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Evaluation of the factors affecting the activity of sakacin C2 against E. coli in milk
Food Control 30 (2013) 453e458
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Evaluation of the factors affecting the activity of sakacin C2 against E. coli in milk Yurong Gao*, Dapeng Li, Xiaoyan Liu Food College of Heilongjiang Bayi Agricultural University, High and New Tech Development Zone, Daqing 163319, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2012 Received in revised form 11 July 2012 Accepted 12 July 2012
Sakacin C2 is a novel, broad spectrum bacteriocin secreted by Lactobacillus sake C2 isolated from Chinese traditional fermented cabbage. Effects of milk fat, emulsifiers, preservatives and homogenization on the activity of sakacin C2 against Escherichia coli ATCC 25922 in milk were evaluated by determined the changes of viable cell counts of E. coli ATCC 25922 after inoculation (105 CFU/ml) then storage at 4 C. Milk fat in pasteurized and homogenized milk products (low fat milk and whole milk) decreased the activity of sakacin C2 against E. coli ATCC 25922. Addition of 5 ml/ml of Tween 80 decreased the effect of sakacin C2 against E. coli ATCC 25922, but 5 mg/ml of lecithin increased the effect against E. coli ATCC 25922 in pasteurized and homogenized whole milk. Nisin and ε-Polylysine significantly increased the effect of sakacin C2 against E. coli ATCC 25922 in pasteurized and homogenized whole milk. Homogenization interfered with the activity of sakacin C2 against E. coli ATCC 25922 in pasteurized skim milk and whole milk. This study might lay the groundwork for the application of sakacin C2 as bio-preservative in milk and dairy products. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Sakacin C2 Bacteriocin E. coli Antimicrobial activity
1. Introduction Escherichia coli is a Gram-negative, rod-shaped bacterium which is one of the main organisms in the mammalian intestinal tract (Erichson & Doyle, 2007). There have been many reports about the discovery of E. coli-contaminated milk and milk products available to the consumer (Adesiyun, 1994; Hahn, 1996, Chapter 4; Soomro, Arain, Khaskheli, & Bhutto, 2002). E. coli is widely accepted as an indicator of fecal contamination and is generally found in unsanitary conditions of foods such as milk and other dairy products (Soomro et al., 2002). E. coli strains cannot only cause food decomposition, some serotypes such as E. coli O157:H7 can also cause food poisoning and gastrointestinal disease originated from animal origin foods including milk and milk products (Vogt & Dippold, 2005). Bacteriocins are antimicrobial peptides produced by bacteria and bacteriocins from lactic acid bacteria (LAB) are generally recognized as safe (GRAS) biopreservatives (Guiname, Cotter, Hill, & Ross, 2005). The use of bacteriocins produced by LAB in the preservation of foods including milk and milk products has now become a promising technology (Arauz, Jozala, Mazzola, & Penna, 2009). Sakacin C2 is a novel bacteriocin with a broad inhibitory spectrum secreted by Lactobacillus sake C2 isolated from Chinese traditional fermented cabbage (Gao, Jia, Gao, & Tan, 2010). Sakacin * Corresponding author. Tel.: þ86 459 6818360; fax: þ86 459 6819235. E-mail address: [email protected] (Y. Gao). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.07.013
C2 displayed the activity against some foodborne spoilage and pathogenic bacteria including not only Gram-positive bacteria such as Staphylococcus aureus, Listeria innocua, Streptococcus thermophilus and Bacillus cereus, but also Gram-negative bacteria such as E. coli and Salmonella typhimurium. Moreover, sakacin C2 showed strong tolerance to heat (even at 121 C for 15 min) and low pH (pH 3.0e8.0) (Gao et al., 2010). Gao, Li, Sheng, and Liu (2011) also studied the mode of action of sakacin C2 against E. coli ATCC 25922 and suggested that sakacin C2 depolarized the trans-membrane electrical potential, inserted the cell membrane and disintegrated the cell ultimately. These characteristics strongly suggested its good potential to be used as bio-preservative in food industry. The bacteriocin activity in food matrices may be affected by several factors such as chemical composition, the use of food additives and physical conditions of food (Cleveland, Montville, Nes, & Chikindas, 2001). In milk system, the antimicrobial activities of several bacteriocins against Gram-positive bacteria have been studied (Bizani, Morrissy, Dominguez, & Brandelli, 2008; Jones, 1974; Silva Malheiros, Sant’Anna, Utpott, & Brandelli, 2012; Zapico, de Paz, Medina, & Nunez, 1999). Alpas and Bozoglu (2000) reported that the bacteriocins combination with pressure and heat inhibited S. aureus, Listeria monocytogenes, E. coli and Salmonella in milk. Silva Malheiros, Sant’Anna, Utpott, et al. (2012) reported that the antilisterial activity of nanovesicle-encapsulated antimicrobial peptide P34 in milk. Bizani et al. (2008) reported that the antimicrobial peptide cerein 8A (160 AU/ml) in UHT milk resulted in a decrease of 3 log cycles in viable cells of
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L. monocytogenes within the 14-day period at 4 C. Lauková and Czikkova (1999) reported that enterocin CCM 4231 inhibited the growth of L. monocytogenes and S. aureus in soy milk. Silva Malheiros, Sant’Anna, Souza Barbosab, Brandelli, and Melo Francob (2012) reported that liposome-encapsulated nisin and bacteriocin-like substance (BLS) P34 effectively controlled the growth of L. monocytogenes in Minas frescal cheese. Bacteriocins produced by LAB generally have inhibitory activity against Grampositive bacteria. Sakacin C2 has inhibitory activity against not only many Gram-positive bacteria but also many Gram-negative bacteria (Gao et al., 2010). Bacteriocins produced by LAB generally have inhibitory activity against Gram-positive bacteria. Sakacin C2 has inhibitory activity against not only many Gram-positive bacteria but also many Gram-negative bacteria (Gao et al., 2010). This activity against many Gram-negative bacteria was not frequently discovered in bacteriocins from LAB. There have been few researches reported on effects of chemical composition and processing on the activity of bacteriocin against Gram-negative bacteria in milk products. The objective of the present study was to evaluate the effects of chemical composition including milk fat, the addition of emulsifiers and preservatives, and homogenization on activity of sakacin C2 against E. coli in milk. 2. Materials and methods
320 AU/ml. Cells of E. coli ATCC 25922 were added to these samples and the initial counts were 105 CFU/ml. Cell counts of samples were determined at the start of the experiment and at 2 d intervals until 12 d of storage at 4 C. 2.5. Effects of emulsifiers on the activity of sakacin C2 against E. coli Effects of emulsifiers on the activity of sakacin C2 against E. coli were determined by supplementing with 5 mg/ml lecithin from soy (Sigma, Hong Kong, China) or 5 ml/ml Tween 80 (Sigma) to the pasteurized and homogenized whole milk followed by the addition of sakacin C2 (480 AU/ml). The sample not supplemented with emulsifiers was used as control. All samples were inoculated with E. coli ATCC 25922 (105 CFU/ml) and cell counts were determined during storage. 2.6. Effects of preservatives on the activity of sakacin C2 against E. coli Effects of preservatives on the activity of sakacin C2 against E. coli were determined by supplementing with 5 mg/l nisin (NisinZÒM, Handary SA, Brussels, Belgium) or 50 mg/l ε-polylysine (EpolylyÒ DX, Handary SA, Brussels, Belgium) to the pasteurized and homogenized whole milk followed by the addition of sakacin C2 (320 AU/ml). The sample not supplemented with preservatives was used as control. All samples were inoculated with E. coli ATCC 25922 (105 CFU/ml) and cell counts were determined during storage.
2.1. Bacteria, culture and inoculation E. coli ATCC 25922 was used as indicator strain. The strain was purchased from China General Microbiological Culture Collection Center (CGMCC, Beijing, China) and stored in nutrient broth with 20% (v/v) glycerol at 20 C. Before E. coli ATCC 25922 was inoculated to milk samples, the cells were cultivated in nutrient broth at 37 C and 100 rpm for 12 h. L. sake C2 producing sakacin C2 was stored in MRS broth with 20% (v/v) glycerol at 20 C (Gao et al., 2010). Before used for the partial purification of the bacteriocin, cultures were cultivated without agitation in MRS broth (Qingdao Hope Bio. Technology Co., Ltd, China) at 30 C for 18 h and were then incubated without agitation in the same medium at 30 C for 24 h. 2.2. Preparation of sakacin C2 Sakacin C2 was prepared from the cell-free supernatant of L. sake C2 culture. The culture was partially purified by cold ethanol and column chromatography (Gao et al., 2010). Bacteriocin activity (AU/ml) was determined by the method of agar diffusion using S. aureus ATCC 63589 as indicator described by Xiraphi et al. (2006). 2.3. Milk samples Pasteurized and homogenized skim milk (0% milk fat), low fat milk (1.3% milk fat) and whole milk (3.6% milk fat) were purchased from a local market and produced by Inner Mongolia Yili Industrial Group Co., Ltd (China). Samples of the pasteurized and homogenized or not homogenized skim milk (0% milk fat) and whole milk (3.6% milk fat) were obtained from Daqing Dairy (Daqing, China). The samples of raw whole milk (3.6% milk fat) were obtained from Daqing Dairy, and the pasteurized not homogenized samples were prepared at 80 C for 15 s in this factory.
