Prevention of Aspergillus ochraceus growth on and Ochratoxin a contamination of sausages using ozonated air

Mycotoxigenic moulds can grow on the surface of sausages and reduce the safety of these sausages for consumption. The aim of this study was to prevent the growth of Aspergillus ochraceus and the presence of Ochratoxin A (OTA) on the surface of Milano-type sausages using ozonated air. Spores of A. ochraceus were used to inoculate the casings of the sausages after casing. A portion of the lot (35 samples) was ripened in typical rooms, and another portion (35 samples) was dried and ripened in a separate room that was treated with gaseous ozone. The gas was delivered at night (8 h/day) at a concentration of ∼1 ppm. The temperature and relative humidity during the drying and ripening were the same for both rooms. Our results demonstrate that the gaseous ozone treatment prevented the growth of A. ochraceus and, consequently, the presence of OTA. In contrast, A. ochraceus grew and produced OTA on the untreated sausages. Moreover, the use of ozone did not influence the ripening, physico-chemical parameters, peroxide value or sensorial characteristics of the sausages.     

Effect of selected mould strains on lipolysis in dry fermented sausages

 Several batches of fermented sausages were prepared and inoculated with five strains of moulds belonging to the genera Penicillium and Mucor, selected for their enzymatic activity, and a strain of Penicillium nalgiovense obtained as a commercial starter culture. Control batches were manufactured similary, but without inoculation. Sausages were produced with either artificial or natural casings, and very similar results were obtained with both. The evolution of the different lipidic fractions was monitored, paying special attention to the free fatty acid (FFA) fraction. Two strains presented a high lipolytic activity as indicated by the rise in FFA in comparison to control batches. In conclusion, not all the mould strains contributed to the lipolytic process occurring during sausage ripening and, therefore, not all of these had a positive effect on the final quality of the product. If moulds are to be used for this purpose it is, therefore, necessary to carefully select suitable moulds beforehand. Received: 25 September 1998 / Revised version: 4 February 1999     

Mould starter cultures for dry sausages-selection, application and effects

The use of moulds on sausage surfaces can lead to both desirable and undesirable effects. The pursued effects are mainly related to successful production or consumer appeal. The undesirable effects are usually connected to growth of undesirable moulds. Most importantly, moulds may produce highly toxic secondary metabolites and β-lactams. Inoculations of sausages with moulds were traditionally done with the indigenous flora of the processing plants, the so-called houseflora, which was mainly composed of penicillia and aspergilli. The gradual shift in sausage production from small local producers to large-scale factories and increasing awareness of the risks for consumer safety has paved the way for industrialised procution of mould starter cultures. The industrialized starter cultures are carefully selected among hundreds of candidates going through multi-stage concepts including several analytical and biochemical investigations. While the technological aspects of mould inoculations are fairly resolved, its influence on prodution of secondary metabolites is yet emerging. Moulds produce enzymes for the degredation of lipid- and protein-matter, but studies show that the proteolytic and lipolytic capabilities differs significantly both between strains and is highly dependent on media, pH and temperature. Only few studies have addressed the aroma impact of mould growth on sausages, but information from related areas like milk based systems or sausage models still gives a quite clear picture of which components are important and their possible routes of information. One example is that methyl ketones play significant roles in the flavour of Blue cheeses. Here the presence and production of methyl ketones is ascribed to the β-oxidation activities of moulds and therefore investigations in sausages flavour have successfully focused on confirming this relationship and its aroma influence. Sausage producers are interested in using the established knowledge on aroma formation in sausages for improving aroma and texture but also for shortening ripening periods or even expanding shelf life. Addition of concentrates of enzymes have yielded some positive results but it is clear that the use of enzymes as additives in sausages production is not straightforward. To ensure optimal consumer safety starter cultures should be applied to achieve maximal controll over the mould population. Starter strains should under no circumstances show pathogenic or toxigenic signs in neither chemical or biological test. Additionally they should not be able to produce antibiotics.     

