CC BY
14
Tadeusz Grega, Maria Walczycka, Dorota Kalicka, Faber Bartlomiej, Marek Sady, Dorota Najgebauer-Lejko ©
Department of Animal Product Technology, Food Technology Faculty, University of Agriculture in Krakow, Poland
LEVELS OF PATHOGENS IN COW'S RAW MILK PRODUCTS DURING
CHILLED STORAGE
Abstract. The aim of work was assessment of hygiene of raw milk products (cream, quark like cheese-tvorog, butter) offered in direct sale and stored in homelike chilled conditions in the perspective of Polish and European Standards.
The research was performed on raw milk products bought at Krakow's market stalls. The assessment was done on fresh material (just after buying) and after chilled storage of 7 days It was shown that milk products were in disagreement with norms in concern of their microbiological quality. It was ascribed mainly to high level of contamination with mastitis microflora and with microorganisms originating from environment. During chilled storage milk products quality was getting worse mainly due to psychrotrophic microflora. The statistically high significant influence of season of the year was stated. The quality of milk products was worse during summer. Raw milk products originated from ill cows - mastitis and also contained undesirable microflora what led to their bad quality influenced also by lack of proper chilling conditions at storage and sell in stalls.
Key words: milk, milk products, pathogenic bacteria, direct sale
Introduction
Food offered for direct sale at open air markets in Krakow (Poland) gathers big attention and popularity recently among such products meaningful part are raw cow's milk products. It is worth to be underlined that despite not doubtful advantages (composition, organoleptic characteristics) they contain many microorganisms and among them also pathogens as reported Faber (2006). This food, after buying, is stored by consumers in chilled conditions. This experiment focused on to what extent chilled storage in home refrigerator could influence microbiological load of above described raw cow's milk products.
Material and methods
Experimental material were raw cow's milk products originating from market stall of Krakow (Poland), assessed from 2000 to 2006 during autumn-winter and spring-summer seasons. The subsequent product were assessed: butter, cream, tvarog (quarg like cheese) in 1st and 7th day of chilled storage in standard chilling conditions (5-10°C)
There were estimated chosen physico-chemical parameters of above products: protein content, fat content, ammonium nitrogen content, FFA (free fatty
© Tadeusz Grega, Maria Walczycka, Dorota Kalicka, Faber Bartlomiej, Marek Sady, Dorota Najgebauer-Lejko, 2008
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acids)content (according to Huert-Gonzalez &Wilbey 2001). All products were also subjected to microbiological analysis. The assessments of microbial load of milk products were performer according to Polish Norms and EU Standards: generals (sampling way, storage and dilution of samples) ISO 6887-1:1999, PN-93 A-86034/01; PN-93 A-86034/02; PN-93 A-86034/03; Total aerobic count at 30oC: PN-93 A-86034/04, ISO 4833:2003; Lactic acid bacterial count at 25oC: IDF 149A:1997, ISO 15214:1998; Psychrotrophic bacteria count at 6oC: PN-A-86034/06: 1993,; Heat resistant bacteria count at 45oC: PN-A-86034/05: 1993; Coliforms count at 30oC: ISO 4832:2006, ISO 4831:2006 PN-93 A-86034/09; PN-93 A-86034/10; Yeasts and moulds count at 20oC; PN-93 A-86034/07.
Also the pathogens of udder origin (Staphylococcus aureus; Streptococcus medicus; Streptococcus epidermis; Streptococcus agalactiae; Streptococcus uberis; Arcanobacter pyogenes) and environmental origin (Salmonellae sp., Listeria sp.: Micrococcus sp.)were estimated. The estimation was done in State Veterinary Institute in Krakow (certified for pathogens incubation) according to: ISO 4832:2006, ISO 4831:2006 PN-93 A-86034/09; PN-93 A-86034/10.
Obtained data were analysed with Statistica v.7.0. There were calculated arithmetic means with standard deviations and importance of differences between them estimated with ANOVA with chilled storage time as main factor. Post hoc analysis was performed with Duncan test.
Results and discussion
Raw milk products are stored in chilled conditions and it should be estimated the changes ongoing in lowered temperature in their composition and first of all in microflora load and eventual pathogenic.
Expected and wanted microflora of milk products consists mainly of Lactobacillus lactis ssp. lactis, Lactobacillus lactis ssp. cremoris, Lactobacillus lactis ssp. lactis biovar. Diacetylactis according to Walstra (1999).
Concerning data presented in Table 1 the speed of lypolysis of milk products changed - large delay.
Table 1.
