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Novel combinations of chitosan, carnocin and sulphite for the preservation of chilled pork sausages
Meat Science 62 (2002) 165–177 www.elsevier.com/locate/meatsci
Novel combinations of chitosan, carnocin and sulphite for the preservation of chilled pork sausages S. Rollera,*, S. Sagooa, R. Boarda, T. O’Mahonyb, E. Capliceb, G. Fitzgeraldb, M. Fogdenc, M. Owenc, H. Fletcherc a School of Applied Science, South Bank University, 103 Borough Road, London SE1 0AA, UK National Food Biotechnology Centre and Department of Microbiology, University College Cork, Western Road, Cork, Ireland c Meat and Livestock Commission, PO Box 44, Winterhill House, Snowdon Drive, Milton Keynes MK6 1AX, UK
b
Received 20 August 2001; accepted 20 November 2001
Abstract The aim of this study was to develop novel preservation systems for fresh pork sausages based on combinations of chitosan (polymeric b-1,4-N-acetylglucosamine) carnocin (a bacteriocin produced by Carnobacterium piscicola) and low concentrations of sulphite. Two pilot-scale trials showed that 0.6% chitosan combined with low sulphite (170 ppm) retarded the growth of spoilage organisms more effectively (3–4 log cfu/g) than high levels (340 ppm) of sulphite alone at 4 C for up to 24 days. Microbial counts for frozen sausages showed that the preservative efficacy of the chitosan/sulphite combination was maintained following frozen storage. Carnocin did not protect sausages from spoilage but in a challenge trial, it reduced the numbers of Listeria innocua by up to 2.0 log cfu/g in the first 5 days of chill-storage. Sulphite was degraded rapidly within the first 3 days of storage in all the sausages that contained only this preservative but levels decreased less rapidly and persisted for longer in the presence of chitosan. Results of Quantitative Descriptive Analysis using 31 trained panellists reflected the gradual deterioration of all the sausages during storage. The batch containing chitosan and sulphite deteriorated less rapidly and was judged to be more acceptable for a longer period than all the other batches. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pork sausages; Chitosan; Carnocin; Sulphite; Bacteriocin; Preservation; Meat spoilage
1. Introduction British pork sausages contain at least 65% meat (determined by UK legislation), fat, rusk (a type of breadcrumb) and spices in a collagen casing. In the UK, sodium metabisulphite is used to inhibit microbial spoilage of raw sausages and to achieve a shelf life of about 15 days at chill temperatures from the day of manufacture (Dowdell & Board, 1971; Surkiewicz, Johnston, Elliott, & Simmons, 1972). Sulphite is an elective agent; it inhibits the growth of Gram-negative bacteria and selects a flora that is fermentative in character and includes both Gram-positive bacteria and yeasts (Banks, Dalton, Nychas, & Board, 1985). The latter are responsible for producing acetaldehyde, which binds sulphite and causes
* Corresponding author. Tel. +44-207-815-7961; fax: +44-207815-7999. E-mail address: [email protected] (S. Roller).
loss of antimicrobial activity (reviewed in Banks, Nychas, & Board, 1987). Sulphites have a long history of safe use in meat products. Exposure to sulphites (although not specifically via foods preserved with them) has been linked, however, with the exacerbation of asthmatic and other respiratory conditions in some sensitive individuals (reviewed in Simon, 1998; BIBRA, 1999). In 1995, an EC Council Directive was issued specifying a detailed list of foods to which sulphites can be added and setting maximum permitted levels of addition. In the USA, sulphiting agents are not permitted in meats or in foods recognised as a source of Vitamin B1 and since 1986 have been banned from use in fresh fruit and vegetables (Anon., 1986). Some consumers regard the deliberate addition of sulphites and/or any other chemical preservatives to foods as a form of adulteration. Yet, a low level of sulphite is required to give fresh pork sausages the ‘‘bloom’’ (pink colouring) that is attractive to the purchaser. There is thus a clear need to develop novel preservation systems
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00243-1
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based on low levels of sulphite (to inhibit Gram-negative bacteria and maintain desirable sensorial properties) together with an additional preservative that is active against yeasts and Gram-positive bacteria. Chitosan, a deacetylated form of chitin, is a polysaccharide found in the shells of crabs and shrimp. Chitosan consists of polymeric 1,4-linked 2-amino-2deoxy-b-D-glucose. A number of applications for chitosan have been proposed including heavy metal chelation in wastewater treatment, as a hypocholesterolemic agent, and as dietary fibre (Shahidi, Arachchi, & Jeon, 1999). As chitosan exhibits antimicrobial activity against a range of foodborne microorganisms in the laboratory, it has attracted attention as a potential food preservative of natural origin (Roller and Covill, 1999, 2000; Rhoades & Roller, 2000; Tsai, Wu, & Su, 2000; Shahidi et al., 1999). The reported Minimimum Inhibitory Concentrations (MICs) vary widely from 0.01 to 1.0% and yeasts tend to be more sensitive than bacteria (Knowles & Roller, 2001; Roller & Covill, 2000; Rhoades & Roller, 2000; Seo, Mitsuhashi, & Tanibe, 1992; Sudarshan, Hoover, & Knorr, 1992; Tsai & Su, 1999; Tsai et al., 2000; Wang, 1992). Antimicrobial efficacy demonstrated in the laboratory is not always realized in foods due to the highly reactive nature of the polycationic chitosan, which interacts readily with proteins, fats and other anionic substances in foods (Rhoades & Roller, 2000). Nevertheless, some successful applications, especially against spoilage yeasts in juices and emulsified sauces, have been reported (Roller & Covill, 1999, 2000). In meat products, slight inhibition ( 1–2 log cfu/g) of total microbial growth in refrigerated beef patties has been reported in the presence of 1.0% but not 0.5 or 0.2% chitosan (Darmadji & Izumimoto, 1994). Bacteriocins produced by lactic acid bacteria, including pediocin, sakacin and nisin, are naturally occurring antimicrobial peptides that inhibit a range of Grampositive foodborne pathogens and spoilage organisms (Jack, Tagg, & Ray, 1995). The genetic determinants of bacteriocin production, their mode of action and efficacy in laboratory systems have been studied extensively in the last 15 years (reviewed by de Vuyst & Vandamme, 1993; Ennahar, Sashihara, Sonomoto, & Ishizaki, 2000; Hill, 1995; Holz & Stahl, 1995; Hoover & Steenson, 1993; McAuliffe, Ross, & Hill, 2001; Ray & Daeschel, 1994). The antilisterial activity of many bacteriocins is well-known (Ennahar, Deschamps, & Richard, 2000; Leroy & de Vuyst, 2000; Ray & Miller, 2000). Although these biopreservatives have the potential to protect some foods from spoilage, their application in raw or processed meat products is limited because binding with meat particles and fat may cause loss of activity (Aymerich, Artigas, Garriga, Monfort, & Hugas, 2000; Yang & Ray 1994). Nisin has nevertheless been approved for use in processed meat products such as vacuum-packed frankfurters in which spoilage by lactic
acid bacteria can reduce shelf life. A number of investigators have reported the use of bacteriocin-producers in starter cultures for fermented sausages (reviewed in Wessels, Jelle, & Ness, 1998). Preliminary studies have indicated that chitosan may act synergistically with sodium metabisulphite against Lactobacillus viridescens, a common cause of spoilage of cured meat products (Roller, 2001). The bacteriocin carnocin 124, produced by a meat isolate, Carnobacterium piscicola, has been purified to homogeneity and its biochemical and antimicrobial properties have been characterised (O’Mahony, 2001). The main aim of this study was to assess the feasibility of using chitosan and carnocin in combination with low concentrations of sulphite to control the microbial spoilage of fresh pork sausage.
2. Materials and methods 2.1. Chemicals Chitosan glutamate was obtained from Pronova (Drammen, Norway). It contained 42% glutamate and had a deacetylation range of 75–85% (manufacturer’s data). All microbiological media and diluents were from Oxoid (Basingstoke, UK). Sodium metabisulphite was from Fisher Scientific (Loughborough, UK). All other chemicals were from Sigma Chemical Co (Poole, Dorset, UK), unless otherwise indicated. 2.2. Bacteriocin production and determination of antilisterial activity Carnobacterium piscicola NFBC124, isolated from a chicken portion (Vaughan et al., 1994), was used. It produced the antimicrobial peptide carnocin 124, which is virtually identical to piscicolin 126 (Fitzgerald, Harmark, Selleck, Hillier, & Davidson, 1999; Jack et al., 1996; O’Mahony, 2001). Bacteriocin concentration was determined by the spot assay method of Hoover and Steenson (1993). Serial two-fold dilutions of cell-free supernatants from a culture of the producer organism were spotted in 5 ml aliquots onto a Tryptone Soya Agar/ 0.5% yeast extract plate (TSAYE) previously overlaid with 5 ml of TSAYE /0.75% agar containing 5 105 cfu/ml of Listeria innocua CNRZ 4202 and incubated at 30 C for 2 days. Arbitrary units per millilitre (AU/ml) were calculated as the reciprocal of the highest dilution that gave a clear zone of inhibition on the indicator lawn. A bacteriocin-negative variant of C. piscicola was isolated by repeated sub-culturing of the producer strain in TSBYE until spot-testing of filtered supernatant from an overnight culture confirmed that a bacteriocin-negative phenotype had been selected. API CH50 kits (BioMerieux, France) were used to confirm that the fermentation profile of the bacteriocin-negative
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variant was the same as that of the parent strain. The production of carnocin could be induced in the variant by the inclusion of 0.2% cell free supernatant from the producer strain, as shown previously (O’Mahony, 2001; Saucier, Poon, & Stiles, 1995). 2.3. Production of fermented milk powder containing bacteriocin Fermented milk powders were produced on a pilot scale from 50 l of 10% Reconstituted Milk Powder supplemented with 0.5% yeast extract (RSMYE). The medium was adjusted to pH 8 before sterilization. The bacteriocin-producing and non-producing variant of C. piscicola were grown in deMan Rogosa and Sharpe broth (MRSB, adjusted to pH 7.5 with 10M NaOH) at 30 C for 18 h and were added to the RSMYE at a level of 1%. The fermentation (30 C) was allowed to proceed for approximately 24 h until the pH reached 5.6. The fermentate was stored for 24 h at 4 C; during this period it was tested for bacteriocin activity as described above. Upon confirmation of either presence or absence of antilisterial activity, the fermentate was flash pasteurized at 75 C for 15 s and subsequently evaporated under vacuum at 60 C until the total solids reached 25%. The condensate was spray dried (inlet temperature 85 C, outlet temperature 185 C). A sample of the resultant powder was resuspended in sterile distilled water and tested again for antimicrobial activity and pH. The powder was stored at 4 C and checked periodically for antilisterial activity.