Volatile compound generation in dry fermented sausages by the surface inoculation of selected mould species

The effect of the inoculation of dry fermented sausage surface with an atoxigenic, proteolytic and lipolytic strain of Mucor racemosus, Penicillium aurantiogriseum and Penicillium camemberti on the volatile composition was studied. The analysis of the headspace volatile compounds using gas chromatography/mass spectrometry enabled the identification of 55 volatiles. The study showed that every mould species produced a different volatile profile which was also different from that of the control sausages. Compounds derived from amino acid catabolism, i.e. branched aldehydes and the corresponding alcohols, were produced in higher amounts in sausages inoculated with Penicillium spp. On the other hand, volatiles coming from the microbial esterification were related to sausages inoculated with M. racemosus. The development of the fungal mycelia on the sausage surface protected lipids from oxidation, thus giving rise to fewer lipid oxidation products in the inoculated sausages.     

Application and assessment of extruded edible casings manufactured from pectin and gelatin/sodium alginate blends for use with breakfast pork sausage

Casings formed from pectin (PN) containing 2.5% and 5% corn oil (CO) and olive oil (OO) and gelatine/sodium alginate blends (GSAB) containing 2.5% CO and OO respectively were used for sausage manufacture. Mechanical properties and water content of casings were assessed prior to application. Following sausage manufacture, product quality and shelf-life evaluation were assessed in terms of sensory attributes, instrumental colour, moisture loss and lipid oxidation. All manufactured casings were of good quality and initially produced intact and stable sausage products. However, with time, shrinkage of products occurred where GSAB casings were used. Sensory analysis of sausages showed that PN casings were more preferred to GSAB casings for sausage manufacture. CIE colour analysis supported sensory evaluation. Water losses from sausages using GSAB casings were lower compared to sausages using PN casings due to GSAB casings having higher ability to trap and absorb water compared to PN casings. Lipid oxidation developed over time for all sausage products, however, sausages manufactured using GSAB casings containing both CO and OO at 2.5% had better oxygen barrier properties compared to sausages manufactured using PN containing the same concentration of emulsified oils. Sausages manufactured using casings containing OO were more prone to lipid oxidation than those using CO. Importantly, sausages manufactured using PN casings containing 5.0% CO were extremely stable to lipid oxidation over a six day storage period and significantly, were <1 on the TBARS numbers scale.     

Comparison of Bacteriological Status During Ripening of Traditional Fermented Sausages Filled into Different Diameter Artificial Casings

The ripening process of fermented sausages is affected by diameter and type of sausage casings, and depends on changes in the microflora, important from hygienic and technological aspects. The aim of this study was to compare the bacteriological status sausages which were stuffed in artificial collagen sausage casings of different diameters (35 mm and 60 mm) during ripening and drying. The sausage stuffing was the same, as was the uncontrolled ripening conditions. In bigger diameter sausages, significantly higher average total bacterial count, enterobacteria and lactic acid bacteria counts were found than in smaller diameter sausages.     

Moulds and mycotoxins detected in the regional speciality fermented sausage ‘slavonski kulen’ during a 1-year production period

ABSTRACT

This study investigated the surface-to-interior mycotoxin contamination of the regional speciality meat product ‘slavonski kulen’ that occurred during a 12-month period and identified moulds present on its surface during the final production stage. In total, 15 pieces of slavonski kulen were produced and sampled at production months 0, 3, 6, 9 and 12 (three samples per piece). Concentrations of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) were determined using the ELISA method, while the identification of moulds made use of both traditional and molecular methods. A 12-month-long production of dry-fermented sausages under controlled conditions that did not include product cleaning at any point caused significant mycotoxin contamination, with determined mean AFB₁ and OTA concentrations of 11.79 ± 2.34 and 16.13 ± 3.32 µg kg^–1, respectively. Significantly higher mycotoxin levels were observed in the external layers of the products in comparison with their interior and, even more so, central parts. In total, five Penicillium species (P. polonicum, P. terrigenum, P. solitum, P. jugoslavicum and P. corylophilum) and two Aspergillus species (A. versicolor and A. sydowii) were isolated. Moulds responsible for the production of AFB₁ and OTA in the later production stages had probably been overgrown by non-toxicogenic moulds, so that the former did not last until the end of the sausages’ production process and were therefore not identified.     

Effect of trisodium phosphate on slip and textural properties of hog and sheep natural sausage casings

The defective gliding of certain natural casings during the stuffing of sausages is an important problem in the meat processing industry. The gliding behaviour of (defective) hog and sheep casings was assessed with a newly developed instrument, and by a technologist during the stuffing of sausages. Casings were treated with 0.01 M trisodium phosphate; control casings were untreated. Cooked and smoked sausages were made in hog casings treated or untreated with phosphate and subjected to compression tests. In all cases the treatment with phosphate clearly facilitated the gliding of the casings over the test pipes, as compared to the control casings. The instrument to measure the casing gliding properties did not provide reliable information about the actual stuffing of sausages. The phosphate-treated casings had a lower shear force than the control casings after being used as skins for cooked and smoked sausages. If confirmed, the finding that mild phosphate treatment can diminish the force required to shear a casing will be of interest to the sausage industry because the toughness of certain hog casings is considered a problem.     