Changes in chosen parameters of raw milk products during chilled storage (n = 200; x ± SD)
Parameters Product
Cream
1st day of storage 7 th day of storage
Total fat (%) 26,02 + 3,92a 22,16 + 2,99a
Free fatty acids 0,91 + 0,10a 1,45 + 0,26a
Butter
1st day of storage 7 th day of storage
Total fat (%) 81,16 + 4,05a 77,16 + 5,12a
Free fatty acids 1,25 + 0,08a 2,16 + 1,02a
Tvorog
1st day of storage 7 th day of storage
Total protein (%) 12,87 + 1,42a 9,99 + 0,79a
Ammonium nitrogen(%) 12,64 + 0,91a 18,96 + 1,01a
A - the mean value with the same letter in rows differ significantly with P<0,01 a - the mean value with the same letter in rows differ significantly with P<0,05
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That parameter changes were statistically significant for milk product obtained on milk fat basis (butter and cream) during 7 days chilled storage. That phenomenon was probably due to lowering of certain microflora levels in the products. Also the same experimental factor (changes in microflora with storage time) had statistically meaningful influence on lowering of total protein content with parallel growth of ammonium nitrogen levels in chilled stored cheese - tvorog (Tablel). Above mentioned changes could be ascribed to growth of psychrotrophic microflora which showed both proteolitic and lipolitic activities what was confirmed by data presented in Tables 2, 3, 4, 5.
Table 2.
The influence of chilled storage conditions on microbiological quality of butter _(n = 200; x ± SD)__
Parameter 1st day 7th day
Total aerobic bacteria count (log cfu/cm3) 6,31± 5,49 a 6,48 ± 5,6 a
Lactobacillaceae count (log cfu/cm3) 5,29 ± 4,26 a 5,22± 4,32 a
Psychrotrophic bacteria count (log cfu/cm3) 5,69 ± 4,30 A 5,78 ± 4,49 A
Heat resistant bacteria count (log cfu/cm3) 2,13 ± 2,08 a 2,03 ± 2,26 a
Coliforms count (log cfu/cm3) 3,63 ± 3,03 A 3,32 ± 2,89 A
Yeasts and moulds count (log cfu/cm3) 4,83 ± 4,04 a 4,85 ± 3,00 a
A - the mean value with the same letter in rows differ significantly with P<0,01 a - the mean value with the same letter in rows differ significantly with P<0,05
The results showed that total aerobic bacteria count and psychrothrophic bacteria count were growing whereas loads of acidifying bacteria, heat resistant bacteria, coliforms, yeasts and moulds were falling down.
Table 3.
The influence of chilled storage conditions on microbiological quality of cream _(n = 200; x ± SD)__
Parameter 1st day 7th day
Total aerobic bacteria count (log cfu/cm3) 5,86 ± 5,03 A 5,99 ± 5,23 A
Lactobacillaceae count (log cfu/cm3) 4,46 ± 3,39 A 4,21 ± 3,49 A
Psychrotrophic bacteria count (log cfu/cm3) 4,19 ± 3,68 A 4,33 ± 3,72 A
Heat resistant bacteria count (log cfu/cm3) 2,01 ± 1,04 a 1,93 ± 0,95 a
Coliforms count (log cfu/cm3) 3,67 ± 3,00 A 3,33 ± 2,96 A
Yeasts and moulds count (log cfu/cm3) 4,79 ± 4,26 A 4,67 ± 4,21 A
A - the mean value with the same letter in rows differ significantly with P<0,01 a - the mean value with the same letter in rows differ significantly with P<0,05
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Table 4.
The influence of chilled storage conditions on microbiological quality of tvorog _cheese (n = 200; x ± SD) _
Parameter 1st day 7th day
Total aerobic bacteria count 5,22 ± 5,05 a 5,46 ± 5,26 a
(log cfu/cm3)
Lactobacillaceae count 4,46 ± 3,85 a 4,25 ± 3,79 a
(log cfu/cm3)
Psychrotrophic bacteria count 4,31 ± 3,91 A 4,57 ± 3,96 A
(log cfu/cm3)
Heat resistant bacteria count 1,71 ± 0,95 A 1,58 ± 0,90 A
(log cfu/cm3)
Coliforms count (log cfu/cm3) 2,62 ± 1,57 a 2,59 ± 1,61 a
Yeasts and moulds count 4,75 ± 3,99 a 4,66 ± 3,94 a
(log cfu/cm3)
A - the mean value with the same letter in rows differ significantly with P<0,01
a - the mean value with the same letter in rows differ significantly with P<0,05
The temperature of chilled storage showed to be statistically significant. There is growing an interest in functional milk products in Poland, produced with traditional methods.
But most of accessible data concerns products produced with industrial methods and usage of pasteurised milk as raw material. Bochdziewicz et al. (2004) during assessing of quality of Polish cheeses (tvarog) stated that water content In those products was ranging from 74 to 74,5%. The acidity of those cheeses was influenced by fat content in the product and ranged from 4,53 to 4,55. The fat level also influenced sensory score obtained by cheeses during organoleptic assessment.
