1045 Journal of Food Protection, Vol. 62, No. 9, 1999, Pages 1045±1049 Copyright Q, International Association of Milk, Food and Environmental Sanitarians Research Note Numbers and Species of Motile Aeromonads during the Manufacture of Naturally Contaminated Spanish Fermented Sausages (Longaniza and Chorizo) JUAN P. ENCINAS, CEÂ SAR J. GONZAÂ LEZ, MARIÂA-LUISA GARCIÂA-LOÂ PEZ, AND ANDREÂ S OTERO* Department of Food Hygiene and Food Technology, Veterinary Faculty, University of LeoÂn, E-24071, LeoÂn, Spain Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1045/2007730/0362-028x-62_9_1045.pdf by guest on 02 October 2021 MS 98-157: Received 30 June 1998/Accepted 5 March 1999 ABSTRACT The prevalence of aeromonads in the initial mixes of 14 batches of chorizo and longaniza obtained from three small- and . middle-sized factories was 78.5%, with counts ranging from 1.00 to 4.47 log10 CFU/g. Only 2 of 10 mixture samples prepared at a large and modern processing plant yielded these organisms, with levels below 1 log10 CFU/g. The hygienic status of factories signi®cantly affected incidence and counts. Of 39 presumptive isolates from glutamate starch penicillin agar, 36 were con®rmed as motile aeromonads and allocated to Aeromonas hydrophila (n 5 24), A. veronii biovar sobria (n 5 10), and A. caviae (n 5 2). All of them were beta-hemolytic and capable of growing at 58C. Regardless of initial contamination, aeromonads were rapidly inactivated during the early stages of manufacture. Certain motile aeromonads can cause gastroenteritis in of naturally contaminated chorizo and longaniza, to deter- humans (4, 17). These organisms constitute a heteroge- mine the effect of certain factors (i.e., hygienic status of neous group of strains able to grow at refrigeration tem- the factory, casing diameter, etc.) on contamination rate and perature. This and other characteristics (i.e., ability to grow aeromonads populations, and to identify isolates to species anaerobically) enable them to become a component of the level. spoilage micro¯ora of high pH raw meat, especially when MATERIALS AND METHODS packed under a modi®ed atmosphere (6). Aeromonas spp. are ubiquitous, and strains possessing virulence-associated Sausages. Three lots of longaniza and 21 lots of ®ve chorizo properties are readily found in pork, often in relatively high varieties produced by four different manufacturers were studied. numbers (12, 24). Although it has been suggested that salt Three of the factories were of small and medium size (regarding content and other parameters may prevent growth of aero- the number of employees and the level of production) and used monads in some pork products (24), little information is more traditional (artisanal) procedures (no additives other than available on the behavior of these bacteria during manu- curing salts, no starter, scarce control of drying conditions, and no analytical control of meat and fat). The fourth factory was a facture and drying of fermented sausages. large-sized factory using highly developed (industrial) technolog- Spanish processing factories produce nearly 100,000 ical processes (addition of carbohydrates and sorbates, use of start- tons of ``chorizo'' per year (20). This dry sausage is made er culture, narrow control of drying conditions, and analytical con- from chopped lean meat (mainly pork or a mixture of pork trol of meat and fat). According to data on visual inspection of and beef) and pork fat seasoned with paprika, garlic, oreg- plants and processes, the hygienic statuses of factories one, two, ano, salt, etc. (19). Over 30 different varieties of chorizo, and three were very similar to each other; all were of a lower many with regional characteristics, are manufactured by level than that of factory four. both large enterprises and small plants (19). The initial mixture of all lots of sausages contained lean pork Longaniza is the generic term for a fermented sausage (70 to 75%), pork back fat (25 to 30%), sodium chloride (2 to made from ®nely minced lean and fat pork mixed together 3%), sodium nitrite (100 ppm), paprika (1 to 2%), garlic (0.5 to , with spices and seasonings. Longaniza is made in a manner 0.8%), and oregano ( 0.01%). Garlic (dehydrated bulbs of Allium sativum) and oregano (dried and ground leaves of Origanum spp.) similar to chorizo, but it is thinner and the ends are tied were from several Spanish provinces. Both hot and mild varieties together like a horseshoe (19). The varieties (at least nine) of paprika (dried and ground ripe fruit of Capsicum annum) were also have regional characteristics, and production is esti- from La Vera region (CaÂceres, Spain). mated at 13,000 tons. In factory one, longaniza mixture (without starter culture) The aims of this study were to investigate numbers of was stored at 48C for 24 h, stuffed into natural pork casings (20 Aeromonas spp. present during the manufacture and drying to 22 mm in diameter), smoked in closed rooms where oak was being burned in a perforated container (temperature of rooms dur- * Author for correspondence. Tel: 134987291286; Fax: 134987291284; ing smoking estimated at 25 to 308C), and matured in well-ven- E-mail: [email protected]. tilated rooms under natural climatic conditions. 