RESEARCH NOTE

ENTEROBACTERZACEAE IN ICE CREAM SOLD AT RETAIL IN TENERIFE, SPAIN

CRISTOBALINA RODRiGUEZ-ALVAREZ', ARTURO HARDISSON, ROBERTO ALVAREZ, ANGELES ARIAS and ANTONIO SIERRA

Department of Preventive Medicine & Public Health University of La Laguna,

Tenerife, SPAIN

Accepted for Publication December 26, 1993

ABSTRACT

Ice cream is a dairy product which favors the growth of microorganisms and can be exposed to contamination during handling while being sold at the retail market.

R e presence of microorganisms belonging to the Enterobacteriaceae family was studied in 67 ice cream samples in the province of Santa Cruz de Tenerife. All the samples showed positive counts, of which 82.1 % revealed microbiol counts of >log 3 - < log 5 CFU/g. The isolated species identified (as percent of total) were: Klebsiella pneumoniae, 34.8; Enterobacter aerogenes, 19.3; Enterobacter cloacae, 1 I. 1 ; Enterobacter aglomerans, 11.6; Escherichia coli and Salmonella arizonae, 2.6; Citrobacter freundii, I .3; and Serratia marcenscens, 0.7.

INTRODUCTION

Research aimed at identifying the different species of the Enterobacteriaceae family is not a common practice in food analyses. However, since this bacterial family is used as a marker of hygiene in processed foods that may include a large number of bacteria, it becomes almost necesary to determine the health

'To whom correspondence should be addressed: Dr. Cristobalina Rodriguez-Alvarez, Citedra de Medicina Preventiva y Salud Xblica, Facultad de Medicina, Universidad de La Laguna, Tenerife, Islas Canarias, Espafia. FAX: 34 22 603407.

Journal of Food Quality 17 (1994) 423-427. All Rights Reserved. Copyright 1994 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 423

424 C. RODRIGUEZ-ALVAREZ, ET AL.

risk involved (Cousins and Marlatt 1990; Matushek et al. 1992). Ice cream provides a favorable environment for the growth and/or survival of microorgan- isms. Furthermore, when at retail, it may be exposed to contamination during handling (Ubach et al. 1990).

Therefore, this report is directed towards examining ice cream sold in Santa Cruz de Tenerife (Canary Islands, Spain) for microbial counts, isolation and identification of the bacteria belonging to the family Enterobacteriaceae.

MATERIALS AND METHODS

A total of 67 samples of strawberry, vanilla, chocolate and malaga ice cream sold at the retail outlets were collected in Santa Cruz de Tenerife and analyzed. The samples were placed in sterile containers and transferred, under isothermal refrigeration, to the laboratory.

To achieve uniformity, the samples were kept at room temperature for an hour for softening.

The following Methods for the Examination of Dairy Products (Richardson 1985) and as recommended by the International Commission on Microbiological Specifications for Foods (I.C.M.S.F. 1983) were used:

Total Counts of Enterobacteriaceae

After decimal dilution, and in duplicate, 1 ml of decimal dilution was placed in sterile petri dishes. Following this, 15 ml of Oxoid Violet Red Bile Glucose Agar (VRBG) (Hampshire, England), tempered at 45-47C, was poured in, each plate carefully mixed and allowed to harden on a flat surface. Each plate was then covered with 10 ml of the same sterile medium and incubated for 18-24 h at 37C.

The violet red colonies, surrounded by violet precipitate, were counted. This precipitate (bile salts) is due to acid production from the glucose. The characteristic colonies were multiplied by the dilution factor, to obtain the number of Enterobacteriaceae totals/ml of ice cream.

Isolation and Identification

A 25 ml sample was inoculated into flasks containing OXOID Triptone Soya Broth (TSB) for resuscitation of metabolically injured microorganisms present. The flasks were kept at room temperature for 2 h and were shaken at regular

ENTEROBACTERIACEAE IN ICE CREAM 425

intervals. Then 10 ml was inoculated into tubes containing simple OXOID Mossel EE enriching broth, followed by incubation at 37C for 24 h. Tubes with positive bacterial growth were vigorously shaken and by means of a loop were spread onto OXOID Violet Red Bile Glucose Agar plates and incubated at 37C, for 24 h.

The isolated colonies were identified as the species of Enterobacterieae by means of the automated system AutoScan 4, (Baxter, Spain) using the Neg Bp Combo Panel Type 21 (Jorgensen 1991).

RESULTS AND DISCUSSION

The percentages of positive microbial counts, grouped in log values, of all the samples analyzed are shown in Fig. 1. Incidence was 100% positive between levels of 2 log 2 - 5 <log 5 CFU/g, and 82.07% between levels of 2 log 3 - <log 5 CFU/g.