in fresh pork sausages. In both, the sausages were prepared according to a formulation and method supplied by the Meat and Livestock Commission (MLC), UK. In Trial 1, eight batches, each of 10 kg of sausages were prepared according to the formulations given in Table 1. The pork and fat were minced through a 5-mm plate. The dry ingredients (rusk, seasoning, skim milk powder, chitosan and/or sodium metabisulphite, as appropriate) were mixed separately and added to the meat mixture with continuous mixing. During mixing, chilled water was added slowly to the meat mixture until absorbed. The mixture was minced again through the 5-mm plate and mixed until even distribution achieved. The sausage mix was filled into Devro small diameter casing to make sausages of 56 g each. Four sausages ( 225 g) were placed into a cellophane-wrapped pack. A 10-kg batch yielded approximately 44 packs of sausages. Microbiological analyses (see later) were done on sausages stored at 4 C for 24 days. Batches of defrosted frozen sausages from Trial 1 were used to train the sensory panel (see later). In the second trial, carried out essentially as described earlier, 15-kg batches of sausages were prepared according to the formulations shown for the first five batches in Table 1 except that the fermented milk powder was replaced by a commercial skim milk powder. The sulphite content of the sausages was also determined periodically by the Monier Williams/HPLC method (Banks et al., 1987; Monier-Williams, 1927). Samples of sausages from Trial 2 were frozen and defrosted as required for sensory analysis (see later). 2.5. Sausage sampling method and microbiological analyses
2.4. Chitosan, carnocin and sulphite combinations in fresh pork sausages Two trials were carried out to test the preservative efficacy of chitosan, carnocin and sulphite combinations
The Public Health Laboratory Service (UK) method (Roberts, Hopper, & Greenwood, 1995) was used to determine the size and composition of the microbial
Table 1 Chitosan, sulphite and carnocin content of fresh pork sausages prepared in Trials 1 and 2 Batch No. and Name
1 Control 2 High sulphite 3 Low sulphite 4 Chitosan 5 Chitosan+low sulphite 6 Carnocin 7 Carnocin+chitosan 8 Carnocin+low sulphite a
Content (%)b Sodium metabisulphite (final sulphite conc.)
Chitosan glutamate
Carnocin-negative skim milk powdera
Carnocin-positive skim milk powder (final carnocin conc.)
0 0.045 (340 ppm) 0.0225 (170 ppm) 0 0.0225 (170 ppm) 0 0 0.0225 (170 ppm)
0 0 0 0.6 0.6 0 0.6 0
2.0 2.0 2.0 2.0 2.0 0 0 0
0 0 0 0 0 2.0 (320 AU/g) 2.0 (320 AU/g) 2.0 (320 AU/g)
Carnocin-negative fermented skim-milk powder was used in all Batches in Trial 1 and in Batches 6–8 in Trial 2. The fermented powder was replaced by a commercial skim milk powder in Batches 1–5 in Trial 2. b All sausages contained 42.5% lean pork, 25.0% pork fat, 8.0% bland rusk (RHM), 2.5% seasoning 40910 (containing 60% salt, Kerry Ingredients) and 20.0% chilled tap water.
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flora of sausages. Samples (25 g) were taken from two sausages in each pack of four. Each sausage was sliced transversely into several sections; the end sections were discarded. Several slices (casing was not removed) were selected randomly from the middle and end to make up 25 g. The 25-g sample was added to 225 ml of Maximum Recovery Diluent (MRD) and homogenised with a stomacher for 2 min. A sample (1 ml) of the slurry was diluted serially in MRD in duplicate and appropriate dilutions were spread-plated (0.1 ml in duplicate) on the following: Plate Count Agar (PCA) for total viable count (incubated at 30 C for 2 days); deMan Rogosa Sharpe Agar (MRSA) for lactic acid bacteria (30 C for 3 days); Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (25 C for 5 days) and Violet Red Bile Glucose Agar (VRBGA) for enteric organisms (37 C for 2 days). In the second trial, 1 ml of a 1:10 sausage slurry was used to prepare pour plates of VRBGA in order to increase the sensitivity limit of the method. The oxidase test and Gram-staining of selected colonies from PCA were done according to Roberts et al. (1995). For pH determination, a further 25 g of sausage was homogenised in 225 ml of distilled water for 2 min and the pH of the slurry measured. 2.6. Challenge trial with Listeria innocua in carnocincontaining sausage mix Additional mixtures of sausage meat were manufactured using the formulation for Batch 1 (Control, no preservative added) and Batch 6 (Carnocin) in Table 1. Listeria innocua CNRZ 4202 was added to the sausage mix at a level of 5.5 105 cfu/g immediately after manufacture and the 100-g samples of the mixture were placed in 125 ml polypropylene containers and stored at 4 C. Microbiological analyses were done periodically for up to 15 days. Samples were taken in triplicate and plated out in triplicate on Listeria Selective Agar (LSA). Residual levels of carnocin were determined periodically for up to 8 days according to the method of Coffey et al. (1998). 2.7. Sensory evaluation The sausage formulation was adapted from a traditional recipe. The modifications were intended to permit effective sensory evaluation without destroying the essential features of this type of meat product by eliminating strong flavouring ingredients and using bland meat and rusk. Sausages were individually blast-frozen at 28 C for 2 h and stored frozen at 30 C until required. The sausages from Trial 1 were defrosted for 24 h at 3 C and brought to approximately 16 C before assessment by a group of 11 males and 20 females. The panellists were presented with six specimens of either raw or cooked sausage (see later) for evaluation under controlled
conditions. They were unaware of the specific purpose of the experiment. Sausages were cut in half longitudinally and one half was displayed per panellist, cut again transversely so that both the internal and external surfaces could be presented. Sausages were displayed on transparent plastic trays with white paper lining under white fluorescent lighting. A balanced-block experimental design was used throughout. Odour attributes were assessed first, followed by overall acceptability and appearance. Quantitative Descriptive Analysis (Stone, Sidel, Oliver, Woolsey, & Singleton, 1974) was used to obtain nine attributes to describe the odour of both raw and cooked sausages from Trial 1 by selecting and aggregating the most frequently used of the fifty descriptors mentioned by the panellists. The panellists were then trained to recognise standardised odour attributes. The intensity of each attribute was assessed for each sausage tested on a 0–4 scale and similarly, the overall acceptability in terms of odour and appearance was assessed on a 1–7 scale. Sausages from each batch in Trial 2 were thawed and held at 7 C as a group, specimens being removed from the group and examined at intervals for up to 12 days. Panellists (six per occasion) were taken from the trained pool described above. Cooked sausages were grilled for 10–12 min, positioned approximately 15 cm (12–20 cm) from the heating element and rotated every 3 min through 90 C, until they reached a temperature of 102 C. They were then stored at 45 C for approximately 10 min before assessment. Each panellist received one-half of a sausage from a group of three to assess.
3. Results 3.1. Production of carnocin The carnocin-positive fermented milk powder contained 16,000 AU/g, as determined against L. innocua, while the carnocin-negative control powder displayed no inhibitory activity against this target organism. The antimicrobial activity was conserved from fermentation to the dried powder and for up to 12 months during storage at 4 C (data not illustrated). 3.2. Chitosan, carnocin and sulphite combinations in sausages: Trial 1 The results (Fig. 1 a–c) show that total counts, lactic acid bacteria and yeasts in all the sausages stored at 4 C were very similar for 24 days with the single exception of Batch 5. This batch contained the combination of 0.6% chitosan and low sulphite (170 ppm SO2). Differences in counts of up to 3 log cfu/g were noted between Batch 5
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Fig. 1. Viable counts and pH of fresh pork sausages containing chitosan, carnocin and sulphite (Trial 1) and stored at 4 C for 24 days. Viable numbers were determined on Plate Count Agar (PCA) for total viable counts (a), deMan Rogosa Sharp Agar (MRSA) for lactic acid bacteria (b) and Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (c). Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, !); Batch 4 Chitosan (0.6%, !); Batch 5 Chitosan and low sulphite (&); Batch 6 Carnocin (320 AU/g, &); Batch 7 Carnocin and chitosan (^); Batch 8 Carnocin and low sulphite (^). Data points are means of quadruplicate counts0.10 log cfu/g (max.) for Fig. 1a–c and of duplicate pH determinations ( 0.05 pH unit) for Fig. 1d.
