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Food Microbiology 26 (2009) 587–591
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Food Microbiology
journal homepage: www.elsevier .com/locate/ fm
Combined effects of chitosan and MAP to improve the microbialquality of amaranth homemade fresh pasta
M.A. Del Nobile a,b,*, N. Di Benedetto a, N. Suriano a, A. Conte a, M.R. Corbo a,b, M. Sinigaglia a,b
a Department of Food Science, University of Foggia, Via Napoli 25, 71100 Foggia, Italyb BIOAGROMED – Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualita,Universita degli Studi di Foggia, Via Napoli 52, 71100 Foggia, Italy
a r t i c l e i n f o
Article history:Received 7 October 2008Received in revised form27 March 2009Accepted 28 March 2009Available online 5 April 2009
Keywords:AmaranthChitosanMAPMicrobiological qualityGluten-free pasta
* Corresponding author. Department of Food ScienNapoli 25, 71100 Foggia, Italy. Tel./fax: þ39 881 589 2
E-mail address: [email protected] (M.A. Del N
0740-0020/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.fm.2009.03.012
a b s t r a c t
In this work a study on the combined effects of chitosan and modified atmosphere packaging (MAP) toimprove the microbiological quality of amaranth-based homemade fresh pasta is presented. In particular,two different chitosan concentrations were combined to three different MAP conditions and testedagainst the following spoilage microorganisms: mesophilic bacteria, Staphylococcus spp., yeasts, mouldsand total coliforms. Their viable cell concentrations were monitored for about 2 months at 4 �C. Resultssuggest that there is a combined effect between MAP and chitosan in delaying the microbial quality lossof pasta during storage. Moreover, it was also found that among the tested MAP conditions, thecombination of 30:70 N2:CO2 is the most efficient, promoting an extension of the microbial acceptabilitylimit beyond two months.
� 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The diffusion of pasta throughout the world and the popularityof both traditional and non-conventional products have provideda tangible impulse to the development of new technologies topreserve the food quality. For its high water content (30% max),fresh pasta is an easily perishable foodstuff. Its spoilage is due toboth the metabolic activity of microorganisms (bacteria, yeasts,moulds) that can grow in the product, and to various enzymaticactivities (Bacci et al., 2004; Zardetto, 2005). The preservingcapacity of fresh pasta is essentially linked to the level of themicrobial count found in the product at the end of the process andto the efficacy of methods used to avoid proliferation, such as theuse of barriers to prevent a new contamination of sanitized productand the adoption of barriers to restrict the growth of microorgan-isms surviving to sanitization (modified atmosphere packaging,vacuum packaging and cooling) (Piergiovanni, 1995; Sinigagliaet al., 1995).
Different national laws dealing with food additives rule the use ofchemical preservatives and bacteriostatics in fresh pasta production
ce, University of Foggia, Via42.
obile).
All rights reserved.
(FDA, 2006). However, nowadays, Western society appears to beexperiencing a trend of ‘‘green’’ consumerism (Eriksson, 2004)desiring fewer synthetic food compounds and products witha smaller impact on the environment. Therefore, new methods tomake food safe and with natural image are required (Burt, 2004). Apossible alternative is represented by the use of active agents fromplant, animal or microbial origin (Altieri et al., 2005; Bevilacqua et al.,2007; Conte et al., 2007; Singh et al., 2002). Among the antimicro-bials available, a compound that has received considerable attentionfor commercial applications is the chitosan (No et al., 2002, 2007;Park et al., 2002; Rhoades and Roller, 2000; Roller and Covill, 1999,2000; Shahidi et al., 1999; Sudarshan et al., 1992). It is a non-toxicbiopolymer derived by deacetylation of chitin. It has attracted muchattention in various fields as a result of its biological activity, which isantimicrobial against a wide range of food-borne filamentous fungi,yeast and bacteria (No et al., 2002; Yoshihiko et al., 2003; Sagoo et al.,2002; Tsai et al., 2002), antitumor (Cai-qin et al., 2002; Sinha et al.,2004), hypocholesterolemic (Gallaher et al., 2002; Sinha et al., 2004)and biodegradable (Xie et al., 2001).
