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«Improving food safety of sprouts and cold-smoked salmon by physical and biological preservation methods Dissertation zur Erlangung des Grades eines ...»

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Institut für Lebensmitteltechnologie

Fachgebiet: Allgemeine Lebensmitteltechnologie und –mikrobiologie

Prof. Dr. W. P. Hammes

Improving food safety of sprouts and cold-smoked

salmon by physical and biological preservation methods


zur Erlangung des Grades eines Doktors

der Naturwissenschaften

(Dr. rer. nat.)

der Fakultät Naturwissenschaften

der Universität Hohenheim

vorgelegt von

Alexander Weiss

aus Karlsruhe

Die vorliegende Arbeit wurde am 14.02.2007 von der Fakultät Naturwissenschaften der Universität Hohenheim als „Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften“ angenommen.

Tag der mündlichen Prüfung: 30.03.2007 Dekan: Prof. Dr. H. Breer Berichterstatter, 1. Prüfer: Prof. Dr. W. P. Hammes Mitberichterstatter, 2. Prüfer Prof. Dr. R. Carle

3. Prüfer: Priv. Doz. Dr. C. Hertel Meiner Familie gewidmet Contents General introduction Chapter 1 1 Outline Chapter 2 30 Thermal seed treatment to improve the food safety Chapter 3 32 status of sprouts Efficacy of heat treatment in the reduction of Chapter 4 43 – salmonellae and Escherichia coli O157:H on alfalfa, mung bean and radish seeds used for sprout production Characterization of the microbiota of sprouts and their Chapter 5 55 potential for application as protective cultures Lactic acid bacteria as protective cultures against Chapter 6 76 Listeria spp. on cold-smoked salmon Concluding remarks Chapter 7 88 Summary Chapter 8 96 Zusammenfassung Chapter 9 99 Lebenslauf

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Chapter 1 General introduction In the past decades the consumption of convenient food products has increased dramatically.

The consumption of 18 kg of convenience food per capita in Germany in 1985 has more than doubled up to 2005 (Anonymous, 2006). The sales for the sector of fresh-cut produce in the US for example have grown steadily from $5 billion in 1994, $6 billion in 1997, $12 billion in 2003 up to $15 billion in 2005 (IFPA, 2006). “Ready-to-eat-“(RTE) food belongs to one category of the group of convenience food which is defined as “intended to be eaten as purchased without further preparation by the consumer, particularly without additional cooking (FDA, 2001)”. The most problematic products thereof are those intended to be eaten raw. In general they can also be allotted to the group of minimally processed foods. Rolle and Chism (1997) define minimally processing of fruits and vegetables as operations that include washing, selecting, peeling, slicing etc. and that keep the food as a living tissue. Examples for RTE food of animal origin are seafood (e.g. fish salads, smoked fish), soft cheese and fermented sausages, examples of plant origin are vegetables (e.g. salads, seed sprouts) and fruits.

Based on the fact that in products eaten raw no germ reduction step is commonly applied, the consumer may be exposed to increased risks due to microbial contamination. Among these organisms Listeria monocytogenes, salmonellae or enteropathogenic E. coli (EHEC) are of primary importance. (Nguyen and Carlin, 1994; FDA 2001; FAO/WHO, 2003).

The ensurance of a continuous hygiene concept “from farm to fork”, based on Good Agricultural Practice (GAP) and Good Manufacturing Practice (GMP), is an exclusively means to obtain the product safety to a highest degree.

Due to the current situation mentioned above, it seems necessary to give special attention to the group of RTE foods. This coincides with initiatives of the FDA, WHO, as well as the European Commission (FDA 2001; WHO, 2002; Anonymous, 2002a). Especially in the U.S.

it is described in details. Federal food safety regulatory agencies consider the control of listeriosis in RTE food a priority initiative with the aim to achieve the 50% reduction by 2005.

Furthermore the federal government established a goal of working with industry and consumers to achieve an additional reduction in listeriosis of 50% by 2010 (DHSH, 2000;

CFSAN, 2001). In Europe the product group of fruits and vegetables eaten raw was included in a coordinated program for the food control in 2002 due to a recommendation (2002/66/EG)

Chapter 1 2

of the EU-committee (Anonymous, 2002b). Such concepts are in correspondence with the definition for Food Safety Objects (FSO), which have the focus on the target after analysing the problem (ICMSF, 2002).