Quality properties of sausage made with gamma-irradiated natural pork and lamb casing

Quality properties in emulsion-type sausage stuffed into irradiated natural casing were studied. Fresh salted and semidried natural pork and lamb casing was washed and irradiated at 0, 3, and 5 kGy by gamma-ray and emulsion-type pork sausage (Brattella Weiss Wurst) was manufactured. The sausage was stored in a 4°C refrigerator. The numbers of total aerobic bacteria, Enterococcus and coliform bacteria in the irradiated natural casing or sausage prepared from irradiated casing were significantly decreased or eliminated compared to those of the nonirradiated control. The D(10) values of total aerobic bacteria of the pork and lamb casing were 0.87 and 0.92 kGy, respectively. The vacuum-packaged sausages made with irradiated casings had a higher 2-thiobarbituric acid reactive substances value than that of the nonirradiated controls only at 5-day with pork casing and at 10-day with lamb casing. The total working force for shear of the sausages was decreased in both irradiated casings but the sensory evaluation showed no difference. Therefore, the gamma irradiation was a useful technique to sanitize the natural pork and lamb casings and to extend shelf-life, primarily microbial quality, of the sausage made with natural casings.     

The mycobiota of three dry-cured meat products from Slovenia

The surface mycobiota of three types of Slovenian dry-cured meat products were isolated from a total of 75 items of product that were sampled periodically during the drying/ripening stage of processing. The predominant filamentous fungal genus isolated was Penicillium. Eurotium spp., Aspergillus versicolor and Cladosporium spp. were isolated from only two of the products. Eight Penicillium species were identified. Penicillium nordicum was recovered frequently. Penicillium nalgiovense was recovered less frequently, from one product only (a salami), while a yet-to-be described species Penicillium “milanense” was isolated from 21 items. The other penicillia were rarely isolated. Of the isolated and identified species, those that can produce mycotoxins are: A. versicolor, Penicillium brevicompactum, Penicillium chrysogenum, P. nordicum, and Penicillium polonicum. Their growth on dry-cured meat products is undesirable.     

Fermented Sausage Casings

Casing is part of sausages, giving it shape, size and integrity, but also having a role in volumetric, structural and chemical changes which occur in sausage during different production phases. For fermented sausages, natural or artificial casings can be used. Artificial casings have an advantage from the hygienic point of view, because microbiological contamination is negligible, storage at low temperatures unnecessary, and there is no problem with product spoilage during storage and transport. Today, artificial sausage casings are a better choice for production of large diameter sausages, while they are equivalent to natural casings for production of small diameter sausages.     

Quantification of <Emphasis Type="Italic">Penicillium nalgiovense</Emphasis> on Dry-Cured Sausage ‘Salchichón’ Using a SYBR Green-Based Real-Time PCR

To evaluate the effective implantation of a specific protective culture of Penicillium nalgiovense, a real-time quantitative PCR (qPCR) using SYBR Green methodology was developed. Two specific primers were designed on the basis of the published partial sequences of the Internal Transcribe Spacer (ITS)1–5.8S-ITS2 region of various strains of P. nalgiovense. Using the developed method, a PCR product of 51 bp with a T _m value 81.34 °C was detected. T _m values of the amplified product allowed specific differentiation between P. nalgiovense and the remaining mould species tested. The developed qPCR method was tested on inoculated slices of dry-cured sausage (‘salchichón’) showing an efficiency of 97.24 %, a R ² value of 0.99 and a detection limit of P. nalgiovense of 1 log colony-forming units (cfu)/cm². The qPCR method demonstrated that the protective strain of P. nalgiovense grew and competed against an ochratoxin A (OTA)-producing Penicillium verrucosum strain on commercial dry-cured sausage. This qPCR method provides a specific, accurate and sensitive detection and quantification of P. nalgiovense on dry-cured sausage salchichón in order to estimate its colonization during their processing. This assay will improve strategies to prevent and control unwanted mould colonization and OTA risk in dry-cured meat commodities.     