2.7. Effects of homogenization and heat treatment on the activity of sakacin C2 against E. coli Effects of homogenization on the activity of sakacin C2 against E. coli were determined by adding sakacin C2 (240 AU/ml) and E. coli ATCC 25922 (105 CFU/ml) to the pasteurized and homogenized (200 bar) skim milk (0% milk fat) and whole milk (3.6% milk fat). Samples not supplemented with sakacin C2 were used as control. The survival of E. coli ATCC 25922 after treatments was determined. Effects of heat treatment on the activity of sakacin C2 against E. coli were determined by adding sakacin C2 (240 AU/ml) and E. coli ATCC 25922 (105 CFU/ml) to the raw whole milk (3.6% milk fat) and the pasteurized not homogenized raw whole milk. Sample of raw whole milk not supplemented with sakacin C2 was used as control. The survival of E. coli ATCC 25922 after treatments was determined. 2.8. Bacteria counting E. coli Chromogenic medium (Qingdao Hope Bio. Technology Co., Ltd, China) was used for the enumeration of E. coli ATCC 25922. The liquor samples (0.1 ml) of the undiluted or diluted samples using normal saline (0.85% (m/v) NaCl) were spread directly on the surface of prepoured plates and then were incubated at 30 C for 48 h. 2.9. Data analysis All experiments were repeated three times and in duplicate. Microsoft Excel (version 2003) was used to analyze the data. Statistical analysis was analyzed with the software SAS 8.1 (SAS Institute Inc., Cary, NC, USA). Differences were considered significant at P < 0.05. 3. Results and discussion
2.4. Effects of milk fat on the activity of sakacin C2 against E. coli 3.1. Effects of milk fat on the activity of sakacin C2 against E. coli Sakacin C2 preparation was added to 50 ml of the pasteurized and homogenized skim milk (0% milk fat), low fat milk (1.3% milk fat) and whole milk (3.6% milk fat) to the final concentration of
Effects of sakacin C2 (320 AU/ml) on the growth of E. coli ATCC 25922 in the pasteurized and homogenized skim milk, low fat milk
Y. Gao et al. / Food Control 30 (2013) 453e458
and whole milk during storage at 4 C are shown in Fig. 1. The cell counts of E. coli ATCC 25922 in the pasteurized and homogenized skim milk, low fat milk and whole milk not supplemented with sakacin C2 all increased from 5 to about 7 log CFU/ml after 12 d of storage at 4 C. The mode of action of sakacin C2 against E. coli ATCC 25922 is considered as bactericidal action (Gao et al., 2011). Adding 320 AU/ml of sakacin C2 to the pasteurized and homogenized skim milk, the cell number of E. coli ATCC 25922 decreased to below detectable levels (<10 CFU/ml) after 4 d and maintained until to 12 d (Fig. 1). Adding 320 AU/ml of sakacin C2 to the pasteurized and homogenized low fat milk, the cell number of E. coli ATCC 25922 decreased to 1.92 log CFU/ml after 4 d and below 3 log CFU/ml until to 12 d (Fig. 1). But in the pasteurized and homogenized whole milk, the counts of E. coli ATCC 25922 decreased to 3.05 log CFU/ml after 4 d and then recovered growth to 5.79 log CFU/ml after 12 d of storage at 4 C (Fig. 1). These results suggested that the maximum activity against E. coli presented in the pasteurized and homogenized skim milk (0% milk fat), but reduced activity presented in the milk with 1.3% and 3.6% fat. These results in this study were similar to those reported by Jung, Bodyfelt, and Daeschel (1992) and Chollet, Sebti, Martial-Gros, and Degraeve (2008). Jung et al. (1992) observed that the antilisterial activity of nisin decreased by 33% in skim milk, by 50% in milk with 1.2% fat and by 88% in milk with 12.9% fat. Chollet et al. (2008) reported that the activity of nisin against Kocuria rhizophila significantly decreased when the fat in gel attained 30% (w/w). Bacteriocins react to the cell membrane of sensitive organism. Bacteriocins firstly bind to the charged groups of phospholipids of cell membrane, then insert the membrane and leading to the depolarization of trans-membrane electrical potential, efflux of materials (Gao et al., 2011; Moll, Roberts, Konings, & Driessen, 1996). These negative effects of fat on the activity of bacteriocins may be caused by bacteriocins adsorption onto fat globules, as a result, the amount of bacteriocins acting with the cell membrane of target organisms reduced. Therefore, in this study the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized low fat milk (1.3% fat) and whole milk (3.6%) significantly decreased compared to that in the pasteurized and homogenized skim milk. 3.2. Effects of emulsifiers on the activity of sakacin C2 against E. coli Effects of emulsifiers (Tween 80 and lecithin) on the activity of sakacin C2 (480 AU/ml) against E. coli ATCC 25922 in the pasteurized and homogenized whole milk were summarized in Table 1. In the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in milk sample supplementing with lecithin (5 mg/ml) were 3.51 and 6.76 log CFU/ml, and the cell counts were 2.61 and 3.93 log CFU/ml
6
Log CFU/ml
5 4 3 2 1 0 0
2
4
6
8
10
12
Time (d) Fig. 1. Effects of sakacin C2 on the growth of E. coli ATCC 25922 in the pasteurized and homogenized skim milk (,), low fat milk (>) and whole milk (6) during the 12 d of storage at 4 C.
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Table 1 Effects of emulsifiers on the activity of sakacin C2 against E. coli in the pasteurized and homogenized whole milk. Treatment
Whole Whole Whole Whole
milk milk milk milk
Cell counts at 4 C for 2 d (log CFU/ml) (control)a þ sakacin C2 þ sakacin C2 þ Tween 80 þ sakacin C2 þ lecithin
5.54 2.61 2.08 3.51
0.31 0.17 0.14 0.26
Cell counts at 4 C for 12 d (log CFU/ml) 7.52 3.93 2.12 6.76
0.31 0.26 0.35 0.29
a Initial cell counts of E. coli ATCC 25922 in pasteurized and homogenized whole milk were 4.96 log CFU/ml.
in those without lecithin after stored for 2 and 12 d (Table 1). Therefore, lecithin decreased the effect against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. In the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in milk sample supplementing with Tween 80 were 2.08 and 2.12 log CFU/ml, and the cell counts were 2.61 and 3.93 log CFU/ml in those without Tween 80 after stored for 2 and 12 d (Table 1). Therefore, the addition of Tween 80 (5 ml/ml) increased the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. Lecithin is an anionic lipophilic emulsifier and is widely used in human food, animal feed, and pharmaceuticals. Moreover, it has been proved that soy-derived lecichin has significant function on lowering cholesterol (Iwata, Kimura, Tsutsumi, Furukawa, & Kimura, 1993). The result of Henning, Metz, and Hammes (1986) showed that lecithin helped to form stable complex between nisin and zwitterionic phospholipids. The data shown in Table 1 was in agreement with this theory. It could be concluded that the formation of stable complex between sakacin C2 and zwitterionic phospholipids of lecithin decreased the effect of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. Tween 80 is a nonionic emulsifier extensively used in foods, particularly in milk products such as ice cream. Adding Tween 80 to milk products prevents proteins from coating the fat globule (Goff, 1997). Goff, Liboff, Jordan, and Kinsella (1987) reported that Tween 80 decreased the amount of protein bound to milk fat droplets observed by electron microscopy. In our study, the milk fat significantly decreased the effect of sakacin C2 against E. coli in milk. Therefore, it could be concluded that Tween 80 can also decrease the bacteriocin absorbed by milk fat and thereby increased the effect of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. 3.3. Effects of preservatives on the activity of sakacin C2 against E. coli Effects of nisin on the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk were summarized in Fig. 2. In the pasteurized and homogenized whole milk, the supplement of nisin (5 mg/l) displayed no significant effect on the survival of E. coli ATCC 25922 during the 12 d of storage at 4 C (p > 0.05) (Fig. 2). But, the cell counts of E. coli ATCC 25922 in the presence of both nisin and sakacin C2 were 3.56 log CFU/ml less than that in the presence of sakacin C2 alone after stored at 4 C for 12 d. These results strongly suggested that nisin significantly increased the effect of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk (Fig. 2). As a polycyclic antibacterial peptide against Gram-positive spoilage and pathogenic bacteria, nisin is extensively used in processed cheese, meats, beverages, etc. However, nisin cannot inhibit Gram-negative bacteria such as E. coli and S. typhimurium (SobrinoLopez & Martin-Belloso, 2006). The outer membrane of Gram-
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8 7 Log CFU/ml
6 5 4 3 2 1 0 0
2
4
6
8
10
12
Time (d) Fig. 2. Effects of nisin on the activity of sakacin C2 against E. coli in the pasteurized and homogenized whole milk. The cell counts of E. coli ATCC 25922 in the pasteurized and homogenized whole milk (), whole milk in the presence of nisin (B), whole milk in the presence of sakacin C2 (,), whole milk in the presence of both nisin and sakacin C2 (6) during the 12 d of storage at 4 C.
