Selected characteristics of micrococci isolated from Spanish dry fermented sausages

The flora development of two batches of Spanish dry sausages has been studied. Lactic acid bacteria and organisms growing on Mannitol—Salt agar increased during the first five days, remaining at constant levels afterwards. From Mannitol—Salt agar, 629 strains were isolated. The maximum recovery of Gram-positive, catalase-positive cocci was between 5 and 15 days of ripening and after 20 days no micrococci were detected. All isolates of nitrate reducing micrococci (42 strains) showed an acceptable growth at temperatures (19–25°C) and water activities (0.93–0.95) of the sausages during the fermentation step. From the quantitative determination of the nitrate reduction ability, six strains were selected as potential starters for industrial sausage production.     

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     

Characteristics of lactobacilli isolated from dry fermented sausages

A study of the microflora of Spanish dry fermented sausages showed a similar pattern to that observed in other sausages during ripening. Lactic acid bacteria and organisms growing on Mannitol-salt-agar increased during the first five days, remaining at constant levels afterwards. From MRS agar, 368 strains were isolated and characterized. All Lactobacillus strains (343) could be separated into three groups according to their reactions in the API-50 L system. The biochemical characteristics of the Lactobacillus strains analyzed were those typical of isolates from sausages and other meat products. The extracellular and intracellular lipolytic activities of some strains of the three groups were also studied. It is concluded that lipolytic activity of lactobacilli is mainly directed against mono- and diglycerides and short-chain fatty acid triglycerides.     

Effect of selected mould strains on the sensory properties of dry fermented sausages

The effect of one Mucor and two Penicillium strains (Penicillium-3 and Penicillium-6) on the development of the sensory properties of dry fermented sausages has been studied. These strains were previously isolated from Spanish fermented sausages and selected for their proteolytic and lipolytic activity. Several experimental batches were prepared: one of them was non-inoculated and considered as control batch, three were inoculated with the selected strains, and the last one was inoculated with a commercial starter culture of P. nalgiovense. The pleasantness of the sensory attributes of the different batches were determined using an unstructured line scale of 10 cm. A triangular test was also carried out. Apart from their external appearance, batches inoculated with Mucor and Penicillium-3 had the highest scores for all the sensory attributes studied. The batch inoculated with P. nalgiovense had the lowest scores and similar to the control batch but its external appearance was considered to be the most pleasant. The batch inoculated with Penicillium-6 presented intermediate characteristics. A relation between these results and the enzymic activity of the moulds was established. The three strains selected for this study are proposed to be incorporated in a commercial starter culture because they improve the sensory properties of these meat products.     

Technological activities of Staphylococcus carnosus and Staphylococcus simulans strains isolated from fermented sausages

The aim of this study was to determine the technological properties of 2 strains of Staphylococcus simulans (Ssm12, Ssm21) and 4 strains of S. carnosus (SC28, SC31, SC54 and SC55) for the selection of a potential starter cultures to employ in the processing of dry fermented sausages. The strains were studied to evaluate nitrate reductase, proteolytic, lipolytic, decarboxylase and antioxidant activities as well as growth ability at different temperatures, pH and NaCl concentrations. Nitrate reductase activity was determined at 15, 20 and 30 °C. By spectrophotometric method all the strains were able to reduce nitrate to nitrite at the different temperatures but these results were not confirmed by the agar plate method. Antioxidant and lipolytic activities were evaluated by spectrophotometric assay. All the strains showed antioxidative enzymes superoxide dismutase (SOD) and catalase whereas all appeared unable to hydrolyse pork fat. Proteolytic activity was determined by agar plate method, spectrophotometric assay (OPA) and sodium dodecyl sulphate gel-electrophoresis (SDS–PAGE) and all strains appeared to be able to hydrolyse sarcoplasmic proteins but not myofibrillar proteins. Finally, all the strains grew at 15 and 20 °C, in presence of 10%, 15% and 20% of NaCl and at pH 5.0 and 5.5 and were unable to produce histamine, cadaverine and putrescine. The results showed that all strains studied possess useful technological activities that would make them eligible as a good starter cultures for fermented sausages.     

Evaluation of different systems for the identification of Bacillus strains isolated from Spanish fermented sausages

Sixty-nine isolates obtained during the manufacture and ripening of Spanish fermented sausages were identified to species level using the Vitek Bacillus biochemical card, the dichotomous key and table proposed by Berkeley et al. ((1984). In Methods in Microbiology, Vol. 16. Academic Press, London, p. 291), morphological and physiological tests and the API 20E miniaturized system. None of the tested systems was entirely satisfactory and the final identification was mainly done on the basis of cellular morphology and the table of test results. Our isolates belonged to the species: B. subtilis (37), B. megaterium (22), B. pumilus (5), B. circulans (3) and unidentified (2). Forty-five cultures (65.2%) were accurately identified with the dichotomous key. A similar figure for the Vitek Bacillus biochemical card was 36%. The results of the API 20E system were very reproducible, especially those of the Voges-Proskauer test. Most of the strains of B. megaterium were misidentified as B. subtilis with the dichotomous key. On the other hand, a high percentage of the cultures belonging to B. subtilis were misidentified as B. megaterium with the Vitek system.     

Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages

Lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities under conditions that eliminated the effects of low pH and hydrogen peroxide. From 720 isolates tested 119 were inhibitory to Lactobacillus fermentum CECT285. The isolates showing the largest inhibitory activity exhibited an antagonistic effect against several other lactobacilli and the selected foodborne pathogens Staphylococcus aureus and Listeria monocytogenes. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. The isolates were tentatively characterized as Lactobacillus sake. One of the isolates, L. sake 148 was chosen for further study. The compound excreted by L. sake 148 was active against various lactobacilli and several Gram-positive foodborne bacteria, but not against the Gram-negative bacteria tested. The antagonistic effects were almost eliminated by treatment with proteases, whereas they were heat resistant and bacteriostatic rather than bacteriocidal.     

从自然发酵风干肠中分离出的葡萄球菌生产性能的研究

从自然发酵风干肠中分离出五株葡萄球菌,通过菌种的耐亚硝酸盐能力、耐酸能力、生长温度和最适生长温度、蛋白质及脂肪降解活性等实验,对其生产性能进行研究。结果表明,木糖葡萄球菌(Staphylococcu sxylosus,S2)能在0.015‰亚硝酸盐中生长;耐pH为3的酸性环境;不耐65℃的高温,烘烤后可被灭活;30～35℃内旺盛生长;具有降解蛋白质和脂肪的能力,符合作为肉制品发酵剂的要求。      

Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits

In this study, three starter formulations including Lactobacillus curvatus and Staphylococcus xylosus strains selected in vitro on the basis of their lipolytic and proteolytic activities were employed for the manufacture of traditional fermented sausages of southern Italy. Microbial population, proteolysis, lipolysis, changes in free amino acids (FAA) and free fatty acids (FFA) and development of characteristic taste and flavor of the final product were investigated. Proteolysis and lipolysis were observed in sausages inoculated with proteolytic and lipolytic S. xylosus coupled with L. curvatus, while the sausage started with only S. xylosus without lactobacilli was identical to the non-inoculated control, indicating that the proteolysis could be due to both microbial activity and endogenous proteases activated by the decrease in pH. The statistical analysis applied to the instrumental and sensory data showed that there was an effect of the starter used on the characteristics of the sausage obtained. In particular, the control samples showed very close features different from the sausages obtained by adding starter cultures. Finally, analyzing the sensory parameters the sausages ripened without starter addition and those started without the L. curvatus AVL3 showed similar features indicating an influence of the presence of the lactobacilli on the final organoleptic quality of the sausages. An appropriate choice of a combination of strains in a starter formulation is fundamental to obtain products of the expected quality.     

从风干肠中分离出的三株乳酸菌发酵性能的研究

对从自然发酵风干肠中分离出的三株乳酸菌的发酵性能进行研究,主要包括菌种的耐盐性试验、耐亚硝酸盐试验、生长温度的测定、产酸速率的测定、蛋白质及脂肪降解活性试验、拮抗性试验。结果表明:分离乳酸菌1号菌的耐热性强,可能在烘烤后继续发挥产酸效力,因此不适合作为发酵剂使用;2号菌、3号菌则可在6%的食盐浓度下和0.15g/kg的亚硝酸钠下生长,在65℃以上完全不生长,产酸速度快,不分解蛋白质和脂肪,相互之间无拮抗作用,完全符合肉制品发酵剂的要求。     

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.     

Characterization of the headspace volatile compounds of selected Spanish dry fermented sausages

The volatile profile of three different Spanish dry fermented sausages (`salchichón', `fuet' and `chorizo') was studied using dynamic capillary gas–liquid chromatography/mass spectrometry (GC–MS) headspace analysis. Complex mixtures of esters, ketones, alcohols, aldehydes and terpenes characterized retail samples of each of these products. Esters were dominant components of the volatile compounds from one type of salchichón (salchichón 2), terpenes of the other type (salchichón 1) and of one type of fuet (fuet 2). Common to both types of fuet was a number of ketones and alcohols. `Chorizo' had similar high numbers of esters as `salchichón 2' but also had three sulphur-containing compounds which are known components of garlic and are used as an ingredient in chorizo.     

