Antagonistic Potential of Chilli Pepper associated Bacillus amyloliquefaciens SBCHGBA1 against aflatoxigenic Aspergillus flavus and Aspergillus parasiticus

The Burkholderia cepacia complex (BCC) is capable of remaining viable in low-nutrient environments and harsh conditions, posing a contamination risk in non-sterile pharmaceutical products as well as a challenge for detection. To develop optimal recovery methods to detect BCC, three oligotrophic media were evaluated and compared with nutrient media for the recovery of BCC from autoclaved distilled water or antiseptic solutions. Serial dilutions (10-1 to 10-12 CFU/ml) of 20 BCC strains were inoculated into autoclaved distilled water and stored at 6°C, 23°C and 42°C for 42 days. Six suspensions of Burkholderia cenocepacia were used to inoculate aqueous solutions containing 5 µg/ml and 50 µg/ml chlorhexidine gluconate (CHX) and 10 µg/ml benzalkonium chloride (BZK), and stored at 23°C for a further 199 days. Nutrient media such as Tryptic Soy Agar (TSA) or Tryptic Soy Broth (TSB), oligotrophic media (1/10 strength TSA or TSB, Reasoner's 2nd Agar [R2A] or Reasoner's 2nd Broth [R2AB], and 1/3 strength R2A or R2AB) were compared by inoculating these media with BCC from autoclaved distilled water and from antiseptic samples. The recovery of BCC in water or antiseptics was higher in culture broth than on solid media. Oligotrophic medium showed a higher recovery efficiency than TSA or TSB for the detection of 20 BCC samples. Results from multiple comparisons allowed us to directly identify significant differences between TSA or TSB and oligotrophic media. An oligotrophic medium pre-enrichment resuscitation step is offered for the United States Pharmacopeia (USP) proposed compendial test method for BCC detection.

Foodborne illnesses have been a heavy burden in the United States and globally. Many medicinal herbs have been cultivated in the US and many of which contain antimicrobial compounds with the potential to be used for food preservation. Methanol/water extracts of pomegranate peel (“PP”, Punica Granatum L.), Chinese gallnut (“CG”, Galla chinensis), Forsythia fruit (“FF”, Forsythia suspensa) and Baikal skullcap root (“BS”, Scutellaria baicalensis) were tested for antimicrobial activity using the agar diffusion assay on tryptic soy agar (TSA) and microdilution assay in tryptic soy broth (TSB). CG and PP extracts showed good to excellent inhibitory effect against Vibrio parahaemolyticus and Listeria monocytogenes in both assays, with a minimum inhibitory concentration (MIC) range from 0.04 to 5 mg/mL. BS had moderate inhibitory effects against V. parahaemolyticus with an MIC of 5 mg/mL in TSB, and against L. monocytogenes with an MIC of 20 mg/mL on TSA. CG was analyzed using LC-MS and fractionated using HPLC. The major components were identified as gallic acid, digallic acid, methyl gallate, and gallotannins (oligo-galloyl-D-glucose, nGG, n = 1~10). Six fractions (I - VI) were collected and their antibacterial activities were tested against L. monocytogenes, and V. parahaemolyticus both on TSA and in TSB. On TSA, fraction III, IV and V inhibited V. parahaemolyticus but no fraction inhibited L. monocytogenes. In TSB, all fractions inhibited V. parahaemolyticus and fractions II - V inhibited L. monocytogenes. Future studies are needed to investigate the effects of medicinal plants on food products.

Growth Media of Escherichia coli Does Not Affect Its Survival in Soil under Static Conditions

This study was concerned on the preparation of several antigens on different types of culture media like brain heart infusion, tryptic soy agar and broth. A comparison was carried out among the prepared antigens using skin test in non- vaccinated guinea pigs which showed negative results. Another trials were conducted using sensitized guinea pigs with live attenuated anthrax vaccine. After three weeks of immunization, skin test was conducted and all antigens revealed positive skin reactions. The highest means were recorded by the dilution 1/40 on the brain heart infusion agar with the mean of erythema of 16.38 mm and a difference of thickness 3.44 mm, while on tryptic soy broth the mean of erythema was 14.14 mm and the difference of thickness was 3.43 mm. The mean of erythema and thickness were 10.21 mm and 2 mm subsequently observed on the dilution 1/40 on tryptic soy agar. The antigen gained from brain heart infusion gave a mean of erythema of 9.5 mm and 3.02 mm thickness. The results mentioned above indicated that the best antigens for the diagnosis and evaluation of anthrax vaccines are the antigen prepared on tryptic soy broth and the two diluted antigens as well.

