Lactobacilli MRS Agar Research Articles

Antimicrobial Effects of EcoCal<sup>®</sup> and GF Bactostop<sup>®</sup> Formulated in Emulsified Sausages against Lactic Acid Bacteria

In this study we evaluated the antimicrobial effects of EcoCal® (calcium oxide) and GF Bactostop ® (organic acids mix) in sausages during storage at 10oC. The sausages were formulated with 0.1% EcoCal® (0.1ECO), 0.1% EcoCal®+0.5% GF Bactostop® (0.1ECO+0.5GF), 0.2% EcoCal® (0.2ECO), and 0.2% EcoCal®+0.5% GF Bactostop® (0.2ECO+0.5GF). Total aerobic and lactic acid bacteria in the sausages were enumerated on tryptic soy agar and Lactobacilli MRS agar, respectively, during storage at 10oC for 10 weeks. The 0.1ECO+0.5GF showed the most effective antimicrobial effects on the sausages, and 0.1ECO showed the second most effective antimicrobial effect. Total aerobic bacterial cell counts gradually increased in the control, 0.2ECO, and 0.2ECO+0.5GF groups, but cell growth was generally inhibited in 0.1ECO by approximately day 42 (P<0.05) and 0.1ECO+0.5GF by approximately day 49 (P<0.05). Lactic acid bacterial cell counts gradually increased in the control, 0.2ECO, and 0.2ECO+0.5GF groups, but the lactic acid bacteria growth was inhibited in 0.1ECO by approximately day 49 (P<0.05) and in 0.1ECO+0.5GF by approximately day 64. These results suggest that using 0.1% EcoCal®+0.5% GF Bactostop® in sausage formulation is useful for inhibiting lactic acid bacteria growth, thereby extending the shelf-life of the sausage product.

Lactic acid bacterial diversity in Brie cheese focusing on salt concentration and pH of isolation medium and characterisation of halophilic and alkaliphilic lactic acid bacterial isolates

Latently existing non-starter lactic acid bacteria (NSLAB) were isolated from the French Brie de Meaux cheese using 12 isolation media with different NaCl concentrations and pH. Lactobacilli were frequently isolated using lactobacilli MRS agar, generally used for LAB isolation. Halophilic NSLAB (Tetragenococcus halophilus and Tetragenococcus koreensis) and slightly alkaliphilic NSLAB (Vagococcus lutrae), not detected using lactobacilli MRS agar or PCR-DGGE, were isolated using saline or alkaline media at log 6 to 8 cfu g−1 in the cheese rind. Their NaCl and pH optima were close to those of their isolation conditions. Phylogenetic independence of the T. halophilus isolate and genome characteristics and growth behaviour that allow T. koreensis and V. lutrae to survive in cheese rind via aerobic production of acetate from lactose were revealed. Thus, general laboratory isolation procedures should include various NaCl concentrations and pH conditions to capture a diversity of NSLAB in a living state from cheese.

Investigating the Etiology of Increased Incidence of Sour Knuckles in Commercial Beef Processing Facilities

ObjectivesCattle weights have increased during the last couple of decades and have not always been accompanied by improvements in facility capabilities and management. Alongside quality issues of color, tenderness, and water holding capacity, issues such as sour muscles and bone taints are now appearing with great frequency in the meat industry. Development of off-flavor/sourness in deep muscles such as knuckles (vastus femoris, vastus lateralis, vastus medialis, and rectus femoris) has been a long-standing issue in the beef industry, however, has not been well characterized. Therefore, the objective of this study was to investigate the cause, and characterize sour odor associated with beef knuckles using microbial, odor panel, and gas chromatography-mass spectrometric (GC–MS) analyses.Materials and MethodsKnuckles (n = 10) identified as having no sour odor (control), slight odor, or severe odor were collected from the fabrication line of a commercial beef processing plant. Sponge samples of synovial fluid and femur surface of the round were also obtained at the time of collection, for determination of anaerobic sporeformer counts. The collected knuckles were transported on ice to the laboratory where they were aseptically separated into two halves, with one half destined for microbial, odor, and GC–MS analyses on the day of collection (Day 0) and the other half for the same analyses after 35 d of vacuum packaged storage at 2°C (Day 35). For microbial analysis, 15 g of tissue was excised from the muscle surface and was analyzed for aerobic plate counts (Petrifilm Aerobic Count plates) and lactic acid bacteria counts (Lactobacilli MRS agar). Samples (5 g) for GC–MS were held at –80°C until analysis. The remainder of the sample was diced and used for trained odor panels. Data were analyzed using the ANOVA function in R (v. 3.5.1.), with a significance level of α = 0.05. Upon finding significant differences (P &lt; 0.05) the means function was used to determine differences between groups.ResultsIrrespective of sourness classification of the knuckles, similar (P &gt; 0.05) anaerobic sporeformer counts were obtained for the synovial fluid and femur surface. Additionally, muscle tissue samples from control, slightly sour and severely sour knuckles had similar (P &gt; 0.05) aerobic plate counts and lactic acid bacteria counts. Odor panelists identified differences (P &lt; 0.05) for all attributes between control and sour knuckles (slight and severe) on Day 0. Similarly, on Day 35, differences (P &lt; 0.05) were observed between control, slightly sour, and severely sour knuckles for all attributes, with severe receiving the highest score for all categories. GC–MS results showed no differences (P &gt; 0.05) between control and sour knuckles for propionic, butyric, isobutyric, and acetonic acid.ConclusionMicrobiological analysis found no differences in culturable organisms between control, slight, and severely sour knuckles on Day 0 or Day 35. However, odor panelists were able to identify differences between control and sour knuckles even after 35 d in vacuum packaging. GC–MS analysis did not indicate a statistical difference in the abundance of volatiles between the treatments, probably due to high variations within treatment groups.