A survey of antibiotic resistance in Micrococcaceae isolated from Italian dry fermented sausages

The transfer of bacteria that are resistant to antimicrobial agents or resistance genes from animals to humans via the food chain is increasingly a problem. Therefore, it is important to determine the species and the numbers of bacteria involved in this phenomenon. For this purpose, 148 strains of microstaphylococci were isolated from three types of Italian dry fermented sausages. Eight of 148 strains belonged to the genera Kocuria and Micrococcus. The remaining 140 strains belonged to 11 different species of the genus Staphylococcus. The species most frequently isolated was Staphylococcus xylosus, followed by Staphylococcus saprophyticus and Staphylococcus aureus. Antibiotic resistance levels differed among the species and depended on the strain origin. Microstaphylococci were generally susceptible to beta-lactams, but 12 strains were resistant to methicillin, 8 were resistant to oxacillin, and 9 were resistant to penicillin G. No resistance was observed for aminoglicosides and cephalosporines. Many strains were resistant to sulfonamide, colistin suphate, tetracyclin, and bacitracin. Two strains of S. aureus, four strains of S. xylosus, and one strain of Staphylococcus sciuri were able to grow in the presence of 8 microg of vancomycin per g, but all strains were susceptible to teicoplanin. Twenty-two microstaphylococci were resistant to at least five of the tested antibiotics. The multiresistant strain S. aureus 899 was unaffected by eight antibiotics, including vancomycin and methicillin, indicating that a more prudent use of antibiotics in animal husbandry and better hygienic conditions during production should be encouraged because they can play a major role in reducing the incidence of such multiresistant microorganisms and the possible spread of the genetic elements of their resistance.     

Yeast populations on Spanish fermented sausages

Yeast populations on 24 lots of Spanish fermented sausages, made by four factories (F1, F2 and F4, artisanal; F3, industrial) were investigated throughout manufacture and the influence of different variables evaluated. In addition, 41 yeast strains were identified at the species level using two miniaturised systems: ATB32C (API System) and Vitek Yeast Biochemical Card (Vitek YBC). Levels of yeasts found in the sausage mixture (mean counts around 4 log units/g) were similar to those described by other authors. In sausages from factories F1 and F2, a further increase was noted, reaching 5.5 log units/g after fermentation. Counts subsequently decreased to 3.6 and 5 log units/g, respectively. In sausages from factories F3 and F4, decreasing counts were observed from the beginning, particularly in sausages from F3, where yeasts were almost absent in the finished product. Type of manufacture and sausage diameter, were the variables most influencing yeast counts. Debaryomyces hansenii (teleomorph of C. famata) was the dominant species, being found at all stages of manufacture. Trichosporon ovoides (formerly T. beigelii), Yarrowia lipolytica (perfect form of C. lipolytica), C. intermedia/curvata, C. parapsilosis, C. zeylanoides and Citeromyces matritensis (teleomorph of C. globosa) were also present. Direct identification was possible only with 50% of the total of strains investigated, although a higher number of strains was identified using the API than the Vitek YBC system.     

Accelerated ripening of dry fermented sausages

Research on accelerated ripening of dry fermented sausages started in the early 1990s. Fermented sausages manufacture is a very important part of meat industry in many countries and the acceleration of ripening would result in a reduction of the storage time and would increase the profit margin and the competitiveness of the end product. The different strategies that have been assayed with this purpose include ripening at elevated temperature, use of genetically modified starter bacteria, addition of enzymes and addition of slurry systems. Over the last decade, numerous studies have been carried out, especially on the addition of enzymes. The aim of this paper is to review and update the knowledge on this topic. The more recent approaches in this field, such as the use of microbial extracts, are also presented.     

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.     

Effect of selected yeast strains on the sensory properties of dry fermented sausages

This study examines the role of yeasts in the sensorial characteristics of dry fermented sausages. A total of 172 yeast strains were isolated from commercially obtained dry fermented sausages and their enzymatic activities studied. Those most active against meat proteins and pork fat were selected. Experimental sausages were manufactured and the yeasts inoculated onto the surface or into the mince. Control batches were manufactured similarly, but without inoculation. After ripening, the composition of the protein and lipid fractions were studied. The sensory properties of the products were evaluated by trained judges. Despite the enzymatic activity of the yeasts, the organoleptic characteristics of the sausages—irrespective of inoculation site—did not differ significantly from those of the controls. The influence of yeasts on the ripening of dry fermented sausages is, therefore, doubtful.     

风干香肠中乳酸菌的分离鉴定及益生特性研究

由风干香肠中分离纯化得到的3株乳酸菌ORC2、QM7和QM8,均能产乳酸、革兰氏染色阳性、接触酶阴性。经系统发育分析,结合菌落形态、细胞形态、生化反应试验,确定菌株ORC2和QM7为肠膜明串珠菌,QM8为柠檬明串珠菌。乳酸菌ORC2、QM7和QM8均能降低培养基中的胆固醇,且对·OH、DPPH·和O-2·有一定的清除能力,其清除率都与细胞浓度呈正相关。在细胞浓度为5×108CFU/mL时,菌株ORC2对·OH的清除率最高,为65.36%;菌株QM8对DPPH·的清除率最高,为61.21%;而对O-2·的清除率最高的菌株为QM7,达到75.70%。菌株QM7在培养96 h能清除最多的胆固醇,清除率为51.2%。     

干发酵香肠中的生物胺

综述干发酵香肠中生物胺产生的条件,国外干发酵香肠中常见生物胺的种类及含量,与生物胺积累有关的微生物以及影响生物胺产生的理化因素,并提出了控制干发酵香肠中生物胺积累的措施。