Due to the increase in celiac disease and allergic reactions/intolerances to prolamine and gluteline, demand for gluten-freeproducts is rising. In the last years, in fact, different grain fromdurum wheat have been used, as partial or total substitutes, toproduce non-conventional pasta with healthy characteristics ordirected to specific targets (Kasarda, 2001). Amaranth is one of the
M.A. Del Nobile et al. / Food Microbiology 26 (2009) 587–591588
oldest grain crop ever known. It is classified among pseudocerealsfor content of saccharides (62.0%) slightly lower than that ofcommon cereals, even though with a higher digestibility. The moststriking characteristic of amaranth is the lack of prolamine andgluteline that allow its use in gluten-free products (Teutonico andKnorr, 2007).
The interest in gluten-free flours combined to increasing need ofsafe methods for food preservation is a valid reason to promoteresearch on new formulation of amaranth-based homemade freshpasta. In particular, the combined effects of chitosan and modifiedatmosphere packaging conditions on the microbial quality ofselected gluten-free fresh product were investigated in this work.
2. Materials and methods
2.1. Raw materials
Amaranth-based flour was provided by Molino Bongiovanni(Mondovı, Cuneo, Italy). Chitosan (Danisco, Braband, Denmark) wastested in two concentrations: 2000 mg/kg and 4000 mg/kg of freshpasta.
2.2. Pasta production
Amaranth flour and tap water (30% v/w) were mixed to preparepasta dough. Before mixing chitosan with pasta dough, workingactive solutions were prepared dissolving chitosan in lactic acidsolution (1.38% v/v). These solutions were added to the dough,separately, to obtain final concentrations of 2000 mg/kg and4000 mg/kg of pasta (CHT2000 and CHT4000). As controls, pastasample without antimicrobial (CNT) and pasta sample with lacticacid solution (0.42% v/w) (CNT-LAC), were also prepared. Pasta wasobtained in form of Spaghetti by a fresh pasta-maker (Pastamatic,Simac 1400N, Treviso, Italy) equipped with head bronze. Thekneading time was 15 min. All the pasta samples were packaged inhigh-barrier plastic film [Nylon/Polyethylene, 95 mm (Tecnovac, SanPaolo D’Argon, Bergamo, Italy)] by means of S100-Tecnovacequipment. An amount of pasta equal to 30 g was arranged in eachbag. Film barrier properties are reported in the following, asspecified by the manufacturer: oxygen transmission rate equal to50 cm3 m�2 day�1 atm�1 calculated at 23 �C and 75% RelativeHumidity (RH) and water vapour transmission rate equal to2.6 g m�2 day�1 calculated at 23 �C and 85% RH. The bags were170 mm � 250 mm long. The samples were packaged in air and inprotective atmosphere and stored at 4 �C. In particular, 80:20, 0:100and 30:70 N2:CO2 combinations were used as gas mixture in thepackaging. Microbiological analyses and determination of pH weremade during 2 storage months, details of which are given below.
2.3. Microbiological analyses
Aliquots of 10 g of each sample were diluted with 90 ml ofa sterile saline solution (0.90% NaCl) and homogenized in a stom-acher bag through a stomacher Lab-Blender 400 (PBI International,Milan). Serial dilutions in sterile saline solution were plated ontoappropriate media. The media and the conditions were thefollowing: Plate Count Agar (PCA) incubated at 30 �C for 48 h foraerobic mesophilic bacteria; Violet Red Bile Agar (VRBA) incubatedat 37 �C for 24 h for total coliforms; Baird-Parker Agar, supple-mented with egg yolk tellurite emulsion, incubated at 37 �C for 48 hfor Staphylococcus spp. In order to confirm the presence of Staph-ylococcus spp catalase test and microscopic observation (Axi-oskop20, Zeiss, Germany) were carried out. Malt Extract Agar,acidified to pH 4.5 by sterile solution of citric acid 1:1 (w/v) incu-bated at 25 �C for 5 days for moulds and Sabouraud Dextrose Agar
supplemented with chloramphenicol (0.1 g l�1) (C. Erba, Milan,Italy) incubated at 25 �C for 48 h for yeasts. All media andsupplements were from Oxoid (Milan, Italy). All the analyses wereperformed twice, on two different batches. The measurement ofpH was performed on the homogenized product by a pH-metre(Crison, Barcelona, Spain).
