RAPID ENUMERATION OF CLOSTRIDIAL SPORES IN RAW MILK SAMPLES USING AN IMPEDIMETRIC METHOD

MAURO FONTANA’, STEFANO BUSJELLO’, STEFANO BISOTTr-‘, GIUSTO DALLORTO’, GABRIELE UNGER’, GERJJNDE SCHWAMEIS’,

UDO JUTERSCHNIG3, BRIGITTE UNGER3, HERBERT MASANIGER4 and MANFRED SCHINKINGER3s5

‘CQlab Laboratory BIRAGHI S.P.A. 1-12030 Cavallermaggiore (CN}, Italy

’Quality Assurance BIRAGHI S.P.A. I-12030 Cavallermaggiore (CN), Italy

3R&D Laboratory, SY-LAB Gerate GmbH Tullnerbachstrasse 61 -65

A-301 I Neupurkersdoe Austria

4Quality Assurance, Kamtnemilch A-9800 SpittaUDrau, Austria

Accepted for Publication June 26, 2002

ABSTRACT

Lute spoilage of cheese is due to gas formation during lactic acid fermentation by spore-forming, gram-positive, anaerobic clostridia of the species Clostridium tyrobutyricum, Clostridium butyricum and Clostridium sporogenes. Since small numbers of such clostridial spores readily cause considerable losses in cheese production, spore numbers of fewer than 100 spores/liter must be determined reliably. Until recently, the only reliable method available was the time-consuming (7 days) and cumbersome Most Probable Number Method (MPN). The objective of this study was to examine the feasibility of using impedance technology as an alternative method for the enumeration of clostridial spores. Three topifreen replicates of 7.5-12.0 mL samples were tested using an impedimetric method with and without the addition of Oxyrase to generate anaerobic conditions within the impedance measurement cells. Results were obtained in less than 48 h. Data derived from the rapid impedance method were statistically comparable to those obtained using the reference method (MPN) .

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Journal of Rapid Methods and Automation in Microbiology 10 (2002) 107-1 16. All Rights Reserved. OCopyrighl 2002 by Food & Nutrition Press, Inc.. Trurnbull. Connecticut. 107

108 M. FONTANA E T A .

INTRODUCTION

Clostridial spores are primarily responsible for “late blowing”, a special kind of spoilage of long ripening cheese. As a result, these clostridial spores are of particular interest for producers of such cheese. Although the clostridial strains effecting cheese spoilage are nonpathogenic, gas production causes the cheese to increase in size or even explode, resulting in high economic losses for the producers (meter et al. 1984). This is a major problem in Italy since approximately 30% of total raw milk are used for the production of Grana-like cheese and an additional 10% for the production of other types of middle or long maturation cheese (Lodi 1993). Numerous researchers have verified that both intensity and speed in appearance of late blowing cheese depend on the number of clostridial spores present in the raw milk, and that even very low numbers can cause this phenomena if acidity, moisture and salt content are not well-balanced during maturation (Botazzi 1983; Bani et al. 1991; Spolaor and Rudello 199 1).

Clostridial spores contaminating crops mainly originate from soil. Contamination levels of soil are known to change seasonally, showing distinct peaks during rainy periods. If ingested with feed, clostridial spores have the potential to survive and may be present in cattle feces. Clostridial spores will then proliferate in forages of high carbohydrate content if the pH value is not quickly lowered to an adequate level of acidity. If proper sterile procedures are not followed, the milk may become contaminated during the milking process.

It is in the interest of manufacturers of raw cheese to know the spore contamination level of their starting milk product. The spore contamination level is important not only for the reasons mentioned previously but also due to the fact that in many areas spore content is a parameter used for milk grading that determines the level of the respective payment. Moreover, a rapid and reliable determination of spore content is a proper means to enhance cheese producers’ flexibility in reducing spore levels in contaminated milk by a mechanical (bactofugation) or chemical (lysozyme) treatment.

The most popular method to detect low numbers of spores (less than 100 spores/liter) is the MPN (most probable number) method (Bergere and Sivela 1990). Although widely used, this method is lengthy and laborious. Aside from the MPN method, only a few alternatives are available. A highly specific PCR method detects low numbers of C. tyrobutyn’curn; however, quantification based on that technology is not possible (Herman et al. 1995; Kiljn ef al. 1995). Although this test method is rapid and specific, it is not commonly used in cheese processing plants. Recently a simple double tube system has been reported for the enumeration of C. tyrobutyricum (Schmidt et al. 2000). This test method generates results within two to four days.

