Folia Microbiol. 53 (5), 423–426 (2008)
http://www.biomed.cas.cz/mbu/folia/
Effect of Growth Temperature and pH on the Aminopeptidase Activity of Pseudomonas putida, P. fluorescens and Flavobacterium odoratum; the 4-Nitroaniline Test Is Reliable L.A. LOPEZ-TOMASa, J.A. ORDOÑEZb, C. MEDIAVILLAa, J.L. RODRIGUEZ-MARINa, P. SARMIENTOa, A. ZAMORAa, G. GARCIA DE FERNANDOb* aServicio de Bromatología e Higiene de los Alimentos, Centro Militar de Veterinaria de la Defensa, Ministerio de Defensa,
28024 Madrid, Spain
bDepartamento de Nutrición, Bromatología y Tecnología de Alimentos, Facultad de Veterinaria, 28040 Madrid, Spain
e-mail
[email protected] Received 17 January 2008 Revised version 17 April 2008
ABSTRACT. No significant difference (p > 0.05) was observed in the specific aminopeptidase activity (SAA) developed by Pseudomonas fluorescens, P. putida and Flavobacterium odoratum either growing at pH 5.0–6.5 or at 7 and 12 °C. Nevertheless, a significant difference was found when comparing the SAA of these organisms. The SAA of F. odoratum was lower than those of pseudomonads. The 4-nitroaniline test is reliable to estimate the G– load of fresh food products.
Abbreviations SAA(s)
specific aminopeptidase activity(ies)
TSB
tryptone soy broth
The 4-nitroaniline test estimates the amount of G–-bacteria present in a sample (Perez de Castro et al. 1988; Lopez-Tomas et al. 2006), being an alternative and rapid method to the traditional plate counts. This makes it a valuable tool to determine the relevant microbial load of refrigerated products, especially fresh meat, fish and milk (Perez de Castro 1989; Alvarado et al. 1992; Dainty 1996), since G–-psychrotrophs are the main bacteria reponsible for the spoilage of refrigerated products of animal origin maintained in aerobiosis. Beef and pork usually have a pH of ≈5.7 one day after slaughter (Tarrant and Sherington 1980) or 5.8 (Beltran et al. 1997; von Lengerken et al. 2002), while poultry (chicken or turkey) usually has a higher pH, around 5.9–6.0 (Lyon and Buhr 2001). Other foods are characterized by pH >6.5 in normal conditions, for instances milk (Sherbon 1988) and fish (Love 1997). The term “refrigeration” covers a broad temperature interval, from just 20 °C, which became undetectable at 27 °C. It can be hypothesized that the magnitude of the SAA generated could depend on the growth conditions of the bacteria. Therefore, the aim of this work was to study the effect of pH and temperature conditions during bacterial growth on the aminopeptidase activity of G–-bacteria usually implicated in the spoilage of refrigerated foods aerobically stored. MATERIAL AND METHODS Microorganisms: Pseudomonas putida CECT 324, P. fluorescens CECT 378 and Flavobacterium odoratum CECT 998, all provided by the Spanish Collection of Culture Type (CECT). *Corresponding author.
