Screening Method to Evaluate Amino Acid-Decarboxylase Activity of Bacteria Present in Spanish Artisanal Ripened Cheeses
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cheese Manufacturing
2.2. Strain Isolation
2.3. Preparation of Decarboxylase Media
2.4. Assessment of Amino Acid Decarboxylase Activity
2.5. Confirmation of Amino Acid Decarboxylase Activity by HPLC
2.6. Analytical Validation of the Qualitative Microplate Method of Amino Acid Decarboxylase Activity
2.7. Identification of Strains with Decarboxylase Activity
3. Results and Discussion
3.1. Validation of the Qualitative Microplate Method of Amino Acid Decarboxylase Activity
3.2. Amino Acid Decarboxylase Activity of the Control Strains
3.3. Biogenic Amine Production by Isolates from Goat’s and Ewe’s Milk Cheeses
3.3.1. Total Amino-Acid Decarboxylase Activity of the Isolated Strains
3.3.2. Specific Amino Acid Decarboxylase Activity of the Isolated Strains
3.3.3. Identification of BA-Producing Strains
3.4. Consequences of HHP Treatments on the Formation of BA
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Reagent | Low Nitrogen Decarboxylase Broth (LND) | Low Glucose Decarboxylase Broth (LGD) | Adjusted pH |
---|---|---|---|
Tryptone | 0.125 | 0.25 | |
Yeast extract | 0.125 | 0.25 | |
NaCl | 0.25 | 0.25 | |
CaCO3 | 0.01 | 0.01 | |
Pyridoxal-5-phosphate | 0.03 | 0.03 | |
Glucose | 0.05 | 0.001 | |
Bromocresol purple | 0.01 | 0.01 | |
All amino acids | 1.0 | 1.0 | 5.5 |
l-Lysine | 1.0 | 1.0 | 5.0 |
l-Ornithine | 1.0 | 1.0 | 5.5 |
l-Histidine | 1.0 | 1.0 | 5.7 |
l-Tyrosine | 0.25 | 0.25 | 5.5 |
Broth | Data of ROC Analysis | Ornithine | Lysine | Histidine | Tyrosine |
---|---|---|---|---|---|
LND | AUC | 0.999 (0.974–1.00) | 0.992 (0.961–1.00) | 0.737 (0.659–0.806) | 0.980 (0.943–0.996) |
Optimal cut-off (mg/L) | 10 | 15 | 50 | 25 | |
Sensitivity at optimal cut-off (%) | 98.53 | 98.68 | 65.38 | 92.25 | |
Specificity at optimal cut-off (%) | 100 | 93.15 | 91.75 | 100 | |
PPV (%) | 100 | 83.7 | 81 | 100 | |
NPV (%) | 99.0 | 98.6 | 83.2 | 66.7 | |
FN at cut-off | 3 | 6 | 11 | 1 | |
FP at cut-off | 1 | 1 | 17 | 6 | |
LGD | AUC | 0.907 (0.849–0.948) | 0.935 (0.883–0.969) | 0.592 (0.509–0.672) | 0.989 (0.956–0.999) |
Optimal cut-off (mg/L) | 15 | 10 | 50 | 20 | |
Sensitivity at optimal cut-off (%) | 84.37 | 88 | 30 | 93.06 | |
Specificity at optimal cut-off (%) | 97.5 | 97.6 | 100 | 100 | |
PPV (%) | 90 | 88 | 100 | 100 | |
NPV (%) | 95.8 | 97.6 | 90.3 | 37.5 | |
FN at cut-off | 5 | 5 | 2 | 0 | |
FP at cut-off | 5 | 3 | 14 | 8 |
Strain | PU | CA | HI | TY | |||||
---|---|---|---|---|---|---|---|---|---|
Broth | DMA | HPLC | DMA | HPLC | DMA | HPLC | DMA | HPLC | |
