use of natural compounds to improve the microbial stability of amaranth-based homemade fresh pasta

6
Use of natural compounds to improve the microbial stability of Amaranth-based homemade fresh pasta M.A. Del Nobile a, b, * , N. Di Benedetto a , N. Suriano a , A. Conte a , C. Lamacchia a, b , M.R. Corbo a, b , M. Sinigaglia a, b a Department of Food Science, University of Foggia, Via Napoli, 25 – 71100 Foggia, Italy b 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, 25 – 71100 Foggia, Italy article info Article history: Received 2 April 2008 Received in revised form 26 September 2008 Accepted 9 October 2008 Available online 18 October 2008 Keywords: Amarnath Antimicrobial compounds Microbiological quality Pasta Shelf life abstract A study on the use of natural antimicrobial compounds to improve the microbiological stability of refrigerated amaranth-based homemade fresh pasta is presented in this work. In particular, the anti- microbial activity of thymol, lemon extract, chitosan and grapefruit seed extract (GFSE) has been tested against mesophilic and psychrotrophic bacteria, total coliforms, Staphylococcus spp., yeasts and moulds. A sensory analysis on both fresh and cooked pasta was also run. Results suggest that chitosan and GFSE strongly increase the microbial acceptability limit of the investigated spoilage microorganisms, being the former the most effective. Thymol efficiently reduces the growth of mesophilic bacteria, psychrotrophic bacteria and Staphylococcus spp., whereas it does not affect, substantially, the growth cycle of total coliforms. Lemon extract is the less effective in preventing microbial growth. In fact, it is able to delay only total mesophilic and psychrotrophic bacterial evolution. From a sensorial point of view no signifi- cant differences were recorded between the control samples and all the types of loaded amaranth-based pasta. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction The current range of ‘‘pasta’’ products is relatively vast, even if these products vary widely in terms of shape, colour, composition, storage, requirements and use. In the last years, different grain from durum wheat have been used (as partial or total substitutes) in production of non-conventional pasta with healthy characteristics or directed to specific targets such as people following a celiac diet (Kasarda, 2001). Amaranth is one of the oldest grain crop ever known. It is classified among pseudocereals for its content of saccharides (62.0%) slightly lower of that of common cereals even if with a higher digestibility. The seeds contain 7–8% of fat, 12.5–18% of protein content including all essential aminoacids with about 5% lysine and 4.4% sulfur aminoacids, which are limiting in other grains. At the same time it does not contain gluten protein (prola- mine and gluteline) and this is a reason of using Amaranth in gluten-free based products (Teutonico and Knorr, 2007). The diffusion of pasta throughout the world and the popularity of both traditional product and non-conventional products have provided a tangible impulse to the development of production technologies and storage techniques. For its high water content (30% max), fresh pasta is a foodstuff easily perishable. Its spoilage is due to both the metabolic activity of microorganisms (bacteria, yeasts, moulds) that can easily grow in the product and to various enzymatic activities (Guerzoni et al., 1994; Di Fabio et al., 1995). The preserving capacity of fresh pasta is essentially due to the level of the microbial count found in product at the end of the process and to the efficacy of methods used to avoid proliferation, such as the use of barriers to prevent a new product contamination and the adoption of barriers to restrict the growth of microorganisms surviving to sanitation (modified atmosphere packaging, cooling). Different national laws rule the use of chemical preservatives and bacteriostatics in fresh pasta production (FDA, 2006). Nowadays, however, Western society appears to be experiencing a trend of ‘‘green’’ consumerism (Smid and Gorris, 1999) desiring fewer synthetic food additives and products with a smaller impact on the environment. Therefore, new methods to make food safe, which have a natural image, are required (Burt, 2004). A possible alternative is the use of natural active agents such as essential oils and chitosan. Essential oils (EOs) are aromatic oily liquids obtained from plant material that exert a very pronounced antimicrobial activity. * Corresponding author. Department of Food Science, University of Foggia, Via Napoli, 25 – 71100 – Foggia, Italy. Tel./fax: þ39 881 589 242. E-mail address: [email protected] (M.A. Del Nobile). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm 0740-0020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2008.10.003 Food Microbiology 26 (2009) 151–156

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Page 1: Use of natural compounds to improve the microbial stability of Amaranth-based homemade fresh pasta

lable at ScienceDirect

Food Microbiology 26 (2009) 151–156

Contents lists avai

Food Microbiology

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

Use of natural compounds to improve the microbial stabilityof Amaranth-based homemade fresh pasta