and all the other batches (Fig. 1). The effect was particularly evident in the first 7–10 days of storage at 4 C, i.e. within the expected shelf life for sulphited, chilled sausages. Subsequently, growth in Batch 5 occurred at similar rates to those in all the other sausages but the size of the populations at all sampling points up to 24 days was always less in Batch 5 than in the other batches. The pH of all the sausages (Fig. 1d) was around 5.9–6.3 up to 7 days of chill storage, followed thereafter by an acid drift to around pH 5.0–5.5. As judged by the oxidase and Gram tests on colonies taken from PCA, 340 ppm sulphite selected initially a
Gram-positive flora whereas 170 ppm failed to suppress the growth of the Gram-negative bacteria. After Day 13, both Gram-negative and Gram-positive organisms were isolated from all the sausages. This was taken as evidence of the loss of free sulphite from those batches, which contained this preservative. In sausages containing 0.6% chitosan (Batch 4), Gram-negative bacteria predominated up to and including Day 3 but thereafter both Gram-negative and Gram-positive bacteria were isolated. The flora in sausages containing carnocin (Batch 6) was predominantly Gram-positive on Day 1; thereafter, both Gram-positive and Gram-negative
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Table 2 Viable counts in pork sausages from Trial 1 stored frozen for 30 days and defrosted at 4 C for 24 ha Batch No. and content
1 Control 2 High sulphite (340 ppm) 3 Low sulphite (170 ppm) 4 Chitosan (0.6%) 5 Chitosan+low sulphite 6 Carnocin (320 AU/g) 7 Carnocin+chitosan 8 Carnocin+low sulphite
Viable count (log cfu/g)
pH
Total count on PCAb
Lactic acid bacteria on MRSA
Yeast and moulds on OGYEA
5.9 5.3 4.8 5.4 3.5 5.9 5.3 6.0
4.7 4.6 3.8 4.6 2.5 4.3 3.6 4.4
3.8 3.4 3.3 3.5 2.2 4.0 3.5 3.9
6.4 6.2 6.1 6.1 6.3 6.6 6.4 6.6
a
Counts are means of quadruplicate counts0.10 log cfu/g (maximum). Up to 30 colonies on PCA were selected from each sausage batch and tested for oxidase and Gram reactions. All were oxidase-negative and Gram-positive. b
organisms were isolated. No Gram-negative bacteria were detected in Batch 1 (containing no added preservatives) until Day 10. A comparison of counts in sausages frozen for 30 days (Table 2) with those in sausages chill-stored for 1 day (Fig. 1) showed that there was an increase in numbers of about 1 log cfu/g in some batches following frozen storage. The reasons for this increase are not known. However, direct comparisons of counts in products stored under different conditions must be made with caution. The viable counts and pH values for the frozen sausages (Table 2) showed that the numbers of organisms in Batch 5 (containing the combination of chitosan and low sulphite) were up to 2 log cfu/g lower than in any of the other batches. No Gram-negative organisms were detected on PCA in any of the batches frozen for 30 days. Whilst training of the sensory panel was taking place, some qualitative observations regarding the appearance and odour of sausages were made. Sausages containing chitosan appeared coarser in texture than all others. Some seepage of liquid (‘‘bleeding’’) was observed in some sausages containing chitosan after 16 days or longer at chill temperatures. It is possible that chitosan caused meat particles to contract and express water as happens during the production of fermented sausages. If so, chitosan-containing sausages would have a more ‘‘particulate’’ appearance. Sausages containing carnocin (Batches 6, 7 and 8) lost their bloom (pink colour) and had a yellowish hue.
consistent with the rapid loss of carnocin activity to 50% within 1 day and less than 10% of the original activity within 5 days of storage. 3.4. Chitosan and sulphite combinations in sausages: Trial 2 The results of the second trial (Fig. 3 a–c) essentially confirmed those of the first. In Batch 5, containing the combination of 0.6% chitosan and 170 ppm sulphite, there was initial inactivation of microorganisms on the first day, followed by growth that was consistently below that observed in the other batches for the duration of the trial (21 days). After 10 days of chill storage, the total counts in Batch 5 were nearly 4 log cfu/g lower than in the control containing no preservatives (Batch
3.3. Challenge trial A challenge trial using the strain of L. innocua with known in vitro sensitivity to carnocin was done in sausage mix with the same composition as Batch 6 (Table 1). Carnocin caused a kill of about 1.5–2.0 log cfu/g in the first 5 days of chill-storage (Fig. 2). Subsequently, the rate of die-off of listeria was similar in the carnocintreated sausage mix as in the carnocin-free control,
Fig. 2. Survival of Listeria innocua in sausage mix containing 2% fermented skim milk powder with 320 AU carnocin (&) and without carnocin (*). Bar chart indicates % carnocin activity remaining in the sausage mix. No activity was detected on Days 12 and 15.
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1). After 15 days of storage, the differences in total counts was still evident although the magnitude was smaller (2 log cfu/g). The total counts for sausages containing high sulphite (Batch 2) and chitosan alone (Batch 4) were approximately 1 log lower than in the control sausages
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between 6 and 9 days storage but this was reduced to a difference of less than 0.5 log cfu/g by Day 15 and no difference by the end of the trial on Day 21 (Fig. 3a). The VRBGA counts (Fig. 3d) showed that high sulphite (Batch 2) and the combination of chitosan with low
Fig. 3. Viable counts (a–d), pH (e) and total sulphite content (f) in fresh pork sausages containing chitosan and sulphite (Trial 2) and stored at 4 C for 21 days. Viable numbers were determined on Plate Count Agar (PCA) for total viable counts (a), deMan Rogosa Sharp Agar (MRSA) for lactic acid bacteria (b), Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (c) and Violet Red Bile Glucose Agar (VRBGA) for enteric bacteria (d). Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, !); Batch 4 Chitosan (0.6%, !); Batch 5 Chitosan and low sulphite (&). Data points are means of quadruplicate counts0.10 log cfu/g (maximum) for Fig. 3a– d and of duplicate determinations for Fig. 3e and f.
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sulphite (Batch 5) selectively inactivated Gram-negative bacteria to a level where they were undetectable by the plate-count method. As in Trial 1, a gradual decline in pH from about 6.0–6.2 down to about 4.6–5.0 occurred over the 21 days of chill storage in Trial 2 (Fig. 3e). Gram-stains and oxidase tests indicated that Grampositive organisms predominated up to Days 9 and 15 in sausages containing low and high levels of sulphite, respectively. Subsequently, both Gram-negative and Gram-positive bacteria were isolated from those batches, suggesting breakdown of sulphite. A mixed flora of both Gram-positive and Gram-negative organisms was isolated from Batches 1 (Control) and 4 (Chitosan only). Notably, the microbial flora of sausages containing the chitosan/sulphite combination was predominantly Gram-positive throughout the trial. Sulphite breakdown was confirmed by quantitative analyses of both total and free sulphite in the sausages. The total sulphite content remaining in the sausages is shown in Fig. 3f. The analytical results demonstrated that sulphite was degraded rapidly within the first 3 days of storage in all the batches that had this preservative added to them. However, in the presence of chitosan, sulphite levels decreased less rapidly in the first 3 days and persisted at a higher level for longer (21 days). The free sulphite content of the sausages was approximately 20–30% that of their total sulphite content in all the batches and the rate of breakdown (results not illustrated) reflected that for total sulphite. The microbial counts obtained from defrosted sausages incubated at 7 C for 10 days are shown in Fig. 4. The results showed similar trends to those observed with fresh-chilled sausages and demonstrated that the preservative efficacy of the chitosan/sulphite combination was maintained following frozen storage. Like the fresh-chilled sausages, the pH of the defrosted sausages decreased from around 6.0–6.5 to below 5.0 after 10 days of chill-storage (results not illustrated). As in freshchilled sausages, sulphite levels persisted longer in the batch containing the chitosan/sulphite combination than in that containing low levels of sulphite (170 ppm) only. After 7 days of storage, the total sulphite content in Batches 2 (high sulphite, 340 ppm), 3 (low sulphite, 170 ppm) and 5 (0.6% chitosan and 170 ppm low sulphite) were 160, 60 and 90 ppm, respectively; after 14 days, the levels were 80, 30 and 80 ppm, respectively. Free sulphite was detected at a level approximately onethird that of the total sulphite and reflected the rate of breakdown seen with total sulphite. 3.5. Sensory evaluation Quantitative Descriptive Analysis (Stone et al., 1974) was used to obtain the 9 odour attributes shown in Table 3. These attributes were selected and aggregated from the 50 most frequently used descriptors mentioned
by the 31 panellists used in this study to describe the odour of the raw and cooked sausages. The panellists were trained to recognise standardised odour attributes and to assess their intensity on a 0–4 scale and similarly the overall acceptability in terms of odour and appearance on a 1–7 scale as shown in Table 4 and Fig. 5. The development of unacceptable odours and the changes in visual acceptability of the raw sausages with time are shown in Fig. 4e and f. Principal Component diagrams were computed for the odour attribute distribution with time (Fig. 5) and the variations of distribution are presented in Fig. 6a–c. The sensory results confirmed the gradual deterioration of all the sausages over a period of 12 days. However, the batch containing the combination of chitosan and low sulphite deteriorated less rapidly and was judged as more acceptable than all the other batches, including the control batch containing high sulphite (Fig. 4e and f; Fig. 6a–c). When presented with cooked sausages on Days 3, 8, 10 and 12, the trained panellists scored all the batches as ‘‘extremely acceptable’’ or ‘‘moderately acceptable’’ in terms of development of unfavourable odours (scores 1 and 2, respectively, Table 4) and there was little change during the course of the 12-days trial (results not illustrated). In terms of visual acceptability, the cooked sausages deteriorated slightly from being ‘‘slightly acceptable’’ and ‘‘moderately acceptable’’ on Day 3, to being ‘‘neither acceptable nor unacceptable’’ on Day 12 but again, there were no differences between the batches. The sensorial and microbiological differences between the raw batches of sausages were not reflected in the cooked products in terms of odour and appearance.
4. Discussion Darmadji and Izumimoto (1994) reported that chitosan at a concentration of 1.0% reduced microbial counts by an average of 1–2 log cfu/g (total numbers and pseudomonads, staphylococci, coliforms, Gramnegative bacteria and micrococci) in minced beef patties stored at 4 C for 10 days. Microbial counts were determined on only two occasions: Day 0 and Day 10. At lower chitosan concentrations (0.2 and 0.5%) and higher storage temperature (30 C), no inhibition of growth was observed. The number of viable organisms present in the meat used by Darmadji and Izumimoto (1994) at the start of their experiment was high (> 107cfu/g). This initial level of microbial contamination would not be acceptable for minced meat sold as such under the European Community Hygiene Directive (EC Council Directive, 1994). This specifies that the aerobic mesophilic count in minced meat is unacceptable if it exceeds 5 106 cfu/g in one or more samples out of every five taken. Youn, Park, Kim, and Ahn
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Fig. 4. Viable counts (a–d) and sensory properties (e and f) of pork sausages (Trial 2) containing chitosan and sulphite and stored at 7 C for 12 days after defrosting. Viable numbers were determined on Plate Count Agar (PCA) for total viable counts (a), deMan Rogosa and Sharpe Agar (MRSA) for lactic acid bacteria (b), Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (c) and Violet Red Bile Glucose Agar (VRBGA) for enteric bacteria (d). Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, !); Batch 4 Chitosan (0.6%, !); Batch 5 Chitosan and low sulphite (&). Data points are means of quadruplicate counts0.10 log cfu/g (maximum) for Figs. 4a–d and of at least 6 panellists scores each for Fig. 4e and f.
