M17 Broth Research Articles

Lactobacillus brevis BK11의 증식과 항균물질 생산을 위한 최적 배양조건 및 prebiotics의 영향

숙성된 백김치로부터 얻은 Lactobacillus brevis BK11의 증식과 항균물질 생산에 대한 배양용 배지 종류, 공기 조성, 초기 pH, 배양온도 및 시간과 프리바이오틱스의 영향을 조사하였다. L. brevis BK11의 증식과 항균물질 활성은 BHI와 M17 배지 보다는 MRS 배지 상에서 더 높게 나타났으며, 배지의 초기 pH 6.0에서 생산량이 최대에 이르렀다. 혐기적 조건보다는 호기적 및 미호기적 조건에서 배양했을 때와 <TEX>$25^{\circ}C$</TEX> 보다는 <TEX>$30^{\circ}C$</TEX>와 <TEX>$37^{\circ}C$</TEX> 온도에서 배양했을 때 균 성장과 항균물질 생산에 유리하였다. BK11 균주는 배양 24시간 만에 정지기에 도달하였고, 36시간 후부터는 생균수가 감소되었고, 배양상등액과 박테리오신 용액의 항균 활성은 <TEX>$37^{\circ}C$</TEX>, 24-30시간 배양했을 때 가장 높았다. 세포수와 유산 및 박테리오신 생산은 프락토올리고당 1-2% 첨가한 경우에 가장 높았으나, 이뉼린과 라피노오스는 균 증식에 별다른 도움을 주지 못했다. 결과적으로 L. brevis BK11의 항균물질 생산은 세포수와 관계 있었으며, 이러한 최적 조건 하에서 배양한 경우 Helicobacter pylori ATCC 43504의 성장을 효과적으로 저해할 수 있다. Lactobacillus brevis BK11 obtained from Baikkimchi was selected to study the effects of culture medium, initial pH, atmosphere composition, incubation temperature and time, and prebiotics on growth and production of antimicrobial substances. Growth and antimicrobial substances production of L. brevis BK11 were significantly higher in MRS broth than in BHI or M17 broth. The production of cell mass, lactic acid, and bacteriocin by BK11 strain was at maximum in MRS broth adjusted to pH 6.0. Aerobic and microaerobic conditions were favored cell growth and antimicrobial substances production than anaerobic condition. Biomass and lactic acid production and antimicrobial substances activity of BK 11 were significantly better at 30 and <TEX>$37^{\circ}C$</TEX> than at <TEX>$25^{\circ}C$</TEX>. Growth of the strain BK11 entered the stationary growth stage at 24 h after inoculation, and decreased after 36 h. Antimicrobial activities of cell-free culture supernatant and bacteriocin solution were highest when cultured in MRS broth with an initial pH 6.0 for 24-30 h at <TEX>$37^{\circ}C$</TEX>. In addition, the highest cell number and lactic acid and bacteriocin production were recorded in the presence of 1 and 2% (w/v) fructooligosaccharide (FOS), however, inulin and raffinose did not affect biological and physicochemical characteristics and antimicrobial activities of L. brevis BK11 cultures. According to these results, production of antimicrobial substances by L. brevis KB11 was closely associated with cell density. Under optimal conditions for antimicrobial substances production, L. brevis BK11 effectively inhibited the growth of Helicobacter pylori ATCC 43504.

Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage

We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LB+) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer.

Identification and Characterization of Lactobacilli from Rajshahi Traditional Curds

ABSTRACT The popular food, dairy products like curds are consumed by many people in its various forms in Bangladesh. Infact, these different types of curds are made without real respect for basic standards of technology and hygiene. This situation will be improved if lactic ferments are well-identified and their synergy taken into consideration. Not much information has been reported about the Lactobacilli species responsible for curd production in Bangladesh. In this context, this study was conducted to identify and characterize lactic acid bacteria in curds to provide scientific data needed for improvement of the fermented product. Twenty samples were collected from the producers of curds in the supermarket of Shaheb Bazar, Rajshahi. The culture media used were Czapek Dox (pH 5.3), M17, MRS and nutrient broth and agar for the investigation of lactic acid bacteria. The identified lactic acid bacteria were Lactobacilli including Lactobacillus casei (sample 1), Lactobacillus delbrueckii (sample 3), Lactobacillus plantarum (sample 4) and Lactobacillus helveticus (sample 6) based on morphological characteristics, catalase activities, milk coagulation, growth at different temperature, growth in medium with different NaCl concentrations, growth at Original Research Article

