Food Microbiology 28 (2011) 526e536

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Manufacture and characterization of functional emmer beverages fermented

by selected lactic acid bacteria
Rossana Coda, Carlo Giuseppe Rizzello*, Antonio Trani, Marco Gobbetti
Dipartimento di Biologia e Chimica Agro-Forestale ed Ambientale, University of Bari, 70126 Bari, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Autochthonous lactic acid bacteria from emmer flour were screened based on the kinetic of acidification
Received 9 July 2010 and used to ferment beverages containing emmer flour, emmer gelatinized flour, and emmer malt at
Received in revised form percentages ranging 5e30% (wt/wt). Preliminarily, the concentration of raw flour and malt was selected
28 October 2010
based on sensory analysis. Different protocols were set up for the manufacture of four different beverages
Accepted 2 November 2010
which used Lactobacillus plantarum 6E as the starter. Emmer beverages were mainly differentiated based
Available online 12 November 2010
on the concentration of organic acids, carbohydrates, amino acids, dietary fibers, vitamins, antioxidant
and phytase activities, and volatiles and sensory profiles. Wheat flour bread was used as the control to
Keywords:
Emmer
determine the hydrolysis index (HI ¼ 100), as an indirect estimation of the glycemic index. The beverage
Cereal beverages made with 30% (wt/wt) of gelatinized flour showed an HI of 56%, its viscosity was improved by using an
Lactic acid bacteria EPS-producing strain and it allowed the survival of the potential probiotic Lactobacillus rhamnosus SP1 at
Probiotics cell density of ca. 5  108 cfu/ml throughout storage at 4  C. Among the exploited biotechnological
options, this latter beverage could be considered as a promising novel functional food.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction (e.g. tchoukoutou, jnard), non-alcoholic beverages (e.g. uji, ben-

saalga), porridges (e.g. mawè), and cooked gels (e.g. kenkey, idli,
Cereals are considered one of the most important sources of and mifen) (Nout, 2009). During the last decade, consumer vege-
dietary carbohydrates, proteins, vitamins, minerals and fibers for tarianism and nutritional problems (e.g., cholesterol content and
people all over the world. Nevertheless, the nutritional quality of lactose intolerance) led to an increasing demand for non-dairy milk
some cereals and the sensory properties of their products are substitutes with high acceptance and functionality (Prado et al.,
sometimes inferior or poor compared to other staple-foods 2008). Cereal-based beverages could have a huge potential either
(Blandino et al., 2003). to fulfill the consumer demand for non-dairy beverages or as
A variety of technologies (e.g., cooking, sprouting and milling) potential vehicles for functional compounds such as antioxidants,
are used for cereal processing but fermentation still remains the dietary fiber, minerals, probiotics and vitamins (Kreisz et al., 2008).
best choice for improving the nutritional, sensory and shelf-life European ancient cereals such as einkorn, emmer and spelt
properties (Mattila-Sandholm, 1998). This is the main reason why were staple-foods for the human population since more than
a large proportion of cereals is processed into foods and beverages 10,000 years (Kòvacs, 2008). Currently, the increasing interest
by fermentation prior to consumption (Nout, 2009). Although towards ancient grains stimulated investigations about their use in
several preparations remain a house art, especially in African industrial productions of bread, pasta and biscuits as an alternative
countries, raw grain materials and/or type of fermentation are the to the common wheat products. Because of the valuable nutritional
main criteria to classify cereal-based fermented drinks. Indeed, and/or physiological properties, and the low-input techniques used
lactic acid bacteria (Lactobacillus, Pediococcus spp.), Enterobacter for its management, a large variety of emmer-based products are
spp., yeasts (Candida, Debaryomyces, Endomycopsis, Hansenula, now available in the market including flour, bread, breakfast
Pichia, Saccharomyces, and Trichosporon spp.), and filamentous cereals, pasta and crackers (Buerli, 2006; Marques et al., 2007). The
fungi (Amylomyces, Aspergillus, Mucor and Rhizopus spp.) are mainly nutritional potential of ancient grains was characterized and they
involved in the manufacture of cereal-based alcoholic beverages were also shown to be suitable for sourdough biotechnology
(Ruibal-Mendieta et al., 2005; Coda et al., 2010). Based on these
findings, rediscovered ancient grains (e.g., emmer) may be
* Corresponding author. considered as suitable substrates for the manufacture of novel and
E-mail address: rizzello@agr.uniba.it (C.G. Rizzello). functional beverages.

0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2010.11.001
 R. Coda et al. / Food Microbiology 28 (2011) 526e536 527

This study aimed at manufacturing and characterizing the washed twice in 50 mM phosphate buffer (4  C, pH 7.0), and re-
physical, chemical, functional, and sensory properties of non- suspended in the tap water used for making beverages.
alcoholic emmer beverages fermented with selected autochtho- After beverage pasteurization, total bacteria were determined
nous lactic acid bacteria. on Plate Count Agar (PCA, Oxoid) at 30  C for 48 h; yeasts were
determined on Yeast extract-Peptone-Dextrose agar (YPD, Oxoid),
2. Materials and methods added of 150 ppm chloramphenicol, at 30  C for 72 h; total enter-
obacteria were determined on Violet Red Bile Glucose Agar
2.1. Bacterial strains and culture media (VRBGA, Oxoid) at 37  C for 24 h.