Bombella apis sp. nov., an acetic acid bacterium isolated from the midgut of a honey bee.

As part of a study to investigate the microbial diversity in the intestine of Apis mellifera, we isolated strain MRM1T from the midgut. MRM1T was a Gram-stain-negative, strictly aerobic, non-motile, non-spore forming and rod-shaped bacteria. Creamy beige-coloured colonies were circular with entire margins in Lactobacilli MRS agar. The strain grew at 25-37 °C (optimum, 30-37 °C) and at a pH range of 4.0 to 9.0 (optimum pH, 7.0-8.5). The strain tolerated 0-1 % (w/v) NaCl (optimal growth occurred in the absence of NaCl). On the basis of the results of a phylogenetic analysis based on the 16S rRNA gene sequences, we determined that MRM1T represents a member of the genus Bombella with the highest sequence similarity to Bombella intestini LMG 28161T (98.8 %). The major quinone was Q10, and dominant fatty acids (>10 %) were C19 : 0cyclo ω8c (33.6 %), C16 : 0 (22.2 %), C18 : 1ω7c (15.9 %) and C14 : 0 (12.5 %). The polar lipid profile of MRM1T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified phospholipid and four unidentified lipids. The DNA G+C content of MRM1T was 59.5 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, MRM1T represents a novel species of the genus Bombella, for which the name Bombella apis sp. nov. is proposed with the type strain MRM1T (=KCTC 52452T=JCM 31623T).

Selective Enumeration of Lactobacillus acidophilus for Probiotic Formulations

Probiotic microorganisms have been utilized for many years in food processing, preservations and for nutraceutical health benefits. There are a large number of pharmaceutical formulations containing probiotic microorganisms available in the market worldwide. Majority of these organisms are the members specifically from genera Lactobacillus, Lactococcus and Bifidobacterium. A number of media have been proposed for selective or differential enumeration of lactobacilli and bifidobacteria in mixed populations. The development of selective medium using different types of ingredients and antibiotic are well practiced. In the present study we have developed a selective medium for the enumeration of Lactobacillus acidophilus by utilizing the antibiotic ofloxacin resistance of the organism in Lactobacilli MRS-agar. Samples were analyzed by determining viable counts in reference Lactobacilli MRS agar and selective Lactobacilli MRS-ofloxacin agar media. The results were statistically analyzed to evaluate the precision, accuracy, reproducibility and selectivity of the method developed. Thus MRS containing ofloxacin medium can be used for selective enumeration of Lactobacillus acidophilus strains available in commercial formulations containing Bifidobacterium species.

