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Investigation of reduction and tolerance capability of lactic acid bacteria isolated from kimchi against nitrate and nitrite in fermented sausage condition
Meat Science 97 (2014) 609–614
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Investigation of reduction and tolerance capability of lactic acid bacteria isolated from kimchi against nitrate and nitrite in fermented sausage condition Hyun-Dong Paik a, Joo-Yeon Lee b,⁎ a b
Division of Animal life Science, Konkuk University, Seoul 143-701, Republic of Korea Korea Livestock Products HACCP Accreditation Service, Anyang 8-dong, Gyeonggi-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 July 2013 Received in revised form 12 September 2013 Accepted 24 March 2014 Available online 2 April 2014 Keywords: Kimchi Lactic acid bacteria Fermented sausage Starter culture Nitrate Nitrite
a b s t r a c t Lactobacillus brevis KGR3111, Lactobacillus curvatus KGR 2103, Lactobacillus plantarum KGR 5105, and Lactobacillus sakei KGR 4108 isolated from kimchi were investigated for their potential to be used as starter culture for fermented sausages with the capability to reduce and tolerate nitrate/nitrite. The reduction capability of tested strains for nitrate was not dramatic. All tested strains, however, showed the capability to produce nitrite reductase with the reduction amount of 58.46–75.80 mg/l of NO− 2 . L. brevis and L. plantarum showed nitrate tolerance with the highest number of 8.71 log cfu/ml and 8.81 log cfu/ml, and L. brevis and L. sakei exhibited nitrite tolerance with the highest number of 8.24 log cfu/ml and 8.25 log cfu/ml, respectively. As a result, L. brevis, L. plantarum, and L. sakei isolated from kimchi showed a tolerance against nitrate or nitrite with a good nitrite reduction capability, indicating the satisfaction of one of the selection criteria to be used as starter culture for fermented sausages. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fermented sausages are produced from raw meat and a few additives, such as spices, starter culture, and nitrite curing salt. Unlike milk and malt, raw meats cannot be pasteurized and are therefore highly susceptible to the growth of undesirable microorganisms. Moreover, fermented sausages are generally manufactured without any heat treatment throughout the process of fermentation, ripening, and drying where the typical chemical, biochemical, physical and microbiological characteristics are developed (Flores & Bermell, 1996; Kunz, 1994; Luecke, 1997). Considering the general tendency that most fermented sausages are not heated prior to consumption and are usually stored without refrigeration, the selection of favorable conditions that encourage the specific growth and development of desirable and safe microflora and strict control of the growth of spoiling bacteria, including pathogenic strains, are essential for the microbial stability and shelf-life extension of fermented sausages (Bacus & Brown, 1981; Leistner, 1985; Luecke, 1997). Leistner (1984) suggested the sequence hurdle technology model to meet the safety requirements of fermented sausage with consideration of its typical characteristics of additives
⁎ Corresponding author at: Korea Livestock Products HACCP Accreditation Service, Anyang 430-731, Republic of Korea. Tel.: +82 31 390 5246; fax: +82 31 465 6698. E-mail address: [email protected] (J.-Y. Lee).
http://dx.doi.org/10.1016/j.meatsci.2014.03.013 0309-1740/© 2014 Elsevier Ltd. All rights reserved.
and processing. Among the hurdles, preservatives such as nitrite curing salt take the first hurdle before other critical hurdles, such as competitive flora, reduced pH as well as water activity are developed. Nitrite and also nitrate are used as necessary curing agents in the production of fermented sausages by their antimicrobial effects (Hausschild, Hilsheimer, Jarvis, & Raymond, 1982), especially against Clostridium botulinum (Christiansen, Tomkin, Shaparis, Johnston, & Kautter, 1975; Collins-Thompson, Chang, Davidson, Larmond, & Pivnick, 1974) and Staphylococcus aureus (Labots, 1976) even their health risks with mutagenic effect forming nitrosamines (Lundberg, Weitzberg, Cole, & Benjamin, 2004). In Europe, for fermented sausages and ham curing agents are still used either solely as saltpeter (potassium nitrate, E 251) or sodium nitrate (E 252) or in combination with potassium nitrite (E 249) as regulated in Directive 2006/52/EC (Hammes, 2012). For such an antimicrobial effect, nitrate should first reduce into nitrite by the incorporation with microbial reductase. Nitrite can be reduced and can release nitrogen monoxide (NO) that might act as a bactericidal agent by blocking sulphydryl groups having active center of nonheme iron–sulphur proteins that are essential for electron transport, enzyme activity, and energy production as proposed by Tompkin, Christiansen, and Shaparis (1978). NO produced from nitrate and nitrite by reduction has also an important role as curing color agent by formation of pink nitrosomyoglobin as well as antioxidant (Giddings, 1977; Hammes, 2012). The most efficient nitrate reducing organisms are staphylococci and micrococci (Gøtterup et al., 2008). Some meat lactic acid bacteria
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(LAB) have also been reported to possess nitrate reductases and hemedependent or heme-independent nitrite reductases (Hammes, Bantleon, & Min, 1990; Wolf, Arendt, Pfaehler, & Hammes, 1990). Such a capacity of LAB to possess both nitrate and nitrite reductase activities is regarded as one of the selection criteria for LAB to be used starter culture in fermented sausage production (Ammor & Mayo, 2007). In the previous studies, kimchi (a common term of Korean traditional foods produced with vegetables by LAB fermentation) microorganisms were investigated for their potential utility as a substitute for starter culture for fermented sausages (Lee & Kunz, 2005, 2006). The LAB integrated via the addition of kimchi into meat mixture indicated the potential utility to be substituted for starter culture by showing good adaptation to the new habitat of fermented sausage condition, reaching maximum numbers of 8.65–8.80 log cfu/g after 1–2 days of fermentation (Park & Lee, 2012). Lee, Kim, and Kunz (2006) isolated the majority of LAB from kimchi during its fermentation and then they were identified as Lactobacillus brevis KGR 3111, Lactobacillus curvatus KGR 2103, Lactobacillus plantarum KGR 5105, and Lactobacillus sakei KGR 4108. These LAB were then investigated for their suitable properties, such as adaptability, growth as well as acidity profile, for use as starter culture in fermented sausage conditions. Among the tested LAB, L. curvatus, L. plantarum, and L. sakei showed relatively good potential to be used as starter culture in sausage production by showing good growth as well as souring properties. In this study, L. brevis KGR 3111, L. curvatus KGR 2103, L. plantarum KGR 5105, and L. sakei KGR 4108 isolated from kimchi were investigated for their potential to be used as starter culture of fermented sausages by having the capability to reduce nitrate and nitrite. Nitrite and the released product such as NO are well known as the agents that interfere with the bacterial growth because of its mutagenic effects. Nitrous acids remove amino acids of DNA bases such as adenine, guanine and cytosine, causing their false match and replication. Therefore, one of the desirable characteristics of a starter used in the sausage production is the tolerance to the presence of at least 100 mg/kg of sodium nitrite (Roca & Incze, 1990). Accordingly, the selected strains were evaluated for their reduction and tolerate of nitrate and nitrite in fermented sausage condition. For this study, model media designed as like the fermented sausage and added with potassium nitrate or sodium nitrite were used to exclude the influence of other contaminants. 2. Materials and methods 2.1. Keeping and preparation of LAB strains isolated from kimchi for the tests Each strain of four LAB species, L. brevis KGR 3111, L. curvatus KGR 2103, L. plantarum KGR 5105, L. sakei KGR 4108, isolated from kimchi and identified in the previous study (Lee et al., 2006) was used for the tests. The strains of four LAB species were kept at −72 °C in a Microbank (Microbank™ Mast Diagnostica, Laboratorium-Praeparate GmbH, UK), a sterile vial containing porous beads which serve as carriers for microorganisms. For the tests, the beads of each strain of Microbank were incubated for 2 days at 30 °C in the test tubes containing MRS broth (Merck, Germany). After the second inoculation, 50 ml of the suspension was centrifuged at 3000 ×g (4 °C) for 20 min. The collected cells were washed in a sterile physiological saline solution (0.9% NaCl solution) twice. These harvested cells were re-suspended in saline solution. Then the absorbance was measured at 550 nm and the solution was diluted to 106 log cfu/ml medium with the help of the corresponding standard curves as shown in Fig. 1. 2.2. Preparation of model-media and inoculation The basic model-medium used in this study was composed to simulate the substantial conditions of meat mixtures employed for
