Use of bacteriocin-producing starter cultures of Lactobacillus plantarum and Lactobacillus curvatus in production of ostrich meat salami

Ostrich meat salami was produced by using Lactobacillus plantarum strain 423 and Lactobacillus curvatus strain DF126. The strains produce the bacteriocins plantaricin 423 and curvacin DF126, respectively. The specific activity of plantaricin 423 in MRS broth at 30?°C increased as the pH decreased from 6.5 to 3.5, but activity subsequently decreased. The activity of curvacin DF126 increased under the same conditions, but remained stable for the duration of the growth cycle. Maximum curvacin DF126 and plantaricin 423 activity levels were recorded at a culture pH of around 4. The spectra of antimicrobial activity recorded for plantaricin 423 and curvacin DF126 were similar. Neither of the two bacteriocins inhibited the growth of Micrococcus sp. MC50 and did not have any inhibitory effect on either of the producer strains. Curvacin DF126 and plantaricin 423 inhibited the growth of L. monocytogenes in salami meat. However, after 15 h of fermentation the viable count of L. monocytogenes LM1 increased, probably due to a decrease in activity of the bacteriocins and/or the development of resistant bacterial cells. This is the first report on the inhibition of L. monocytogenes in ostrich meat salami by using bacteriocinogenic starter cultures.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Hydrolysis of muscle myofibrillar proteins by Lactobacillus curvatus and Lactobacillus sake

Proteolytic enzyme activities of whole cells and cell free extracts (CFE) of Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were characterised using synthetic chromogenic compounds and myofibrillar proteins as substrates. The hydrolytic action was monitored by SDS-PAGE and reverse phase-HPLC analyses. The CFE of L. sake partially contributed, together with muscle enzymes, to the initial hydrolysis of myofibrillar proteins. Whole-cells of both L. curvatus and L. sake generated peptides considered important for cured-meat taste. The peptide mapping, resulting from the action on the substrates assayed, revealed a profile of extra and intracellular enzymes. Both strains expressed strong amino acid metabolism.     

Inhibition of Yersinia enterocolitica by Lactobacillus sake strains of meat origin

The growth of Yersinia enterocolitica at 4, 8, 15 and 24°C, in mixed cultures with Lactobacillus sake strains previously isolated from Spanish dry fermented sausages was investigated. Growth of Y. enterocolitica was affected by L. sake strains at all temperatures studied. The inhibition was higher as the incubation temperature increased. L. sake 148, a bacteriocinogenic strain, was less inhibitory to Y. enterocolitica growth than L. sake 23, a stronger lactic acid producer strain. The low pH and the lactic acid produced by the lactobacilli seem to be major factors contributing to the inhibition of Y. enterocolitica strains.     

Inhibition of Listeria monocytogenes by Lactobacillus sake strains of meat origin

The ability of two Lactobacillus sake strains of meat origin to inhibit the growth of Listeria monocytogenes at 4, 8, 15, 24 and 32°C in a conventional liquid media was investigated. Growth of L. monocytogenes was affected by Lac. sake strains at all temperatures. The inhibition was higher at 15, 24 and 32°C than at refrigeration temperatures. The inhibitory activity of both lactobacilli was similar perhaps due to the fact that Lac. sake 148 produces a bacteriocin inhibitory to L. monocytogenes, while Lac. sake 23 is a strong lactic acid producer. The antagonism exhibited by the lactobacilli on the L. monocytogenes strains seems to display a bacteriostatic rather than a bacteriocidal effect.     

Biogenic amines in meat inoculated with Lactobacillus sake starter strains and an amine-positive lactic acid bacterium

 Formation of biogenic amines in minced meat inoculated with two Lactobacillus sake starter culture strains and an amine-positive lactic acid bacterium (G-106) was studied. The effects of these starter cultures against the formation of biogenic amines were dependent on the kind of decarboxylating microorganisms present in the raw material and the effects were different for each amine. Starter strains maintained the microbiological quality of the meat kept at 20°C for 7 days, and inhibited the formation of putrescine and cadaverine. They also inhibited the formation of phenylethylamine caused by G-106 when it was present at an initial level of 10² colony-forming units (CFU)/g. However, the formation of tyramine was not affected and the formation of histamine was increased when starters were used in samples inoculated with G-106. Received: 4 December 1996 / Revised form: 27 January 1997     

