C, 7 min); amplification (40 cycles of 940C/1 min at 530C/1 min at 650C/8 min); a final elongation (at 650C/16 min). To highlight amplicons, it was performed agarose gel electrophoresis in 1x TBE using 1.5% agarose gel. Migration was performed for 4 h at 5 V/cm.

2.8. Statistical analysis Statistical analysis was performed using the statistics package of Microsoft Office 10.

3. Results and discussions

3.1. Characteristics of rye sourdoughs fermented by Lactobacillus plantarum After 24 h fermentation and reported to the control sourdough, P9.A rye SD (DY 160)

registered 16.5 times increased LAB cell number and 4 times for TTA, and a decreasing of pH with 1.7 units. Fermentation characteristics of P9.A rye SD were higher than for P18.A rye SD. Lactic and acetic acids analysis during fermentation (at 0, 45 or 90 minutes) of the wheat dough obtained by adding 2% rye SD showed that the P9.A caused a 17.5% increase of lactic acid, and 16% of acetic acid, reported to control wheat dough and 11.7% of lactic acid related to P18.A wheat dough. 3.2. Synergistic effect of rye sourdough and calcium propionate or essential oils on the viability of the microorganisms in wheat doughs

In the first stage 372 colonies from DY 160 bread dough were tested to determine their ability to produce catalase. This first test allowed quantification of LAB catalase-negative and yeasts catalase-positive, at 0 and 90 min fermentation of bread dough (figure 1).

Control bread doughs (CD, without rye SD or preservatives) were dominated by yeasts (7.90 log10 CFU/g) at 90 min of fermentation.

In bread doughs fermented by P9.A or P18.A rye SD, addition of calcium propionate or essential oils caused an inhibition of LAB and yeast populations (figure 1). However, the addition of 0.1% CaP resulted in an increased number of LAB and yeasts viable cells. Subinhibitory levels of preservatives (0.1% CaP) can lead to stimulation of microorganism growth (S. MARIN & al. [25]; K.I. SUHR and P.V. NIELSEN [26]).

Comparing the bread doughs fermented by the addition of P9.A or P18.A rye SD, the number of LAB cells didn’t show significant variations (p>0.05), all samples having about 8 log10 CFU/g. However, it was observed that LAB in P9.A bread dough were slightly inhibited compared with P18.A bread dough after kneading, because the fermentation parameters of P9.A rye SD may have a stress effect. In doughs with DY 160, the number of LAB and yeasts are moderately fluctuated, increasing water content (DY) will increase growth of LAB and yeasts cells number, which are in accordance with other data reported in scientific literature (A. GIANOTTI & al. [40]; R. Di CAGNO & al. [10]). Since rye sourdough and wheat doughs had the same DY 160, increasing of LAB number in rye sourdough and moderate fluctuation in wheat dough proved the importance of ash content of flour (1% for rye flour and 0.65% for wheat flour).

The P9.A bread dough showed a slight increase in the number of the LAB after 90 min of fermentation for samples with CaP (0.1% or 0.2%, w/w) or ORE. CaP has no activity against yeasts, with limited activity against bacteria, but it is known as a potent inhibitor of fungi (E. LÜCK and M. JAGER [22]). Adding GAR and ONI essential oils resulted in a decrease in the number of LAB cells. Bread doughs with added P18.A rye SD were less sensitive to the addition of essential oils (almost constant LAB number) comparative with P9.A sample. The addition of ONI resulted in inhibition of LAB and yeast in both cases (figure 1).

Quantification of the number of yeast colonies allowed the determination of CaP and EOs influence on yeast cells used to obtain bread. The use of fermented SD does not affect the viability of yeast and hence no fermentation process of bread. CaP and ORI slightly decrease the viability of yeast cells after 90 min fermentation, while the ONI and GAR



stimulate the growth of yeast cell. For bread doughs fermented with P18.A rye SD, quantification of the number of yeasts did not reveal variations. 3.3. Influence of calcium propionate and essential oils on the microorganism’s metabolism (TTA, pH, organic acids) in wheat doughs

After kneading, lactic and acetic acids were not detected either in the classic control dough, or in the dough with P9.A or P18.A rye SD and CaP. This situation may be because CaP in the presence of lactic and acetic acids brought by P9.A or P18.A rye SD, decompose and form calcium lactate (E 327) and calcium acetate (E 263) which are food additives (preservatives) acting mainly against yeasts and fungi (drugs.com, efsa.europa.eu). CaP stimulates LAB and yeasts viability but it decreases TTA, increases pH, and stimulate lactic acid for P9.A bread dough (figure 2). The preservatives had a negative influence on the fermentative capacity of yeasts and quality indicators of bread (volume, height, porosity and moisture), but prolonged its shelf life.