2.4. Sensorial evaluation
In order to highlight sensorial differences between the controland the pasta enriched with chitosan, uncooked and cookedspaghetti were subjected to sensory evaluation just after theirproduction. Each type of pasta was cooked, separately, in a cookercontaining about 4000 ml of tap water at 100 �C. The analyses wereperformed in isolated booths in a standard taste panel kitchen. Allthe samples were submitted in a single session to a panel of 8trained tasters for estimation of color, aroma and overall accept-ability of the uncooked pasta and adhesiveness, bulkiness, firm-ness, elasticity, color, aroma, taste and overall acceptability of thecooked ones. To this aim, a nine-point hedonic rating scale, where 1corresponded to extremely unpleasant and 9 to extremely pleasant,was used.
2.5. Modelling of microbial data
To quantitatively determine the effectiveness of chitosan andMAP in inhibiting the growth of spoilage microorganisms understudy, the time at which the viable cell concentration reached itsacceptability limit was calculated according to the Gompertzequation, as re-parameterized by Corbo et al. (2006):
logðNðtÞÞ¼logðNmaxÞ�A$exp��exp
��ðmmax$2:71Þl�MAL
A
�þ1��
þA$exp��exp
��ðmmax$2:71Þl�t
A
�þ1��
ð1Þ
where N(t) is the viable cell concentration (CFU/g) at time t, A isrelated to the difference between the decimal logarithm ofmaximum bacteria growth attained at the stationary phase and thedecimal logarithm of the initial cell load concentration (CFU/g),mmax is the maximal specific growth rate (Dlog[CFU/g]/day), l is thelag time (day), t is the time (day), MAL is the microbial acceptabilitylimit (day), Nmax is the maximum allowable cell load concentration(CFU/g). As any microbiological legal specification for homemadefresh pasta is reported, directive limits suggested by MinisterialHealth Decree 32 (1985) for egg and stuffed pasta were used. Forthis reason, the above equation was used for mesophilic bacteria,total coliforms and Staphylococcus spp. data that exceeded themaximum allowable cell load concentration during the observationperiod. To this aim, the value of Nmax was set to 106 CFU/g formesophilic bacteria and 104 CFU/g for total coliforms and Staphy-lococcus spp.
For all the other microbial data, a different approach was used toquantitatively determine the combined effects of chitosan and MAPon fresh pasta. In particular, the viable cell concentration at the endof the observation period (54th day) was calculated according toa modified version of the above-mentioned Equation (1):
logðNðtÞÞ ¼logðN54Þ�A$exp��exp
��ðmmax$2:71Þl�54
A
�þ1��
þA$exp��exp
��ðmmax$2:71Þl�t
A
�þ1��
ð2Þ
where the new parameter N54 was introduced as the viable cellconcentration (CFU/g) after 54 days of storage.
Table 1Mean values of log(NMesophilic
54 ) obtained by fitting Equation (2) to experimental data,along with their standard deviation.
Mesophilic bacteria 30:70 N2:CO2 80:20 N2:CO2 0:100 N2:CO2
CNT 3.47 � 0.02A, x 5.25 � 0.16B, x 4.72 � 0.15C, x
M.A. Del Nobile et al. / Food Microbiology 26 (2009) 587–591 589
2.6. Statistical analysis
The values of fitting parameters (MAL and log(N54)) weresubmitted to one-way analysis of variance (ANOVA) and toTukey’s testthrough the software ‘‘Statistica for Windows’’ (Statsoft, Tulsa, OK).
CNT-LAC 3.55 � 0.03A, x 5.41 � 0.14B, x 4.51 � 0.17C, x
CHT2000 3.33 � 0.03A, y 3.71 � 0.02B, y 3.44 � 0.02C, y
CHT4000 3.44 � 0.04A, x 3.70 � 0.02B, y 3.60 � 0.02C, y
Values in the same row with different letters (A, B, C) are significantly different(P < 0.05); values in the same column with different letters (x, y) are significantlydifferent (P < 0.05).