Lactobacillus plantarum 6E, Lb. plantarum 10E, Weissella confusa 2.2. Manufacture of cereal beverages
12E and Lactobacillus brevis 20S, previously isolated from emmer or
spelt flours and selected based on the kinetics of acidification (Coda Emmer grains and flour were purchased from a local supplier
et al., 2010); Weissella cibaria WC4, W. cibaria WC3, W. cibaria WC9, (Molino del Salento, Maglie, Italy). The characteristics of wholemeal
Lb. plantarum PL9 and Pediococcus pentosaceus PP1, previously flour were: moisture, 15.0%; protein (N  5.70), 15.1% of dry matter
isolated from wheat sourdoughs and selected for the capacity of (d m); fat, 2.5% of d m; ash, 1.9% of d m; and carbohydrates, 65.4% of
synthesizing exopolysaccharides (EPS) (Di Cagno et al., 2006); and d m, respectively.
the potential probiotic strain Lactobacillus rhamnosus SP1 (supplied Clean emmer grains (100 g) were placed in perforated nylon
by Sacco Srl, Cadorago, CO, Italy) were used as starters for bags and steeped for 20 h in running tap water at 28e30  C (Beta
fermentation. Except for Lb. rhamnosus SP1, all bacterial strains et al., 1995). After steeping, grains were immersed in 2% sodium
belonged to the Culture Collection of the Dipartimento di Biologia e hypochlorite solution for 10 min and then rinsed 5 times with
Chimica Agro-Forestale ed Ambientale, University of Bari, Italy. excess of water. Grains were germinated at 28  C, 95% RH, for 5 days
Strains were cultivated in modified MRS (mMRS), prepared with in a germinator equipped with a humidifier. The germinated grains
1% (wt/v) maltose and 5% (v/v) fresh yeast extract, final pH of 5.6 were dried in a forced-air oven at 50  C for 24 h. The dried malt was
(Oxoid Ltd, Basingstoke, Hampshire, England). Fresh yeast extract cleaned, and the roots and shoots were removed by hand using
was prepared re-suspending baker’s yeast (60 g) in deionized water a corrugated, rubber surface.
(300 ml). After sterilization (120  C for 20 min), the suspension was The protocols for making emmer beverages are described in
centrifuged at 6000 g for 20 min, and the supernatant was Fig. 1. Three technology options, which included the use of emmer
recovered and added to mMRS prior to sterilization (Coda et al., flour, emmer flour subjected to gelatinization and emmer malt,
2010). Only Lb. rhamnosus SP1 was propagated in MRS. When were considered. The following types of fermented beverages were
used for cereal beverage fermentation, lactic acid bacteria were produced: F5, F10, and F15 (containing 5, 10, and 15%, wt/wt, of
cultivated until the late exponential phase of growth was reached emmer flour, respectively); GF5, GF10, GF15, and GF30 (containing
(ca. 10 h), harvested by centrifugation at 9000 g for 10 min at 4  C, 5, 10, 15 and 30%, wt/wt, of emmer gelatinized flour, respectively);

Fig. 1. Protocols for making emmer beverages.

528 R. Coda et al. / Food Microbiology 28 (2011) 526e536

and M5, M10, and M15 (containing 5, 10, and 15%, wt/wt, of emmer system (GE Healthcare, Buckinghmshire, UK) equipped with an
malt, respectively). Preliminarily, the amount of emmer flour or Aminex HPX-87H column (ion exclusion, Biorad, Richmond, CA),
emmer malt employed for the beverages were screened based on and an UV detector operating at 210 nm (organic acids and acetoin)
sensory analysis. Lactic acid bacteria strains were inoculated at the or a refractive index detector (Perkin Elmer 200a) operating at
initial cell density of ca. 5  107 cfu/ml. When the synthesis of EPS 32  C (carbohydrates and ethanol). Elution was at 60  C, with a flow
was assayed, beverages were added of 10% sucrose (Di Cagno et al., rate of 0.6 ml/min, using H2SO4 10 mM as mobile phase (Zeppa
2006). All the beverages were pasteurized at 63  C for 30 min, with et al., 2001; Davis et al., 2009).
the only exception of a GF30 added with the potential probiotic Lb. Total and individual free amino acids were analyzed by a Bio-
rhamnosus SP1 at the initial cell density of ca. 5  109 cfu/ml (GF30P). chrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge
Science Park, England) with a Na-cation-exchange column (20 by
2.3. Determination of pH, total titratable acidity (TTA) and kinetics 0.46 cm internal diameter) as described by Rizzello et al., (2010a).
of acidification
2.7. Phytase activity
The values of pH were determined on-line by a pHmeter (Model
507, Crison, Milan, Italy) with a food penetration probe. Total Phytase activity was measured in terms of inorganic ortho-
titratable acidity (TTA) was determined on 10 g of beverage phosphate released from the phytic acid by phytase (Shimizu,
homogenized with 90 ml of distilled water and expressed as the 1992). The reaction mixture, containing 150 ml of extract and
amount (ml) of 0.1 M NaOH to get pH of 8.3. 600 ml of substrate (3 mM Na-phytate in 0.2 M Na-acetate, pH 4.0),
Kinetics of acidification were modelled according to the Gom- was incubated at 45  C. The reaction was stopped by adding 750 ml
pertz equation as modified by Zwietering et al. (1990): y ¼ k þ A exp of 5% trichloroacetic acid. The released inorganic phosphate was
{exp[(Vmax$e/A)(lt) þ 1]}; where y is the acidification extent measured by adding 750 ml of color reagent, prepared daily by
expressed as dpH/dt (units of pH/h); at the time t; k is the initial mixing four volumes of 1.5% (wt/vol) ammonium molybdate in 5.5%
level of the dependent variable to be modelled (pH units); A is the (vol/vol) sulphuric acid solution and one volume of a 2.7% (wt/vol)
difference in pH (units) between inoculation and the stationary ferrous sulphate solution. The absorbance was measured at
phase (DpH); Vmax is the maximum acidification rate expressed as 700 nm. One unit (U) of phytase activity was defined as the amount
dpH/h; l is the length of the lag phase expressed in hours; and t is of enzyme required to liberate 1 nmol of phosphate per min under
the time. The experimental data were modelled through the non- the assay conditions.
linear regression procedure of the statistic package Statistica per
Windows (Statsoft, Tulsa, Oklahoma, USA). 2.8. Total phenols and antioxidant activity