After 90 min of fermentation, classic dough (CD) presented 0.749 mg/g lactic acid and 0.177 mg/g acetic acid in a proportion of 4:1. The addition of preservatives (CaP, respectively EOs) had a slightly negative effect on LAB metabolism, lowering the total amount of organic acids by 11.4% for bread dough with P9.A rye SD and CaP, and 7.1% for bread dough with P9.A rye SD and EOs. CaP inhibited acetic acid production, but has stimulated increasing the lactic acid amount (figure 3). Bread dough with P9.A rye SD had more lactic acid in the presence of CaP (41.3%) than for EOs (25.1%), with values significantly higher (p < 0.05). CaP showed an inhibitory effect against LAB metabolism, leading to a lower value of TTA and increasing pH (figure 2), even if it increased LAB and yeast viability (figure 1).

EOs led to an obvious decrease in pH value of bread dough with the addition of P9.A rye SD is comparative to P18.A rye SD (figure 2). Analyzing the organic acids produced in bread dough with rye SD and EOs it was observed that EOs inhibited the production of acetic acid, especially in P9.A bread dough (figure 3). The decrease in LAB number for GAR and ONI essential oils is correlated with the lower amount of acetic acid in the case of P9.A bread dough after 90 min fermentation (average 7.900 µg/g for P9.A and 9.207 µg/g for P18.A) (figure 2). Compared to the total amount of organic acids it was observed that the level of the acetic acid is lower for P9.A bread dough (figure 3). Even if acetic acid has an antifungal activity stronger than lactic acid, it has a negative effect on viability and fermentation capacity of yeasts, producing undesirable effects on quality parameters of bread. Antifungal activity of LAB may be explained by the capacity of producing organic acids (lactic, acetic, tartaric, oxalic etc.) but also compounds like fatty acids, bacteriocins, cyclic dipeptides and other (V. GAGIU & al. [9]; U. SCHILLINGER and J.V. VILLARREAL [8]).

3.4. Quantification of the cells number for each LAB strains from bread doughs based on biochemical and genetic profile

The distinction between Lactobacillus plantarum C71, C78 and C81 strains was made based on biochemical characteristics determined by API 50 CHL test. Based on these tests, it was found that rhamnose is sugar that can be metabolized by strains C71 and C78 and cannot be metabolized by C81 strain. To make the distinction between Lactobacillus plantarum C71 and C78 strains, 41 strains of bacteria grown on medium with rhamnose were subjected to molecular analysis rep-PCR. In the first stage chromosomal DNA isolation was performed. DNA extracted samples analyzed by electrophoretic and spectrophotometric methods showed optimal purity and concentration (data not showed) for PCR analysis. Following amplification, amplicons were checked by electrophoresis, yielding electrophoretic profiles (figures 4 - 6). Rep-PCR profiles (figures 4 - 6) revealed that 24 LAB strains presented similar profile to with Lactobacillus plantarum C78 strain, and 17 strains presented similar profile with Lactobacillus plantarum C71 strain.





Correlating the results of biochemical and molecular analyses, it was found that C71 and C81 strains of Lactobacillus plantarum predominate in P9.A bread dough (C71:C78:C81, 3:1:2), while Lactobacillus plantarum C78 and C71 strains predominate in P18.A bread dough (C71:C78:C81, 2:3:1). This demonstrates that in the bread dough it is kept the same proportion of C78 and C81 strains of Lactobacillus plantarum with inhibitory activity against toxigenic fungi.