3. Results and discussion
The microbial quality decay of amaranth-based homemadefresh pasta loaded with chitosan and packed under three differentMAP conditions was assessed by monitoring the viable cellconcentration of the main spoilage microbial groups such as mes-ophilic bacteria, total coliforms, Staphylococcus spp., yeasts andmoulds. As reported above, two different mathematical approacheswere used depending on the evolution of each microbial group andon the legislation limit taken into account. Chitosan efficientlydelay the growth of mesophilic bacteria in samples packaged in air.For these data the Equation (1) was used and the relative values ofMALMesophilic bacteria obtained from the fitting procedure are:20.39 � 0.78 for CNT, 17.69 � 1.77 for CNT-LAC, 47.12 � 1.52 forCHT2000 and 50.29� 2.54 for CHT4000. These numbers confirmedthe well-known antimicrobial properties of chitosan on Grampositive and negative bacteria (No et al., 2007). In particular, itseffectiveness on wet noodles was also demonstrated. Lee et al.(2000) observed that wet noodles containing chitosan(Mw ¼ 37 kDa, 0.1% or 0.5% dissolved in 1% lactic acid) could bestored more than 80 days compared to 7 days of the control. Inanother study, Lee and No (2002) demonstrated that the samechitosan (Mw ¼ 37 kDa), dissolved in 1% acetic acid and added towheat flour at concentration of 0.0%, 0.17%, 0.35%, 0.52% and 0.7%,was effective in prolonging the shelf life of noodles. In particular,the shelf life of product containing 0.17%, 0.35%, 0.52% and 0.70%chitosan was extended by 1, 2 and 3 days, respectively, compared tothat of the control.
Fig. 1 shows the evolution during storage of mesophilic bacterialpopulation for samples stored under 80:20 N2:CO2. As can beinferred, the cell load did never exceed the selected threshold limit(106 CFU/g) during the entire observation period. For this reason,the experimental data were modelled by using Equation (2). Thesame equation was used for data related to samples stored under30:70 and 0:100 MAP conditions. The obtained NMesophilic
54 valuesare listed in Table 1. As can be seen, samples packaged under 30:70
Fig. 1. Evolution of mesophilic viable cell concentration in fresh pasta samples, withand without chitosan, stored under 80:20 N2:CO2 gas mixture. The curves shown in thefigure were obtained by fitting the Equation (2) to the experimental data. (B) CNT, (:)CNT-LAC, (,) CHT2000, (A) CHT4000. Data are the mean (n ¼ 2) � standarddeviation.
N2:CO2 showed the lowest NMesophilic54 values, suggesting that CO2
efficiently delayed the growth of mesophilic bacteria. It is worthnoting that CO2 concentration higher than 70% caused an increasein the NMesophilic
54 value. This finding is in agreement with whatreported in the literature by Piergiovanni (1995) who stated theeffectiveness of carbon dioxide on fresh food products. Data listedin Table 1 also highlight that for samples packaged under 80:20 and0:100 N2:CO2, a substantial decrease in the NMesophilic
54 value isobtained for amaranth-based homemade fresh pasta loaded withchitosan. The above evidence suggested that chitosan acted ina combined way with MAP to delay the growth of mesophilicbacteria. It is noteworthy that the lactic acid was not responsible forthe chitosan antimicrobial efficiency. As a matter of fact, thedifferences between CNT and CNT-LAC were very small.