2.4. Microbiological analysis Extracts were prepared by weighing 5 g of sample and mixing
with 50 ml of 80% methanol. The mixture was purged with nitrogen
The number of presumptive lactic acid bacteria was estimated stream, mixed for 30 min and centrifuged at 6000 rpm for 20 min.
by plating on MRS or mMRS agar media (Oxoid) at 30  C for 48 h. Extracts were transferred into test tubes, purged with nitrogen
DNA was extracted from colonies of the highest plate dilutions in stream and stored at ca. 4  C before analysis.
mMRS or MRS and used for RAPD-PCR analysis as described by Analysis of total phenols was according to the method of
Minervini et al., (2009). RAPD-PCR analysis was carried out as Slinkard and Singleton (1997). Gallic acid was the standard. The
described by Coda et al. (2010), using the primers P7 (50 reaction mixture contained 20 ml of beverage extract, 100 ml of
AGCAGCGTGG 30 ), and M13 (50 -GAGGGTGGCGGTTCT-30 ) (Invi- Folin-Ciocalteu reagent (Sigma Chemical Co.) and 1.58 ml of
trogen, Milan, Italy) (De Angelis et al., 2006). distilled water. 300 ml of saturated sodium carbonate solution were
added after few min. The mixture was incubated at 20  C for 2 h
2.5. Viscosity and total dry matter and the absorbance at 765 nm was determined. The concentration
of total phenols was calculated as gallic acid equivalent.
The apparent viscosity was measured on approximately 35 ml of The free radical scavenging capacity was determined using the
fermented beverage using the sine wave vibro-viscometer A&D SV- stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH_) as reported by
10 (A&D Company Ltd., Japan), which measures viscosity by Yu et al. (2002, 2003). The antioxidant reaction was started by
detecting the driving electric current needed to resonate two transferring 1 ml of beverage extract into a test tube, containing
sensor plates at a constant frequency of 30 Hz and amplitude of less 4 ml of 80% methanol and 1 ml of freshly prepared DPPH_ solution.
than 1 mm. Viscosity measurements were carried out on beverages The final concentration of DPPH_ in the reaction mixture was
previously adapted at 25  C for 30 min. Total dry matter was 100 mmol. The reaction was monitored by reading the absorbance at
determined on 100 ml of beverage at 105  C for 24 h (AOAC, 1985). 517 nm every 2 min for 30 min. A blank reagent was used to study
stability of DPPH_over the test time. The absorbance measured after
2.6. Carbohydrates, organic acids, ethanol and free amino acids 10 min was used for the calculation of the mmol DPPH_scavenged by
beverage extract. The kinetic of the antioxidant reaction in the
Water/salt-soluble extracts from beverages were prepared presence of the extract was also determined over 30 min and
following the method of Weiss et al. (1993). An aliquot of beverage compared with butylated hydroxytoluene (BHT) at the concentra-
(containing 7.5 g of flour) was diluted with 30 ml of 50 mM tion of 75 ppm as the antioxidant reference.
TriseHCl (pH 8.8), held at 4  C for 1 h, vortexing at 15-min intervals,
and centrifuged at 20,000 g for 20 min. The supernatant, con- 2.9. In vitro starch hydrolysis
taining the water/salt-soluble fraction, was filtered through a Mil-
lex-HA 0.22-mm pore size filter (Millipore Co., Bedford, MA) and The analysis of starch hydrolysis mimicked the in vivo digestion
used for analyses. of starch. Aliquots of beverages, containing 1 g of starch, were given
Carbohydrates, organic acids, acetoin, and ethanol contained in in randomized order to 10 volunteers. The analysis was carried out
the water/salt-soluble extracts were determined by High Perfor- as described by Liljeberg et al. (1996). The glucose content was
mance Liquid Chromatography (HPLC), using an ÄKTA Purifier measured with Enzy Plus D-Glucose kit (Diffchamb Västra Frölunda,
 R. Coda et al. / Food Microbiology 28 (2011) 526e536 529

Sweden). Factor conversion from glucose to starch was 0.9. The rate HP-Innowax 60 m  0.25 mm nominal diameter  0.25 mm of film
of starch digestion was expressed as the percentage of potentially thickness (Agilent, Santa Clara, CA, USA) and coupled with a mass
available starch hydrolyzed at different times (30, 60, 90, 120 and spectrometer 5975C (Agilent). Helium was the carrier gas at
180 min). The hydrolysis curves were obtained with the equation a constant flow of 1 ml/min. Separation conditions were: start oven
described below, using the software STATISTICA 7.0. Hydrolysis temperature 40  C for 4 min, ramp to 220 at 8  C/min, held for
curves follow a first order equation: C ¼ CN$(1ekt) where C is the 15 min. Acquisition parameters were: full scan mode from 15 to
concentration at t time, CN is the equilibrium concentration, k is the 260 Da, MS source 230  C, MS Quad 150  C, energy 70 eV, emission
kinetic constant and t is the chosen time (De Angelis et al., 2007). 34 mA.
Peak identification was carried out comparing retention times
2.10. Determination of dietary fiber concentration with those of standards (Sigma Chemical Co.). Computer matching
with the reference mass spectra of NIST and Wiley libraries was
Pepsin solution was prepared dissolving 100 mg of pepsin (2000 carried out. The identification of some compounds was carried out
FIP-U/g Sigma Chemical Co.) per ml of 0.08 M HCleKCl buffer, pH only by matching with the reference mass spectra.
1.5. Pancreatin solution was prepared dissolving 5 mg of pancreatin
(Sigma Chemical Co.) per ml of 0.1 M of phosphate buffer pH, 7.5. a- 2.13. Sensory analyses
Amylase solution was prepared dissolving 120 mg of a-amylase
(17.5 IU amylase/mg, Sigma Chemical Co.) in 1 ml of 0.1 M Triz- Two different protocols were used for sensory analysis. The first
maemaleate buffer, pH 6.9. The amyloglucosidase solution (14 IU/ protocol (Luckow et al., 2006) used 10 non trained panellists. Acidic,
mg, Roche, Mannheim, Germany) in 0.2 M sodium acetate buffer, cereal and sweet were considered as sensory attributes for flavor
pH 4.75, was used. and taste, using a scale from 0 to10. The evaluation of sensory
The concentration of fiber was determined as described by Goñi attributes was discussed with the assessors during the introductory
et al., (2009). In detail, 100 ml of beverages, concentrated 2.5 fold
through a Speed-Vac centrifuge at 35  C (Thermo Scientific, Wal-
Table 1
tham, MA), were submitted to enzymatic hydrolysis. First, samples Sensory attributes of cereal beverages, their abbreviations and descriptions.
were treated with 20 ml of HCleKCl buffer pH 1.5 (pH checked) and
0.5 ml pepsin solution. After 40 min at 40  C in water bath with Characteristic Abbreviation Definition

