Microbiological events during commercial meat fermentations.

Microbiological developments during industrial meat fermentations (salami), made with and without commercial starter cultures, were followed at two factories in Germany and Italy. In the German product microbial growth was evident only for the first 48 h, followed by a gradual decline in numbers of most micro-organisms. The pH fell from 5.8 to 4.8 in the 28 d required for production. In Italy a similar situation was seen, except that a second period of bacterial growth began around 15 d, coincident with the appearance of intentional surface mould growth which reversed the pH fall, the final pH being 6.2. The German starter culture was a mixture of Lactobacillus plantarum and Staphylococcus carnosus, whereas in Italy only Staph. carnosus was used. The strain of Lact. plantarum used did not grow in the German product whereas the Staph. carnosus grew well in both products to form a substantial proportion of the final microflora.     

Staphylococcus carnosus bacteriophages isolated from salami factories in Germany and Italy

Two phages lysing strains of Staphylococcus carnosus , an organism used as a starter culture for salami production, were isolated from factories in Germany and Italy. Morphologically they show the C1 morphotype and are unrelated to the only other known Staph. carnosus phage. The phages were physiologically and morphologically similar but showed differences in their structural proteins and DNA restriction patterns. Their genomes consisted of linear double stranded DNA with a genome size of 19 kb. The phages lysed a wide range of Staph. carnosus strains from commercial meat starter cultures as well as the DSM type strain. Despite the presence of these phages, the products were normal from the point of view of colour, texture and flavour.     

Characterization of catalase and superoxide dismutase in Staphylococcus carnosus 833 strain

AIMS: Staphylococcus carnosus, used as starter culture in fermented meat products, decreases the level of volatiles arising from lipid oxidation. To analyse its antioxidant capacities, catalase and superoxide dismutase (SOD) were characterized. METHODS AND RESULTS: Catalase and SOD activities were measured with spectrophotometric methods and visualized on non-denaturing polyacrylamide gels. The corresponding sod gene was identified by PCR. Southern hybridizations and enzymatic analyses showed that there was a single catalase and a single SOD in Staph. carnosus 833 strain. The gene encoding the Staph. carnosus SOD was found to encode a protein closely related to SOD requiring manganese. Catalase and SOD levels increased in mid-log phase. Only catalase was induced by oxygen, nitrate or nitrite while glucose induced neither enzyme. Metal ion limitation increased catalase and decreased SOD activities. CONCLUSION: Staph. carnosus synthesizes both enzymes in conditions encountered in sausage manufacturing. These results could explain the antioxidant properties of Staph. carnosus starter culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of the antioxidant properties of Staphylococci will allow a more rational use of these starters in meat fermented products.     

Bacterial growth in plant tissue culture media

C. LEIFERT AND W.M. WAITES. 1992. When Murashige and Skoog's liquid plant medium was inoculated with 10 different bacterial species in the absence of plants only Bacillus subtilis showed significant growth. The numbers of Lactobacillus plantarum, Pseudomonas maltophilia, Erwinia carotovora and Staphylococcus saprophyticus decreased rapidly and were not detected at 28 d.
Bacillus subtilis, Lact. plantarum, Ps. maltophilia, Erw. carotovora and Staph. saprophyticus grew and persisted in the same medium in the presence of Delphinium plants, while only Lact. plantarum and Erw. carotovora grew and persisted in the presence of Hemerocallis plants.
Hemerocallis plants lowered the pH of media from 5.6 to about 3.9 while Delphinium plants increased it to about 5.9 within 7 d after subculturing on fresh media. The pH drop in Hemerocallis media is thought to prevent the growth and persistence of bacteria such as B. subtilis, Staph. saprophyticus and Ps. maltophilia , which were found to be more sensitive to low pH than Lact. plantarum and Erw. carotovora. Bacterial growth in the medium altered the pH, reduced the plant growth and/or resulted in plant death.     

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.     

Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T

Lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T, a new amylolytic L(+) lactic acid producer, was investigated and compared with starch fermentation by Lact. plantarum A6. At non-controlled pH, growth and lactic acid production from starch by Lact. manihotivorans LMG 18010T lasted 25 h. Specific growth and lactic acid production rates continuously decreased from the onset of the fermentation, unlike Lact. plantarum A6 which was able to grow and convert starch product hydrolysis into lactic acid more rapidly and efficiently at a constant rate up to pH 4.5. In spite of complete and rapid starch hydrolysis by Lact. manihotivorans LMG 18010T during the first 6 h, only 45% of starch hydrolysis products were converted to lactic acid. When pH was maintained at 6.0, lactic acid, amylase and final biomass production by Lact. manihotivorans LMG 18010T increased markedly and the fermentation time was reduced by half. Under the same conditions, an increase only in amylase production was observed with Lact. plantarum A6. When grown on glucose or starch at pH 6.0, Lact. manihotivorans LMG 18010T had an identical maximum specific growth rate (0.35 h(-1)), whereas the maximum rate of specific lactic acid production was three times higher with glucose as substrate. Lactobacillus manihotivorans LMG 18010T did not produce amylase when grown on glucose. Based on the differences in the physiology between the two species and other amylolytic lactic acid bacteria, different applications may be expected.     

The antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10(8) cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10(6)-10(7) cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus. In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5.5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10⁸ cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10⁶–10⁷ cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus . In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5·5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli: overexpression, purification, and characterization of the enzymatically active C-terminal domain of enzyme IImtl equivalent to enzyme IIImtl

The extreme C-terminus (Ser-490 to Lys-637) of the Escherichia coli EIImtl was subcloned to test structural and mechanistic proposals about the existence of an EIII-like domain in this enzyme. Oligonucleotide-directed mutagenesis was used to produce a unique NcoI restriction site and, at the same time, to change Ser-490 into methionine in a flexible region in front of the proposed EIII-like domain. The 16-kDa C-terminal domain (CI) was overexpressed in Escherichia coli, purified, and analyzed in vitro for catalytic activity in the presence of an EIImtl mutated at its first phosphorylation site, His-554 (EII-H554A). The results presented show that this domain can be expressed as a structurally stable, enzymatically active entity which is able to restore the PEP-dependent phosphorylation activity of the mutant EIImtl-H554A to 25% of wild-type levels. To demonstrate the EIII activity of the CI domain in a more direct way, we also substituted it for EIIImtl in the Staphylococcus carnosus system. The CI domain was active in transferring the phosphoryl group to Staph. carnosus EII; however, it was 6.5 times less active compared to Staph. carnosus EIIImtl itself. EIIImtl from Staph. carnosus, on the other hand, was able to substitute for the isolated C-terminal domain in the E. coli mannitol phosphorylation assay; however, it appeared to be 2 or 3 times less effective.     

Screening of lactic starter from Tunisian fermented vegetables and application for the improvement of caper (Capparis spinosa) fermentation through an experimental factorial design

This study aims at designing a lactic starter for caper fermentation isolated from Tunisian fermented vegetables to improve the process and produce consistent and high-quality product. In this study, the lactic starter was isolated by exploring the lactic acid bacteria (LAB) of Tunisian artisanal fermented vegetables. Identification was carried out by partial 16S rRNA gene sequencing. Screening was based on salt tolerance and antagonistic activities against Escherichia coli ATCC 10536 and Enterococcus faecalis ATCC 10541. Caper fermentation was optimized through a full factorial experimental design (23), by exploring three factors: starter inoculum size, NaCl concentration, and acetate content. Differences in pH values, Total aerobic mesophilic bacteria and LAB counts between the beginning and end of fermentation are selected as responses and corresponding regression coefficients were calculated. The lactic microbiota is mainly represented by Lactobacillus plantarum group. Based on salt tolerance and antimicrobial activity, the strain Lactobacillus plantarum F3 was selected as starter for caper fermentation. The effect of NaCl concentration, acetate content, and inoculum size on acidity, total aerobic mesophilic bacteria count, and LAB count after 1 week and 1 month of caper fermentation was studied. Depending on the fermentation time, either 1 week or 1 month, the initial conditions should comprise 0% acetate, 108 CFU/mL inoculum, and 5% NaCl for 1 week against 5% acetate, 107 CFU/mL inoculum, and 10% NaCl for 1 month lasting caper fermentation. A protocol for caper fermentation was set up ensuring hygienic quality and LAB viability. Lb. plantarum F3 was selected as lactic starter for caper fermentation, and initial fermentation conditions were optimized through a full factorial design. This work has shown loss in LAB viability after 1 week of fermentation. Based on results obtained, an optimized fermentation protocol was set up. This protocol ensures LAB survival and high hygienic quality of the product.     