Molecular analysis showed that Lactobacillus plantarum C71 and C81 strains might be stimulated by CaP (0.1%, 0.2%) and ORI, while Lactobacillus plantarum C78 and C71 strains may be less sensitive to EOs. In the ripe doughs, Lactobacillus plantarum is more tolerant and represent approx. 90% of bacterial microbiota (due to transport and metabolism abilities), being the most important producer of lactic acid (P. DAMIANI & al. [19]) and favoring Saccharomyces cerevisiae growth (A. HANSEN & al. [20]; A. GOBBETTI & al. [21]). Also, Lactobacillus plantarum is dominant in sourdoughs with DY 160 (R. Di CAGNO & al. [10]).

Following the case studies conducted on microbiological analysis of bread with 2% (w/w) rye sourdough and CaP (0.1%, 0.2%, w/w), essential oils (ONI, GAR, ORE 0.1%, v/w) at 400 ppm concentration, it was found they do not substantially alter the LAB and yeasts viability. Biochemical and molecular analyses of LAB strains isolated from bread dough samples revealed a genetic and biochemical stability of strains, and keeping the proportions they were added in the rye dough.

Figure 4. Rep-PCR profile of LAB strains isolated from rhamnose environment

1–4. Samples, 5. Control strain C71; 6. Control strain C78; 7-14. Samples, 15. Control strain C81



16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

31 32 33 34 35 36 37 38 39 40 41 42 43 44



4. Conclusions Wheat dough with the best fermentation parameters (LAB and yeast populations,

amount of lactic acid) had the addition of the rye sourdough fermented by P9.A mixed inoculum of Lactobacillus plantarum. Rye sourdoughs fermented with Lactobacillus plantarum allowed accumulation of compounds with antifungal activity (lactic and acetic acids) that may increase the shelf life of bread. The intensity of the antifungal effect depends on the proportion of individual organic acids in total organic acids; a higher percentage of acetic acid leads to obtaining a prolonged shelf life of bread. Also, calcium lactate and acetate formed in case of rye sourdough and CaP potentiate the delay of bread molding, since they are natural food preservatives formed during fermentation. ONI and ORI essential oils had less effect on the LAB and yeast populations compared to CaP (0.2%), but had a similar effect of increasing lactic acid and lowering acetic acid. Molecular analysis of LAB strains isolated from dough samples reveal the genetic and biochemical stability of the Lactobacillus plantarum strains and keeping the proportions they were added in the rye dough. Biochemical results correlated with a longer delay of molding for bread with CaP demonstrates that CaP has an antifungal effect stronger than ORI, ONI and GAR essential oils. 5. Acknowledgments

This work had benefited from financial support through the contract 51-005/2007 ’’Integrated system to decrease fungal and mycotoxins contamination in baking industry, to increase food safety (MYCREDPAN)’’, by the Romanian R&D Programme - Partnerships in Priority S&T Domains.

References

1. P. LIAQAT. Food Microbiology. National Book Foundation. Islamabad (1988). 2. I. C. GEREZ, I.M. TORINO, G. ROLLAN, F.G. de VALDEZ. Prevention of bread mould spoilage by

using lactic acid bacteria with antifungal properties. Food Control 20, 144–148 (2009). 3. D.K.D. DALIÉ, A.M. DESCHAMPS, F. RICHARD-FORGET. Lactic acid bacteria – Potential for

control of mould growth and mycotoxins: A review. Food Control 21(4), 370–380 (2010). 4. A. LAITILA, H-L. ALAKOMI, L. RAASKA, T. MATTILA-SANDHOLM, A. HAIKARA. Antifungal

activities of two Lactobacillus plantarum strains against Fusarium moulds in vitro and in malting of barley. Journal of Applied Microbiology 93(4), 566–576 (2002).

5. A.C. OUWEHAND. Antimicrobial components from lactic acid bacteria. In Lactic Acid Bacteria. Microbiology and Functional Aspects ed. Salminen, S. and von Weight, A., 139 – 159. New York: Marcel Dekker (1998).

6. M.L. NIKU-PAAVOLA, A. LAITILA MATTILA, T. SANDHOLM, A. HAIKARA. New types of antimicrobial compound produced by Lactobacillus plantarum. Journal of Appl. Microbiology 86, 29–35 (1999).