There are specific problems that can be assigned to each of this product type. The microbial load of foods depends in principle on their bacterial contamination, possibilities of decontamination and the properties of the products which allow the bacteria to survive, to grow or to die. In the following the product related problematic and the state of knowledge will be described.

Microbiological safety of sprouts

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Within ready-to-eat foods of plant origin, differences exist between salads, fruits and sprouts with regard to the development of the native microbial load. The microbial load on salads and fruits is that present at the time of harvesting. Through processing (e.g. skinning, cutting, slicing etc.) the plant parts, dust as well as the bacterial load will be removed. Basically the flora from the field is of hygienic relevance for salads and fruits, a psychtrophic flora (responsible for spoilage) may be added during processing (e.g. water). Botanically the term sprout describes the plant part developing on the hypocotyle from the seed leaves. Mung bean sprouts for example are botanically not sprouts, but consist only of hypocotyle and seed leaves.

During manufacturing of sprouts, however, the bacterial load will increase because the conditions under which seeds are sprouted (2–7 days of sprouting, temperatures of 20–40°C and optimum water activity (near 1.0) are ideal for bacterial proliferation. At the first day of sprouting the total bacterial counts increase by 2 to 3 log units and attain a maximum level after 2 days (Fu et al., 2001). The native flora on the seeds is therefore of hygienic relevance, during sprouting the mesophilic bacteria will grow, psychrotrophic ones may be added during washing. The potential growth of human pathogens such as salmonellae and E. coli O157:H7 in these microbial communities is of major concern as seed sprouts have been implicated in several outbreaks of foodborne diseases, mainly caused by salmonellae and Escherichia coli O157: H7 (Table 3). The largest outbreak involved more than 6000 persons in Japan, and was associated with the consumption of radish sprouts. Therefore, in sprout production, the assurance of the absence of pathogens on seeds can be regarded as the critical control point, as defined by the Codex Alimentarius Commission (Anonymous, 1993).

Many studies have been performed to decontaminate seeds using methods such as irradiation, UV light, pulsed electric or magnetic fields, high pressure, heat treatments as well as disinfectants (FDA, 2001). Seeds have been soaked, dipped, sprayed and fumigated with a wide range of chemical compounds. Especially chlorine has been extensively tested (Jacquette et. al., 1996; Beuchat et al., 2001; Holliday et. al, 2001; Montvillle and Schaffner, 2004), and further agents used in studies were gaseous acetic acid ( Delaquis et al., 1999), ammonia (Himathongkham et al., 2001) calcinated calcium (Bari et al., 2003) and electrolyzed oxidizing water (Kim et al., 2003). Among the methods mentioned above, washing is of major importance. In Table 4 the efficacy of washing agents to decontaminate the seed are compiled.

To minimize the risk of food poisoning, it has been recommended by the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) to achieve a 5-log reduction of Table 3 Examples of outbreaks of foodborne diseases associated with raw seed sprouts

–  –  –

pathogens on seeds used for sprout production (NACMCF, 1999). As it had been shown that treatment with 20,000 ppm calcium hypochlorite may be adequate (Beuchat et al., 2001; Fett, 2002), this type of treatment is in the US commonly used for seed disinfection. However, in certain countries such as Germany, the application of chlorine or other disinfectants for the production of organic food is not accepted. An alternative is a hot water treatment of the seeds. Such decontamination step can be seen as a fist hurdle to ensure the food safety of sprouts.

A second hurdle may be the application of biological methods such as the use of antagonistic plant ingredients as well as the addition of protective cultures. These may have a sustainable effect to prevent the growth of organism during the production process.

In general, biopreservation consists of the following principle: 1) the use of organisms to control growth of spoilage flora as well as pathogenic species 2) the use of microbial antagonistic compounds to control microbial growth and negatively affect viability of pathogens 3) natural plant defence principle such as microbial attack-induced resistance.

Finally, non-pathogenic microorganisms that can compete with pathogens for physical space and nutrients (Parish et al., 2003).

There are a few published reports on the use of biocontrol agent, such as protective cultures, to prevent growth of human pathogens on sprouts. In studies described by del Campo et al.

(2001) and Palmai and Buchanan (2002), the efficiency of selected strains in model media have been tested. Enomoto (2004) studied the effect of Enterobacteria against Pseudomonas fluorescens. Up to now no isolates from sprouts or the sprouting environments were effective in praxi to prevent the growth of pathogens in the course of the sprouting process.

