MEAT SCIENCE

Meat Science 71 (2005) 249255 www.elsevier.com/locate/meatsci

Microbiological and sensory changes in Morcilla de Burgos preserved in air, vacuum and modied atmosphere packaging
lez-Ferna ndez a, Eva M. Santos b, Ana M. Diez a, Consuelo Gonza a a,* Isabel Jaime , Jordi Rovira
a

Department of Biotechnology and Food Science, University of Burgos, Pza Misael Ban uelos s/n, 09001 Burgos, Spain b micas, Universidad Auto noma del Estado de Hidalgo, Unidad Universitaria, Centro de investigaciones Qu Crta Pachuca-Tulancingo Km 4.5. 42076 Pachuca, Hidalgo Received 24 July 2004; received in revised form 16 March 2005; accepted 19 March 2005

Abstract Morcilla de Burgos is the most popular blood sausage in Spain. Traditionally, this product is distributed and sold without packaging in the local market. To extend its shelf-life and expand the market, dierent packaging methods have been employed and compared: morcilla stored in air (without packaging), in vacuum and in modied atmosphere packaging (MAP) using three dierent CO2 concentrations 30%, 50% and 80% and balanced with N2. Total viable count (TVC), psychrotrophs, lactic acid bacteria (LAB), pseudomonads, enterobacteria, moulds and yeasts, enterococci, Staphylococcus aureus, and sulte reducing clostridia were analysed during storage at 4 C. Sulte-reducing clostridia, pathogenic staphylococci, and enterococci were not detected in any sample. In air-stored morcilla a signicant increase in all microbial groups was observed during storage. Pseudomonads were the predominant microorganisms reaching a population higher than 8 log cfu/g after 27 days of storage. On the other hand, a decrease in pH was noticed in MAP and in vacuum packaged morcilla (pH 4.73) during storage. At the same time, LAB becomes the predominant species in all these packaged samples. The rest of the microbiota did not grow during storage. In morcilla packed with 50% and 80% of CO2, counts of pseudomonads and enterobacteria were lower than found in the vacuum packs. Sensory analysis showed that shelf-life of morcilla stored in air did not exceed 17 days, while samples packed under vacuum and with 30% CO2 were acceptable until 22 days of storage. Morcillas packaged with 50% and 80% CO2 were sensorially acceptable for 32 days. 2005 Elsevier Ltd. All rights reserved.
Keywords: Morcilla; Blood sausage; Vacuum packaging; Modied atmosphere packaging; Lactic acid bacteria

1. Introduction Morcilla de Burgos is a popular cooked blood sausage produced in the region around Burgos, in the north of Spain. It is made of onion, rice (sometimes precooked), animal fat (mainly lard and tallow), blood and dierent spices according to the local procedure, stued in natural pork or beef casings and boiled for

Corresponding author. Tel.: +34 947 258814; fax: +34 947 258831. E-mail address: jrovira@ubu.es (J. Rovira).

about 1 h at 9095 C. The physicochemical and sensory characteristics of this product have been previously de lez-Ferna ndez, Jaime, & Rovira, scribed (Santos, Gonza 2003). Nowadays, producers of morcilla are interested in extending the shelf-life of this product in order to increase the potential market and satisfy consumer demands. Modication of the extrinsic parameters of the product ecosystem, like temperature and gaseous atmosphere, is a common practice compared with the use of chemical preservatives. Vacuum packaging has so far been the most widely used packaging technique for