In Fig. 2 Staphylococcus spp. cell growth cycle for pasta samplespackaged under ordinary atmosphere conditions is shown. Thesedata highlight that only the control sample goes beyond thethreshold limit, confirming literature data dealing with the effec-tiveness of chitosan on Staphylococcus spp. (Darmadji and Izumi-moto, 1994). Therefore, a quantitative determination of therecorded antimicrobial effect was obtained by fitting Equation (1)to the experimental data relative to CNT and CNT-LAC. The calcu-lated MALStaphylococcus were 16.28 � 1.34 and 19.14 � 1.29 for CNTand CNT-LAC samples under air and 15.03 � 2.98 and 23.06 � 1.67for the same samples packaged under 80:20 N2:CO2. Samplespackaged under 0:100 MAP conditions showed MALStaphylococcus
value of 31.07 � 2.91 and 26.48 � 10.01 for CNT and CNT-LAC,respectively. Conversely, the pasta samples without chitosanpackaged under 70:30 N2:CO2, as well as all the samples containingchitosan, packaged both under ordinary and modified atmosphere,did not exceed the microbial acceptability level. Thus, as above,
Fig. 2. Evolution of Staphylococcus spp. concentration in fresh pasta samples, with andwithout chitosan, packaged under ordinary atmosphere conditions. The curves shownin the figure were obtained by fitting the Equation (2) to the experimental data. (B)CNT, (:) CNT-LAC, (,) CHT2000, (A) CHT4000. Data are the mean (n ¼ 2) � standarddeviation.
Table 2Mean values of log(NStaphylococcus
54 ) obtained by fitting Equation (2) to experimentaldata, along with their standard deviation.
Staphylococcusspp.
Air 30: 70N2:CO2
80:20N2:CO2
0:100N2:CO2
CNT – 2.16 � 0.09x – –CNT-LAC – 2.22 � 0.14x – –CHT2000 2.10 � 0.13A, x <2 2.47 � 0.42B, x 3.42 � 0.2C, x
CHT4000 2.24 � 0.02A, x <2 2.74 � 0.19B, x 2.97 � 0.14B, y
Values in the same row with different letters (A, B, C, D) are significantly different(P < 0.05); data in the same column with different letters (x, y) are significantlydifferent (P < 0.05). – for log(NStaphylococcus
54 ) higher than 4.
Table 3Mean values of log(NColiforms
54 ) obtained by fitting Equation (2) to total coliformsexperimental data, along with their standard deviation.
Total coliforms 30: 70 N2:CO2 80:20 N2:CO2 0:100 N2:CO2
CNT <1 – 3.56 � 0.17x
CNT-LAC <1 2.97 � 0.21A 3.45 � 0.37B, x
CHT2000 <1 <1 2.42 � 0.18y
CHT4000 <1 <1 2.46 � 0.40y
Values in the same row with different letters (A, B, C, D) are significantly different(P < 0.05); values in the same column with different letters (x, y, k, z) are signifi-cantly different (P < 0.05). – for log(NColiforms
54 ) higher than 4.
M.A. Del Nobile et al. / Food Microbiology 26 (2009) 587–591590
Equation (2) was fitted to these experimental data. The results fromthe fitting process are listed in Table 2. As can be inferred fromtheNStaphylococcus
54 values reported in table, there is a combined effectbetween chitosan and MAP in retarding the microbial quality loss offresh pasta, and the best performance was recorded by using chi-tosan, at whatever concentration, and 70:30 gas mixture.
As regards total coliforms, only the CNT sample packaged underboth ordinary atmosphere and 80:20 N2:CO2 went over the totalcoliforms threshold limit. Thus, for these samples it was possible tocalculate the following MALColiforms: 9.53 � 1.01 for CNT in air and54.36 � 2.44 for control pasta under 80:20. For all the othersamples whose microbial cell load never reached 104 CFU/g,Equation (2) was used. The low concentration of such microbialindicator organisms may suggest that adequate processing andpost-processing conditions were adopted during homemade pastaproduction (Sinigaglia et al., 1995). As an example, Fig. 3 shows theevolution during storage of viable cell concentration of total coli-forms in refrigerated amaranth-based homemade fresh pastapackaged under 0:100 N2:CO2. The obtained NColiforms
54 values arelisted in Table 3. Data listed in the table show that contrary to whatobserved for the other cases, lactic acid affected the growth ofcoliforms. In fact, it has been reported that Gram positive bacteria,as lactic acid bacteria, at pH 5 are not killed but normally grow (Gilland Badoni, 2004; Knarreborg et al., 2002); whereas, the coliformbacteria viability at pH 5 is greatly affected by organic acids. Benzoicacid, fumaric acid and lactic acid are particularly effective, whereasbutyric acid, formic acid and propionic acid are less importantantimicrobial compounds. Data listed in Table 3 suggest that 30:70is the most efficient MAP mixture and combined to chitosan it cancontribute substantially to preserve the microbial quality of freshpasta.