constant shaking, 20 ml Trizmaemaleate buffer pH 6.9 (pH Odor

Overall intensity OI The odor perceived immediately
checked) and 5 ml a-amylase solution were added. Samples were
of odor
incubated at 37  C, for 3 h. Finally, sodium acetate buffer pH 4.7 was Toasted odor TO The odor related to toasted/coke cereal
added followed by 0.4 ml of amyloglucosidase solution, and incu- evacuate before mixing
bation was at 60  C for 45 min under constant shaking. After Flavor
enzyme treatments, samples were transferred into dialysis tubes Overall intensity FI The flavor evaluated orally after mixing
(cut off 12,400 Da) and dialyzed against water for 48 h at 37  C. of flavor the sample with a spoon
Retentates were freeze-dried for gravimetric determination of Toasted flavor TF The flavor related to toasted cereal
dietary fibers. Basic tastes
Sweet bSW Taste on the tongue stimulated by sugars
2.11. Determination of hydrosoluble vitamins Bitter bBT Taste associated with caffeine and quinine
Sour/Acid bSO Taste associated with lactic acid
Astringent bAS Mouth drying. The complex of drying,
Hydrosoluble vitamins were determined by HPLC (Anyakora puckering and shrinking sensations in the
et al., 2008), using an ÄKTA Purifier system (GE Healthcare) lower oral cavity causing contractions of
equipped with a C18 Xterra column (Waters Inc., Mississauga, USA) the body tissue in the mouth
and an UV detector at 220 nm. The mobile phase was 70% buffer A Others
(0.941 g hexane sulphonic acid, 10 ml acetic acid, 990 ml distilled Artificial AR Non-specific, often used to describe
water) and 30% methanol (HPLC grade). The analysis was carried imitation products
Earthy ER Tasting dirty and musty
out in isocratic mode at a flow rate of 0.8 ml/min, at 25  C.
Dairy DA A flavor of condensed, sweet milk
Hydrosoluble vitamins were extracted as described by Aslam et al. Cereal CE A flavor of cereal
(2008) with some modifications. Ten milliliters of each beverage Savory SV A meaty, fleshy, beany flavor
were added of 25 ml of buffer A, homogenized, and kept on shaking After taste
water bath at 70  C for 40 min. Thereafter, the samples were cooled Sweet aSW A lingering sweet syrupy flavor
down, added of trifluoroacetic acid (TFA, final concentration 0.05%) Bitter aBT A lingering bitter flavor that hits the top
and filtered (0.22 mm). of the tongue
Earthy aER A lingering dirty, earthy, musty flavor
Sour/Acid aSO A lingering acidic, tangy flavor
2.12. Solid phase micro-extraction (SPME)/gas-chromatography
(GC)/mass-spectrometry(MS) analysis Oral texture
Particles PR Particles presence and bits
Uniformity of UF The uniformity of mass after drink
Volatile compounds from beverages were extracted by SPME. mass
The analysis of volatile compounds was carried out using SPME
Manual texture
vials of 12 ml (Supelco, Bellafonte, PA, USA) filled with 2 ml of Thickness TH The force required to stir the sample
samples containing 15% of NaCl. After 30 min of equilibration time, with spoon
the extraction was performed using a fiber 50/30 mm, DVB/CAR/ Adherence to AD The amount of the sample adhering to the
PDMS (Supelco), exposed in the static headspace of vials for 40 min spoon spoon evacuate by taking a spoonful of
sample and turning the spoon over
at 37  C. The volatile compounds were then desorbed by directly
inserting the fiber for 4 min into the injector port (splitless mode Appearance
for 1 min) maintained at 220  C of a gas chromatographer 6850 Darkness of the DK
color
(Agilent, CA, United States), equipped with a capillary column,
530 R. Coda et al. / Food Microbiology 28 (2011) 526e536

Table 2
Sensory analysis (Luckow et al., 2006) of emmer beverages fermented with Lactobacillus plantarum 6E.

F5 F10 F15 GF5 GF10 GF15 GF30 M5 M10 M15

Flavor
Acidic 6.2  0.2 6.2  0.4 7.0  0.2 6.2  0.3 7.5  0.5 9.4  0.5 7.4  0.1 7.4  0.4 7.6  0.4 8.0  0.2
Cereal 6.3  0.2 6.4  0.3 7.2  0.4 7.4  0.4 7.5  0.4 8.4  0.3 7.5  0.3 7.0  0.2 8.0  0.5 9.4  0.4
Sweet 5.0  0.3 6.4  0.5 7.0  0.3 4.5  0.3 5.2  0.3 4.5  0.5 6.5  0.5 6.0  0.3 5.0  0.3 5.0  0.2

Taste
Acidic 6.0  0.2 7.0  0.3 7.2  0.4 5.0  0.3 6.4  0.4 6.6  0.2 6.8  0.2 5.5  0.2 6.6  0.5 7.0  0.2
Cereal 6.3  0.3 6.4  0.2 7.0  0.3 6.5  0.3 7.2  0.3 7.3  0.4 7.5  0.2 8.3  0.4 8.5  0.3 8.6  0.4
Sweet 5.5  0.3 5.2  0.2 5.8  0.2 6.0  0.2 7.2  0.2 6.5  0.2 7.5  0.2 6.4  0.4 6.3  0.3 6.0  0.2

Sensory attributes were scored by using a scale 0e10. Protocols for making emmer beverages are described in Fig. 1. F5, F10, and F15 contained 5, 10, and 15%, wt/wt, of emmer
flour respectively; GF5, GF10, GF15, and GF30 contained 5, 10, 15, and 30%, wt/wt, of gelatinized emmer flour, respectively; M5, M10 and M15 contained 5, 10, and 15%, wt/wt,
of emmer malt, respectively. Data are the means of three independent experiments  standard deviation (n ¼ 3) twice analyzed.