negative bacteria is a complex barrier system for many antimicrobial agents such as bacteriocins, which normally have a deleterious effect on the inner membrane (Alakomi et al., 2000). Sakacin C2 is a bacteriocin with activity against not only Gram-positive bacteria but also Gram-negative bacteria. Synergistic effect of sakacin C2 in combination with nisin against E. coli ATCC 25922 may be the result of sakacin C2 altering the out membrane, rendering the outer membrane permeable, thereby nisin being able to act on the inner membrane. Synergistic effects of nisin in combination with pediocin AcH and curvaticin 13 against Gram-positive bacteria have been reported (Bouttefroy & Millière, 2000; Hanlin, Kalchayand, Ray, & Ray, 1993). However, synergistic action of nisin and other bacteriocins from LAB against Gram-negative bacteria have not been reported. These results may imply that the combined use of two antimicrobial agents may be advantageous to produce a synergistic effect what make the individual antimicrobial agent effective at low concentration even when indictor strain is sensitive to one antimicrobial agent but not sensitive to another. Effects of ε-Polylysine on the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk are summarized in Fig. 3. Adding 50 mg/l of ε-Polylysine in the 8 7
Log CFU/ml
6
pasteurized and homogenized whole milk had not significant inhibitory effect on E. coli ATCC 25922 during the 12 d of storage at 4 C (p > 0.05) (Fig. 3). The cell counts of E. coli ATCC 25922 in the presence of sakacin C2 were 5.99 log CFU/ml after storage at 4 C for 12 d, and that in the presence of both sakain C2 and ε-Polylysine rapidly decreased to below detectable level (<10 CFU/ml) after stored at 4 C for 6 d (Fig. 3). These data showed the significantly synergistic effects of sakacin C2 in combination with ε-Polylysine against E. coli ATCC 25922 in the pasteurized and homogenized whole milk (Fig. 2). ε-Polylysine is natural antimicrobial agent produced by Streptomyces albulus which have activity against a wide range of microorganisms including bacteria (both Gram-positive and Gram-negative bacteria), yeast and mould (Yoshida & Nagasawa, 2003). ε-Polylysine is generally recognized as safe (GRAS) preservative used in a variety of foods including milk and dairy products. Moreover, ε-Polylysine has strong stability including pH (4.0e10.0) and heat (120 C for 20 min) (Neda, Sakurai, Takahashi, Ashiuchi, & Ohgushi, 1999). Sakacin C2 and ε-Polylysine exerted their activity against E. coli in different ways. The mode of action of ε-Polylysine was believed to firstly absorb onto the cellular membrane of bacterial cells, strip off the membrane and result in the abnormal distribution of the protoplasm (Shima, Matsuoka, Iwamoto, & Sakai, 1984). Sakacin C2 is believed to firstly depolarize the trans-membrane electrical potential, make the surface of cells wrinkle and disintegrate the cell ultimately (Gao et al., 2011). The synergistic effects of different antimicrobial agents may be achieved by the different mechanisms of action or sites of activity (Badaoui Najjar, Kashtanov, & Chikindas, 2007). The synergistic effects of sakacin C2 in combination with ε-Polylysine against E. coli ATCC 25922 in this study is also in accordance with this hypothesis. The multiple hurdle approach was extensively used to achieve optimal antimicrobial activity and decreased the amount of antimicrobial agents used in foods (Leistner, 2000). 3.4. Effects of homogenization and heat treatment on the activity of sakacin C2 against E. coli in milk Effects of homogenization on the activity of sakacin C2 against E. coli in the pasteurized skim milk and whole milk is summarized in Table 2. In the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in the pasteurized and homogenized skim milk were 2.56 and 4.18 log CFU/ml, and those in the pasteurized but unhomogenized skim milk were 1.92 and 2.83 log CFU/ml after storage at 4 C for 2 d and 12 d (Table 2). Similarly, in the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in the pasteurized and homogenized whole milk were 4.08 and 6.87 log CFU/ml, and Table 2 Effects of homogenization and heat treatment on the activity of sakacin C2 against E. coli in milk.
5 4
Treatment
3 2 1 0 0
2
4
6
8
10
12
Time (d) Fig. 3. Effects of ε-polylysine on the activity of sakacin C2 against E. coli in the pasteurized and homogenized whole milk. The cell counts of E. coli ATCC 25922 in the whole milk (), whole milk in the presence of ε-polylysine (B), whole milk in the presence of sakacin C2 (,), whole milk in the presence of both ε-polylysine and sakacin C2 (6) during the 12 d of storage at 4 C.
Homogenization Control Unhomogenized skim milk þ sakacin C2 Homogenized skim milk þ sakacin C2 Unhomogenized whole milk þ sakacin C2 Homogenized whole milk þ sakacin C2 Heat treatment Control Raw whole milk þ sakacin C2 Pasteurized not homogenized raw whole milk þ sakacin C2
Cell counts at 4 C for Cell counts at 4 C for 2 d (log CFU/ml) 12 d (log CFU/ml) 5.34 0.27 1.92 0.14
7.66 0.31 2.83 0.12
2.56 0.12
4.18 0.11
2.38 0.18
3.96 0.22
4.08 0.16
6.87 0.34
5.68 0.21 2.66 0.16 3.64 0.20
7.82 0.36 3.41 0.18 4.88 0.23
Y. Gao et al. / Food Control 30 (2013) 453e458
those in the pasteurized but unhomogenized whole milk were 2.38 and 3.96 log CFU/ml after storage at 4 C for 2 d and 12 d (Table 2). These results strongly suggested that homogenization of milk caused the loss in the activity of sakacin C2 against E. coli. Homogenization of milk caused the changes of fat globules and milk proteins (Fox & McSweeney, 1998, Chapter 3). After homogenization, the average diameter of fat globules decreased and their number and surface area increased, and the distribution of milk proteins changed (Fox & McSweeney, 1998, Chapter 3). Thus, the amount of bacteriocin adsorption onto fat globules and milk proteins was increased and thereby reduced the bacteriocin to act with target organisms. Lakamraju, McGuire, and Daeschel (1996) also reported that bacteriocin absorption to certain surfaces may interfere with the inhibitory activity of nisin. The results in our paper exhibited that homogenized interfere with the antimicrobial activity of sakacin C2 against E. coli in the pasteurized skim milk and whole milk. Effects of heat treatment on the activity of sakacin C2 against E. coli in raw whole milk were summarized in Table 2. Addition of sakacin C2 in raw whole milk and pasteurized not homogenized raw whole milk significantly decreased the viable cell count of E. coli compared with that in the samples without sakacin C2 (P < 0.05). Moreover, in presence of sakacin C2, the viable cell counts in pasteurized not homogenized raw whole milk were significantly more than raw whole milk (P < 0.05). From these results, it can be concluded that pasteurized decreased the inhibitory activity of sakacin C2 in whole milk. Raikos, Kapolos, Farmakis, Koliadima, and Karaiskakis (2009) reported that the mean particle diameter of fat droplets increased after heat treatment in full-fat and semi-fat milk. Therefore, the superficial area of fat droplets increased after heat treatment. After addition of bacteriocins, the amount of bacteriocins adsorption on fat globule increased, as a result, the activity of sakacin C2 against E. coli reduced after whole milk pasteurized. Sakacin C2 was a novel bacteriocin with a broad inhibitory spectrum secreted by L. sake C2 isolated from Chinese traditional fermented cabbage. Sakacin C2 exhibited a broad antimicrobial activity. It could significantly inhibit many gram-positive bacteria including Listeria innocua and S. aureus, and moderately against some gram-negative bacteria such as E. coli and S. typhimurium in nutrient broth (Gao et al., 2010). From this result, it may be concluded that sakacin C2 may have stronger inhibitory to the gram-positive pathogenic bacteria such as Listeria spp. and S. aureus than gram-negative pathogenic bacteria such as E. coli. The activity against Gram-negative bacteria of sakacin C2 was not frequently seen in the bacteriocins from LAB. The inhibitory of bacteriocins from LAB against Gram-positive pathogenic bacteria such as Listeria spp. and S. aureus in dairy foods have been extensively reported. Therefore, the present study was focus on evaluating the use of sakacin C2 to control the growth of E. coli in milk. 4. Conclusion It can be first concluded that sakacin C2 have stronger inhibitory activity against E. coli in milk from this study. Moreover, the results presented in this study also strongly suggested that the activity of sakacin C2 against E. coli in milk was dependent on the chemical composition and the additives supplemented in the foods, and the treatment. Therefore, sakacin C2 may be have good application prospect for inhibiting the growth of E. coli in milk and dairy products. Acknowledgments This study was funded by Young Science Mainstay Subject of the Education Department of Heilongjiang Province (1155G38).
