The Microbiology of Sweet Water and Glycol Cooling Systems Used in HTST Pasteurizers in Fluid Milk Processing Plants in the United States

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1 799 Journal of Food Protection, Vol. 5, No., Page (November 989) Copyright International Aociation of Milk, Food and Environmental anitarian The Microbiology of weet Water and Glycol Cooling ytem Ued in HTT Pateurizer in Fluid Milk Proceing Plant in the United tate A. A. TRANTZ, E. A. ZOTTOLA*, R. L. PETRAN, B. J. OVERDAHL, and L. B. MITH Department of Food cience and Nutrition, Univerity of Minneota, 4 Eckle Avenue, t. Paul, MN 558 (Received for Publication March, 989) ABTRACT Refrigerated water and water/glycol mixture ued in high temperature hort time (HTT) pateurizer have been implicated a potential ource of poilage and pathogenic bacteria in milk. Thi tudy wa carried out to determine the incidence of bacteria in thee cooling ytem. weet water and glycol coolant from 68 fluid milk plant were ampled to determine the incidence of pychrotroph, meophile, coliform, and almonellae. A modified mot probable number (MPN) technique wa performed uing ml of coolant in lactoe broth (LB, Difco). ample were incubated for 7 d at C or for 48 h at 7 C. MPN veel poitive for growth at 7 C were ued to inoculate elective enrichment, differential and biochemical confirmation media for almonellae, and brilliant green bile broth (BGB, Difco) for enumeration of coliform. In addition, 5 L of each ample were paed through.45 (j.m filter (Millipore). The filter were incubated in LB. The previouly decribed procedure for the detection of almonellae wa ued. The procedure of Lovett et al. (7) wa ued to examine each ample for the preence of literia. The population of organim that grew at and 7 C varied greatly from plant to plant. MPN value ranged from <. to >/ ml of coolant. When preent, coliform were uually at low level. Eight ample had coliform MPN value >./ ml of coolant. almonella typhimurium wa iolated from one weet water ample uing the filtration procedure. No Literia were iolated. The ue of anitizer in dairy coolant wa not aociated with low microbial population in the coolant ample. Thi tudy ugget that coolant ued in HTT pateurizer may erve a a reervoir for bacterial contamination of pateurized milk. Coolant ued in high temperature hort time (HTT) pateurizer are refrigerated or weet water, or a mixture of approximately % propylene glycol and 7% water. Thee coolant often contain varying level of milk olid. rut, ediment, and other debri. Cooling media contami- 'Publihed a paper No. 694 of the contribution erie of the Minneota Experiment tation baed on reearch conducted under Project 8-56 upported by Hatch fund and in part by a grant from the National Dairy Promotion and Reearch Board. nated with low level of nutrient could upport the growth or urvival of undeirable organim. The Grade A Pateurized Milk Ordinance (PMO) (Anon., 985) require that recirculated cooling water ued in milk proceing plant be teted emi-annually. An MPN of <. coliform/ ml of cooling water or < coliform/ ml by a membrane filter method i the limit pecified in the PMO. When coliform population exceed thi limit, the water upply i to be phyically inpected and neceary correction made until ubequent ample are in compliance. The PMO doe not indicate what method are to be ued to obtain atifactory ample. The PMO alo require that preure differential mut be maintained in the regenerator ection of HTT pateurizer o that, in the event of leakage, flow will be from the heated milk into the raw milk. There are no federal regulation governing the preure in the ection of pateurizer where cooling take place. Three of eight diary plant that reponded to a urvey by Zottola and mith () maintained higher preure on the coolant than on the milk. A urvey of weet water and glycol coolant ued in Minneota dairy plant (4) reported recovery of high level of pychrotrophic bacteria from both type of coolant. Although no coliform were recovered from glycol coolant, approximately one-third of the weet water ample demontrated coliform contamination. It wa uggeted that coolant erve a a ource of poilage organim in dairy product and that pin hole, crack, and other defect in the HTT plate could allow contamination of pateurized milk by weet water and glycol coolant. The objective of the preent reearch were to determine the microbiological quality of dairy cooling ytem and to determine if dairy coolant could erve a a reervoir for microbial contamination of pateurized milk. MATERIAL AND METHOD Two urvey were conducted. The firt involved grade A fluid milk plant in the North Central tate of Minneota, North Dakota, and outh Dakota. The National urvey involved 5 plant throughout the United tate. JOURNAL OF FOOD PROTECTION, VOL. 5, NOVEMBER 989

