Combined effects of chitosan and MAP to improve the microbial quality of amaranth homemade fresh pasta
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against the following spoilage microorganisms: mesophilic bacteria, Staphylococcus spp., yeasts, moulds
combination of 30:70 N2:CO2 is the most efcient, promoting an extension of the microbial acceptabilitylimit beyond two months.
the wional pent o
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
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 specic targets (Kasarda, 2001). Amaranth is one of the
* Corresponding author. Department of Food Science, University of Foggia, ViaNapoli 25, 71100 Foggia, Italy. Tel./fax: 39 881 589 242.
Contents lists availab
Food Microbiology 26 (2009) 587591E-mail address: firstname.lastname@example.org (M.A. Del Nobile).preserve 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 efcacy 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 (modied atmosphere packaging,
possible alternative is represented by the use of active agents fromplant, animal ormicrobial 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 elds as a result of its biological activity, which isantimicrobial against a wide range of food-borne lamentous fungi,yeast and bacteria (No et al., 2002; Yoshihiko et al., 2003; Sagoo et al.,Keywords:AmaranthChitosanMAPMicrobiological qualityGluten-free pasta
The diffusion of pasta throughoutof both traditional and non-conventa tangible impulse to the developm0740-0020/$ see front matter 2009 Elsevier Ltd.doi:10.1016/j.fm.2009.03.012 2009 Elsevier Ltd. All rights reserved.
orld and the popularityroducts have providedf new technologies to
(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). AAvailable online 5 April 2009
suggest 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, theAccepted 28 March 2009and total coliforms. Their viable cell concentrations were monitored for about 2 months at 4 C. ResultsCombined effects of chitosan and MAPquality of amaranth homemade fresh p
M.A. Del Nobile a,b,*, N. Di Benedetto a, N. Suriano a
aDepartment of Food Science, University of Foggia, Via Napoli 25, 71100 Foggia, ItalybBIOAGROMED Istituto per la Ricerca e le Applicazioni Biotecnologiche per la SicurezUniversita` 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 2009
a b s t r a c t
In this work a study on thimprove the microbiologictwo different chitosan co
journal homepage: wwAll rights reserved.improve the microbialta
. Conte a, M.R. Corbo a,b, M. Sinigaglia a,b
la Valorizzazione dei Prodotti Tipici e di Qualita`,
mbined effects of chitosan and modied atmosphere packaging (MAP) toality of amaranth-based homemade fresh pasta is presented. In particular,trations were combined to three different MAP conditions and tested
le at ScienceDirect
elsevier .com/locate/ fm
icroldest grain crop ever known. It is classied 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 ours 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 modiedatmosphere 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 our was provided by Molino Bongiovanni(Mondov`, Cuneo, Italy). Chitosan (Danisco, Braband, Denmark) wastested in two concentrations: 2000 mg/kg and 4000 mg/kg of freshpasta.
2.2. Pasta production
Amaranth our 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 nal 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 lm [Nylon/Polyethylene, 95 mm (Tecnovac, SanPaolo DArgon, 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, asspecied by the manufacturer: oxygen transmission rate equal to50 cm3 m2 day1 atm1 calculated at 23 C and 75% RelativeHumidity (RH) and water vapour transmission rate equal to2.6 g m2 day1 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-mentedwith egg yolk tellurite emulsion, incubated at 37 C for 48 hfor Staphylococcus spp. In order to conrm the presence of Staph-ylococcus spp catalase test and microscopic observation (Axi-oskop20, Zeiss, Germany) were carried out. Malt Extract Agar,acidied to pH 4.5 by sterile solution of citric acid 1:1 (w/v) incu-
M.A. Del Nobile et al. / Food M588bated at 25 C for 5 days for moulds and Sabouraud Dextrose Agarsupplemented with chloramphenicol (0.1 g l1) (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, rm-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):
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 specic 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 specication 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 10
6 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 andMAPon fresh pasta. In particular, the viable cell concentration at the endof the observation period (54th day) was calculated according toa modied version of the above-mentioned Equation (1):
where the new parameter N was introduced as the viable cell
obiology 26 (2009) 58759154
concentration (CFU/g) after 54 days of storage.
2.6. Statistical analysis
The values of tting parameters (MAL and log(N54)) weresubmitted toone-wayanalysis of variance (ANOVA) and toTukeys testthrough the software Statistica for Windows (Statsoft, Tulsa, OK).
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 efcientlydelay 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 tting procedure are:
N2:CO2 showed the lowest NMesophilic54 values, suggesting that CO2
efciently delayed the growth of mesophilic bacteria. It is worthnoting that CO2 concentration higher than 70% caused an increasein the NMesophilic54 value. This nding 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 N
Mesophilic54 value is
Table 1Mean values of log(NMesophilic54 ) obtained by tting 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, xCNT-LAC 3.55 0.03A, x 5.41 0.14B, x 4.51 0.17C, xCHT2000 3.33 0.03A, y 3.71 0.02B, y 3.44 0.02C, yCHT4000 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 signicantly different(P < 0.05); values in the same column with different letters (x, y) are signicantlydifferent (P < 0.05).
Fig. 2. Evolution of Staphylococcus spp. concentration in fresh pasta samples, with andwithout chitosan, packaged under ordinary atmosphere conditions. The curves shownin the gure were obtained by tting the Equation (2) to the experimental data. (B)
M.A. Del Nobile et al. / Food Microbiology 26 (2009) 587591 589Fig. 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 thegure were obtained by tting the Equation (2) to the experimental data. (B) CNT, (:)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 conrmedthe 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 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 37 kDa), dissolved in 1% acetic acid and added towheat our 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%chitosanwas 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 rea...