Effects of selected natural preservatives on the mycelial growth and ochratoxin A production of the food-related moulds Aspergillus westerdijkiae and Penicillium verrucosum

ABSTRACT

The present study examines the influence of the natural preservatives carvacrol, eugenol, trans-cinnamaldehyde and the essential oil (EO) Origanum vulgare on ochratoxin A (OTA) production and the mycelial growth of two food-related moulds, Penicillium verrucosum and Aspergillus westerdijkiae, by broth macro-dilution assay for 21 days. With the addition of ½ minimum inhibitory concentration (MIC) carvacrol, eugenol and O. vulgare EO, the mycelial dry weight of both moulds decreased significantly over the whole incubation period of 7, 14 and 21 days. Trans-cinnamaldehyde slightly stimulated the growth of A. westerdijkiae and P. verrucosum at 14 and 21 days of incubation. Growth inhibition did not accompany inhibition of OTA production. Although the growth of both moulds was inhibited after the addition of ½ MIC carvacrol, eugenol and O. vulgare EO, the OTA production of the strong mycotoxin producer A. westerdijkiae was stimulated. Only trans-cinnamaldehyde inhibited the production of OTA with the addition of ½ MIC. P. verrucosum produced significantly less OTA than A. westerdijkiae, and its mycotoxin production was almost completely inhibited by the addition of ½ MIC of the natural preservatives.     

Microbial and chemical changes in 'Sobrasada' during ripening

‘Sobrasada’ is a raw-cured product typical of Mallorca (Balearic Islands). Throughout its ripening process the product, like other raw-cured sausages, undergoes a series of chemical and microbial changes which lead to the formation of its desirable final characteristics.In the ‘sobrasada’ studied, the breeds, feeding and casings used in the Balearic Islands were employed. The evolution of different groups of microorganisms was followed: mesophylic aerobic microorganisms, lactic acid bacteria, Enterobacteriaceae, yeasts and moulds, group D Streptococcus, proteolytic microorganisms and lipolytic microorganisms. It was found that the predominant flora, from the beginning to the end, was made up of lactic acid bacteria. Conversely, Enterobacteriaceae disappeared as ripening went on, and the rest of the microbial groups studied underwent little variation along the process.An important characteristics of the product is the fast fall of pH, from values near 6·0 to values around 5·3, in the first week.     

Reduction of Clostridium sporogenes spore outgrowth in natural sausage casings using nisin

Preservation of natural sausage casings using dry salt or saturated brine is regarded as sufficient to inactivate vegetative pathogenic non-spore-forming bacteria present on the casings. Although the outgrowth of bacterial spores is prevented by salt or saturated brine preservation, these spores will remain present and develop into vegetative cells when conditions are more favourable. To prevent subsequent outgrowth additional preservation measures should be implemented. In the experiments described the use of nisin was evaluated to reduce outgrowth of spores in desalinated casings. The bacteriocin nisin was chosen because of its known efficacy against spore-forming bacteria and their spores in various foodstuffs. Clostridium spore suspensions (Clostridium sporogenes, ATCC 3584) were used in two concentrations to inoculate three nisin concentrations (10, 50, 100 μg/mL) in water containing gamma-irradiated casings. Additionally, the binding of nisin to casings, using ¹⁴C-labeled nisin Z and subsequent availability of nisin were evaluated. Results demonstrate that nisin is partly reversibly bound to casings and can reduce the outgrowth of Clostridium spores in the model used by approximately 1 log₁₀ (90%). However, the biological relevance of these results needs to be determined further by conducting industrial trials before any recommendation can be made on the practical implementation of nisin in the preservation of natural sausage casings.     

春草莓表面霉菌鉴定及其致腐能力研究

目的调查春草莓表面霉菌的总体状况,对主要致腐真菌进行致腐能力和产毒素研究,为草莓的保鲜及食用安全评价提供理论依据。方法对60份春草莓样品进行霉菌检测,分离霉菌采用ITS序列测序方法进行鉴定;利用高效液相色谱法及酶联免疫法对腐烂草莓及真菌发酵液中赭曲霉毒素A(ochratoxin A,OTA)和展青霉素(patulin,PAT)进行检测,对出现率较高的霉菌进行草莓复接试验,判断其致腐能力。结果健康草莓表面的霉菌计数对数值3.5~5.5的占样品总数的83%;毛霉菌属、枝孢菌属和青霉菌属在草莓中出现率较高;由这3类霉菌致草莓腐烂样品和其发酵液均检测不出真菌毒素OTA和PAT,复接试验中毛霉属真菌在36 h在草莓表面形成明显菌斑,72 h可形成明显菌斑而更大面积软腐。结论草莓表面存在大量且多种霉菌,霉菌侵染草莓是导致其腐烂主要诱因,毛霉属霉菌占主要生态位但青霉菌属、枝孢菌属霉菌更易导致其软腐,草莓表面霉菌不会产生真菌毒素而对草莓进行二次污染。     