training sessions and with the aim to select the fermentation value of Vmax (0.76  0.01 dpH/h, overall range 0.76e0.40 dpH/h)
conditions for making emmer beverages, 7.0 was considered as the were found for Lb. plantarum 6E. Based on these results, Lb. plan-
optimal score. Lower or higher values than 7.0 were considered tarum 6E was used as starter to ferment the emmer beverages
weakly or excessively pronounced, respectively. The second according to the protocols described in Fig. 1.
protocol (LapvetelaKinen and Rannikko, 2000; Luckow and After manufacture, the 10 beverages were assayed for sensory
Delahunty, 2004) considered a vocabulary for odor and flavor properties (Table 2). Within beverages made with emmer flour (F),
attributes. References that could be used to remind panellists about the highest scores for flavor and taste were found for F15. Regarding
the quality of each attribute were identified (Table 1). The gelatinized flour (GF), the beverages differed mainly for acidic and
descriptive sensory analysis was carried out once the training was cereal flavor. Both these attributes were the highest for GF15
completed. Beverages were served in white polystyrene cups (Table 2). Compared to GF15, scores for acidic and cereal flavor were
(40 ml in a 120 ml cup), and were labeled randomly with selected lower for GF30 (Table 2) presumably for the masking effect due to
codes. Beverages were served at room temperature (20  C) to better the different thickness. No significant (P > 0.05) differences were
differentiate odors and flavors, and to facilitate the characterization found between GF10 and GF30, for each of the attributes. In the
and comparison of each sample. Each assessor received 2 samples case of malt beverages (M), acidic flavor and taste were the highest
for each beverage; 3 independent experiments were carried out. for M10 and M15 (Table 2). Malt beverages also received the highest
score for cereal taste. Based on the above results and considering
2.14. Statistical analysis 7.0 as the optimal score, beverages F15, GF10, GF30 and M5 were
selected for further analyses.
Data were subjected to one-way ANOVA; pair-comparison of
treatment means was achieved by Tukey’s procedure at P < 0.05, 3.2. Physical, chemical and nutritional characteristics
using the statistical software Statistica for Windows (Statistica 7.0,
Windows). Descriptive sensory attributes were analyzed with After 4 h of fermentation at 30  C, F15, GF10, and M5 had values
Principal Component Analysis (PCA) using the software XLStat 2010 of pH in the range of 3.95e3.99, whereas GF30 reached the pH of
(Addinsoft USA, New York, NY) (Dijksterhuis, 1997). 3.99 after 5 h (Table 3). The cell number of presumptive lactic acid
bacteria at the end of fermentation was ca. 2.5  108 cfu/ml, without
3. Results differences between the 4 beverages. Pasteurization decreased the
cell number of presumptive lactic acid bacteria to ca. 2.0  106 cfu/ml
3.1. Selection of starters and beverages for all the beverages. After pasteurization, enterobacteria and yeasts
were not found in 10 g of each sample and, as estimated by plating
Strains Lb. plantarum 6E, Lb. plantarum 10E, W. cibaria 12E and on PCA, total bacteria were ca. 2.5  107 cfu/ml.
Lb. brevis 20S, previously isolated from emmer and spelt flour (Coda After pasteurization, the physical, chemical, functional and
et al., 2010), were singly used as starters for beverage fermentation sensory characteristics of the emmer beverages were determined
(10%, wt/wt, of emmer flour, F10) and screened based on the (see below). No differences were found after 30 days of storage at
kinetics of acidification. Strains reached the value of pH 4.0 after ca. 4  C (data not shown).
4 h of fermentation at 30  C, with the exception of Lb. brevis 20S Titratable acidity significantly (P < 0.05) differentiated the
which needed ca. 8 h. The values of DpH were ca. 1.84. The shortest beverages, ranging from 1.0  0.12 to 8.6  0.16 ml of 0.1 M NaOH/
value of l (0.27  0.02 h, overall range 0.27e3.07 h) and the highest 10 g. Viscosity significantly (P < 0.05) differentiated the beverages

Table 3
Physical and chemical features of selected emmer beverages fermented with Lactobacillus plantarum 6E.

Beverages pHt0 pHtf TTA Viscosity Dry matter Sugars (mM) Organic acids (mM) Acetoin Ethanol
(mPa s) (%) (mM) (mM)
Maltose Glucose Fructose Lactic Acetic
F15 5.84  0.05 3.95  0.03 3.5  0.11 1.53  0.08 2.56  0.08 2.7  0.2 3.9  0.1 1.7  0.1 27.0  0.2 0.7  0.1 1.3  0.1 13.0  0.1
GF10 5.98  0.04 3.99  0.02 2.3  0.09 3.69  0.05 9.03  0.05 10.2  0.1 3.9  0.1 2.3  0.1 18.0  0.2 0.3  0.1 0.8  0.1 nf
GF30 5.90  0.02 3.99  0.02 8.6  0.16 30.5  0.18 26.77  0.07 27.5  0.2 14.4  0.1 6.7  0.1 56.8  0.2 0.8  0.1 1.3  0.1 nf
M5 6.21  0.04 3.96  0.05 1.0  0.12 1.35  0.04 1.01  0.03 1.4  0.1 1.0  0.1 0.9  0.1 21.3  0.2 0.7  0.1 0.8  0.1 nf

Protocols for making emmer beverages are described in Fig. 1. F15 contained 15%, wt/wt, of emmer flour; GF10 contained 10%, wt/wt, of gelatinized emmer flour; GF30
contained 30%, wt/wt,of gelatinized emmer flour; and M5 contained 5%, wt/wt, of emmer malt. Data are the means of three independent experiments  standard deviation
(n ¼ 3) twice analyzed. nf, not found.
 R. Coda et al. / Food Microbiology 28 (2011) 526e536 531

Fig. 2. Concentration of free amino acids (FFA) and amino acid derivatives (mg/kg) of selected emmer beverages: F15 (15%, wt/wt, emmer flour), GF10 (10%, wt/wt, gelatinized
emmer flour), GF30 (30%, wt/wt, gelatinized emmer flour) and M5 (5%, wt/wt, emmer malt). Protocols for making emmer beverages are described in Fig. 1. Data are the means of
three independent experiments twice analyzed. Bars of standard deviations are also represented.