0309-1740/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.03.028

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cooked products (Borch, Kant-Muermans, & Blixt, 1996; Korkeala, Lindroth, Suihko, Kuhmonen, & Penttila , 1985; Samelis, Kakouri, & Rementzis, 2000). However, it presents some disadvantages, such as liquid exudation and deformation of the products. Because of this, modied atmosphere packaging (MAP) has become more common and applied to dierent cooked meat products (Ahvenainen, Kivikataja, & Skytta , 1990; Hitlian & Hotchkiss, 1987; Pexara, Metaxopoulos, & Drosinos, 2002). However, ambiguous results have been obtained with regard to the longer shelf-life of MAP meat products comparing to vacuum packed ones (Blickstad & Molin, 1983; Pexara et al., 2002; Samelis & Georgiadou, 2000). CO2/N2 atmospheres are particularly suitable for the preservation of cooked and cured meat products, mainly because of the strong inhibition of growth of the great majority of microorganisms (Borch et al., 1996). Blood sausages are traditional meat products and their consumption is quite popular in many European countries, although the ingredients used dier according to the producing region. Few data are available concerning to the packaging of these products and their sensory and microbiological quality during storage (Korkeala et al., 1985). The aim of the present work was to study the microbiological and sensory quality of morcilla de Burgos preserved in air, vacuum and modied atmosphere packaging during storage at 4 C.

ples were placed in oxygen and water impermeable bags CN300 (Cryovac Grace S.A, Sealed Air Corporation, Barcelona, Spain) with an oxygen transmission rate of 15 cm3/m2/24 h/atm at 23 C and 0% RH and a water vapour transmission rate of 2 g/m2/24 h at 38 C and 90% RH. One of the batches was vacuum packed in an EVT-7CD packaging machine (Tecnotrip, Tarrasa, Spain). The remaining three groups were back ushed in the same equipment connected to a three gas component mixer (WITT-Gasetechnik, Witten, Germany) with the following atmospheres 30% CO2/70% N2 (30/70), 50% CO2/50% N2 (50/50) and 80% CO2/20% N2 (80/ 20). A headspace of 34 l/kg of sample was provided in order to avoid packaging collapse due to the decrease of CO2 concentration during storage (Gill & Penney, 1988; McMullen & Stiles, 1991). Packages were stored in a refrigerated incubator at 4 1 C. The experiment was repeated three times. 2.3. Sampling procedure For each experiment 124 samples were used. Two morcillas were taken directly after cooking for microbiological analysis and pH measurement (day 0), while the rest followed the cooling process. On day 1, two morcillas were also randomly selected for microbiological analysis and pH measurement before packaging. During storage two packages from each trial (air, vacuum and MAP 30/70, 50/50 and 80/20) were randomly selected for the pH, microbiological and sensory analyses every ve days from the 2nd to the 47th day (12 analyses per trial). 2.4. Analysis of gas composition

2. Materials and methods 2.1. Samples and processing of the sausages Morcillas stued in natural beef casings made by one producer were selected for the dierent preservation experiments. The formulation included raw onion (55%), rice (18%), lard (18%), blood (9%), salt and different spices (paprika, black pepper and oregano). The chopped onion and fat were mixed with the rice, salt, spices and blood and the sausage emulsion was stued into 3545 mm natural beef casings that were preserved with salt and rinsed in clear water prior to use. No nitrite was added to the formulation. The blood sausages were then transferred to a cooking vessel and boiled in water at 9596 C for around 1 h (day 0). After cooking, morcillas were air cooled overnight at room temperature (810 C). 2.2. Packaging of the products On day 1, samples made the previous day were individually packed according to the preservation procedure and transported under refrigeration to the laboratory. Samples were divided into ve batches, being one paper wrapped (air storage control), while the rest of the sam-

Oxygen and carbon dioxide concentrations in the headspace gases of the morcilla packages, except the air and vacuum packages, were measured with a digital O2 and CO2 analyser Combi check 9800-1 (PBI Dansensor A/S, Ringsted, Denmark). Thirty millilitre gas samples were drawn by the needle of the analyser through a septum glued onto the surface of the pack. Detection limits for O2 and CO2 were 0.01% and 0.1%, respectively. 2.5. pH measurement pH was measured by blending 25 g of product with 225 ml of distilled water for 2 min. A digital pH-meter Micro pH 2000 (Crison, Barcelona, Spain) was used for the measurement. 2.6. Microbial analyses Twenty-ve gram slices of morcilla (including the skin) were taken aseptically and homogenised with 225