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Growth medium: TSB (CM0129; Oxoid); when necessary, the pH was adjusted by adding appropriate amounts of 0.1 mol/L HCl or NaOH, sterilizing the medium (15 min, 121 °C) afterwards. Culture conditions. Each species was grown at pH of 5.0, 5.5, 6.0 and 6.5, maintaining the temperature at 7 °C. On the other hand, cultures were also grown at 2, 7 and 12 °C in TSB, pH 6.0. Cultures were incubated until a biomass was generated giving an absorbance of, at least, 0.8 UA at 600 nm. After this, an aliquot of the culture was taken and diluted with 0.1 mol/L Tris buffer (pH 8.0), to achieve an absorbance between 0.6 and 0.7 UA at 600 nm. Aminopeptidase activity. To detect this activity in cultures, aliquots of independent samples of 8, 2 or 0.4 mL were taken, and poured in test-tubes containing 24, 18 or 19.6 mL of 0.1 mol/L Tris buffer (pH 8.0), achieving dilutions of 1 : 4, 1 : 10 or 1 : 50. From each dilution, 6 mL were taken and the aminopeptidase activity was determined using L-alanine-4-nitroanilide as substrate and reading the A390 following the method of Perez de Castro et al. (1988). Arbitrary measurement of SAA gives an indication of the enzymic activity per cell. It is estimated from the quotient between the aminopeptidase activity (A390) and the microbial concentration that develops it (A600). Statistical treatment. Experimental data were analyzed by using the statistical analysis tools of Microsoft Excel program. Comparison of variances between the two groups was performed by the t-test, while the ANOVA procedure was used for the variance comparison of >2 groups. The SAAs at different pH were analyzed by the ANOVA procedure at α = 0.05. The SAAs at different temperature were analyzed by the t-test at α = 0.05. The comparison of SAAs of the three bacterial species was carried out by the ANOVA procedure at α = 0.001. RESULTS AND DISCUSSION Effect of pH on SAA. Table I shows the means and analysis of variance data for the SAA of the organisms studied. The means are from the sixteen values obtained at each pH value, i.e. from 16 independent samples. As the experimental F is lower than the critical F ( p > 0.05), the null hypothesis can be accepted and it may be concluded that the culture medium pH has no influence on the aminopeptidase activity developed. Table I. Effect of the growth medium (TSB) pH on the SAA developed by P. putida, P. fluorescens and F. odoratum at 7 °Ca Organism
pH
SAA average
Variance
F valueb
p
P. putida
5.0 5.5 6.0 6.5
1.58 1.40 1.70 1.71
0.0724 0.0284 0.3814 0.0585
2.49
0.07
P. fluorescens
5.0 5.5 6.0 6.5
1.56 1.75 1.88 1.78
0.2367 0.5064 0.1875 0.1638
1.02
0.39
F. odoratum
5.0 5.5 6.0 6.5
1.45 1.37 1.24 1.36
0.1345 0.0393 0.1473 0.0840
1.14
0.34
an = 16, 4 from each dilution (1 : 1, 1 : 4, 1 : 10 and 1 : 50) from independent samples. bThe critical F value (α = 0.05) is 2.76. An experimental F value > critical F value would indicate significant differences.
Effect of temperature on SAA. The effect of the temperature growth on the SAA of the organisms investigated is shown in Table II. As the experimental Student’s t-test is lower than the critical t ( p > 0.05), the null hypothesis can be accepted and it may be deduced that growth temperature (7 and 12 °C) did not affect the aminopeptidase production. Data on bacterial growth were also obtained at 2 °C but, unfortunately, growth was not sufficient to reach the minimum level (A600 = 0.6) required to calculate the SAA, and
THE 4-NITROANILINE TEST OF AMINOPEPTIDASE ACTIVITY 425
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could not be treated statistically. Nonetheless, the specific activities developed at 2 °C, from smaller cellular concentrations, do not greatly differ from those developed at higher temperatures (Table II). Table II. Effect of the incubation temperature on the specific aminopeptidase activity developed by Pseudomonas putida, P. fluorescens and Flavobacterium odoratum in TSB at pH 6.0 Temperature, °C
n
SAA average
Variance
t valuea
p
P. putida
7 12 2
32 32 13
1.55 1.66 1.58
0.2214 0.1156 0.2741
1.12
0.26
P. fluorescens
7 12 2
32 32 18
1.81 1.72 1.70
0.3396 0.1164 0.3291
0.78
0.44
F. odoratum
7 12 2
32 32 15
1.30 1.39 1.33
0.0945 0.1123 0.1852
1.13
0.26
Microorganism
aThe critical t value (α = 0.05) is 2.0. An experimental t value > critical t value would indicate significant differences.
Comparison of the SAAs (Table III). P. fluorescens develops a higher activity than P. putida, and this is more active than F. odoratum. The analysis of variance shows that the SAA of, at least, one of the three species, was significantly different ( p < 0.001). Paired comparisons were made of the SAA of the three species studied and the analysis of variance concluded that comparison of the SAA of F. odoratum with those of the two Pseudomonas spp. revealed significant differences ( p < 0.001). However, analysis of the 96 values obtained for each species shows that the mean value of the SAA of F. odoratum (1.37) represents almost 80 % of the value of the SAA of Ps. fluorescens (1.74) and almost 85 % of the value of SAA of P. putida (1.62). In conclusion, in spite of the significant differences, actually the absolute values of SAA differed in the worst case by ≈20 %. Table III. Comparison of the SAA developed in P. putida, P. fluorescens and F. odoratum grown at 7 and/or 12 °C in TSB adjusted at different pHa Microorganism
SAA average
Variance
F valueb
p
P. putida P. fluorescens F. odoratum
1.62 1.74 1.37
0.1347 0.2197 0.1047
22.27
critical F value indicates significant differences.