2 8 TRANTZ, ZOTTOLA, PETRAN, OVERDAHL, AND MITH ample collection North Central urvey. Approximately 8 L of coolant ued in HTT pateurizer were collected from grade A fluid milk plant by project peronnel. Plant peronnel provided information on the coolant and anitizer uage. An attempt wa made to obtain coolant both before and after it had paed through the cooling ection of the pateurizer; the deign of everal dairie precluded collection of both type of ample. ample were collected aeptically and tranported on ice to our laboratory for analye within. National urvey. terile ample bottle to collect 8 L of coolant and inulated hipping container were hipped to 5 dairie in 7 tate. Included with each hipment were intruction on ampling and hipping procedure and a ample information heet which wa to be filled out at the time of ample collection. Information wa requeted on coolant type, anitizer ued in the coolant, and preure maintained in the HTT. No attempt wa made to obtain coolant before and after it had paed through the cooling ection of the HTT. Eight L of coolant wa collected aeptically by plant peronnel and hipped to our laboratory in inulated container by an overnight expre ervice. Analye were initiated within h of receipt of the coolant. ample analyi Growth at 7"C. ample were analyzed uing a modified -tube mot probable number (MPN) technique. A total of ml of ample wa ued. One hundred ml were aeptically tranferred to L flak containing terile triple-trength LB (Difco); ml were ued to inoculate three 5 x 5 mm crew-cap tet tube containing double-trength LB; and ml wa ued to inoculate three 6 x 5 mm tet tube containing ingle trength LB. If the coolant wa glycol, an appropriate amount of terile water wa added to the ample to decreae the glycol concentration to le than 6%. The amount of water to add wa determined from the information on glycol concentration received from the plant upplying the ample. Zottola and mith () indicated that high concentration of glycol may inhibit recovery of microorganim. When the National urvey ample were analyzed, the need to dilute the glycol wa avoided by inoculating the and ml aliquot of ample into the volume of. trength LB which would reult in a final glycol concentration of 6%. MPN veel were incubated at 7 C for to 48 h. Enumeration of coliform. One ml from flak or tube howing growth and ga production at 7 C wa ued to inoculate BGLB. BGLB tube poitive for broth growth and ga after 48 h at 7 C were conidered coliform-poitive. Enumeration of almonellae. One ml of the broth from 7 C MPN veel indicating growth at 7 C wa ued to inoculate elenite-cytine (Difco) and tetrathionate broth (Difco) which were incubated at 7 C. Thoe elective broth howing growth after h were treaked onto bimuth ulfite (Difco), xyloe lyine deoxycholate (Difco), and brilliant green agar (Difco) and were incubated at 7 C for -48 h. Colonie with reaction typical for almonellae were ued to inoculate triple ugar iron (Difco) and lyine iron agar (Difco) lant. Reultant iolate with almonella characteritic were erotyped by the Univerity of Minneota Veterinary Pathobiology Laboratory. Filtration procedure. Becaue very low level of almonellae were expected, a membrane filtration technique wa ued. Five liter of ample were paed through.45 urn filter (Millipore Corp., Bedford, MA). The filter were incubated in LB at 7 C for -48 h. The previouly decribed procedure for detection of almonellae wa ued. Growth at W C. The MPN of pychrotrophic organim in the coolant ample wa determined a previouly decribed for growth at 7 C, except MPN veel were incubated at C for 7 d. Detection o/literia monocytogene. The procedure of Lovett et al. (7) wa ued to examine each coolant ample for the preence of Literia, monocytogene. Identification of iolate. Iolated colonie were treaked on trypticae oy agar (TA, Difco) from