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Effects of salt concentration on Chinese sauerkraut fermentation
LWT - Food Science and Technology 69 (2016) 169e174
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Effects of salt concentration on Chinese sauerkraut fermentation Tao Xiong a, b, *, Junbo Li a, b, Fan Liang c, Yanping Wang a, b, Qianqian Guan a, b, Mingyong Xie a a
State Key Laboratory of Food Science & Technology, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China College of Food Science, Nanchang University, Nanchang, 330047, PR China c College of Food Science and Technology, Hunan Agricultural University, Changsha, 410128, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2015 Received in revised form 21 December 2015 Accepted 26 December 2015 Available online 29 December 2015
The aim of the study was to determine the effects of salt concentration on traditional sauerkraut fermented spontaneously. Lactic acid bacteria (LAB), fungi and Escherichia coli (E. coli) in the brine were analyzed in the three kinds of sauerkraut. The contents of sugars (sucrose, glucose, fructose) and organic acids (lactic acid, acetic acid) in the brine and inside the cabbage were monitored by high-performance liquid chromatography (HPLC). In addition, the pH value was monitored in the brine. Results demonstrated that sucrose and glucose were consumed and fructose was accumulated gradually during fermentation. The whole fermentation process was dominated by LAB and a considerable accumulation of lactic acid was observed both in cabbage and brine at the end of fermentation. Salt concentration had a significant effect on sauerkraut fermentation at early stage. The LAB population and metabolic rate was reduced and the yield of lactic acid decreased with the increase of salt concentration. Suitable salt concentration can effectively inhibit the reproduction of fungi and E. coli. In comparison, high salt concentration delayed the maturation of sauerkraut and inhibited the metabolism of LAB. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Chinese sauerkraut Salt concentration Lactic acid bacteria Fermentation Metabolism
1. Introduction It was recorded that Chinese sauerkraut (also known as “Pao Cai”) originated from Zhou Dynasty 3000 years ago (Zhang, 1994) and spread to South Korea in the 5th century (Ma, 2010). Chinese sauerkraut, as a typical brine-salted vegetable fermented by lactic acid bacteria (LAB), is widely consumed in China (Yan, Xue, Tan, Zhang, & Chang, 2008). Unlike kimchi which uses direct salting to withdraw juice from the cabbage (dry salting), traditional Chinese sauerkraut is anaerobic, fermented in brine with a low salt concentration (2%e10%) by the indigenous microorganism on the raw cabbage (Chen, 2007). LAB plays an important role during fermentation, because they contribute to sensory characteristics and preservation (Holzapfel, 1995). Fermentation of sauerkraut can be divided into hetero-fermentation and homo-fermentation phase, and species and quantity of LAB varies with fermentation stage. The initial phase was hetero-fermentation dominated by Leuconostoc citreum, Leuconostoc mesenteroides and Weissella
* Corresponding author. State Key Laboratory of Food Science & Technology, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China. E-mail address: [email protected] (T. Xiong). http://dx.doi.org/10.1016/j.lwt.2015.12.057 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
€nen, 2008), and koreensis et al. (Park et al., 2010; Wiander & Ryha then gradually transited to homo-fermentation phase, dominated by Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus sakei et al. (Kim et al., 2002; Plengvidhya, Breidt, Lu, & Fleming, 2007). It was also reported that the fermentation process of Chinese sauerkraut with 4% salt also experienced hetero-fermentative and successively homofermentative phase (Xiong, Guan, Song, Hao, & Xie, 2012). Salt concentration of sauerkraut had an effect on saline taste and microbial structure of brine so as to directly or indirectly affect the quality and flavor of sauerkraut. High salt concentration can better inhibit the growth of spoilage bacteria in brine, meanwhile, the first hetero-fermentative phase was absent, due to the intolerance towards salt of Leuconostocs (Cagno et al., 2009; Wouters et al., 2013). Now, many researches about the effect of salinity on utilization ratio of sugar and acid production during olive and cucumber fermentation have been reported (Efstathios & Constantinos, 2006; Frederico et al., 2005; Lu, Fleming, & Mcfeeters, 2001; McFeeters & rez-Díaz, 2010). The water activity and osmotic pressure of brine Pe with different combinations of salt in Spanish olive fermentation were described in detail (Panagou, Hondrodimou, Mallouchos, & Nychas, 2011). However, there is little information regarding the influence of salt concentration on the fermentation of Chinese
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sauerkraut. The aim of this study was to determine the effect of salt concentration on sauerkraut fermentation. The changes of pH, LAB, fungi and Escherichia coli in brine as well as the contents of organic acid, sugar, and sucrose in cabbage and brine under different salt concentrations were examined.
