Food: Journal of technology and food hygiene No. 366, 2005.

pag 62-69 1 DETERMINATION OF THE MICROBIOLOGICAL QUALITY OF RAW MILK AND IN A handmade Quesi llo COOPERATIVE IN A RURAL AREA CENTRO-SUR DE CHILE † Microbiological DETERMINATION OF QUALITY OF RAW MILK AND FRESH CHEESE HANDCRAFT Elaborate IN A RURAL COOPERATIVE OF WOMEN IN CENTRAL-SOUTH REGION OF CHILE Garcés, R.1 *, Brown, C. 2, Hair, M.3, Orellana, A. 3, Brandl. E 4, Lopez, J.L.5 ¢ To the memory of Prof. Dr. Juan Luis López Fernández (1953-2004). 1. Food Dept. of Animal Origin. Agency for Health and Food Safety (AGES). Technikerstrasse 70, A-6020 Innsbruck, Austria 2. Institute of Science and Food Technology. Faculty of Agricultural Sciences. Universidad Austral de Chile, Valdivia, Chile 3. Unit of Hygiene and Milk Quality. Faculty of Veterinary Medicine. Universidad de Conc epción, Chillán, Chile ¢ Visiting Professor, accredited specialist in food hygie ne. DAAD Convention, Free University of Berlin, Germany 4. Institute of Hygiene and Milk Technology and Food Science, University of Veterinary Medicine - Vienna . Vienna, Austria 5 Department. Animal Production. Faculty of Veterinary Medicin e. University of Las Palmas de Gran Canaria. Gran Canaria, Spain * Author for co rrespondence: rgarces@mail.com, @ ages.at rene.garces-Avilez † Shortly ABSTRACT It describes The way and Working Conditions of Rural Women in R elationship to milk handling and elaboration of fresh cheese. Total mesophilic b acteria in raw milk in high summer Were more Than in winter (P <0.05). Enterobac teriacea bacteria in Both seasons of the year Were above international recommend ations. High bacterial counts of Both groups are a clear and unequivocal signal of high occurrence of environmental contamination and manipulation under Inadequ ate hygiene conditions. In summer as in winter, Gram-negative bacteria in fresh cheese Were Allowed above the maximum limit (E. coli) or absinthe (Salmonella). With Respect to S. aureus, This Was Also above the recommended ranges and During the time this study Was Not Appeared as a food-borne Risk. L. Was monocytogenes absent in all fresh cheese sample. Contrary to What May Have Been Expected, bac terial counts (milk and fresh cheese) Were more Than high in summer in winter. K ey words: Milk quality, fresh cheese, Farmers woman, seasonal Influence article we describe briefly how and working conditions of rural women in relation to mil king and milk handling and manufacturing of cheese. By comparing the total mesop hilic bacterial count in milk was observed in summer counts were higher in a sta tistically significant (P <0.05) than in winter. Enterobacteriaceae counts at bo th stations was well above international recommendations. High bacterial counts of both groups are a clear signal and Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 2 Clear indication of the high occurrence of pollution and handling conditions of hygiene. Quesillo In Gram-negative organisms, both summer and winter, were found above the maximum limit (E. coli) or absent (Salmonella). Regarding S. aureus, is also presented on the ranges accepted that time would not be in risk of food poisoning. Was not found in L. monocytogenes. Contrary to expectations, the bact erial count (milk and cheese) was higher in summer than in winter. Keywords: Qua lity of milk, cheese, rural, seasonal effect INTRODUCTION Within the definition

of food security, food safety is one of the key concepts. The approach "from far m to fork" is sometimes understood as applicable only to eating establishments a nd food business, often forgetting that, especially in developing countries ther e are many small producers, who obtain for example in the case, milk of their ow n animals and use it for household consumption and also for the manufacture of f resh artisan cheeses for either the sale or consumption, thus forming an unprote cted market, informal and virtually no health checks. String cheese with the che ese are traditional cheese Chanco, Chile, whose consumption is important and com mon in this country (Brown et al., 2002, 2003). String cheese can be defined as a cheese made with pasteurized milk, fresh (not matured), with high humidity (65 0 g / kg) and about 130 g / kg fat. Is obtained by coagulating enzyme only and n o development of acidity in the process, its physical properties€chemical and se nsory-specific are described in the 2494 Chilean Standard (INN, 2000), also can be added to the traditional fresh cheeses (without use of ultrafiltration) is sh ort life (Brown, 1999). At