Efficacy of sodium metabisulphite for control of Aspergillus flavus and aflatoxin B1 contamination in vitro and in chilli powder and whole red chillies

Effect of Hydrogen Peroxide and Sodium Chloride on Enumeration of Thermally Stressed Cells of Staphylococcus aureus

Inoculation Preparation Affects Survival of Salmonella enterica on Whole Black Peppercorns and Cumin Seeds Stored at Low Water Activity

Some Mold can proliferate in food to produce mycotoxin such as alphatoxin producing strain, Aspergillus flavus. Previous studies showed that the alphatoxin can cause cell mutation to induce hepatic cancers. Chemical preservative can be used in foodstuffs to inhibit mold proliferation. However, this method can let the chemical preservative remained to induce allergy and even harmful to health. Up to now, many studies found natural-non-toxic antimicrobial metabolites (biopreservatives) can be produced from some potential bacteria. Therefore, using biopreservatives to replace chemical preservatives against alphatoxin producing mold is important issue today. The Bacillus-like bacteria were isolate from health chicken intestines. After fermentation and acid precipitation, the crude CFS(cell free supernatant) extracts from various bacteria were obtained. These extracts from various bacteria can been determined whether can inhibit alphatoxin-produced fungi (Aspergillus flavus) by using agar well diffusion method. Thus the potential bacteria having anti-fungi activities were selected by agar well diffusion method. The potential bacteria strain can be further identified by 16S rRNA sequence, RAPD(random amplified polymorphic DNA) and API CH 50B method. The data from 16S rRNA sequence analysis reveal that the potential bacteria strain was Bacillus family. However the 16S rDNA analysis can't discriminate between Bacillus amyloliquefaciens and Bacillus subtilis. Thus the potential strain was further analyzed by using API CH 50 B biochemical identification system. The above result indicated the potential strain has 95% similarity to the Bacillus amyloliquefaciens. Next, analysis of RAPD pattern among the potential strain and reference strains of Bacillus amyloliquefaciens and Bacillus subtilis, the result demonstrated the potential bacteria strain, isolated from health chicken intestines with inhibition of alpha toxin producing fungi potential is Bacillus amyloliquefaciens.

Commercial biological indicator spore strips in glassine envelopes, produced by three manufacturers, were evaluated by fraction-negative procedures after being heated at 121.0 +/- 0.05 degrees C. Only one type of spore strip met the manufacturer's specifications. The strips of one manufacturer were further evaluated by fraction-negative and survivor curve-plate count procedures after being heated under several conditions (enclosed in glassine envelopes, in trypticase soy broth plus 0.0015% bromocresol purple, in Trypticase soy broth alone in Water for Injection, directly); Trypticase soy broth plus bromocresol purple and tryptic soy agar, respectively, were used as recovery media. The heating condition affected the D-value of the spore strip. Recovery procedures also had an effect; in all cases, the D-values obtained from the survivor curve tests were larger than those obtained from fraction-negative tests carried out under the same conditions. To determine if the differences in D-values between the two evaluation procedures were caused by the recovery media, we evaluated, by both methods, one type of spore strip heated directly and in glassine envelopes, using tryptic soy agar plus bromocresol purple and Trypticase soy broth plus 1.5% agar, respectively, as the recovery media. The survivor curve results showed that for both enclosed and unenclosed spore strips, there was a marked difference between the two recovery media; however, there was no difference when fraction-negative tests were used.