RAPID ENUMERATION OF SPORES IN RAW MILK 109

Impedance technology is a well-known, versatile application that is commonly used as an alternative to conventional microbiological methods (Silley 1997). Oxyrase enzyme has been extensively tested by Fung and colleague (Ali et al. 1991; Fung et al. 1994; Yu and Fung 1991) in culture media to generate an anaerobic environment and Oxyrase was tested in this study along with impedance technology. The aim of the present study was to adapt an impedi- metric detection method for the semiautomated enumeration of clostridial spores in raw milk whereby results were desired within 48 h.

MATERIALS AND METHODS

Milk Samples

One hundred twenty-five raw, partially skimmed or mechanically treated milk samples were used to establish the basic calibration. They were taken from large storage tanks containing milk used for routine cheese production (BIRAGHI S.P.A., Cavallermaggiore, Italy). Sample aliquots of 250 mL were collected and analyzed on-site the same day. If the samples were stored and transported, the temperature was maintained at 2-8C and analysis took place within 24 h. The sampling period was one year. Twenty-five additional milk samples from the same plant as well as 29 samples from an Austrian cheese processing plant were utilized for statistical method comparison. In addition, 48 samples were collected at the Austrian plant to test the possibility of adapting the use of Oxyrase with the impedance method using disposable measuring cells. Sample aliquots of 250 mL were collected, stored and transported at 2-8C and analyzed within 24 h.

MF" Method

The MPN method was performed according to Fryer and Halligan (1976) using 5 replicates of 3 serial 10-fold dilutions. Raw milk quantities of 1 .O mL, 0.1 mL and 0.01 mL were analyzed. For partially skimmed and bactofugated milk, 10.0 mL, 1.0 mL and 0.1 mL sample volumes were used. For 17 Italian milk samples, the MPN method was additionally verified at an accredited reference laboratory (AGRILAB S.r.1. Reg. Madonna dei Prati, 318 12044 Centallo, Italy) analyzing identical samples on the same day. The MPN procedure conducted by the external Italian laboratory and the investigation of the Austrian samples were based on the analyses of 3 replicates of 3 serial 10- fold dilutions using the sample quantities specified above.

110 M. FONTANA E T A .

Impedance Method

A BacTrac 4100 system based on impedance splitting technology (SY-LAB Gerate GmbH, Austria) was used. BacTrac measurements were performed aerobically in 10.0 mL reusable measuring cells at 37C for 48 h at 10-min intervals. “Impedance splitting” refers to the fact that two independent values are measured concurrently: the impedance of the media (M-value) and the impedance of the electrochemical bilayer on the surface of the electrodes (E- value). The relative change in impedance over the course of time is registered with reference to the value at the start of the measurement. The point of time when a rising growth curve surpasses a defined threshold is recorded as Impedance Detection Time (IDT). A 5 % relative change in impedance of the E- value was the threshold used for evaluation. A modified C. tyrobutyricum media formulation, BiMedia 665A-BIRAGHI; Cavallennaggiore, Italy, was used for the impedance experiments.

For every sample, 5 replicates (7.5 mL sample and 2.5 mL sterile BiMedia 665A) were analyzed in 10.0 mL reusable BacTrac measuring cells. For mechanically treated (Bactofuge) milk samples, 15 replicates of 7.5 mL samples were analyzed to obtain higher precision. The media was sterilized for 15 min at 121C immediately before addition of the milk samples. Aliquots of 14.0-15.0 mL of the milk samples were pasteurized at 85.5C for 15 min in a water bath to inactivate vegetative cells and stimulate spore germination. After pasteuriza- tion, the samples were rapidly cooled to 37C using a water bath and transferred to the BacTrac measuring cells. The samples were cautiously transferred in order to prevent excessive amounts of oxygen in the medium often caused by vigorous pouring. The cells were subsequently agitated gently to mix the contents.

Forty-eight of the Austrian samples were analyzed in the same manner as the Italian samples, but after the addition of 0.2 mL Oxyrase (Oxyrase Inc., Mansfield) to the 10.0 mL sample and medium solution contained in the measuring cells. Using 20.0 mL BacTrac measuring cells, these samples were additionally analyzed in parallel using 3 replicates of 12.0 mL samples mixed with 4.0 mL BiMedia 665A supplemented with 0.32 mL Oxyrase.

Data Analysis

The BacTrac was initially calibrated to convert detection times to spore counts by comparing the MPN results of 125 samples obtained by the external laboratory to the respective BacTrac results generated by BIRAGHI according to the following procedure: The arithmetic mean (2) and the standard deviation (sd) of the BacTrac detection times obtained for the 5 replicates of every sample were calculated. For samples giving no detection time (negative results), a detection time of 48.01 h was assumed for all calculations. Samples with

RAPID ENUMERATION OF SPORES IN RAW MILK 111

detection times beyond k 1 s, were not included in the data set used for calibration. Individual results for samples with identical MPN numbers were further grouped together and the arithmetic mean of the group (X) was calculated. A correlation diagram was created by plotting the results in a bi- dimensional graph where the x-axis represented the group mean of BacTrac detection times and the y-axis, the base-10 logarithms (log) of spore numbers obtained from MPN evaluation. The relationship between BacTrac detection times and spore counts was in accordance with linear regression.