L. brevis | LGD | (−) | 1.11 | (−) | ND | ND | ND | (+) | 109.8 |
L. casei | LGD | (−) | 0.78 | (−) | ND | ND | ND | (+) | 77.14 |
S. epidermidis | LND | (−) | 0.85 | (−) | ND | (+) | 46.48 | (−) | ND |
Medium | BAP | PU | CA | HI | TY | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
± | + | ++ | +++ | ± | + | ++ | +++ | ± | + | ++ | +++ | ± | + | ++ | +++ | ||
VRBG | 25 | 1 | 2 | 17 | 4 | 0 | 0 | 17 | 7 | 8 | 10 | 4 | 0 | 3 | 6 | 9 | 0 |
BPA | 15 | 1 | 1 | 3 | 3 | 0 | 0 | 4 | 3 | 4 | 4 | 1 | 0 | 2 | 2 | 7 | 0 |
KF | 89 | 5 | 3 | 22 | 4 | 13 | 0 | 22 | 9 | 19 | 21 | 4 | 0 | 4 | 8 | 71 | 0 |
M-17 | 98 | 7 | 2 | 5 | 0 | 7 | 1 | 2 | 1 | 14 | 6 | 2 | 0 | 5 | 6 | 69 | 0 |
MRS | 72 | 2 | 2 | 2 | 3 | 0 | 0 | 2 | 3 | 7 | 0 | 0 | 0 | 5 | 8 | 50 | 0 |
Total | 299 | 16 | 10 | 49 | 14 | 20 | 1 | 47 | 23 | 52 | 41 | 11 | 0 | 19 | 30 | 206 | 0 |
Identification | N | PU | CA | HI | TY | ||||
---|---|---|---|---|---|---|---|---|---|
Gram Negative | |||||||||
Hafnia alvei | 4 | 4 | 537.7–889.54 | 2 | 641–1001.30 | 4 | 30.43–95.68 | 4 | 22.44–151.54 |
Enterobacteriaceae | 1 | 1 | 1037.52 | 1 | 1173.24 | 1 | 111.43 | 1 | 73.41 |
Gram Positive | |||||||||
Staphylococcus cohni subsp. cohni | 1 | - | 1 | 1.50 | 1 | 1.57 | 1 | 25.78 | |
Staphylococcus warneri | 2 | 2 | 69.29–753.55 | 2 | 240.94–694.22 | 1 | 88.63 | 1 | 65.50 |
Staphylococcus capitis | 1 | 1 | 310.30 | 1 | 246.37 | 1 | 95.13 | - | |
Staphylococcus lentus | 2 | 1 | 23.44 | 2 | 5.41–19 | - | 2 | 39.58–191.69 | |
Staphylococcus hominis | 2 | 1 | 890.96 | 1 | 998.20 | 1 | 91.63 | 2 | 102.42–245.22 |
Enterococcus faecalis | 8 | 8 | 39.66–884.27 | 8 | 32.44–972 | 6 | 25.62–92.35 | 8 | 327.5–477.20 |
Enterococcus durans | 2 | - | - | - | 2 | 337.40–357.44 | |||
Enterococcus avium | 1 | - | 1 | 26.52 | 1 | 56.51 | 1 | 24.88 | |
Enterococcus faecium | 6 | 3 | 19.68–1113.80 | 3 | 1.32–1281.50 | 3 | 20.22–111.38 | 6 | 9.91–366.47 |
Lactococcus lactis subsp. lactis | 10 | 1 | 7.25 | 1 | 7.50 | 2 | 21.24–33.38 | 10 | 198.77–450.77 |
Pediococcus pentosaceus | 1 | - | - | 1 | 33.44 | 1 | 55.71 | ||
Lactobacillus brevis | 5 | - | - | 1 | 22.51 | 5 | 212.58–519.52 | ||
Lactobacillus plantarum | 3 | 1 | 22.41 | - | 1 | 24.5 | 3 | 307.62–528.45 | |
Lactobacillus paracasei subps. paracasei | 1 | - | - | - | 1 | 307.03 | |||
Leuconostoc spp. | 3 | 2 | 252.88–749.05 | 2 | 14.47–28.83 | 2 | 22.74–25.75 | 3 | 330.01–417 |
Bacillus macerans | 1 | - | - | - | 1 | 418.10 | |||
Bacillus licheniformis | 1 | - | - | - | 1 | 403.07 |
Identification | N | PU | CA | HI | TY | ||||
---|---|---|---|---|---|---|---|---|---|
Gram Negative | |||||||||
Escherichia coli | 2 | 2 | 746–857.48 | 2 | 762.35–983.93 | 2 | 41.37–74.12 | 2 | 21.7–281.48 |
