الفهرس 
REVIEW OF LITERATURFungal spoilage of food and feedOnly 14 pages are availabe for public view
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Abstract
This study was performed to investigate the antifungal activity of lactic acid bacterial (LAB) strains. Seven bacterial strains were tested against four fungal isolates that were isolated from infected corn grains and identified as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Rhizopus nigricans.
* The fungal biomass inhibition method was used as a pre-screening test: All tested lactic acid bacterial strains inhibited the growth of the four fungal isolates to varying extents except lactobacillus plantarum NRRL B-448 strain. L. plantarum ATCC 14917 achieved the highest inhibition percentage (88.4%) against R. nigricans. also, Bifidobacterium lactis (Bb12) achieved the highest inhibition percentage (87.75%) against A. parasiticus. while, Streptococcus thermophiles ATCC-3 achieved the highest inhibition percentages (86.8%) and (86%) against A. flavus and R. niger, respectively.
* The aflatoxin-producing Aspergilli were detected by the black-light method: It was found that two fungal strains were positive AFs (as they produce blue fluorescence which associated with the aflatoxin production). They include Aspergillus parasiticus, produced both B and G aflatoxins while the other was Aspergillus flavus, and produced only B aflatoxins.
A total of twelve LAB strains were screened for antifungal activity against two aflatoxin-producing Aspergilli. The tested LAB strains were Lactobacillus acidophilus ATCC 20552, Lactobacillus acidophilus NRRL-B-4495, Lactobacillus acidophilus LA3, Lactobacillus acidophilus, Lactobacillus plantarum ATCC 14917, Lactobacillus casei DSM 20011, Lactobacillus rhamnosus, Bifidobacterium bifidium, Bifidobacterium longum 15708, Bifidobacterium lactis (Bb12), Streptococcus thermophiles, Streptococcus thermophiles ATCC-3.
*The antifungal activity was performed by three methods: in the Overlay method, the maximum inhibitions of A. flavus and A. parasiticus were obtained by S. thermophiles ATCC-3. Streptococcus thermophiles ATCC-3 was very highly active (++++) against A. flavus and have moderate activity (++) against A. parasiticus. Bifidobacterium lactis (Bb12) and Lactobacillus casei DSM 20011 were highly active (+++) against A. flavus and have moderate activity (++) against A. parasiticus.
While, in the Well diffusion method, the maximum inhibition zones against A. flavus were obtained by B. lactis Bb12(38.0 mm) and B. longum (37.0 mm) and in case of A. parasiticus, the maximum inhibition zones was obtained by B. lactis Bb12 (35.0 mm) followed by B. longum 15708(33.0 mm). In case of using the disc diffusion assay B. longum achieved the maximum inhibition zones against A. flavus (11.0 mm) and A. parasiticus (7.5 mm).
Whereas, in the Agar dilution method, the maximum inhibitions of A. flavus were shown by B. lactis Bb12 (70.3%) followed by L. casei DSM 20011 (57.77 %) and S. thermophiles ATCC-3 (57.46 %), respectively and in case of A. parasiticus the maximum inhibitions were obtained by B. bifidium (84.59%) followed by B. lactis Bb12 (82.66%) and S. thermophiles ATCC-3 (68.69%).
*Assessment of MIC of selected LAB strains examined against both A. flavus and A. parasiticus using two methods
1. Assessment of the MIC of lactic acid bacterial strains by the fungal biomass inhibition method: It was observed that increasing of the supernatant concentration from 10 % to 25 % was accompanied with a decrease in the dry weights of the tested fungi. But, in case of using supernatant concentrations from 50 % to 75 %, there is no any detectable growth for both of the tested fungal isolates. The highest antifungal activity was obtained by B. lactis (Bb12) which has the MIC at supernatant concentration of 10% against both, A. flavus and A. parasiticus. A moderate antifungal activity was detected by both L. acidophilus NRRL-B-4495 and L. plantarum ATCC 14917, which have the MIC at supernatant concentration of 20 % against A. flavus, while only L. acidophilus NRRL-B-4495 has MIC at supernatant concentration of 20 % against A. parasiticus.