2.4.3. Statistical analysis Data was represented as mean values (n ¼ 4) ± standard deviation of means. Analysis of variance (ANOVA) was performed on the data obtained every 12 h, followed by Student's t test using SPSS 20. Differences were considered significant at p < 0.05. Origin8.6 software was used for mapping.
2. Materials and methods
3. Results and discussion
2.1. Materials
3.1. Changes of pH value during Chinese sauerkraut fermentation
Fresh cabbage and other auxiliary materials were purchased from a local supermarket in Nanchang, Jiangxi Province, China.
T, F, E represent for 2%, 5%, 8% salt concentration sauerkraut, further referred to as T, F, E. Error bars represent the standard deviation. The pH is a critical indicator of fermentation progress, and its drop occurred mainly due to lactic acid, the metabolism by LAB (Adams, 1990; Kandler, 1983). As shown in Fig. 1, the initial pH values of sauerkraut with different salt concentrations (2%, 5%, 8%, w/v) were between 6.35 and 6.50. The pH values decreased sharply at first and followed by a slowly decrease to a stable level, reducing to 4 in the 1st, 2nd and 3.5th day, respectively. Salt had a significant (P < 0.05) influence on pH values at the first 48 h in fermentation, the lower the salt concentration was, the faster the pH decreased, which may be because the acid producing ability of LAB was restrained gradually with the increasing of salt concentration mez et al., 2012). In contrast, salt had no significant (Rodríguez-Go (P > 0.05) effect on the changes of pH at the end of fermentation, but the lower salt concentration resulted in the lower pH.
Cabbages were cut into small pieces (2e3 cm 6e8 cm), then the pieces of cabbage were washed and drained as the raw material of sauerkraut. Fermentation were carried out in 5 L ceramic jars, each containing 1 kg sliced cabbage pieces and auxiliary materials, including crystal sugar (4%), hot red pepper (4%), garlic (3%), ginger (2%) and Chinese prickly ash (1.5%) (All percentages were calculated by the volume of sterile water). Salt (2%, 5%, and 8%, w/v) and sugar (4%) were dissolved in 2000 mL sterile water, saline solutions at the three concentrations were prepared as mentioned above. Finally cold sterile water was added into sauerkraut jar that was then water sealed. The sauerkraut jars were kept at ambient temperature (20e25 C) during experiments. 2.3. Sampling During the fermentation, brine (10 mL) and cabbage (10 g) samples were withdrawn aseptically every 12 h for 7 days, the sauerkraut jars were shaken before each sampling. A part of the brine was used for measurements of pH value and analysis of microbiological changes, the other part was stored at 20 C for HPLC analysis. The cabbage samples were pulped and diluted 10 times, 5 mL diluent was taken and then stored at 20 C for HPLC rez-Díaz, 2010). analysis (McFeeters & Pe 2.4. Analytical methods 2.4.1. Microbiological analysis 1 mL brine was aseptically added into 9 mL sterile saline (0.85% NaCl, w/v), appropriately diluted, 100 mL brine of 3 suitable gradient dilutions were respectively coated on the following flat plate, each with a parallel coating. Violet Red Bile Dextrose agar (VRBDA) for Enterobacteriaceae, the agar media were incubated at 37 C for 24 h, LAB on MRS incubated at 37 C for 48 h (Efstathios & Constantinos, 2006; Wang, Ren, Liu, Zhu, & Wang, 2013), Fungi on Yeast Extract Peptone Dextrose Medium agar (YPD) incubated at 25 C for 72 h. To avoid bacterial growth, YPD was supplemented with chloramphenicol (0.1 g L1; SigmaeeAldrich) (Wendy, Ilenys, rez, 2012). & Pe 2.4.2. Determination of organic acids, sugar and pH value HPLC (Model 1200, Agilent, USA) was used to determine the concentration of sugars and organic acids. For HPLC analysis, the brine and cabbage slurry samples were thawed and centrifuged at 10000 g for 10 min, then filtered through 0.22 mm membrane (Xiong, Li, Guan, Peng, & Xie, 2014). The pH value of the brine samples was measured using a pH meter (PHS-25, Shanghai Precision Scientific Instruments Company, China).
