Survival of Listeria monocytogenes Inoculated in Retail Yogurt ...

World Academy of Science, Engineering and Technology 41 2010Survival of Listeria monocytogenes Inoculatedin Retail Yogurt ProductsYu-Hsien Hsieh, Jiang-Gong Liu, and J.C. HwangAbstract—Commercial products classified as low-fat yogurt (Y1),full-fat yogurt (Y2), AB-yogurt (Y3), and lactoferrin-fortified yogurt(Y4) using four Listeria monocytogenes as a cocktail inoculum. Thesamples were stored at 4–7℃ and 22℃. The ability of L.monocytogenes to survive at different storage temperatures has beenwidely explored since the temperature-related abuse of food productsoften occur food products are commonly not maintained at theappropriate temperature. Our results indicate that all four retail yogurtproducts studied herein supported the growth of L. monocytogenes atrefrigeration temperatures (4–7℃). AB-yogurt andlactoferrin-fortified yogurt, however, significantly inhibited thegrowth of L. monocytogenes in this temperature range. Changes in thepH, Brix, Baume, and total soluble solids (TSS) of retail yogurt andmilk products caused by changes in the number of lactic acid bacteria,effectively control L. monocytogenes growth at low temperatures.Results of this study significantly contribute to is very important inrisk assessment studies, which generally consider only the presence orabsence of the pathogen.Keywords—Listeria monocytogenes, Yogurt, Survival,Microbiologicial quality, RetailI. INTRODUCTIONOGURT is a popular soft drink item and food for breakfastYin oriental societies for centuries. The production ofyogurt is so quite easy that it is custom-made and sold in manybreakfast restaurants and stores in Taiwan. Listeriamonocytogenes can survive and grow in yogurt products at atemperature of 4 °C, thus the storage of yogurt inside a generalrefrigerator cannot successfully prevent the growth of Listeriamonocytogenes which is psychrotrophic. Moreover, many newyogurt-based products have been formulated to attract thepublic attention and consumption. The ingredients orcomponents-added utilized to improve or fortify the functionalproperties of yogurt products include lactoferrin-fortified,AB-yogurt, barley fiber-added, mixing with yam orpearl-barley. These improvements or innovations willabsolutely modify the physical and chemical properties (i.e.viscosity) of yogurt; however, how these changes or impacts onthe growth and survival of Listeria monocytogenes remainsunclear. Thus, this investigation aimed to determine theYu-Hsien Hsieh is with the Department of Chemical Engineering andMaterials Science, Yuan-Ze University, Nei-li, Taiwan, ROC.(e-mail:d9223009@hotmail.com)Jiang-Gong Liu is with the Department of Food Science, Yuanpei University,Hsin-Chu 300, Taiwan, ROC(e-mail: jg.liu@msa.hinet.net)Professor J-C Hwang is with the Department of Chemical Engineering andMaterials Science, Yuan-Ze University, Nei-li, Taiwan, ROC.(e-mail:d9223009@hotmail.com)survival of Listeria monocytogenes inoculated in retail yogurtproducts with fortified functional propertiesII. MATERIALS AND METHODS2.1 Organisms and preparation of inoculumA four-strain cocktail of Listeria monocytogenes consistingof (i) BCRC 14848 and 15334 (purchased from Food IndustryResearch and Development Institute, Hsin-Chu, Taiwan). (ii)Scott A (clinical isolate, serotype 4b) and V7 strains (milkisolate, serotype 1) obtained from the National Laboratory ofFoods and Drugs, Department of Health, Executive Yuan, ROC(Taipei, Taiwan) was used in this study.2.2 Fate of Listeria monocytogenes in yogurt samplesInoculation procedure: Portions (approximately 10g) ofsamples were aseptically weighted and placed individually inpolyethylene bags. Each bag was inoculated with 0.5 ml of theListeria monocytogenes cocktail suspension at high (c.a.3x10 8CFU/ ml) or low inoculum (c.a.3x10 4 CFU/ml). The inoculumwas thoroughly distributed by external hand massage for 1 min.These bags were sealed with several folds and incubated atroom (22℃) and refrigerated (4℃) temperatures, respectively.Then triplicate samples were periodically withdrawn formicrobiological and chemical analysis.III. RESULTS AND DISCUSSIONSIn Taiwan, promotions of many new yogurt and milkproducts emphasize the benefits of product fortification forconsumer health. Four yogurt products (drinks) with differentcompositions were studied, and product fortification andgrowth of L. monocytogenes in these commercial drinks werediscussed. These 4 products were low-fat yogurt (Y1), full-fatyogurt (Y2), AB-yogurt (Y3), and lactoferrin-fortified yogurt(Y4).Table 1 shows the physical and chemical analysis of theretail yogurt products used in this study. Depending on theadded ingredients, Y1 contains the highest levels ofoligosaccharides (58.9 mg/100 ml) and carbohydrates (16.2g/100 ml) and lowest levels of proteins (2.1 g/100 ml). The pHof all 4 products was 3.94. The physical and chemicalproperties of the products also vary due to the differentingredients. The effects of storage temperature on lactic acidbacterial counts in retail yogurt products at 22°C and 4–7°C areshown in Table 2. The growth of L. monocytogenes at 22°C ishigher than that at 4–7°C, indicating that refrigeration cancontrol the growth of the bacterium. Y4 had the lowest lacticacid bacterial counts at 22°C and 4–7°C among the 4 yogurtproducts; this may be due to the fortification with lactoferrin,which inhibits the growth of L. monocytogenes.305

World Academy of Science, Engineering and Technology 41 2010Tables 3 shows the results of a sample that received a higherinoculum, 3 × 108 CFU/ml and lower inoculum, 3 × 104CFU/ml. Following Bacterial counts increased with increasingculture time. Decrease in Listeria counts was more evident at22°C (-0.16~-7.1) than at 4–7°C (-0.8~-1.0). Y3 showed theleast decrease of all samples (22°C, -1.6; 4–7°C, -0.9). In Table3 (lower inoculum, 3 × 104 CFU/ml), the decrease at 22°C wasmore obvious (22°C, -0.8 ~-4.2; 4–7°C, -0.7~ -1.8). The impactof higher inoculum content on the decrease of bacterial countwas more evident at 22°C than at 4–7°C. Regardless of theamount of inoculum used, the Y3 sample showed the leastreduction in Listerial counts, suggesting that this product maybe more conducive to listerial growth than the other products.IV. CONCLUSIONSThe results for the 4 commercial yogurt products used in thisstudy confirm that food processors should not rely mainly onpasteurization and fermentation to ensure full protection andsafety of yogurt milk products. Some food-grade additives mayalso be used along with GMP. Lactoferrin proved to beeffective in controlling the growth of L. monocytogenes in bothAB-yogurt (Y3) and yogurt with lactoferrin ingredient (Y4).Moreover, addition of a fortifying ingredient to yogurt-milkproducts may play a beneficial role in keeping yogurt acidityfrom decreasing to very low levels during storage, thuspreventing the deterioration of its sensory attributes andextending its shelf life.This study clearly demonstrated that L. monocytogenes cangrow in all commercial yogurt-milk products, and moreimportantly, fortified yogurt products have some essentialnutrients that are good for human health. Milk products withfortifying ingredients need to be stored at suitable temperaturesto avoid spoilage and slight changes in pH, physical andchemical properties, lactic acid bacterial counts, and Brix andTSS values owing to L. monocytogenes contamination shouldbe noted regularly.TablesV. TABLESTABLE I PHYSICAL AND CHEMICAL ANALYSIS OF RETAIL YOGURT PRODUCTS INTHIS STUDYTable 1 Physical and chemical analysis of different yogurt products in this studyCalorie (Kcal/100mL) a78.6866479Protein (g/100mL) a 2.13.33.72.9Fat (g/100mL) a 0.62.502.4Carbohydrate (g/100mL) a 16.212.512.211.4Sodium (mg/100mL) a 46.357109.845Oligosacchorride (mg/100mL) a 58.9N.D.N.D.N.D.Lactoferrin (μ g/100mL) aN.D.N.D.N.D.1000Baume (%) b 15±011±012±1010±0°Brix (%) b 18±013±017.8±0.2914.9±0.1pH b 3.94±0.014.36±0.044.16±0.024.27±0.01Viscosity(cps) b 26.3±1.01575±2518.3±1.077.8±8.5Lactics strains inoculated a unknownBifidobacterium longumBifidobacteriumunknownLactobacillus acidophilusLactobacillus acidophilusLactics No.