Effect of a post-packaging pasteurization process on inactivation of a Listeria innocua surrogate in meat products

Effects of surface pasteurization on inactivation of Listeria innocua were investigated. Surface temperature, monitored during post-packaging pasteurization, was used to predict the lethality of L. monocytogenes. Temperatures reached 70°C for lean and fat sausages within 9 min of treatment. An inoculation study validated the efficacy of post-processing and the thermal lethality of L. monocytogenes. Pre-cooked sausage and ham, inoculated with approximately 10⁷ CFU/cm² of L. innocua, were heated to a surface temperature of 70°C. Numbers of L. innocua were reduced by 7 log on surface-inoculated sausage. Guidelines for safe, ready-to-eat meat products are provided for small scale meat processors.     

Presence of ochratoxin A on the surface of dry-cured Iberian ham after initial fungal growth in the drying stage

Accumulation of ochratoxin A (OTA) on the surface and to a 0.5 cm depth of dry-cured Iberian ham after initial fungal growth was investigated. For this, 20 dry-cured Iberian hams from the drying stage showing incipient fungal growth on the surface were analyzed. In addition, the presence of OTA-producing molds was examined on the surface of the hams by real-time quantitative PCR (qPCR) based on the otanpsPN gene. Quantification of specific OTA-producing molds, such as Penicillium nordicum and Penicillium verrucosum was also achieved on the hams by specific qPCR methods. Ten of 20 dry-cured hams showed OTA at higher levels than those established by legal regulation. OTA was even detected in the deep section of hams. OTA-producing molds ranged from 1.5 to 7.3 log cfu/cm². Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum.     

Evaluation of natural hog casings modified by surfactant solutions combined with lactic acid by response surface methodology

The effects of soy lecithin concentrations (X₁, 1:25–1:30), soy oil concentrations (X₂, 0%–2.5%), residence time for surfactant solutions (X₃, 60–90 min) and lactic acid concentrations (X₄, 18–21 ml/kg) on technological properties of hog casings were evaluated by response surface methodology (RSM). The burst pressure, maximum rupture force, and elongation of both uncooked treated casings (UTC) and cooked treated casings (CTC) were determined. The histological structures of UTC and CTC were observed by light microscopy and transmission electron microscope and images illustrated that casings became more porous after treatments. Polynomial regression models on burst pressure of UTC (P < 0.05) and CTC (P < 0.05), maximum rupture force of UTC (P < 0.01) and elongation of CTC (P < 0.01) were established. Sausage was made using modified casing with the best resistance of burst pressure or rupture force. Bursting during immersion vacuum cooling did not happen to sausages made from these modified casings.     

Effects of composite surface coating and pre-drying on the properties of kabanosy dry sausage

Coating of dry sausages with renewable materials could be an alternative to vacuum packaging. In this study kabanosy dry sausage was coated with a composite emulsion and stored for 7 or 15 days at 4–6 °C. Effects of different emulsion formulas (0.5 or 1% w/w of kappa-carrageenan and 5 or 10% w/w of glycerol) and pre-drying of coated sausages (at 50 °C for 1.5 h) were investigated. Carrageenan concentration had a significant effect (P ≤ 0.05) on the amount of emulsion adsorbed on the sausage surface but little influence on the barrier properties of the coatings. At both glycerol concentration levels, coatings had no visible cracks and were easily removed from the sausage surface after 7 and 15 days of storage. The colour values of coatings (L*, a*, and b*) changed along with the decreasing water activity during storage. Pre-drying of coated sausages reduced peeled product weight loss after storage. The financial analysis showed that among coatings tested the best proved to be the emulsion containing (w/w): 5% glycerol, 5% gelatin, 0.5% carrageenan, 20% lard, 20% beeswax, and 50% water.