Numerous studies have been performed to improve the food safety of sprouts, which were described above. None led to satisfactory results. The combination of a hot water treatment of seeds and the use of protective cultures was regarded as a more efficient method. The topic of this study was to prove the hypotheses.

Microbiological safety of cold-smoked salmon

Cold-smoked salmon belongs together with fish salads and other smoked fish products to the group of ready-to-eat seafood. Food of animal origin is in general subjected to a greater hygienic risk than food of plant origin, as it may contain bacteria that cause zoonotic diseases.

The native microflora on water fish and shellfish is dominated by psychrotrophic or psychrophilic gram-negative bacteria belonging to the genera Pseudomonas, Moraxella,

Chapter 1 9

Acinetobacter, Shewanella and Flavobacteria (Liston, 1990). Gram positive organisms such as Bacillus, Micrococcus, Clostridium and Corynebacterium have also been found in varying proportions. Lactic acid bacteria are seldom part of the dominant flora, but are present at levels of 10-1000 cfu/g (Rachman et al., 2004). The microbial flora of freshly harvested farmed fish is similar to the flora found on wild fish. However, as the microflora is a reflection of the environment, it is not surprising that aqua cultured fish are more likely to be contaminated with certain non-indigenous species, because of the closer proximity of fish farms to human or animal populations and their waste (Shewan, 1977). Huss et al. (1995a) have shown that L. monocytogenes is frequently isolated from fresh water and polluted seawater, but not from virtually unpolluted ocean or spring water.

Listeria monocytogenes is a short (0.5 µm in diameter by 1 to 2 µm long) gram positive, nonspore-forming rod. Infections of humans can result in listerioses, a disease whose symptoms include septicaemia, meningitis and abortion. Cells of L. monocytogenes, ingested with the food, cross the barrier of the intestinal tract and are then engulfed by white blood cells and transported to different parts of the body. The bacteria are able to grow and multiply within cells and spread directly from cell to cell. All strains of L. monocytogenes appear to be pathogenic but their virulence, as defined in animal studies, varies substantially (Farber, 2002). Listerioses is an opportunistic infection that most often affects those with severe underlying disease, pregnant women and the elderly. Important characteristics of this organism are its ability to grow at temperatures of 1-45°C, at pH-values of 4.3-9.5, at water activity of 0.90, and in salt concentrations higher than 10% (Farber et al., 1992;

Tienunggoon et al., 2000) The microflora of a fish product such as cold-smoked salmon reflects the indigenous flora itself, the microflora of the processing environment as well as the conditions during manufacturing and storage. The manufacturing steps of cold-smoked fish often reduce the number of microorganisms exert a strong influence on the composition of the microflora.

Generally, Pseudomonades and Shewanella putrefaciens become predominant during aerobic storage conditions (Gram et al. 1987), whereas vacuum or CA-conditions favour the growth of lactic acid bacteria, psychrotrophic Enterobacteriaceae or marine vibrios (Gram and Huss, 2000). Among foodborne pathogens that may be present on cold-smoked salmon, L.

monocytogenes is of special concern. Several studies have reported a prevalence of 6-36% in cold smoked salmon and other RTE fish products (Ben Embarek, 1994; Lyhs et al., 1998;

Dominguez et al., 2001; Becker et al., 2002; Farber, 2002; Gombas et al., 2003; FDA, 2003;

Nakamura et al., 2004).

Chapter 1 10

The experience has shown that contamination mostly occurs after catching, mainly at handling on board, and later along the production chain. Colburn et al. (1990) found Listeria species in 81% of freshwater samples and 30% of estuarine cost water samples. Due to the studies of Eklund et al. (1995) and Rorvik (2000), initial and most important source of contamination in processing plants is the surface (skin, mucus, tail head) and the intestinal cavity of the fish at the time of decapitation, peeling and filleting (Dauphin et al., 2001;

Hoffmann et al., 2003). In contrast to these findings, Bagge-Ravn et al. (2003), Gall et al.

(2004), Lappi et al. (2004a) as well as Nakamura et al. (2006) showed in their studies that Listeria monocytogenes during production process appears to be a major source of finished product contamination. The strains persist at plants, proliferate in the environment during warmer seasons, and contaminate products during manufacturing processes (Vogel et al., 2001).

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