2. Assessment of the MIC of lactic acid bacterial strains by the agar dilution method: The results of MIC value showed that A. flavus exhibited the highest susceptibility (52.8 %) towards the supernatant of both B. lactis (Bb12) and L. acidophilus NRRL-B- 4495 with MIC values at 250 µl and 1000 µl. Also, A. parasiticus showed the highest susceptibility (52.2 %) towards the supernatant of L. acidophilus NRRL-B- 4495 with MIC value at 250 µl. Also, The cell-free supernatants of both S. thermophiles ATCC- 3 and L. plantarum ATCC 14917 have a moderate antifungal activity and have exhibited MIC values at a conc. of 1000 µl against both of A. flavus (31.9 and 40.4 %, respectively) and A. parasiticus ( 30.8 and 27.8 %, respectively).
*Determination of the antifungal metabolites produced by the LAB strains as follow:
1. HPLC analysis: It was observed from the results of the total content of the identified organic acids that L. rhamnosus was the highest strain in the production of total organic acids (6.898 mg/ ml) after 48hrs. Then L. casei DSM 20011 (4.717 mg/ ml) after 16hrs followed by S. thermophiles (4.694 mg/ ml) after 24hrs. Also, it was observed that the main and the highest organic acids produced by LAB strains were the lactic acid (2.64 mg/ ml) and the acetic acid (1.72 mg/ ml) from L. rhamnosus after 48 hrs. These organic acids were followed by succinic acid (1.12 mg/ ml) from L. plantarum ATCC 14917 after 24 hrs and propionic acid (1.04 mg/ ml) from S. thermophiles ATCC-3 after 48 hours. While the lowest organic acids produced by L. casei DSM 20011was mallic acid (0.024 mg/ ml).
2. Quantitative determination of the titratable acidity showed that, the highest total organic acids produced (38.28 mg/mL) after 48 hours was by L. plantarum ATCC 14917, followed by L. acidophilus ATCC 20552 (24.77 mg/mL). While, the lowest total organic acids was produced (4.50 mg/mL) by L. casei DSM 20011after 48hrs.
3. Quantitative determination of H2O2 revealed that, all tested LAB strains produced hydrogen peroxide at minimal concentrations ranging between 0.00816 mg/mL to 0.03266 mg/mL in the extracellular extracts. The maximum amount of H2O2 was produced (0.03266 mg/mL) by L. plantarum ATCC 14917and the lowest amount of H2O2 was produced (0.00816 mg/mL) by L. rhamnosus.
4. Quantitative determination of diacetyl produced was ranged from (4.304 mg/mL) to (34.432 mg/mL) in the extracellular extracts: The highest diacetyl was produced (34.432 mg/mL) by B. lactis (Bb12), followed by S. thermophiles ATCC-3 (25.824 mg/mL) after 48hrs. While, the lowest diacetyl was produced (4.304 mg/mL) by L. rhamnosus.
5. Quantitative determination of total protein concentration: The protein concentration is carried out by spectrophotometric analysis using Biuret method. The highest concentrations of protein was produced (3.69 mg/dl) was by Streptococcus thermophiles ATCC-3 followed by Lactobacillus rhamnosus (3.41 mg/dl). While the least protein concentrations was obtained (0.61 mg/dl) in the cells- free supernatant of Lactobacillus acidophilus.
* By determination of the physical characteristics of antifungal metabolites produced by the LAB strains as follow:
1. The effect of temperature: It was observed that the optimum antifungal activity for all tested LAB strains was recorded at 30°C. As the inhibitory activity of the antifungal substances were decreased with the increase in temperature. However, the cell-free supernatants of L. Acidophilus LA3, L. Plantarum ATCC14917and L. casei DSM 20011 were resistant and have antifungal potentials at different temperatures against A. flavus and A. parasiticus, showing thermal stability at temperatures range from 60 to 100 °C. However, these strains lost their activity after treatment at 121°C for 15 min. Also, the cell-free supernatants of both S. thermophiles ATCC-3 and S. thermophiles sp, were thermal stable when exposed to the temperature up to 60°C. But above these conditions, they were sensitive (80-100°C and autoclaving at 121°C for 15 min).