Other authors found differences in the SAA. Pérez de Castro et al. (1988) and Pérez de Castro (1989), working with Pseudomonas strains DC-7 and JU-7 of “cluster” 1, DC-5 and P-4 of “cluster” 2 and NT-19 and P-2 of “cluster” 3 (Shaw and Latty 1984), demonstrated that some of these strains presented a different degree of SAA, although they did not find any relationship between the degree of activity and belonging to a specific cluster. Crucial factors in microbial development (such as pH and temperature) therefore do not affect the aminopeptidase activity, and this ability is more conditioned by the species than by the medium in which bacteria grow. The 4-nitroaniline test will not be affected by the growth conditions of the G–-microbial contaminants of the sample. However, if the microbiota of a given sample is composed of only one species, an error could possibly arise in estimating the microbial load of such sample, although this would be minimum and often temporary. This work has been supported by projects AGL2005-01239/ALI and S-0505/AGR-0314.
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REFERENCES ALVARADO F.J., GONZALEZ C., WONG C., GUTIERREZ L., SEPULVEDA J., KUMATE J.: Identification of Vibrio cholerae O1 by flow cytometry. Revista Latinoamericana de Microbiología 36, 283–293 (1994). BELTRAN J.A., JAIME I., SANTOLARIA P., SAÑUDO C., ALBERTI P., RONCALES P.: Effect of stress-induced high post mortem pH on protease activity and tenderness of beef. Meat Sci. 45, 201–207 (1997). DAINTY R.H.: Chemical/biochemical detection of spoilage. Internat.J.Food Microbiol. 33, 19–34 (1996). FONCHY E., MORIN A., RODRIGUEZ N., MÜLLER B., CHALIER P.: Effect of growth temperature on hydrolytic and esterifying activities from Pseudomonas fragi CRDA 037 grown on whey. Appl.Environ.Microbiol. 65, 3114–3120 (1999). FRANK H.A., REID A., SANTO L.M., LUM N.A., SANDLER S.T.: Similarity in several properties of psychrophilic bacteria grown at low and moderate temperatures. Appl.Microbiol. 24, 571–574 (1972). VON LENGERKEN G., MAAK S., WICKE M.: Muscle metabolism and meat quality of pigs and poultry. Veterinar.Zootechn. 20, 82–86 (2002). LOPEZ-TOMAS L.A., ORDOÑEZ J.A., GARCIA DE FERNANDO G.D.: The p-nitroaniline test to asses the bacterial microbiota of raw ground meat aerobically stored. Meat Sci. 72, 222–228 (2006). LOVE R.M.: Biochemical dynamics and the quality of fresh and frozen fish, pp. 1–31 in G.M. Hall (Ed.): Fish Processing Technology, 2nd ed. Blackie Academic & Professional, London 1997. LYON C.E., BUHR R.J.: Bases bioquímicas de la textura de la carne, pp. 111–143 in R.I. Richardson, G.C. Mead (Eds): Ciencia de la Carne de Ave. Acribia, Zaragoza 2001. PÉREZ DE CASTRO B.: Desarrollo de una prueba rápida y sencilla para estimar el grado de frescura de la carne y leche. PhD Thesis. Facultad de Veterinaria, Universidad Complutense, Madrid 1989. PÉREZ DE CASTRO B.P., ASENSIO M.A., SANZ B., ORDOÑEZ J.A.: A method to assess the bacterial content of refrigerated meat. Appl. Environ.Microbiol. 54, 1462–1465 (1988). SHAW B.G., LATTY J.B.: A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source test. J.Appl.Bacteriol. 57, 59–67 (1984). SHERBON J.W.: Physical properties of milk, pp. 409–460 in N.P. Wong (Ed.): Fundamentals of Dairy Chemistry, 3rd ed. AVI Book, New York 1988. TARRANT P.V., SHERINGTON J.: An investigation of ultimate pH in the muscle of commercial beef carcasses. Meat Sci. 4, 287–297 (1980).