3.2. Microbiological changes during fermentation As shown in Fig. 2A, the original population of LAB were 3.14e3.34 log CFU/mL and then sharply increased at the first day in three fermentation, the concentration of T and F exceeded log8.0 CFU/mL at 1st and 1.5th day respectively. However, the growth of LAB in E was relatively slow, only reaching to 8.02 log CFU/mL in the 3rd day, probably due to the initiating strain of fermentation mainly was hetero-fermentative LAB with short metabolic cycle, poor salt tolerance and acid resistance (Wouters et al., 2013; Xiong et al., 2012). In the mid-late fermentation, the amount of LAB remained stable above 8.0 log CFU/ml, probably because the dominant LAB was homo-fermentative with strong salt-tolerance and acid-resistance (Chorianopoulos, Boziaris, rez-Díaz, 2010). Stamatiou, & Nychas, 2005; McFeeters & Pe Therefore, salt had a significant inhibitory effect on the growth of LAB in brine at early stage of fermentation, but the effect was not
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significant on the late fermentation of sauerkraut (Choi et al., 2003). It was reported that pectinase produced by fungi softened vegetables and led to deterioration in flavor (Etchells, Bell, Monroe, Masley, & Demain, 1958). Fungi was regarded as undesirable microorganism in sauerkraut fermentation (Wouters et al., 2013; Zhao & Ding, 2008). As shown in Fig. 2B, initial populations of fungi were about 3.75e4.00 log CFU/mL and reduced to an undetectable amount before the ripe of sauerkraut, but the specific growth rate was affected by the amount of salt. It was found that the fungi in T reached to 5.35 log CFU/mL in the 0.5th day, while that in F reached to 4.67 log CFU/mL in the 1st day, then both decreased gradually until disappeared. However, the amount of fungi in E was maintained at 3e4 log CFU/mL for 5 days, then rapidly decreased till uncountable in the 6.5th day. Heterofermentative LAB and yeast consumed sugar to produce carbon dioxide forming anaerobic environment in brine (Wendy et al., 2012; Xiong et al., 2014). It is presumed that the anaerobic environment, decreased pH, as well as elevated acidity may make fungi disappear. Salt was found to have a significant (p < 0.05) influence on the amount of fungi during fermentation. Compared with the other two salt concentration, 5% (w/v) salt not only reduced the peak of fungi, but also made fungi disappear firstly. As shown in Fig. 2C, the initial content of E. coli in three kinds of sauerkraut were 4.38e4.54 log CFU/mL, the growth curve of E. coli was similar to the fungi. The amount of E. coli in T and F rose to the
peak in the 36th hour and then decreased gradually, but E. coli in F passed through the peak and disappeared earlier than that in T. E. coli in E were inhibited and had no significant changes in cell population till the 5th day, and then sharply decreased. Results showed that salt had an obvious inhibitory effect on E. coli in Chinese sauerkraut, probably due to the combined effect of brine pH, LAB and salt concentration. It was also reported that the growth of E. coli was inhibited by lactic acid, bacteriocins and phenyllactic acid produced by lactic acid bacteria (Kim, Song, Yook, Ryu, & Byun, 2004; Li et al., 2015). 3.3. Changes in levels of sugars and organic acids during fermentation 3.3.1. Changes in levels of sugars during fermentation Crystal sugar, mainly containing sucrose as well as a small amount of glucose and fructose (Xiong et al. 2014), was added to the fermentation as initial carbon source. As shown in Fig. 3A, the content of sucrose in cabbage was quite different from that in brine, sucrose in brine quickly decreased while the increasing concentration of sucrose in cabbage was relatively low when fermentation started. When the content of sucrose in brine decreased to the same as that in cabbage, the content of sucrose inside and outside the cabbage synchronously declined, and total sucrose was mostly consumed in the end. However, salt had a significant (p < 0.05)