M.A. Del Nobile a,b,*, N. Di Benedetto a, N. Suriano a, A. Conte a, C. Lamacchia a,b, 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, 25 – 71100 Foggia, Italy

a r t i c l e i n f o

Article history:Received 2 April 2008Received in revised form26 September 2008Accepted 9 October 2008Available online 18 October 2008

Keywords:AmarnathAntimicrobial compoundsMicrobiological qualityPastaShelf life

* Corresponding author. Department of Food ScienNapoli, 25 – 71100 – Foggia, Italy. Tel./fax: þ39 881 5

E-mail address: [email protected] (M.A. Del N

0740-0020/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.fm.2008.10.003

a b s t r a c t

A study on the use of natural antimicrobial compounds to improve the microbiological stability ofrefrigerated amaranth-based homemade fresh pasta is presented in this work. In particular, the anti-microbial activity of thymol, lemon extract, chitosan and grapefruit seed extract (GFSE) has been testedagainst mesophilic and psychrotrophic bacteria, total coliforms, Staphylococcus spp., yeasts and moulds. Asensory analysis on both fresh and cooked pasta was also run. Results suggest that chitosan and GFSEstrongly increase the microbial acceptability limit of the investigated spoilage microorganisms, being theformer the most effective. Thymol efficiently reduces the growth of mesophilic bacteria, psychrotrophicbacteria and Staphylococcus spp., whereas it does not affect, substantially, the growth cycle of totalcoliforms. Lemon extract is the less effective in preventing microbial growth. In fact, it is able to delayonly total mesophilic and psychrotrophic bacterial evolution. From a sensorial point of view no signifi-cant differences were recorded between the control samples and all the types of loaded amaranth-basedpasta.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The current range of ‘‘pasta’’ products is relatively vast, even ifthese products vary widely in terms of shape, colour, composition,storage, requirements and use. In the last years, different grain fromdurum wheat have been used (as partial or total substitutes) inproduction of non-conventional pasta with healthy characteristicsor directed to specific targets such as people following a celiac diet(Kasarda, 2001).

Amaranth is one of the oldest grain crop ever known. It isclassified among pseudocereals for its content of saccharides(62.0%) slightly lower of that of common cereals even if witha higher digestibility. The seeds contain 7–8% of fat, 12.5–18% ofprotein content including all essential aminoacids with about 5%lysine and 4.4% sulfur aminoacids, which are limiting in othergrains. At the same time it does not contain gluten protein (prola-mine and gluteline) and this is a reason of using Amaranth ingluten-free based products (Teutonico and Knorr, 2007).

ce, University of Foggia, Via89 242.obile).

All rights reserved.

The diffusion of pasta throughout the world and the popularityof both traditional product and non-conventional products haveprovided a tangible impulse to the development of productiontechnologies and storage techniques. For its high water content(30% max), fresh pasta is a foodstuff easily perishable. Its spoilage isdue to both the metabolic activity of microorganisms (bacteria,yeasts, moulds) that can easily grow in the product and to variousenzymatic activities (Guerzoni et al., 1994; Di Fabio et al., 1995). Thepreserving capacity of fresh pasta is essentially due to the level ofthe microbial count found in 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 product contamination and theadoption of barriers to restrict the growth of microorganismssurviving to sanitation (modified atmosphere packaging, cooling).Different national laws rule the use of chemical preservatives andbacteriostatics in fresh pasta production (FDA, 2006).

Nowadays, however, Western society appears to be experiencinga trend of ‘‘green’’ consumerism (Smid and Gorris, 1999) desiringfewer synthetic food additives and products with a smaller impact onthe environment. Therefore, new methods to make food safe, whichhave a natural image, are required (Burt, 2004). A possible alternativeis the use of natural active agents such as essential oils and chitosan.

Essential oils (EOs) are aromatic oily liquids obtained from plantmaterial that exert a very pronounced antimicrobial activity.