the sausage production (Table 2). The basic composition of modelmedium was made of meat extract (Fluka, Germany) and as fermenting sugar, D(+)-glucose (Merck, Germany) was added. To investigate the influence of nitrate and nitrite on the growth of LAB from kimchi and their capability of reducing nitrate and nitrite, 0.6 g/l of potassium nitrate (Merck, Germany) and 0.15 g/l of sodium nitrite (Merck, Germany) were added into the basic model-medium, respectively. The start pH condition of model-media was adjusted by the addition of 0.5 N HCl into 5.8 (Hechelmann, 1985; Koch, 1982). The modelmedia were autoclaved for 20 min at 121 °C and 1.2 bar. To avoid Maillard reactions owing to heat treatment, glucose was sterilized separately and added aseptically to the medium after cooling. After cooling, the media were inoculated with approximately 106 log cfu/ml of each strain and the inoculates were distributed evenly. 2.3. Fermentation of model-media and sampling procedure The model-media formulated in 250 ml or 500 ml Erlenmeyer flasks and inoculated with each LAB of four references (Table 1) were fermented at 25 °C for 120 h. During the fermentation for 120 h, the sampling was performed in duplicate after 0, 4, 8, 12, 16, 24, 36, 48 and then every 24 h. The medium was shortly mixed with a sterile magnetic stirrer and then 1 ml for the determination of cfu and 1 ml for the enzymatic tests for nitrate and nitrite reduction. 2.4. Determination of nitrate and nitrite reduction by reference LAB The capability of reference LAB isolated from kimchi to reduce nitrate or nitrite was evaluated by determining their residue amount in each model-medium. The quantitative evaluation was carried out with the cuvette tests of the Boehringer Mannheim Company (Germany) according to the enclosed instructions. A test-tube containing 1 ml sample solution from each model-medium was first led in water bath at 80 °C for 15 min to stop enzymatic processes and then centrifuged (Biofuge 17RS, Heraeus Sepatech, USA) at 10,000 g for at least 1 min at room temperature (25 °C). The supernatant was filtered with filter-paper No. 595 1/2 (Whatman, Germany) in a funnel. The supernatant was kept at − 72 °C in Safe-Lock tubes (Eppendorf-Netheler-Hanz GmbH, Germany) until used for the different enzymatic analysis. The test for nitrate was based on the photometric measurement of NADPH. In the presence of the enzyme nitrate reductase, nitrate is reduced to nitrite by NADPH. The amount of NADPH oxidized during the reaction is linearly proportional to the amount of nitrate. The amount of reduced nitrate was determined by measuring the decrease in NADPH by means of its light absorbance at 340 nm. The determination of nitrite is based on measuring the light absorbance of the red-violet diazo dye. Nitrite reacts with sulphanilamide and N-(1-naphthyl)-ethylene-diamine dihydrochloride to give a red-violet diazo dye. The diazo dye is measured on the basis of its absorbance in the visible range at 540 nm. The reduction of nitrate/nitrite was calculated by percentage of their start amount, 100.02 mg/l for nitrate (calculated as mass percentage of 61.328% of NO− 3 in KNO3) and 367.97 mg/l for nitrite (calculated as mass percentage of 66.679% of NO − 2 in NaNO 2 ), respectively. 2.5. Determination of nitrate and nitrite tolerance of reference LAB The tolerance of reference LAB (Table 1) against nitrate and nitrite was estimated by determining their evolution in viable cell counts during the fermentation. The changes of viable cell number of test LAB were evaluated every 0, 4, 8, 12, 16, 24, 48 and then every 24 h. To evaluate the growth rate of the corresponding LAB, the generation time (n) was calculated. The generation time means the time it takes a population to double during exponential growth
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611
L. plantarum KGR5105
L. brevis KGR3111 8.0E+08 3.5E+08
6.0E+08
2.5E+08
5.0E+08
cfu/ml
cfu/ml
y = 1E+09x R² = 0.8992
7.0E+08
y = 6E+08x R² = 0.9983
3.0E+08
2.0E+08 1.5E+08
4.0E+08 3.0E+08
1.0E+08
2.0E+08
5.0E+07
1.0E+08 0.0E+00
0.0E+00 0
0.2
0.4
0.6
0
Density (O.D.)
0.4
0.6
Density (O.D.)
L. sakei KGR 4108
L.curvatus KGR2103 6.0E+07
4.0E+08
5.0E+07
3.5E+08
y = 4E+07x R² = 0.9636
y = 3E+08x R² = 0.9762
3.0E+08
cfu/ml
4.0E+07
cfu/ml
0.2
3.0E+07
2.5E+08 2.0E+08 1.5E+08
2.0E+07
1.0E+08 1.0E+07
5.0E+07 0.0E+00
0.0E+00 0
0.5
1
1.5
Density (O.D.)
0
0.5
1
1.5
Density (O.D.)
Fig. 1. Standard curves of dependency between optical density and cell number of L. brevis KGR3111, L. curvatus KGR2-13, L. plantarum KGR5105, and L. sakei KGR4108.
(Schlegel, 1985). From the logarithmical values of cfu the generation time (n) is calculated by the following equation: n¼
logN− logN0 log2
N N0
ð1Þ
the number of cells at time t the number of cells at time 0
3. Results and discussion 3.1. Nitrate reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi After some authors have reported that lactic acid bacteria involved in meat fermentation exhibit nitrate/nitrite reductase activities contributing
Table 1 Composition of model-medium batches. Categories
Contents
g/l
Basic medium
Meat extracts D(+)-Glucose NaCl K2HPO4 · 3H2O MgSO4 · 7H2O Glutamate
12.0 10.0 20.0 2.0 0.15 0.5
Nitrate medium
Basic medium Potassium nitrate Basic medium Sodium nitrite
Nitrite medium
0.6 0.15
the development of the characteristic pink curing color (Coretti, 1958; Hugas & Monfort, 1997; Wolf & Hammes, 1988; Wolf et al., 1990), their enzymatic activities were suggested as selective criteria for LAB to be used as starter culture in the production of fermented sausages (Buckenhueskes, 1993). In the present work, the activities of nitrate/ nitrite reduction of the lactic acid bacteria strains, L. brevis KGR3111, L. curvatus KGR 2103, L. plantarum KGR 5105, and L. sakei KGR 4108, isolated from kimchi were investigated. Nitrate is still often used as an essential additive in fermented sausage manufacture with a slow decrease of pH-values and prolonged ripening periods despite of the trend toward preferred use of nitrite by the industry (Hotchkiss & Cassens, 1987). In order to achieve the effects with nitrate in the production of fermented sausages, it first has to be reduced to nitrite. This reaction can only take place in the presence of enzymes, i.e. nitrate reductase that is produced by microorganisms. Interestedly, the activities of nitrate reductase were detected by some lactic acid bacteria involved in meat fermentation (Wolf & Hammes, 1988). Coretti (1958) has shown that some strains of L. plantarum reduced nitrate in 0.05% nitrate-broth. Several of the investigated strains reduced the entire nitrate added in 5–8 days. Such an active nitrate reduction by LAB isolated from kimchi was not detected in the present study as all tested strains showed only a slight nitrate reduction. L. brevis even looked as if it reduced hardly nitrate at all, with a negligible decrease in nitrate of 3.77% (13.9 mg/l) of original amount at the end of phase of fermentation as shown in Fig. 2. L. plantarum, L. sakei and L. curvatus from kimchi reduced nitrate of 14.84% (54.61 mg/l), 18.06% (66.46 mg/l), and 18.06% (66.46 mg/l) of original amount at the end of fermentation, respectively. These nitrate reduction was mainly achieved during the initial phase of fermentation whereas hardly any further nitrate was reduced after 12 h. The reason for this may have been the pH decrease in the medium influencing the activity of the nitrate reductase. The initial pH value of media was adjusted with
%
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100 95 90 85 80 75 70 65 60 55 50 0
4
8
12
24
48
72
96
120
Time [h] L.sake
L.curvatus
L.plantarum
L.brevis
Fig. 2. Nitrate reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi.