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effect on sucrose content of sauerkraut in the early fermentation stage. The lower the salt concentration was, the higher the content pez, Dura nQuintana, & of LAB in brine was (Bautista, Arroyo Lo ndez, 2010). Hetero-fermentative LAB, which can Garrido Ferna produce high-activity dextransucrase, catalyzing decomposition of sucrose (Hyun, Dong, & Nam, 2007), accounted for the majority of LAB in brine (Xiong et al., 2012). Sucrose content of Tb rapidly decreased while that in Tc increased slowly, probably due to the weak inhibition on the growth of microorganisms by high water activity as well as the slow diffusion of sucrose from Tb to Tc caused by low osmotic pressure (Frederico et al. 2005; Panagou et al. 2011). In contrast, sucrose content in Fc and Ec with higher salt concentration increased rapidly at the initial 36 h. In addition, the consuming rate of sucrose slowed down in late fermentation, as a result of the metabolism of homo-fermentative LAB, which was inhibited by the accumulation of lactic acid and the decrease of nchez, Rejano, Montan ~ o, & brine pH (Chorianopoulos et al., 2005; Sa de Castro, 2001). At the end of fermentation, E had the lowest coefficient of sucrose utilization, and sucrose remained the most inside and outside the cabbage. As shown in Fig.3B, at the early stage of the fermentation, the content of glucose in cabbage was 118.1 ± 4.2 mM and quickly decreased, while glucose concentration in brine was 11 ± 0.5 mM
and increased comparatively slowly. There were two possible sources of glucose in the brine, one may be permeation from the cabbage to the brine; the other may be the transformation of sucrose in the brine (McFeeters et al., 2010; Chorianopoulos et al., 2005). In comparison, the content of glucose in cabbage decreased the most but increased the least in Tb. These results suggested that glucose was consumed by microorganism, which grew the fastest among three fermentation. In the mid-late period of fermentation, the concentration of glucose tended to be stable and the concentration of glucose in the cabbage was slightly higher than that in the brine. Although glucose is a preferred energy source over sucrose and other monosaccharide by many microorganisms (Lu et al., 2001), the residual glucose determined in the study was high, presumably because the presence of sucrose reduced glucose utilization (Fasina, Fleming, & Thompson, 2002). The initial content of fructose in cabbage was much higher than that in the brine. The content of fructose decreased slowly throughout the fermentation in cabbage while fructose concentration increased rapidly in brine at the first 60 h of fermentation and then gradually leveled off. As shown in Fig. 3C, the sum amount of fructose in cabbage and brine was 103.8e125.7 mM finally, which was higher than the starting amount 93.7 ± 2.0 mM. These results indicated that the utilization rate of fructose was relatively
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low and more fructose was transformed than consumed in the brine. Fructose was not preferred carbon source of microorganisms in Chinese sauerkraut, a similar result was reported by other researchers in cucumber juice fermentation (Lu et al., 2001). 3.3.2. Changes in levels of main organic acids during fermentation The stability of the strains was directly influenced by the content of lactic acid, the main organic acid in sauerkraut (Xiong et al., 2014). As shown in Fig. 4A, the level of lactic acid increased slowly in the first 48 h and accumulated rapidly in the following 96 h, followed by a slow increase in the brine. An interesting phenomenon was observed that the content of lactic acid in cabbage was higher than in brine at the first periods, but lower at the last. While LAB reached a concentration of 108 CFU/mL in the second day and sucrose was quickly consumed at the same time, the amount of lactic acid was small. These results might be caused by two reasons, one is that fermentation was dominated by hetero-fermentative LAB at the early stage, which was poor in acid production; the other is that some microbes in brine can utilize sucrose but cannot produce lactic acid, such as yeast, mold and pathogenic bacteria (Wendy et al., 2012). In contrast, the fermentation was dominated by homo-fermentative lactic bacteria, which had better acidtolerance and acid-production ability in mid-late phase, but the accumulation of lactic acid in the last day was slow. This may be that the capacity of acid was affected by the accumulation of lactic acid and Hþ and LAB need more energy to maintain the stability of acid production (Lolkema, Poolman, & Konings, 1995; Wu et al. 2012). Salt had a significant impact on the production of lactic acid throughout fermentation in sauerkraut, the higher the salt concentration was, the lower the lactic acid production was (Zhao & Ding, 2008). Hetero-fermentative LAB produced equimolar amount of lactic acid, CO2 and acetic acid under aerobic