Page 2: Use of natural compounds to improve the microbial stability of Amaranth-based homemade fresh pasta

M.A. Del Nobile et al. / Food Microbiology 26 (2009) 151–156152

Usually compounds with phenolic groups are most effective (Dor-man and Deans, 2000), even if minor components play a significantrole in the whole antimicrobial effect (Lattaoui and Tantanoui-Elaraki, 1994; Lambert et al., 2001). Their antimicrobial propertieswere investigated by ‘‘in vitro’’ and ‘‘in vivo’’ assay, demonstratingtheir effect against natural spoilage bacteria and against food bornepathogens (Singh et al., 2002; Burt, 2004; Altieri et al., 2005;Bevilacqua et al., 2007; Conte et al., 2007).

Chitosan is a natural non-toxic biopolymer derived by deace-tylation of chitin. It has attracted much attention in various fields asa result of its biological activity, which is antimicrobial (No et al.,2002; Yoshihiko et al., 2003), antitumor (Suzuki et al., 1986),hypocholesterolemic functions (Sugano et al., 1992), biocompati-bility, and biodegradability (Xie et al., 2001). However, its activitydepends on several factors such as the kind of chitosan (deacety-lation degree, molecular weight) used, the pH of the medium, thetemperature, the presence of numerous components of food, etc.(Devlieghere et al., 2004).

To our knowledge, no work is reported on the microbiologicalstability of amaranth-based fresh pasta. The influence of somepotential inhibiting ingredients, such as thymol, grapefruit seedextract (GFSE), lemon extract and chitosan on the microbiologicalquality of fresh pasta is addressed in this work. In particular, the cellloads of the main specific spoilage bacteria responsible for freshpasta safety and consumer acceptance were monitored for a periodof about 25 days to establish the microbial acceptability limit of theinvestigated food product.

2. Materials and methods

2.1. Raw materials

Amaranth flour was provided by Molino Bongiovanni (Mondovı,Cuneo, Italy). Four natural origin antimicrobial compounds, thymol(Sigma, Poole, UK), grapefruit seed extract (GFSE) (Biocitro, Probenas.l, Zaragoza, Spain), lemon extract (Spencer Food Industrial,Amsterdam, The Netherlands), and chitosan (Danisco, Braband,Denmark), tested in concentration of 2000 mg/Kg of fresh pasta,were studied. For each compound, working active solutions wereprepared and respectively mixed to the pasta dough. In order toenhance its water solubility, thymol was dissolved in ethyl alcohol(95%) and then diluted with distilled water (50%, v/v), chitosan inlactic acid solution (1% v/v), whereas GFSE and lemon extract,readily soluble in water, were dissolved in distilled water. All thestock solutions were freshly prepared before using, then sterilizedby filtering through membranes (0.20 mm pore size; Minisart,Sartorius, Goettingen, Germany).

2.2. Pasta production

Amaranth flour and tap water (30% v/w) were mixed to preparepasta dough. The active solutions were separately added to the doughto obtain a final concentration of 2000 mg/Kg of pasta, for eachnatural compound used. As control, pasta sample without antimi-crobials (Cnt), pasta sample with lactic acid solution (0.3%) (Cnt–lac),pasta sample with ethanol (0.16%) (Cnt–eth) were also prepared.‘‘Tagliatelle’’ were obtained by a pasta maker (Pastamatic, Simac1400 N, Treviso, Italy), equipped with head bronze. The kneadingtime was 15 min. All pasta samples (30 g) were packed in high-barrier plastic film [Nylon/Polyethylene, 95 mm (Tecnovac, San PaoloD’Argon, Bergamo, Italy)] by means of S100-Tecnovac equipment.Film barrier properties are reported in the following as specified bythe manufacturer: CO2 permeance 3.26�10�19 mol m m�2 s�1 Pa�1,O2 permeance 9.23�10�19 mol m m�2 s�1 Pa�1, water vapor trans-mission rate 1.62�10�10 kg m�2 s�1. The bags were 170 mm�250 mm long. The samples were packaged in air and stored at 4 �C.

Microbiological analyses and determination of pH were made during25 storage days, 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 (AMB) and at 5 �C for 1 week for psy-chrotrophic bacteria; Violet Red Bile Agar (VRBA) incubated at 37 �Cfor 24 h for total coliforms; Baird-Parker Agar, supplemented withegg yolk tellurite emulsion, incubated at 37 �C for 48 h for Staph-ylococcus spp.; Malt Extract Agar, acidified to pH 4.5 trough a sterilesolution of citric acid 1:1 (v/v), incubated at 25 �C for 5 days formoulds; Sabouraud Dextrose Agar, added with 0.2 g/l chloram-phenicol (C. Erba, Milan, Italy), incubated at 25 �C for 48 h foryeasts. All media and supplements were from Oxoid (Milan, Italy).All microbiological analyses were performed twice on two differentbatches.