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ml of sterile Ringers solution (Oxoid, Basingstoke, UK) for 2 min in a sterile plastic bag in a lab blender (Stomacher 400, Seward, London, UK). Serial decimal dilutions in sterile Ringers solution (Oxoid) were prepared and duplicate 1 ml or 0.1 ml samples of appropriate dilutions were poured or spread onto total count and selective agar plates. Total viable count (TVC) was determined on Plate Count Agar (PCA, Oxoid), incubated at 30 C for 48 h; psychrotrophs were determined on PCA but incubated at 7 C for 10 days; lactic acid bacteria (LAB) on MRS Agar (Oxoid), incubated anaerobically in 6% CO2, at 30 C for 23 days; pseudomonads on Pseudomonads Agar Base (Oxoid) with CFC supplement (Oxoid), incubated at 25 C for 48 h; enterobacteria on Violet Red Bile Glucose agar (VRBGA, Oxoid), overlayed with 5 ml of the same medium and incubated at 37 C for 24 h; yeasts and molds on Sabouraud Chloramphenicol Agar (Biokar Diagnostics, Beauvais, France), incubated at 25 C for 5 days; enterococci on Slanetz and Bartley Medium (Oxoid), incubated at 37 C for 48 h; Baird Parker microbiota on Baird-Parker Agar Base (Oxoid) with Egg Yolk Tellurite Emulsion (Oxoid), incubated at 37 C for 48 h, sulte-reducing clostridia in tubes containing 20 ml of melted TSN Agar (Tryptone Sulte Neomycin, Biokar), inoculated with 1 ml from the rst dilution, cooled rapidly in ice-water and overlayed with 2 ml of sterile medium to exclude oxygen, and incubated at 37 C for 24 h. Suspect black colonies from Baird Parker agar were tested for positive coagulase reaction (Difco, Michigan, USA). For experimental purposes, the lowest detection limit of the above techniques was 102 cfu/g except for total viable count, psychrotrophs, enterobacteria and sultereducing clostridia whose limit was 10 cfu/g.

External appearance, slice appearance, odour and taste were scored on a 5-point hedonic scale as follows: 5 = excellent, 4 = good, 3 = acceptable, 2 = fair and 1 = unacceptable (Kotzekidou & Bloukas, 1996). When low scores were given the reason had to be stated. On the other hand, for o odours, 1 corresponded to absence or minimum intensity and 5 to maximum intensity of each parameter. 2.8. Statistical analyses Microbial, sensory and pH data were statistically analysed using ANOVA procedures. Where appropriate, means were ranked using the Least Signicance Difference test (LSD0.05). Data analyses were conducted using the statistical package Statgraphics Plus for Windows ver. 4.0.

3. Results The CO2 concentrations of all gas mixtures decreased by 34% during storage, due to the solubilisation of CO2 in the product (data no shown). Initially headspace oxygen concentrations of the CO2/N2 mixtures were below 0.7 % on day 1 and gradually increased to values approaching 1.3% at the end of the study. The mean initial pH of the morcillas before packaging was 6.31 0.05 and it signicantly decreased (P < 0.05) with storage, regardless of the packaging method, but being more rapid in samples packed under vacuum (Fig. 1). Morcillas stored in air showed the least decrease during storage, reaching a pH of 5.73 at day 47. The pH of the MAP products decreased to values around

6.5

2.7. Sensory evaluation Sensory analysis was performed in two sessions by a ve-member trained sensory panel. Two open-discussion sessions and 10 training sessions were held to familiarise the judges with the attributes to be evaluated and the scale to use, respectively. Evaluation was always done with regard to initial characteristics of the fresh product prior to packing. Sensory analysis was omitted, when the product was considered unt for consumption. External appearance of the unopened packages and o-odours during the opening of the pack were evaluated in the rst block. For the second block, when microbiological samples were taken, morcillas were cut in 1-cm thick slices and evaluated at room temperature for slice appearance, and odour in randomised order. Then the samples were heated in a microwave (Panasonic, Matsushita Electric, UK) to a core temperature of 7075 C and assessed for taste.
6

5.5 pH 5 4.5 4 0 5 10 15 20 25 30 Storage time 35 40 45 50

Fig. 1. Mean pH values of morcillas stored in air (r), under vacuum (j) or under modied atmosphere containing 30% CO2 (e), 50% CO2 (h) and 80% CO2 (n).