5.8 as similar as the start condition of fermented sausages (Hechelmann, 1985; Koch, 1982). The pH values of the media containing L. plantarum, L. sakei, and L. curvatus from kimchi reached 5.47–5.76 within 12 h of fermentation as previously reported (Lee et al., 2006). This was ascertained in a study by Coretti (1958) who found that some strains of L. plantarum reduced nitrate in vitro when cultivated in media with high pH and little sugar. Kandler and Weiss (1986) found also some strains of L. plantarum reducing nitrate under low glucose concentration and pH at 6.0 or higher. This explains why nitrate is preferentially used for dry sausages with slow and long ripening time (Luecke, 1997). 3.2. Nitrite reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi
%
In order to investigate the nitrite reductase activity of the investigated strains, the remaining of nitrite content was frequently measured during a period of 120 h. The results were calculated as a percentage of the start concentration as presented in Fig. 3. Nitrite reduction is regarded as a rare property in LAB. However, some authors have shown that nitrite reductases are present in LAB strains involved in meat fermentation (Collins-Thompson & Rodriquez Lopez, 1981; Dodds & Collins-Thompson, 1984; Wolf & Hammes, 1988; Wolf et al., 1990). Hammes et al. (1990) produced fermented sausage of high sensory quality using a mixed starter culture containing exclusively lactobacilli (Lactobacillus pentosus, L. sakei and Lactobacillus farciminis). Several strains of L. sakei and L. farciminis in their work could reduce nitrite independently from heme and release NO and N2O during the de-nitrification process, which were involved in the mechanisms of nitrosomyoglobin formation. In the present work, most tested strains showed nitrite reductase capabilities with the remnants of nitrite ranged from 24.21 to 41.55% of the start concentration (100.02 mg/l) after 120 h fermentation. There were only very little reduction of nitrite up to 12 h in all batches. The nitrite reductase 100 90 80 70 60 50 40 30 20 10 0
activities of the strains began after 24 h. From this point of time the reduction of nitrite in the medium inoculated with L. sakei was especially remarkable. In 24 h of fermentation, L. sakei reduced 31.71% of nitrite added, corresponding to 31.72 mg/l of NO− 2 , whereas the other strains reduced 3.91–12.90%. After 120 h fermentation, L. sakei, L. curvatus, L. brevis, and L. plantarum reduced 60.54 mg/l, 75.81 mg/l, 63.83 mg/l, 58.46 mg/l of nitrite (NO − 2 ) treated. The most powerful reduction activity of nitrite was observed in the batch inoculated with L. plantarum, whereas the weakest activity was at the batch inoculated with L. curvatus. These results agree with those of the work by Gøtterup et al. (2008) who have found L. plantarum, L. sakei, and L. farciminis as the LAB having nitrate/nitrite reducing potential. Nitrite is regarded as toxic due to its formation of N-nitroso compounds in food matrix as well as in the human body (Hotchkiss & Cassens, 1987; Tricker & Preusmann, 1987). Furthermore, the reactive intermediate compounds released from nitrite are known to be toxic for a variety of bacteria including not only pathogens but also desirable microorganisms (Castellani & Niven, 1955; Perigo, Whiting, & Bashford, 1967). Therefore, the reduction of nitrite content in food matrix is regarded as detoxification mechanism (Coleman, Newman, CornishBowden, & Cole, 1978) and is one of the important strategies of producers. In this regard, the nitrite reducing ability of kimchi LAB in this study suggests a good potential utility as starter culture of kimchi LAB in the production of fermented sausages. 3.3. Nitrate and nitrite tolerance of L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi The tolerance of L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi against nitrate and nitrite was determined by evaluating their growth profile in the model-media treated with nitrate or nitrite (Table 2). The effects of nitrate and nitrite addition on the growth rate of the investigated strains are illustrated in more detail by the comparison between the generation time of the corresponding strains in the media with or without nitrate (Fig. 4) or nitrite (Fig. 5). In the batches treated with nitrate, L. brevis, L. curvatus, and L. plantarum had a lag phase with the slowdown in their growth or even reduced viable counts during the first 4 h of fermentation as shown in Table 2. In particular, L. curvatus was influenced by nitrate negatively on the length of the lag phase with an increase for 4 h than without nitrate. In other side, the adaptation of L. sakei to modelmedia was facilitated by nitrate addition by showing no lag phase. There was an effect of the nitrate to retard the cell division of tested strains with the increased generation time with an exception of L. brevis. Unlike the other strains, L. brevis indicated facilitated growth rate with a decreased generation time under the condition with nitrate as compared to the control (Fig. 4). The highest number of L. brevis was also improved to 8.71 log cfu/ml by the nitrate added as compared Table 2 Growth profile of L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi in the model-media treated with nitrate or nitrite. Hours
0
4
12
24
48
72
96
120
Time [h] L. sake
L. curvatus
L. brevis
L. plantarum
Fig. 3. Nitrite reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi.
0 4 8 12 24 48 72 96 120
L. brevis
L. curvatus
L. plantarum
L. sakei
CB
NaB
NiB
CB
NaB
NiB
CB
NaB
NiB
CB
NaB
NiB
6.00 6.08 6.57 7.22 7.94 8.18 8.04 7.89 8.08
6.00 6.03 –⁎ 7.24 8.34 8.63 8.54 8.71 8.54
6.00 6.00 6.03 6.65 7.08 8.17 8.16 8.24 7.81
6.00 6.08 6.19 6.63 7.16 7.11 6.82 6.61 6.46
6.00 5.75 5.88 6.16 6.67 6.93 6.81 6.55 –
6.00 5.73 5.81 6.33 7.06 6.93 6.78 6.64 6.18
6.00 5.98 6.42 7.26 8.32 8.51 8.41 7.99 7.26
6.00 6.06 6.66 7.33 7.97 8.13 8.81 7.23 7.22
6.00 5.12 6.23 6.56 7.88 8.26 8.15 7.47 7.77
6.00 6.04 6.54 6.90 6.63 8.17 7.99 7.11 7.28
6.00 6.45 6.95 6.83 7.61 7.99 8.10 7.72 –
6.00 6.30 6.86 7.15 8.04 8.25 7.68 6.47 –
L. brevis: Lactobacillus brevis KGR 3111; L. curvatus: Lactobacillus curvatus KGR 2103; L. plantarum: Lactobacillus plantarum KGR 5105; L. sakei: Lactobacillus sakei KGR 4108; CB = control batch investigated in basic medium; NaB = batch investigated in nitrate medium; NiB = batch investigated in nitrite medium; ⁎not tested.
Time [h]
H.-D. Paik, J.-Y. Lee / Meat Science 97 (2014) 609–614
8 7 6 5 4 3 2 1 0
CB NaB
L.brevis 3.23 2.58
L.curvatus 5.57 7.08
L.plantarum 2.58 3.15
L.sake 3.63 4.49
Fig. 4. Effect of potassium nitrate added into model medium designed for fermented sausage on the generation time of lactic acid bacteria isolated from kimchi. CB: control batch investigated in basic medium; NaB: batch investigated in nitrate medium.