conditions via 6P-gloconate pathway (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003). It was also found that a short acetic acid fermentation by acetic acid bacteria in early stage of fermentation in Chinese sauerkraut (Xiong, Peng, Li, Li, & Guan, 2015). Appropriate concentration of acetic acid can effectively improve the sensory properties of sauerkraut (Oliveira, Perego, Oliveira, & Converti, 2012). As shown in Fig. 4B, the level of acetic acid changed slightly faster from the 1.5th day to 4th day, then tended to be stable until the end of fermentation. The acetic acid content in cabbage was higher than that in the brine at the first day, which might be that the cabbage contains a high amount of acetic acid. Moreover, the content of acetic acid was the highest in the brine with 2% salt and varied
depending on different concentrations of salt in sauerkraut. The highest accumulation of acetic acid in T may benefit from its highest concentration of LAB from the first day to 4th day, during which fermentation was dominated by hetero-fermentative LAB (Xiong et al., 2012). In addition, the combination of acetic acid and ethanol, producing ethyl acetate, can enhance the flavor of sauerkraut. 4. Conclusion This study examined the impact of different salt concentrations (2%, 5%, 8%, w/v) on the fermentation profiles of Chinese traditional sauerkraut. Results showed that salt concentration mainly affected the early stage of sauerkraut fermentation significantly. Different salt concentrations resulted in the different osmotic pressure, water activity and strains structure as well as microbial metabolism and the rate of exchange substances (Wouters et al., 2013). Research has shown that 2% (w/v) salt accelerated the maturation of the sauerkraut, it can not effectively inhibit the growth of harmful microorganisms. In contrast, 5% salt can make fungi and E. coli in F passed through the peak and disappeared earlier. 8% salt in E not only delayed the maturation of sauerkraut but also minimized the utilization of sucrose and slowed down the metabolism of LAB. Meanwhile, although the emergence of the peak of fungi and E. coli was avoided, it failed to make them disappear rapidly. Taken together, 5% salt concentration can improve the quality of sauerkraut in traditional fermentation. To provide a reliable basis for the industrial production of Chinese sauerkraut, future researches in the field will include detailed experimental designs that will assess the effect of added salt on sensory, flavor and shelf life, in addition, accelerate the demise of harmful microorganisms in low salt sauerkraut by enhancing the hetero-fermentation. Acknowledgments The financial supports from the National Natural Science Foundation of China (NSFC, Project No. 31560449) and the National High Technology Research Development Key Program of China (863 Key Program, 2011AA100904), State Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-ZZB201309 and No. SKLF-ZZA-201303) are gratefully acknowledged. References Adams, M. R. (1990). Topical aspects of fermented foods. Trends in Food Science &
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calcium chloride instead of sodium chloride. Journal of Food Science, 75, C291eC296. Oliveira, R. P. S., Perego, P., Oliveira, M. N., & Converti, A. (2012). Growth, organic acids profile and sugar metabolism of Bifidobacterium lactis in co-culture with Streptococcus thermophilus: the inulin effect. Food Research International, 48, 21e27. Panagou, E. Z., Hondrodimou, O., Mallouchos, A., & Nychas, G. J. E. (2011). A study on the implications of NaCl reduction in the fermentation profile of Conservolea natural black olives. Food Microbiology, 28, 1301e1307. Park, J. M., Shin, J. H., Lee, D. W., Song, J. C., Suh, H. J., Chang, U. J., et al. (2010). Identification of the lactic acid bacteria in kimchi according to initial and overripened fermentation using PCR and 16S rRNA gene sequence analysis. Food Science and Biotechnology, 19, 541e546. Plengvidhya, V., Breidt, F., Lu, Z., & Fleming, H. P. (2007). DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations. Applied and Environmental Microbiology, 73, 7697e7702. mez, F., Bautista-Gallego, J., Romero-Gil, V., Arroyo-Lo pez, N. F., GarRodríguez-Go ndez, A., & García-García, P. (2012). Effects of salt mixtures on rido-Ferna Spanish green table olive fermentation performance. LWT e Food Science and Technology, 46, 56e63. nchez, A. H., Rejano, L., Montan ~ o, A., & de Castro, A. (2001). Utilisation at