Biogenic amines in meat inoculated with Lactobacillus sake starter strains and an amine-positive lactic acid bacterium

 Formation of biogenic amines in minced meat inoculated with two Lactobacillus sake starter culture strains and an amine-positive lactic acid bacterium (G-106) was studied. The effects of these starter cultures against the formation of biogenic amines were dependent on the kind of decarboxylating microorganisms present in the raw material and the effects were different for each amine. Starter strains maintained the microbiological quality of the meat kept at 20°C for 7 days, and inhibited the formation of putrescine and cadaverine. They also inhibited the formation of phenylethylamine caused by G-106 when it was present at an initial level of 10² colony-forming units (CFU)/g. However, the formation of tyramine was not affected and the formation of histamine was increased when starters were used in samples inoculated with G-106. Received: 4 December 1996 / Revised form: 27 January 1997     

New insertion sequence in Lactobacillus fructivorans strains isolated from spoiled sake

A genomic subtraction between Lactobacillus fructivorans ATCC 8288(T) and an alcoholophilic strain of L. fructivorans (ATCC 15435 strain H-1) was performed. Subtractive DNA fragments were identified to be parts of a 1468-bp insertion sequence, a few copies of which were present with in four alcoholophilic strains tested. The insertion sequence, which we named ISLfr1, seems to belong to the ISL3 family. For three alcoholophilic strains, primer walk sequencing revealed that ISLfr1 was inserted into the ORF, which showed homology to accC2 of Lactobacillus plantarum WCFS1. accC2 in one strain seemed to be nonfunctional because of deletion and frameshift mutations, although ISLfr1 was not found in this gene. Although accC2 encodes acetyl-CoA carboxylase, which is essential for lipid metabolism, L. fructivorans was found to have a homolog of accC2. Nonfunctional accC2 might be involved in producing the unusually long acyl chains observed only in alcoholophilic L. fructivorans. From a food engineering standpoint, it appears that the concomitant PCR amplification of ISLfr1 and rDNA is useful for the specific detection of alcoholophilic L. fructivorans.     

Intraspecific diversity of Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, and Leuconostoc mesenteroides associated with vacuum-packed meat product spoilage analyzed by randomly amplified polymorphic DNA PCR

The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA-restriction fragment length polymorphism profiles. The RAPD-M13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mesenteroides, 6 in L. sakei, 12 in L. plantarum, and 6 in L. curvatus. Of these, six profiles corresponding to L. mesenteroides and one corresponding to L. sakei were found in both products. The Shannon-Weaver diversity index (H'), calculated according to RAPD-M13 and RAPD-T3 profiles during storage, revealed that most profiles appeared only in single samplings in both products, indicating a high strain substitution rate during chilled storage of vacuum-packed meat products. When bloating appeared, only one profile corresponding to L. mesenteroides subsp. dextranicum was present throughout the storage period.     

Production of three anti-listerial peptides by Lactobacillus curvatus in MRS broth

Three novel bioactive peptides (BAP1–3) from Lactobacillus curvatus CWBI-B28 were isolated and purified using de Man, Rogosa and Sharp (MRS) broth by a three-step protein purification protocol including ammonium sulfate precipitation, hydrophobic C₁₈ Sep-Pak column and reverse-phase high performance liquid chromatography (RP-HPLC). This procedure allowed the recovery of chromatographically pure antimicrobial peptides with the yields of 19%, 10% and 15% of BAP1, BAP2 and BAP3, respectively. The respective apparent molecular masses as determined by tricine-SDS-polyacrilamide gel electrophoresis were 6365, 3426 and 3496. N-terminal amino acid sequencing of the BAPs and comparison of their sequences with those of international data bases indicated that BAP1 is more likely to be a casein-derived bioactive protein produced upon hydrolysis of the tryptone present in MRS broth by Lb. curvatus CWBI-B28 during active growth. However, the identity of BAP2 and BAP3 could not be determined with certainty; yet, they would be novel bacteriocins not fitting in any of the known classes of bacteriocins. Therefore, this strain would have the ability to produce intrinsic antimicrobial substances and also release bioprotective peptides from milk-proteins upon cultivation in milk or casein-containing food products due to its proteolytic activity. Thus, Lb. curratus CWBI-B28 possesses a good potential to be used in food preservation and safety.     