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References Adesiyun, A. A. (1994). Bacteriological quality and associated public health risk of pre-processed bovine milk in Trinidad. International Journal of Food Microbiology, 21(3), 253e261. Alakomi, H. L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., & Helander, I. M. (2000). Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Applied Environmental Microbiology, 66(5), 2001e2005. Alpas, H., & Bozoglu, F. (2000). The combined effect of high hydrostatic pressure, heat and bacteriocins on inactivation of foodborne pathogens in milk and orange juice. World Journal of Microbiology and Biotechnology, 16, 387e392. Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science and Technology, 20(3e4), 146e154. Badaoui Najjar, M., Kashtanov, D., & Chikindas, M. L. (2007). Epsilon-poly-L-lysine and nisin A act synergistically against gram-positive food-borne pathogens Bacillus cereus and Listeria monocytogenes. Letters in Applied Microbiology, 45(1), 13e18. Bizani, D., Morrissy, J. A. C., Dominguez, A. P. M., & Brandelli, A. (2008). Inhibition of Listeria monocytogenes in dairy products using the bacteriocin-like peptide cerein 8A. International Journal of Food Microbiology, 121(2), 229e233. Bouttefroy, A., & Milliére, J. B. (2000). Nisin-curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313. International Journal of Food Microbiology, 62(1e2), 65e75. Chollet, E., Sebti, I., Martial-Gros, A., & Degraeve, P. (2008). Nisin preliminary study as a potential preservative for sliced ripened cheese: NaCl, fat and enzymes influence on nisin concentration and its antimicrobial activity. Food Control, 19(10), 982e989. Cleveland, J., Montville, T. J., Nes, I. F., & Chikindas, M. L. (2001). Bacteriocins: safe, natural antimicrobials for food preservation. International Journal of Food Microbiology, 71(1), 1e20. Erichson, M. C., & Doyle, M. P. (2007). Food as a vehicle for transmission of Shiga toxin-producing Escherichia coli. Journal of Food Protection, 70(10), 2426e2449. Fox, P. F., & McSweeney, P. L. H. (1998). Dairy chemistry and biochemistry (1st ed.). London: Thomson Science. Gao, Y., Jia, S., Gao, Q., & Tan, Z. (2010). A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage. Food Control, 21(1), 76e81. Gao, Y., Li, D., Sheng, Y., & Liu, X. (2011). Mode of action of sakacin C2 against Escherichia coli. Food Control, 22(5), 657e661. Goff, H. D. (1997). Colloidal aspects of ice cream-a review. International Dairy Journal, 7(6), 363e373. Goff, H. D., Liboff, M., Jordan, W. K., & Kinsella, J. E. (1987). The effects of polysorbate 80 on the fat emulsion in ice cream mix: evidence from transmission electron microscopy studies. Food Microstructure, 6(2), 193e198. Guiname, C. M., Cotter, P. D., Hill, C., & Ross, R. P. (2005). Microbial solutions to microbial problems; lactococcal bacteriocins for the control of undesirable biota in food. Journal of Applied Microbiology, 98(6), 1316e1325. Hahn, G. (1996). Bacteriological quality of raw milk. Brussels (Belgium): International Dairy Federation. Hanlin, M. B., Kalchayand, N., Ray, P., & Ray, B. (1993). Bacteriocins of lactic acid bacteria in combination have greater antibacterial activity. Journal of Food Protection, 56(3), 252e255. Henning, S., Metz, R., & Hammes, W. P. (1986). New aspects for the application of nisin to food products based on its mode of action. International Journal of Food Microbiology, 3(3), 135e141. Iwata, T., Kimura, Y., Tsutsumi, K., Furukawa, Y., & Kimura, S. (1993). The effect of various phospholipids on plasma lipoproteins and liver lipids in hypercholesterolemic rats. Journal of Nutritional Science and Vitaminology, 39(1), 63e71. Jones, L. W. (1974). Effect of butterfat on inhibition of Staphylococcus aureus by nisin. Canadian Journal of Microbiology, 20(9), 1257e1260. Jung, D.-S., Bodyfelt, F. W., & Daeschel, M. A. (1992). Influence of fat and emulsifiers on the efficacy of nisin in inhibiting Listeria monocytogenes in fluid milk. Journal of Dairy Science, 75(2), 387e393. Lakamraju, M., McGuire, J., & Daeschel, M. (1996). Nisin adsorption and exchange with selected milk proteins and silanized silica surfaces. Journal of Colloid and Interface Science, 178(2), 495e504. Lauková, A., & Czikkova, S. (1999). The use of enterocin CCM 4231 in soy milk to control the growth of Listeria monocytogenes and Staphylococcus aureus. Journal of Applied Microbiology, 87, 182e186. Leistner, L. (2000). Basic aspects of food preservation by hurdle technology. International Journal of Food Microbiology, 55(1), 181e186. Moll, G. N., Roberts, G. C. K., Konings, W. N., & Driessen, A. J. M. (1996). Mechanism of lantibiotic-induced pore-formation. Antonie van Leeuwenhoek, 69(1), 185e191. Neda, K., Sakurai, T., Takahashi, M., Ashiuchi, M., & Ohgushi, M. (1999). Twogeneration reproduction study with teratology test of e-poly-L-lysine by dietary administration in rats. Japanese Pharmacology & Therapeutics, 27(7), 1139e1159. Raikos, V., Kapolos, J., Farmakis, L., Koliadima, A., & Karaiskakis, G. (2009). The use of sedimentation field-flow fractionation in the size characterization of bovine milk fat globules as affected by heat treatment. Food Research International, 42(5e6), 659e665. Shima, S., Matsuoka, H., Iwamoto, T., & Sakai, H. (1984). Antimicrobial action of epsilon-poly-L-lysine. The Journal of Antibiotics (Tokyo), 37(11), 1449e1455.
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Silva Malheiros, P.da., Sant’Anna, V., Souza Barbosab, M. de, Brandelli, A., & Melo Francob, B. D. G.de. (2012). Effect of liposome-encapsulated nisin and bacteriocin-like substance P34 on Listeria monocytogenes growth in Minas frescal cheese. International Journal of Food Microbiology, 156(3), 272e277. Silva Malheiros, P.da., Sant’Anna, V., Utpott, M., & Brandelli, A. (2012). Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk. Food Control, 23(1), 42e47. Sobrino-Lopez, A., & Martin-Belloso, O. (2006). Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin. Journal of Food Protection, 69(2), 345e353. Soomro, A. H., Arain, M. A., Khaskheli, M., & Bhutto, B. (2002). Isolation of Escherichia coli from raw milk and milk products in relation to public health sold under market conditions at Tandojam Pakistan. Journal of Nutrition, 1(3), 151e152.
Vogt, R. L., & Dippold, L. (2005). Escherichia coli O157:H7 outbreak associated with consumption of ground beef, JuneeJuly 2002. Public Health Reports, 120(2), 174e178. Xiraphi, N., Georgalaki, M., Van Driessche, G., Devreese, B., Van Beeumen, J., & Tsakalidou, E. (2006). Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442. Antonie van Leeuwenhoek, 89(1), 19e26. Yoshida, T., & Nagasawa, T. (2003). Epsilon-poly-L-lysine: microbial production, biodegradation and application potential. Applied Microbiology and Biotechnology, 62(1), 21e26. Zapico, P., de Paz, M., Medina, M., & Nunez, M. (1999). The effect of homogenization of whole milk, skim milk and milk fat on nisin activity against Listeria innocua. International Journal of Food Microbiology, 46(2), 151e157.