made with gelatinized flour, especially GF30 (Table 3). Values of dry highest sharp drop of the DPPH color intensity, was found for GF30,
matter, carbohydrates and organic acids of GF30 were ca. 3 times indicating the rapid and high capacity to quench DPPH radical.
higher than those of GF10. As the consequence of the centrifuga- After 10 min of reaction, the remaining color intensity of DPPH was
tion, dry matter of F15 and M5 was markedly lower than that of 51.3  0.12, 47.7  0.09, 41.5  0.10 and 29.2  0.24% for GF10, M5,
GF10 (Table 3). Compared to GF10 and M5, the concentration of F15 and GF30, respectively. Phytase activity significantly (P < 0.05)
acetoin was higher in F15 and GF30 (Table 3), while ethanol was differed between beverages (Table 4). It was the highest in GF30.
only found in F15. The highest concentration of total free amino The level of phytase activity in GF10, M5, and, F15 resulted 37, 53,
acids (FAA) was found for M5 (336.0  11 mg/l) followed by GF30, and 67% of that found in GF30, respectively. Hydrosoluble vitamins
15F and GF10 (224.6  9, 115.1  7 and 68.0  10 mg/l, respectively). thiamin (B1) and niacin (PP) were detected in all the beverages.
Glu, Val, Met, Ile, Leu, Trp, Arg and Pro were the FAA found at the Thiamin was found at concentrations of 7.0  0.2, 8.1  0.2,
highest concentrations, especially in M5 and GF30 (Fig. 2). 22.4  0.3, and 3.0  0.2 mg/l in F15, GF10, GF30, and M5, respec-
Based on the technological protocols used for making emmer tively. The concentration of niacin was 10.0  0.3, 14.1  0.2,
beverages, GF30 and GF10 showed the highest level of dietary 43.0  0.2, and 5.0  0.1 mg/l for F15, GF10, GF30, and M5,
fibers (Table 4). No significant (P > 0.05) differences were found respectively.
between F15 and M5. Under the conditions of this study, 80% Emmer beverage GF30 was further characterized for the rate of
methanol extracts from beverages (Ragaee et al., 2006) were used in vitro starch hydrolysis. The hydrolysis of starch at 30e180 min
to determine the concentration of total phenols and antioxidant and the values of hydrolysis index (HI) are shown in Fig. 4. After
properties. The concentration of total phenols of GF30 was ca. 3 180 min, the percentages of hydrolyzed starch were 62.1 and 35.0%
times higher compared to the other beverages (Table 4). The anti- for wheat bread (control) and GF30, respectively. In agreement, the
oxidant properties were determined based on the scavenging value of HI for GF30 was 56%.
activity towards DPPH radical. DPPH radical without antioxidants
or beverage extracts were stable over the time (Fig. 3). According to 3.3. EPS enrichment
previous studies (Ragaee et al., 2006), the color intensity of DPPH_
showed a logarithmic decline when in the presence of BHT. The Preliminarily, five EPS-producing lactic acid bacteria were singly
used as starters for fermentation of GF30 beverage. Except for the
use of W. cibaria WC9, an increase of the viscosity was found for
almost all the GF30 beverages (40.5  1.2 to 117.4  2.4 mPa s)
Table 4
Concentrations of dietary fiber (g/l) and total phenols (mM gallic acid), and phytase
compared to GF30 fermented with Lb. plantarum 6E. The highest
activity of selected emmer beverages fermented with Lactobacillus plantarum 6E. value of viscosity was determined by the addition of W. cibaria
WC4. Compared to GF30 fermented with W. cibaria WC4 alone,
Beverages Dietary fiber Total phenols Phytase
(g/l) (mM gallic acid) activitya (U)
the co-fermentation with Lb. plantarum 6E and W. cibaria WC4 in
the presence of 10% (wt/wt) sucrose did not cause variation of the
F15 0.23  0.03 0.32  0.01 0.58  0.02
GF10 11.32  0.04 0.31  0.02 1.10  0.02 viscosity.
GF30 38.48  0.02 0.95  0.02 1.75  0.02
M5 0.34  0.02 0.28  0.04 0.82  0.03 3.4. Volatile compounds and sensory analysis
Protocols for making emmer beverages are described in Fig. 1. F15 contained 15%,
wt/wt, of emmer flour; GF10 contained 10%, wt/wt, of gelatinized emmer flour; GF30 Aiming at further differentiating the beverages, volatile
contained 30%, wt/wt, of gelatinized emmer flour; and M5 contained 5%, wt/wt, of compounds and sensory analyses were carried out. Table 5 shows
emmer malt. Data are the means of three independent experiments  standard
deviation (n ¼ 3) twice analyzed.
the profile of volatile compounds according to chemical classes. A
a
One unit (U) of phytase activity was defined as the amount of enzyme required total of 45 compounds were identified. Alcohols were the most
to liberate 1 nmol of phosphate per min under the assay conditions. abundant compounds, ranging from ca. 36.0e53.4% of the total
532 R. Coda et al. / Food Microbiology 28 (2011) 526e536

Fig. 3. Kinetics of the scavenging activity of BHT (75 ppm), and emmer beverages (F15, 15%, wt/wt, emmer flour; GF10, 10%, wt/wt, gelatinized emmer flour; GF30, 30%, wt/wt,
gelatinized emmer flour; and M5, 5%, wt/wt, emmer malt) towards DPPH radical. Protocols for making emmer beverages are described in Fig. 1. The concentration of DPPH in the
reaction mixture and control (DPPH) was 100 mmol. Data are the means of three independent experiments twice analyzed. Bars of standard deviations are also represented.