A pH Control System Based on Malate Decarboxylation for the Cultivation of Lactic Acid Bacteria

Most species of lactic acid bacteria decarboxylate l-malate to lactate and CO(2) if an energy source such as glucose is present. A proton is taken up in the reaction, which prevents pH decreases in the growth medium caused by lactic acid production from glucose fermentation. MRS broth (pH 7.0) (Difco Laboratories) containing 10 mM glucose and various concentrations of l-malate (0, 25, 50, 75, and 100 mM) was used to cultivate Lactobacillus plantarum. After 72 h at 37 degrees C, all malate was decarboxylated and all glucose was fermented, with resultant final pH values of 4.5, 6.3, 6.9, 7.3, and 7.5, respectively. When d-malate (which cannot be decarboxylated) was substituted for l-malate, the final pH values were 4.5, 5.2, 5.6, 5.8, and 5.9. By varying the ratios of glucose to l-malate in the growth medium, it was possible to obtain pH values which were lower, the same, or higher than the initial pH values. In contrast, buffers such as phosphate only retard decreases in pH. l-Malate, when compared with K(2)PO(4) on an equal molar basis, provided greater resistance to decreases in pH. Higher specific growth rates were observed for L. plantarum and Leuconostoc mesenteroides when l-malate rather than K(2)PO(4) was incorporated into the growth medium.     

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.     

A Staphylococcus aureus-specific oligonucleotide probe derived from 16S rRNA gene sequences

Genes encoding 16S rRNA were sequenced from 16 species of Staphylococcus. Sequence analysis highlighted a potential Staph. aureus -specific region and a complementary oligonucleotide probe was synthesized and its specificity tested. Northern blotting indicated molecular specificity, and dot blots to RNA from Staph. aureus, Staph. capitis, Staph. caprae, Staph. carnosus, Staph. caseolyticus, Staph. cohnii, Staph. epidermidis, Staph. gallinarum, Staph. haemolyticus, Staph. hominis, Staph. hyicus, Staph. saprophyticus, Staph. sciuri, Staph. simulans, Staph. warneri and Staph. xylosus indicated species-specificity.     

Effect of growth parameters on the residual activity of Lactobacillus plantarum after drying

In order to select optimal conditions for the production of dry and active starter cultures it is important to determine the influence of growth conditions on the residual activity of dried bacteria. The influence of medium composition, pH during growth, continuous vs batch reactor and growth phase was studied on the residual activity of Lactobacillus plantarum after drying. The effect of high sodium chloride concentrations during growth on the residual activity of Lact. plantarum after drying was measured. The samples were dried by convection and in a fluidized bed. Bacteria with the highest residual activity after drying were produced in batch or chemostat with pH-controlled growth using enriched or diluted MRS medium. The presence of 1 or 1·25 mol l⁻¹ NaCl during growth resulted in a decreased residual activity after drying. Variations in growth conditions (application of stress) generally did not result in higher residual activities after drying.     

Citrate metabolism by Lactobacillus plantarum isolated from orange juice

The behaviour of Strains of Lactobacillus plantarum isolated from fermented orange juice and Lact. plantarum DSM 20174 was studied in the presence of citrate. When used as sole carbon source, citrate scarcely supported the growth of the bacteria. It was shown to enhance the growth of Lact. plantarum in glucose media. Under acid conditions (pH 4·0–5·0), 1 mol of citrate yielded 1·7 mol of acetate as sole major final metabolite with release of CO₂ in the gas phase.     