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Table 3 Sensory attributes developed for Quantitative Descriptive Analysis (QDA) of pork sausages Sensory attributes of sausages Fermenting Sausagey Vinegary
Musty Sour Winey
Rancid Sweet Yeasty
Table 4 Measurement scales developed for sensory attributes (Quantitative Descriptive Analysis, odour and appearance) of sausages QDA
Odour, appearance
Value Meaning
Value
Meaning
0 1 2 3
Non-detectable Weak Moderate Strong
1 2 3 4
4
Very strong
5 6 7
Extremely acceptable Moderately acceptable Slightly acceptable Neither acceptable nor unacceptable Slightly unacceptable Moderately unacceptable Extremely unacceptable
(1999) have reported inactivation of up to 2 log cfu/g of the total microbial flora in a sausage meat immediately upon addition of 0.35 or 0.5% chitosan; however, after storage at 30 C for just 1 day, total counts in the chitosan-treated sausage were the same as in the control. Storage temperatures other than 30 C were not studied by these authors (Youn et al., 1999). Unlike these studies, which offered little scope for the use of chitosan as a
preservative in meat products, the results in Figs. 1, 3 and 4 show that chitosan, in combination with low levels of sulphite, could be used effectively to inhibit microbial spoilage in the British fresh sausage. In recent years, some success with the application of bacteriocins in meat products, especially with respect to inhibition of L. monocytogenes, has been reported although this has usually been with production in situ in fermented products such as salami in which acidification is not a problem (Schillinger, Geisen, & Holzapfel, 1996; Schillinger, Kaya, & Lu¨cke, 1991). Lacticin 3147 in combination with sodium citrate or sodium lactate has been reported as an effective biopreservative in fresh pork sausage (Scannell, Ross, Hill, & Arendt, 2000). Nisin added at a level of 400 and 800 IU/g has been shown to extend the lag phase of L. monocytogenes inoculated into minced buffalo meat and stored at 4 C by 4 and 16 days, respectively (Pawar, Malik, Bhilegaonkar, & Barbuddhe, 2000). Although carnocins have been described in the literature as possessing a broad inhibitory spectrum (Jack, et al., 1996; Mathieu, Michel, Lebrihi, & Lefebre, 1994; O’Mahony, 2001; Stoffels, Sahl, & Gudmundsdottir, 1993), in our study the inclusion of a carnocin-containing fermented ingredient in fresh pork sausage failed to influence the rate of spoilage (Fig. 1). There are three possible explanations for this. Firstly, the initial level of bacteriocin added may have been insufficient, as reported previously in studies with bacteriocin-producing Lac. sake (Buncic et al., 1997). Secondly, it is possible that bacteriocin-resistant bacteria filled the ecological niche provided by the action of carnocin on more sen-
Fig. 5. Principal Component odour attribute distribution for raw pork sausages.
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Fig. 6. Principal Component odour attribute distribution replicas for pork sausages containing chitosan and sulphite after 1, 8 and 10 days of storage at 7 C. Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, ~); Batch 4 Chitosan (0.6%, ~); Batch 5 Chitosan and low sulphite (&).
sitive species. Finally, a number of other ingredients in the sausage mix, namely proteases, fats and sulphite may have had a negative impact on the activity of carnocin. The rapid loss in residual carnocin activity in the first 3 days of storage (Fig. 2) was similar to the 70% loss in nisin activity in raw meat during storage at 5 C for 4 days (Chung et al., 1989). Like nisin, carnocin may be susceptible to degradation by sodium metabisulphite, as reported by Thomas, Clarkson, and Delves Broughton (2000). According to microbiological criteria developed in the late 1990s, it is recommended that the total and yeast counts of raw sausages should not exceed 105 and 104 cfu/g, respectively, immediately following manufacture under Good Manufacturing Practice (GMP, IFST 1999). The total and yeast counts on the day of manufacture in all the batches of sausages prepared in this study were well within these recommendations (Figs. 1 and 3). In Trial 2, the total and yeast counts exceeded the 107 and 106 cfu/g recommended as the maximum acceptable
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levels by microbiological criteria for meat (IFST, 1999) in all the Batches except Batch 5 (containing the combination of chitosan and low sulphite) by Day 6 of storage at 4 C (Figs. 3 a and c). Notably, these criteria were not exceeded in the combination batch (Batch 5) until after Day 18 of the trial. It can be calculated, therefore, that the shelf life of the sausages was extended nearly three times by the presence of the combination of chitosan and sulphite. The relatively short shelf life of the sulphited sausages in both trials (as evidenced by the few days needed to reach unacceptable levels of both total and yeast counts) can be explained by the relatively low levels of addition of sulphite in this study (170 and 340 ppm at the time of production). Under British legislation (Anon., 1995), the maximum permitted level of SO2 in fresh sausage is 450 ppm at the point of sale. Due to considerable loss of preservative during manufacture and distribution, manufacturers routinely add up to 650 ppm sulphite during manufacture (Wilson, 1981). Most commercial sausages contain between 180 to 240 ppm total sulphite when analysed within 1–2 d of manufacture (Nick Walsh, RSSL, personal communication), consistent with higher levels having been added during production to compensate for rapid initial sulphite breakdown. The sulphite content of British pork sausages is determined traditionally by a method based on the collection of SO2 released from a sample suspended in boiling acid (Monier-Williams, 1927). Consequently, the sulphite content of sausages is conventionally expressed as ppm of free and/or total SO2. However, at the relatively neutral pH of sausages, the amounts of SO2 formed from metabisulphite are negligible. At the pH of sausages prepared in our study ( pH 6 on day of manufacture), sodium metabisulphite would have dissociated into bisulphite (HSO3 ) and sulphite (SO23 ) in a 94:6 ratio (Banks et al., 1987). Furthermore, it has been shown that sulphite is effective against microorganisms only when present in the free (unbound) form and that it is most potent at low temperatures. The free sulphite content and consequently the antimicrobial efficacy in sausages is known to diminish rapidly with time as a consequence of binding with acetaldehyde produced by spoilage yeasts (reviewed in Banks et al., 1987). It is well known that sulphite is most active against Gram-negative microorganisms, particularly the Enterobacteriaceae (Banks et al., 1987). In the present study, the shapes of the microbial and pH curves, the Gramstaining and oxidase results, the VRBGA counts and the residual sulphite analyses in the sausage batch containing the chitosan/sulphite combination (Batch 5) all suggest that selective inactivation and inhibition of Gram-negative bacteria had occurred. The mechanism of preservation by the chitosan/sulphite combination may be two-fold. Firstly, chitosan may have acted as a ‘‘slow-release’’ agent for sulphite,
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particularly free sulphite, thereby preventing its premature degradation or irreversible binding with other ingredients in the sausage. With a pKa of 6.3, approximately one-half of the chitosan present in the sausage on the day of manufacture would have been highly positively charged and could have interacted readily but reversibly with the negatively charged sulphite moieties. Secondly, any unbound chitosan may have selectively inhibited the yeast flora, thereby preventing acetaldehyde production and the consequent inactivation of sulphite by binding. The efficacy of the chitosan/sulphite combination could be explained therefore on the basis of protection from sulphite breakdown by chitosan. The sensory results demonstrated that the addition of chitosan to sausages would not lead to off-odours and that the appearance would not be rendered objectionable, either of which could potentially lead to rejection by the consumer. Chitosan has recently been affirmed as Generally Recognised as Safe (GRAS) by the US FDA (2001), thus removing some of the regulatory restrictions on its use in foods (Anon., 2001). Further work on novel combination systems such as the chitosan/sulphite combination described here would be facilitated by greater understanding of the mode of action of novel antimicrobials like chitosan on microorganisms. Finally, additional studies in real food systems are needed to assess the commercial potential of this natural antimicrobial compound.
Acknowledgements The following organizations are gratefully acknowledged for funding this work: European Commission (Contract FAIR CT96–1066), Meat and Livestock Commission UK, Danisco (Aplin and Barrett Ltd., UK), CPC (UK) Ltd., DSM Food Specialties (Netherlands) and Norsk Hydro (Norway).
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Schillinger, U., Kaya, M., & Lucke, F. K. (1991). Behaviour of Listeria monocytogenes in meat and its control by a bacteriocin producing strain of Lactobacillus sake. Journal of Applied Bacteriology, 70, 473–478. Seo, H., Mitsuhashi, K., & Tanibe, H. (1992). Antibacterial and antifungal fiber blended by chitosan. In C. J. Brine, P. A. Sandford, & J. P. Zikakis (Eds.), Advances in chitin and chitosan (pp. 34–40). New York: Elsevier Applied Science. Shahidi, F., Arachchi, J. K. M., & Jeon, Y. (1999). Food applications of chitin and chitosans. Trends in Food Science and Technology, 10, 37–51. Simon, R. A. (1998). Update on sulphite sensitivity. Allergy, 53, 78– 79. Stoffels, G., Sahl, H. G., & Gudmundsdottir, A. (1993). Carnocin U149, a potential biopreservative produced by Carnobacterium piscicola, large scale purification and activity against various Grampositive bacteria including Listeria sp. International Journal of Food Microbiology, 20, 199–210. Stone, H., Sidel, J. L., Oliver, S., Woolsey, A., & Singleton, R. C. (1974). Sensory evaluation by quantitative descriptive analysis. Food Technology, 28(11), 24–33. Sudarshan, N. R., Hoover, D. G., & Knorr, D. (1992). Antibacterial action of chitosan. Food Biotechnology, 6, 257–272. Surkiewicz, B. F., Johnston, R. W., Elliott, R. P., & Simmons, R. E. (1972). Bacteriological survey of fresh pork sausage produced at establishments under federal inspection. Applied Microbiology, 23, 515–520. Thomas, L. V., Clarkson, M. R., & Delves-Broughton, J. (2000). Nisin. In A. S. Naidu (Ed.), Natural food antimicrobial systems (pp. 463–524). Boca Raton, FL, USA: CRC Press. Tsai, G. J., & Su, W.-H. (1999). Antibacterial activity of shrimp chitosan against Escherichia coli. Journal of Food Protection, 62, 239– 243. Tsai, G. J., Wu, Z.-Y., & Su, W.-H. (2000). Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. Journal of Food Protection, 63, 747–752. Vaughan, E. E., Caplice, E., Looney, R., O’Rourke, N., Coveney, H., Daly, C., & Fitzgerald, G. F. (1994). Isolation from food sources of lactic acid bacteria that produced antimicrobials. Journal of Applied Bacteriology, 76, 118–123. Wang, G.-H. (1992). Inhibition and inactivation of five species of foodborne pathogens by chitosan. Journal of Food Protection, 55, 916–919. Wessels, S., Jelle, B., & Nes, I. F. (1998). Bacteriocins of the lactic acid bacteria: an overlooked benefit for food. Copies from: Danish Toxicology Centre, 2 Kogle Alle, DK-2970 Horsholm, Denmark. Wilson, N.R.P. (1981). Sausages and other comminuted meat products. In Meat and meat products (pp. 164–181). Applied Science Publications. Yang, R., & Ray, B. (1994). Prevalence and biological control of bacteriocin producing psychrotrophic leuconostocs associated with spoilage of vacuum packaged processed meats. Journal of Food Protection, 57, 209–217. Youn, S.-K., Park, S.-M., Kim, Y.-J., & Ahn, D.-H. (1999). Effect on storage property and quality in meat sausage by added chitosan. Journal of Chitin and Chitosan, 4, 189–194.
