Genomic analysis of Pediococcus starter cultures used to control Listeria monocytogenes in turkey summer sausage.

The pulsed-field technique of clamped homogeneous electric field electrophoresis was employed to characterize and size genomic DNA of three pediocin-producing (Ped+) and two non-pediocin-producing (Ped-) strains of Pediococcus acidilactici. Comparison of genomic fingerprints obtained by digestion with the low-frequency-cleavage endonuclease AscI revealed identical restriction profiles for four of the five strains analyzed. Summation of results for 10 individually sized AscI fragments estimated the genome length to be 1,861 kb for the four strains (H, PAC1.0, PO2, and JBL1350) with identical fingerprints. Genomic analysis of the pediocin-sensitive, plasmid-free strain P. acidilactici LB42 with the unique fingerprint revealed nine AscI fragments and a genome length of about 2,133 kb. Ped- (JBL1350) and Ped+ (JBL1095) starter cultures (one each) were used to separately prepare turkey summer sausage coinoculated with a four-strain Listeria monocytogenes mixture (ca. 10(5) CFU/g). The starter cultures produced equivalent amounts of acid during fermentation, but counts of L. monocytogenes were reduced to a greater extent in the presence of the Ped+ starter culture (3.4 log10 unit decrease) than in the presence of the Ped- starter culture (0.9 log10 unit decrease). Although no listeriae were recovered from sausages following the cook/shower, appreciable pediocin activity was recovered from sausages prepared with the Ped+ strain for at least 60 days during storage at 4 degrees C. The results of this study revealed genomic similarities among pediococcal starter cultures and established that pediocins produced during fermentation provide an additional measure of safety against listerial proliferation in turkey summer sausage.     

Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages

AIM: Study of the effectiveness of in situ bacteriocin production by lactic acid bacteria (LAB) to control Listeria monocytogenes in dry-fermented sausages. METHODS AND RESULTS: Two bacteriocin-producing strains: Lactococcus lactis subsp. lactis LMG21206 and Lactobacillus curvatus LBPE were grown in a pilot scale fermentor and lyophilized to be directly used in dry sausage fermentation. A commercial starter culture (Bel'meat SL-25) not inhibitory to L. monocytogenes (Bac- starter) was mixed (1 : 1) with each of the two lyophilized bacteriocin-producing strains to obtain starters active against the pathogen (Bac+ starter). Anti-Listeria effectiveness of the Bac+ starters was studied in dry-fermented sausages. The meat batter was experimentally contaminated with a mixture of four different strains of L. monocytogenes (10(2)-10(3) CFU g(-1)). The results showed that L. monocytogenes did not grow in any of the contaminated batches, but no significant decrease (P > 0.05) was observed either in the positive control (no added starter culture) or in samples fermented with the Bac- starter culture during the fermentation period and up to 15 days of drying. When the Bac+ starter contained Lb. curvatus LBPE, cell counts of L. monocytogenes decreased to below the detectable limit (<10 CFU g(-1)) after 4 h of fermentation and no survivors could be recovered by enrichment beyond day 8 of drying. When the Bac+ starter culture containing Lc. lactis LMG21206 was used, a decrease in Listeria counts to below the detectable limit was achieved after 15 days of drying. CONCLUSIONS: The bacteriocin-producing strains studied may be used as adjunct cultures for sausage fermentations to control the occurrence and survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Addition of the Bac+ strains, especially the Lb. curvatus strain would provide an additional hurdle to enhance the control of L. monocytogenes in fermented meat products.     

Plasmid transfers by conjugation and electroporation in Pediococcus acidilactici

W.J. KIM, B. RAY AND M.C. JOHNSON. 1992. Plasmid profiles of wild and mutant strains of Pediococcus acidilactici M showed that a 53.7 kb plasmid (pPR72) encodes the sucrose hydrolysis trait ( Suc +) and an 11.1 kb plasmid encodes the bacteriocin production trait ( Pap +). Neither of these plasmids encode traits involving fermentation of other carbohydrates, antibiotic resistance or resistance to bacteriocin. Broad host-range plasmids (pAMβ1 and pIP501) from Enterococcus faecalis and plasmid pPR72 from Ped. acidilactici were conjugally transferred by filter mating into two strains of Ped. acidilactici. Four plasmids, ranging in size from 4.4 to 53.7 kb, were also transferred into Ped. acidilactici strains by electroporation. Optimum transformation of the 4.4 kb plasmid, pGK12, was obtained at a DNA concentration of 1 μg/220 μl. The same amount of DNA gave lower transformation frequencies as the plasmid size increased. Results of these studies indicated that both conjugation and electroporation can be used to transfer plasmid-linked traits in Ped. acidilactici strains.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