Polyamines Affect Activity of Aminopeptidases from Lactobacillus sake

The effects of polyamines (agmatine, cadaverine, putrescine, spermidine and spermine) on the activity of the main aminopeptidases (AP I and AP II) from Lactobacillus sake were determined. Concentrations in the range of 1 mM caused 6–25% inhibition of AP I except for spermine (45% inhibition). Higher polyamine levels (5–10 mM), except for putrescine, exerted a stronger inhibition (20–60%) on AP I. Agmatine and putrescine also reduced AP II activity (6–25%) at concentrations of 0.1–10 mM while cadaverine did not have a notable effect. Spermidine and spermine stimulated (15–70%) the activity of AP II at concentrations >1 mM. Thus, control of polyamine levels is important because they are a potential hazard for human health and also because of their effects on aminopeptidase activity.     

Purification and characterization of a dipeptidase from Lactobacillus curvatus DPC2024

A dipeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-sephacel, phenyl sepharose, chelating sepharose fast flow and MonoQ. The purified dipeptidase was a monomer of ∼52 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme was optimally active at pH 8 and 50°C and retained ∼10% of its maximum activity after pre-heating for 10 min at 70°C. The enzyme was a metallopeptidase, strongly inhibited by 0.1 mM ethylenediaminetetraacetic acid and o-phenanthroline and reactivated by a number of divalent metal ions. The enzyme was also inhibited by p-chloromercuribenzoate and β-mercaptoethanol. The enzyme was a strict dipeptidase, capable of hydrolysing a range of dipeptides but not tri-, tetra- or pentapeptides, p-nitroanilide derivatives of amino acids nor N- and C-terminal-blocked dipeptides. The N-terminal amino acid sequence of the first 20 residues showed significant homology with dipeptidases from Lactobacillus delbrueckii subsp. lactis DSM 7290 and Lactococcus lactis subsp. cremoris MG1363.     

Sakacin M, a bacteriocin-like substance from Lactobacillus sake 148.

The antagonistic activity of Lactobacillus sake 148 was evaluated during its growth on complex broth media and in a semisynthetic defined medium (SDM) with various supplements. The antagonistic activity was a growth-associated property, being detected and quantified when L. sake 148 was grown at either 4, 8, 16, 25 or 32 degrees C. The concentrated culture supernatant of L. sake 148 was subjected to purification by lyophilization and gel filtration. The purification procedure resulted in a small increase in its specific activity (7-fold) and in a low recovery of the original inhibitory activity (8%). Gel filtration analysis of the partially purified activity on Sephadex G-50 revealed an apparent molecular weight of 4640. The partially purified antagonistic activity of L. sake 148 was destroyed by treatment with proteolytic enzymes. However, the antagonistic activity was resistant to heat, having D-values at 121, 135 and 150 degrees C of 23.8, 17.4 and 15.2 min, respectively.     

Effect of the use of autochthonous Lactobacillus curvatus, Lactobacillus plantarum and Staphylococcus xylosus strains on microbiological and biochemical properties of the Sardinian fermented sausage

In this study, a starter culture was tentatively made by testing autochthonous Lactobacillus and Staphylococcus strains, isolated from traditional ‘Salsiccia Sarda’ sausage. Three sausage-making trials were carried out using an association of Staphylococcus xylosus + Lactobacillus curvatus (A), Staphylococcus xylosus + Lactobacillus plantarum (B), and Lactobacillus plantarum alone (C). Microbiological profiles, free amino acids, biogenic amines, and free fatty acids were evaluated after 21 days of ripening. Sausages A and B showed the highest number of lactic acid bacteria that produced a rapid acidification of substrates and reduced the number of spoilage microorganisms. Sausages made with L. plantarum alone or together with S. xylosus had the highest content of free amino acids, whereas the one produced with S. xylosus + L. curvatus showed the lowest content of total biogenic amines and best acceptance score. No significant differences among samples were found as to the free fatty acids concentration.     

Enhancing the antilisterial effect of Lactobacillus curvatus CWBI-B28 in pork meat and cocultures by limiting bacteriocin degradation

This work focused on Listeria monocytogenes growth inhibition and growth rebound in raw and cooked pork meat inoculated with Lactobacillus curvatus strains. During storage of raw meat homogenates in the presence of the bacteriocin-producing strain Lactobacillus curvatus CWBI-B28wt, the Listeria monocytogenes cfu count was initially reduced to an undetectable level, but a growth rebound occurred after two weeks, coinciding with loss of 70% of the bacteriocin activity present at the end of week 2. The Listeria growth rebound was suppressed when proteolysis of bacteriocin was countered by the absence of proteases (bacteriocin addition to cooked meat) or the presence of 1% soy flour (added to provide competing substrates). Further experiments confirmed that bacteriocin is sensitive to the action of proteolytic enzymes isolated from both Lactobacillus curvatus CWBI-B28wt and the meat matrix. Bacteriocin proteolysis thus emerges as a cause of Listeria growth rebound.     