2.4. pH evaluation

The measurement of pH on the homogenised product wasperformed twice on two different batches by using a pH-meter(Crison, Barcelona, Spain).

2.5. Sensory analysis

Both uncooked and cooked fresh pasta were subjected tosensory evaluation to know if it was possible to distinguish thecontrol sample from the other samples containing the activecompounds. Uncooked pasta samples were submitted in a group ata single session to a panel of 8 trained tasters for estimation ofcolour, aroma and overall acceptability using a nine-point hedonicrating scale (Rathi et al., 2004), where 1 corresponded to extremelyunpleasant and 9 to extremely pleasant. Pasta samples with andwithout active compounds, cooked at optimal cooking time (OCT),were also submitted to the same judges to evaluate the adhesive-ness, bulkiness, firmness, elasticity, color, aroma, taste and overallacceptability. To cook pasta each sample (100 g) was immersed,separately, in a cooker containing about 2000 ml of tap water at100 �C until reaching the optimal cooking. The OCT for the differentpasta types were evaluated previously by cooking each pastasample at different times and submitting the cooked products tojudges that were asked to indicate for each type the best OCT. Theanalyses were performed in isolated booths in a standard tastepanel kitchen.

2.6. Modeling of microbial data

To quantitatively determine the effectiveness of the activecompounds in slowing down microbial growth, the time at whichthe viable cell concentration reached its acceptability limit wascalculated according to the Gompertz equation as re-parameterizedby Corbo et al. (2006):

logðNðtÞÞ¼logðNmaxÞ�Aexp��exp

��ðmmax2:71Þl�MAL

A

�þ1��

þAexp��exp

��ðmmax2:71Þl�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 of

Page 3: Use of natural compounds to improve the microbial stability of Amaranth-based homemade fresh pasta

Fig. 1. Evolution during storage at 4 �C of mesophilic bacteria in packaged fresh pastawith and without active compounds. The curves shown in the above figurewere obtained by fitting the Equation (1) to the experimental data. (B) (---) Cnt, (,)(d $ $ d) GFSE, (A) (dd) Thymol, (6) (d d) Chitosan, (:) (d $ d) Lemon extract.Data are the mean (n¼ 2)� standard deviation.

M.A. Del Nobile et al. / Food Microbiology 26 (2009) 151–156 153

maximum bacterial 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), Nmax is the maximum cell loadconcentration (CFU/g), MAL is the microbial acceptability limit(day) (i.e., the time at which N(t) is equal to Nmax). In the case oftotal mesophilic and psychrotrophic bacterial count the value ofNmax was set to 106 CFU/g, whereas, in the case of total coliformsand Staphylococcus spp. Nmax was set to 104 CFU/g (MinisterialHealth Decree 32, 1985).

To determine the microbial acceptability limit for sole chitosan-loaded sample, the following expression was fitted to the experi-mental data related to the psychrotrophic bacteria:

for t < t* logðNðtÞÞ ¼ A1g1 þ�

logðNmaxÞ � A2g3

�þ A2g2

�A1 expn� exp

nh�m1

max2:71

l1�tA1

iþ 1

oo

for t � t* logðNðtÞÞ ¼ logðNmaxÞ

�A2g3 þ A2 exp�� exp

���m2

max2:71l

2�ðt�t*ÞA2

�þ 1��

8>>>>>>>>>><>>>>>>>>>>:

(2)

where:

g1 ¼ exp

(� exp

("�m1

max2:71�l1 � t*

A1

#þ 1

));

g2 ¼ exp

(� exp

("�m2

max2:71�l2

A2

#þ 1

));

g3 ¼ exp

(� exp

("�m2

max2:71�l2 � ðMAL � t*Þ

A2

#þ 1

));

A1 and A2 are related to the difference between the decimallogarithm of maximum bacteria growth attained at the stationaryphase and the decimal logarithm of the initial cell load concentration(CFU/g) for the first and the second Gompertz expression, respec-tively; m1

max and m2max are the maximal specific growth rate for the

first and the second Gompertz expression, respectively (Dlog[CFU/g]/day); l1 and l2 are the lag time for the first and the second Gom-pertz expression, respectively (day). Equation (2) was obtained bycombining two adjacent Gompertz expressions as re-parameterizedby Corbo et al. (2006). In particular, the first is aimed to describe themicroorganism death kinetic, which takes place at very beginning ofstorage (t< t*), whereas the second describes the psychrotrophicgrowth kinetic during the second stage (t> t*). As above, the value ofNmax was set to 106 CFU/g (Ministerial Health Decree 32, 1985).