Production of Biogenic Amines by Lactic Acid Bacteria Isolated from Uzicka Sausages

The aim of this study was to monitor production of seven biogenic amines (Cadaverine – CAD, Putrescine – PUT, Spermine – SPE, Spermidine – SPD, Histamine – HIS, Tyramine – TYR and Tryptamine – TRY) in selected 24 lactic acid bacteria (LAB) strains. The decarboxylase activity of the microorganisms was studied in growth medium after 24h cultivation. The ability of 24 LAB isolates cultivated in MRS broth and M17 broth supplement with 0.5% glucose to produce biogenic amines was assessed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The investigation showed that LAB isolated from Uzicka sausage are not significant producers of biogenic amines in vitro.

Effects of metal ions on growth, β-oxidation system, and thioesterase activity of Lactococcus lactis

The effects of divalent metal ions (Ca2+, Mg2+, Fe2+, and Cu2+) on the growth, β-oxidation system, and thioesterase activity of Lactococcus lactis were investigated. Different metal ions significantly influenced the growth of L. lactis: Ca2+ and Fe2+ accelerated growth, whereas Cu2+ inhibited growth. Furthermore, Mg2+ inhibited growth of L. lactis at a low concentration but stimulated growth of L. lactis at a high concentration. The divalent metal ions had significant effects on activity of the 4 key enzymes of the β-oxidation system (acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and thiolase) and thioesterase of L. lactis. The activity of acyl-CoA dehydrogenases increased markedly in the presence of Ca2+ and Mg2+, whereas it decreased with 1mmol/L Fe2+ or 12mmol/L Mg2+. All the metal ions could induce activity of enoyl-CoA hydratase. In addition, 12mmol/L Mg2+ significantly stimulated activity of L-3-hydroxyacyl-CoA dehydrogenase, and all metal ions could induce activity of thiolase, although thiolase activity decreased significantly when 0.05mmol/L Cu2+ was added into M17 broth. Inhibition of thioesterase activity by all 4 metal ions could be reversed by 2mmol/L Ca2+. These results help us understand the effect of metal ions on the β-oxidation system and thioesterase activity of Lactococcus lactis.

Enterocin HZ produced by a wildEnterococcus faeciumstrain isolated from a traditional, starter-free pickled cheese

Bacteriogenic Enterococcus faecium HZ was identified by using biochemical (Strep-API 20, API-50 CHL, fatty acid profile) and 16S rRNA analysis (99·99%). Ent. faecium HZ was sensitive to clinically important antibiotics such as vancomycin, and did not have gelatinase and haemolysis activities. Enterocin HZ, a bacteriocin from Ent. faecium HZ, was sensitive to papain and tyripsin, but resistant to pepsin, lipase, catalase, α-amylase, organic solvents, detergents, ß-mercaptoethanol, and heat treatment (90°C/30min). It was biologically active at pH 2·0-9·0 and synthesised at the highest level in MRS or M17 broth at 32 or 37°C with an inoculum amount of 0·1-0·5% and an initial pH of 6·0-7·0. Enterocin HZ production reached maximum level at middle and late logarithmic phase and its molecular weight was ∼4·5kDa. It was active against some Gram-positive foodborne bacteria. Ent. faecium HZ or its bacteriocin enterocin HZ is a good candidate to be studied as a food biopreservative since enterocin HZ showed strong bactericidal activity against Listeria monocytogenes in UHT milk and also Ent. faecium HZ grew very well in milk and produced enterocin HZ at maximum level.

Escherichia coli Nissle 1917-derived factors reduce cell death and late apoptosis and increase transepithelial electrical resistance in a model of 5-fluorouracil-induced intestinal epithelial cell damage

We evaluated the capacity for supernatants (SNs) derived from Escherichia coli Nissle 1917 (EcN), cultured under different growth conditions, to prevent 5-fluorouracil (5-FU)-induced intestinal epithelial cell damage. EcN was cultured in: Luria Bertani (LB) broth, tryptone soya broth (TSB), de Man Rogosa Sharpe (MRS) broth, and M17 broth supplemented with 10% (v/v) lactose solution (M17). Intestinal epithelial cells (IEC-6) were treated with the following EcN SNs: LB+, TSB+, MRS+, and M17+ in the presence and absence of 5-FU (1.5 or 5 μM). Cell viability, apoptotic activity and cell monolayer permeability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and transepithelial electrical resistance (TER) assays, respectively. 5-FU significantly reduced cell viability (P < 0.05) at both 24 and 48 h. However, only EcN SN produced from LB and M17 growth media significantly decreased cell death induced by 5-FU (by approximately 10% after 24 and 48 h; and 10% after 24 h, respectively [P < 0.05]). When measured by flow cytometry all EcN SNs in the presence of 5-FU increased the proportion of viable cells (by 3–5% for 24 h, 3–7% for 48 h, P < 0.05) and reduced late-apoptotic cells after 24 and 48 h, compared with 5-FU control. Moreover, all EcN SNs significantly reduced the disruption of IEC-6 cell barrier function induced by 5-FU by 7–10% (P < 0.05), compared with DMEM control. We conclude that EcN derived factors could potentially reduce the severity of intestinal mucositis.