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5.40 and no dierences occurred in pH between the three gas mixtures (P > 0.05) during storage. Sulte-reducing clostridia, pathogenic staphylococci and enterococci were not detected in any sample. Fig. 2 shows the microbiological results of air-stored and vacuum packaged morcilla during storage, and Fig. 3 shows the TVC and LAB counts from the air, vacuum and MAP packed samples. Both gures show the trends of the dierent microbial populations analysed during storage, it must be taken into account that at each sampling, two dierent packages were analysed, and each datum shows the average of three experiments. Growth on TVC plates were the only counts detected on day 0, while the rest of microbial populations examined were absent. However, on the day of packaging (day 1) the

9 8 7 6
Lo g cfu/g

5 4 3 2 1 0 0 5 10 15 20 25 30 35 Storage time (days) 40 45 50

a
9 8 7 6

9 8 7 6
Lo g cfu/g

Log cfu/g

5 4 3

5 4 3 2

1
1

0
0 0 5 10 15 20 25 30 35 40 45 50

0
b

10

15

20 25 30 35 Storage time (days)

40

45

50

Storage time (days) 9 8 7 6

Fig. 3. Development of TVC (a) and LAB (b) counts on morcillas stored at 4 C in air (r), under vacuum (j) or under modied atmosphere containing 30% CO2 (e), 50% CO2 (h) and 80% CO2 (n).

5 4 3 2 1 0 0 5 10 15

20 25 30 35 Storage time (days)

40

45

50

Fig. 2. Development of TVC (r), LAB (j), pseudomonads (m), psychrotrophs (e), enterobacteria (h), yeast and moulds (n) on morcillas stored in air (a) and under vacuum (b).

population of LAB, pseudomonads, yeast and moulds was about 3 log cfu/g, while enterobacteria counts reached 1 log cfu/g. A signicant increase in these groups was observed during storage of the air-stored samples (P < 0.05). After 27 days, the number of pseudomonads was higher than 8 log cfu/g and slightly higher than counts for LAB, yeast and moulds. Growth of enterobacteria signicantly increased but at a much slower rate (P < 0.05) than the other microbial groups, reaching values of 5 log cfu/g on day 49. On the other hand, a considerable increase in the TVC, psychrotrophs and LAB counts was observed in vacuum, the numbers being similar after seven days while other bacteria tested were restricted (Fig. 2). In the case of MAP packaged samples, LAB again grew rapidly, but numbers were less than those found in the vacuum packaged samples. No dierence in the growth rates of LAB was found (P > 0.05) between the three gas

Lo g cfu/g

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mixtures, although LAB counts, in packages with 30% CO2, were slightly higher than the counts found in samples packaged under 50% and 80% CO2 (Fig. 3). The rest of the microbiological groups showed a similar behaviour to that described for the vacuum packaged morcillas although pseudomonads and enterobacteria counts were less than the numbers found in the vacuum packs, especially in morcillas packaged under 50% and 80% of CO2, where enterobacteria were very low. The method of packaging and storage time had signicant eects (P < 0.05) on sensory properties. All sensory parameters dramatically decreased during storage, except o-odours, which increased, especially in vacuum packaged morcilla (Table 1). In general, morcillas stored in 50% and 80% CO2 presented better scores than morcillas vacuum-packaged and packaged under 30% CO2 and these presented better (P < 0.05) scores than those stored in air. In the case of morcillas stored in air, the appearance was not acceptable after 12 days due to the drying