the industry level is produced mainly by a continuous system, which, briefly, in the process involves a) the concentration of milk by ultrafiltration, b) aseptic packaging line and strict control of production, so that its life is substantially enlarged. The production of curd fresh in this A ndean country is an activity that, although unquantified, has been on the rise i n recent years as a source of income for families with fewer resources, with the particularity of the female population has been the which has enhanced this act ivity is known that in emerging developing countries women typically receive les s education than men. In this regard the World Bank concludes that if women rece ive the same education as their agricultural production would increase by 7% to 22% and the FAO for its part says that farmers receive only 5% of extension serv ices to worldwide. Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 3 The objective of this study was to determine in a preliminary way and before he could bring to market a safe product and certified by the regional health author ity, the hygienic quality of fresh cheeses produced in two seasons of the year b y opposing a group of women farmers grouped in a marketing cooperative cheese in the city San Carlos, Province of Ñuble, Chile. The specific objectives were: a) determine the microbiological quality of raw milk used as raw material for the production of curd and b) determine the microbiological quality of fresh cheeses made in traditional way. General Background In the city of San Carlos, Province of Ñuble (36 º 00'-38 º 30'S), ten farmers came together giving rise to a coope rative for the legal sale (with a resolution of the health authority) of quesill o each elaborated on their homes. The climatic conditions in the area include ra infall exceeding 1,200 mm, with an average temperature between 12 ° C and 15 ° C . Summer is characterized by dry (5% of annual rainfall) and hot (absolute maxim um> 34 ° C measured in the shade). In the winter there are lower temperatures (< 0 ° C), also focusing more than 50% of annual rainfall. MATERIAL AND METHODS For the purpose of studying the quality of raw milk and cheese, were carried out in spection visits and sampling visits to each of the producers. The aim of the fir st was a visual inspection of the facility, observe the handling of raw milk and the process of preparing the cheese. Also was used to apply a small questionnai re to obtain general information of each farmer and the resources available and their mode of application. Inspection visits (one for each season to study) were made both at the beginning of the austral summer and winter. Sampling visits we re made every 15 days, adding up to: 6 samplings in summer (60 samples of milk a nd cheese) and six in winter (60 samples of milk and cheese). This latter type o f visits were intended to collect raw milk and cheese. In the first and only vis it of inspection by season also took advantage of making the first sampling of m ilk and fresh cheeses of the season. Material Samples of milk The reference proc edure considered for the sample of raw milk was the Decision 91/180/EEC. At each visit, took a sample of milk (100 mL) from a tub or container used for processi

ng cheese, and had no farm milk tank refrigerated storage of raw milk. The sampl es were taken from the middle of the tub or container and only after the total m ilk for the preparation of which was accumulated there and before it was heated. Prior to sample collection stirred the contents of the tub or container, for at least 30 seconds with a Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 4 aluminum bucket capacity 50 mL, with handle 40 cm long and sterilized by autocla ving. This in turn were used to collect and deposit the milk sample in sterile g lass bottles of 200 mL capacity. The number of milk samples for total mesophilic count (TMC), to estimate the total average value of the study period, was made according to the recommendations made by Directive 92/46/EEC.€Samples of curd sa mples number (n) per group (number of cheeses produced on the day of sampling) w as determined following the procedure described in FIL-IDF 50B: 1985. The n valu e is 5, which in this study corresponded to about 20% of the cheeses produced in each day. Samples were obtained aseptically and only once all the product produ ced that day was transferred to the storage site designed for that purpose in ea ch farm, which in all cases corresponded to the home refrigerator, next to food consumption familiar. Sterile knife was used by direct flame. Were cut wedge-sha ped (from surface inwards) of approximately 100 g / cheese, and were introduced into sterilized glass jars autoclaving. Microbiological Methods of milk and both cheeses and milk samples of cheeses were placed immediately in a cooler and con tinued during transport to a maximum temperature of 4 º C. The samples were proc essed within six hours of collection. 