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Evaluation of the factors affecting the activity of sakacin C2 against E. coli in milk Yurong Gao*, Dapeng Li, Xiaoyan Liu Food College of Heilongjiang Bayi Agricultural University, High and New Tech Development Zone, Daqing 163319, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2012 Received in revised form 11 July 2012 Accepted 12 July 2012
Sakacin C2 is a novel, broad spectrum bacteriocin secreted by Lactobacillus sake C2 isolated from Chinese traditional fermented cabbage. Effects of milk fat, emulsifiers, preservatives and homogenization on the activity of sakacin C2 against Escherichia coli ATCC 25922 in milk were evaluated by determined the changes of viable cell counts of E. coli ATCC 25922 after inoculation (105 CFU/ml) then storage at 4 C. Milk fat in pasteurized and homogenized milk products (low fat milk and whole milk) decreased the activity of sakacin C2 against E. coli ATCC 25922. Addition of 5 ml/ml of Tween 80 decreased the effect of sakacin C2 against E. coli ATCC 25922, but 5 mg/ml of lecithin increased the effect against E. coli ATCC 25922 in pasteurized and homogenized whole milk. Nisin and ε-Polylysine significantly increased the effect of sakacin C2 against E. coli ATCC 25922 in pasteurized and homogenized whole milk. Homogenization interfered with the activity of sakacin C2 against E. coli ATCC 25922 in pasteurized skim milk and whole milk. This study might lay the groundwork for the application of sakacin C2 as bio-preservative in milk and dairy products. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Sakacin C2 Bacteriocin E. coli Antimicrobial activity
1. Introduction Escherichia coli is a Gram-negative, rod-shaped bacterium which is one of the main organisms in the mammalian intestinal tract (Erichson & Doyle, 2007). There have been many reports about the discovery of E. coli-contaminated milk and milk products available to the consumer (Adesiyun, 1994; Hahn, 1996, Chapter 4; Soomro, Arain, Khaskheli, & Bhutto, 2002). E. coli is widely accepted as an indicator of fecal contamination and is generally found in unsanitary conditions of foods such as milk and other dairy products (Soomro et al., 2002). E. coli strains cannot only cause food decomposition, some serotypes such as E. coli O157:H7 can also cause food poisoning and gastrointestinal disease originated from animal origin foods including milk and milk products (Vogt & Dippold, 2005). Bacteriocins are antimicrobial peptides produced by bacteria and bacteriocins from lactic acid bacteria (LAB) are generally recognized as safe (GRAS) biopreservatives (Guiname, Cotter, Hill, & Ross, 2005). The use of bacteriocins produced by LAB in the preservation of foods including milk and milk products has now become a promising technology (Arauz, Jozala, Mazzola, & Penna, 2009). Sakacin C2 is a novel bacteriocin with a broad inhibitory spectrum secreted by Lactobacillus sake C2 isolated from Chinese traditional fermented cabbage (Gao, Jia, Gao, & Tan, 2010). Sakacin * Corresponding author. Tel.: þ86 459 6818360; fax: þ86 459 6819235. E-mail address: [email protected] (Y. Gao). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.07.013
C2 displayed the activity against some foodborne spoilage and pathogenic bacteria including not only Gram-positive bacteria such as Staphylococcus aureus, Listeria innocua, Streptococcus thermophilus and Bacillus cereus, but also Gram-negative bacteria such as E. coli and Salmonella typhimurium. Moreover, sakacin C2 showed strong tolerance to heat (even at 121 C for 15 min) and low pH (pH 3.0e8.0) (Gao et al., 2010). Gao, Li, Sheng, and Liu (2011) also studied the mode of action of sakacin C2 against E. coli ATCC 25922 and suggested that sakacin C2 depolarized the trans-membrane electrical potential, inserted the cell membrane and disintegrated the cell ultimately. These characteristics strongly suggested its good potential to be used as bio-preservative in food industry. The bacteriocin activity in food matrices may be affected by several factors such as chemical composition, the use of food additives and physical conditions of food (Cleveland, Montville, Nes, & Chikindas, 2001). In milk system, the antimicrobial activities of several bacteriocins against Gram-positive bacteria have been studied (Bizani, Morrissy, Dominguez, & Brandelli, 2008; Jones, 1974; Silva Malheiros, Sant’Anna, Utpott, & Brandelli, 2012; Zapico, de Paz, Medina, & Nunez, 1999). Alpas and Bozoglu (2000) reported that the bacteriocins combination with pressure and heat inhibited S. aureus, Listeria monocytogenes, E. coli and Salmonella in milk. Silva Malheiros, Sant’Anna, Utpott, et al. (2012) reported that the antilisterial activity of nanovesicle-encapsulated antimicrobial peptide P34 in milk. Bizani et al. (2008) reported that the antimicrobial peptide cerein 8A (160 AU/ml) in UHT milk resulted in a decrease of 3 log cycles in viable cells of
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L. monocytogenes within the 14-day period at 4 C. Lauková and Czikkova (1999) reported that enterocin CCM 4231 inhibited the growth of L. monocytogenes and S. aureus in soy milk. Silva Malheiros, Sant’Anna, Souza Barbosab, Brandelli, and Melo Francob (2012) reported that liposome-encapsulated nisin and bacteriocin-like substance (BLS) P34 effectively controlled the growth of L. monocytogenes in Minas frescal cheese. Bacteriocins produced by LAB generally have inhibitory activity against Grampositive bacteria. Sakacin C2 has inhibitory activity against not only many Gram-positive bacteria but also many Gram-negative bacteria (Gao et al., 2010). Bacteriocins produced by LAB generally have inhibitory activity against Gram-positive bacteria. Sakacin C2 has inhibitory activity against not only many Gram-positive bacteria but also many Gram-negative bacteria (Gao et al., 2010). This activity against many Gram-negative bacteria was not frequently discovered in bacteriocins from LAB. There have been few researches reported on effects of chemical composition and processing on the activity of bacteriocin against Gram-negative bacteria in milk products. The objective of the present study was to evaluate the effects of chemical composition including milk fat, the addition of emulsifiers and preservatives, and homogenization on activity of sakacin C2 against E. coli in milk. 2. Materials and methods
320 AU/ml. Cells of E. coli ATCC 25922 were added to these samples and the initial counts were 105 CFU/ml. Cell counts of samples were determined at the start of the experiment and at 2 d intervals until 12 d of storage at 4 C. 2.5. Effects of emulsifiers on the activity of sakacin C2 against E. coli Effects of emulsifiers on the activity of sakacin C2 against E. coli were determined by supplementing with 5 mg/ml lecithin from soy (Sigma, Hong Kong, China) or 5 ml/ml Tween 80 (Sigma) to the pasteurized and homogenized whole milk followed by the addition of sakacin C2 (480 AU/ml). The sample not supplemented with emulsifiers was used as control. All samples were inoculated with E. coli ATCC 25922 (105 CFU/ml) and cell counts were determined during storage. 2.6. Effects of preservatives on the activity of sakacin C2 against E. coli Effects of preservatives on the activity of sakacin C2 against E. coli were determined by supplementing with 5 mg/l nisin (NisinZÒM, Handary SA, Brussels, Belgium) or 50 mg/l ε-polylysine (EpolylyÒ DX, Handary SA, Brussels, Belgium) to the pasteurized and homogenized whole milk followed by the addition of sakacin C2 (320 AU/ml). The sample not supplemented with preservatives was used as control. All samples were inoculated with E. coli ATCC 25922 (105 CFU/ml) and cell counts were determined during storage.
2.1. Bacteria, culture and inoculation E. coli ATCC 25922 was used as indicator strain. The strain was purchased from China General Microbiological Culture Collection Center (CGMCC, Beijing, China) and stored in nutrient broth with 20% (v/v) glycerol at 20 C. Before E. coli ATCC 25922 was inoculated to milk samples, the cells were cultivated in nutrient broth at 37 C and 100 rpm for 12 h. L. sake C2 producing sakacin C2 was stored in MRS broth with 20% (v/v) glycerol at 20 C (Gao et al., 2010). Before used for the partial purification of the bacteriocin, cultures were cultivated without agitation in MRS broth (Qingdao Hope Bio. Technology Co., Ltd, China) at 30 C for 18 h and were then incubated without agitation in the same medium at 30 C for 24 h. 2.2. Preparation of sakacin C2 Sakacin C2 was prepared from the cell-free supernatant of L. sake C2 culture. The culture was partially purified by cold ethanol and column chromatography (Gao et al., 2010). Bacteriocin activity (AU/ml) was determined by the method of agar diffusion using S. aureus ATCC 63589 as indicator described by Xiraphi et al. (2006). 2.3. Milk samples Pasteurized and homogenized skim milk (0% milk fat), low fat milk (1.3% milk fat) and whole milk (3.6% milk fat) were purchased from a local market and produced by Inner Mongolia Yili Industrial Group Co., Ltd (China). Samples of the pasteurized and homogenized or not homogenized skim milk (0% milk fat) and whole milk (3.6% milk fat) were obtained from Daqing Dairy (Daqing, China). The samples of raw whole milk (3.6% milk fat) were obtained from Daqing Dairy, and the pasteurized not homogenized samples were prepared at 80 C for 15 s in this factory.