surface, and after 17 days the other sensory attributes reached values below the acceptable level (less than 3 on the hedonic scale). At this point, the judges detected visible mouldiness, slightly putrid aroma, mouldy taste and dehydration. On the contrary, vacuum and modied atmosphere packaging led to a very sour odour on opening the bags and a sour odour and taste of the samples. These eects were more intense in vacuum packaged morcillas than in MAP samples (P < 0.05). In fact, the appearance of the vacuum packed samples and those with 30% CO2 was below the acceptability level after 22 and 17 days, respectively, because of vacuum loss, blowing of certain packs, and the presence of milky exudates and slime formation in the case of vacuum samples, and microbial colony spots on the surface of the gas packaged products as well as the presence of slime. Samples packed in 50% and 80% CO2 presented sporadically visible microbial colonies from the 27th day, although the appearance of sausages packaged under 80% CO2 was

Table 1 Eect of packaging type and storage time on dierent sensory parameters Sensory parameter Time storage 2 Appearance Air Vacuum 30/70 50/50 80/20 O-odours Air Vacuum 30/70 50/50 80/20 Slice appearance Air Vacuum 30/70 50/50 80/20 Odour Air Vacuum 30/70 50/50 80/20 Taste Air Vacuum 30/70 50/50 80/20
af A A

7 3.41c 4.27e B 4.88c B 4.50f B 4.88d

B A

12 2.45b 3.52d C 4.75c C 4.75f C 4.75cd

B A

17 2.05b 3.35cd A 2.63b BC 3.75e C 4.13c

B A

22
A B

27 1.00a 2.50b BC 2.63b B 2.50c C 3.13b

B A

32 NT A 1.75a A 2.13ab A 1.88b B 3.13b NT C 4.75e AB 2.25b B 2.50b A 1.75a NT A 3.07a A 3.50bc A 3.10bc A 3.10ab NT A 1.32a B 2.20ab B 2.60abc B 2.60ab NT A 1.50a B 2.40a B 3.00bc B 2.80b

37 NT NT A 1.88a A 2.13bc A 2.13a NT NT AB 2.63b A 1.75a B 3.13b NT NT A 2.60a A 2.90ab A 2.80a NT NT A 2.40ab A 2.40ab A 2.50a NT NT A 2.67a A 2.50ab A 2.67b

42 NT NT AB 1.75a A 1.00a B 1.88a NT NT A 3.63c A 2.88b A 3.88b NT NT A 2.50a A 2.40a A 2.80a NT NT A 1.80a A 1.90a A 2.20a NT NT A 2.20a A 2.10a A 1.80a