Method for reliable isolation of Lactobacillus sakei strains originating from Tunisian seafood and meat products

In Tunisia, several food products derived from meat or seafood are naturally processed, without any addition of bacterial starters. Such fermented, dried-cured, salted, or marinated products, as well as the raw meat or fish may thus provide a source to isolate the natural microflora colonizing such environments. We isolated lactic acid bacteria from a representative range of flesh-foods sold or manufactured in different parts of Tunisia, and selectively searched for Lactobacillus sakei, a lactic acid bacterium potentially useful as starter or protective culture. Eighty six (86) strains were isolated from various seafood (anchovy, sardine, sole, mullet, and octopus), or meat (pork, veal, beef, sheep, chicken, and turkey) products that were either fresh, or transformed by different traditional processes. Several methods were used in order to develop a rapid and reliable protocol for the direct identification of L. sakei. Amplified ribosomal DNA restriction analysis (ARDRA) classified the various isolates into 9 distinct groups. Search for the presence of the L. sakei specific katA gene indicated that all positive isolates were grouped in the same ARDRA group. Sequencing of 16S rDNA confirmed those isolates as L. sakei. Those 22 different L. sakei strains represent 25.6% of the total isolates, while other isolates found in the different ARDRA groups were tentatively ascribed to Lactobacillus plantarum, Lactococcus lactis/garviae, Enterococcus avium, Streptococcus parauberis, Hafnia alvei, Pediococcus pentosaceus, and Lactobacillus curvatus through 16S rDNA sequencing. A fast and reliable method to isolate and discriminate L. sakei from complex food environments is proposed.     

Technological and safety properties of Lactobacillus plantarum strains isolated from a Tunisian traditional salted meat

A total of 17 strains of Lactobacillus plantarum, isolated from a Tunisian traditional salted meat and identified by biochemical and molecular methods, were characterized according to their technological properties including acidifying, antimicrobial and enzymatic activities as well as antibiotic resistance in order to select the most suitable for use as starter cultures for the production of fermented sausages. All the strains studied showed good acidifying activity and were able to reduce the pH to less than 4.3 in 72, 48 and 24h at 15, 25 and 37°C respectively. The majority of strains displayed antimicrobial activities against Salmonella arizonae, Staphylococcus aureus, Pseudomonas aeuroginosa and Escherichia coli, however characterization of the antimicrobial substances showed that none of the strains could produce bacteriocins. All the L. plantarum strains were able to hydrolyze casein, whereas none of them was found to possess lipolytic activity. The majority of strains of L. plantarum were resistant to tetracycline, erythromycin, rifampicin, ampicillin and penicillin G.     

Proximate,amino acid and mineral composition of ostrich meat

The proximate composition, amino acid composition and mineral composition of three different muscles from the legs of seven ostriches were determined. Results indicated that components analyzed remained relatively constant between different muscles. Ostrich meat is characterized by an extremely low intramuscular fat content. Values for water, protein, ash, amino acid and mineral contents are in agreement with values obtained for beef and chicken.     

Sakacin Q produced by Lactobacillus curvatus ACU-1: Functionality characterization and antilisterial activity on cooked meat surface

This work was conducted to evaluate the antilisterial activity of sakacin Q produced by Lactobacillus curvatus ACU-1 on the surface of cooked pork meat. A genetic re-characterization of the producer strain and a study of the structural genes involved in bacteriocin production were carried out as complementary data. Studies indicated that the bacteriocin was not attached to the producer cells favoring pre-purifications steps. Bacteriocin effectiveness was not compromised by adsorption to meat and fat tissues. Several ways of dispensing the bacteriocin onto the meat surface, namely cell culture, cell free supernatant (CFS), a mixture of both and freeze-dried reconstituted CFS, were investigated. The use of the latter was the most effective one to control Listeria growth within studied systems. L. curvatus ACU-1 and its bacteriocin presented promising technological characteristics that made them suitable for meat biopreservation.