Time [h]
with 8.14 log cfu/ml in the medium without nitrate (Table 2). Considering the result within this study, it is ascertained that nitrate per se does not have inhibitory action on LAB as reviewed by Hammes (2012). L. brevis did show only a negligible decrease in nitrate as much as 3.77% of start amount in medium in this study, and therefore the medium inoculated with L. brevis contained more than 96.25 mg/l of nitrate throughout the fermentation period showing a resistance against nitrate. L. plantarum reached to the highest count of 8.81 log cfu/ml that was higher than that in the control (8.51 log cfu/ml) in spite of its increased generation time due to nitrate (Table 2). L. sakei and L. curvatus were influenced negatively by nitrate on their growth rate and their highest numbers. The most negative influence of nitrate on the growth rate was found in the batch inoculated with L. curvatus with an increase in generation time of 1.51 h (Fig. 4). The highest count of L. curvatus was 6.93 log cfu/ml, which was smaller than its highest count in the medium without nitrate (7.16 log cfu/ml) as shown in Table 2. In spite of concerns over the role of nitrite in the formation of nitrosamines and its mutagenic effect, there has been considerable interest in the value of this additive as an antimicrobial agent. Nitrite is one of the agents that interfere with the bacterial growth (Schlegel, 1985; White, 1975). This interest was focused mainly on the control of undesirable microorganisms such as Clostridium botulinum (Christiansen et al., 1975; Collins-Thompson et al., 1974), and S. aureus (Labots, 1976). LAB have been previously reported to be resistant to nitrite (Dodds & Collins-Thompson, 1984; Shank, Silliker, & Harper, 1962; Skjelkvåle & Tjaberg, 1974). However, inhibitive effects of nitrite on the growth of LAB in cured meat products have also been shown (Matsuoka, Furukawa, Takahashi, & Yamanaka, 1994). As mentioned above, it was found that nitrite as well as nitrate per se does not have inhibitory action on LAB (Dykhuizen et al., 1996; Pichner, Hechelmann, Steinstueck, & Gareis, 2006). The reactive intermediate compounds that converted from nitrate or nitrite such as N2O3, ONOO−, RS-NO are regarded to interfere with the functions of proteins (enzymes), membranes and DNA of target molecules and structures via Nnitrosylation, S-nitrosylation, nitrosyl-heme formation, disulphide
7 6 5 4 3 2 1 0 CB NiB
L.brevis 3.23 5.62
L.curvatus 5.57 4.55
L.plantarum 2.58 3.84
L.sake 3.63 6.43
613
formation and lipid peroxidation (Lundberg et al., 2004; Møller & Skibsted, 2002). In such a mechanism, however, lactobacilli are found to exhibit increased resistance (Dykhuizen et al., 1996). Among the tested strains isolated from kimchi within the present work, L. sakei, L. brevis, and L. plantarum were negatively influenced on their growth rates with the higher generation times as compared to that of the control as shown in Fig. 5. The most negative effect of nitrite on the initial growth rate was observed in the batch inoculated with L. plantarum by showing a reduction in numbers during the first 4 h as compared to that in the medium without nitrite (Table 2). However, L. brevis, L. plantarum, and L. sakei exhibited their resistance to the presence of nitrite by showing comparable to or even higher cell number than those in the medium without nitrite. For example, L. brevis had a highest number of 8.24 log cfu/ml which was slightly higher than that in the control (8.14 log cfu/ml). This tendency was observed also at the batch inoculated with L. sakei with the higher number of 8.25 log cfu/ml as compared to 8.17 log cfu/ml of the control (Table 2). As a matter of fact, it is expected that the nitrite tolerance of kimchi LAB is related to their nitrite reduction ability, since the reduction of nitrite content in the food matrix is regarded as detoxification mechanism improving the tolerance of bacteria against nitrite (Coleman et al., 1978). L. curvatus was sensitive to nitrite even though it showed the best nitrite reduction ability. Furthermore, L. brevis, L. plantarum, and L. sakei reduced nitrite were quite resistant to nitrite. Therefore, the results within the present work suggest that the nitrite resistance of kimchi LAB was not directly related to their ability to reduce nitrite, which agrees with the suggestion by Dodds and Collins-Thompson (1984).
4. Conclusion This study was conducted to investigate the potential utility of LAB isolated from kimchi, L. brevis KGR3111, L. curvatus KGR 2103, L. plantarum KGR 5105, and L. sakei KGR 4108, as starter culture in the production of fermented sausages with the capability to reduce and tolerate nitrate and nitrite added. The reduction capability of tested strains for nitrate was not dramatic, but nevertheless all tested strains reduced nitrate for 14.84–18.06% (54.61–66.46 mg NO− 3 /l) from the initial amount in 120 h. The nitrite reduction began after 24 h and from this point of time all tested strains showed the capability to produce nitrite reductase with the remnants of nitrite ranged from 24.21 to 41.55% of the start concentration, corresponding the reduction amount of 58.46–75.80 mg/l of NO− 2 . Nitrate and nitrite added had a negative effect on the growth of tested strains by increasing the generation time. The initial growth rate of L. curvatus, L. brevis, L. plantarum, and L. sakei was negatively influenced by the nitrate treated with the increased generation time. L. brevis, L. plantarum, and L. sakei were also influenced by the nitrite added with the decreased growth rate. Among them, however, L. brevis and L. plantarum showed the improved highest number of 8.71 log cfu/ml and 8.81 log cfu/ml in the case of nitrate treatment as compared to 8.18 log cfu/ml and 8.51 log cfu/ml without any treatment, respectively, indicating their tolerance against nitrate. The nitrite tolerance was found in the batches inoculated with L. brevis and L. sakei with the improved highest cell umber of 8.24 log cfu/ml and 8.25 log cfu/ml as compared to 8.18 log cfu/ml and 8.17 log cfu/ml in the control batches, respectively. As a result, L. brevis, L. plantarum, and L. sakei isolated from kimchi showed a tolerance against nitrate or nitrite with a good nitrite reduction `, indicating the satisfaction of one of the selection criteria for lactic acid bacteria to be used starter culture for fermented sausages.
Acknowledgments Fig. 5. Effect of sodium nitrite added into model medium designed for fermented sausage on the generation time of lactic acid bacteria isolated from kimchi. CB: control batch investigated in basic medium; NiB: batch investigated in nitrite medium.
The study presented in this paper was supported by the FriedrichEbert-Stiftung, Germany with a grant for Joo-Yeon Lee.
