Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

Production of Enterotoxin A in Milk

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 10(6)Staphylococcus aureus cells per ml. When similar milks were inoculated with 10(4)S. aureus cells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 10(6)S. aureus cells per ml and incubated at 35 C. Generally, a concentration of 5 x 10(7)S. aureus cells per ml of milk was reached before enterotoxin A was detected.     

Microbiology of Lebanon Bologna

Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 10(4)/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also.     

Effect of pH and Oxygen Tension on Staphylococcal Growth and Enterotoxin Formation in Fermented Sausage

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 × 10⁷ cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.     

Microbial ecology of the soppressata of Vallo di Diano, a traditional dry fermented sausage from southern Italy, and in vitro and in situ selection of autochthonous starter cultures

The microbial ecology of "soppressata of Vallo di Diano," a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus, S. succinus, and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus, L. sakei, and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.     

Effect of Selected Lactic Acid Bacteria on Growth of Staphylococcus aureus and Production of Enterotoxin

Representative strains of 15 species of lactic acid bacteria were examined for their ability to influence growth of Staphylococcus aureus and production of enterotoxin in associative culture. Among the organisms used as effectors the streptococci were most inhibitory, followed by Pediococcus cerevisiae. The lactobacilli and Leuconostoc citrovorum were not inhibitory to growth and only slightly inhibitory to production of enterotoxin. Enterotoxin was detected in all cultures in which the population of S. aureus reached 8 x 10(7) per ml. At lower S. aureus populations no enterotoxin was detected after incubation for 48 h. Mechanisms of inhibition of growth and enterotoxin production by S. aureus strain 243 grown in association with Streptococcus lactis A64 or P. cerevisiae 10791 in APT broth were investigated. Competition for vital nutrients, especially niacin and biotin, and probably production of hydrogen peroxide contribute to inhibition. Production of lactic acid appears to inhibit growth of S. aureus in the early but not the late stages of incubation.     

Elimination of Escherichia coli O157:H7 from fermented dry sausages at an organoleptically acceptable level of microencapsulated allyl isothiocyanate

Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log(10) CFU/g Pediococcus pentosaceus and 6 log(10) CFU/g Staphylococcus carnosus) and a five-strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log(10) CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at < or =26 degrees C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13 degrees C for at least 25 days. The water activity (a(w)), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a(w) from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log(10) CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log(10) CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists.     

Survival of Salmonellae During Pepperoni Manufacture

Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10(4) salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10(3)/g, S. dublin could not be recovered from starter culture-fermented pepperoni after 14 days of drying but persisted in the natural flora-fermented sausage. S. typhimurium (initial count, 10(4)/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar survival patterns in beef pork, or beef-pork pepperoni. Heating salmonellae contaminated beef-pork pepperoni (after fermantation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

A quantitative study of enterotoxin production by sheep milk staphylococci.

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Staphylococcal enterotoxin production in the presence of non-enterotoxigenic staphylococci.

Enterotoxigenic Staphylococcus aureus strains were grown with a non-enterotoxigenic strain in laboratory medium, in milk, and in ham. Differences in pigmentation were used to differentiate the enterotoxigenic strains from the non-enterotoxigenic ones. Enterotoxin was detectable in milk when the colony counts of the non-enterotoxigenic strain were 15 to 20 times greater than those of the enterotoxigenic ones and in ham when the ratio was 60 to 77:1. Enterotoxin was detectable in milk when the enterotoxigenic strains reached counts of 10(7) colony-forming units per ml and in ham when the counts reached 10(8) colony-forming units per ml. It may be necessary in some food poisoning outbreaks to examine many isolates (up to 50 or 60) for enterotoxin production to be able to detect the enterotoxigenic staphylococci.     

Growth and enterotoxin A production by Staphylococcus aureus S6 in Manchego type cheese

Milk (from cow, goat and sheep) was inoculated with Staphylococcus aureus strain S6, which is generally considered to be a strong enterotoxin B producer and a weak enterotoxin A producer. It was then used to make Manchego type cheese as prepared industrially. Two concentrations of starter culture (1% and 0.1%) were tested. Staphylococcal growth was good in both but better in the more dilute culture. Staphylococcal enterotoxin B was not detected at any stage of the ripening process of any cheese tested. However enterotoxin A was detected in both starter concentrations, reaching as high as 769 ng/100 g of cheese in the 0.1% starter batches.     

Improvement of Raw Sausage Fermentation by Stress-Conditioning of the Starter Organism Lactobacillus sakei

Effective growth and high acidification activity during meat fermentation are key characteristics of starter lactobacilli to ensure hygienic safety and sensory quality of the product. In this study, we demonstrated that the performance of Lactobacillus sakei in sausage fermentation can be improved by preinoculation treatments with sublethal heat, cold, and salt stress. Sausages were produced and inoculated with stress-treated cells of L. sakei 23 K (pLPV111) and the isogenic mutant of the class III heat-shock repressor CtsR, which was previously shown to exhibit improved growth in fermenting sausages. The pH values of sausages fermented with stressed cells attained defined threshold values in a distinctly shorter time than those inoculated with unstressed cells. In particular, the cold-stressed cells (4 degrees C) reduced the pH to 5.0 within approximately 40 hours compared with approximately 70 hours for untreated cells. This enhanced acidification activity of the cold-stressed cells was consistent with an increased growth rate. Growth studies in culture medium showed that stress-treated cells with improved performance did not exhibit this advantage when exposed to curing salt, one of the major stressors at the beginning of sausage fermentation. Preinoculation stress treatment is a promising way to improve the effectiveness of meat starter lactobacilli.     

Growth and enterotoxin A production by Staphylococcus aureus S6 in Manchego type cheese

(from cow, goat and sheep) was inoculated with Staphylococcus aureus strain S6, which is generally considered to be a strong enterotoxin B producer and a weak enterotoxin A producer. It was then used to make Manchego type cheese as prepared industrially. Two concentrations of starter culture (1% and 0.1%) were tested. Staphylococcal growth was good in both but better in the more dilute culture. Staphylococcal enterotoxin B was not detected at any stage of the ripening process of any cheese tested. However enterotoxin A was detected in both starter concentrations, reaching as high as 769 ng/100 g of cheese in the 0.1% starter batches.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Elimination of Escherichia coli O157:H7 from Fermented Dry Sausages at an Organoleptically Acceptable Level of Microencapsulated Allyl Isothiocyanate

Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log₁₀ CFU/g Pediococcus pentosaceus and 6 log₁₀ CFU/g Staphylococcus carnosus) and a five-strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log₁₀ CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at ≤26°C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13°C for at least 25 days. The water activity (a_w), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a_w from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log₁₀ CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log₁₀ CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Isolation and characterization of a virulent bacteriophage from Staphylococcus carnosus

Abstract A virulent bacteriophage, øSK311, was isolated from Staphylococcus carnosus , an organism used as a starter culture for the production of dry sausage. Electron microscopic studies revealed that this bacteriophage showed some morphological similarities with the Escherichia coli phages T4 and λ. The host range of øSK311 extends over 9 different staphylococcal species. A phage-resistant mutant of S. carnosus could be isolated. Cells of this mutant exhibited a capsule-like structure. The DNA of øSK311 possesses a G + C content of 31.4 mol% and appears to be highly modified.