(CFU)/ml aY110 8 /mla:data obtained from the product content label10 7 /mlb:results expressed as a mean of three replicates for each analysisY2Streptococcus lactis10 8 /mlY3Y410 8 /ml*Low-fat yogurt (Y1), Full-fat yogurt (Y2), AB-yogurt (Y3), Lactoferrin fortified ingredientsyogurt (Y4)TABLE II EFFECT OF STORAGE TEMPERATURE ON LACTIC ACID BACTERIALCOUNTS IN RETAIL YOGURT PRODUCTS AT 22 AND 4-7℃Storage (day)0123456Y18.6±0.4 b8.7±0.1 c8.9±0.1 b8.7±0.4 b8.8±0.2 b8.9±0.2 b8.2±0.2 c22℃Y29.5±0.1 a9.7±0.2 a9.7±0.1 a10.2±0.4 a9.5±0.1 a9.5±0.1 a9.6±0.0 aY37.8±0.3 c8.9±0.1 b8.8±0.3 b7.6±0.2 c7.1±0.2 c7.3±0.2 c8.7±0.2 b*The same letter is non-significant at rowsY47.7±0.1 c6.7±0.1 d6.8±0.0 c7.1±0.0 d7.2±0.1 c7.2±0.0 c7.4±0.1 dY17.8±0.2 c8.3±0.4 b8.0±0.2 b9.4±0.2 a8.1±0.1 c8.2±0.1 b8.8±0.2 bY29.5±0.1 a9.3±0.3 a9.1±0.1 a9.1±0.1 b9.1±0.1 a9.1±0.1 a9.8±0.2 a4- 7℃Y38.2±0.2 b8.7±0.1 b7.6±0.2 c7.4±0.2 c8.7±0.2 b8.5±0.4 b8.9±0.1 bTABLE III EFFECT OF STORAGE TEMPERATURE ON GROWTH OF L.monocytogenes CULTURES INOCULATED IN RETAIL YOGURT PRODUCTSStorage period (day)0Y14.2±0.1 a22℃Y23.8±0.2 cY34.3±0.1 aY44.0±0.0 bY14.2±0.1 a4- 7℃Y2Y33.8±0.2 c4.3±0.1 aY47.7±0.1 c6.8±0.1 c6.7±0.1 d6.6±0.1 d6.6±0.1 d6.8±0.2 c6.8±0.1 cY44.0±0.0 b13.4±0.0 b1.9±0.1 d4.0±0.1 a2.7±0.0 c4.0±0.1 a3.5±0.1 b3.8±0.1 a3.3±0.2 cREFERENCES[1] Hsieh, Y. H., Liu, J. G., Hung, S. C. & Hwang, J. C. (2010). The ability ofListeria spp. to survive in retail milk with fortified ingredients.International Journal of Food Microbiology, (Under review)[2] Liu, J. G., & Lin, T. S. (2008). Survival of Listeria monocytogenesinoculated in retail soymilk products. Food control, 19, 862-869.[3] Liu, J. G., Lin, T. S., & Lin, W. N. (2010). Evaluating the growth ofListeria monocytogenes that has been inoculated into tofu containingbackground microflora Food control, (Inpress).[4] Jenssen, H. & Hancock, R. E. W. (2009). Antimicrobial properties oflactoferrin. Biochimie, 519 91, 19-29.[5] Rocourt, J. (1996). Risk factors for listeriosis. Food Control, 7, 195-202.[6] Hsieh, Y. H., Liu, J. G., Y. M. & Hwang, J. C. (2010). Survival of Listeriamonocytogenes inoculated in retail yogurt roductions at 22°C and 4-7°Cin Taiwan. International Journal of Food Microbiology, (Under review)234562.0±0.3 b0.0±0.0 d0.0±0.0 c0.0±0.0 c0.0±0.0 c1.9±0.2 b1.0±0.3 c0.0±0.0 c0.0±0.0 c0.0±0.0 c3.9±0.1 a3.9±0.1 a3.4±0.1 a3.6±0.1 a3.5±0.1 a1.8±0.2 b1.8±0.1 b1.6±0.1 b1.0±0.3 b1.9±0.1 b4.1±0.1 a3.9±0.1 a3.5±0.2 b3.5±0.3 b3.3±0.2 b3.5±0.0 c2.4±0.1 b2.3±0.2 c2.5±0.2 c2.4±0.1 c3.8±0.2 b4.0±0.1 a3.8±0.1 a3.4±0.1 a3.6±0.2 aTABLE IV.EFFECT OF STORAGE TEMPERATURE ON GROWTH OF LISTERIALCOUNTS INOCULATED ( HIGHER INOCULUM, 3X10 8 CFU/ML) IN YOGURTPRODUCTS AT 22 AND 4-7℃Storage period (day)0123Y17.1±0.0 a5.4±0.0 c5.0±0.0 c3.2±0.2 c22℃Y26.9±0.1 b4.4±0.1 d2.8±0.2 d0.0±0.0 dY37.2±0.1 a6.3±0.1 a6.4±0.0 a6.6±0.3 aY47.1±0.1 a6.0±0.3 b5.8±0.0 b5.2±0.1 bY17.1±0.0 a7.1±0.1 a7.0±0.1 a7.1±0.1 aY26.9±0.1 b6.0±0.1 c6.5±0.2 b6.5±0.1 c4- 7℃Y37.2±0.1 a7.1±0.1 a7.1±0.0 a6.9±0.1 b2.6±0.1 d2.1±0.1 c2.1±0.0 c2.0±0.2 c2.2±0.1 cY47.1±0.1 a6.5±0.2 b6.3±0.1 c6.2±0.0 d42.1±0.0 c0.0±0.0 d6.4±0.1 a5.1±0.1 b6.7±0.1 a6.1±0.1 c6.4±0.0 b6.0±0.1 c50.0±0.0 c0.0±0.0 c6.3±0.1 a4.2±0.2 b6.4±0.1 a6.1±0.0 b6.1±0.2 b6.0±0.0 b60.0±0.0 c0.0±0.0 c5.6±0.1 a3.3±0.2 b6.3±0.1 a6.1±0.0 b6.3±0.1 a6.1±0.0 b306