Novel combinations of chitosan, carnocin and sulphite for the preservation of chilled pork sausages S. Rollera,*, S. Sagooa, R. Boarda, T. O’Mahonyb, E. Capliceb, G. Fitzgeraldb, M. Fogdenc, M. Owenc, H. Fletcherc a School of Applied Science, South Bank University, 103 Borough Road, London SE1 0AA, UK National Food Biotechnology Centre and Department of Microbiology, University College Cork, Western Road, Cork, Ireland c Meat and Livestock Commission, PO Box 44, Winterhill House, Snowdon Drive, Milton Keynes MK6 1AX, UK
b
Received 20 August 2001; accepted 20 November 2001
Abstract The aim of this study was to develop novel preservation systems for fresh pork sausages based on combinations of chitosan (polymeric b-1,4-N-acetylglucosamine) carnocin (a bacteriocin produced by Carnobacterium piscicola) and low concentrations of sulphite. Two pilot-scale trials showed that 0.6% chitosan combined with low sulphite (170 ppm) retarded the growth of spoilage organisms more effectively (3–4 log cfu/g) than high levels (340 ppm) of sulphite alone at 4 C for up to 24 days. Microbial counts for frozen sausages showed that the preservative efficacy of the chitosan/sulphite combination was maintained following frozen storage. Carnocin did not protect sausages from spoilage but in a challenge trial, it reduced the numbers of Listeria innocua by up to 2.0 log cfu/g in the first 5 days of chill-storage. Sulphite was degraded rapidly within the first 3 days of storage in all the sausages that contained only this preservative but levels decreased less rapidly and persisted for longer in the presence of chitosan. Results of Quantitative Descriptive Analysis using 31 trained panellists reflected the gradual deterioration of all the sausages during storage. The batch containing chitosan and sulphite deteriorated less rapidly and was judged to be more acceptable for a longer period than all the other batches. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pork sausages; Chitosan; Carnocin; Sulphite; Bacteriocin; Preservation; Meat spoilage
1. Introduction British pork sausages contain at least 65% meat (determined by UK legislation), fat, rusk (a type of breadcrumb) and spices in a collagen casing. In the UK, sodium metabisulphite is used to inhibit microbial spoilage of raw sausages and to achieve a shelf life of about 15 days at chill temperatures from the day of manufacture (Dowdell & Board, 1971; Surkiewicz, Johnston, Elliott, & Simmons, 1972). Sulphite is an elective agent; it inhibits the growth of Gram-negative bacteria and selects a flora that is fermentative in character and includes both Gram-positive bacteria and yeasts (Banks, Dalton, Nychas, & Board, 1985). The latter are responsible for producing acetaldehyde, which binds sulphite and causes
* Corresponding author. Tel. +44-207-815-7961; fax: +44-207815-7999. E-mail address: [email protected] (S. Roller).
loss of antimicrobial activity (reviewed in Banks, Nychas, & Board, 1987). Sulphites have a long history of safe use in meat products. Exposure to sulphites (although not specifically via foods preserved with them) has been linked, however, with the exacerbation of asthmatic and other respiratory conditions in some sensitive individuals (reviewed in Simon, 1998; BIBRA, 1999). In 1995, an EC Council Directive was issued specifying a detailed list of foods to which sulphites can be added and setting maximum permitted levels of addition. In the USA, sulphiting agents are not permitted in meats or in foods recognised as a source of Vitamin B1 and since 1986 have been banned from use in fresh fruit and vegetables (Anon., 1986). Some consumers regard the deliberate addition of sulphites and/or any other chemical preservatives to foods as a form of adulteration. Yet, a low level of sulphite is required to give fresh pork sausages the ‘‘bloom’’ (pink colouring) that is attractive to the purchaser. There is thus a clear need to develop novel preservation systems
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00243-1
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based on low levels of sulphite (to inhibit Gram-negative bacteria and maintain desirable sensorial properties) together with an additional preservative that is active against yeasts and Gram-positive bacteria. Chitosan, a deacetylated form of chitin, is a polysaccharide found in the shells of crabs and shrimp. Chitosan consists of polymeric 1,4-linked 2-amino-2deoxy-b-D-glucose. A number of applications for chitosan have been proposed including heavy metal chelation in wastewater treatment, as a hypocholesterolemic agent, and as dietary fibre (Shahidi, Arachchi, & Jeon, 1999). As chitosan exhibits antimicrobial activity against a range of foodborne microorganisms in the laboratory, it has attracted attention as a potential food preservative of natural origin (Roller and Covill, 1999, 2000; Rhoades & Roller, 2000; Tsai, Wu, & Su, 2000; Shahidi et al., 1999). The reported Minimimum Inhibitory Concentrations (MICs) vary widely from 0.01 to 1.0% and yeasts tend to be more sensitive than bacteria (Knowles & Roller, 2001; Roller & Covill, 2000; Rhoades & Roller, 2000; Seo, Mitsuhashi, & Tanibe, 1992; Sudarshan, Hoover, & Knorr, 1992; Tsai & Su, 1999; Tsai et al., 2000; Wang, 1992). Antimicrobial efficacy demonstrated in the laboratory is not always realized in foods due to the highly reactive nature of the polycationic chitosan, which interacts readily with proteins, fats and other anionic substances in foods (Rhoades & Roller, 2000). Nevertheless, some successful applications, especially against spoilage yeasts in juices and emulsified sauces, have been reported (Roller & Covill, 1999, 2000). In meat products, slight inhibition ( 1–2 log cfu/g) of total microbial growth in refrigerated beef patties has been reported in the presence of 1.0% but not 0.5 or 0.2% chitosan (Darmadji & Izumimoto, 1994). Bacteriocins produced by lactic acid bacteria, including pediocin, sakacin and nisin, are naturally occurring antimicrobial peptides that inhibit a range of Grampositive foodborne pathogens and spoilage organisms (Jack, Tagg, & Ray, 1995). The genetic determinants of bacteriocin production, their mode of action and efficacy in laboratory systems have been studied extensively in the last 15 years (reviewed by de Vuyst & Vandamme, 1993; Ennahar, Sashihara, Sonomoto, & Ishizaki, 2000; Hill, 1995; Holz & Stahl, 1995; Hoover & Steenson, 1993; McAuliffe, Ross, & Hill, 2001; Ray & Daeschel, 1994). The antilisterial activity of many bacteriocins is well-known (Ennahar, Deschamps, & Richard, 2000; Leroy & de Vuyst, 2000; Ray & Miller, 2000). Although these biopreservatives have the potential to protect some foods from spoilage, their application in raw or processed meat products is limited because binding with meat particles and fat may cause loss of activity (Aymerich, Artigas, Garriga, Monfort, & Hugas, 2000; Yang & Ray 1994). Nisin has nevertheless been approved for use in processed meat products such as vacuum-packed frankfurters in which spoilage by lactic
acid bacteria can reduce shelf life. A number of investigators have reported the use of bacteriocin-producers in starter cultures for fermented sausages (reviewed in Wessels, Jelle, & Ness, 1998). Preliminary studies have indicated that chitosan may act synergistically with sodium metabisulphite against Lactobacillus viridescens, a common cause of spoilage of cured meat products (Roller, 2001). The bacteriocin carnocin 124, produced by a meat isolate, Carnobacterium piscicola, has been purified to homogeneity and its biochemical and antimicrobial properties have been characterised (O’Mahony, 2001). The main aim of this study was to assess the feasibility of using chitosan and carnocin in combination with low concentrations of sulphite to control the microbial spoilage of fresh pork sausage.