1. Milk - RMT (CFU / mL): After decimal di lutions, was placed 1 mL of each dilution in Petri dishes (91/180/EEC). Then eac h plate was added to 15-20 mL of plate count agar cast (47-50 º C). Inoculum was homogenized with the agar plate using a circular motion and swing. It was left to stand until the agar solidified, then added a second layer (approx. 10 mL) of the same agar. Once set it reversed the plates and incubated at 35 º C, 48 h. T otal Enterobacteriaceae (CFU / mL): After decimal dilutions, 1 mL was deposited each dilution in Petri dishes. Then each plate was added to 15-20 mL of Agar VRBG mol ten (47-50 ° C). Inoculum was homogenized with the agar plate using a circular m otion and swing. It was left to stand until the agar solidified: then added a se cond layer (approx. 10 ml) agar VRBG. Once set it reversed the plates and incuba ted at 37 ° C, 18-24 h. To confirm that the colonies were of Enterobacteriaceae were tested by the cytochrome oxidase (positive test: violet). Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 5 2. Quesillos The preparation of the primary dilution (from solid sample) used in the laboratory was technically equivalent to ISO 6887 (1983). Basically for the enumeration of RMT, total Enterobacteriaceae, E. coli and S. aureus preparing d ecimal dilutions were made on aseptically, individual pieces of curd sample and sample were placed in a stomacher bag to make 10 g. After the bag was added 90 m L of peptone water. The mixture was homogenized in the Stomacher (2-5 min). Acco rding to the expected number of microorganisms and on the basis of initial dilut ion (10-1), the decimal dilutions of 10-2 to 10-6. - RMT (CFU / g): After decima l dilutions, was placed 1 mL of each dilution in Petri dishes, sterile empty (tw o plates per dilution). Then each plate was added to 15-20 mL of plate count aga r cast (47-50 º C). Inoculum was homogenized with the agar plate using a circula r motion and swing. It was left to stand until the agar solidified, then added a second layer (approx. 10 mL) of the same agar. Once set it reversed the plates and incubated at 35 ° C, 48 h - Enterobacteriaceae total (CFU / g): From decimal

dilutions, was placed 1 mL of each dilution in Petri plates empty and sterile ( two plates dilution). Then each plate was added to 15-20 mL of Agar VRBG molten (47-50 ° C). Inoculum was homogenized with the agar plate using a circular motio n and swing. It was left to stand until the agar solidified: then added a second layer (approx. 10 mL) VRBG agar. Once set it reversed the plates and incubated at 37 ° C, 18-24 h. To confirm that the colonies were of Enterobacteriaceae were tested by the cytochrome oxidase (positive test: violet). - E. Coli (MPN / g): a) Presumptive test: sowing in tubes, three series, with CLS from the decimal di lutions. It was incubated at 37 ° C and readings made at 24 and 48 hours. We sel ected positive tubes (turbidity + gas) and continued with the confirmatory phase , b) confirmatory test: a sample with a sterile loop, and planted immediately in EC broth tubes The inoculated tubes were incubated (44.5 ° C 1824 h). Reaction was considered positive when there was growth (turbidity) and gas evolution in t he hood Durham.€All tubes confirmed as positive were used for isolation, TW afte r planting (44 ° C, 24 h, water bath) for re-planting in EMB agar and biochemica l testing for indole production. - S. aureus coagulase-positive (CFU / g): 0.1 m L were seeded in each decimal dilution in duplicate on Petri dishes containing a gar Baird-Parker. Plates were invested and incubated (37 ° C, 48 hours). Were se lected for counting the plates containing between 20-200 colonies typical (black bright and clear halo of 25 mm, among other features). Once these colonies coun ted, was confirmed by the coagulase test. Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 6 Were seeded in tubes, in BHI broth and incubated (37 ° C, 18-24 h). Subsequently 0.1 mL of the previous crop was added to a tube with 0.3 mL of rabbit plasma ED TA was reconstituted and incubated (37 ° C, 6 h). The reaction is positive when the clot formed is firm. - Salmonella (presence in 25 g): a) for enrichment in n onselective liquid medium: were taken, aseptically, individual pieces of the sam ple of cheese, weighing between 4 and 8 g and were placed in sterile packaging ( Stomacher bag) to complete 25 g. After the bag was added approx. 