On-line monitoring of important organoleptic methyl-branched aldehydes during batch fermentation of starter culture Staphylococcus xylosus reveal new insight into their production in a model fermentation

A small fermentor (55 mL) was directly interfaced to a membrane inlet mass spectrometer for continuous on-line monitoring of oxygen and volatile metabolites during batch fermentations of the starter culture Staphylococcus xylosus. Using this technique, we were able to correlate production of the very important flavor compounds 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal with various growth conditions. We found that the aldehydes were present in the culture broth only as transient metabolites. They were produced in the exponential growth phase, reached a maximum concentration when the culture became anaerobic, and then they rapidly disappeared from the culture medium. This general pattern was observed for three different strains of S. xylosus and S. carnosus. Small amounts of inoculum or increased exposure to oxygen were found to favor production of the aldehydes as a result of a longer aerobic growth period. Growing S. xylosus under conditions resembling those in a fermented sausage revealed that NaCl (5%) increased aldehyde production considerably, whereas KNO(3) (0.03%) or NaNO(2) (0.03%) had little effect. A lowering of pH from 7.2 to 6.0 reduced cell density, but had a minor affect on aldehyde production.     

Microbial quality and direct PCR identification of lactic acid bacteria and nonpathogenic Staphylococci from artisanal low-acid sausages

Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment, Lactobacillus sakei and Lactobacillus curvatus were detected in 11.8% of the samples, and Lactobacillus plantarum and Staphylococcus xylosus were detected in 17.6%. Enriched samples allowed the detection of L. sakei and S. xylosus in all of the samples (100%) and of Enterococcus faecium in 11.8% of the sausages. The percentages of L. curvatus, L. plantarum, Staphylococcus carnosus, and Staphylococcus epidermidis varied depending on the sausage type. L. curvatus was detected in 80% of fuets and in 57% of chorizos. L. plantarum was found in 50% of fuets and 100% of chorizos. S. epidermidis was detected in only 11.8% of fuets, and S. carnosus was detected in only 5.9% of chorizos. Lactococcus lactis, Staphylococcus warneri, and Staphylococcus simulans were not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts of Enterobacteriaceae and did not contain any of the food-borne pathogens assayed.     

Influence of autochthonous starter cultures on microbial dynamics and chemical-physical features of traditional fermented sausages of Basilicata region

In this study, a combination of a Lactobacillus sakei strain and a Staphylococcus equorum strain was used as autochthonous starter for an experimental production of Basilicata fermented sausages. The influence of starter addition on the safety and quality parameters and microbiological and chemical-physical properties of the products was investigated. Microbial counts of safety indicators were lower in the samples with the addition of starter culture, and, at the end of ripening, Enterobacteriaceae and Gram negative bacteria were detected only in samples made without the starter addition. The addition of starter led to a final product with better microbiological and chemical-physical features, and a positive effect on flavor and aroma compounds formation by a good proteolytic and lipolytic activities. The use of autochthonous starter cultures allows to obtain products with the organoleptic characteristics expected and steady in time and to standardize the production process, improving the safety and quality, but preserving the essential character of the product.     

Production of L- AND D-lactate by Pediococcus pentosaceus in batch and steady-state continuous culture

The fast growth and acid production of a strain of Pediococcus pentosaceus , used as a starter culture in the production of dry sausages, was dependent on the presence of acetate. In a batch culture on a mixture of glucose and sucrose both sugars were consumed simultaneously. Similar growth rates and product yields were obtained on glucose and sucrose, d - AND l -lactate were produced via a D- and L-lactate dehydrogenase (LDH), respectively, and no racemase was present. In batch cultures about 15% of the lactic acid produced was the D-isomer, whereas in a sucrose-limited, continuous culture the fraction of D-lactic acid increased with decreasing dilution rate. The results are discussed in relation to the two LDH activities.