2. Materials and methods 2.1. Chemicals Chitosan glutamate was obtained from Pronova (Drammen, Norway). It contained 42% glutamate and had a deacetylation range of 75–85% (manufacturer’s data). All microbiological media and diluents were from Oxoid (Basingstoke, UK). Sodium metabisulphite was from Fisher Scientific (Loughborough, UK). All other chemicals were from Sigma Chemical Co (Poole, Dorset, UK), unless otherwise indicated. 2.2. Bacteriocin production and determination of antilisterial activity Carnobacterium piscicola NFBC124, isolated from a chicken portion (Vaughan et al., 1994), was used. It produced the antimicrobial peptide carnocin 124, which is virtually identical to piscicolin 126 (Fitzgerald, Harmark, Selleck, Hillier, & Davidson, 1999; Jack et al., 1996; O’Mahony, 2001). Bacteriocin concentration was determined by the spot assay method of Hoover and Steenson (1993). Serial two-fold dilutions of cell-free supernatants from a culture of the producer organism were spotted in 5 ml aliquots onto a Tryptone Soya Agar/ 0.5% yeast extract plate (TSAYE) previously overlaid with 5 ml of TSAYE /0.75% agar containing 5 105 cfu/ml of Listeria innocua CNRZ 4202 and incubated at 30 C for 2 days. Arbitrary units per millilitre (AU/ml) were calculated as the reciprocal of the highest dilution that gave a clear zone of inhibition on the indicator lawn. A bacteriocin-negative variant of C. piscicola was isolated by repeated sub-culturing of the producer strain in TSBYE until spot-testing of filtered supernatant from an overnight culture confirmed that a bacteriocin-negative phenotype had been selected. API CH50 kits (BioMerieux, France) were used to confirm that the fermentation profile of the bacteriocin-negative
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variant was the same as that of the parent strain. The production of carnocin could be induced in the variant by the inclusion of 0.2% cell free supernatant from the producer strain, as shown previously (O’Mahony, 2001; Saucier, Poon, & Stiles, 1995). 2.3. Production of fermented milk powder containing bacteriocin Fermented milk powders were produced on a pilot scale from 50 l of 10% Reconstituted Milk Powder supplemented with 0.5% yeast extract (RSMYE). The medium was adjusted to pH 8 before sterilization. The bacteriocin-producing and non-producing variant of C. piscicola were grown in deMan Rogosa and Sharpe broth (MRSB, adjusted to pH 7.5 with 10M NaOH) at 30 C for 18 h and were added to the RSMYE at a level of 1%. The fermentation (30 C) was allowed to proceed for approximately 24 h until the pH reached 5.6. The fermentate was stored for 24 h at 4 C; during this period it was tested for bacteriocin activity as described above. Upon confirmation of either presence or absence of antilisterial activity, the fermentate was flash pasteurized at 75 C for 15 s and subsequently evaporated under vacuum at 60 C until the total solids reached 25%. The condensate was spray dried (inlet temperature 85 C, outlet temperature 185 C). A sample of the resultant powder was resuspended in sterile distilled water and tested again for antimicrobial activity and pH. The powder was stored at 4 C and checked periodically for antilisterial activity.
in fresh pork sausages. In both, the sausages were prepared according to a formulation and method supplied by the Meat and Livestock Commission (MLC), UK. In Trial 1, eight batches, each of 10 kg of sausages were prepared according to the formulations given in Table 1. The pork and fat were minced through a 5-mm plate. The dry ingredients (rusk, seasoning, skim milk powder, chitosan and/or sodium metabisulphite, as appropriate) were mixed separately and added to the meat mixture with continuous mixing. During mixing, chilled water was added slowly to the meat mixture until absorbed. The mixture was minced again through the 5-mm plate and mixed until even distribution achieved. The sausage mix was filled into Devro small diameter casing to make sausages of 56 g each. Four sausages ( 225 g) were placed into a cellophane-wrapped pack. A 10-kg batch yielded approximately 44 packs of sausages. Microbiological analyses (see later) were done on sausages stored at 4 C for 24 days. Batches of defrosted frozen sausages from Trial 1 were used to train the sensory panel (see later). In the second trial, carried out essentially as described earlier, 15-kg batches of sausages were prepared according to the formulations shown for the first five batches in Table 1 except that the fermented milk powder was replaced by a commercial skim milk powder. The sulphite content of the sausages was also determined periodically by the Monier Williams/HPLC method (Banks et al., 1987; Monier-Williams, 1927). Samples of sausages from Trial 2 were frozen and defrosted as required for sensory analysis (see later). 2.5. Sausage sampling method and microbiological analyses
2.4. Chitosan, carnocin and sulphite combinations in fresh pork sausages Two trials were carried out to test the preservative efficacy of chitosan, carnocin and sulphite combinations
The Public Health Laboratory Service (UK) method (Roberts, Hopper, & Greenwood, 1995) was used to determine the size and composition of the microbial
Table 1 Chitosan, sulphite and carnocin content of fresh pork sausages prepared in Trials 1 and 2 Batch No. and Name
1 Control 2 High sulphite 3 Low sulphite 4 Chitosan 5 Chitosan+low sulphite 6 Carnocin 7 Carnocin+chitosan 8 Carnocin+low sulphite a
Content (%)b Sodium metabisulphite (final sulphite conc.)
Chitosan glutamate
Carnocin-negative skim milk powdera
Carnocin-positive skim milk powder (final carnocin conc.)
0 0.045 (340 ppm) 0.0225 (170 ppm) 0 0.0225 (170 ppm) 0 0 0.0225 (170 ppm)
0 0 0 0.6 0.6 0 0.6 0
2.0 2.0 2.0 2.0 2.0 0 0 0
0 0 0 0 0 2.0 (320 AU/g) 2.0 (320 AU/g) 2.0 (320 AU/g)
Carnocin-negative fermented skim-milk powder was used in all Batches in Trial 1 and in Batches 6–8 in Trial 2. The fermented powder was replaced by a commercial skim milk powder in Batches 1–5 in Trial 2. b All sausages contained 42.5% lean pork, 25.0% pork fat, 8.0% bland rusk (RHM), 2.5% seasoning 40910 (containing 60% salt, Kerry Ingredients) and 20.0% chilled tap water.
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flora of sausages. Samples (25 g) were taken from two sausages in each pack of four. Each sausage was sliced transversely into several sections; the end sections were discarded. Several slices (casing was not removed) were selected randomly from the middle and end to make up 25 g. The 25-g sample was added to 225 ml of Maximum Recovery Diluent (MRD) and homogenised with a stomacher for 2 min. A sample (1 ml) of the slurry was diluted serially in MRD in duplicate and appropriate dilutions were spread-plated (0.1 ml in duplicate) on the following: Plate Count Agar (PCA) for total viable count (incubated at 30 C for 2 days); deMan Rogosa Sharpe Agar (MRSA) for lactic acid bacteria (30 C for 3 days); Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (25 C for 5 days) and Violet Red Bile Glucose Agar (VRBGA) for enteric organisms (37 C for 2 days). In the second trial, 1 ml of a 1:10 sausage slurry was used to prepare pour plates of VRBGA in order to increase the sensitivity limit of the method. The oxidase test and Gram-staining of selected colonies from PCA were done according to Roberts et al. (1995). For pH determination, a further 25 g of sausage was homogenised in 225 ml of distilled water for 2 min and the pH of the slurry measured. 2.6. Challenge trial with Listeria innocua in carnocincontaining sausage mix Additional mixtures of sausage meat were manufactured using the formulation for Batch 1 (Control, no preservative added) and Batch 6 (Carnocin) in Table 1. Listeria innocua CNRZ 4202 was added to the sausage mix at a level of 5.5 105 cfu/g immediately after manufacture and the 100-g samples of the mixture were placed in 125 ml polypropylene containers and stored at 4 C. Microbiological analyses were done periodically for up to 15 days. Samples were taken in triplicate and plated out in triplicate on Listeria Selective Agar (LSA). Residual levels of carnocin were determined periodically for up to 8 days according to the method of Coffey et al. (1998). 2.7. Sensory evaluation The sausage formulation was adapted from a traditional recipe. The modifications were intended to permit effective sensory evaluation without destroying the essential features of this type of meat product by eliminating strong flavouring ingredients and using bland meat and rusk. Sausages were individually blast-frozen at 28 C for 2 h and stored frozen at 30 C until required. The sausages from Trial 1 were defrosted for 24 h at 3 C and brought to approximately 16 C before assessment by a group of 11 males and 20 females. The panellists were presented with six specimens of either raw or cooked sausage (see later) for evaluation under controlled
conditions. They were unaware of the specific purpose of the experiment. Sausages were cut in half longitudinally and one half was displayed per panellist, cut again transversely so that both the internal and external surfaces could be presented. Sausages were displayed on transparent plastic trays with white paper lining under white fluorescent lighting. A balanced-block experimental design was used throughout. Odour attributes were assessed first, followed by overall acceptability and appearance. Quantitative Descriptive Analysis (Stone, Sidel, Oliver, Woolsey, & Singleton, 1974) was used to obtain nine attributes to describe the odour of both raw and cooked sausages from Trial 1 by selecting and aggregating the most frequently used of the fifty descriptors mentioned by the panellists. The panellists were then trained to recognise standardised odour attributes. The intensity of each attribute was assessed for each sausage tested on a 0–4 scale and similarly, the overall acceptability in terms of odour and appearance was assessed on a 1–7 scale. Sausages from each batch in Trial 2 were thawed and held at 7 C as a group, specimens being removed from the group and examined at intervals for up to 12 days. Panellists (six per occasion) were taken from the trained pool described above. Cooked sausages were grilled for 10–12 min, positioned approximately 15 cm (12–20 cm) from the heating element and rotated every 3 min through 90 C, until they reached a temperature of 102 C. They were then stored at 45 C for approximately 10 min before assessment. Each panellist received one-half of a sausage from a group of three to assess.