2. The effect of pH: It was observed that, the highest antifungal activity obtained was at pH range from 4.0 to 6.0 and the antifungal activity decreases as the pH increase. At pH (2.0), a complete loss of the antifungal activity was observed for all strains of Lactic acid bacteria. A complete loss of activity was observed at pH (8.0) and above except for L. Acidophilus LA3, S. thermophiles ATCC-3, S. thermophiles sp, and L. casei DSM 20011. Also, L. Plantarum ATCC14917, B. lactis (Bb12) and B. bifidium had antifungal activities at pH (8.0) against both A. flavus and A. parasiticus. While, at pH range from 8.0 to 12.0, it was observed that all tested LAB strains had no antifungal activity at the alkaline environment.
3. The effect of Enzymes: A complete inactivation of the antifungal of the cell-free supernatants was observed for certain strains such as L. Acidophilus LA3, L. Plantarum ATCC 14917, L. casei DSM 20011, S. thermophiles ATCC-3, S. thermophiles sp, B. lactis (Bb12) and B. bifidium after treatment with proteolytic enzymes (pepsin and trypsin). This indicated that the antifungal substances are protein in nature or at least has a proteinaceous activator. However, the treatment of the cell-free supernatants of the other LAB strains with the proteolytic enzymes did not result any changes of the antifungal activity. This indicating that the antifungal substance produced was not bacteriocin or has a proteinaceous activator. Also, it was observed that the treatment of cell-free supernatants of all LAB strains with catalase enzyme did not result any changes of the antifungal activity. This is indicating that the hydrogen peroxide did not account for the observed inhibition.
4. The effect of Surfactants: Almost all the surfactants used had no effect on the antifungal activity of the cell-free supernatant of the tested LAB strains in the concentrations used except the effect of Sodium Dodecyl Sulphate (SDS). As the SDS has stimulatory effect to the antifungal activity of the cells-free supernatant of the LAB strains against A. flavus and A. parasiticus.
This study was performed to investigate the antifungal activity of lactic acid bacterial (LAB) strains. Seven bacterial strains were tested against four fungal isolates that were isolated from infected corn grains and identified as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Rhizopus nigricans.
* The fungal biomass inhibition method was used as a pre-screening test: All tested lactic acid bacterial strains inhibited the growth of the four fungal isolates to varying extents except lactobacillus plantarum NRRL B-448 strain. L. plantarum ATCC 14917 achieved the highest inhibition percentage (88.4%) against R. nigricans. also, Bifidobacterium lactis (Bb12) achieved the highest inhibition percentage (87.75%) against A. parasiticus. while, Streptococcus thermophiles ATCC-3 achieved the highest inhibition percentages (86.8%) and (86%) against A. flavus and R. niger, respectively.
* The aflatoxin-producing Aspergilli were detected by the black-light method: It was found that two fungal strains were positive AFs (as they produce blue fluorescence which associated with the aflatoxin production). They include Aspergillus parasiticus, produced both B and G aflatoxins while the other was Aspergillus flavus, and produced only B aflatoxins.
A total of twelve LAB strains were screened for antifungal activity against two aflatoxin-producing Aspergilli. The tested LAB strains were Lactobacillus acidophilus ATCC 20552, Lactobacillus acidophilus NRRL-B-4495, Lactobacillus acidophilus LA3, Lactobacillus acidophilus, Lactobacillus plantarum ATCC 14917, Lactobacillus casei DSM 20011, Lactobacillus rhamnosus, Bifidobacterium bifidium, Bifidobacterium longum 15708, Bifidobacterium lactis (Bb12), Streptococcus thermophiles, Streptococcus thermophiles ATCC-3.
*The antifungal activity was performed by three methods: in the Overlay method, the maximum inhibitions of A. flavus and A. parasiticus were obtained by S. thermophiles ATCC-3. Streptococcus thermophiles ATCC-3 was very highly active (++++) against A. flavus and have moderate activity (++) against A. parasiticus. Bifidobacterium lactis (Bb12) and Lactobacillus casei DSM 20011 were highly active (+++) against A. flavus and have moderate activity (++) against A. parasiticus.