225 mL of BPW. Homogenized in Stomacher (3-4 min) and incubated at 37 ° C for 24 h, b) selectiv e enrichment in liquid media: b.1) The time for incubation in nonselective mediu m, extracted with a pipette mL of culture and placed in tube containing 10 mL of Müller-Kauffmann broth. Incubated at 43 º C for 18 to 24 h; b.2) in parallel wi th the above, was placed 1 mL of culture of 10 mL of selenite-cystine broth and incubated at 37 ° C for 18-24 h, c ) Isolation differential solid media: From th e crops grown in different selective liquid media, were seeded in duplicate on B S agar agar and BGA. It was incubated at 37 ° C for 24 h (agar BGA) and 48 h (ag ar BS) d) Testing biochemistry: Those suspected colonies of Salmonella grown bot h on agar BGA (pink, transparent, red halo) and agar BS (black or brown, clear e dge) were planted in TSI agar (37 ° C, 24 h) and LIA agar (37 ° C, 24-48 h) e) C hecking biochemistry final suspect colonies were seeded on nutrient agar in Petr i dishes . It was incubated at 37 ° C for 18-24 h and made an API 20E gallery. W hen the API 20E identification corresponded with Salmonella, serological confirm ation was performed; f) Serological identification: He dropped a drop of reagent control over a marked area of a slide and a drop of reactive antibodies in anot her area of the slide marked . With a sterile loop was collected and a portion o f culture was mixed with reagent control drop and the drop of reagent antibodies . He mingled with the handle for 15-20 seconds and then slide gently rotated (ci rcular motion) for 2 minutes and observe the presence or absence of agglutinatio n - L. monocytogenes (presence in 25 g): a) enrichment in liquid medium: were ta ken, aseptically, individual pieces of the sample of cheese, weighing between 4 and 8 g and were placed in sterile packaging (Stomacher bag) to complete 25 g, t hen the bag was added to 225 mL of Fraser broth at a concentration half. The mix ture was homogenized in the Stomacher (3-4 min) and was incubated (30 ° C, 24 h) , b) Isolation differential between ALOA: It was deposited in duplicate 0.1 mL o f Fraser broth on ALOA plates. Spread the inoculum with sterile glass loop and l

et the plates (15 min). Cultures were incubated (30 ° C, 24 h). The suspected co lonies L. monocytogenes grown on ALOA (blue-green light, opaque halo) were trans ferred to nutrient agar for confirmation and were carried incubation (31 ° C, 24 h), c) Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 7 Monoconfirm Listeria test: identification of Listeria spp. and differentiation b etween L. monocytogenes and other Listeria species. Statistics For statistical a nalysis we used the multivariate GLM procedure of SPSS V.11.0 (2001), where the dependent variables were RMT and total Enterobacteriaceae in raw milk and fresh cheeses as fixed factors season (summer vs. Winter ) and the treatment of milk ( raw milk vs. processed milk in cheese). Running the ANOVA procedure was the leve l of significance (P) for both fixed factors and their interaction. The selected model was the full factorial so that the analysis took into account all the eff ects of the factors.€RESULTS AND DISCUSSION Descriptive milking, milk handling a nd processing of cheese. It is noteworthy that in all cases the cows are owned b y the farmers, which is manual milking and milk until the production of fresh ch eeses are kept in the refrigerator (fridge) in the household. In a joint study u nder the same conditions and on the same samples of raw milk used in the present study reports on somatic cell count (SCC) in both seasons. In winter, the value of SCC was 360 700 cells / mL and in summer 412 500 cells / mL (P> 0.05). In th is study, the pH of the cheeses in both winter and summer remained in the range 6.2 to 6.6. It is also interesting to note that in the cooperative any of its me mbers have suffered from infectious disease (diagnosed clinically) such as typho id, hepatitis, tuberculosis and cholera. In addition to the time of initiating t he study none of them was in possession of a valid food handlers. Only 40% of th e producers he washed his hands before the start of milking and only 20% made su re to do a cleansing of the udder and teats with a damp cloth and then a proper drying with a towel or