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.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Inhibition of Listeria in dry fermented sausages by the bacteriocinogenic Lactobacillus sake CTC494

Lactobacillus sake CTC494 isolated from a naturally fermented sausage, produced an antibacterial agent active against selected strains of Listeria monocytogenes and L. innocua. The agent was bacteriolytic against L. monocytogenes and sensitive to proteolytic enzymes; it was identified as a bacteriocin and was designated as sakacin K. The ability of Lact. sake CTC494 to inhibit the growth of listeria, compared to a bacteriocinogenic negative control strain, was examined in a model sausage system and in dry fermented sausages. In dry fermented sausages Lact. sake CTC494 was able not only to suppress the growth of listeria but to diminish their number by 1.25 log compared to the non-bacteriocinogenic control strain. Thus, Lact. sake CTC494 has proved to be a good starter culture providing good organoleptical and sensorial qualities to the product and can be employed as a bioprotective starter culture in fermented meat products.     

Monitoring of staphylococcal starters in two French processing plants manufacturing dry fermented sausages

AIMS: The growth and survival of Staphylococcus xylosus and Staphylococcus carnosus were monitored during sausage manufacture in two processing plants. METHODS AND RESULTS: The gram-positive, catalase-positive cocci isolated from the processing plants F10 and F11 were identified by Staphylococcus-specific PCR and species-specific oligonucleotide array. In the inoculated products with starter cultures, 90% of staphylococcal strains isolated in F10 were identified as S. xylosus and 10% as S. carnosus. In F11, 77% were identified as S. xylosus and 20% as S. carnosus. Staphylococcus xylosus dominated the staphylococcal microbiota while S. carnosus survived during the process. The pulse-field gel electrophoresis analysis revealed that all S. xylosus and S. carnosus strains isolated corresponded to the starter strains inoculated. The two starter strains of S. xylosus co-dominated in the isolates from sausages of F11, whereas the strain with pattern A1 was dominant in the isolates from sausages of F10. In the environments, no S. carnosus and S. xylosus were found, whereas Staphylococcus equorum and Staphylococcus saprophyticus were the main species isolated. CONCLUSIONS: This work highlighted the domination of S. xylosus starter strains, which showed a strong capacity to grow during sausage process, while S. carnosus survived during the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Successful implantation of starter cultures is obviously a prerequisite for their contribution to sensorial qualities. Thus, the monitoring of the growth and the survival of S. xylosus and S. carnosus are required to guarantee a well-adapted starter culture. This study revealed that the two Staphylococcus species are suitable for manufacturing sausages in processing plants with very different capacities of production.     

Reduction of Escherichia coli O157:H7 by stimulated Pediococcus acidilactici

This study was conducted to determine if stimulating the growth of meat starter culture (Pediococcus acidilactici) in a laboratory medium (Brain Heart Infusion broth +2.3% NaCl + 1.5% sucrose; LBHI) and during meat fermentation would control Escherichia coli O157:H7. In LBHI medium without P. acidilactici, the numbers of E. coli O157:H7 increased from 4.00 to 8.34 log10 cfu ml-1, whereas in the presence of P. acidilactici (approximately 6.0 log10 cfu ml-1) in LBHI, LBHIM (LBHI + 0.005% MnSO4), LBHIO (LBHI + 0.3 unit ml-1 Oxyrase), and LBHIMO (LBHI + M + O), the numbers of E. coli O157:H7 increased from 4.00 to 8.05, 7.50, 7.99, and 6.50 log10 cfu ml-1, respectively, after incubation at 40 degrees C for 15 h. During salami fermentation, the numbers of E. coli O157:H7 changed from 7.00 to 6.40 and 5.10 log10 cfu g-1 without and with P. acidilactici (approximately 7.0 log10 cfu g-1), respectively. Stimulated P. acidilactici by M, O, and MO further reduced the number of E. coli O157:H7 from 7.00 to 4.00, 4.80, and 3.65 log10 cfu g-1, respectively. The combination of MO was a better growth stimulator for P. acidilactici, which controlled E. coli O157:H7 in both systems (P < 0.05).     