While, in the Well diffusion method, the maximum inhibition zones against A. flavus were obtained by B. lactis Bb12(38.0 mm) and B. longum (37.0 mm) and in case of A. parasiticus, the maximum inhibition zones was obtained by B. lactis Bb12 (35.0 mm) followed by B. longum 15708(33.0 mm). In case of using the disc diffusion assay B. longum achieved the maximum inhibition zones against A. flavus (11.0 mm) and A. parasiticus (7.5 mm).
Whereas, in the Agar dilution method, the maximum inhibitions of A. flavus were shown by B. lactis Bb12 (70.3%) followed by L. casei DSM 20011 (57.77 %) and S. thermophiles ATCC-3 (57.46 %), respectively and in case of A. parasiticus the maximum inhibitions were obtained by B. bifidium (84.59%) followed by B. lactis Bb12 (82.66%) and S. thermophiles ATCC-3 (68.69%).
*Assessment of MIC of selected LAB strains examined against both A. flavus and A. parasiticus using two methods
1. Assessment of the MIC of lactic acid bacterial strains by the fungal biomass inhibition method: It was observed that increasing of the supernatant concentration from 10 % to 25 % was accompanied with a decrease in the dry weights of the tested fungi. But, in case of using supernatant concentrations from 50 % to 75 %, there is no any detectable growth for both of the tested fungal isolates. The highest antifungal activity was obtained by B. lactis (Bb12) which has the MIC at supernatant concentration of 10% against both, A. flavus and A. parasiticus. A moderate antifungal activity was detected by both L. acidophilus NRRL-B-4495 and L. plantarum ATCC 14917, which have the MIC at supernatant concentration of 20 % against A. flavus, while only L. acidophilus NRRL-B-4495 has MIC at supernatant concentration of 20 % against A. parasiticus.
2. Assessment of the MIC of lactic acid bacterial strains by the agar dilution method: The results of MIC value showed that A. flavus exhibited the highest susceptibility (52.8 %) towards the supernatant of both B. lactis (Bb12) and L. acidophilus NRRL-B- 4495 with MIC values at 250 µl and 1000 µl. Also, A. parasiticus showed the highest susceptibility (52.2 %) towards the supernatant of L. acidophilus NRRL-B- 4495 with MIC value at 250 µl. Also, The cell-free supernatants of both S. thermophiles ATCC- 3 and L. plantarum ATCC 14917 have a moderate antifungal activity and have exhibited MIC values at a conc. of 1000 µl against both of A. flavus (31.9 and 40.4 %, respectively) and A. parasiticus ( 30.8 and 27.8 %, respectively).
*Determination of the antifungal metabolites produced by the LAB strains as follow:
1. HPLC analysis: It was observed from the results of the total content of the identified organic acids that L. rhamnosus was the highest strain in the production of total organic acids (6.898 mg/ ml) after 48hrs. Then L. casei DSM 20011 (4.717 mg/ ml) after 16hrs followed by S. thermophiles (4.694 mg/ ml) after 24hrs. Also, it was observed that the main and the highest organic acids produced by LAB strains were the lactic acid (2.64 mg/ ml) and the acetic acid (1.72 mg/ ml) from L. rhamnosus after 48 hrs. These organic acids were followed by succinic acid (1.12 mg/ ml) from L. plantarum ATCC 14917 after 24 hrs and propionic acid (1.04 mg/ ml) from S. thermophiles ATCC-3 after 48 hours. While the lowest organic acids produced by L. casei DSM 20011was mallic acid (0.024 mg/ ml).
2. Quantitative determination of the titratable acidity showed that, the highest total organic acids produced (38.28 mg/mL) after 48 hours was by L. plantarum ATCC 14917, followed by L. acidophilus ATCC 20552 (24.77 mg/mL). While, the lowest total organic acids was produced (4.50 mg/mL) by L. casei DSM 20011after 48hrs.