7. A. CORSETTI, M. GOBETTI, J. ROSSI, P. DAMIANI. Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Applied Microbiology and Biotechnology 50, 253–256 (1998).

8. U. SCHILLINGER, J.V. VILLARREAL. Inhibition of Penicillium nordicum in MRS medium by lactic acid bacteria isolated from foods, Food Control 21(2), 107-111 (2010).

9. V. GAGIU, F. ISRAEL-ROMING, N. BELC, R. DIMA. Inhibitory activity of Lactobacillus plantarum strains and calcium propionate on spoilage fungi. Romanian Biotechnological Letters 18(2), 8214-8220 (2013).

10. R. Di CAGNO, E. PONTONIO, S. BUCHIN, M. DE ANGELIS, A. LATTANZI, F. VALERIO, M. GOBBETTI, M. CALASSO. Diversity of the lactic acid bacteria and yeast microbiota switching from firm to liquid sourdough fermentation. Applied and Environmental Microbiology 80(10), 3161–3172 (2014).

11. P. SKANDAMIS, K. KOUTSOUMANIS, K. FASSEAAS, G.J.E. NYCHAS. Inhibition of oregano essential oil and EDTA on E. coli O157:H7. Ital. J. Food Sci. 13, 55–65 (2001).



12. A. VERA, V. RIGOBELLO, Y. DEMARIGNY. Comparative study of culture media used for sourdough lactobacilli. Food Microbiology 26, 728–733 (2009).

13. Ö. SIMSEK, A.H. ÇON, S. TULUMOĞLU. Isolating lactic starter cultures with antimicrobial activity for sourdough processes. Food Control 7(4), 263–270 (2006).

14. M. KOCKOVÁ, P. GEREKOVÁ, Z. PETRULÁKOVÁ, E. HYBENOVÁ, E. ŠTURDÍK, V. ĽUBOMÍR. Evaluation of fermentation properties of lactic acid bacteria isolated from sourdough. Acta Chimica Slovaca 4(2), 78–87 (2011).

15. A. CORSETTI, L. SETTANI, S. VALMORRI, M. MASTRANGELO, S. GIOVANNA. Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentation. Food Microbiology 24, 592–600 (2007).

16. T. ZOTTA, P. PIRAINO, A. RICCIARDI, P.L.H. MCSWEENEY, E. PARENTE. Proteolysis in Model Sourdough Fermentations. J. Agric. Food Chem. 54, 2567-2574 (2006).

17. R. CODA, A. CASSONE, C.G. RIZZELLO, L. NIONELLI, G. CARDINALI, M. GOBBETTI. Antifungal Activity of Wickerhamomyces anomalus and Lactobacillus plantarum during Sourdough Fermentation: Identification of Novel Compounds and Long-Term Effect during Storage of Wheat Bread. Appl. Environ. Microbiol. 77(10), 3484–3492 (2011).

18. S. WECKX, ROEL VAN DER MEULEN, D. MAES, I. SCHEIRLINCK, G. HUYS, P. VANDAMME, L. DE VUYST. Lactic acid bacteria community dynamics and metabolite production of rye sourdough fermentations share characteristics of wheat and spelt sourdough fermentations. Food Microbiology 27, 1000-1008 (2010).

19. P. DAMIANI, M. GOBBETTI, L. COSSIGNANI, A. CORSETTI, M.S. SIMONETTI, D J. ROSSI. The sourdough microflora. Characterization of hetero- and homofermentative lactic acid bacteria, yeast and their interactions on the basis of the volatile compounds produced. Lebensm. Wiss. Technol. 29, 63-70 (1996).

20. A. HANSEN, B. LUND, J.M. LEWIS. Flavour production and acidification of sourdough relation to starter culture and fermentation temperature. Lebensm. Wiss. Technol. 22, 145–149 (1989).

21. M. GOBBETTI, A. CORSETTI, J. ROSSI. Maltose-fructose co-fermentation by Lactobacillus brevis subsp. Lindneri CB1 fructose-negative strain. Appl. Microbiol. Biotechnol. 42, 939-944 (1995).

22. E. LÜCK, M. JAGER. Antimicrobial Food Additives: Characteristics, Uses, Effects 2nd (second) rev. and enlarge edition by Lück, Erich; Jager, Martin published by Springer (1997).