Similar investigations were leaded by Smietana et al. (2003). The results were subsequent: water content: 71,03 - 76,42 %; and in dry solids - total protein content: 12,89 - 14,31%; fat content: 6,12 - 7,05%; lactose content: 3,74 - 4,38 %; ash content: 0,84 - 1,12%. As final conclusion was said that above listed changes in cheeses composition were mainly due to cheeses' production method.
It should be underlined that tvarog assessed in this experiment was fulfilling State demands concerning this type of product (PN - 91/A-86300), as basic composition and same chosen physico-chemical characteristics go. Water content ranged from 70 to 76 %; fat content on dry mass basis ranged from 15 to 42%; acidity ranged from 80 to 115.
Whereas microbiological quality and safety was disqualifying concerning above mentioned Polish Norm. The number of moulds should not exceed 500 cfu/g; yeasts 10 000 cfu/g; the coagulase-positive staphylococci should be not present in 0,1g of product and coliforms number lower that 0,001 in 1 g of product.
Smietana et al. (2003) also estimated the microbiological quality of tvorog cheeses available In supermarkets. Total aerobic count in fresh cheeses ranged from
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2,7 to 9,0 x 103. Whereas after 14 days of chilled storage it ranged from 1,9 to 4,1 x 104. Yeasts, moulds and coliforms were not present in those products.
Proteolitic and lipolitic changes in dairy products were reported by Sady (2003) and Regula (2005). According to Sady (2003) levels of FFA in bioyoghurts stored from 1 to 14 days ranged from 9,14 to 14,29 meq/cm3. The levels of that parameter in the same period of storage for regular yoghurts ranged from 7,92 to 12,62 meq/cm3 .
Regula, (2005) estimated the level of N-ammonium produced in milk fermented drinks (kefir, yoghurt, bioyoghurt, acidified milk) as 9,53 - 9,75 mg/100g in dependence with storage time (1-14 days). Panfil-Kuncewicz and Laniewska-Trokenheim (2006) searched for contamination of tvorog cheese by L. moncytogenes, Enterococcus sp., coli rods and E. coli during storage (7-21 days) in 5oC. They showed that with ongoing storage time the amount of L. moncytogenes load diminished when the levels of other bacteria did not changed statistically. Similarly Steinka and Kukulowicz (2005) estimated the survival of enterococci during acidifying of milk, in milk curds and in tvarog cheeses stored in chilled conditions at presence of Staph. aureus and yeasts (Candida sp.). They showed that with the initial low contamination of tvorog cheeses with enterococci (2-4 log cfu/g) the reduction of those bacteria after 14 days of storage was not big and did not exceed 1 log cfu/g. The observation of behaviour of enterococci population in tvorog cheeses vacuum packed stored revealed their average growth as 0,82 log cfu/g starting from the second day of storage. storage The data showed that enterococci were very stable in curds and in tvorog cheeses despite they were the monoculture or live in companion of staphylococci and/or yeasts. (Steinka and Kukulowicz, 2005).
It has to be underlined that raw milk products had severe microbiological contamination what was influence by large contamination of raw material (milk) , traditional way of production without effective antimicrobial heat treatment (pasteurisation).
Lactobacillaceae did not show enough antagonistic activity against psychrotrophic microflora (especially against fungi imperfecti). Bacteriocins produced by some species of lactic acid bacteria are strong antibacterial proteins showing severe killing abilities against pathogens as Staph. aureus, Enterococcus faecalis and Listeria monocytogenes (Nerbrink et al. 1999; Van Schaik et al., 1999).
Conclusion
Concluding this experiment connected with assessment of raw milk products it has to be stated that their microbiological loads with presence of pathogens makes them dangerous and unserviceable in accordance with Polish State and European Union food low regulations.
References
1. Bochdziewicz K., Smietana Z., Rutkowska Z., 2004. Charakterystyka cech sensorycznych i reologicznych polskiego twarogu. IX Sesja Naukowa, UWM Olsztyn, 199-206.
2. Faber B., 2006. Ocena jakosci mleka krowiego oraz jego przetworow znajduj^cych si$ w sprzedazy bezposredniej. Praca doktorska AR Krakow.
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3. Huert-Gonzalez L., Wilbey R.A, 2001. Determination of free fatty acids produced in filled-milk emulsion as result of the lypolytic activity of lactic acid bacteria. Food Chemistry, 72, 301-307.
4. IDF 149A:1997. Dairy starter cultures lactic acid bacteria (LAB) standard of identity.
5. ISO 4831:2006 Microbiology of food and animal feeding stuffs -- Horizontal method for the detection and enumeration of coliforms -- Most probable number technique.
6. ISO 4832:2006 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of coliforms -- Colony-count technique
7. ISO 4833:2003 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of microorganisms -- Colony-count technique at 30 degrees C .