2.7. Statistical analysis

The mean values of the Microbial Acceptability Limit (MAL) andthe sensorial data were submitted to one-way analysis of variance(ANOVA) and to Tukey’s test through the software ‘‘Statistica 7.1 forWindows’’ (Stasoft, Tulsa, OK).

3. Results and discussion

The microbiological quality of Amaranth-based home-madefresh pasta loaded with natural compounds stored at 4 �C for about25 days has been assessed by monitoring the viable cell concen-tration of mesophilic and psychrotrophic bacteria, total coliforms,

Staphylococcus spp., yeasts and moulds. Regarding the last twomicrobial groups, the viable cell concentrations were below theexperimental detection limit for the entire storage period, both incontrol samples and in pasta containing the active compounds. Theobtained results on the other investigated microbial groups arepresented, separately in the following.

3.1. Mesophilic bacteria

Fig. 1 shows the evolution of total mesophilic bacteria duringstorage at 4 �C in amaranth-based homemade fresh pasta loadedwith thymol, chitosan, lemon extract and GFSE, respectively, and inthe control samples. As can be inferred, the investigated activeagents affected substantially the microorganism growth cycle. Ithas long been recognized that several EOs have antimicrobialproperties (Boyle, 1955; Shelef et al., 1984; Nychas, 1995; Tuley deSilva, 1996) and the relatively recent growth of interest in greenconsumerism has led to a renewal scientific interest in thesesubstances.

The curves shown in Fig. 1 were obtained by fitting Equation (1)to the experimental data. As can be seen, the above equationsatisfactorily describes the trend of data. The MAL values obtainedfrom fitting process are listed in Table 1. It’s worth noting that thereare no statistically significant differences between the controlsamples, suggesting that at concentration used in this work bothethanol and lactic acid did not affect the growth cycle of the mes-ophilic population. In addition, data listed in Table 1 highlight thata substantial increase in the MAL value was obtained for theamaranth-based pasta loaded with the selected natural preserva-tives. In particular, chitosan was the most efficient in slowing downthe growth of this spoilage microbial group. The antimicrobialeffectiveness of GFSE was practically the same as that of thymol,whereas lemon extract was the less effective agent in delaying themicrobial growth. These results agree with literature data. No et al.(2007) have been reviewed numerous examples of chitosanapplications to improve food quality and prolong shelf life. Themechanism of the antimicrobial activity of chitosan has not yetbeen fully elucidated, but several hypotheses have been proposed.The most feasible hypothesis is a change in microbial cell perme-ability due to interactions between the positively charges of chi-tosan and the negatively charged of microbial cell membranes (Noet al., 2007). Lee et al. (2002) also investigated the effects of

Page 4: Use of natural compounds to improve the microbial stability of Amaranth-based homemade fresh pasta

Table 1MAL values obtained by fitting Equations (1) and (2) to microbiological experimentaldata, along with their standard deviations.

MAL[day]

Mesophilicbacteria

Psychrotrophicbacteria

Totalcoliforms

Staphylococcusspp.

Cnt 1.32� 0.32A 3.57� 0.90A – 1.58� 0.21A

Cnt–eth 1.38� 0.33A 3.49� 0.18A – 1.33� 0.17A

Cnt–lac 1.18� 1.18A 3.34� 0.21A – 1.42� 0.17A

GFSE 11.4� 0.85C 12.3� 1.08D 4.38� 1.12B 14.8� 4.19B

Thymol 9.48� 2.11C 8.62� 1.04C 1.20� 0.31A 12.1� 1.88B

Lemonextract

5.43� 0.93B 5.77� 1.97B – 3.76� 0.56A

Chitosan 17.7� 1.03D >25 16.1� 1.78C >25

– Means MAL equal to zero.Data in each column with different superscript letter are statistically different(P< 0.05).