paper (individual cow). Only 30% had milk quickly and wit h the closed vessel to the place of storage. Table 1 summarizes the conditions f or the preparation of cheese. It is important to mention that none of the cases remained constant temperature heating of milk, pasteurization way to get a slow, 63 ° C for 30 min (U.S. PHS / FDA, 1999). Moreover, only 60% of respondents had a thermometer to monitor the temperature of heating the milk and the percentage less than half used it at some point in the development. This is obviously a ri sk, since milk is a good growth medium for many microorganisms due to their high water content, its neutral pH and its wide range of available nutrients (Doyle, 1997), and inadequate pasteurization milk for cheese making can cause poisoning Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 8 (Jay, 2002) as well as failure to achieve the objective of pasteurization of mil k recontamination from the environment is always a risk to control (Eneroth et a l., 2000). Moreover, all the respondents (10 farmers) used wooden utensils durin g processing. This also may have contributed to increased milk recontamination o r contamination and / or cheeses, by the difficulty of making an easy and thorou gh cleaning of wooden utensils. This, it is implicit in article 18 of the Chilea n Health Act (Food Health Regulations, 1996) and described by Brown and Astete ( 1991). Table 1 shows both RMT and total Enterobacteriaceae in milk samples of cu rd and in the austral summer and winter. When comparing performance against RMT in milk, it is noted that in summer counts were higher than in winter, which was reflected in differences statistically significant (P <0.05) than in winter. The absolute values obtained were averages of 65 x 106 CFU / mL compared with 8 x 106 CFU / mL, for summer a

nd winter respectively. Both counts are well above international guidelines (92/ 46/EEC DC, 1992). RMT These high values do not match those reported for fresh mi lk in small farms in Europe (Specker, 1996; Kloppert et al., 1997, Sonntag, 1998 , Friedl, 2001) but are within the ranges previously reported in Chile and for t his same area for Garcés (1998). For its part, total Enterobacteriaceae counts i n both seasons is also well above international guidelines (Milch-Ghetto-Verordn ung, 1980; VO Milch, 1995) and local (Ministerio de Salud Chile, 1997), with abs olute values 2x103 CFU / mL compared to 6x103 CFU / mL in winter and summer resp ectively. High bacterial counts of both groups are a clear and unequivocal signa l of the high occurrence of pollution and handling in poor hygienic conditions ( Kenyon, 1978; Bramley and McKinnon, 1990; Jayarao and Wang, 1999). This milk doe s not meet one of the essential quality requirements provided for treatment and processing of dairy products (Brown and Molina, 1991; Cersovsky et al., 1982; To rnadijo et al., 1998). This was corroborated by direct observation during visits to farms. There was no proper handling of fresh milk,€collect this in plastic c ontainers that did not meet the minimum requirements of washing, disinfection, a nd maintenance. The transport of raw milk from the stable to the place of manufa cture of the cheeses was not the most appropriate, because in most cases was slo w and in containers without lids and places that are walkable and exposed to dir t, dust, mud and flies. As already mentioned, there was an RMT better in winter than in summer (P <0.05) and the same trend in total Enterobacteriaceae counts ( P = 0.065). The above results could be because in the winter dirt (mud, manure, feed residues, etc..) Attached to the udder and adjacent parts of animals is mor e obvious or visible in summer as this time the high temperatures of the geograp hical area as Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 9 already described in general background, where the study was done quickly caused it to dry the udder and adjacent parts allowing debris falling crude dirt givin g an appearance of greater cleanliness, therefore not carried out a thorough cle aning of the udder . If we add to the above, the factor of availability and qual ity of water used, as clearly described in the introduction, lower in summer, re sulted in increased pollution and consequently a lower quality of milk in the su mmer, what This coincides with that expressed by Garcés (2001). Regarding RMT an d enumeration of coliforms in cheese, both have high values. The RTM and total E nterobacteriaceae counts do not meet European standards (92/46/EEC DC, 1992) and although only