The Influence of Mild Pulsed Electric Field (PEF) on Acid Tolerance, Bile Tolerance, Growth and Protease Activity of the Dairy Culture Bacteria Lactobacillus acidophilus LA-K

The objective of this study was to determine the influence of mild PEF conditions on acid tolerance, growth, bile tolerance and protease activity of L. acidophilus LA-K. The treatments were positive square unipolar pulse width of 3 µs, pulse period of 0.5 second, voltage of 1 kV/cm, delay time of 20 µs and flow rate of 60 ml/min at 40.5°C PEF treatment temperature. The control was passed through the PEF system at the same flow rate (60 ml/min) without receiving any pulsed electric field condition. The acid tolerance was determined every 30 minutes for 120 minutes of incubation in acidified MRS broth at pH 2. Growth of the culture was determined hourly for 32 hours of incubation at 37°C in MRS broth. The bile tolerance was determined hourly for 16 hours of incubation in MRS-Thio broth supplemented with 0.3% (w/v) Oxgall (bovine bile) and 0.2% sodium thioglycolate. Samples were plated in duplicates using Lactobacilli MRS agar. The petriplates were incubated anaerobically at 37C for 48 hours. Protease activity was determined by o-phthaldialdehyde (OPA) spectrophotometric assay at 0, 12, 24, 36 and 48 hours of incubation of inoculated skim milk at 40°C three replications were conducted. The experimental design was repeated measurements on complete randomized block, replications were the blocks. Lactobacillus acidophilus subjected to mild PEF conditions as well as the control were acid tolerant until the end of the 120 minutes of incubation but there was a significant (P&lt;0.0001) decrease in the viable bacterial counts after each incubation time of 30 minutes. Mild PEF conditions studied significantly improved acid tolerance of the bacterium. The mild PEF treated culture reached the logarithmic phase of the growth an hour earlier than the control. Lactobacillus acidophilus LA-K exhibited tolerance to the bile conditions and exhibited similar growth patterns in the presence or absence of bile acids but the bacterium reached the stationary phase after 12 hours of incubation in bile conditions compared to 16 hours of incubation in the absence of bile conditions. Mild Pulsed Electric Field conditions had insignificant effect on bile tolerance of L. acidophilus LA-K. Mild Pulsed Electric Field treatment significantly (P&lt;0.0001) enhanced the protease activity of the bacterium compared to the control.

The effect of mild Pulsed Electric Field (PEF) conditions on acid tolerance, bile tolerance, growth and protease activity of the Lactobacillus delbrueckii ssp. bulgaricus LB-12.

Pulsed electric field (PEF) processing involves the application of pulses of voltage for less than one second to fluid products placed between two electrodes. So this study was conducted for the quantitative assessments of mild PEF conditions on acid tolerance, growth, bile tolerance and protease activity of Lactobacillus delbrueckii ssp. bulgaricus LB-12. The control and PEF treated samples were prepared by inoculating 10 ml of freshly thawed culture of Lactobacillus delbrueckii ssp. bulgaricus LB-12 into 990 ml of sterile 0.1% (wt/v) peptone water and treated in a pilot plant PEF system (OSU-4M). The treatments were positive square unipolar pulse width of 3 μs, pulse period of 0.5 second, voltage of 1 kV/cm, delay time of 20 μs and flow rate of 60 ml/min at 40.5°C. The control was passed through the PEF system (60 ml/min) without receiving any pulsed electric field condition. The acid tolerance was determined every 30 minutes for 120 minutes of incubation in acidified MRS broth at pH 2. Growth was determined hourly for 25 hours of incubation at 37°C in MRS broth. The bile tolerance was determined hourly for 16 hours of incubation in MRS-Thio broth . Samples were plated in duplicates using pH modified Lactobacilli MRS agar. The petriplates were incubated anaerobically at 43C for 72 hours. Protease activity was determined by o-phthaldialdehyde (OPA) UV- spectrophotometric assay at 0, 12, 24, 36 and 48 hours of incubation of conducted. inoculated skim milk at 40°C. Three replications were The control. The stationary phase of the bacterium was between hours 10 and 18. Moreover significant experimental design was repeated measurements on complete randomized block, Replications were the blocks. Data were analyzed using Proc Mixed model of Statistical Analysis System (SAS).The viability of the control Lactobacillus delbrueckii ssp. bulgaricus LB-12 was lost after 30 minutes of incubation in acidified MRS broth (pH 2), whereas, the bacterium subjected to mild PEF treatment was acid tolerant until the end of 120 minutes of incubation. Mild PEF significantly improved acid tolerance of Lactobacillus delbrueckii ssp. bulgaricus LB-12. and the growth reached the logarithmic phase an hour earlier than the decrease

Evaluation of reactive oxygen species generating AirOcare system for reducing airborne microbial populations in a meat processing plant

