1045

Journal of Food Protection, Vol. 62, No. 9, 1999, Pages 1045±1049 Copyright ᮊ, International Association of Milk, Food and Environmental Sanitarians

Research Note Numbers and Species of Motile Aeromonads during the Manufacture of Naturally Contaminated Spanish Fermented (Longaniza and )

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 Ͼ 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 ϭ 24), A. veronii biovar sobria (n ϭ 10), and A. caviae (n ϭ 2). All of them were beta-hemolytic and capable of growing at 5ЊC. 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 , 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 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 , 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 Ͻ with spices and seasonings. Longaniza is made in a manner 0.8%), and ( 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 4ЊC 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: ϩ34987291286; Fax: ϩ34987291284; ing estimated at 25 to 30ЊC), 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 10ЊCor 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 14ЊC). 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 20ЊC). 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 26ЊC 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 18ЊC 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 Ͼ 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- Њ AG, Darmstadt, Germany) agar (15) incubated 24 h at 28 C. tected numbers higher than 3.64 log CFU/g. In agreement Plates were examined for yellow colonies surrounded by a trans- 10 with the literature, raw pork samples were frequently con- parent halo (15), which were recorded as presumptive Aeromonas spp. For lactic acid bacteria (LAB), deMan, Rogosa, and Sharpe taminated with aeromonads and sometimes had high colony agar (Oxoid, Basingstoke, UK) pour plates overlaid with the same counts (5, 18, 24, 25). Because several workers (10, 28) medium were used. After incubation at 30ЊC for 3 days, ®ve col- have reported a low incidence of these bacteria in pig's onies from each counting plate were randomly selected for con- feces, intestinal carriage cannot explain their high preva- ®rmation purposes. Gram-positive, catalase-negative, nonsporing lence on pork. Carcass wash water is considered the most rods and cocci were considered LAB. likely source (5, 28); however, Gill and Jones (7) demon- strated that, in pig slaughtering plants, aeromonads grew Identi®cation of aeromonads. One to ®ve characteristic Aeromonas colonies from each of the 25 positive samples were well in the accumulated detritus of the dehairing equipment, selected, puri®ed, gram-stained, and differentiated from related or- with the organisms being spread throughout the dressing ganisms on the basis of the following tests: oxidase, glucose fer- and breaking lines, where they grew further. Therefore, if mentation, growth in nutrient broth without added NaCl, resis- the slaughtering plant operations and processing equipment tance to vibriostatic agent O/129 (Oxoid), DNase production on sanitation are inadequate, the possibility of pig carcass con- DNase agar (Oxoid), and acid from m-inositol (Sigma Chemical tamination appears to be high. Furthermore, the ubiquitous Co., St. Louis, Mo., USA). Reactions in Aeromonas hydrophila nature of these bacteria suggests that faulty sanitary prac- medium (14), motility, and growth at 5 and 37ЊC were also ex- tices at the meat processing industries introduce new op- amined. portunities for contamination. In this study, the hygienic For the identi®cation of 36 of the 39 isolates, the following status and technological conditions of processing plants ex- phenotypic tests were carried out: Voges±Proskauer; gelatinase erted a major in¯uence on the contamination rate and aero- production; acid from L-arabinose, sucrose, D-mannitol, and sali- Ͻ Ͻ cin (Sigma); aesculin (Sigma) hydrolysis; ornithine (Sigma) de- monads counts (P 0.001 after mixing, and P 0.01 at 3 days). We assumed that the poor control of raw materials carboxylase; H2S production from L-cysteine (Sigma); utilization of ammonium citrate (Merck); beta-hemolysis (sheep blood; Ox- (especially meat and fat), the inadequate sanitation mea- oid); and antibiotic sensitivity (cephalothin, colistin, carbenicillin, sures, and the initial handling and processing conditions J. Food Prot., Vol. 62, No. 9 AEROMONADS IN SPANISH SAUSAGES 1047

TABLE 1. Changes in pH, lactic acid (LA), lactic acid bacteria (LAB), and aeromonads counts during the manufacture and drying of chorizo and longaniza Samples Sausages Sampling positive for analyzeda (n) time (days) pH LAb LABc Aeromonadsd aeromonads