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Labots, H. (1976). Effect of nitrite on the development of Staphylococcus aureus in fermented sausages. Proc. 2nd int. Symp. Nitrite Meat Prod., Zeist (pp. 21–27). Lee, J. Y., Kim, C. J., & Kunz, B. (2006). Identification of lactic acid bacteria isolated from kimchi and studies on their suitability for application as starter culture in the production of fermented sausages. Meat Science, 72(3), 437–445. Lee, J. Y., & Kunz, B. (2005). The antioxidant properties of baechu-kimchi and freeze-dried kimchi-powder in fermented sausages. Meat Science, 69, 741–747. Lee, J. Y., & Kunz, B. (2006). Kimchi comparable with starter culture. Physicochemical and sensory characteristics of fermented sausage manufactured with kimchi. Fleischwirtschaft International, 3, 39–46. Leistner, L. (1984). Hurdle Technology fuer die Herstellung stabiler Fleischerzeugnisse. Mitteilungsblatt der BAFF, 84, 5882–5889. Leistner, L. (1985). Allgemeines ueber Rohwurst und Rohschinken. Mikrobiologie und Qualität von Rohwurst und Rohschinken. Kulmbacher Reihe Band, 5. (pp. 1–29). Kulmbach: Insititut für Mikrobiologie, Toxikologie und Histologie der Bundesanstalt für Fleischforschung. Luecke, F. K. (1997). Fermented sausages. In B. J. B. Wood (Ed.), (2nd ed.)Microbiology of fermented foods, Vol. 2. (pp. 441–483). London: Blackie Academic & Professional. Lundberg, J. O., Weitzberg, E., Cole, J. A., & Benjamin, N. (2004). Nitrate, bacteria and human health. Nature Reviews Microbiology, 2, 593–602. Matsuoka, A., Furukawa, N., Takahashi, T., & Yamanaka, Y. (1994). Utilization of lactic acid bacteria from kefir grains as starter cultures for fermented ground eat products. Animal Science and Technology, 65, 732–737. Møller, J. K. S., & Skibsted, L. H. (2002). Nitric oxide and myoglobins. Chemical Reviews, 102, 1167–1178. Park, Y. S., & Lee, J. Y. (2012). The effect of kimchi on the microbiological stability of fermented sausages. Meat Science, 92, 721–727. Perigo, J. A., Whiting, E., & Bashford, T. E. (1967). Observations on the inhibition of vegetative cells of Clostridium sporogenes by nitrite which has been autoclaved in a laboratory medium. Discussed in the context of sub-lethally processed cured meats. Journal of Food Technology, 2, 377–397. Pichner, R., Hechelmann, H., Steinstueck, H., & Gareis, M. (2006). Shigatoxin producing Escherichia coli (STEC) in conventionally and organically produced salami products. Fleischwirtschaft, 10, 112–114. Roca, M., & Incze, K. (1990). Fermented sausages. Food Reviews International, 6, 91–118. Schlegel, H. G. (1985). Allgemeine Mikrobiologie (6 Auflage ). Stuttgart, New York: Georg Thieme Verlag. Shank, J. L., Silliker, J. H., & Harper, R. H. (1962). The effect of nitric oxide on bacteria. Applied Microbiology, 10, 185–189. Skjelkvåle, E. R., & Tjaberg, T. B. (1974). Comparison of salami sausage produced with and without addition of sodium nitrite and sodium nitrate. Journal of Food Science, 39, 520–524. Tompkin, R. B., Christiansen, L. N., & Shaparis, A. B. (1978). Enhancing nitrite inhibition of Clostridium botulinum with isoascorbate in perishable canned cured meat. Applied Environmental Microbiology, 35(1), 59–61. Tricker, A. R., & Preusmann, R. (1987). Influence of cysteine and nitrate on the endogenous formation of N-nitrosoamino acids. Cancer, 67, 95–100. White, J. W. (1975). Relative significance of dietary source of nitrate and nitrite. Journal of Agriculture and Food Chemistry, 33, 886–891. Wolf, G., Arendt, E. K., Pfaehler, U., & Hammes, W. P. (1990). Heme-dependent and hemeindependent nitrite reduction by lactic acid bacteria results in different N-containing products. International Journal of Food Microbiology, 10, 323–330. Wolf, G., & Hammes, W. P. (1988). Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli. Archives of Microbiology, 149, 220–224.
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Meat Science journal homepage: www.elsevier.com/locate/meatsci
Investigation of reduction and tolerance capability of lactic acid bacteria isolated from kimchi against nitrate and nitrite in fermented sausage condition Hyun-Dong Paik a, Joo-Yeon Lee b,⁎ a b
Division of Animal life Science, Konkuk University, Seoul 143-701, Republic of Korea Korea Livestock Products HACCP Accreditation Service, Anyang 8-dong, Gyeonggi-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 July 2013 Received in revised form 12 September 2013 Accepted 24 March 2014 Available online 2 April 2014 Keywords: Kimchi Lactic acid bacteria Fermented sausage Starter culture Nitrate Nitrite
a b s t r a c t Lactobacillus brevis KGR3111, Lactobacillus curvatus KGR 2103, Lactobacillus plantarum KGR 5105, and Lactobacillus sakei KGR 4108 isolated from kimchi were investigated for their potential to be used as starter culture for fermented sausages with the capability to reduce and tolerate nitrate/nitrite. The reduction capability of tested strains for nitrate was not dramatic. All tested strains, however, showed the capability to produce nitrite reductase with the reduction amount of 58.46–75.80 mg/l of NO− 2 . L. brevis and L. plantarum showed nitrate tolerance with the highest number of 8.71 log cfu/ml and 8.81 log cfu/ml, and L. brevis and L. sakei exhibited nitrite tolerance with the highest number of 8.24 log cfu/ml and 8.25 log cfu/ml, respectively. As a result, L. brevis, L. plantarum, and L. sakei isolated from kimchi showed a tolerance against nitrate or nitrite with a good nitrite reduction capability, indicating the satisfaction of one of the selection criteria to be used as starter culture for fermented sausages. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fermented sausages are produced from raw meat and a few additives, such as spices, starter culture, and nitrite curing salt. Unlike milk and malt, raw meats cannot be pasteurized and are therefore highly susceptible to the growth of undesirable microorganisms. Moreover, fermented sausages are generally manufactured without any heat treatment throughout the process of fermentation, ripening, and drying where the typical chemical, biochemical, physical and microbiological characteristics are developed (Flores & Bermell, 1996; Kunz, 1994; Luecke, 1997). Considering the general tendency that most fermented sausages are not heated prior to consumption and are usually stored without refrigeration, the selection of favorable conditions that encourage the specific growth and development of desirable and safe microflora and strict control of the growth of spoiling bacteria, including pathogenic strains, are essential for the microbial stability and shelf-life extension of fermented sausages (Bacus & Brown, 1981; Leistner, 1985; Luecke, 1997). Leistner (1984) suggested the sequence hurdle technology model to meet the safety requirements of fermented sausage with consideration of its typical characteristics of additives
⁎ Corresponding author at: Korea Livestock Products HACCP Accreditation Service, Anyang 430-731, Republic of Korea. Tel.: +82 31 390 5246; fax: +82 31 465 6698. E-mail address: [email protected] (J.-Y. Lee).
http://dx.doi.org/10.1016/j.meatsci.2014.03.013 0309-1740/© 2014 Elsevier Ltd. All rights reserved.
and processing. Among the hurdles, preservatives such as nitrite curing salt take the first hurdle before other critical hurdles, such as competitive flora, reduced pH as well as water activity are developed. Nitrite and also nitrate are used as necessary curing agents in the production of fermented sausages by their antimicrobial effects (Hausschild, Hilsheimer, Jarvis, & Raymond, 1982), especially against Clostridium botulinum (Christiansen, Tomkin, Shaparis, Johnston, & Kautter, 1975; Collins-Thompson, Chang, Davidson, Larmond, & Pivnick, 1974) and Staphylococcus aureus (Labots, 1976) even their health risks with mutagenic effect forming nitrosamines (Lundberg, Weitzberg, Cole, & Benjamin, 2004). In Europe, for fermented sausages and ham curing agents are still used either solely as saltpeter (potassium nitrate, E 251) or sodium nitrate (E 252) or in combination with potassium nitrite (E 249) as regulated in Directive 2006/52/EC (Hammes, 2012). For such an antimicrobial effect, nitrate should first reduce into nitrite by the incorporation with microbial reductase. Nitrite can be reduced and can release nitrogen monoxide (NO) that might act as a bactericidal agent by blocking sulphydryl groups having active center of nonheme iron–sulphur proteins that are essential for electron transport, enzyme activity, and energy production as proposed by Tompkin, Christiansen, and Shaparis (1978). NO produced from nitrate and nitrite by reduction has also an important role as curing color agent by formation of pink nitrosomyoglobin as well as antioxidant (Giddings, 1977; Hammes, 2012). The most efficient nitrate reducing organisms are staphylococci and micrococci (Gøtterup et al., 2008). Some meat lactic acid bacteria