2.7. Effects of homogenization and heat treatment on the activity of sakacin C2 against E. coli Effects of homogenization on the activity of sakacin C2 against E. coli were determined by adding sakacin C2 (240 AU/ml) and E. coli ATCC 25922 (105 CFU/ml) to the pasteurized and homogenized (200 bar) skim milk (0% milk fat) and whole milk (3.6% milk fat). Samples not supplemented with sakacin C2 were used as control. The survival of E. coli ATCC 25922 after treatments was determined. Effects of heat treatment on the activity of sakacin C2 against E. coli were determined by adding sakacin C2 (240 AU/ml) and E. coli ATCC 25922 (105 CFU/ml) to the raw whole milk (3.6% milk fat) and the pasteurized not homogenized raw whole milk. Sample of raw whole milk not supplemented with sakacin C2 was used as control. The survival of E. coli ATCC 25922 after treatments was determined. 2.8. Bacteria counting E. coli Chromogenic medium (Qingdao Hope Bio. Technology Co., Ltd, China) was used for the enumeration of E. coli ATCC 25922. The liquor samples (0.1 ml) of the undiluted or diluted samples using normal saline (0.85% (m/v) NaCl) were spread directly on the surface of prepoured plates and then were incubated at 30 C for 48 h. 2.9. Data analysis All experiments were repeated three times and in duplicate. Microsoft Excel (version 2003) was used to analyze the data. Statistical analysis was analyzed with the software SAS 8.1 (SAS Institute Inc., Cary, NC, USA). Differences were considered significant at P < 0.05. 3. Results and discussion
2.4. Effects of milk fat on the activity of sakacin C2 against E. coli 3.1. Effects of milk fat on the activity of sakacin C2 against E. coli Sakacin C2 preparation was added to 50 ml of the pasteurized and homogenized skim milk (0% milk fat), low fat milk (1.3% milk fat) and whole milk (3.6% milk fat) to the final concentration of
Effects of sakacin C2 (320 AU/ml) on the growth of E. coli ATCC 25922 in the pasteurized and homogenized skim milk, low fat milk
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and whole milk during storage at 4 C are shown in Fig. 1. The cell counts of E. coli ATCC 25922 in the pasteurized and homogenized skim milk, low fat milk and whole milk not supplemented with sakacin C2 all increased from 5 to about 7 log CFU/ml after 12 d of storage at 4 C. The mode of action of sakacin C2 against E. coli ATCC 25922 is considered as bactericidal action (Gao et al., 2011). Adding 320 AU/ml of sakacin C2 to the pasteurized and homogenized skim milk, the cell number of E. coli ATCC 25922 decreased to below detectable levels (<10 CFU/ml) after 4 d and maintained until to 12 d (Fig. 1). Adding 320 AU/ml of sakacin C2 to the pasteurized and homogenized low fat milk, the cell number of E. coli ATCC 25922 decreased to 1.92 log CFU/ml after 4 d and below 3 log CFU/ml until to 12 d (Fig. 1). But in the pasteurized and homogenized whole milk, the counts of E. coli ATCC 25922 decreased to 3.05 log CFU/ml after 4 d and then recovered growth to 5.79 log CFU/ml after 12 d of storage at 4 C (Fig. 1). These results suggested that the maximum activity against E. coli presented in the pasteurized and homogenized skim milk (0% milk fat), but reduced activity presented in the milk with 1.3% and 3.6% fat. These results in this study were similar to those reported by Jung, Bodyfelt, and Daeschel (1992) and Chollet, Sebti, Martial-Gros, and Degraeve (2008). Jung et al. (1992) observed that the antilisterial activity of nisin decreased by 33% in skim milk, by 50% in milk with 1.2% fat and by 88% in milk with 12.9% fat. Chollet et al. (2008) reported that the activity of nisin against Kocuria rhizophila significantly decreased when the fat in gel attained 30% (w/w). Bacteriocins react to the cell membrane of sensitive organism. Bacteriocins firstly bind to the charged groups of phospholipids of cell membrane, then insert the membrane and leading to the depolarization of trans-membrane electrical potential, efflux of materials (Gao et al., 2011; Moll, Roberts, Konings, & Driessen, 1996). These negative effects of fat on the activity of bacteriocins may be caused by bacteriocins adsorption onto fat globules, as a result, the amount of bacteriocins acting with the cell membrane of target organisms reduced. Therefore, in this study the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized low fat milk (1.3% fat) and whole milk (3.6%) significantly decreased compared to that in the pasteurized and homogenized skim milk. 3.2. Effects of emulsifiers on the activity of sakacin C2 against E. coli Effects of emulsifiers (Tween 80 and lecithin) on the activity of sakacin C2 (480 AU/ml) against E. coli ATCC 25922 in the pasteurized and homogenized whole milk were summarized in Table 1. In the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in milk sample supplementing with lecithin (5 mg/ml) were 3.51 and 6.76 log CFU/ml, and the cell counts were 2.61 and 3.93 log CFU/ml
6
Log CFU/ml
5 4 3 2 1 0 0
2
4
6
8
10
12
Time (d) Fig. 1. Effects of sakacin C2 on the growth of E. coli ATCC 25922 in the pasteurized and homogenized skim milk (,), low fat milk (>) and whole milk (6) during the 12 d of storage at 4 C.
455
Table 1 Effects of emulsifiers on the activity of sakacin C2 against E. coli in the pasteurized and homogenized whole milk. Treatment
Whole Whole Whole Whole
milk milk milk milk
Cell counts at 4 C for 2 d (log CFU/ml) (control)a þ sakacin C2 þ sakacin C2 þ Tween 80 þ sakacin C2 þ lecithin
5.54 2.61 2.08 3.51
0.31 0.17 0.14 0.26
Cell counts at 4 C for 12 d (log CFU/ml) 7.52 3.93 2.12 6.76
0.31 0.26 0.35 0.29
a Initial cell counts of E. coli ATCC 25922 in pasteurized and homogenized whole milk were 4.96 log CFU/ml.
in those without lecithin after stored for 2 and 12 d (Table 1). Therefore, lecithin decreased the effect against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. In the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in milk sample supplementing with Tween 80 were 2.08 and 2.12 log CFU/ml, and the cell counts were 2.61 and 3.93 log CFU/ml in those without Tween 80 after stored for 2 and 12 d (Table 1). Therefore, the addition of Tween 80 (5 ml/ml) increased the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. Lecithin is an anionic lipophilic emulsifier and is widely used in human food, animal feed, and pharmaceuticals. Moreover, it has been proved that soy-derived lecichin has significant function on lowering cholesterol (Iwata, Kimura, Tsutsumi, Furukawa, & Kimura, 1993). The result of Henning, Metz, and Hammes (1986) showed that lecithin helped to form stable complex between nisin and zwitterionic phospholipids. The data shown in Table 1 was in agreement with this theory. It could be concluded that the formation of stable complex between sakacin C2 and zwitterionic phospholipids of lecithin decreased the effect of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. Tween 80 is a nonionic emulsifier extensively used in foods, particularly in milk products such as ice cream. Adding Tween 80 to milk products prevents proteins from coating the fat globule (Goff, 1997). Goff, Liboff, Jordan, and Kinsella (1987) reported that Tween 80 decreased the amount of protein bound to milk fat droplets observed by electron microscopy. In our study, the milk fat significantly decreased the effect of sakacin C2 against E. coli in milk. Therefore, it could be concluded that Tween 80 can also decrease the bacteriocin absorbed by milk fat and thereby increased the effect of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk. 3.3. Effects of preservatives on the activity of sakacin C2 against E. coli Effects of nisin on the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk were summarized in Fig. 2. In the pasteurized and homogenized whole milk, the supplement of nisin (5 mg/l) displayed no significant effect on the survival of E. coli ATCC 25922 during the 12 d of storage at 4 C (p > 0.05) (Fig. 2). But, the cell counts of E. coli ATCC 25922 in the presence of both nisin and sakacin C2 were 3.56 log CFU/ml less than that in the presence of sakacin C2 alone after stored at 4 C for 12 d. These results strongly suggested that nisin significantly increased the effect of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk (Fig. 2). As a polycyclic antibacterial peptide against Gram-positive spoilage and pathogenic bacteria, nisin is extensively used in processed cheese, meats, beverages, etc. However, nisin cannot inhibit Gram-negative bacteria such as E. coli and S. typhimurium (SobrinoLopez & Martin-Belloso, 2006). The outer membrane of Gram-
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8 7 Log CFU/ml
6 5 4 3 2 1 0 0
2
4
6
8
10
12
Time (d) Fig. 2. Effects of nisin on the activity of sakacin C2 against E. coli in the pasteurized and homogenized whole milk. The cell counts of E. coli ATCC 25922 in the pasteurized and homogenized whole milk (), whole milk in the presence of nisin (B), whole milk in the presence of sakacin C2 (,), whole milk in the presence of both nisin and sakacin C2 (6) during the 12 d of storage at 4 C.