F1 (3)e 0 5.82 0.24 7.31 Ϯ 0.60 4.47 Ϯ 0.01 3 3 5.89 0.24 6.88 Ϯ 0.13 2.80 Ϯ 0.96 3 11 5.04 0.34 7.60 Ϯ 0.05 Ðg 18 5.13 0.48 7.15 Ϯ 0.04 Ð 25 4.83 NDf 8.79 Ϯ 0.06 Ð 32 5.10 ND 8.57 Ϯ 0.02 Ð F2A (3) 0 5.69 0.35 5.66 Ϯ 0.39 1.47 Ϯ 1.20 2 3 5.73 0.26 6.10 Ϯ 0.35 1.47 Ϯ 1.30 2 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 11 5.06 0.31 8.27 Ϯ 0.19 Ð 18 5.06 0.46 8.60 Ϯ 0.23 Ð 25 4.80 ND 9.05 Ϯ 0.44 Ð 32 5.19 ND 8.54 Ϯ 0.17 Ð F2B (3) 0 5.65 0.31 5.51 Ϯ 0.72 1.37 Ϯ 0.91 2 3 5.77 0.23 5.65 Ϯ 0.97 1.01 Ϯ 0.40 2 11 5.02 0.32 8.46 Ϯ 0.17 Ð 18 5.12 0.46 8.20 Ϯ 0.20 Ð 25 4.80 ND 9.08 Ϯ 0.15 Ð 32 5.01 ND 8.60 Ϯ 0.30 Ð F3 (5) 0 5.92 0.27 5.61 Ϯ 0.32 3.62 Ϯ 1.31 5 3 5.98 0.25 5.60 Ϯ 0.15 3.73 Ϯ 1.57 3 11 5.01 0.30 8.40 Ϯ 0.05 Ð 18 5.16 0.31 8.79 Ϯ 0.54 Ð 25 4.80 ND 8.95 Ϯ 0.07 Ð 32 ND 0.39 ND Ð F4A (5) 0 5.76 0.27 7.52 Ϯ 0.91 Ͻ11 3 5.94 0.23 7.14 Ϯ 0.61 Ͻ11 11 4.54 ND 9.01 Ϯ 0.78 Ð 18 4.60 0.52 9.34 Ϯ 0.68 Ð 25 4.58 ND 9.38 Ϯ 0.53 Ð 32 4.57 0.69 9.03 Ϯ 0.35 Ð F4B (5) 0 5.96 0.30 7.81 Ϯ 0.75 Ͻ11 3 5.88 0.32 7.87 Ϯ 0.59 Ͻ11 11 ND ND ND Ð 18 4.68 0.41 9.40 Ϯ 0.86 Ð 25 4.76 ND 8.37 Ϯ 0.24 Ð 32 4.27 0.54 9.05 Ϯ 0.55 Ð a F1, factory 1, longaniza; F2A, factory 2, chorizo picante; F2B, factory 2, chorizo dulce; F3, factory 3; F4A, factory 4, chorizo en barra; F4B, factory 4, chorizo en vela. b LA, lactic acid (%). c Ϯ log10 CFU/g (mean standard deviation). d Ϯ log10 CFU/g (mean standard deviation of positive lots). e Number of lots. f ND, not determined. g Ͻ Ð, undetectable levels ( 0.7 log10 CFU/g). were responsible for both the high incidence and numbers (17, 26). Other variables such as paprika variety had no obtained in samples from factories one and three. signi®cant (P Ͼ 0.05) effect. Signi®cant (P Ͻ 0.001) effects of casing diameter on Unlike factories one, two, and three, factory four yield- aeromonads levels were detected after three days. Casing ed only two (20%) positive mixture samples, with aero- diameter is important in controlling ultimate pH values of monad levels below 1 log10 CFU/g. The good hygienic and fermented sausages (1), but, after 3 days, the pHs of our technological conditions of this processing plant and the samples were not signi®cantly (P Ͼ 0.05) affected by this addition of lactic acid bacteria might not be the only rea- parameter. Reduced O2 levels seem to be more likely re- sons for this great difference; the potential contribution of sponsible for this effect, because the sensitivity of aero- added sorbates should be considered. Published reports (9, monads to NaNO2 increases under anaerobic conditions 22) demonstrate that Aeromonas spp. are sensitive to po- 1048 ENCINAS ET AL. J. Food Prot., Vol. 62, No. 9 tassium sorbate and that low temperatures and salt increase 2. Callister, S. M., and W. P. Agger. 1987. Enumeration and character- the effectiveness of this preservative. As factory four was ization of Aeromonas hydrophila and Aeromonas caviae isolated situated 200 km from our laboratory, refrigerated (7ЊC) from grocery store produce. Appl. Environ. Microbiol. 53:249±253. 3. Encinas, J. P. 1993. Identi®cacioÂn de riesgos microbioloÂgicos y pun- mixture samples containing sorbate levels of 1,000 ppm, tos crõÂticos durante la elaboracioÂn y maduracioÂn de permitted by Spanish law until 1997 (21), were shipped out fermentados. Ph.D. thesis. Universidad de LeoÂn, LeoÂn, Spain. under refrigerated conditions and examined up to 12 h after 4. Farmer III, J. J., M. J. Arduino, and F. W. Hickman-Brenner. 