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(LAB) have also been reported to possess nitrate reductases and hemedependent or heme-independent nitrite reductases (Hammes, Bantleon, & Min, 1990; Wolf, Arendt, Pfaehler, & Hammes, 1990). Such a capacity of LAB to possess both nitrate and nitrite reductase activities is regarded as one of the selection criteria for LAB to be used starter culture in fermented sausage production (Ammor & Mayo, 2007). In the previous studies, kimchi (a common term of Korean traditional foods produced with vegetables by LAB fermentation) microorganisms were investigated for their potential utility as a substitute for starter culture for fermented sausages (Lee & Kunz, 2005, 2006). The LAB integrated via the addition of kimchi into meat mixture indicated the potential utility to be substituted for starter culture by showing good adaptation to the new habitat of fermented sausage condition, reaching maximum numbers of 8.65–8.80 log cfu/g after 1–2 days of fermentation (Park & Lee, 2012). Lee, Kim, and Kunz (2006) isolated the majority of LAB from kimchi during its fermentation and then they were identified as Lactobacillus brevis KGR 3111, Lactobacillus curvatus KGR 2103, Lactobacillus plantarum KGR 5105, and Lactobacillus sakei KGR 4108. These LAB were then investigated for their suitable properties, such as adaptability, growth as well as acidity profile, for use as starter culture in fermented sausage conditions. Among the tested LAB, L. curvatus, L. plantarum, and L. sakei showed relatively good potential to be used as starter culture in sausage production by showing good growth as well as souring properties. In this study, L. brevis KGR 3111, L. curvatus KGR 2103, L. plantarum KGR 5105, and L. sakei KGR 4108 isolated from kimchi were investigated for their potential to be used as starter culture of fermented sausages by having the capability to reduce nitrate and nitrite. Nitrite and the released product such as NO are well known as the agents that interfere with the bacterial growth because of its mutagenic effects. Nitrous acids remove amino acids of DNA bases such as adenine, guanine and cytosine, causing their false match and replication. Therefore, one of the desirable characteristics of a starter used in the sausage production is the tolerance to the presence of at least 100 mg/kg of sodium nitrite (Roca & Incze, 1990). Accordingly, the selected strains were evaluated for their reduction and tolerate of nitrate and nitrite in fermented sausage condition. For this study, model media designed as like the fermented sausage and added with potassium nitrate or sodium nitrite were used to exclude the influence of other contaminants. 2. Materials and methods 2.1. Keeping and preparation of LAB strains isolated from kimchi for the tests Each strain of four LAB species, L. brevis KGR 3111, L. curvatus KGR 2103, L. plantarum KGR 5105, L. sakei KGR 4108, isolated from kimchi and identified in the previous study (Lee et al., 2006) was used for the tests. The strains of four LAB species were kept at −72 °C in a Microbank (Microbank™ Mast Diagnostica, Laboratorium-Praeparate GmbH, UK), a sterile vial containing porous beads which serve as carriers for microorganisms. For the tests, the beads of each strain of Microbank were incubated for 2 days at 30 °C in the test tubes containing MRS broth (Merck, Germany). After the second inoculation, 50 ml of the suspension was centrifuged at 3000 ×g (4 °C) for 20 min. The collected cells were washed in a sterile physiological saline solution (0.9% NaCl solution) twice. These harvested cells were re-suspended in saline solution. Then the absorbance was measured at 550 nm and the solution was diluted to 106 log cfu/ml medium with the help of the corresponding standard curves as shown in Fig. 1. 2.2. Preparation of model-media and inoculation The basic model-medium used in this study was composed to simulate the substantial conditions of meat mixtures employed for
the sausage production (Table 2). The basic composition of modelmedium was made of meat extract (Fluka, Germany) and as fermenting sugar, D(+)-glucose (Merck, Germany) was added. To investigate the influence of nitrate and nitrite on the growth of LAB from kimchi and their capability of reducing nitrate and nitrite, 0.6 g/l of potassium nitrate (Merck, Germany) and 0.15 g/l of sodium nitrite (Merck, Germany) were added into the basic model-medium, respectively. The start pH condition of model-media was adjusted by the addition of 0.5 N HCl into 5.8 (Hechelmann, 1985; Koch, 1982). The modelmedia were autoclaved for 20 min at 121 °C and 1.2 bar. To avoid Maillard reactions owing to heat treatment, glucose was sterilized separately and added aseptically to the medium after cooling. After cooling, the media were inoculated with approximately 106 log cfu/ml of each strain and the inoculates were distributed evenly. 2.3. Fermentation of model-media and sampling procedure The model-media formulated in 250 ml or 500 ml Erlenmeyer flasks and inoculated with each LAB of four references (Table 1) were fermented at 25 °C for 120 h. During the fermentation for 120 h, the sampling was performed in duplicate after 0, 4, 8, 12, 16, 24, 36, 48 and then every 24 h. The medium was shortly mixed with a sterile magnetic stirrer and then 1 ml for the determination of cfu and 1 ml for the enzymatic tests for nitrate and nitrite reduction. 2.4. Determination of nitrate and nitrite reduction by reference LAB The capability of reference LAB isolated from kimchi to reduce nitrate or nitrite was evaluated by determining their residue amount in each model-medium. The quantitative evaluation was carried out with the cuvette tests of the Boehringer Mannheim Company (Germany) according to the enclosed instructions. A test-tube containing 1 ml sample solution from each model-medium was first led in water bath at 80 °C for 15 min to stop enzymatic processes and then centrifuged (Biofuge 17RS, Heraeus Sepatech, USA) at 10,000 g for at least 1 min at room temperature (25 °C). The supernatant was filtered with filter-paper No. 595 1/2 (Whatman, Germany) in a funnel. The supernatant was kept at − 72 °C in Safe-Lock tubes (Eppendorf-Netheler-Hanz GmbH, Germany) until used for the different enzymatic analysis. The test for nitrate was based on the photometric measurement of NADPH. In the presence of the enzyme nitrate reductase, nitrate is reduced to nitrite by NADPH. The amount of NADPH oxidized during the reaction is linearly proportional to the amount of nitrate. The amount of reduced nitrate was determined by measuring the decrease in NADPH by means of its light absorbance at 340 nm. The determination of nitrite is based on measuring the light absorbance of the red-violet diazo dye. Nitrite reacts with sulphanilamide and N-(1-naphthyl)-ethylene-diamine dihydrochloride to give a red-violet diazo dye. The diazo dye is measured on the basis of its absorbance in the visible range at 540 nm. The reduction of nitrate/nitrite was calculated by percentage of their start amount, 100.02 mg/l for nitrate (calculated as mass percentage of 61.328% of NO− 3 in KNO3) and 367.97 mg/l for nitrite (calculated as mass percentage of 66.679% of NO − 2 in NaNO 2 ), respectively. 2.5. Determination of nitrate and nitrite tolerance of reference LAB The tolerance of reference LAB (Table 1) against nitrate and nitrite was estimated by determining their evolution in viable cell counts during the fermentation. The changes of viable cell number of test LAB were evaluated every 0, 4, 8, 12, 16, 24, 48 and then every 24 h. To evaluate the growth rate of the corresponding LAB, the generation time (n) was calculated. The generation time means the time it takes a population to double during exponential growth
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611
L. plantarum KGR5105
L. brevis KGR3111 8.0E+08 3.5E+08
6.0E+08
2.5E+08
5.0E+08
cfu/ml
cfu/ml
y = 1E+09x R² = 0.8992
7.0E+08
y = 6E+08x R² = 0.9983
3.0E+08
2.0E+08 1.5E+08
4.0E+08 3.0E+08
1.0E+08
2.0E+08
5.0E+07
1.0E+08 0.0E+00
0.0E+00 0
0.2
0.4
0.6
0
Density (O.D.)
0.4
0.6
Density (O.D.)
L. sakei KGR 4108
L.curvatus KGR2103 6.0E+07
4.0E+08
5.0E+07
3.5E+08
y = 4E+07x R² = 0.9636
y = 3E+08x R² = 0.9762
3.0E+08
cfu/ml
4.0E+07
cfu/ml
0.2
3.0E+07
2.5E+08 2.0E+08 1.5E+08
2.0E+07
1.0E+08 1.0E+07
5.0E+07 0.0E+00
0.0E+00 0
0.5
1
1.5
Density (O.D.)
0
0.5
1
1.5
Density (O.D.)
Fig. 1. Standard curves of dependency between optical density and cell number of L. brevis KGR3111, L. curvatus KGR2-13, L. plantarum KGR5105, and L. sakei KGR4108.