FIG. 1. LOG TOTAL COUNT OF ENTEROBACTERIACEAE PER G ICE CREAM SOLD AT THE RETAIL MARKET RESULTS ARE REPORTED AS POSITIVE PERCENT

We believe that this high number of Enterobacteriaceae is mainly due to the difficulty of maintaining low microbial counts in products sold at the retail

426 C. RODRfGUEZ-ALVAREZ. ET AL.

stores. Agusti (1990) reported that the principal causes of high bacterial load are due to exposure to air and the water used to wet the scoops when dispensing the product.

Our results are similar to those presented in this country, by Izquierdo et al. (1984) and Sinchez et al. (1986) who obtained positive counts of Enterobac- teriaceae in practically all of the samples analyzed. Thus, we conclude that, in spite of the fact that the majority of these microorganisms are not generally associated with pathogenicity, their significance in foods may become important when present in large numbers.

As percent of total FIG. 2. THE SPECIES ENTEROBACTERIACEAE FAMILY IDENTIFIED IN

ICE CREAM SAMPLES SOLD AT THE RETAIL STORES RESULTS ARE REPORTED AS PERCENT OF TOTAL

The percentages from the samples are recorded in Fig. 2 as percent of total microbial species isolated.

The majority of the isolates (45.79%) belonging to the genus Klebsiella as follows: K. pneumoniae (34.83%), K. ozenae (7.09%) and K. oxytocca (3.87 %). This was followed by the genus Enterobacter (47.09%) and the species E. aerogenes (19.35%), E. cloacea (16.13%) and E. agglomerans (11.61%). E. coli was found at a low level of 2.59% of all the strains identified.

In the same sample, up to three or four genuses were identified, but this was not always the case with the high counts. We wish to point out the high incidence of fecal coliforms (99.31 %); of these, 47.33% were K. pneumoniae, and only 2.66% E . coli, the sole fecal indicator listed in the Health regulations

ENTEROBACTERIACEAE IN ICE CREAM 421

of this country (Ministerio de Relaciones con las Cortes y Secretaria del Gobierno 1983).

Miguel et al. (1985) and Sanchez et al. (1986), reported a high incidence of fecal coliforms in ice cream. We believe that the significance of fecal coliforms is becoming more and more important, since they are an indicator of high incidence of contamination, although lacking the specific pathogenicity of E. coli, which must be considered.

REFERENCES

AGUSTi, C. 1990. Carga microbiana en el helado. Origen y control. Aliment. Eguipos Tecnol. Jul-Agos, 109-1 12.

COUSINS, D.L. and MARLATT, F. 1990. An evaluation of a conductance method for the enumeration of Enterobacteriaceae in milk. J. Food Prot. 53, 568-570.

Int. Commission on Microbiological Specifications for Foods. (I.C.M.S.F.). 1983. Microorganismos de 10s alimentos. 1. TCcnicas de analisis microbiol- ogico, pp. 105-281, Acribia, Zaragoza.

IZQUIERDO, J. , GRIFOL, F. and LUCENA, F. 1984. Campaiia de control microbiolbgico-sanitario de helados. Aliment. 154, 29-3 1.

JORGENSEN, J.H. 1991. Antibacterial susceptibility tests: automated or instrument-based methods. In Manual of Clinical Microbiology, 5th Ed., A. Balows, K.L. Hausler and Shadomy, eds. pp. 1166-1172, American Society for Microbiology, Washington, DC.

MATUSHEK, M.G., CURIALE, M.S., McALLISTER, J.S. and FOX, T.L. 1992. Comparison of various plating procedures for the detection and enumeration of coliforms in ice cream and ice milk. J. Food Prot. 55,

MIGUEL, A., PEREZ, M. and UBACH, M. 1985. Estudio de la contaminacion bacteriana de 10s helados artesanales de Palma de Mallorca. Aliment. 160, 73-77.

Ministerio de Relaciones con las Cortes y Secretaria del Gobierno. 1983. REAL DECRETO 670 de la Presidencia del Gobierno por el que se aprueba la Reglamentacid TCcnico-Sanitaria para la elaboracih, circulacidn y comercio de helados. BOE no. 78, Apr. 2.

MOSSEL, D.A.A. and MORENO, B. 1985. Microbiologia de 10s alimentos. Fundamentos ecologicos para garantizar y comprobar la inocuidad y calidad de 10s alimentos, pp. 123-171, Acribia, Zaragoza.

RICHARDSON, G.A. (ed.) 1985. Standard Methods for Examination of Dairy Products, 15th Ed., pp. 89-150, American Public Health Assoc., Washing- ton, DC.

113-115.

428 C. RODRiGUEZ-ALVAREZ, E T A L

SANCHEZ, J.C., MOLINA, R. and GONZALEZ, A. 1986. Control higiknico sanitario'de 10s helados de venta a granel en Albacete. Aliment. 97, 93-98.

UBACH, M., MIGUEL, A. and PUIG, P. 1990. Comparaci6n entre el mktodo rlpido-1 de la A.O.A.C. y el mCtodo oficial para el recuento de E. coli en helados. Cent. Invest. Control Calidad (C.I.C.C.). 6, 30-33.