highly significant differences were detected (P <0.01) between su mmer count over the winter to figure Total Enterobacteriaceae, but not for RTM, this was due to the high variability observed in both winter and summer in the R MT (over 100% coefficient of variation). To explain this behavior could be based on the points made for raw milk compounded with the handling and storage of che ese, which gives these count as high. Total Enterobacteriaceae, with values stil l too high, continue to behave differently from RMT (Table 2) with a clear decre ase in summer and winter stagnation. This could be attributed to the quality of the wash water, referred to as a critical control point for both quality and qua ntity, provides a significant number of coliforms, as observed during the inspec tion visit that rural women tended to washing udders and utensils of the dairy, more winter than in summer. The water used at this time on the farm was not drin king, occupying the coming of canals or wells, these are poorly built, poorly se aled, etc.., Which can lead to a crawl and introduction of hazardous material, f ecal some cases, into the water source. Table 2 shows that Gram-negative organis ms, both summer and winter are above the maximum allowed in E. coli and Salmonel la absent, although it is wise to remember that the two bacteria are also associ ated, when present in raw milk, with reduced quality (Jayarao and Wang, 1999) or major food poisoning (Rohrbach et al., 1992; Steele et al., 1997). In this stud y, E. coli indicates poor condition of cleanliness and management and / or impro per storage of the product. This was corroborated by direct observation during i

nspections and responses to the questionnaire. Regarding the count of these bact eria in the cheeses, it is likely that if he had made a count back in outlets su ch as the analysis would have shown significantly higher counts at registration. Anyway, the count of E. coli is above internationally accepted ranges (DC 92/46 /EEC, 1992). The quantitative determination of S. aureus in food is done in orde r to establish its potential to cause food poisoning and demonstrate post-proces sing contamination (Bennett and Lancette, 1998). Between the years 1993-2002 in Chile reported 48 outbreaks of food poisoning Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 10 staphylococcal 504 people affected. In the aforementioned outbreaks, dairy produ cts had a strong responsibility, 56.52% of cases (INPPAZ, 2002). In this study, S. aureus showed levels above the internationally accepted ranges (DC 92/46/EEC, 1992). The counts presented in Table 2,€for the moment not constitute a risk of food poisoning and it is recognized that concentrations are usually required 10 6 cells of S. aureus per gram of food for forming toxin sufficient to cause pois oning (CDC, 2000). The absence of Salmonella in compliance with European regulat ions (92/46/EEC DC, 1992) and could indicate that the temperature range reached by the milk for the manufacture of fresh cheeses (in the pseudo pasteurization o r heat treatment made milk) theoretically exceeded 50 ° C, destroying it (ICMSF, 1996) have existed, allowing you to keep away, for now, the presence of this ba cteria. Moreover, the absence of L. monocytogenes in the cheeses sampled also co mplies with European regulations (92/46/EEC DC, 1992) and persisting absence wou ld avoid a high risk of poisoning or disease outbreaks among consumers (Rijpens et al., 1997). Sometimes has been associated the absence of L. monocytogenes to low or no presence of carriers of it (Kerr et al., 1993). This can not be discus sed in this study, since a study of these farmers to determine their suitability or otherwise as food handlers and / or carriers of communicable infectious dise ases was not raised as an objective of this work. In short, the fresh cheeses pr oduced by this group of farmers is deficient more and no less important, so it i s urgent to correct the high count RMT happens during the processing of milk in fresh cheeses whatever the season that concerned, given the implications on huma n health. Turn should clarify the source of contamination with Enterobacteriacea e high total and implement a management plan and overall improvement, probably s hould start with training for farmers, research and improvement of water quality improvement for places the preparation of cheeses as well as constant monitorin g of the same operation of this mini-company and thus avoid that they may lose t he license for which health will undoubtedly at the expense of family income to cut off a source of money coming from the work contributed by the peasant housew ife. Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 11 REFERENCES Bramley, A. J., McKinnon, C. H. 1990. The microbiology of raw milk. P ages 163-208 in Dairy Microbiology. 2nd ed. R. K. Robinson, ed. Vol 1. Elsevier Appl. Sci, New York, NY. Bennett, R.W. and G. A. Lancette. 1998. Bacteriological Analytical Manual. 8th ed. (Revision A). FDA. AOAC International. USA. Brito, C . and Astete, A. 1991. Cheese Bulletin No. 5: Field Dairies hygienic handling of their personal attachments and winemaker. Universidad Austral de Chile - Chile. 18p Agriculture Min. Brito, C. and L. Molina H. 1991. Quality of milk for cheese . El Campesino. National Agricultural Soc (Chile). XXII :18-22. Brito, C. 1999. Technology development of fresh and aged cheeses. In: R. A. Garcés (Editor) "Pos tgraduate Course Quality of Milk and Milk Products." 5-9 April. The Master of Ve terinary Science. Universidad de Concepción. Fac Vet Med. Chillán, Chile. Brito, C., Mendez, P., Molina, L.H., Pinto, M. Development of Chanco cheese using a re

duced-fat process of homogenization in milk. Agro Sur 2002. 30 (1): 68-79. Brito , C., Manriquez, X., Molina, LH, Pinto, M. Study of Chanco cheese ripening of re duced-fat, made with homogenized milk. Arch Latinoam. Nutr. 2003. 53: 299-305. C DC (Center for Disease Control). 2000. Guidelines for Confirmation of FoodborneDisease Outbreaks. Morb. Mort. W. Report. 49:54-62. Cersovsky, H., Sonntag, S., Johst, F. 1982. Quality specifications for raw milk and methods for their determ ination. In "Manufacture of dairy products." Ed Acribia, Zaragoza. pp 3-55. Comm ission Decision 91/180/EEC of 14 February 1991 laying down certain methods of an alysis and testing of raw milk and heat-treated milk Official Journal L 093, 13/ 04 / 1991, p. 1-48 Council Directive 92/46/EEC of 16 June 1992 laying down healt h rules concerning the production and marketing of raw milk, heat-treated milk a nd milk products Official Journal L 268, 14.9.1992, p.1-31. Eneroth, A., Ahrné, S., Molin, G. 2000 Contamination routes of Gram-negative spoilage bacteria in th e production of pasteurised milk, Evaluated by randomly amplified polymorphic DN A (RAPD). International Dairy Journal. 10:325-331. FIL-IDF 100B: 1991. Milk and milk products Enumeration of microorganisms (Colony count technique at 30 ° C).€ Friedl, M. 2001. Hygienische von Hart-und Sicherheit aus Rohmilch Halbhartkaesen thermisierter oder Milch. Dissertation. Dr. Med Vet. Aus dem Institut für und M ilchhygiene Milchtechnologie, Veterinärmedizinische Universität Wien. Garcés, R. 1998. Hygiene of Milk Production in Chilean Dairy Farms: Investigation and Asse ssment According To the Council Directive 92/46/EEC. Dissertation. Dr. Med Vet. Aus dem Institut für und Milchhygiene Milchtechnologie, Veterinärmed. Universitä t Wien. Garcés, R. 2000. Hygiene of milk production in goats and cattle campuses : Measurement and research tool based on Directive 92/46/EEC (Preliminary Result s). Annals of the Royal Academy of Veterinary Sciences in eastern Andalusia. 13: 145-164. Garcés, R. 2001. HACCP Milk Producers and small scale (1-06). In: Repor t of the FAO E-mail Conference on "Small Scale Milk Collection and Processing in Developing Countries" FAO. 29 May - 28 July. Animal Production Service & Animal Production and Health Division Editor. FAO, Rome, 71 pp. INN, 2000. Dairy-Chees e, fresh-Requirements. Norma Chilena 2494. National Standards Institute, Chile. International Commission on Microbiological Specifications for Food (ICMSF). 199 6. Microorganisms in Foods. 5. Microbiological specifications of food pathogens. University of Toronto Press. Jay, J. 2002. Food Microbiology. 4 E. Editorial Ac ribia, Zaragoza. 615 pp. Jayarao, B. M., Wang, L. 1999. A study on the Prevalenc e of Gram-negative bacteria in Bulk Tank Milk. J. Dairy Sci 82, 2620-2624. Kenyo n, J. P. 1978. The Problem of coliforms. Dairy Industries 27, 32-34. Kerr, K. G. , Birkenhead, D., Scale, K., Major, J., Hawkey, P. M. 1993. Prevalence of Lister ia spp on the hands of food workers. J. Food Protec. 56, 525-527. Kloppert, B., Wolter, W., Zschock, M., Stojanowic, V. 1997. Rohmilchqualität in Hessischer Mil cherzeugerbetrieben mit Milch-Ab-Hof - Abgabe Direktvermarktung oder unter Berüc ksichtigung der bakteriologischen Besonderer Beschaffenheit. 38. Arbeitstagung d es Arbeitsgebietes "Lebensmittelhygiene" vom 29.09.-10.02.1997 in Garmisch-Parte nkirchen. Chile Ministry of Health. 