After establishment of the calibration, 25 Italian and 29 Austrian samples were analyzed in parallel using MPN and the calibrated Bactrac impedance method. The two methods were then statistically compared using a Student's t- Test for paired results.

RESULTS AND DISCUSSION

The BacTrac calibration was based on results for the following 9 spore groups (based on MPN results): 9300 spores/liter (n=4), 4300 spores/liter (n=8), 2400 sporedliter (n=3), 2300 spores/liter (n=21), 900 spores/liter (n=3), 30 spores/liter (n=2), 25 sporedliter (n=6), 5 spores/liter (n=5) and 0 spores/liter (n= 16), respectively. Plotting the mean of the BacTrac detection time (x-axis) against the log of spores/liter (MPN result of the group) resulted in the calibration graph shown in Fig. 1. Using linear regression, the following relationship between the BacTrac detection time and sporedliter was established:

Spores/liter (log) = -0.1028 * t + 5.3448 with a r2 of 0.98 and a s,, = 0.12 log.

Figure 1 shows the linear relationship between the results obtained from the MPN method and the BacTrac impedance detection time over the range of 0 to lo4 spores/liter. This calibration was used to transform the detection times (mean of 5 or 15 replicates) for all the samples analyzed into the corresponding spore numbedliter.

For statistical method comparison, a log MPN result of 2 was aligned to MPN results with a value of < 300. Using the 54 results as shown in Table 1, a statistical method comparison of a two-sided Student's t-Test was performed based on the hypothesis that the difference of the mean is equal to zero (H, = 0). With t = 1.094, the assumption Ho = 0 proves valid since the requirement It I < = tcrit (".,) is fulfilled for t = 1.094 and tCrit (n=53) = 2.006 on a 95% significance level. The calculated probability value p (t < =tcrit) was 0.279.

112 M. FONTANA ETAL..

BacTrac calibration

4.5 4

3.5 3

2.5 2

1.5 1

0.5

0 10 20 30 40 50 60

Detection time BacTrac (hours)

FIG. 1 . RELATIONSHIP BETWEEN MPN METHOD AND BACTRAC DETECTION TIME FOR ESTABLISHMENT OF LINEAR RELATIONSHIP; BACTRAC CALIBRATION LINE

Table 1 specifies results obtained using the aforementioned calibration to transform the impedance detection times (hours, mean of 5 replicates) into spores per liter for an

additional 54 milk samples (25 Italian and 29 Austrian samples).

The results of the 48 Austrian milk samples analyzed with the addition of Oxyrase to maintain anaerobic conditions within the measuring cells are shown in Table 2 (BacTrac method A). The results shown for BacTrac method B are based on the modified BacTrac method using only 3 replicates and 12.0 mL, of each sample.

Forty-three result pairs were statistically analyzed for the comparison of the MPN method with BacTrac method A (standard procedure using 5 replicates and the addition of Oxyrase) and 41 result pairs were used for comparison of the MPN method with BacTrac method B (modified BacTrac method using 3 replicates of 12.0 mL samples and the addition of Oxyrase). The Student’s t-Test results obtained for comparison of MPN with BacTrac method A and MPN with BacTrac method B were t = -0.935; tcr,r(n.,=42) = 2.019; p (t< =tcrit) = 0.355 and t = -0.032; tcrit (n.1=40) = 2.021; p(t< =tcrit) = 0.947, respectively. Therefore, the criteria for acceptance of the null hypothesis are met in both cases. The results obtained with BacTrac methods A and B do not differ on the 95% significance level from those obtained with the reference method.

