Production of organic acids by Lactobacillus strains in three different media

Ten strains of Lactobacillus (Lb). casei, Lb. rhamnosus, Lb. plantarum, Lb. paracasei and Lb. curvatus species were chosen to determine the production of organic acids after cultivation in skimmed milk, MRS broth and Jerusalem artichoke (JA) medium. The highest acidity was obtained in MRS broth and the weakest acidification was found in skimmed milk. Lb. casei Shirota produced the highest amount and Lb. rhamnosus VT1 the lowest amount of substances being estimated as titratable acidity. All strains produced lactic acid in the investigated broth and most of the strains produced acetic acid in MRS broth except Lb. curvatus 2768 and Lb. casei Shirota, in JA broth except Lb. paracasei SF1 and in skimmed milk except Lb. casei 2750, Lb. curvatus 2768, Lb. curvatus 2775 and Lb. casei Shirota. All strains, except Lb. plantarum 01, produced butyric acid in MRS broth. Beside the lactic and acetic acids, formic, citric, succinic and glutamic acids were also produced in MRS broth; formic and succinic acids were produced in skimmed milk and succinic acid in JA broth. Some strains showed change in their fermentation profile from homofermentative to mix-acid fermentation in milk. The antifungal efficiency of the lactic and acetic acid in the amount produced by lactobacilli was investigated. None of the investigated aspergilli were inhibited. The inhibitory effect of acids against Fusarium increased unequivocally with the increasing concentration. The study pointed at the dissimilarity of organic acid production of Lactobacillus strains, which was considerably influenced by the media.     

Modelling contributes to the understanding of the different behaviour of bacteriocin-producing strains in a meat environment

Bacteriocins from lactic acid bacteria are potent antibacterial agents that enable the producer cells to selectively and efficiently inhibit a part of the competing background flora, including several spoilage bacteria and foodborne pathogens. The use of bacteriocinogenic lactic acid bacterium strains, either as starters or as protective cultures, is very limited in the food industry, possibly because many questions about the in situ functionality of such strains remain unanswered. A modelling approach was developed to study the functionality of lactic acid bacterium bacteriocin producers in a meat environment. The kinetic properties of three potential bacteriocin-producing starter cultures for sausage fermentation, namely Lactobacillus sakei CTC 494, Lactobacillus curvatus LTH 1174, and Lactobacillus amylovorus DCE 471 were compared in MRS, used as a meat simulation medium. Moreover, the application possibilities of the bacteriocin-producing Enterococcus faecium RZS C5 strain were evaluated. It turned out that growth and bacteriocin production by Lb. sakei CTC 494 and Lb. curvatus LTH 1174 are adapted to the meat environment, whereas Lb. amylovorus DCE 471 is kinetically not able to dominate the meat fermentation process. Furthermore, the use of bacteriocin-producing enterococci as functional cocultures seems to be attractive.     

Partial Characterization of Nine Bacteriocins Produced by Lactic Acid Bacteria Isolated from Cold-Smoked Salmon with Activity against Listeria monocytogenes

Nine LAB bacteriocin-producers, isolated from vacuum-packaged cold-smoked salmon (CSS), were phenotypically and genotypically identified as Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Enterococcus faecium, and Pediococcus acidilactici. Their bacteriocins were partially characterized. The antimicrobial spectrum was determined against Listeria monocytogenes, E. faecalis, E. faecium, and Staphylococcus aureus. The molecular size of bacteriocins ranged from 2.8 to 4.5 kDa. They were inactivated by treatment with proteolytic enzymes but not by lipolytic or glycolytic enzymes. Maximal activity against L. monocytogenes ranged between 800 and 10000 AU/mL at pH 6.5. Most of the bacteriocins maintained full activity in a pH range of 2.0 to 8.0 but were partially or completely inactivated at pH 10.0. After heating at 60°C and 100°C, only two bacteriocins from Lb. curvatus strains partially lost activity. All bacteriocins showed a narrow spectrum of activity and a high anti-listerial activity, which is characteristic of the class IIa bacteriocins. Isolated bacteriocin-producing LAB could be used successfully in the bio-preservation of CSS and development of new potential bio-preservatives for CSS active against L. monocytogenes.     