1046 ENCINAS ET AL. J. Food Prot., Vol. 62, No. 9 Six batches of chorizo were obtained from factory two. The and ampicillin; AntibioÂticos S.A., LeoÂn, Spain) (4, 17, 27). These sausage mixture (no starter added) was held for 48 h at 108Cor 36 isolates were also inoculated into the API20E and API20NE lower, ®lled into 30- to 40-mm-diameter natural pork casings, kits (BiomeÂrieux EspanÄa S.A., Madrid, Spain) (29). The ``suicide smoked as in factory one, and placed in climate chambers (70 to phenomenon'' was investigated as described by Namdari and Bot- 80% relative humidity [RH] and 10 to 148C). Three of these batch- tone (23). Adscription to species was carried out according to the es were ``chorizo picante'' (seasoned with hot paprika), while the schemes of Farmer et al. (4) and Kirov (17). remaining batches were ``chorizo dulce'' (seasoned with mild pa- prika). pH and acidity. pH values were measured by inserting the Five lots of nonsmoked chorizo dulce produced by factory spear electrode of a pH meter (Crison 501, Barcelona, Spain) into three were also studied. These sausages (30 to 40 mm in diameter) the samples (batter and center of sausages). Recorded values of were produced by traditional procedures (natural fermentation and pH were the mean of readings in two different locations within drying during wintertime at room temperatures, estimated at 10 the mixture or within the sausage. The con®guration and amount to 208C). Because of the lack of both the smoking process and of lactic acid were determined enzymatically by using D-lactate the lack of control of RH and temperature during ripening, a sig- and L-lactate dehydrogenase (Boehringer GmbH, Mannheim, Ger- ni®cant surface fungal growth was allowed to develop. many). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/9/1045/2007730/0362-028x-62_9_1045.pdf by guest on 02 October 2021 Starter culture (Pediococcus damnosus and Carnobacterium Statistical analysis. The in¯uence (on pH values and on mi- piscicola, prepared daily by the manufacturer), dextrose (0.2%), crobial counts) of the main technological characteristics varying and potassium sorbate (1,000 ppm) were added to the initial sau- between lots of sausages was statistically estimated by Student's sage mixture prepared in factory four. The product was fermented t test (comparison of means, after log transformation in microbial at 24 to 268C and 80 to 90% RH for 1 to 2 days and then dried counts), which was performed with an SPSS/PC software pack- under controlled conditions (14 to 188C and RH progressively age, version 1.0, SPSS Inc., Chicago, Ill. Selected variables were: decreasing to 60 to 70%). This large and modern enterprise pro- (i) type of process (artisanal versus industrial), (ii) smoking, (iii) duced two types of chorizo, the main differences being the casing sausage diameter (thin [30 to 40 mm] versus thick [50 to 80 mm]), diameter and shape. From this factory, ®ve lots of ``chorizo en and (iv) type of paprika (hot versus mild). barra'' (80 mm in diameter) and ®ve lots of ``chorizo en vela'' (50 mm in diameter) were analyzed. RESULTS AND DISCUSSION Sampling and microbiological analysis. From each lot, 200 Data obtained (Table 1) indicate that sausage mixtures to 300 g of product was removed from the initial sausage mix (chorizo and longaniza) prepared in the small- and middle- (day 0) and from the sausages at 3, 11, 18, 25, and 32 days after the preparation of the initial mixture. Samples were homogenized sized manufacturing plants (factories one to three) had a by blending 100 g of the product (the sausages without casings) high overall incidence of aeromonads, with 78.5% of sam- with 400 ml of sterile buffered peptone water (0.1% w/v) con- ples being positive. Table 1 also shows that the initial mean taining Tween 80 (1% w/v) in a Stomacher Model 400 (A. J. levels of these bacteria in contaminated lots from these . Seward & Co., Ltd., London, UK) for 2 min. Further 10-fold plants ranged from 1.00 to 4.47 log10 CFU/g. The prev- dilutions were prepared in the same diluent and plated (1 ml dis- alence of contamination is similar to that found by Okrend tributed over 3 plates) on appropriate media. Aeromonad counts et al. (24) in ground pork (80%) and by Ibrahim and Mac were determined on glutamate starch penicillin (GSP; E. Merck Rae (12) on different cuts of pork (74%); the former de- 8 AG, Darmstadt, Germany) agar (15) incubated 24 h at 28 C.