3. Results 3.1. Production of carnocin The carnocin-positive fermented milk powder contained 16,000 AU/g, as determined against L. innocua, while the carnocin-negative control powder displayed no inhibitory activity against this target organism. The antimicrobial activity was conserved from fermentation to the dried powder and for up to 12 months during storage at 4 C (data not illustrated). 3.2. Chitosan, carnocin and sulphite combinations in sausages: Trial 1 The results (Fig. 1 a–c) show that total counts, lactic acid bacteria and yeasts in all the sausages stored at 4 C were very similar for 24 days with the single exception of Batch 5. This batch contained the combination of 0.6% chitosan and low sulphite (170 ppm SO2). Differences in counts of up to 3 log cfu/g were noted between Batch 5
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Fig. 1. Viable counts and pH of fresh pork sausages containing chitosan, carnocin and sulphite (Trial 1) and stored at 4 C for 24 days. Viable numbers were determined on Plate Count Agar (PCA) for total viable counts (a), deMan Rogosa Sharp Agar (MRSA) for lactic acid bacteria (b) and Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (c). Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, !); Batch 4 Chitosan (0.6%, !); Batch 5 Chitosan and low sulphite (&); Batch 6 Carnocin (320 AU/g, &); Batch 7 Carnocin and chitosan (^); Batch 8 Carnocin and low sulphite (^). Data points are means of quadruplicate counts0.10 log cfu/g (max.) for Fig. 1a–c and of duplicate pH determinations ( 0.05 pH unit) for Fig. 1d.
and all the other batches (Fig. 1). The effect was particularly evident in the first 7–10 days of storage at 4 C, i.e. within the expected shelf life for sulphited, chilled sausages. Subsequently, growth in Batch 5 occurred at similar rates to those in all the other sausages but the size of the populations at all sampling points up to 24 days was always less in Batch 5 than in the other batches. The pH of all the sausages (Fig. 1d) was around 5.9–6.3 up to 7 days of chill storage, followed thereafter by an acid drift to around pH 5.0–5.5. As judged by the oxidase and Gram tests on colonies taken from PCA, 340 ppm sulphite selected initially a
Gram-positive flora whereas 170 ppm failed to suppress the growth of the Gram-negative bacteria. After Day 13, both Gram-negative and Gram-positive organisms were isolated from all the sausages. This was taken as evidence of the loss of free sulphite from those batches, which contained this preservative. In sausages containing 0.6% chitosan (Batch 4), Gram-negative bacteria predominated up to and including Day 3 but thereafter both Gram-negative and Gram-positive bacteria were isolated. The flora in sausages containing carnocin (Batch 6) was predominantly Gram-positive on Day 1; thereafter, both Gram-positive and Gram-negative
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Table 2 Viable counts in pork sausages from Trial 1 stored frozen for 30 days and defrosted at 4 C for 24 ha Batch No. and content
1 Control 2 High sulphite (340 ppm) 3 Low sulphite (170 ppm) 4 Chitosan (0.6%) 5 Chitosan+low sulphite 6 Carnocin (320 AU/g) 7 Carnocin+chitosan 8 Carnocin+low sulphite
Viable count (log cfu/g)
pH
Total count on PCAb
Lactic acid bacteria on MRSA
Yeast and moulds on OGYEA
5.9 5.3 4.8 5.4 3.5 5.9 5.3 6.0
4.7 4.6 3.8 4.6 2.5 4.3 3.6 4.4
3.8 3.4 3.3 3.5 2.2 4.0 3.5 3.9
6.4 6.2 6.1 6.1 6.3 6.6 6.4 6.6
a
Counts are means of quadruplicate counts0.10 log cfu/g (maximum). Up to 30 colonies on PCA were selected from each sausage batch and tested for oxidase and Gram reactions. All were oxidase-negative and Gram-positive. b
organisms were isolated. No Gram-negative bacteria were detected in Batch 1 (containing no added preservatives) until Day 10. A comparison of counts in sausages frozen for 30 days (Table 2) with those in sausages chill-stored for 1 day (Fig. 1) showed that there was an increase in numbers of about 1 log cfu/g in some batches following frozen storage. The reasons for this increase are not known. However, direct comparisons of counts in products stored under different conditions must be made with caution. The viable counts and pH values for the frozen sausages (Table 2) showed that the numbers of organisms in Batch 5 (containing the combination of chitosan and low sulphite) were up to 2 log cfu/g lower than in any of the other batches. No Gram-negative organisms were detected on PCA in any of the batches frozen for 30 days. Whilst training of the sensory panel was taking place, some qualitative observations regarding the appearance and odour of sausages were made. Sausages containing chitosan appeared coarser in texture than all others. Some seepage of liquid (‘‘bleeding’’) was observed in some sausages containing chitosan after 16 days or longer at chill temperatures. It is possible that chitosan caused meat particles to contract and express water as happens during the production of fermented sausages. If so, chitosan-containing sausages would have a more ‘‘particulate’’ appearance. Sausages containing carnocin (Batches 6, 7 and 8) lost their bloom (pink colour) and had a yellowish hue.
consistent with the rapid loss of carnocin activity to 50% within 1 day and less than 10% of the original activity within 5 days of storage. 3.4. Chitosan and sulphite combinations in sausages: Trial 2 The results of the second trial (Fig. 3 a–c) essentially confirmed those of the first. In Batch 5, containing the combination of 0.6% chitosan and 170 ppm sulphite, there was initial inactivation of microorganisms on the first day, followed by growth that was consistently below that observed in the other batches for the duration of the trial (21 days). After 10 days of chill storage, the total counts in Batch 5 were nearly 4 log cfu/g lower than in the control containing no preservatives (Batch
3.3. Challenge trial A challenge trial using the strain of L. innocua with known in vitro sensitivity to carnocin was done in sausage mix with the same composition as Batch 6 (Table 1). Carnocin caused a kill of about 1.5–2.0 log cfu/g in the first 5 days of chill-storage (Fig. 2). Subsequently, the rate of die-off of listeria was similar in the carnocintreated sausage mix as in the carnocin-free control,
Fig. 2. Survival of Listeria innocua in sausage mix containing 2% fermented skim milk powder with 320 AU carnocin (&) and without carnocin (*). Bar chart indicates % carnocin activity remaining in the sausage mix. No activity was detected on Days 12 and 15.
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1). After 15 days of storage, the differences in total counts was still evident although the magnitude was smaller (2 log cfu/g). The total counts for sausages containing high sulphite (Batch 2) and chitosan alone (Batch 4) were approximately 1 log lower than in the control sausages
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between 6 and 9 days storage but this was reduced to a difference of less than 0.5 log cfu/g by Day 15 and no difference by the end of the trial on Day 21 (Fig. 3a). The VRBGA counts (Fig. 3d) showed that high sulphite (Batch 2) and the combination of chitosan with low
Fig. 3. Viable counts (a–d), pH (e) and total sulphite content (f) in fresh pork sausages containing chitosan and sulphite (Trial 2) and stored at 4 C for 21 days. Viable numbers were determined on Plate Count Agar (PCA) for total viable counts (a), deMan Rogosa Sharp Agar (MRSA) for lactic acid bacteria (b), Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (c) and Violet Red Bile Glucose Agar (VRBGA) for enteric bacteria (d). Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, !); Batch 4 Chitosan (0.6%, !); Batch 5 Chitosan and low sulphite (&). Data points are means of quadruplicate counts0.10 log cfu/g (maximum) for Fig. 3a– d and of duplicate determinations for Fig. 3e and f.
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sulphite (Batch 5) selectively inactivated Gram-negative bacteria to a level where they were undetectable by the plate-count method. As in Trial 1, a gradual decline in pH from about 6.0–6.2 down to about 4.6–5.0 occurred over the 21 days of chill storage in Trial 2 (Fig. 3e). Gram-stains and oxidase tests indicated that Grampositive organisms predominated up to Days 9 and 15 in sausages containing low and high levels of sulphite, respectively. Subsequently, both Gram-negative and Gram-positive bacteria were isolated from those batches, suggesting breakdown of sulphite. A mixed flora of both Gram-positive and Gram-negative organisms was isolated from Batches 1 (Control) and 4 (Chitosan only). Notably, the microbial flora of sausages containing the chitosan/sulphite combination was predominantly Gram-positive throughout the trial. Sulphite breakdown was confirmed by quantitative analyses of both total and free sulphite in the sausages. The total sulphite content remaining in the sausages is shown in Fig. 3f. The analytical results demonstrated that sulphite was degraded rapidly within the first 3 days of storage in all the batches that had this preservative added to them. However, in the presence of chitosan, sulphite levels decreased less rapidly in the first 3 days and persisted at a higher level for longer (21 days). The free sulphite content of the sausages was approximately 20–30% that of their total sulphite content in all the batches and the rate of breakdown (results not illustrated) reflected that for total sulphite. The microbial counts obtained from defrosted sausages incubated at 7 C for 10 days are shown in Fig. 4. The results showed similar trends to those observed with fresh-chilled sausages and demonstrated that the preservative efficacy of the chitosan/sulphite combination was maintained following frozen storage. Like the fresh-chilled sausages, the pH of the defrosted sausages decreased from around 6.0–6.5 to below 5.0 after 10 days of chill-storage (results not illustrated). As in freshchilled sausages, sulphite levels persisted longer in the batch containing the chitosan/sulphite combination than in that containing low levels of sulphite (170 ppm) only. After 7 days of storage, the total sulphite content in Batches 2 (high sulphite, 340 ppm), 3 (low sulphite, 170 ppm) and 5 (0.6% chitosan and 170 ppm low sulphite) were 160, 60 and 90 ppm, respectively; after 14 days, the levels were 80, 30 and 80 ppm, respectively. Free sulphite was detected at a level approximately onethird that of the total sulphite and reflected the rate of breakdown seen with total sulphite. 3.5. Sensory evaluation Quantitative Descriptive Analysis (Stone et al., 1974) was used to obtain the 9 odour attributes shown in Table 3. These attributes were selected and aggregated from the 50 most frequently used descriptors mentioned
by the 31 panellists used in this study to describe the odour of the raw and cooked sausages. The panellists were trained to recognise standardised odour attributes and to assess their intensity on a 0–4 scale and similarly the overall acceptability in terms of odour and appearance on a 1–7 scale as shown in Table 4 and Fig. 5. The development of unacceptable odours and the changes in visual acceptability of the raw sausages with time are shown in Fig. 4e and f. Principal Component diagrams were computed for the odour attribute distribution with time (Fig. 5) and the variations of distribution are presented in Fig. 6a–c. The sensory results confirmed the gradual deterioration of all the sausages over a period of 12 days. However, the batch containing the combination of chitosan and low sulphite deteriorated less rapidly and was judged as more acceptable than all the other batches, including the control batch containing high sulphite (Fig. 4e and f; Fig. 6a–c). When presented with cooked sausages on Days 3, 8, 10 and 12, the trained panellists scored all the batches as ‘‘extremely acceptable’’ or ‘‘moderately acceptable’’ in terms of development of unfavourable odours (scores 1 and 2, respectively, Table 4) and there was little change during the course of the 12-days trial (results not illustrated). In terms of visual acceptability, the cooked sausages deteriorated slightly from being ‘‘slightly acceptable’’ and ‘‘moderately acceptable’’ on Day 3, to being ‘‘neither acceptable nor unacceptable’’ on Day 12 but again, there were no differences between the batches. The sensorial and microbiological differences between the raw batches of sausages were not reflected in the cooked products in terms of odour and appearance.