Physicochemical factors differentially affect the biomass and bacteriocin production by bovine Enterococcus mundtii CRL1656

Bovine Enterococcus mundtii CRL1656 (Centro de Referencia para Lactobacilos Culture Collection) produces an anti-Listeria and anti-Streptococcus dysgalactiae bacteriocin identified as mundticin CRL1656. The strain and its bacteriocin are candidates to be included in a beneficial product to prevent bovine mastitis as an alternative to antimicrobial agents. To optimize the production of biomass and mundticin CRL1656 by E. mundtii CRL1656, a complete 3×24 factorial design was applied. The effect of culture medium, initial pH, inoculum size, incubation temperature, and agitation conditions on biomass and bacteriocin production was evaluated simultaneously. Growth parameters were determined using the modified Gompertz model. A nonlinear model was used to estimate the effects of the variables on growth parameters. Bacteriocin production was analyzed using a linear mixed model. Optimal biomass and mundticin CRL1656 production by E. mundtii CRL1656 were obtained in different conditions. Maximal growth was recorded in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or M17 broths, pH 6.5, 5.0% inoculum, 30°C, with agitation. However, bacteriocin titers were higher in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or de Man-Rogosa-Sharpe (MRS) broths, pH 6.5, 30°C, both with or without agitation. Knowledge of the optimum conditions for growth and bacteriocin production of E. mundtii CRL1656 will allow the obtainment of high levels of biomass and mundticin CRL1656 as bioingredients of potential products to prevent bovine mastitis.

Effect of fatty acids on the β-oxidation system and thioesterase of Lactococcus lactis subspecies lactis

The influence of fatty acids on the β-oxidation system and thioesterase of Lactococcus lactis was investigated in this study. The results showed that fatty acids (C8:0–C16:0) significantly inhibited the growth of Lactococcus lactis, and laurate (C12:0) had the highest bactericidal effects. We detected the maximum activity of β-oxidation at different incubation times (8, 12, and 18h) to be 6.460, 7.751, and 8.203, respectively, and the maximum activity of thioesterase at different incubation times (8, 12, and 18h) to be 19.498, 27.180, and 12.800, respectively. Fatty acids were seen to induce the β-oxidation system and activity of thioesterase; decanoic acid (C10:0) and palmitic acid (C16:0) were also seen to induce the β-oxidation system of Lactococcus lactis, but the induced ability was significantly different. Octanoic acid (C8:0) and palmitic acid (C16:0) were seen to induce thioesterase activity in Lactococcus lactis. When 1mM palmitic acid (C16:0) was added to M17 broth, the activity of thioesterase increased 5-fold after 2min; however, adding octanoic acid (C8:0) changed the activity little. Evidence showed that the ability to induce the β-oxidation system and thioesterase activity was related to the fatty acids’ chain lengths.

Nisin Z-Producing Lactococcus lactis Subsp. Lactis GYl32 Isolated from Boza

In this study, bacteriocin-producing Lactococcus lactis subsp. lactis GYL32 strain was isolated from Boza. The GYL32 strain inhibited not only related strains but also gram-positive bacteria, such as Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. The antimicrobial substance was inactivated after treatment with proteinase K and α-chymotrypsin but not other enzymes. Bacteriocin remained active after 2 h of incubation at pH 2–11. It was stable to heat after treatment at 100C for 20 min. The bacteriocin produced by GYL32 was characterized as nisin by different pH, enzyme and heat treatments. The sequence analysis result showed that it is nisin Z, and its genetic determinants are encoded on genomic DNA. The maximum antimicrobial activity (5,120 AU/mL) was recorded after 8 h in M17 broth. Tricine-SDS-PAGE analysis showed that its molecular weight 6,700 Da. L. lactis subsp. lactis GYL32 strain may be used as a protective culture for manufacturing fermented products. Practical Applications Nisin is the best-studied bacteriocin produced by some Lactococcus lactis strains and has been used for food preservation in over 50 countries. The present study describes isolation and characterization of nisin Z-producing L. lactis strain isolated from traditional cereal-based fermented food, which is Boza from Turkey. The results of this study suggest that nisin Z producer L. lactis subsp. lactis GYL32 strain may be used as a starter culture for improving the food safety of the fermented products.