M.A. Del Nobile et al. / Food Microbiology 26 (2009) 151–156154

chitosan with different molecular weights (Mw¼ 1, 5, 30, and120 kDa) on the shelf life of wheat bread. The bread containing 30or 120 kDa chitosan at 0.1% concentration showed 10–103 CFU/g ofviable cells while the control bread without chitosan reveled106 CFU/g after 8 days of storage at room temperature. Moreover,chitosan was also tested on rice cake (Lee et al., 2000), soybean curd(Lee et al., 2001), soybean sprouts (Lee and Rhee, 1999; No et al.,2003), starch jelly (Moon et al., 1997; Lee and No, 2001) and noodle(No et al., 2007).

Fig. 2 shows the pH plotted as a function of storage time forboth the control samples and the natural compounds loadedpasta. A marked decrease in pH value was detected for the controlpasta (from 6.5 to 4.0) after only four days of storage at 4 �C. Onthe contrary, for samples added with antimicrobials, a slightvariation in the pH values during the storage time was observed.In particular, pH ranged from 6.5 to about 5.0 for lemon extractadded pasta samples, and from 6.5 to about 5.6 when thymol,GFSE or chitosan were added to pasta dough. The data shown inFig. 2 are in agreement with that reported in Fig. 1, suggesting thatthe observed changes in pH could be associated to microbialgrowth. In fact, microbial metabolism determines a greatproduction of acids, although the final products of the fermenta-tion differ according to the genera (Bevilacqua et al., 2007). It isworth noting that according to the above trend of pH, the detectedantimicrobial activity can’t be ascribed to a reduction in thepH value.

Fig. 2. Evolution during storage at 4 �C of pH in packaged fresh pasta with and withoutactive compounds. The curves shown in the above figure were used to highlight thetrend of data. (B) (---) Cnt, (,) (d $ $ d) GFSE, (A) (dd) Thymol, (6) (d d)Chitosan, (:) (d $ d) Lemon extract. Data are the mean (n¼ 2)� standard deviation.

3.2. Psychrotrophic bacteria

The evolution during storage at 4 �C of psychrotrophic viablecell concentration in amaranth-based home-made fresh pasta withand without antimicrobial compounds is shown in Fig. 3. As above,equation (1) was fitted to the experimental data, with the exceptionof data relative to pasta with chitosan. In fact, for this latter samplea sharp decrease in cell concentration below the experimentaldetection limit was monitored at the early stage of storage. Psy-chrotrophic viable cell loads went up to the detection limit after 10days of storage, reaching the stationary phase after about 18 days.To determine the microbial acceptability limit for these sample theEquation (2) was fitted to the experimental data.

MAL values obtained by the fitting process are also listed inTable 1. As can be observed, a substantial increase in MAL valueswas recorded for pasta with active compounds, as compared to thecontrol samples. As for mesophilic bacteria, chitosan loaded pastashowed the highest MAL value among the investigated naturalcompounds (higher than 25 days, which was the observation limitin this study). Concerning the other natural preservatives, MALdecreased according to the following order: GFSE> thy-mol> lemon extract. The mechanism of action of the EOs has notbeen studied in great detail (Lambert et al., 2001). Considering thelarge number of different groups of chemical compounds of EOs, itis most likely that their antibacterial activity is not attributable toone specific mechanism but that there are several targets in the cell(Skandamis and Nychas, 2001; Carson et al., 2002). An importantcharacteristic of EOs and their components is their hydrophobicity,which enables them to partition in the lipids of the bacterial cellmembrane and mitochondria, disturbing the structures andrendering them more permeable (Sikkema et al., 1994). Leakage ofions and other cell contents can then occur (Oosterhaven et al.,1995; Gustafson et al., 1998; Helander et al., 1998; Cox et al., 2000;Ultee et al., 2002).

3.3. Total coliforms

Fig. 4 shows the evolution during storage at 4 �C of viable cellconcentration of total coliforms in amaranth-based fresh pasta. Alsoin this case Equation (1) was fitted to the experimental data, and theresults are also listed inTable 1. Also for this microbial group chitosanloaded pasta had a MAL value noticeably higher than that of thecontrol samples, suggesting that the above natural polysaccharide

Fig. 3. Evolution during storage at 4 �C of psychrotrophic bacteria in packaged freshpasta with and without active compounds. The curves shown in the above figure wereobtained by fitting the Equation (1) or (2) to the experimental data. (B) (---) Cnt, (,)(d $ $ d) GFSE, (A) (dd) Thymol, (6) (d d) Chitosan, (:) (d $ d) Lemon extract.Data are the mean (n¼ 2)� standard deviation.