3. Quantitative determination of H2O2 revealed that, all tested LAB strains produced hydrogen peroxide at minimal concentrations ranging between 0.00816 mg/mL to 0.03266 mg/mL in the extracellular extracts. The maximum amount of H2O2 was produced (0.03266 mg/mL) by L. plantarum ATCC 14917and the lowest amount of H2O2 was produced (0.00816 mg/mL) by L. rhamnosus.
4. Quantitative determination of diacetyl produced was ranged from (4.304 mg/mL) to (34.432 mg/mL) in the extracellular extracts: The highest diacetyl was produced (34.432 mg/mL) by B. lactis (Bb12), followed by S. thermophiles ATCC-3 (25.824 mg/mL) after 48hrs. While, the lowest diacetyl was produced (4.304 mg/mL) by L. rhamnosus.
5. Quantitative determination of total protein concentration: The protein concentration is carried out by spectrophotometric analysis using Biuret method. The highest concentrations of protein was produced (3.69 mg/dl) was by Streptococcus thermophiles ATCC-3 followed by Lactobacillus rhamnosus (3.41 mg/dl). While the least protein concentrations was obtained (0.61 mg/dl) in the cells- free supernatant of Lactobacillus acidophilus.
* By determination of the physical characteristics of antifungal metabolites produced by the LAB strains as follow:
1. The effect of temperature: It was observed that the optimum antifungal activity for all tested LAB strains was recorded at 30°C. As the inhibitory activity of the antifungal substances were decreased with the increase in temperature. However, the cell-free supernatants of L. Acidophilus LA3, L. Plantarum ATCC14917and L. casei DSM 20011 were resistant and have antifungal potentials at different temperatures against A. flavus and A. parasiticus, showing thermal stability at temperatures range from 60 to 100 °C. However, these strains lost their activity after treatment at 121°C for 15 min. Also, the cell-free supernatants of both S. thermophiles ATCC-3 and S. thermophiles sp, were thermal stable when exposed to the temperature up to 60°C. But above these conditions, they were sensitive (80-100°C and autoclaving at 121°C for 15 min).
2. The effect of pH: It was observed that, the highest antifungal activity obtained was at pH range from 4.0 to 6.0 and the antifungal activity decreases as the pH increase. At pH (2.0), a complete loss of the antifungal activity was observed for all strains of Lactic acid bacteria. A complete loss of activity was observed at pH (8.0) and above except for L. Acidophilus LA3, S. thermophiles ATCC-3, S. thermophiles sp, and L. casei DSM 20011. Also, L. Plantarum ATCC14917, B. lactis (Bb12) and B. bifidium had antifungal activities at pH (8.0) against both A. flavus and A. parasiticus. While, at pH range from 8.0 to 12.0, it was observed that all tested LAB strains had no antifungal activity at the alkaline environment.
3. The effect of Enzymes: A complete inactivation of the antifungal of the cell-free supernatants was observed for certain strains such as L. Acidophilus LA3, L. Plantarum ATCC 14917, L. casei DSM 20011, S. thermophiles ATCC-3, S. thermophiles sp, B. lactis (Bb12) and B. bifidium after treatment with proteolytic enzymes (pepsin and trypsin). This indicated that the antifungal substances are protein in nature or at least has a proteinaceous activator. However, the treatment of the cell-free supernatants of the other LAB strains with the proteolytic enzymes did not result any changes of the antifungal activity. This indicating that the antifungal substance produced was not bacteriocin or has a proteinaceous activator. Also, it was observed that the treatment of cell-free supernatants of all LAB strains with catalase enzyme did not result any changes of the antifungal activity. This is indicating that the hydrogen peroxide did not account for the observed inhibition.
4. The effect of Surfactants: Almost all the surfactants used had no effect on the antifungal activity of the cell-free supernatant of the tested LAB strains in the concentrations used except the effect of Sodium Dodecyl Sulphate (SDS). As the SDS has stimulatory effect to the antifungal activity of the cells-free supernatant of the LAB strains against A. flavus and A. parasiticus.