1997. Food Health Regulations D.S. 977/77. O fficial Journal of the Republic of Chile. April 29, 1997. Santiago, Chile. ISO 6 887-1983. (E) Microbiology General guidance for the preparation of dilutions for microbiological Examination. International Organization for Standardization. Ri jpens, N. P., Jannes, G., Herman, L. M. F. 1997. Incidence of Listeria spp. and Listeria monocytogenes in ready-to-eat chicken and turkey products Determined by PCR and hybridization Line Probe Assay. J. Food Protec. 60, 548-550 Rohrbach, B .W., Draughon, F. A., Davidson, P. M., Oliver, S. P. 1992. Monocytogenes Prevale nce of Listeria, Campylobacter jejuni, Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 12 Yersinia enterocolitica and Salmonella in Bulk Tank Milk: Risk Factors and Risk of Human Exposure. J. Food Protec. 55, 93-97. Sonntag, S. 1997. Milchqualität Förderung ohne Ende?. Neue Landwirtschaft. 62-65. Specker, M. 1996. Untersuchung en zum Vorkommen von Listeria, Salmonella, Campylobacter und staphylokokken in R

ohmilch in Land Brandenburg. Dissertation. FU Berlin. SPSS 11.0 for Windows stat istical software, 2001. SPSS Inc. Headquarters, 233 S Chicago, Illinois 60606. S teele, M. L., McNab, WB, Poopo, C., Griffiths, W., Chen, S., Degrandis, S. A., F ruhner, L. C., Larkin, C. A., Lynch, J. A., Odumeru, J. A. 1997. Survey of Ontar io Bulk Tank Raw Milk for Food-Borne Pathogens. J. Food Protec. 60, 1341-1346. T ornadijo, M. E., Marra, A. I., Garcia Fontan, M. C., Prieto, B., Carballo, J. 19 98. The quality of milk for cheese manufacture: Chemical Quality. Cienc. Tecnol. Aliment. 2, 79-91. U.S. PHS / FDA (United States Public Health Service / Food a nd Drug Administration). 1999. Grade''A''Pasteurized Milk Ordinance, 1999 revisi on. Public Health Service / Food and Drug Administration Publication No. 229. Ve rordnung über die Bezahlung und der Güteprüfung Anlieferungsmilch (Milch-Güte-Ve rordnung) vom 09. Juli 1980, BGBl. l Nr.75 S.1150 Verordnung über Hygiene-und Mi lch Qualitätsanforderungen an und auf Erzeugnisse Milchbasis (Milch VO) vom 24.a pril 1995, BGBl. l Nr.95 S. 544. TABLE AND TABLES Table 1. Summary of main conditions encountered during the prep aration of cheese, and affect the hygienic quality of the final product. Items 1 . Prior to the preparation of fresh cheeses milk is heat treated? 2 Maintain a t emperature> 63 º C for a time less than 30 minutes? 3. "Wash hands before beginn ing the preparation of fresh cheeses? 4. Do you use for work clothing (boots, ha t, mask, apron, gloves)? 5.€Do you use wooden utensils during the preparation? 6 Do the wash cloths used ", and boils disinfected after use? 7. Method of cleani ng wooden utensils: Boil? 8. "Disinfect with bleach all equipment used in the da iry? 9. Cleaning metal utensils employed during the preparation of fresh cheeses : brushing, use detergent, warm water, etc..? 10. 11. Yes% 70 0 50 0 100 70 40 9 0 40 No% 30 100 * 50 100 0 30 60 10 60 60 0 After the preparation of fresh cheeses: Cleaning and disinfection of the dairy, 40 removing remains of curds and dirt from floors, walls and ceiling? Once devel oped the cheeses are kept in the refrigerator "?. 100 * Only 60% had thermometer for temperature control of milk during processing. Food: Journal of technology and food hygiene No. 366, 2005. pag 62-69 13 Table 1. Average count of total mesophilic count (log10) and total Enterobacteri aceae (log10) in samples of raw milk cheeses produced in two seasons in San Carl os, Chile. Summer Winter P RMT (CFU / mL) (CFU / mL) (CFU / g) (CFU / mL) 7,81 a 3.78-8.47 b 4.04 a 6,90 a 3.30 to 8.26 b * NS ** 0.065 Milk RMT E.T

Quesillo E.T 3.60 to RMT: total mesophilic count. ET: total Enterobacteriaceae NS: P <0.05, **: P <0.01 Different letters in the same column, P rage counts of E. coli (log10), S. aureus (log10), Salmonella onocytogenes in samples taken from two seasons in San Carlos, E. coli 1 S. aureus (CFU / g) 2 Salmonella (25 g) Absence Absence 3 L. monocytogenes (25 g) Absence Absence 4 (MPN / g) Summer Winter 2.94 to 2.64 b 3.47 to 3.07 b Different letters in the same column, P <0.01 1 2 3 CLS (37 º C, 24-48 h), EC (4 4.5 º C, 18-24 h); reseeding EMB; Indol. Baird-Parker (37 º C, 48 hours), BHI (3 7 ° C, 18-24 h) reconstituted rabbit plasma EDTA (37 ° C, 6 h) Müller-Kauffmann (43 º C, 18-24 h), Selenite-Cystine (37 º C, 18-24 h) BGA (37 º C, 24 h), BS (37 º C, 48 h), TSI (37 ° C, 24 h), LIA (37 ° C, 24-48 h); Fraser Broth API 20E 4 1 / 2 (30 º C, 24 h); ALOA (30 º C, 24 h) monoconfirm Listeria test not significant *: <0.05 Table 2. Ave and L. quesillos m Chile.