23. M.B. LIEWEN, E.H. MARTH. Growth of sorbate - resistant and – sensitive strains of Penicillium roqueforti in the presence of sorbate. Journal of Food Protection 48(6), 525–529 (1985).

24. P. LAVERMICOCCA, F. VALERIO, A. VISCONTI. Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products. Appl. Environ. Microbiol. 69(1), 634 – 640 (2003).

25. S. MARIN, V. SANCHIS, D. SANZ, A.J. RAMOS, R. CANEL, N. MAGAN. Control of growth and fumonisin B1 production by Fusarium proliferatum isolates in moist maize with propionate preservatives. Food Addit. Contam. 16(12), 555–563 (1999).

26. K.I. SUHR, P.V. NIELSEN. Effect of weak acid preservatives on growth of bakery product spoilage fungi at different water activities and pH values. International Journal of Food Microbiology 95(1), 67–78 (2004).

27. L.B. BULLERMAN. Effects of potassium sorbate on growth and ochratoxin production by Aspergillus ochraceus and Penicillium species. Journal of Food Protection 48, 162–165 (1985).

28. M. TZATZAKIS, A.M. TSATSAKIS, A. LIAKOU, D.J. VAKALOUNAKIS. Effect of common food preservatives on mycelium growth and spore germination on Fusarium oxysporum. J. Environm. Sci. Heath B 35(4), 527–537 (2000).

29. L.A.M. RYAN, F. DAL BELLO, E.K. ARENDT. The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread. International Journal of Food Microbiology 125(3), 274-278 (2008).

30. A. SADEGHI. The secret of sourdough; a review of miraculous potentials of sourdough in bread shelf life. Biotechnology 7(3), 413–417 (2008).

31. L.L. BARRER-NECHA, C. GARDUÑO-PIZAÑA, L.J. GARCIA-BARRERA. In vitro antifungal activity of essential oils and their compounds on mycelial growth of Fusarium oxysporum f. sp. gladioli (Massey) Snyder and Hansen. Plant Pathology Journal 8(1): 17-21 (2009).

32. R.S. FARAG, Z.Y. DAW, F.M. HEWEDY, G.S.A. EL-BAROTY. Antimicrobial activity of some Egiptian spice essential oils. Journal of Food Protection 52, 665–667 (1989).



33. M. OMYDBEYGI, M. BARZEGAR, Z. HAMIDI, H. NAGHDIBADI. Antifungal activity of thyme, summer savory and clove essential oils against Aspergillus flavus in liquid medium and tomato paste. Food Control 18, 1518–1523 (2007).

34. D.E. CONNER, L.R. BEAUCHAT. Effect of essential oils from plants on growth of food spoilage yeasts. Journal of Food Science 49, 429–434 (1984).

35. P.M. DAVIDSON. Chemical preservatives and naturally antimicrobial compounds. In MP Doyle, LR Beauchat, TJ Montville (Eds.). Food microbiology fundamentals and frontiers (2nd ed., 593–628) Washington C: ASM Press (2001).

36. W. SI, J. GONG, R. TSAO, T. ZHOU, H. YU, C. POPPE, R. JOHNSON, Z. DU. Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology 100(2), 296-305 (2005).

37. K.R. JOHNSON. A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription. Analytical Biochemistry 190(2), 170-174 (1990).

38. A.M. RIDLEY. Genomic Fingerprinting by Application of rep-PCR, Molecular Bacteriology 7, 103-115 (1998).

39. A.D. MAY, L. H. SHARON. DNA fingerprinting of Lactobacillus crispatus strain CTV-05 by repetitive elements sequence-based PCR analysis in a pilot study of vaginal colonization. Journal of Clinical Microbiology 41(5), 1881-1887 (2003).

40. A. GIANOTTI, L. VANNINI, M. GOBBETTI, A. CORSETTI, F. GARDINI, M.E. GUERZONI. Modelling of the activity of selected starters during sourdough fermentation. Food Microbiology 14: 327–337 (1997).

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Synergistic effects of the biological and chemical ...rombio.eu/vol22nr5/8_V_Gagiu_2_(03.10.pdf · Synergistic effects of the biological and chemical preservatives in dough fermentation