Page 5: Use of natural compounds to improve the microbial stability of Amaranth-based homemade fresh pasta

Fig. 4. Evolution during storage at 4 �C of total coliforms in packaged fresh pasta withand without active compounds. The curves shown in the above figure were obtainedby fitting the Equation (1) to the experimental data. (B) (---) Cnt, (,) (d $ $ d) GFSE,(A) (dd) Thymol, (6) (d d) Chitosan, (:) (d $ d) Lemon extract. Data are themean (n¼ 2)� standard deviation.

M.A. Del Nobile et al. / Food Microbiology 26 (2009) 151–156 155

strongly reduced the growth of total coliforms. These resultsconfirmed literature data dealing with the effectiveness of chitosanon coliforms (Darmadji and Izumimoto, 1994). As regards plantextracts, GFSE loaded pasta shows MAL value higher than thecontrols, but much lower than pasta with chitosan. The other twonatural substances investigated in this work did not show anyantimicrobial effect against total coliforms. In fact, no statisticallysignificant differences were observed with respect to the controlsamples. As a fact, components of EOs have been identified aseffective antibacterial agents with very low minimum inhibitoryconcentrations by in vitro test; however, higher concentration isneeded to achieve the same effect in foods (Burt, 2004).

3.4. Staphylococcus spp.

The Staphylococcus spp. cell growth cycle in pasta samples withand without antimicrobial compounds is shown in Fig. 5. As above,Equation (1) was fitted to the experimental data. The obtainedresults are also listed in Table 1. As can be seen, lemon extractloaded pasta did not differ statistically to the control samples,

Fig. 5. Evolution during storage at 4 �C of Staphylococcus spp. in packaged fresh pastawith and without active compounds. The curves shown in the above figurewere obtained by fitting the Equation (1) to the experimental data. (B) (---) Control,(,) (d $ $ d) GFSE, (A) (dd) Thymol, (6) (d d) Chitosan, (:) (d $ d) Lemonextract. Data are the mean (n¼ 2)� standard deviation.

whereas the other natural compounds showed a remarkable higherMAL value, if compared to pasta without preservatives. In partic-ular, chitosan had the highest MAL value among the testedcompounds, whereas GFSE and thymol showed a comparableantimicrobial effect (Burt, 2004), with microbial acceptability limitequal to 14 and 12 days, respectively.

3.5. Sensorial analysis

To the aim of assessing the influence of selected active compoundson the initial sensorial quality of amaranth-based pasta, sensorialanalysis was run on both uncooked and cooked fresh pasta. Panelistswere asked to detect differences between the control samples andthose containing active compounds. Results showed that verycomparable scores were recorded among the different samples foreach sensorial attribute taken into account, suggesting that theinvestigated active agents could be advantageously used to controlthe microbial quality without affecting the sensorial properties.

4. Conclusion

The antimicrobial activity of thymol, lemon extract, chitosanand GFSE loaded in amaranth-based homemade fresh pasta hasbeen tested against four spoilage microbial groups: mesophilic andpsychrotrophic bacteria, total coliforms and Staphylococcus spp.Results point out that chitosan is the most successful among theinvestigated compounds in slowing down the growth of the abovespoilage microorganisms, whereas lemon extract is the less effec-tive. In fact, MAL value as high as 25 days was obtained for chitosan-loaded pasta. GFSE is effective against all the above spoilagemicroorganisms, whereas thymol is able to delay the growth cycleof mesophilic and psychrotrophic bacteria and Staphylococcus spp.

Being some of the selected active compounds highly effectiveagainst the spoilage proliferation, without affecting the sensorialproperties of both fresh and cooked pasta, it is possible to assessthat the technique could be advantageously used to prolong theshelf life of amaranth-based homemade fresh pasta. Results alsohighlight that the above approach could be scaled up to a factorylevel and combined with other packaging techniques, such asmodified atmosphere packaging, to reach higher shelf life.

Acknowledgements

This work was financially supported by Ministero dell’Economiae delle Finanze, Ministero dell’Istruzione, dell’Universita e dellaRicerca Scientifica e Tecnologica e l’Assessorato Bilancio e Pro-grammazione Regione Puglia by the programme ‘‘Accordo di Pro-gramma Quadro in Materia di Ricerca Scientifica della RegionePuglia – Progetto Esplorativo – Title: Innovazione di processo per laproduzione di paste funzionali’’.

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