Bacteriocin-producing lactobacilli in Spanish-style fermented sausages: characterization of bacteriocins

Three bacteriocins were studied. Sakacin K, a bacteriocin from Lactobacillus sake CTC494 was purified to homogeneity by a four-step system involving ammonium sulphate precipitation, binding to a cation exchanger, hydrophobic interaction and reverse phase chromatography in FPLC (fast performance liquid chromatography) system. The peptide sequence was determined by Edman degradation. The first 30 amino acid residues from the N-terminus of sakacin K were identical to those of curvacin A from Lactobacillus curvatus LTH1174 and sakacin A from Lactobacillus sakei Lb706. The structural gene of sakacin K in Lb. sake CTC494 was located on a 60 Kbp plasmid (as has been previously reported) by curvacin A in Lb. curvatus LTH1174 and sakacin A in Lb. sakei Lb706. Plantaricin D, a bacteriocin-like compound isolated from Lb. plantarum CTC305 was purified and sequenced using the same procedure as described for sakacin K. The first 15 amino acid residues of plantaricin D were identical to those obtained from the bacteriocin inducer peptide (termed plantaricin A) of the bacteriocinogenic system in Lb. plantarum C11. The structural gene of the plantaricin D peptide was located on a similar EcoRl chromosomal fragment when compared to plantaricin A in Lb. plantarum C11. These two isolated bacteriocin-like peptides (sakacin K and plantaricin D) were purified from two new different strains obtained from fermented meat, confirming the ecological importance of these substances. Lactobacillus sakei CTC372, the third bacteriocinogenic strain selected in this study from fermented sausages, produced a bacteriocin named sakacin T. The bacteriocin is not found free in the supernatant of the cells. It is active against Listeria monocytogenes and Staphylococcus aureus. The genetic determinant of this bacteriocin was localized in a 84·4 Kbp plasmid by conjugative transfer and curing assays.     

Identification of Lactic Acid Bacteria Isolated from Serbian Traditional Fermented Sausages Sremski and Lemeski Kulen

The microbial composition of Serbian traditional fermented sausages, Sremski and Lemeski kulen, was studied. The dominant lactic acid bacteria in these products were Lactobacillus (77.1 and 54.3%, respectively) and Leuconostoc (20.0 and 22.9%, respectively). Lemeski kulen was characterized by a high percent of Lactococcus (20.0%). The most abundant Lactobacillus species in Sremski and Lemeski kulen was Lb. brevis (61.5 and 57.9%, respectively). Sremski kulen contained Lb. pentosus (11.5%) and Lb. salivarius (7.7%), in contrast to Lemeski kulen which contained Lb. curvatus ssp. curvatus (15.8%). Lb. paracasei, Lb. plantarum and Lb. fermentum were present in similar percentages in both products.     

Characterization of bacteriocin ST8KF produced by a kefir isolate Lactobacillus plantarum ST8KF

Lactobacillus plantarum ST8KF, isolated from kefir, produced a 3.5 kDa bacteriocin (bacST8KF) active against Lb. casei, Lb. salivarius, Lb. curvatus and Listeria innocua. BacST8KF was sensitive to proteolytic enzymes, but stable between pH 2.0 and 10.0, and heat resistant (20 min at 121 °C). BacST8KF did not adsorb to the surface of the producer cell. Maximum activity (25,600 AU mL⁻¹) was recorded in MRS broth with glucose, in MRS broth with glucose replaced by sucrose, and in MRS broth with glucose, supplemented with KH₂PO₄ after 24 h at 30 °C. Tri-ammonium citrate and glycerol in excess of 5.0 g L⁻¹ repressed bacST8KF production. Production of bacST8KF increased from 800 AU mL⁻¹ after 3 h of fermentation in MRS broth at 30 °C to 12,800 AU mL⁻¹ after 9 h and to 51,200 AU mL⁻¹ after 27 h. These results suggest that bacST8KF may be a secondary metabolite and shows that its mode of activity is bacteriostatic.     