Specific Targeting of the Metallophosphoesterase YkuE to the Bacillus Cell Wall Requires the Twin-arginine Translocation System

The twin-arginine translocation (Tat) pathway is dedicated to the transport of fully folded proteins across the cytoplasmic membranes of many bacteria and the chloroplast thylakoidal membrane. Accordingly, Tat-dependently translocated proteins are known to be delivered to the periplasm of Gram-negative bacteria, the growth medium of Gram-positive bacteria, and the thylakoid lumen. Here, we present the first example of a protein, YkuE of Bacillus subtilis, that is specifically targeted by the Tat pathway to the cell wall of a Gram-positive bacterium. The cell wall binding of YkuE is facilitated by electrostatic interactions. Interestingly, under particular conditions, YkuE can also be targeted to the cell wall in a Tat-independent manner. The biological function of YkuE was so far unknown. Our present studies show that YkuE is a metal-dependent phosphoesterase that preferentially binds manganese and zinc.

Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.

Characterization of bacteriocin from Lactococcus isolated from traditional Algerian dairy products

Mesophilic strains of lactic acid bacteria belonging to the genus Lactococcus were isolated from traditional dairy products and selected for their ability to produce antimicrobial substances such as bacteriocins. Six strains: Lactococcus lactis ssp. lactis (strains Lc l1, Lc l3, Lc l5), Lactococcus lactis ssp. cremoris (strains Lc c4, Lc c6) and Lactococcus lactis ssp. lactis biovar diacetylactis (strain Lc d2) were selected for their antagonistic activities against different groups of microorganisms. The most active strain, Lactococcus lactis ssp. lactis (Lc l5), produced its inhibitory substance (Bac5) in M17 broth presenting an important inhibition mainly against Listeria monocytogenes. It had no effect on its producer strain. These properties suggested that the inhibitory substance could be considered as a bacteriocin-like substance. SDS-PAGE analyses indicated that it was a homogeneous protein of higher molecular weight of 67 kDa. The bacteriocin was stable over a wide range of pH and temperature; it was active after autoclaving and was not affected by storage at −20°C for 6 months. Moreover, it was sensitive to proteolytic enzymes confirming the proteinaceous nature of the inhibitory substance.

Utilization of UF-Permeate for Production of β-galactosidase by Lactic Acid Bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.

Effect of Medium Composition and Culture Condition on the Production of Bacteriocin-Like Inhibitory Substances (BLIS) by Lactobacillus Paracasei LA07, a Strain Isolated from Budu

ABSTRACTThe effect of medium composition (type and concentration of carbon and nitrogen sources) and culture conditions (pH, aeration and temperature) on the production of bacteriocin-like inhibitory substances (BLIS) by Lactobacillus paracasei LA07 was studied in shake flask culture. The highest BLIS production was obtained from de Man, Rogosa and Sharpe (MRS) broth, which was 4%, 22%, 51% and 63% higher than those obtained in M17 Broth, Tryptic Soy Broth (TSB), Brain Heart Infusion Broth (BHIB) and Nutrient Broth (NB) respectively. The addition of meat extract (5 g/L) and yeast extract (5 g/L) to MRS broth increased further BLIS production by 5% and 8%, respectively. The production of BLIS was further improved by 11% and 13% through the addition of tryptone (3 g/L) and glucose (10 g/L), respectively. The growth of Lb. paracasei LA07 and BLIS production were not significantly affected by agitation, either in anaerobic or in aerobic conditions. The optimum initial pH and temperature for BLIS production by Lb. paracasei LA07 was pH 8.5 and 30 °C, respectively.