Hafnia alvei | 5 | 5 | 738.50–1049.51 | 5 | 787.20–1180.12 | 4 | 43.52–185.54 | 5 | 48.83–184.79 |
Klebsiella oxytoca | 2 | 2 | 30.63–458.50 | 2 | 493.41–866.96 | 2 | 16.04–27.22 | 2 | 5.01–167.24 |
Citrobacter freundii | 1 | 1 | 67.1 | 1 | 1095.3 | 1 | 45.62 | 1 | 372.38 |
Enterobacteriaceae | 1 | 1 | 832.76 | 1 | 846.95 | 1 | 83.95 | 1 | 69.42 |
Gram Positive | |||||||||
Staphylococcus xylosus | 2 | - | - | 2 | 42.31–68.5 | 2 | 92.09–475.35 | ||
Staphylococcus chromogenes | 1 | 1 | 1142.92 | 1 | 1760.06 | 1 | 31.12 | 1 | 441.40 |
Staphylococcus aureus | 1 | - | - | 1 | 100.54 | - | |||
Enterococcus faecalis | 5 | 3 | 860.29–978.9 | 4 | 35.43–1394.87 | 5 | 20.7–92.98 | 5 | 338.01–461.50 |
Enterococcus durans | 9 | 9 | 12.63–1160.44 | 8 | 13.32–1773.03 | 9 | 30.8–179.32 | 9 | 46.09–747.33 |
Enterococcus faecium | 2 | 2 | 24.89–847.05 | 2 | 877.24–941.69 | 2 | 28.98–29.97 | 2 | 100.54–149.41 |
Enterococcus hirae | 2 | 2 | 552.4–579.25 | 2 | 600.67–615.75 | 2 | 32.36–45.95 | 2 | 108.01–187.58 |
Enterococcus avium | 2 | - | 1 | 15.17 | 1 | 2.18 | 2 | 19.93–434.25 | |
Streptococcus salivarius | 1 | - | - | - | 1 | 742.1 | |||
Lactococcus lactis subsp. lactis | 9 | 3 | 34.39–795.26 | 2 | 14.95–844.13 | 2 | 16.38–30.72 | 9 | 211.93–566.2 |
Lactococcus lactis subsp. cremoris | 4 | 3 | 4.78–37.63 | 3 | 5.23–22.94 | 2 | 11.15–19.03 | 4 | 229.47–406.87 |
Pediococcus pentosaceus | 4 | 3 | 9.91–897.67 | 3 | 10.25–1018.4 | 3 | 27.06–88.78 | 4 | 34.58–411.66 |
Lactobacillus paracasei subsp. paracasei | 6 | 2 | 17.42–45.05 | 1 | 17.18 | 1 | 51.93 | 6 | 365.79–575.73 |
Lactobacillus plantarum | 4 | 1 | 8.58 | 1 | 17.58 | 1 | 26.26 | 4 | 45.1–353.31 |
Lactobacillus brevis | 1 | - | - | - | 1 | 240.08 | |||
Lactobacillus pentosus | 1 | 1 | 50.79 | - | - | 1 | 422.75 | ||
Leuconostoc spp. | 5 | 2 | 11.13–1162.76 | 2 | 22.52–1781.26 | 3 | 29.63–33.82 | 5 | 392.01–626.42 |
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Espinosa-Pesqueira, D.; Roig-Sagués, A.X.; Hernández-Herrero, M.M. Screening Method to Evaluate Amino Acid-Decarboxylase Activity of Bacteria Present in Spanish Artisanal Ripened Cheeses. Foods 2018, 7, 182. https://doi.org/10.3390/foods7110182
Espinosa-Pesqueira D, Roig-Sagués AX, Hernández-Herrero MM. Screening Method to Evaluate Amino Acid-Decarboxylase Activity of Bacteria Present in Spanish Artisanal Ripened Cheeses. Foods. 2018; 7(11):182. https://doi.org/10.3390/foods7110182
Chicago/Turabian StyleEspinosa-Pesqueira, Diana, Artur X. Roig-Sagués, and M. Manuela Hernández-Herrero. 2018. "Screening Method to Evaluate Amino Acid-Decarboxylase Activity of Bacteria Present in Spanish Artisanal Ripened Cheeses" Foods 7, no. 11: 182. https://doi.org/10.3390/foods7110182