Pressure-resistant acclimation of lactic acid bacteria from a natural fermentation product using high pressure

Twenty colonies of Pediococcus acidilactici, Lactobacillus brevis, Lactiplantibacillus plantarum and Latilactobacillus curvatus were isolated from fermentation broth of pickled pepper by using MRS agar. The strains were acclimated using high pressure from 300 to 400 MPa for 1 to 5 min. For one generation of acclimation, the survived cells after first high pressure treatment were then activated by culturing on MRS agar for two days for the next high pressure treatment. After acclimation for 30 generations, the survival ratio of L. plantarum and L. curvatus was increased by 138 and 1222 times at 400 MPa for 1 and 3 min, respectively. Weibull model could appropriately fit the survival curves of L. plantarum and L. curvatus treated by high pressure. The time to the first decimal reduction of these two strains after acclimation was 3.5 and 1.8 times, respectively, longer than before acclimation at 300 MPa. From electron microscopy photographs, the acclimated L. curvatus was more curved, smaller and its cell wall was thicker than the unacclimated L. curvatus. After HPP treatment, L. plantarum and L. curvatus have higher cell integrity, smoother cell surface, more uniform protoplasm and smaller cavities in the cell protoplasm compared with unacclimated strains, suggesting that high pressure acclimation introduced some modification to bacteria intrinsic structures and increased their pressure resistance. Moreover, the fermentation performance including glucose utilization capacity and lactic acid production of the acclimated L. curvatus was respectively improved by 9.6% and 9.4% at 37 °C for 24 h in MRS medium.     

Enterococcus faecium P21: a strain occurring naturally in dry-fermented sausages producing the class II bacteriocins enterocin A and enterocin B

Enterococcus faecium P21 isolated from a Spanish dry-fermented sausage shows a narrow antimicrobial activity against closely related lactic acid bacteria and strong antimicrobial activity against spoilage and foodborne Gram-positive pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and Clostridium botulinum. The antimicrobial activity is produced during growth in MRS broth at 32°C; it is heat resistant (20 min at 80–100°C) and abolished by protease treatment, and withstands exposure to pH 2–11, freeze-thawing, lyophilization and long term storage at −20°C. Purification of the antimicrobial activity by ammonium sulphate precipitation, gel filtration, and cation-exchange, hydrophobic interaction and reverse-phase chromatographies showed that the broad antimicrobial spectrum exerted by E. faecium P21 was indeed due to two peptide bacteriocins. Studies on theirN -terminal amino acid sequences and PCR and DNA analyses revealed that these bacteriocins are identical to the class II bacteriocins enterocin A and enterocin B. The genetic organization of the enterocin A (EntA) operon in E. faecium P21 shows that the bacteriocin structural gene (entA), the immunity gene (entiA) and the induction peptide gene (entF) are clustered and colinearly arranged. Strikingly, this organization was structurally different to that reported for the EntA-producer E. faecium CTC492.     

Development of an active wheat gluten film with Lactobacillus curvatus CRL705 bacteriocins and a study of its antimicrobial performance during ageing

Antimicrobial wheat gluten film was obtained at pilot scale by Lactobacillus curvatus CRL705 bacteriocins inclusion in the film-forming solution. Bacteriocins’ minimum inhibitory concentration for the film activation was 2133 AU cm^?3 (lactocin AL705) and 267 AU cm^?3 (lactocin 705). Mechanical and barrier properties as well as film ageing kinetics were not significantly affected by the addition of bacteriocins. The antimicrobial film performance during ageing was assessed. Film activity against Listeria innocua 7 and Lactobacillus plantarum CRL691 was observed over 50 days of ageing. Even when the release of bacteriocins from the film upon water contact was observed for both bacteriocins at the beginning of the ageing period, and anti-Listeria activity was delivered to the simulant up to the 15th day of ageing, film residual activity for both bacteriocins was observed over 50 days. The results confirm the potential of a gluten film doped with L. curvatus CRL705 bacteriocins as a carrier of bacteriocins to avoid Listeria and lactic acid bacterial growth, thus enhancing quality and safety in foods.     

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.     