randomly elected MPN veel, BGLB tube, and differential plate. Iolate were Gram-tained and identified uing API E, Rapid NFT, and taph Trac identification trip (Analytab Product, Plainview, NY). Organim not identified with identification trip were claified baed on Gram-tain, catalae and oxidae reaction, and carbohydrate fermentation pattern (6). Determination of glycol concentration. A Hewlett-Packard 584 wa ued to determine propylene glycol concentration by ga chromatography. The column wa a 5 m x. mm Carbowax M. Helium wa ued a the carrier ga at a head preure of 5 pi. An injection ize of. ju- with a plit ratio of : wa ued. Quantitation wa performed uing butyl celluloe and propylene glycol in methanol a internal tandard. A flame injection detector wa ued. REULT The number of pychrotrophic and meophilic organim that were recovered from ample obtained in the North Central urvey varied greatly from plant to plant (Table ). Both weet water and glycol coolant demontrated a C and 7 C MPN range from <. to > 68 organim/ ml of coolant. No coliform were recovered from mot ample, however the coliform population from five coolant ample did exceed the PMO limit (Table ). The number of bacteria recovered in the North Central urvey wa lower than reported by Ginn et al. (4). No almonellae or Literia were iolated from any North Central urvey ample. Increaed regulatory activity by the Minneota Department of Agriculture, Dairy Diviion, which included the ampling of cooling media for microorganim during routine inpection probably influenced the reult. Glycol concentration of North Central urvey ample ranged from to 46% (Table ). The ph value of mot North Central urvey coolant were 7.8 or higher. One ample had a ph of 5.8 (Table ). Only three North Central urvey plant routinely ued anitizer in the recirculated cooling water (Table ). Each of the three common type of dairy anitizer, chlorine, iodine, and quanternary ammonium compound, were ued. A comparion of the pychrotrophic tandard plate count (PPC) reult of coolant ytem indicated that the microbiological quality of dairy coolant wa not guaranteed when anitizer were ued (Table ). weet water treated with iodine (ample El 5) had a PPC of /ml of coolant, while weet water ample with no added anitizer (ample F95 and R) had PPC value of JOURNAL OF FOOD PROTECTION. VOL. 5, NOVEMBER 989

3 MICROBIOLOGY OF WEET WATER AND GLYCOL COOLING YTEM 8 TABLE. ummary of MPN reult for North Central urvey coolant ample. MPN/ ml Number of ample with MPN value value C 7 C Coliform < > Total 8 8 8* *Coliform MPN determined on one ample per dairy. TABLE. Comparion of the effect of added anitizer on the pychrotrophic tandard plate count (PPC) of North Central urvey coolant ample. ample Coolant medium anitizer ued ph PPC/ml A8 glycol (%) B4 glycol (4%) C5 glycol (%) El 5 weet water F95 weet water H5 glycol (%) J6 glycol (%) L glycol (9%) N7 glycol (46%) 9 glycol (%) P5 glycol (9%) Q8 glycol (%F) R weet water *Quaternary ammonium iodine chlorine quat* compound < < < < < 8 < 768 and 76/ml of coolant, repectively. Thi uggeted that the addition of antizer to the cold water wa not effective in controlling the pychrotrophic population. The ame wa true when anitizer wa added to the glycol ytem. Glycol coolant with added chlorine (ample HI5) had a PPC of /ml of coolant. Glycol ample CI 5, with no added anitizer, had a PPC value of <l/ml of coolant. The glycol coolant ample that had been treated with quaternary ammonium compound (ample 9) alo had a PPC value of <l/ml of coolant. The mot widely ued method to control microbial population in cooling water ued in the canning indutry i the application of chlorine compound (8). Maintenance of an adequate chlorine reidual may be difficult due to contamination of the coolant by food and fluctuation in ph and temperature (5,8). It i poible that the high ph of the dairy coolant reduce the effectivene of added anitizer. Low temperature, contant agitation caued by continuou pumping, and the organic content will alo complicate control of anitizer level in dairy coolant. The population of pychrotrophic bacteria in the National urvey ample ranged from / ml of coolant to >/ ml of coolant (Table ). There wa no difference between the pychrotrophic population found in the ummer or in the pring. The ame range in population of