peak area. Nine alcohols were commonly found in all the 4 bever- with gelatinized flour were mainly characterized by sweet taste and
ages. 1-Pentanol and 1-hexanol showed the highest peak areas. after taste. In particular, GF10 showed dairy taste, while GF30
Aldehydes mainly characterized the profiles of F15 and GF10 (39.9 resulted more acidic (taste and after taste) and with the more
and 13.8% of the total peak area, respectively). F15 showed the intense flavor.
highest areas of hexanal and benzaldehyde. Among ketones, GF10
showed the highest percentage of the total peak area of 3-hydroxy- 3.5. Survival of potential probiotic strain
2-butanone, while F15 contained high levels of 3-ehtyl-2-methyl-
1,3-hexadiene. Carboxylic acids predominated in M5 and GF30. GF30 beverage was also manufactured by co-inoculating Lb.
Acetic and butanoic acids were found at the highest levels. plantarum 6E and the potential probiotic strain Lb. rhamnosus SP1
Data from the sensory analysis of selected emmer beverages (GF30P), following the protocol of Fig. 1. Pasteurization was
were elaborated through PCA analysis (Fig. 5). The first and second omitted. Compared to GF30 started with Lb. plantarum 6E alone, the
factors explained 52.11 and 34.45% of the total variance. Beverages addition of the potential probiotic strain did not cause significant
were markedly separated in the plane of the biplot. On the left part (P > 0.05) differences of the acidification process (data not shown).
of the plane, F15 was characterized by artificial flavor, while M5 Lb. plantarum 6E and Lb. rhamnosus SP1 were monitored by RAPD-
could be differentiated by toasted and cereal flavor, and intense PCR analysis throughout storage at 4  C. Both strains were detect-
odor. On the right part of the plane the two beverages produced able after 30 days of storage. In particular, Lb. plantarum 6E showed

Fig. 4. Rate of starch hydrolysis following chewing, incubation with pepsin and subsequent incubation with pancreatic a-amylase in a dialysis tubing of wheat flour bread, used as
the control (:), and GF30 (30%, wt/wt, gelatinized emmer flour) beverage (C). Protocols for making emmer beverages are described in Fig. 1. Values for each time point not sharing
the same letters are significantly different from each other at P < 0.05.
 R. Coda et al. / Food Microbiology 28 (2011) 526e536 533

Table 5
Volatile compounds found in selected emmer beverages fermented with Lactobacillus plantarum 6E as determined by solid-phase micro-extraction/gas-chromatography/mass
spectrometry (SPME/GC/MS) analysis. Mean values  SD are calculated as ratio peak area/total peak area percent.

Compounds F15 GF10 GF30 M5

Alcohols
Ethanol 2.84  0.13 0.91  0.01 0.94  0.01 1.51  1.23
Isopropyl Alcohol 0.56  0.05 0.58  0.06 0.59  0.09 e
1-Butanol e e 0.11  0.01 15.36  0.13
1-Butanol, 3-methyl- 1.30  0.62 e 0.58  0.44 8.07  1.82
2-Butanol, 3-methyl-, (S)- e 1.19  0.25 1.18  0.08 e
1-Pentanol 9.77  0.62 7.23  2.33 9.69  0.58 1.84  0.49
1-Penten-3-ol e 0.61  0.35 0.77  0.16 e
1-Penten-3-ol, 4-methyl-a 0.60  0.17 e e e
2-Pentanol 1.85  0.03 e e e
2-Pentanol, 4-methyl-a e e 0.39  0.20 e
2-Penten-1-ol, (Z)-a e e 0.44  0.16 e
1-Hexanol 26.44  1.40 39.85  4.77 37.89  3.43 9.20  1.20
1-Hexanol, 2-ethyl- 8.18  4.54 e e e
2-Hexanol e e 0.12  0.01 e
2-Furanmethanola e e 0.67  0.39 e

Aldehydes
Acetaldehyde 1.19  0.29 e 0.69  0.05 e
Butanal, 3-methyl-a e 1.25  0.15 1.03  0.09 e
Propanal, 3-methoxy-a e 1.93  1.03 e e
Pentanediala e e 7.04  0.08 e
Hexanal 15.24  2.07 4.84  5.25 0.79  0.46 e
Hexanedial, 2-hydroxy-a e e 0.28  0.05 e
2-Octenal, (E)-a e e 6.09  1.05 e
2,4-Decadienala 8.71  1.30 5.78  0.68 e 3.96  2.33
Benzaldehyde 14.74  1.10 e e e

Ketones
Acetone 0.24  0.01 e e 0.96  0.02
2-Butanone 0.46  0.09 e 0.33  0.05 e
2-Butanone, 3-hydroxy-a e 5.35  0.82 0.69  0.57 e
2,3-Butanedione 0.81  0.01 1.07  0.01 0.22  0.01 0.27  0.01
2-Pentanonea e e e 0.81  0.05
1-Penten-3-onea 0.56  0.04 e 0.21  0.03 e
2-Heptanone, 5-methyl-a e 0.23  0.01 e e
Esters
Ethyl Acetatea 1.78  0.21 3.72  0.86 1.25  0.03 2.85  0.48

Oxygenous heterocycle compounds

Furan, 2-ethyl a 0.77  0.03 0.35  0.17 0.51  0.16 e
Furan, 2-pentyl-a 2.20  0.30 0.52  0.21 e 1.36  0.37

Alkanes and alkenes

Pentanea e 1.03  0.09 0.69  0.05 e
1-Pentene, 3-methyl-a e 0.42  0.23 e e
1,3-Hexadiene, 3-ethyl-2-methyl-a 14.37  0.25 e e e
2-Dodecene, (E)-a e 1.18  0.94 e e
3-Dodecene, (E)-a e 3.17  0.99 e e

Terpenes
D-Limonene e e 0.47  0.10 e

Miscellaneous
Ammonia 0.36  0.09 0.51  0.28 0.12  0.41 4.72  2.21
sec-Butylaminea e 0.31  0.17 e e
Trichloromethanea 0.06  0.20 e e e

Carboxylic acids
Acetic acid e 17.49  1.45 13.43  2.94 5.72  0.85
Butanoic acid 0.36  0.16 e e 16.01  3.50
Hexanoic acid 1.11  0.49 e 5.01  0.60 9.76  0.82
Octanoic Acid 2.51  0.31 e 1.50  1.02 e