Fig. 3. Evolution of total coliforms in fresh pasta samples, with and without chitosan,packaged under 0:100 N2:CO2. The curves shown in the figure were obtained by fittingthe Equation (2) to the experimental data. (B) CNT, (:) CNT-LAC, (,) CHT2000, (A)CHT4000. Data are the mean (n ¼ 2) � standard deviation.
The yeasts were not recovered from any investigated samples.Otherwise, a moderate moulds proliferation was found in all thespaghetti samples. No threshold limit has been reported in theliterature for moulds. Therefore, also in this case, Equation (2) wasused. As an example, Fig. 4 shows the moulds evolution duringstorage of all the samples packaged in air. The obtained NMoulds
54values are listed in Table 4. As can be inferred, results similar to thatobtained for mesophilic bacteria has been also observed formoulds. In particular, 30:70 gas mixture was the most efficientmodified atmosphere in holding up the moulds proliferation;moreover, a combined action of chitosan and MAP on mouldsgrowth was also recorded. The results reported in table also high-light that lactic acid is not responsible for any antifungal efficiency.
Similar pH ranges, from 6.3 to 5.8, were measured for all theinvestigated pasta samples, suggesting that it did not changesubstantially during storage, according to other work reported inthe literature (Blasi et al., 2007). For this experimental evidence, thedetected antimicrobial and antifungal activity of chitosan and MAPcannot be ascribed to a reduction in pH (data not shown).
The sensory data (data not shown) recorded by trained panel-lists allowed us to evaluate a possible sensorial impact of the chi-tosan in fresh pasta. The panel test highlighted no differencesbetween pasta with and without chitosan, suggesting that thisnatural preservative could be advantageously combined to MAP asvalid alternative to more expensive food thermal-treatmentscommonly used to prolong the shelf life of pasta.
4. Conclusions
In this study chitosan and MAP were used in combination toimprove the microbiological quality of refrigerated amaranth-
Fig. 4. Evolution of moulds in samples of fresh pasta, with and without chitosan,packaged in air. The curves shown in the figure were obtained by fitting the Equation(2) to the experimental data. (B) CNT, (:) CNT-LAC, (,) CHT2000, (A) CHT4000.Data are the mean (n ¼ 2) � standard deviation.
Table 4Mean values of log(NMoulds
54 ) obtained by fitting Equation (2) to microbial data, alongwith their standard deviation.
Moulds Air 30: 70 N2:CO2 80:20 N2:CO2 0:100 N2:CO2
CNT 7.08 � 0.31A, x <2 3.70 � 0.2B, x 2.00 � 0.4C
CNT-LAC 7.36 � 0.64A, x <2 2.79 � 0.31B, y <2CHT2000 6.10 � 0.35A, y <2 2.20 � 0.07B, k <2CHT4000 5.97 � 0.44y <2 <2 <2
Values in the same row with different letters (A, B, C, D) are significantly different(P < 0.05); values in the same column with different letters (x, y, k, z) are signifi-cantly different (P < 0.05).
M.A. Del Nobile et al. / Food Microbiology 26 (2009) 587–591 591
based fresh pasta. Results suggest that MAP and chitosan can act insynergic mode in controlling the microbial quality loss of refriger-ated fresh pasta during refrigerated storage. Results also highlightthat among the tested MAP conditions 30:70 N2:CO2 is the mostefficient gas mixture to preserve the microbial quality of amaranth-based spaghetti. In fact, chitosan, at whatever concentrations, usedin combination with modified atmosphere can extent the microbialacceptability limit of this homemade fresh pasta beyond twomonths. The interesting results on the sanitary aspect, along withthe very comparable sensorial characteristics recorded for thecontrol and the chitosan-added pasta suggest to scale up this non-thermal preserving method to an industrial level.
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