Formation of conjugated linoleic acid by a Lactobacillus plantarum strain isolated from an artisanal cheese: Evaluation in miniature cheeses

Among 129 lactic acid bacteria previously isolated from raw-milk starter-free cheeses manufactured in Galicia (NW Spain), two strains of Lactobacillus plantarum were definitely recognised as producers of conjugated linoleic acid (CLA). Gas chromatography analysis identified cis-9, trans-11 C18:2 as the predominant CLA isomer formed in MRS broth supplemented with linoleic acid. A centrifugation-based model for the manufacture of miniature cheeses was used to evaluate the formation of CLA by Lb. plantarum L200, the highest producer of CLA in MRS broth. The miniature cheeses made with the addition of the L200 strain showed significantly (P < 0.05) higher contents of cis-9, trans-11 CLA than those of the control cheeses (1.09 versus 0.69 percentage of total fatty acids, respectively). These results suggest that Lb. plantarum L200 strain could be used as an adjunct culture to slightly increase the concentrations of CLA in short-ripened cows' milk cheeses.     

Membrane-active bacteriocins to control Salmonella in foods: Are they the definite hurdle?

According to recent surveys salmonellosis is the main disease caused by foodborne microorganisms in many countries. Although traditional methods to control bacterial contamination in food are effective in controlling Salmonella, a major problem arises with fresh and organic food demanded by the market with little preparation and no chemical additives. Besides the development of new technologies that combine soft physical and chemical treatments in a way that causes minimal changes in food properties, antimicrobial peptides derived from natural sources are presented as an interesting alternative to chemical preservatives. There are many bacteriocins active against Salmonella and they could be used in the food industry alone or in combination with other hurdles to increase the anti-Salmonella effect in the so-called hurdle technology. However, further studies are necessary to demonstrate the safety of new bacteriocins and to improve their activity in different environmental conditions encountered during food processing. Moreover, nisin and pediocin PA-1 already approved as food preservatives by various government agencies, can be used along with other hurdles to control the pathogen in foods. In this paper the use of bacteriocins as hurdles for controlling Salmonella in the future is discussed and analyzed.     

Negative effect of divalent metal cations on production of gassericin T, a bacteriocin produced by Lactobacillus gasseri, in milk-based media

A broad-spectrum bacteriocin named gassericin T is produced by Lactobacillus gasseri LA158 in MRS broth, but is minimally produced in milk-based media. Gassericin T production in MRS broth was specifically inhibited by adding divalent metal cations. Such inhibition of gassericin T production depended on the concentration of divalent cations. The addition of trisodium citrate dihydrate (TSC), which is a food-grade chelator of divalent cations, restored gassericin T production both in modified MRS broth containing divalent cations and in milk-based medium. In modified MRS broth, 50 mmol L⁻¹ TSC promoted an over-production of gassericin T. Furthermore, it was confirmed that TSC had a synergistic antibacterial effect with gassericin T. In this study, a cheese-whey-based medium, supplemented with proteose peptone, TSC, and Tween 80, was developed for gassericin T production. These results may contribute to the effective use of bacteriocins for food preservation.     

Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk

Nine strains of non-starter Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk were selected for their anticlostridial activity. Resistance to thermisation (60 °C, 5 min) and pasteurisation (73 °C, 15 s) was investigated using a submerged-coil apparatus. MRS broth-grown cultures of all nine strains survived thermisation in buffer. The level of resistance to thermisation was strain dependent and lower for freshly grown cells (stationary phase cells) than for resting cells (freshly grown cells kept diluted 10-fold in MRS broth at 17 °C for 6 days). None of the nine Lb. paracasei strains survived or recovered after pasteurisation in buffer when grown in MRS broth, while seven of the nine strains survived pasteurisation in UHT whole milk when grown in milk. Identity of the strains was successfully confirmed during the experiments using repetitive-PCR analysis. The potential of Lb. paracasei strains to survive pasteurisation of cheese milk was demonstrated.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Development of Wide-Spectrum Hybrid Bacteriocins for Food Biopreservation

The food industry demands new procedures and methods to produce minimally processed, ready to eat food with intact nutritional, taste, and flavor properties. The biopreservation and the use of both bacteriocins produced by lactic acid bacteria (LAB) and bacteriocinogenic strains as an alternative to substitute chemical antimicrobial for food preservation became increasingly important in the last two decades. When the new proposed natural preservatives techniques are applied, probiotics food can be obtained and, simultaneously, foodborne pathogens and spoilage contaminants can diminish. However, bacteriocins produced by LAB have a narrow antibacterial spectrum and are inactive against Gram-negative bacteria like Salmonella and the emergent enterohemorrhagic Escherichia coli. Knowing the mechanism of action and the structural features of microcins synthesized by Gram-negative bacteria and with potent antimicrobial activity against the mentioned microorganism, the proposal is to obtain hybrid peptides (microcin–bacteriocin) with broad antimicrobial spectrum. This review explains how the inability of bacteriocins to cross the outer membrane of Gram-negative bacteria unable them to act on the bacteria. It will also be discussed how a hybrid bacteriocin can be obtained.     