meophilic bacteria wa oberved in the MPN/ ml at 7 C. However, almot 5% of the ummer ample had TABLE. ummary ample. MPN/ ml value > Total of MPN reult for National urvey coolant Number of ample with MPN value C 7 C Coliform higher 7 C MPN value than thoe oberved in the pring. ix of the over ample analyzed in the National urvey exceeded the coliform tandard for potable water. The preence of added anitizer appeared to have little, if any, effect on the microbial population of the cooling media (Table 4). weet water ample 8 and 9 to which algicide had been added had very high and very low C MPN value, while the 7 C MPN value were imilar. The coliform population of ample 8 exceeded the limit etablihed in the PMO. Both the C and 7 C MPN value of untreated weet water ample ranged from very low level of recovery to recovery of > 6 organim/ ml of coolant. One weet water ample treated with iodine (ample 7) had very low level of all three type or organim. ample, which had alo been treated with iodine, had a C MPN of >68 organim/ ml of coolant. Glycol coolant with no added anitizer (ample 6) had exceively high level of pychrotrophic, meophilic, and coliform organim. Coliform were not recovered from two untreated glycol ample (ample 6 and ). The addition of anitizer did not appear to control the microflora of the cooling media. A poible explanation of the ineffectivene of the JOURNAL OF FOOD PROTECTION. VOL. 5, NOVEMBER 989

4 8 TRANTZ, ZOTTOLA, PETRAN, OVERDAHL, AND MITH TABLE 4. Comparion of the effect o, ample Coolant* anitizer* Iodine Iodine PH C > > 6 MPN/ ml 7 C.7.9 >. > Coliform Quat G G G > > > >.5.4 >. >. 8 9 Algicide Algicide Keego Anti-bact B * = weet water, G = glycol +Data provided on quetionnaire completed by plant peronnel antizer wa revealed in the repone to quetion on anitizer uage in the dairy plant. Information provided on the uage of anitizer indicated that few of the dairy plant carefully controlled anitizer level. Only four plant added chlorine in repone to teting of the coolant, by either a tandard plate count or determination of reidual chlorine level (Table 5). Generally, anitizer level were maintained by addition of et volume of liquid or dry agent to the coolant at interval that raged from daily to every -6 month. One plant ued chlorine a a anitizer while uing quaternary ammonium compound to adjut the ph of the coolant. The ame indicriminant ue of iodine and quaternary ammonium compound wa noted (Table 5). It i intereting to note that. typhimurium wa iolated from only one ample (Table 4, ample 8) uing the membrane filtration technique. wa added monthly to the contaminated weet water to maintain a level of <5 ppm. The preence of thi organim wa not detected by coliform analyi or by MPN analyi at 7 or C. The weet water had a population of coliform/ ml of coolant,_relatively low number of meophile and a high pychrotrophic population. The four major genera of bacteria iolated from both weet water and glycol were taphylococcu, Peudomona, Bacillu, and Enterohacter (Table 6). Other genera were identified with le frequency (Table 7). Of particular importance wa the iolation of. typhimurium from weet water. Other potential pathogen including Yerinia enterocolitica, Echerichia coli, Aeromona, hydrophila, Vibrio pecie, taphylococcu aureu and Klebiella pneumonia were alo iolated. L. monocytogene wa not iolated from any coolant ample analyzed. However, reearch invetigating the peritence of Literia i imulated milk cooling ytem howed that L. monocytogene wa able to grow at 4 C in dilute olution of nonfat dry milk (NFDM), peptone water, and trypticae oy broth (TB) (9). A improved method for the iolation of literia are developed, it i poible that thi organim may be iolated from dairy cooling ytem. It ha alo been reported that. typhimurium had the ability to urvive for extended period at low temperature (). The train of. typhimurium that wa iolated from an outbreak of almonelloi aociated with the conumption of pateurized milk urvived for more than one year at 7 C C in TB and in.% peptone water. The data obtained in the two urvey, and the iolation and identification of both potential pathogen and JOURNAL OF FOOD PROTECTION, VOL. 5, NOVEMBER 989