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Proteolytic activity of Staphylococcus xylosus strains on pork myofibrillar and sarcoplasmic proteins and use of selected strains in the production of "Naples type" salami

AIMS: The aim of this study was to determine the proteolytic activities of Staphylococcus xylosus strains on sarcoplasmic and myofibrillar proteins in order to evaluate the suitability of selected strains as starter cultures in the processing of a dry fermented pork sausage. METHODS AND RESULTS: The proteolytic activity of 27 strains of Staphylococcus xylosus on sarcoplasmic and myofibrillar proteins was determined by agar plate method, o-phtaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four strains were selected for the formulation of six starter cultures to use in the production of "Naples type" salami. The proteolytic contribution of starters was determined by SDS-PAGE, comparing the protein profile of inoculated sausages with that of uninoculated sausages after 0, 15 and 33 days of ripening. The results showed that the proteolytic activity of some strains, determined by the agar plate method, were not confirmed by electrophoretic and spectrophotometric assays. In fact, of 24 strains of Staphylococcus xylosus able to hydrolyse muscle protein extracts on agar plate, only 12 strains were shown to change SDS-PAGE profile of pork proteins. The SDS-PAGE profile of sarcoplasmic proteins extracted from all sausages showed that the major changes were produced with starters S3, S4 and S5 after 15 days of ripening. Also myofibrillar proteins undergo major changes after 15 days of ripening and the protein profiles showed the same pattern in all samples, except for the sausages produced with starter S4. CONCLUSIONS: The results of this work showed that the muscle protein extracts hydrolysis test is suitable for preliminary screening of Staphylococcus xylosus strains on the basis of their proteolytic activity. However, evaluation of muscle protein hydrolysis in a food model system could then be more appropriate for selecting micro-organisms for use as starter cultures for fermented sausages. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the formulation of starter cultures for the dry fermented sausages production.     

Monoclonal antibody-colony immunoblot method specific for isolation of Pediococcus acidilactici from foods and correlation with pediocin (bacteriocin) production.

BALB/c mice were immunized with broken, heat-killed cells of Pediococcus acidilactici H. After murine cell fusions, one monoclonal antibody (MAb), Ped-2B2, was selected on the basis of its positive reaction with seven of seven strains tested in an enzyme-linked immunosorbent assay with whole cells of P. acidilactici. The MAb Ped-2B2 did not show any cross-reactions with other lactic-acid bacteria or other gram-positive or gram-negative organisms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of surface proteins of P. acidilactici indicated that Ped-2B2 reacted with a protein of 116 kDa. MAb Ped-2B2 was used as a probe to isolate Pediococcus species from fermented-meat products by colony immunoblotting. A total of 18 Ped-2B2-reactive Pediococcus spp. isolates were isolated from eight food samples and assayed for bacteriocin production. All of the isolates produced bacteriocins which were heat stable, proteinaceous, and inhibitory to Lactobacillus plantarum NCDO 955. Biochemical characterization of these isolates indicated that they were all P. acidilactici.     

Monitoring of Staphylococcus xylosus DSM 20266 added as starter during fermentation and ripening of soppressata molisana, a typical Italian sausage

AIMS: "Soppressata molisana", a fermented sausage produced in southern Italy, is commonly obtained without starter addition. However, the use of starter cultures is more and more recommended in meat fermentation processes in order to guarantee stable production performance. In this study, the survival of the Staphylococcus xylosus DSM 20266 was evaluated during the ripening of "soppressata molisana" fermented sausage. METHODS AND RESULTS: The fastest method of RAPD-PCR was employed for discrimination of the added strain from those naturally present during the ripening of the "soppressata molisana". The results obtained were confirmed by analysis of the DNA macrorestriction profile by PFGE. The electrophoretic pattern of bacterial total proteins was also studied, but clear differences between the different strains could not be detected. CONCLUSIONS: The RAPD technique was a valid tool for monitoring Staph. xylosus DSM 20266 in "sopressata molisana". SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility of monitoring the presence of Staph. xylosus strains during the ripening of fermented sausages by a reliable and repeatable technique such as RAPD.     

Monoclonal antibody-colony immunoblot method specific for isolation of Pediococcus acidilactici from foods and correlation with pediocin (bacteriocin) production.

BALB/c mice were immunized with broken, heat-killed cells of Pediococcus acidilactici H. After murine cell fusions, one monoclonal antibody (MAb), Ped-2B2, was selected on the basis of its positive reaction with seven of seven strains tested in an enzyme-linked immunosorbent assay with whole cells of P. acidilactici. The MAb Ped-2B2 did not show any cross-reactions with other lactic-acid bacteria or other gram-positive or gram-negative organisms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of surface proteins of P. acidilactici indicated that Ped-2B2 reacted with a protein of 116 kDa. MAb Ped-2B2 was used as a probe to isolate Pediococcus species from fermented-meat products by colony immunoblotting. A total of 18 Ped-2B2-reactive Pediococcus spp. isolates were isolated from eight food samples and assayed for bacteriocin production. All of the isolates produced bacteriocins which were heat stable, proteinaceous, and inhibitory to Lactobacillus plantarum NCDO 955. Biochemical characterization of these isolates indicated that they were all P. acidilactici.     

Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 or 25 degrees C.

Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.     

Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 or 25 degrees C

Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.     

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.