Optimization of bacteriocin production by Lactobacillus acidophilus AA11, a strain isolated from Egyptian cheese

The aim of this work was to investigate the effect of growth conditions on the maximum activity of bacteriocin production by Lactobacillus acidophilus AA11. The bacteriocin was produced at maximum activity in M17 broth supplemented with 0.5% lactose (M17L). The maximum growth of Lactobacillus acidophilus AA11 was obtained at 37°C, while the optimal temperature for the bacteriocin production was 30°C. The pH of bacterial culture also affected bacteriocin production. The optimal pH for bacterial growth and bacteriocin production was 6.5. Moreover, carbon and nitrogen sources significantly influenced the production of bacteriocin. The bacteriocin yield was at least 4-fold higher in M17 broth supplemented with lactose compared to other carbon sources. The optimal organic nitrogen source for bacteriocin production was yeast extract (4%). The addition of KH2PO4, CaCl2 or NH4PO4 into the medium resulted in significant suppression of bacteriocin; however, incorporation of MnSO4 resulted in an impressive increase in bacteriocin production. No increase in bacteriocin production was recorded in medium supplemented with vitamins. The addition of glycerol to the growth medium decreased bacteriocin production. Consequently, maximum bacteriocin activity was obtained in M17L medium (pH 6.5) supplemented with 4% yeast extract and MnSO4 at 30°C. This optimization of bacteriocin production by modification of environmental growth conditions will greatly benefit efficient commercial application.

Milk Protein Fragments Induce the Biosynthesis of Macedocin, the Lantibiotic Produced by Streptococcus macedonicus ACA-DC 198

The aim of the present work was to study the mode of the induction of the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Macedocin was produced when the strain was grown in milk but not in MRS or M17 broth. No autoinduction mechanism was observed. Production did not depend on the presence of lactose or galactose in the culture medium or on a coculture of the producer strain with macedocin-sensitive or macedocin-resistant strains. Induction seemed to depend on the presence of one or more heat-stable protein components produced when S. macedonicus ACA-DC 198 was grown in milk. The partial purification of the induction factor was performed by a combination of chromatography methods, and its activity was confirmed by a reverse transcription-PCR approach (RT-PCR). Mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analyses of an induction-active fraction showed the presence of several peptides of low molecular mass corresponding to fragments of alpha(S1)- and beta-casein as well as beta-lactoglobulin. The chemically synthesized alpha(S1)-casein fragment 37-55 (2,253.65 Da) was proven to be able to induce macedocin biosynthesis. This is the first time that milk protein degradation fragments are reported to exhibit a bacteriocin induction activity.

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa. Industrial relevance Continuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.

Enumeration of starter cultures during yogurt production using Petrifilm™ AC plates associated with acidified MRS and M17 broths

The efficiency of Petrifilm AC (3M Microbiology, St. Paul, MN, USA) associated with the broths M17 and de Mann-Rogosa-Sharpe (MRS) at pH 5.4 was evaluated to enumerate Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus during the yogurt production. Commercial and reference strains of these microorganisms were experimentally inoculated in nonfat milk and incubated at 42 degrees C for 4 h for yogurt production. At the moment of inoculation and after incubation, aliquots were collected, submitted to dilution using the broths M17 and MRS at pH 5.4, and plated for Strep. salivarius and Lb. bulgaricus enumeration according ISO 9232 and at Petrifilm AC plates, respectively. M17 plates were incubated at 42 degrees C, and MRS plates were incubated at 35 degrees C under anaerobiosis. After 48 h, the formed colonies were enumerated and the counts were compared by correlation and analysis of variance (P<0.05). In addition, colonies were randomly selected from all plates and characterized according to Gram staining and morphology. The obtained results indicated that Petrifilm AC plates associated to M17 and MRS at pH 5.4 can be considered as a suitable alternative for Strep. salivarius and Lb. bulgaricus enumeration during yogurt production, with slight interferences due to the acidity of MRS at the moment of inoculation, and due to the acidity of yogurt at the end of fermentation process. It was also observed that the MRS at pH 5.4 was not sufficiently selective for Lb. delbrueckii enumeration, despite it is indicated by the official protocol from ISO 9232.

Thermal inactivation kinetics ofLactococcus lactissubsp.lactisbacteriophage PLL98-22

Survival curves of Lactococcus lactis subsp. lactis bacteriophage pll98 inactivated by heat were obtained at seven temperature values (50-80 degrees C) in M17 broth and skim milk. Deviations from first-order kinetics in both media were observed as sigmoidal shapes in the survival curves of pll98. An empirical model with four parameters was used to define the thermal inactivation. Number of parameters of the model was reduced from four to two in order to increase the robustness of the model. The reduced model produced comparable fits to the full model. Both the survival data and the calculations done using the reduced model (time necessary to reduce the number of phage pll98 six- or seven- log10) indicated that skim milk is a more protective medium than M17 broth within the assayed temperature range.