Growth phase and growth medium effects on the peptidase activities of Lactobacillus helveticus

The effect of growth phase, growth medium and storage conditions on the peptidase activities of Lactobacillus helveticus DPC 4571 were examined. Cell free extracts (CFEs) were prepared from both mid-log and stationary phase cells grown in either 10% (w/v) reconstituted skim milk (RSM) or MRS pH 5.4 broth. In general, Lb. helveticus DPC 4571 was found to possess a broad range of aminopeptidase, dipeptidase, tripeptidase, endopeptidase and proline-specific peptidase activities. The stability of these peptidase activities in a CFE of Lb. helveticus DPC 4571 stored at 4°C was variable. The specific activities of the majority of these peptidases were significantly (P<0.05) influenced by both growth phase and growth medium. Cells grown to mid-log phase in RSM were found to have the highest level of peptidase activity.     

Antimicrobial activity of Enterococcus faecium L50, a strain producing enterocins L50 (L50A and L50B), P and Q, against beer-spoilage lactic acid bacteria in broth, wort (hopped and unhopped), and alcoholic and non-alcoholic lager beers

Enterococcus faecium L50 produces enterocin L50 (L50A and L50B) (EntL50, EntL50A and EntL50B), enterocin P (EntP) and enterocin Q (EntQ) and displays a broad antimicrobial spectrum against the most relevant beer-spoilage lactic acid bacteria (LAB) (i.e., Lactobacillus brevis and Pediococcus damnosus), which is mainly due to the production of EntL50 (EntL50A and EntL50B). Bacteriocin assays using in vitro-synthesized EntL50 (EntL50A and EntL50B) showed that both individual peptides possess antimicrobial activity on their own, EntL50A being the most active, but when the two peptides were combined a synergistic effect was observed. The only virulence genes detected in E. faecium L50 were efaAfm (cell wall adhesin) and ccf (sex pheromone), and this strain was susceptible to most clinically relevant antibiotics. E. faecium L50 survived but did not grow nor showed antimicrobial activity in hopped and unhopped wort, and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) commercial lager beers. However, when unhopped wort was supplemented with 50% (v/v) MRS broth, E. faecium L50 grew and exerted antimicrobial activity similarly as in MRS broth. The enterocins produced by this strain were bactericidal (5 log decrease) against P. damnosus and Lb. brevis in a dose- and substrate-dependent manner when challenged in MRS broth, wort (hopped and unhopped), and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) lager beers at 32 degrees C, and no bacterial resistances were detected even after incubation for 6-15 days. The enterocins in wort and lager beer (5% ethanol, v/v) withstood the heat treatments commonly employed in the brewing industry during mashing, wort boiling, fermentation, and pasteurization, and retained most of their antimicrobial activity in lager beer (5% ethanol, v/v) after long-term storage at 8 and 25 degrees C.     

Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products

Bioactive peptides are short peptides approximately 2–30 amino acids in length. They are inactive within the sequence of the parent protein and can be released during gastrointestinal digestion, during food processing or by hydrolysis using commercial enzymes. Meat derived peptides have a myriad of bioactive potential including, antioxidant, antimicrobial, anti-thrombotic, ACE-I-inhibitory and cytomodulatory functions. Antioxidant and antimicrobial peptides isolated from meat muscle sources may be used as functional ingredient in food formulations to impart human health benefits and/or improve the shelf life of foods. This review collates information regarding peptidic hydrolysates with antioxidant and antimicrobial properties isolated from vertebrate and invertebrate muscle and by-products, identifying the sources, the isolation and characterisation techniques used, and the methods used to demonstrate these bioactivities in vitro.