Protocols for making emmer beverages are described in Fig. 1. F15 contained 15%, wt/wt, of emmer flour; GF10 contained 10%, wt/wt, of gelatinized emmer flour; GF30
contained 30%, wt/wt, of gelatinized emmer flour; and M5, contained 5%, wt/wt, of emmer malt. Data are the means of three independent experiments  standard deviation
(n ¼ 3) twice analyzed.
a
Compounds identified by matching with the reference mass spectra of NIST and Wiley libraries.

a slight decrease from 2.9  1.0  108 (end of fermentation) to 4. Discussion

1.3  2.0  108 cfu/ml. The cell density of Lb. rhamnosus SP1 slightly
decreased from 5.1  0.9  109 to 8.0  0.7  108 cfu/ml. Compared Traditional cereal fermented beverages or gruels are produced
to GF30 started with Lb. plantarum 6E alone, no significant worldwide (Blandino et al., 2003). In most of the cases, fermenta-
(P > 0.05) differences were found for sensory attributes due to the tion occurs spontaneously and involves mixed cultures of yeasts,
addition of Lb. rhamnosus SP1 (data not shown). bacteria and fungi and the substrates for fermentation are mainly
534 R. Coda et al. / Food Microbiology 28 (2011) 526e536

Fig. 5. Principal component biplot (Dijksterhuis, 1997) of sensory analysis (LapvetelaKinen and Rannikko, 2000 and Luckow and Delahunty, 2004) for selected emmer beverages F15
(15%, wt/wt, emmer flour), GF10 (10%, wt/wt, gelatinized emmer flour), GF30 (30%, wt/wt, gelatinized emmer flour), and M5 (5%, wt/wt, emmer malt). Protocols for making emmer
beverages are described in Fig. 1. The abbreviations used for sensory attributes are reported in Table 1.

raw flour, gelatinized flour, and malted grains. (Nout, 2009). The present in all the beverages which suggested that sugar depletion
chemical and nutritional properties of emmer flours were previ- was not a limiting factor for fermentation. Low levels of acetic acid
ously investigated (Coda et al., 2010) showing some interesting were found in all beverages, probably due to the facultative hetero-
properties for processing. This study aimed at investigating the fermentative metabolism of Lb. plantarum (Gobbetti, 1998). Traces
suitability of emmer grains for making functional beverages. Raw of ethanol were found only in the beverage made with 15% (wt/wt)
and gelatinized flour and malted grains were used as substrates for of unprocessed raw flour, probably due to the activity of endoge-
fermentation that was carried out using selected autochthonous nous yeasts. Cell densities of yeasts up to 2  103 cfu/g are usually
lactic acid bacteria starters. Yeasts were not used as starters for detected in cereal flours (De Vuyst and Neysens, 2005). The
processing since the concentration of ethanol has to be lower than concentration of total free amino acids was the highest in the
0.5% (vol/vol) to get healthy claims (Kreisz et al., 2008). On the beverage made with malted flour. As previously shown (Kunze,
contrary, selected autochthonous strains of lactic acid bacteria 1996), malting of seeds causes the partial hydrolysis of polymers
influenced positively the nutritional and technology properties of through the activities of endogenous enzymes.
emmer flours (Coda et al., 2010). Preliminarily, sensory analysis was Consumption of foods rich in fibers is recommended to prevent
carried out with the purpose to select the most suitable concen- several diseases (De Angelis et al., 2007). The daily consumption of
tration of raw (15%, wt/wt) and gelatinized (10% or 30% wt/wt) dietary fibers, for example, regulates the energy intake and satiety,
flour, and malted grains (5%, wt/wt) to be used. Emmer beverages which would decrease obesity (Lyly et al., 2009). Beverages made
were subjected to pasteurization which is usually applied to non- with gelatinized flour, especially at the highest concentration (30%,
alcoholic beverages to prolong the shelf-life and avoid post-acidi- wt/wt), naturally contained a level of dietary fiber similar to that of
fication processes without nutritional and sensory modifications fortified beverages from different sources (Lyly et al., 2009).
(Osuntogun and Aboaba, 2004). Together with fibers, also polyphenols deserve an increasing
Fermentation with the selected autochthonous Lb. plantarum 6E interest for antioxidant and anti-inflammatory properties (Covas
favored a fast acidification process under all technology conditions et al., 2006). All the selected emmer beverages contained appre-
assayed. Lb. plantarum is largely diffuse in spontaneous and sour- ciable levels of polyphenols, and the antioxidant activity, as esti-
dough fermentation of cereals (Gobbetti et al., 2005), and its mated towards DPPH radical, agreed with the concentration of total
robustness was recently proved (Minervini et al., 2010). Most of the polyphenols. Phytic acid is an anti-nutritional factor which
physical and chemical parameters differentiated the beverages, decreases the dietary bioavailability of minerals such as Caþþ,
especially that manufactured with 30% (wt/wt) of gelatinized flour. Mgþþ, Feþþ, and Znþþ, and basic amino acid group of protein.
Viscosity and dry matter were the highest in the beverages made Fermentation by lactic acid bacteria may result in a more suitable
with gelatinized flour where solid particles were not removed. pH to activate flour endogenous phytases and lactic acid bacteria
Overall, high levels of solids increase the buffering capacity of the may also be a source of phytases (Gobbetti et al., 2005). The phytase
medium which may allow a more intense process of fermentation. activity of the emmer beverages was comparable to that found
The viscosity of the emmer beverage made with the highest (30%, during sourdough fermentation (Di Cagno et al., 2008; Coda et al.,
wt/wt) concentration of gelatinized flour approached that of 2010; Rizzello et al., 2010a and b). Usually, wholemeal cereal
yogurt-like products. It could be further increased by using the EPS- flours contain considerable levels of vitamins of the B complex
producing strain W. cibaria WC4. The concentration of soluble (Lebiedzin ska and Szefer, 2006). Thiamin and niacin were found at
carbohydrates depended on the concentration of flour used and high concentration in the emmer beverages, especially in those
maltose was the most abundant. Residual carbohydrates were manufactured with gelatinized or raw flour. In particular, ca. 170 ml
 R. Coda et al. / Food Microbiology 28 (2011) 526e536 535

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Gaddi, A., de la Torre, R., Mursu, J., Bäumler, H., Nascetti, S., Salonen, J.T., Fitó, M.,
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