negative bacteria is a complex barrier system for many antimicrobial agents such as bacteriocins, which normally have a deleterious effect on the inner membrane (Alakomi et al., 2000). Sakacin C2 is a bacteriocin with activity against not only Gram-positive bacteria but also Gram-negative bacteria. Synergistic effect of sakacin C2 in combination with nisin against E. coli ATCC 25922 may be the result of sakacin C2 altering the out membrane, rendering the outer membrane permeable, thereby nisin being able to act on the inner membrane. Synergistic effects of nisin in combination with pediocin AcH and curvaticin 13 against Gram-positive bacteria have been reported (Bouttefroy & Millière, 2000; Hanlin, Kalchayand, Ray, & Ray, 1993). However, synergistic action of nisin and other bacteriocins from LAB against Gram-negative bacteria have not been reported. These results may imply that the combined use of two antimicrobial agents may be advantageous to produce a synergistic effect what make the individual antimicrobial agent effective at low concentration even when indictor strain is sensitive to one antimicrobial agent but not sensitive to another. Effects of ε-Polylysine on the activity of sakacin C2 against E. coli ATCC 25922 in the pasteurized and homogenized whole milk are summarized in Fig. 3. Adding 50 mg/l of ε-Polylysine in the 8 7
Log CFU/ml
6
pasteurized and homogenized whole milk had not significant inhibitory effect on E. coli ATCC 25922 during the 12 d of storage at 4 C (p > 0.05) (Fig. 3). The cell counts of E. coli ATCC 25922 in the presence of sakacin C2 were 5.99 log CFU/ml after storage at 4 C for 12 d, and that in the presence of both sakain C2 and ε-Polylysine rapidly decreased to below detectable level (<10 CFU/ml) after stored at 4 C for 6 d (Fig. 3). These data showed the significantly synergistic effects of sakacin C2 in combination with ε-Polylysine against E. coli ATCC 25922 in the pasteurized and homogenized whole milk (Fig. 2). ε-Polylysine is natural antimicrobial agent produced by Streptomyces albulus which have activity against a wide range of microorganisms including bacteria (both Gram-positive and Gram-negative bacteria), yeast and mould (Yoshida & Nagasawa, 2003). ε-Polylysine is generally recognized as safe (GRAS) preservative used in a variety of foods including milk and dairy products. Moreover, ε-Polylysine has strong stability including pH (4.0e10.0) and heat (120 C for 20 min) (Neda, Sakurai, Takahashi, Ashiuchi, & Ohgushi, 1999). Sakacin C2 and ε-Polylysine exerted their activity against E. coli in different ways. The mode of action of ε-Polylysine was believed to firstly absorb onto the cellular membrane of bacterial cells, strip off the membrane and result in the abnormal distribution of the protoplasm (Shima, Matsuoka, Iwamoto, & Sakai, 1984). Sakacin C2 is believed to firstly depolarize the trans-membrane electrical potential, make the surface of cells wrinkle and disintegrate the cell ultimately (Gao et al., 2011). The synergistic effects of different antimicrobial agents may be achieved by the different mechanisms of action or sites of activity (Badaoui Najjar, Kashtanov, & Chikindas, 2007). The synergistic effects of sakacin C2 in combination with ε-Polylysine against E. coli ATCC 25922 in this study is also in accordance with this hypothesis. The multiple hurdle approach was extensively used to achieve optimal antimicrobial activity and decreased the amount of antimicrobial agents used in foods (Leistner, 2000). 3.4. Effects of homogenization and heat treatment on the activity of sakacin C2 against E. coli in milk Effects of homogenization on the activity of sakacin C2 against E. coli in the pasteurized skim milk and whole milk is summarized in Table 2. In the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in the pasteurized and homogenized skim milk were 2.56 and 4.18 log CFU/ml, and those in the pasteurized but unhomogenized skim milk were 1.92 and 2.83 log CFU/ml after storage at 4 C for 2 d and 12 d (Table 2). Similarly, in the presence of sakacin C2, the cell counts of E. coli ATCC 25922 in the pasteurized and homogenized whole milk were 4.08 and 6.87 log CFU/ml, and Table 2 Effects of homogenization and heat treatment on the activity of sakacin C2 against E. coli in milk.
5 4
Treatment
3 2 1 0 0
2
4
6
8
10
12
Time (d) Fig. 3. Effects of ε-polylysine on the activity of sakacin C2 against E. coli in the pasteurized and homogenized whole milk. The cell counts of E. coli ATCC 25922 in the whole milk (), whole milk in the presence of ε-polylysine (B), whole milk in the presence of sakacin C2 (,), whole milk in the presence of both ε-polylysine and sakacin C2 (6) during the 12 d of storage at 4 C.
Homogenization Control Unhomogenized skim milk þ sakacin C2 Homogenized skim milk þ sakacin C2 Unhomogenized whole milk þ sakacin C2 Homogenized whole milk þ sakacin C2 Heat treatment Control Raw whole milk þ sakacin C2 Pasteurized not homogenized raw whole milk þ sakacin C2
Cell counts at 4 C for Cell counts at 4 C for 2 d (log CFU/ml) 12 d (log CFU/ml) 5.34 0.27 1.92 0.14
7.66 0.31 2.83 0.12
2.56 0.12
4.18 0.11
2.38 0.18
3.96 0.22
4.08 0.16
6.87 0.34
5.68 0.21 2.66 0.16 3.64 0.20
7.82 0.36 3.41 0.18 4.88 0.23
Y. Gao et al. / Food Control 30 (2013) 453e458
those in the pasteurized but unhomogenized whole milk were 2.38 and 3.96 log CFU/ml after storage at 4 C for 2 d and 12 d (Table 2). These results strongly suggested that homogenization of milk caused the loss in the activity of sakacin C2 against E. coli. Homogenization of milk caused the changes of fat globules and milk proteins (Fox & McSweeney, 1998, Chapter 3). After homogenization, the average diameter of fat globules decreased and their number and surface area increased, and the distribution of milk proteins changed (Fox & McSweeney, 1998, Chapter 3). Thus, the amount of bacteriocin adsorption onto fat globules and milk proteins was increased and thereby reduced the bacteriocin to act with target organisms. Lakamraju, McGuire, and Daeschel (1996) also reported that bacteriocin absorption to certain surfaces may interfere with the inhibitory activity of nisin. The results in our paper exhibited that homogenized interfere with the antimicrobial activity of sakacin C2 against E. coli in the pasteurized skim milk and whole milk. Effects of heat treatment on the activity of sakacin C2 against E. coli in raw whole milk were summarized in Table 2. Addition of sakacin C2 in raw whole milk and pasteurized not homogenized raw whole milk significantly decreased the viable cell count of E. coli compared with that in the samples without sakacin C2 (P < 0.05). Moreover, in presence of sakacin C2, the viable cell counts in pasteurized not homogenized raw whole milk were significantly more than raw whole milk (P < 0.05). From these results, it can be concluded that pasteurized decreased the inhibitory activity of sakacin C2 in whole milk. Raikos, Kapolos, Farmakis, Koliadima, and Karaiskakis (2009) reported that the mean particle diameter of fat droplets increased after heat treatment in full-fat and semi-fat milk. Therefore, the superficial area of fat droplets increased after heat treatment. After addition of bacteriocins, the amount of bacteriocins adsorption on fat globule increased, as a result, the activity of sakacin C2 against E. coli reduced after whole milk pasteurized. Sakacin C2 was a novel bacteriocin with a broad inhibitory spectrum secreted by L. sake C2 isolated from Chinese traditional fermented cabbage. Sakacin C2 exhibited a broad antimicrobial activity. It could significantly inhibit many gram-positive bacteria including Listeria innocua and S. aureus, and moderately against some gram-negative bacteria such as E. coli and S. typhimurium in nutrient broth (Gao et al., 2010). From this result, it may be concluded that sakacin C2 may have stronger inhibitory to the gram-positive pathogenic bacteria such as Listeria spp. and S. aureus than gram-negative pathogenic bacteria such as E. coli. The activity against Gram-negative bacteria of sakacin C2 was not frequently seen in the bacteriocins from LAB. The inhibitory of bacteriocins from LAB against Gram-positive pathogenic bacteria such as Listeria spp. and S. aureus in dairy foods have been extensively reported. Therefore, the present study was focus on evaluating the use of sakacin C2 to control the growth of E. coli in milk. 4. Conclusion It can be first concluded that sakacin C2 have stronger inhibitory activity against E. coli in milk from this study. Moreover, the results presented in this study also strongly suggested that the activity of sakacin C2 against E. coli in milk was dependent on the chemical composition and the additives supplemented in the foods, and the treatment. Therefore, sakacin C2 may be have good application prospect for inhibiting the growth of E. coli in milk and dairy products. Acknowledgments This study was funded by Young Science Mainstay Subject of the Education Department of Heilongjiang Province (1155G38).