4.46d 4.54e A 5.00c A 4.88f A 4.75cd NT A 1.00a A 1.00a A 1.00a A 1.00a

A A

1.05a 2.86bc B 2.50b B 3.00d B 3.00b

NT A 1.39a A 1.00a A 1.00a A 1.00a

A A

NT A 1.67b A 1.00a A 1.00a A 1.00a 3.29c 3.93bc AB 4.20cd B 4.50d AB 4.10c

AB A

NT B 2.85c A 1.00a A 1.00a A 1.00a 2.54b 3.64b B 4.00cd B 3.60c B 3.70bc

B A

NT 3.91d B 2.13b A 1.50a AB 1.75a 2.20b 2.90a B 3.50bc B 3.60c B 3.40ab

B A

NT B 4.64e A 2.25b A 1.75a A 1.88a 1.33a 2.96a B 3.00ab B 3.13bc B 3.25ab

B A

5.00d 5.00d A 4.75d A 5.00d A 5.00d

A A

4.29d 4.14c A 4.50d A 4.70d A 4.20c

A A

5.00e 5.00d A 5.00f A 5.00f A 5.00f

A A

4.21d 4.43cd A 4.80ef A 4.70ef A 4.70ef

AB A

3.64c 4.07c B 4.20de B 4.30ef B 4.10de

B

2.79b 3.07b B 4.10d B 4.00de B 4.10de

A A B

2.20a 1.67a C 3.10c C 3.30cd C 3.60cd

A

1.89a 1.46a B 2.75bc B 3.13bc B 3.25bc

5.00c 5.00f A 5.00b A 5.00f A 5.00f

4.25c 4.04e AB 4.60b B 4.80f AB 4.50ef

3.57b 3.89de A 4.20b A 4.20e A 4.20e

A

2.57a 3.50d B 4.20b B 3.80de B 4.00de

2.45a 2.87c AB 2.90a B 3.40cd B 3.40c

AB

NT A 2.18b A 2.50a B 3.50cd B 3.50cd

NT: not tested. Averages with dierent letter in the same row are dierent (P < 0.05). AD Averages with dierent letter in the same column are dierent (P < 0.05).

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acceptable until the 32nd day. Strong sour odours and tastes, as well as distinctive o-odours, when the packages were opened, were apparent from the 22nd day in the vacuum packages and those packages with 30% CO2. These eects were clearly seen from the 32nd day in morcillas packaged under 50% and 80% CO2. In general no signicant dierences were found for the above-mentioned parameters between sausages stored in 50% and 80% CO2.

dictory results could also be related to the gas/volume ratio and product composition. In our case, it seems that high CO2 concentrations, far from aecting the sensory characteristics and produce discolouration, sour odour and taste and exudate increase (Ahvenainen et al., 1990; Borch et al., 1996), extends the shelf-life of morcilla as a result of the inhibition of spoilage LAB, without aecting sensory properties. In this sense, MAP is more eective in retarding microbial growth and concentrations of 50% of CO2 or higher should be recommended to preserve morcilla de Burgos for longer periods.

4. Discussion The high temperature cooking during processing eliminates the vegetative forms of microorganisms and only bacterial spores survive the treatment. However, handling of the product during cooling leads to postcooking contamination by other bacteria on the surface of the product (Korkeala, Lindroth, Ahvenainen, & Alanko, 1987; Ma kela & Korkeala, 1987). When morcilla is packaged under vacuum or modied atmosphere the typical spoilage microora of aerobic storage, predominantly pseudomonads, is replaced by growth of lactic acid bacteria initially deposited during cooling and manipulation. LAB are responsible for the decrease of pH and sensory spoilage (Blickstad & Molin, 1983; Borch et al., 1996; Korkeala & Bjo rkroth, 1997; Korkeala et al., 1985, 1987; Samelis et al., 2000; von Holy, Holzapfel, & Dykes, 1992). Besides the inhibitory eect on pseudomonads, enterobacteria and moulds, carbon dioxide also delayed the growth of LAB, which are composed mainly of heterofermentative LAB belonging to Weissella viridescens, Leuconostoc mesenteroides and Leuconostoc carnosum species (Santos et al., 2005). This eect was more noticeable in morcillas packaged under 50% and 80% of CO2, which could explain the better sensory scores for the product packaged under both treatments. This is in agreement with the results of Blickstad, Enfors, and Molin (1981), Blickstad and Molin (1983), Gill and Penney (1988), Jeremiah, Gibson, and Argnosa (1994) Bell, Penney, Gilbert, Moorhead, and Scott (1995) where the inhibitory eect of CO2 was seen to aect Gram-positive bacteria although to a lesser extent than Gram-negative bacteria. On the contrary, Samelis and Georgiadou (2000) reported that CO2 packaging was ineective in extending the shelf-life of Greek taverna sausage compared to vacuum packaging, however, Lactobacillus sakei/Lactobacillus curvatus was the predominant spoilage population, which has been reported as being less inhibited by high CO2 concentrations. In the work of Pexara et al. (2002) modied atmosphere packaging did not retard the growth rate of LAB although; in this case the spoilage species belonged to Lactobacillus and Leuconostoc genus. Besides the dierent composition of the spoilage population, the contraAcknowledgements This project was nancially supported by Excma. n de Burgos, the Asociacio n de Fabricantes Diputacio n rede Morcilla de Burgos, and Junta de Castilla y Leo search Project BU 19/02.

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