4. Discussion Darmadji and Izumimoto (1994) reported that chitosan at a concentration of 1.0% reduced microbial counts by an average of 1–2 log cfu/g (total numbers and pseudomonads, staphylococci, coliforms, Gramnegative bacteria and micrococci) in minced beef patties stored at 4 C for 10 days. Microbial counts were determined on only two occasions: Day 0 and Day 10. At lower chitosan concentrations (0.2 and 0.5%) and higher storage temperature (30 C), no inhibition of growth was observed. The number of viable organisms present in the meat used by Darmadji and Izumimoto (1994) at the start of their experiment was high (> 107cfu/g). This initial level of microbial contamination would not be acceptable for minced meat sold as such under the European Community Hygiene Directive (EC Council Directive, 1994). This specifies that the aerobic mesophilic count in minced meat is unacceptable if it exceeds 5 106 cfu/g in one or more samples out of every five taken. Youn, Park, Kim, and Ahn
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Fig. 4. Viable counts (a–d) and sensory properties (e and f) of pork sausages (Trial 2) containing chitosan and sulphite and stored at 7 C for 12 days after defrosting. Viable numbers were determined on Plate Count Agar (PCA) for total viable counts (a), deMan Rogosa and Sharpe Agar (MRSA) for lactic acid bacteria (b), Oxytetracycline Glucose Yeast Extract Agar (OGYEA) for yeasts and moulds (c) and Violet Red Bile Glucose Agar (VRBGA) for enteric bacteria (d). Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, !); Batch 4 Chitosan (0.6%, !); Batch 5 Chitosan and low sulphite (&). Data points are means of quadruplicate counts0.10 log cfu/g (maximum) for Figs. 4a–d and of at least 6 panellists scores each for Fig. 4e and f.
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Table 3 Sensory attributes developed for Quantitative Descriptive Analysis (QDA) of pork sausages Sensory attributes of sausages Fermenting Sausagey Vinegary
Musty Sour Winey
Rancid Sweet Yeasty
Table 4 Measurement scales developed for sensory attributes (Quantitative Descriptive Analysis, odour and appearance) of sausages QDA
Odour, appearance
Value Meaning
Value
Meaning
0 1 2 3
Non-detectable Weak Moderate Strong
1 2 3 4
4
Very strong
5 6 7
Extremely acceptable Moderately acceptable Slightly acceptable Neither acceptable nor unacceptable Slightly unacceptable Moderately unacceptable Extremely unacceptable
(1999) have reported inactivation of up to 2 log cfu/g of the total microbial flora in a sausage meat immediately upon addition of 0.35 or 0.5% chitosan; however, after storage at 30 C for just 1 day, total counts in the chitosan-treated sausage were the same as in the control. Storage temperatures other than 30 C were not studied by these authors (Youn et al., 1999). Unlike these studies, which offered little scope for the use of chitosan as a
preservative in meat products, the results in Figs. 1, 3 and 4 show that chitosan, in combination with low levels of sulphite, could be used effectively to inhibit microbial spoilage in the British fresh sausage. In recent years, some success with the application of bacteriocins in meat products, especially with respect to inhibition of L. monocytogenes, has been reported although this has usually been with production in situ in fermented products such as salami in which acidification is not a problem (Schillinger, Geisen, & Holzapfel, 1996; Schillinger, Kaya, & Lu¨cke, 1991). Lacticin 3147 in combination with sodium citrate or sodium lactate has been reported as an effective biopreservative in fresh pork sausage (Scannell, Ross, Hill, & Arendt, 2000). Nisin added at a level of 400 and 800 IU/g has been shown to extend the lag phase of L. monocytogenes inoculated into minced buffalo meat and stored at 4 C by 4 and 16 days, respectively (Pawar, Malik, Bhilegaonkar, & Barbuddhe, 2000). Although carnocins have been described in the literature as possessing a broad inhibitory spectrum (Jack, et al., 1996; Mathieu, Michel, Lebrihi, & Lefebre, 1994; O’Mahony, 2001; Stoffels, Sahl, & Gudmundsdottir, 1993), in our study the inclusion of a carnocin-containing fermented ingredient in fresh pork sausage failed to influence the rate of spoilage (Fig. 1). There are three possible explanations for this. Firstly, the initial level of bacteriocin added may have been insufficient, as reported previously in studies with bacteriocin-producing Lac. sake (Buncic et al., 1997). Secondly, it is possible that bacteriocin-resistant bacteria filled the ecological niche provided by the action of carnocin on more sen-
Fig. 5. Principal Component odour attribute distribution for raw pork sausages.
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Fig. 6. Principal Component odour attribute distribution replicas for pork sausages containing chitosan and sulphite after 1, 8 and 10 days of storage at 7 C. Key to symbols: Batch 1 Control (*); Batch 2 High sulphite (340 ppm, *); Batch 3 Low sulphite (170 ppm, ~); Batch 4 Chitosan (0.6%, ~); Batch 5 Chitosan and low sulphite (&).
sitive species. Finally, a number of other ingredients in the sausage mix, namely proteases, fats and sulphite may have had a negative impact on the activity of carnocin. The rapid loss in residual carnocin activity in the first 3 days of storage (Fig. 2) was similar to the 70% loss in nisin activity in raw meat during storage at 5 C for 4 days (Chung et al., 1989). Like nisin, carnocin may be susceptible to degradation by sodium metabisulphite, as reported by Thomas, Clarkson, and Delves Broughton (2000). According to microbiological criteria developed in the late 1990s, it is recommended that the total and yeast counts of raw sausages should not exceed 105 and 104 cfu/g, respectively, immediately following manufacture under Good Manufacturing Practice (GMP, IFST 1999). The total and yeast counts on the day of manufacture in all the batches of sausages prepared in this study were well within these recommendations (Figs. 1 and 3). In Trial 2, the total and yeast counts exceeded the 107 and 106 cfu/g recommended as the maximum acceptable
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levels by microbiological criteria for meat (IFST, 1999) in all the Batches except Batch 5 (containing the combination of chitosan and low sulphite) by Day 6 of storage at 4 C (Figs. 3 a and c). Notably, these criteria were not exceeded in the combination batch (Batch 5) until after Day 18 of the trial. It can be calculated, therefore, that the shelf life of the sausages was extended nearly three times by the presence of the combination of chitosan and sulphite. The relatively short shelf life of the sulphited sausages in both trials (as evidenced by the few days needed to reach unacceptable levels of both total and yeast counts) can be explained by the relatively low levels of addition of sulphite in this study (170 and 340 ppm at the time of production). Under British legislation (Anon., 1995), the maximum permitted level of SO2 in fresh sausage is 450 ppm at the point of sale. Due to considerable loss of preservative during manufacture and distribution, manufacturers routinely add up to 650 ppm sulphite during manufacture (Wilson, 1981). Most commercial sausages contain between 180 to 240 ppm total sulphite when analysed within 1–2 d of manufacture (Nick Walsh, RSSL, personal communication), consistent with higher levels having been added during production to compensate for rapid initial sulphite breakdown. The sulphite content of British pork sausages is determined traditionally by a method based on the collection of SO2 released from a sample suspended in boiling acid (Monier-Williams, 1927). Consequently, the sulphite content of sausages is conventionally expressed as ppm of free and/or total SO2. However, at the relatively neutral pH of sausages, the amounts of SO2 formed from metabisulphite are negligible. At the pH of sausages prepared in our study ( pH 6 on day of manufacture), sodium metabisulphite would have dissociated into bisulphite (HSO3 ) and sulphite (SO23 ) in a 94:6 ratio (Banks et al., 1987). Furthermore, it has been shown that sulphite is effective against microorganisms only when present in the free (unbound) form and that it is most potent at low temperatures. The free sulphite content and consequently the antimicrobial efficacy in sausages is known to diminish rapidly with time as a consequence of binding with acetaldehyde produced by spoilage yeasts (reviewed in Banks et al., 1987). It is well known that sulphite is most active against Gram-negative microorganisms, particularly the Enterobacteriaceae (Banks et al., 1987). In the present study, the shapes of the microbial and pH curves, the Gramstaining and oxidase results, the VRBGA counts and the residual sulphite analyses in the sausage batch containing the chitosan/sulphite combination (Batch 5) all suggest that selective inactivation and inhibition of Gram-negative bacteria had occurred. The mechanism of preservation by the chitosan/sulphite combination may be two-fold. Firstly, chitosan may have acted as a ‘‘slow-release’’ agent for sulphite,
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particularly free sulphite, thereby preventing its premature degradation or irreversible binding with other ingredients in the sausage. With a pKa of 6.3, approximately one-half of the chitosan present in the sausage on the day of manufacture would have been highly positively charged and could have interacted readily but reversibly with the negatively charged sulphite moieties. Secondly, any unbound chitosan may have selectively inhibited the yeast flora, thereby preventing acetaldehyde production and the consequent inactivation of sulphite by binding. The efficacy of the chitosan/sulphite combination could be explained therefore on the basis of protection from sulphite breakdown by chitosan. The sensory results demonstrated that the addition of chitosan to sausages would not lead to off-odours and that the appearance would not be rendered objectionable, either of which could potentially lead to rejection by the consumer. Chitosan has recently been affirmed as Generally Recognised as Safe (GRAS) by the US FDA (2001), thus removing some of the regulatory restrictions on its use in foods (Anon., 2001). Further work on novel combination systems such as the chitosan/sulphite combination described here would be facilitated by greater understanding of the mode of action of novel antimicrobials like chitosan on microorganisms. Finally, additional studies in real food systems are needed to assess the commercial potential of this natural antimicrobial compound.
Acknowledgements The following organizations are gratefully acknowledged for funding this work: European Commission (Contract FAIR CT96–1066), Meat and Livestock Commission UK, Danisco (Aplin and Barrett Ltd., UK), CPC (UK) Ltd., DSM Food Specialties (Netherlands) and Norsk Hydro (Norway).
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