(Schlegel, 1985). From the logarithmical values of cfu the generation time (n) is calculated by the following equation: n¼
logN− logN0 log2
N N0
ð1Þ
the number of cells at time t the number of cells at time 0
3. Results and discussion 3.1. Nitrate reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi After some authors have reported that lactic acid bacteria involved in meat fermentation exhibit nitrate/nitrite reductase activities contributing
Table 1 Composition of model-medium batches. Categories
Contents
g/l
Basic medium
Meat extracts D(+)-Glucose NaCl K2HPO4 · 3H2O MgSO4 · 7H2O Glutamate
12.0 10.0 20.0 2.0 0.15 0.5
Nitrate medium
Basic medium Potassium nitrate Basic medium Sodium nitrite
Nitrite medium
0.6 0.15
the development of the characteristic pink curing color (Coretti, 1958; Hugas & Monfort, 1997; Wolf & Hammes, 1988; Wolf et al., 1990), their enzymatic activities were suggested as selective criteria for LAB to be used as starter culture in the production of fermented sausages (Buckenhueskes, 1993). In the present work, the activities of nitrate/ nitrite reduction of the lactic acid bacteria strains, L. brevis KGR3111, L. curvatus KGR 2103, L. plantarum KGR 5105, and L. sakei KGR 4108, isolated from kimchi were investigated. Nitrate is still often used as an essential additive in fermented sausage manufacture with a slow decrease of pH-values and prolonged ripening periods despite of the trend toward preferred use of nitrite by the industry (Hotchkiss & Cassens, 1987). In order to achieve the effects with nitrate in the production of fermented sausages, it first has to be reduced to nitrite. This reaction can only take place in the presence of enzymes, i.e. nitrate reductase that is produced by microorganisms. Interestedly, the activities of nitrate reductase were detected by some lactic acid bacteria involved in meat fermentation (Wolf & Hammes, 1988). Coretti (1958) has shown that some strains of L. plantarum reduced nitrate in 0.05% nitrate-broth. Several of the investigated strains reduced the entire nitrate added in 5–8 days. Such an active nitrate reduction by LAB isolated from kimchi was not detected in the present study as all tested strains showed only a slight nitrate reduction. L. brevis even looked as if it reduced hardly nitrate at all, with a negligible decrease in nitrate of 3.77% (13.9 mg/l) of original amount at the end of phase of fermentation as shown in Fig. 2. L. plantarum, L. sakei and L. curvatus from kimchi reduced nitrate of 14.84% (54.61 mg/l), 18.06% (66.46 mg/l), and 18.06% (66.46 mg/l) of original amount at the end of fermentation, respectively. These nitrate reduction was mainly achieved during the initial phase of fermentation whereas hardly any further nitrate was reduced after 12 h. The reason for this may have been the pH decrease in the medium influencing the activity of the nitrate reductase. The initial pH value of media was adjusted with
%
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100 95 90 85 80 75 70 65 60 55 50 0
4
8
12
24
48
72
96
120
Time [h] L.sake
L.curvatus
L.plantarum
L.brevis
Fig. 2. Nitrate reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi.
5.8 as similar as the start condition of fermented sausages (Hechelmann, 1985; Koch, 1982). The pH values of the media containing L. plantarum, L. sakei, and L. curvatus from kimchi reached 5.47–5.76 within 12 h of fermentation as previously reported (Lee et al., 2006). This was ascertained in a study by Coretti (1958) who found that some strains of L. plantarum reduced nitrate in vitro when cultivated in media with high pH and little sugar. Kandler and Weiss (1986) found also some strains of L. plantarum reducing nitrate under low glucose concentration and pH at 6.0 or higher. This explains why nitrate is preferentially used for dry sausages with slow and long ripening time (Luecke, 1997). 3.2. Nitrite reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi
%
In order to investigate the nitrite reductase activity of the investigated strains, the remaining of nitrite content was frequently measured during a period of 120 h. The results were calculated as a percentage of the start concentration as presented in Fig. 3. Nitrite reduction is regarded as a rare property in LAB. However, some authors have shown that nitrite reductases are present in LAB strains involved in meat fermentation (Collins-Thompson & Rodriquez Lopez, 1981; Dodds & Collins-Thompson, 1984; Wolf & Hammes, 1988; Wolf et al., 1990). Hammes et al. (1990) produced fermented sausage of high sensory quality using a mixed starter culture containing exclusively lactobacilli (Lactobacillus pentosus, L. sakei and Lactobacillus farciminis). Several strains of L. sakei and L. farciminis in their work could reduce nitrite independently from heme and release NO and N2O during the de-nitrification process, which were involved in the mechanisms of nitrosomyoglobin formation. In the present work, most tested strains showed nitrite reductase capabilities with the remnants of nitrite ranged from 24.21 to 41.55% of the start concentration (100.02 mg/l) after 120 h fermentation. There were only very little reduction of nitrite up to 12 h in all batches. The nitrite reductase 100 90 80 70 60 50 40 30 20 10 0
activities of the strains began after 24 h. From this point of time the reduction of nitrite in the medium inoculated with L. sakei was especially remarkable. In 24 h of fermentation, L. sakei reduced 31.71% of nitrite added, corresponding to 31.72 mg/l of NO− 2 , whereas the other strains reduced 3.91–12.90%. After 120 h fermentation, L. sakei, L. curvatus, L. brevis, and L. plantarum reduced 60.54 mg/l, 75.81 mg/l, 63.83 mg/l, 58.46 mg/l of nitrite (NO − 2 ) treated. The most powerful reduction activity of nitrite was observed in the batch inoculated with L. plantarum, whereas the weakest activity was at the batch inoculated with L. curvatus. These results agree with those of the work by Gøtterup et al. (2008) who have found L. plantarum, L. sakei, and L. farciminis as the LAB having nitrate/nitrite reducing potential. Nitrite is regarded as toxic due to its formation of N-nitroso compounds in food matrix as well as in the human body (Hotchkiss & Cassens, 1987; Tricker & Preusmann, 1987). Furthermore, the reactive intermediate compounds released from nitrite are known to be toxic for a variety of bacteria including not only pathogens but also desirable microorganisms (Castellani & Niven, 1955; Perigo, Whiting, & Bashford, 1967). Therefore, the reduction of nitrite content in food matrix is regarded as detoxification mechanism (Coleman, Newman, CornishBowden, & Cole, 1978) and is one of the important strategies of producers. In this regard, the nitrite reducing ability of kimchi LAB in this study suggests a good potential utility as starter culture of kimchi LAB in the production of fermented sausages. 3.3. Nitrate and nitrite tolerance of L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi The tolerance of L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi against nitrate and nitrite was determined by evaluating their growth profile in the model-media treated with nitrate or nitrite (Table 2). The effects of nitrate and nitrite addition on the growth rate of the investigated strains are illustrated in more detail by the comparison between the generation time of the corresponding strains in the media with or without nitrate (Fig. 4) or nitrite (Fig. 5). In the batches treated with nitrate, L. brevis, L. curvatus, and L. plantarum had a lag phase with the slowdown in their growth or even reduced viable counts during the first 4 h of fermentation as shown in Table 2. In particular, L. curvatus was influenced by nitrate negatively on the length of the lag phase with an increase for 4 h than without nitrate. In other side, the adaptation of L. sakei to modelmedia was facilitated by nitrate addition by showing no lag phase. There was an effect of the nitrate to retard the cell division of tested strains with the increased generation time with an exception of L. brevis. Unlike the other strains, L. brevis indicated facilitated growth rate with a decreased generation time under the condition with nitrate as compared to the control (Fig. 4). The highest number of L. brevis was also improved to 8.71 log cfu/ml by the nitrate added as compared Table 2 Growth profile of L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi in the model-media treated with nitrate or nitrite. Hours
0
4
12
24
48
72
96
120
Time [h] L. sake
L. curvatus
L. brevis
L. plantarum
Fig. 3. Nitrite reduction by L. brevis, L. curvatus, L. plantarum, and L. sakei isolated from kimchi.