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References Adesiyun, A. A. (1994). Bacteriological quality and associated public health risk of pre-processed bovine milk in Trinidad. International Journal of Food Microbiology, 21(3), 253e261. Alakomi, H. L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., & Helander, I. M. (2000). Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Applied Environmental Microbiology, 66(5), 2001e2005. Alpas, H., & Bozoglu, F. (2000). The combined effect of high hydrostatic pressure, heat and bacteriocins on inactivation of foodborne pathogens in milk and orange juice. World Journal of Microbiology and Biotechnology, 16, 387e392. Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science and Technology, 20(3e4), 146e154. Badaoui Najjar, M., Kashtanov, D., & Chikindas, M. L. (2007). Epsilon-poly-L-lysine and nisin A act synergistically against gram-positive food-borne pathogens Bacillus cereus and Listeria monocytogenes. Letters in Applied Microbiology, 45(1), 13e18. Bizani, D., Morrissy, J. A. C., Dominguez, A. P. M., & Brandelli, A. (2008). Inhibition of Listeria monocytogenes in dairy products using the bacteriocin-like peptide cerein 8A. International Journal of Food Microbiology, 121(2), 229e233. Bouttefroy, A., & Milliére, J. B. (2000). Nisin-curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313. International Journal of Food Microbiology, 62(1e2), 65e75. Chollet, E., Sebti, I., Martial-Gros, A., & Degraeve, P. (2008). Nisin preliminary study as a potential preservative for sliced ripened cheese: NaCl, fat and enzymes influence on nisin concentration and its antimicrobial activity. Food Control, 19(10), 982e989. Cleveland, J., Montville, T. J., Nes, I. F., & Chikindas, M. L. (2001). Bacteriocins: safe, natural antimicrobials for food preservation. International Journal of Food Microbiology, 71(1), 1e20. Erichson, M. C., & Doyle, M. P. (2007). Food as a vehicle for transmission of Shiga toxin-producing Escherichia coli. Journal of Food Protection, 70(10), 2426e2449. Fox, P. F., & McSweeney, P. L. H. (1998). Dairy chemistry and biochemistry (1st ed.). London: Thomson Science. Gao, Y., Jia, S., Gao, Q., & Tan, Z. (2010). A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage. Food Control, 21(1), 76e81. Gao, Y., Li, D., Sheng, Y., & Liu, X. (2011). Mode of action of sakacin C2 against Escherichia coli. Food Control, 22(5), 657e661. Goff, H. D. (1997). Colloidal aspects of ice cream-a review. International Dairy Journal, 7(6), 363e373. Goff, H. D., Liboff, M., Jordan, W. K., & Kinsella, J. E. (1987). The effects of polysorbate 80 on the fat emulsion in ice cream mix: evidence from transmission electron microscopy studies. Food Microstructure, 6(2), 193e198. Guiname, C. M., Cotter, P. D., Hill, C., & Ross, R. P. (2005). Microbial solutions to microbial problems; lactococcal bacteriocins for the control of undesirable biota in food. Journal of Applied Microbiology, 98(6), 1316e1325. Hahn, G. (1996). Bacteriological quality of raw milk. Brussels (Belgium): International Dairy Federation. Hanlin, M. B., Kalchayand, N., Ray, P., & Ray, B. (1993). Bacteriocins of lactic acid bacteria in combination have greater antibacterial activity. Journal of Food Protection, 56(3), 252e255. Henning, S., Metz, R., & Hammes, W. P. (1986). New aspects for the application of nisin to food products based on its mode of action. International Journal of Food Microbiology, 3(3), 135e141. Iwata, T., Kimura, Y., Tsutsumi, K., Furukawa, Y., & Kimura, S. (1993). The effect of various phospholipids on plasma lipoproteins and liver lipids in hypercholesterolemic rats. Journal of Nutritional Science and Vitaminology, 39(1), 63e71. Jones, L. W. (1974). Effect of butterfat on inhibition of Staphylococcus aureus by nisin. Canadian Journal of Microbiology, 20(9), 1257e1260. Jung, D.-S., Bodyfelt, F. W., & Daeschel, M. A. (1992). Influence of fat and emulsifiers on the efficacy of nisin in inhibiting Listeria monocytogenes in fluid milk. Journal of Dairy Science, 75(2), 387e393. Lakamraju, M., McGuire, J., & Daeschel, M. (1996). Nisin adsorption and exchange with selected milk proteins and silanized silica surfaces. Journal of Colloid and Interface Science, 178(2), 495e504. Lauková, A., & Czikkova, S. (1999). The use of enterocin CCM 4231 in soy milk to control the growth of Listeria monocytogenes and Staphylococcus aureus. Journal of Applied Microbiology, 87, 182e186. Leistner, L. (2000). Basic aspects of food preservation by hurdle technology. International Journal of Food Microbiology, 55(1), 181e186. Moll, G. N., Roberts, G. C. K., Konings, W. N., & Driessen, A. J. M. (1996). Mechanism of lantibiotic-induced pore-formation. Antonie van Leeuwenhoek, 69(1), 185e191. Neda, K., Sakurai, T., Takahashi, M., Ashiuchi, M., & Ohgushi, M. (1999). Twogeneration reproduction study with teratology test of e-poly-L-lysine by dietary administration in rats. Japanese Pharmacology & Therapeutics, 27(7), 1139e1159. Raikos, V., Kapolos, J., Farmakis, L., Koliadima, A., & Karaiskakis, G. (2009). The use of sedimentation field-flow fractionation in the size characterization of bovine milk fat globules as affected by heat treatment. Food Research International, 42(5e6), 659e665. Shima, S., Matsuoka, H., Iwamoto, T., & Sakai, H. (1984). Antimicrobial action of epsilon-poly-L-lysine. The Journal of Antibiotics (Tokyo), 37(11), 1449e1455.
458
Y. Gao et al. / Food Control 30 (2013) 453e458
Silva Malheiros, P.da., Sant’Anna, V., Souza Barbosab, M. de, Brandelli, A., & Melo Francob, B. D. G.de. (2012). Effect of liposome-encapsulated nisin and bacteriocin-like substance P34 on Listeria monocytogenes growth in Minas frescal cheese. International Journal of Food Microbiology, 156(3), 272e277. Silva Malheiros, P.da., Sant’Anna, V., Utpott, M., & Brandelli, A. (2012). Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk. Food Control, 23(1), 42e47. Sobrino-Lopez, A., & Martin-Belloso, O. (2006). Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin. Journal of Food Protection, 69(2), 345e353. Soomro, A. H., Arain, M. A., Khaskheli, M., & Bhutto, B. (2002). Isolation of Escherichia coli from raw milk and milk products in relation to public health sold under market conditions at Tandojam Pakistan. Journal of Nutrition, 1(3), 151e152.
Vogt, R. L., & Dippold, L. (2005). Escherichia coli O157:H7 outbreak associated with consumption of ground beef, JuneeJuly 2002. Public Health Reports, 120(2), 174e178. Xiraphi, N., Georgalaki, M., Van Driessche, G., Devreese, B., Van Beeumen, J., & Tsakalidou, E. (2006). Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442. Antonie van Leeuwenhoek, 89(1), 19e26. Yoshida, T., & Nagasawa, T. (2003). Epsilon-poly-L-lysine: microbial production, biodegradation and application potential. Applied Microbiology and Biotechnology, 62(1), 21e26. Zapico, P., de Paz, M., Medina, M., & Nunez, M. (1999). The effect of homogenization of whole milk, skim milk and milk fat on nisin activity against Listeria innocua. International Journal of Food Microbiology, 46(2), 151e157.