The addition of Oxyrase had no influence on the BacTrac detection times when compared directly to samples analyzed in parallel without Oxyrase (data not shown). Additionally, the results are comparable to the reference method as shown in Table 2. Four samples that did not contain Oxyrase (sample ID 4,21,25 and 27 Table 1) showed false positive impedance detection times;

RAPID ENUMERATION OF SPORES IN RAW MILK

TABLE 1 . DATA DERIVED FROM MPN TO BACTRAC METHOD COMPARISON

27 n' 0 0 88 33.1 28 n' 1700 1700 849 23.5 29 n* 350 350 33 37.2

n' = not determined; IDT = impedance detection time (hours)

113

114 M. FONTANA ETAL.

n' =

Bacl

TABLE 2. DATA DERIVED FROM MPN TO BACTRAC METHOD A AND

BACTRAC RESULTS GENERATED AFTER THE ADDITION

not dete 'rac method B = 3 replicates with I2 mL sample + Oxyrase

i COMPARISON; IF OXYRASE

P lle + Oxyrase;

RAPID ENUMERATION OF SPORES IN RAW MILK 115

however, the parallel samples that contained Oxyrase generated negative results. These results are likely due to the growth of aerobic sporeformers present in the samples. The addition of Oxyrase to maintain anaerobic conditions and suppress aerobic sporeformers will likely prevent false positive detection in impedance measurements.

In summary, the impedimetric method with the addition of Oxyrase proved to generate reliable results that were statistically comparable to those obtained using the reference method (MPN). Moreover, this method resulted in a tremendous decrease in analysis time ( < 48 h). The BacTrac technology offers a real alternative for the enumeration of lactate fermenting clostridial spores by significantly decreasing analysis time and reducing the workload. This method has now replaced the MPN-method at the BIRAGHI S.P.A (Cavallennaggiore, Italy) cheese processing plant.

ACKNOWLEDGMENTS

The precious collaboration with Dr. Mateo Picco Cugno (ABIT Grugliasco Torino, Italy) is gratefully acknowledged.

REFERENCES

ALI, M.S., FUNG, D.Y.C and KASTNER, C.L. 1991. Comparison of rapid methods for isolation of Clostridium pe@nngens in meat. J . Food. Sci.

BANI, P., BERTONI, G., GHILARDELLI, P. and GENNARI, R. 1991. Prime ricerche su alcuni fattori di variazione del contenuto di spore di Clostridium nelle feci delle lattifere. Scienza e Technica latterio casearie. 42/XLJZ, 6,

BERGERE, J.L. and SIVELA, S. 1990. Detection and enumeration of Clostridial spores related to cheese quality - classical and new method. IDF Bulletin No. 252, 18-23.

BOTAZZI, V. 1983. Clostridi e fermentazioni butirriche nei formaggi. Industria del latte. 3, 4-7.

FRYER, T.F. and HALLIGAN, A.C. 1976. The detection of Clostridium tyrobutyricum in milk. N.Z. J. Dairy Sci. Technol. 21, 132.

FUNG, D.Y.C, YU, L.S.L, NIROOMOND, F. and TUITEMWONG, K. 1994. Novel methods to stimulate growth of food pathogens by Oxyrase and related membrane fractions. In Rapid Methods and Automation in Microbiology (R.C. Spencer, E.P. Wright and S.W.B. Newsom, eds.) pp. 3 13-326, Intercept Limited Andover, UK.

56(2), 361-370.

13-15.

116 M. FONTANA ET 4.

HERMAN, L.M.F., DE BLOCK, J.H.G.E. and WAES, G.M.A. 1995. A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk. Appl. Env. Micro. 61, 4141-4146.

KILJN, N., NIEUWENHOF, F.F.J., HOOLWERF, J.D., VAN DER WAALS, C.B. and WEERKAMP, A.T. 1995. Identification of Clostridium tyrobuty- ricum as the causative agent of late blowing in cheese by species-specific PCR amplification. Appl. Env. Micro. 61, 2919-2924.

KLETER, G., LAMMERS, W.L. and VOS, E.A. 1984. The influence of pH and concentration of lactic acid and NaCl on the growth of Clostridium tyrobutyricum in whey and cheese. 2. Experiments in cheese. Neth. Milk

LODI, R. I1 Grana: innovazioni tecnologiche - i difetti. 1993. Industria del Latte XXIX, 2, 89-96.

SCHMIDT, K.A. , THAKUR, R.H., JIANG, G. and FUNG, D.Y.C. 2000. Application of a double tube system for enumeration of Clostridium tyrobutyricum. J. Rapid Methods Automation Microbiology 8, 21-29.

SILLEY, P. and FORSYTHE, S. 1996. Impedance microbiology - a rapid change for microbiologists. J . Appl . Biotechnol. 80, 233-243.

SPOLAOR, D. and RUDELLO, F. 1991. Effeti della salutura anticipate e del condizionamento degli ambienti di maturazione sul controllo delle fermentazioni anomale nei formaggi. Sci. e Techn. latterio casearie. 42,6,

YU, L.S.L. and FUNG, D.Y.C. 1991. Effect of Oxyrase enzyme on Listeria monocytogenes and other facultative anaerobes, J. Food Safety 11, 163- 176.

Dairy J. 38, 31-41.

16-18.