1992. mixing. The genera Aeromonas and Plesiomonas, p. 3012±3045. In A. Bal- The safety of sausages and their shelf life are depen- ows, H. G. TruÈper, M. Dworkin, W. Harder, and K. H. Schleifer (ed.), dent on the synergistic effect of several factors. Table 1 The Prokaryotes. A handbook on habitats, isolation, and identi®ca- tion of bacteria, 2nd ed. Springer Verlag, New York. demonstrates that, irrespective of the initial level, aeromon- 5. Fricker, C. R., and S. Tompsett. 1989. Aeromonas spp. in foods: a ads were destroyed or inactivated during the early stages signi®cant cause of food poisoning? Int. J. Food Microbiol. 9:17± of sausage manufacturing (lactic acid content over 0.30%; 23. pH values less than 5.07). This ®nding may help to explain 6. GarcõÂa-LoÂpez, M. L., M. Prieto, and A. Otero. 1998. The physiolog- results obtained by Gobat and Jemmi (8), who in a survey ical attributes of Gram-negative bacteria associated with spoilage of 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 conducted in Switzerland did not detect these organisms in meat and meat products, p. 1±34. In A. Davies and R. Board (ed.), The microbiology of meat and poultry. Blackie Academic and Pro- any of the 56 raw fermented sausages examined. fessional, London. A wide variety of media have been proposed for the 7. Gill, C. O., and T. Jones. 1995. The presence of Aeromonas, Listeria quantitative detection of motile aeromonads in a range of and Yersinia in carcass processing equipment at two pig slaughtering foods (4), starch ampicillin agar being considered ®rst plants. Food Microbiol. 12:135±141. choice by a number of authors (2, 13, 24, 28). In previous 8. Gobat, P.-F., and T. Jemmi. 1993. Distribution of mesophilic Aero- experiments (3), we proved that GSP agar was the most monas species in raw and ready-to-eat ®sh and meat products in Switzerland. Int. J. Food Microbiol. 20:117±120. effective (sensitivity and selectivity) for recovering these 9. Gram, L. 1991. Inhibition of mesophilic spoilage Aeromonas spp. organisms in fermented sausages. In this study, its ef®cacy on ®sh by salt, potassium sorbate, liquid smoke, and chilling. J. Food was satisfactory, as all but 3 (2 Bacillus and 1 yeast strains) Prot. 54:436±442. of the 39 presumptive isolates selected on the basis of their 10. Gray, S. J. 1984. Aeromonas hydrophila in livestock: incidence, bio- colony appearance were con®rmed as motile aeromonads. chemical characteristics and antibiotic susceptibility. J. Hyg. 92:365± These 36 strains were identi®ed as A. hydrophila (n ϭ 24), 375. ϭ ϭ 11. Hudson, J. A., and K. M. De Lacy. 1991. Incidence of motile aero- A. veronii biovar sobria (n 10), and A. caviae (n 2). monads in New Zealand retail foods. J. Food Prot. 54:696±699. More than one Aeromonas spp. were isolated from some 12. Ibrahim, A., and I. C. Mac Rae. 1991. Incidence of Aeromonas and samples. All of them were beta-hemolytic and capable of Listeria spp. in red meat and milk samples in Brisbane, Australia. growing at 5ЊC. The suicide phenomenon (23) was useful Int. J. Food Microbiol. 12:263±270. in differentiating A. hydrophila from A. veronii biovar sob- 13. International Commission on Microbiological Speci®cations for ria and A. caviae. Foods. 1996. Microorganisms in foods. 5. Microbiological speci®- As in other studies on the incidence of Aeromonas spp. cations of food pathogens. Blackie Academic and Professional, Lon- don. in raw pork (7, 11, 12, 24), A. hydrophila was the predom- 14. Kaper, J., R. J. Seidler, H. Lockman, and R. R. Colwell. 1979. Me- inant species recovered. 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Food Prot., Vol. 62, No. 9 AEROMONADS IN SPANISH SAUSAGES 1049

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