8. ISO 6887-1:1999 Microbiology of food and animal feeding stuffs -- Preparation of test samples, initial suspension and decimal dilutions for microbiological examination -- Part 1: General rules for the preparation of the initial suspension and decimal dilutions
9. ISO 15214:1998 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of mesophilic lactic acid bacteria -- Colony-count technique at 30 degrees C.
10. Nerbrink E., Borach E., Blom H., Nesbakken T.A., 1999. A model based on absorbance data on the growth rate of Listeria monocytogenes and including the effects of pH, NaCl, Na-lactate and Na-acetate. International Journal of Food Microbiology, 47(1-2), 99-109.
11. Panfil - Kuncewicz H., Laniewska-Trokenheim L., 2006. Mikroflora patogenna i zanieczyszczaj^ca w twarogach pakowanych roznymi metodami i przechowywanych w niskich temperaturach. X Mi^dzynarodowa Sesja Naukowa „Post^p w technologii, technice, analityce i organizacji mleczarstwa" Olsztyn 118.
12. PN-91 A-86300/Az3:2001 Mleko i przetwory mleczarskie. Sery twarogowe niedojrzewaj^ce.
13. PN-93 A-86034/01 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Przygotowanie probek i rozcienczen. Zakres normy i postanowienia ogolne
14. PN-93 A-86034/02 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Ogolne zasady badan
15. PN-93 A-86034/03 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Przygotowanie probek i rozcienczen
16. PN-93 A-86034/04 Mleko i przetwory mleczarskie. Badania mikrobiologiczne.Liczba drobnoustrojow tlenowych - oznaczanie metod^ plytkow^ w temperaturze 30°C.
17. PN-93 A-86034/05 Mleko i przetwory mleczarskie Badania mikrobiologiczne Drobnoustroje termofilne - oznaczanie liczby metod^ plytkow^ w temperaturze 55°C.
339
18. PN-93 A-86034/06 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Drobnoustroje psychrotrofowe - oznaczanie liczby metod^ plytkow^ w temperaturze 6,5 i 21°C.
19. PN-93 A-86034/07 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Plesnie i drozdze - oznaczanie metod^ plytkow^ w temperaturze 25oC.
20. PN-93 A-86034/09 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Escherichia coli - wykrywanie obecnosci i oznaczanie najbardziej prawdopodobnej liczby(NPL).
21. PN-93 A-86034/10 Mleko i przetwory mleczarskie. Badania mikrobiologiczne. Enterokoki - Wykrywanie obecnosci, oznaczanie liczby metod^ plytkow^.
22. Regula A., 2005. Wplyw roznych kultur startowych na przemiany hydrolityczne w napojach fermentowanych z mleka owczego. Praca doktorka AR Krakow.
23. Sady M., 2003. Wlasciwosci odzywcze i jakosciowe jogurtow z dodatkiem nasion amarantusa i ziaren owsa. Praca doktorska AR Krakow.
24. Steinka I., Kukulowicz A., Znaczenie enterokokow w ocenie jakosci twarogow. X Mi^dzynarodowa Sesja Naukowa „Post^p w technologii, technice, analityce i organizacji mleczarstwa" Olsztyn 119.
25. Smietana Z., Szpendowski J., Bochdziewicz K., 2003. Charakterystyka tradycyjnego"polskiego twarogu" otrzymywanego wedlug wlasnej nowoczesnej techniki i technologii. Przegl^d Mleczarski, 4, 126-129.
26. Van Schaik W., Gahan C.G.M., Hill C., 1999. Acid-adapted Listeria monocytogenes displays enchanced tolerance against the lantibiocins nisin and lacticin 3147. Journal of Food Protection, 62 (5), 536-539.
27. Walstra P., 1999. Dairy Technology: Principles of Milk Properties and Processes. New York, NY, USA Marcel Dekker Incorporated; p.373.
Summary
The aim of work was assessment of hygiene of raw milk products (cream, quark like cheese-tvorog, butter) offered in direct sale and stored in home-like chilled conditions in the perspective of Polish and European Standards.
The research was performed on raw milk products bought at Krakow's market stalls. The assessment was done on fresh material (just after buying) and after chilled storage of 7 days It was shown that milk products were in disagreement with norms in concern of their microbiological quality. It was ascribed mainly to high level of contamination with mastitis microflora and with microorganisms originating from environment. During chilled storage milk products quality was getting worse mainly due to psychrotrophic microflora. The statistically high significant influence of season of the year was stated. The quality of milk products was worse during summer. Raw milk products originated from ill cows - mastitis and also contained undesirable microflora what led to their bad quality influenced also by lack of proper chilling conditions at storage and sell in stalls
Cmammx nadiümna do peda^ii 13.08.2008
340