The microbial contamination of meat and meat products is of continuing concern to the meat industry and regulatory agencies. Air has been established as a source of microbial contamination in slaughter and processing facilities. The objective of this research was to determine the efficacy of reactive oxygen species (ROS) generating AirOcare equipment in reducing airborne bacteria in a meat processing environment. Bacterial strains found in ground beef were used to artificially contaminate the air using a 6-jet Collison nebulizer. Airborne bacterial populations in the meat processing room were monitored every 24 h at multiple locations using a Staplex 6 stage air sampler. Total aerobic, Gram-negative, and lactic acid bacterial populations were determined by sampling on R2A agar, MacConkey agar and Lactobacilli MRS agar, respectively. Approximately 3 log reductions of lactic acid bacteria and Gram-negative bacteria were observed after 24 h of treatment (p < 0.05) compared to ~1.5 log reduction in the control treatment. Further exposure with ROS significantly reduced lactic acid bacteria and Gram-negative bacteria in the air at 48 and 96 h sampling intervals. These findings reveal that reactive oxygen species treatment using AirOcare unit significantly reduces airborne contamination in a meat processing environment.

Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses

We sought to confirm the presence of halophilic and alkaliphilic lactic acid bacteria (HALAB) of marine origin in cheeses and thus contribute to the understanding of the roles of LAB flora in cheese ripening. We used 7% NaCl glucose-yeast extract-peptone-fish extract broth and agar media (pH 9.5) for pour-plating and enrichment culture for 16 cheese samples produced in six European countries. HALAB were present in 9 of the 16 samples at < 20 --> 10(7) CFU g(-1). In three mould-ripened soft cheeses, HALAB counts ranged from 10(6) to 10(7) CFU g(-1) and were one order (two samples) and six orders (one sample) of magnitude greater than that of nonhaloalkaliphilic, common LAB, as enumerated on lactobacilli MRS agar. The 16S rRNA gene sequences (500 bp) of 51 of the 55 isolates examined were identical or similar to that of Marinilactibacillus psychrotolerans or Alkalibacterium olivapovliticus and related species, all of which are HALAB. HALAB of possible marine origin were present in various soft, semi-hard and semi-soft cheeses and were highly predominant in some mould-ripened cheeses. HALAB of possible marine origin are members of the microflora of various cheeses and, when dominant, may play a role in the ripening of cheeses. Microbial analysis of LAB flora in cheeses should take into consideration the presence of HALAB.

Microbial ecology of fresh pork stored under modified atmosphere at −1, 4.4 and 10°C

The prevalent bacteria on fresh pork packaged in modified atmosphere with elevated CO 2 were determined by selection of representative colonies from the greatest dilution of meat samples. The pork samples were stored in two packaging films of different oxygen permeability at three storage temperatures. Strains were classified and those identified as lactic acid bacteria were screened for production of inhibitory substances. The types of bacteria isolated from samples stored in the two packaging films were similar. Storage temperature influenced the type of bacteria that dominated the microbial population. At 10°C the prevalent microflora consisted of aeromonads, Enterobacteriaceae and lactic acid bacteria but at 4.4 and −1°C, aeromonads, Brochothrix thermosphacta and lactic acid bacteria dominated. Listeriae were detected as part of the prevalent microflora on samples stored at −1°C, but not on samples stored at 4.4 or 10°C. Species of lactic acid bacteria dominating the microflora were influenced by growth medium. The majority of isolates taken from Plate Count agar were carnobacteria whereas those from Lactobacilli MRS agar were homofermentative lactic acid bacteria. Of the 538 lactic acid bacteria isolates screened for production of inhibitory substances, 162 strains showed deferred inhibition toward a range of lactic acid bacteria and nonlactic acid bacteria indicator strains.

Importance of Bile Tolerance of Lactobacillus acidophilus Used as a Dietary Adjunct

Cultures of lactobacilli identified as Lactobacillus acidophilus from the intestinal contents of young calves varied in their ability to grow in broth containing .3% oxgall compared with control broth. Frozen concentrated cultures were prepared from a strain exhibiting low tolerance to bile and from a strain exhibiting high tolerance to bile. Plate counts were comparable from the concentrated cultures before and after frozen storage on lactobacilli MRS agar with and without .15% oxgall. In a feeding trial involving newborn dairy calves supplementation of the diet with the more bile resistant strain of Lactobacillus acidophilus caused greater increases of numbers of facultative lactobabilli in the upper small intestines than did the strain exhibiting lower resistance to bile. It was not possible to determine whether the lactobacilli would prevent intestinal infections in the calves challenged with enteropathogenic Escherichia coli. This portion of the study failed as the challenge with Escherichia coli did not cause infections even in control animals.