0 4 8 12 24 48 72 96 120
L. brevis
L. curvatus
L. plantarum
L. sakei
CB
NaB
NiB
CB
NaB
NiB
CB
NaB
NiB
CB
NaB
NiB
6.00 6.08 6.57 7.22 7.94 8.18 8.04 7.89 8.08
6.00 6.03 –⁎ 7.24 8.34 8.63 8.54 8.71 8.54
6.00 6.00 6.03 6.65 7.08 8.17 8.16 8.24 7.81
6.00 6.08 6.19 6.63 7.16 7.11 6.82 6.61 6.46
6.00 5.75 5.88 6.16 6.67 6.93 6.81 6.55 –
6.00 5.73 5.81 6.33 7.06 6.93 6.78 6.64 6.18
6.00 5.98 6.42 7.26 8.32 8.51 8.41 7.99 7.26
6.00 6.06 6.66 7.33 7.97 8.13 8.81 7.23 7.22
6.00 5.12 6.23 6.56 7.88 8.26 8.15 7.47 7.77
6.00 6.04 6.54 6.90 6.63 8.17 7.99 7.11 7.28
6.00 6.45 6.95 6.83 7.61 7.99 8.10 7.72 –
6.00 6.30 6.86 7.15 8.04 8.25 7.68 6.47 –
L. brevis: Lactobacillus brevis KGR 3111; L. curvatus: Lactobacillus curvatus KGR 2103; L. plantarum: Lactobacillus plantarum KGR 5105; L. sakei: Lactobacillus sakei KGR 4108; CB = control batch investigated in basic medium; NaB = batch investigated in nitrate medium; NiB = batch investigated in nitrite medium; ⁎not tested.
Time [h]
H.-D. Paik, J.-Y. Lee / Meat Science 97 (2014) 609–614
8 7 6 5 4 3 2 1 0
CB NaB
L.brevis 3.23 2.58
L.curvatus 5.57 7.08
L.plantarum 2.58 3.15
L.sake 3.63 4.49
Fig. 4. Effect of potassium nitrate added into model medium designed for fermented sausage on the generation time of lactic acid bacteria isolated from kimchi. CB: control batch investigated in basic medium; NaB: batch investigated in nitrate medium.
Time [h]
with 8.14 log cfu/ml in the medium without nitrate (Table 2). Considering the result within this study, it is ascertained that nitrate per se does not have inhibitory action on LAB as reviewed by Hammes (2012). L. brevis did show only a negligible decrease in nitrate as much as 3.77% of start amount in medium in this study, and therefore the medium inoculated with L. brevis contained more than 96.25 mg/l of nitrate throughout the fermentation period showing a resistance against nitrate. L. plantarum reached to the highest count of 8.81 log cfu/ml that was higher than that in the control (8.51 log cfu/ml) in spite of its increased generation time due to nitrate (Table 2). L. sakei and L. curvatus were influenced negatively by nitrate on their growth rate and their highest numbers. The most negative influence of nitrate on the growth rate was found in the batch inoculated with L. curvatus with an increase in generation time of 1.51 h (Fig. 4). The highest count of L. curvatus was 6.93 log cfu/ml, which was smaller than its highest count in the medium without nitrate (7.16 log cfu/ml) as shown in Table 2. In spite of concerns over the role of nitrite in the formation of nitrosamines and its mutagenic effect, there has been considerable interest in the value of this additive as an antimicrobial agent. Nitrite is one of the agents that interfere with the bacterial growth (Schlegel, 1985; White, 1975). This interest was focused mainly on the control of undesirable microorganisms such as Clostridium botulinum (Christiansen et al., 1975; Collins-Thompson et al., 1974), and S. aureus (Labots, 1976). LAB have been previously reported to be resistant to nitrite (Dodds & Collins-Thompson, 1984; Shank, Silliker, & Harper, 1962; Skjelkvåle & Tjaberg, 1974). However, inhibitive effects of nitrite on the growth of LAB in cured meat products have also been shown (Matsuoka, Furukawa, Takahashi, & Yamanaka, 1994). As mentioned above, it was found that nitrite as well as nitrate per se does not have inhibitory action on LAB (Dykhuizen et al., 1996; Pichner, Hechelmann, Steinstueck, & Gareis, 2006). The reactive intermediate compounds that converted from nitrate or nitrite such as N2O3, ONOO−, RS-NO are regarded to interfere with the functions of proteins (enzymes), membranes and DNA of target molecules and structures via Nnitrosylation, S-nitrosylation, nitrosyl-heme formation, disulphide
7 6 5 4 3 2 1 0 CB NiB
L.brevis 3.23 5.62
L.curvatus 5.57 4.55
L.plantarum 2.58 3.84
L.sake 3.63 6.43
613
formation and lipid peroxidation (Lundberg et al., 2004; Møller & Skibsted, 2002). In such a mechanism, however, lactobacilli are found to exhibit increased resistance (Dykhuizen et al., 1996). Among the tested strains isolated from kimchi within the present work, L. sakei, L. brevis, and L. plantarum were negatively influenced on their growth rates with the higher generation times as compared to that of the control as shown in Fig. 5. The most negative effect of nitrite on the initial growth rate was observed in the batch inoculated with L. plantarum by showing a reduction in numbers during the first 4 h as compared to that in the medium without nitrite (Table 2). However, L. brevis, L. plantarum, and L. sakei exhibited their resistance to the presence of nitrite by showing comparable to or even higher cell number than those in the medium without nitrite. For example, L. brevis had a highest number of 8.24 log cfu/ml which was slightly higher than that in the control (8.14 log cfu/ml). This tendency was observed also at the batch inoculated with L. sakei with the higher number of 8.25 log cfu/ml as compared to 8.17 log cfu/ml of the control (Table 2). As a matter of fact, it is expected that the nitrite tolerance of kimchi LAB is related to their nitrite reduction ability, since the reduction of nitrite content in the food matrix is regarded as detoxification mechanism improving the tolerance of bacteria against nitrite (Coleman et al., 1978). L. curvatus was sensitive to nitrite even though it showed the best nitrite reduction ability. Furthermore, L. brevis, L. plantarum, and L. sakei reduced nitrite were quite resistant to nitrite. Therefore, the results within the present work suggest that the nitrite resistance of kimchi LAB was not directly related to their ability to reduce nitrite, which agrees with the suggestion by Dodds and Collins-Thompson (1984).
4. Conclusion This study was conducted to investigate the potential utility of LAB isolated from kimchi, L. brevis KGR3111, L. curvatus KGR 2103, L. plantarum KGR 5105, and L. sakei KGR 4108, as starter culture in the production of fermented sausages with the capability to reduce and tolerate nitrate and nitrite added. The reduction capability of tested strains for nitrate was not dramatic, but nevertheless all tested strains reduced nitrate for 14.84–18.06% (54.61–66.46 mg NO− 3 /l) from the initial amount in 120 h. The nitrite reduction began after 24 h and from this point of time all tested strains showed the capability to produce nitrite reductase with the remnants of nitrite ranged from 24.21 to 41.55% of the start concentration, corresponding the reduction amount of 58.46–75.80 mg/l of NO− 2 . Nitrate and nitrite added had a negative effect on the growth of tested strains by increasing the generation time. The initial growth rate of L. curvatus, L. brevis, L. plantarum, and L. sakei was negatively influenced by the nitrate treated with the increased generation time. L. brevis, L. plantarum, and L. sakei were also influenced by the nitrite added with the decreased growth rate. Among them, however, L. brevis and L. plantarum showed the improved highest number of 8.71 log cfu/ml and 8.81 log cfu/ml in the case of nitrate treatment as compared to 8.18 log cfu/ml and 8.51 log cfu/ml without any treatment, respectively, indicating their tolerance against nitrate. The nitrite tolerance was found in the batches inoculated with L. brevis and L. sakei with the improved highest cell umber of 8.24 log cfu/ml and 8.25 log cfu/ml as compared to 8.18 log cfu/ml and 8.17 log cfu/ml in the control batches, respectively. As a result, L. brevis, L. plantarum, and L. sakei isolated from kimchi showed a tolerance against nitrate or nitrite with a good nitrite reduction `, indicating the satisfaction of one of the selection criteria for lactic acid bacteria to be used starter culture for fermented sausages.
Acknowledgments Fig. 5. Effect of sodium nitrite added into model medium designed for fermented sausage on the generation time of lactic acid bacteria isolated from kimchi. CB: control batch investigated in basic medium; NiB: batch investigated in nitrite medium.
The study presented in this paper was supported by the FriedrichEbert-Stiftung, Germany with a grant for Joo-Yeon Lee.
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