5 MICROBIOLOGY OF WEET WATER AND GLYCOL COOLING YTEM 8 TABLE 5. anitizer uage in National urvey coolant ample^. anitizer Amount ued gallon quart 5-7 ppm* 5 ppm ppm gallon <5 ppm < ppm ppm ppm lb - ppm 6 lb/ lb water Frequency of ue Every to 6 month if needed after teting time per year Monthly; quat** added to adjut ph When needed a per PC*** Monthly A needed every hour Automatically monitored Daily if PC > TABLE 8. Preure differential maintained in the HTT pateurizer cooling ection of 4 dairy plant. Coolant under lower preure Coolant under greater preure Coolant Milk Coolant Milk > Quat Iodine ppm Etimated ppm gallon gallon ppm gallon Unknown volume pint Monthly Monthly time per week time per week Every 4 day 'Information provided on quetionnaire completed by plant peronnel when coolant ample were collected. indicate part per million. **Indicate quaternary ammonium compound. ***Indicate tandard plate count. TABLE 6. Percent of coolant ample from which the mot frequently iolated organim were recovered. Genu % weet water ample % glycol ample taphylococcu Peudomona Bacillu Enterobacter TABLE 7. Organim iolated with low frequency from dairy coolant ample. Organim Glycol weet water Acinetobacter AeromonalVihrio Alcaligene Brochothrix Citrobacter Enterococcu Echerichia Flavobacterium Klebiella KurthialCaryopanon Micrococcu almonella erratia porolactobacillu Yerinia poilage organim, indicate that the cooling media ued in the HTT pateurizer in a fluid milk proceing plant could erve a a ource of undeirable microorganim in pateurized dairy product. uch contamination could occur if the material eparating the pateurized product from the cooling media i cracked or contain pin hole, and the preure differential permit flow from the cooling media into the milk. Four of 4 National urvey dairy plant that reponded to quetion on preure maintained in the cooling ection of the HTT unit did indicate that coolant were under preure greater than or equal to the preure of the milk (Table 8). To prevent contamination of pateurized product with cooling media, it i eential to maintain proper preure differential. Equipment and intrumentation i available to the indutry to monitor preure in HTT ytem. Cooling ytem that ue water/glycol mixture might encounter fewer problem with microbial contamination if the concentration of glycol in the ytem exceed %. Petran and Zottola (9) reported that L. monocytogene wa not able to grow in glycol coolant with.% added NFDM olid when the propylene glycol concentration wa % or greater. A decline in the population of. typhimurium wa oberved at - C in glycol concentration from to 4%. The higher the glycol concentration, the fater the decline in population (). Microorganim in weet water can be controlled by the addition of chlorine, iodine or quaternary ammonium anitizer. However, the addition mut be continuou and contantly monitored. Corroion of cooling ytem i likely when high level of halogen compound are added to coolant. Indicriminant addition of anitizer doe not enure microbial control. REFERENCE. Airoldi, A. A., and E. A. Zottola Growth and urvival of almonella typhimurium at low temperature. J. Food ci. 5:5-5.. Airoldi, A. A., and E. A. Zottola The urvival of almonella typhimurium in propylene glycol/water mixture. J. Food Prot. 5: Anonymou Grade A Pateurized Milk Ordinance (978 recommendation). Publication 9, U.. Dept. of Health, Education and Welfare, Wahington. DC. JOURNAL OF FOOD PROTECTION, VOL. 5, NOVEMBER 989

6 84 TRANTZ, ZOTTOLA, PETRAN, OVERDAHL, AND MITH 4. Ginn, R. E., W. Coleman, and V.. Packard weet water and glycol coolant a potential ource of dairy product contamination. Dairy Food anitation 5: Grave, R. R., R.. Leniewki, and D. E. Lake Bacterial quality of cannery cooling water. J. Food cience 4: Holt, J. G. (ed.) Bergy' Manual of ytematic Bacteriology, Volume I, II. William and Wilkin, Baltimore. 7. Lovett, J., D. W. France, and J. M. Hunt Literia monocytogene in raw milk: detection, incidence, and pathogenicity. J. Food Prot. 5: Odlaug, T. E., and I. J. Pflug poricidal propertie of chlorine compound', applicability to cooling water for canned food. J. Milk Food Technol. 9: Petran, R., and E. A. Zottola urvival of Literia monocytogene in imulated milk cooling ytem. J. Food Prot. 5: Zottola, E. A., and L. B. mith urvival of almonellae in refrigerated, agitated water and water/glycol mixture. J. Food Prot. 49: JOURNAL OF FOOD PROTECTION, VOL. 5, NOVEMBER 989