Behavior of Acid-Adapted and Unadapted Escherichia coli O157:H7 When Exposed to Reduced pH Achieved with Various Organic Acids

A study was carried out to determine if three strains of Escherichia coli O157:H7 grown (18 h) in Tryptic Soy Broth (TSB) and TSB supplemented with 1.25% glucose (TSBG), i.e. unadapted and acid-adapted cells, respectively, exhibited changes in tolerance to reduced pH when plated on Tryptic Soy Agar (TSA) acidified (pH 3.9, 4.2, 4.5, 4.8, 5.1 and 5.4) with acetic, citric or malic acids. All test strains grew well on TSA acidified with acetic acid at pH > or = 5.4 or malic acid at pH > or = 4.5; two strains grew on TSA acidified with citric acid at pH > or = 4.5, while the third strain grew at pH > or = 4.8. Acid-adapted and control (unadapted) cells differed little in their ability to form visible colonies on TSA containing the same acid at the same pH. However, on plates not showing visible colonies, acid-adapted cells retained higher viability than unadapted cells when plated on acidified TSA. Growth of acid-adapted and control cells of E. coli O157:H7 inoculated into TSB containing acetic acid (pH 5.4 and 5.7) and citric or malic acids (pH 4.2 and 4.5) was also studied. There was essentially no difference in growth characteristics of the two types of cells in TSB acidified at the same pH with a given acid. Tolerance of acid-adapted and control cells on subsequent exposure to low pH is influenced by the type of acidulant. The order of sensitivity at a given pH is acetic > citric > malic acid. When performing acid challenge studies to determine survival and growth characteristics of E. coli O157:H7 in foods, consideration should be given to the type of acid to which cells have been exposed previously, the procedure used to achieve acidic environments and possible differences in response among strains. The use of strains less affected by pH than type of acidulant or vice versa could result in an underestimation of the potential for survival and growth of E. coli O157:H7 in acid foods.

The survival of unheated and heat-stressed (52 degrees C, 30 min) cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) adjusted to various pHs (6.0, 5.4, and 4.8) with lactic acid and various water activities (a(w)s) (0.99, 0.95, and 0.90) with NaCl and incubated at 5, 20, 30, and 37 degrees C was studied. The performance of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), and modified eosin methylene blue agar in supporting colony development of incubated cells was determined. Unheated cells of E. coli O157:H7 grew to population densities of 10(8) to 10(9) CFU ml-1 in TSB (pHs 6.0 and 5.4) at an a(w) of 0.99. Regardless of the pH and a(w) of TSB, survival of E. coli O157:H7 was better at 5 degrees C than at 20 or 30 degrees C. At 30 degrees C, inactivation or inhibition of growth was enhanced by reduction of the a(w) and pH. A decrease in the a(w) (0.99 to 0.90) of TSB in which the cells were heated at 52 degrees C for 30 min resulted in a 1.5-log10 reduction in the number of E. coli O157:H7 cells recovered on TSA; pH did not significantly affect the viability of cells. Recovery was significantly reduced on MSMA when cells were heated in TSB with reduced pH or a(w) for an increased length of time. With the exception of TSB (a(w), 0.90) incubated at 37 degrees C, heat-stressed cells survived for 24 h in recovery broth. TSB (a(w), 0.99) at pH 6.0 or 5.4 supported growth of E. coli O157:H7 cells at 20 or 37 degrees C, but higher numbers of heated cells survived at 5 or 20 degrees C than at 37 degrees C. The ability of unheated and heat-stressed E. coli O157:H7 cells to survive or grow as affected by the a(w) of processed salami was investigated. Decreases of about 1 to 2 log10 CFU g-1 occurred soon after inoculation of salami (pHs 4.86 and 4.63 at a(w)s of 0.95 and 0.90, respectively). Regardless of the physiological condition of the cells before inoculation into processed salami at an a(w) of either 0.95 or 0.90, decreases in populations occurred during storage at 5 or 20 degrees C for 32 days. If present at < or = 100 CFU g-1, E. coli O157:H7 would unlikely survive storage at 5 degrees C for 32 days. However, contamination of salami with E. coli O157:H7 at 10(4) to 10(5) CFU g-1 after processing would pose a health risk to consumers for more than 32 days if storage were at 5 degrees C. Regardless of the treatment conditions, performance of the media tested for the recovery of E. coli O157:H7 cells followed the order TSA > modified eosin methylene blue agar > MSMA.

The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7.

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.

Increased Detection of Acid-Injured Escherichia coli O157:H7 in Autoclaved Apple Cider by Using Nonselective Repair on Trypticase Soy Agar