high pH Sa of starter cultures of lactobacilli for Spanish-style green olive fermentation. international Journal of Food Microbiol, 67, 115e122. Wang, X. H., Ren, H. Y., Liu, D. Y., Zhu, W. Y., & Wang, W. (2013). Effects of inoculating Lactobacillus sakei starter cultures on the microbiological quality and nitrite depletion of Chinese fermented sausages. Food Control, 32, 591e596. rez, D. (2012). Role of selected oxidative yeasts and Wendy, F., Ilenys, M., & Pe bacteria in cucumber secondary fermentation associated with spoilage of the fermented fruit. Food Microbiology, 32, 338e344. €nen, E. L. (2008). Identification of lactic acid bacteria strains Wiander, B., & Ryha isolated from spontaneously fermented sauerkraut and their use in fermentation of sauerkraut and sauerkraut juice in combination with a low NaCl content. Milchwissenschaft, 63, 386e389. Wouters, D., Bernaert, N., Conjaerts, W., Droogenbroeck, B. V., Loose, M. D., & Vuyst, L. D. (2013). Species diversity, community dynamics, and metabolite kinetics of spontaneous leek fermentations. Food Microbiology, 33, 185e196. Wu, C., Zhang, J., Chen, W., Wang, M., Du, G., & Chen, J. (2012). A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance. Applied Microbiology and Biotechnology, 93, 707e722. Xiong, T., Guan, Q. Q., Song, S. H., Hao, M. Y., & Xie, M. Y. (2012). Dynamic changes of lactic acid bacteriaflora during Chinese sauerkraut fermentation. Food Control, 26, 178e181. Xiong, T., Li, X., Guan, Q. Q., Peng, F., & Xie, M. Y. (2014). Starter culture fermentation of Chinese sauerkraut: growth, acidification and metabolic analyses. Food Control, 41, 122e127. Xiong, T., Peng, F., Li, X., Li, J. B., & Guan, Q. Q. (2015). Changes and metabolic characteristics of main microorganisms during Chinese sauerkraut fermentation. Food Science, 36, 158e161. Yan, P. M., Xue, W. T., Tan, S. S., Zhang, H., & Chang, X. H. (2008). Effect of inoculating lactic acid bacteria starter cultures on the nitrite concentration of fermenting Chinese paocai. Food Control, 19, 50e55. Zhang, S. S. (1994). Chinese pickles. Beijing: Higher Education Press. Zhao, D. Y., & Ding, X. L. (2008). Studies on the low-salt Chinese potherb mustard (Brassica juncea, Coss.) pickle. Idthe effect of a homofermentative L(þ)-lactic acid producer Bacillus coagulans on starter culture in the low-salt Chinese potherb mustard pickle fermentation. LWT e Food Science and Technology, 41, 474e482.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effects of salt concentration on Chinese sauerkraut fermentation Tao Xiong a, b, *, Junbo Li a, b, Fan Liang c, Yanping Wang a, b, Qianqian Guan a, b, Mingyong Xie a a
State Key Laboratory of Food Science & Technology, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China College of Food Science, Nanchang University, Nanchang, 330047, PR China c College of Food Science and Technology, Hunan Agricultural University, Changsha, 410128, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2015 Received in revised form 21 December 2015 Accepted 26 December 2015 Available online 29 December 2015
The aim of the study was to determine the effects of salt concentration on traditional sauerkraut fermented spontaneously. Lactic acid bacteria (LAB), fungi and Escherichia coli (E. coli) in the brine were analyzed in the three kinds of sauerkraut. The contents of sugars (sucrose, glucose, fructose) and organic acids (lactic acid, acetic acid) in the brine and inside the cabbage were monitored by high-performance liquid chromatography (HPLC). In addition, the pH value was monitored in the brine. Results demonstrated that sucrose and glucose were consumed and fructose was accumulated gradually during fermentation. The whole fermentation process was dominated by LAB and a considerable accumulation of lactic acid was observed both in cabbage and brine at the end of fermentation. Salt concentration had a significant effect on sauerkraut fermentation at early stage. The LAB population and metabolic rate was reduced and the yield of lactic acid decreased with the increase of salt concentration. Suitable salt concentration can effectively inhibit the reproduction of fungi and E. coli. In comparison, high salt concentration delayed the maturation of sauerkraut and inhibited the metabolism of LAB. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Chinese sauerkraut Salt concentration Lactic acid bacteria Fermentation Metabolism
1. Introduction It was recorded that Chinese sauerkraut (also known as “Pao Cai”) originated from Zhou Dynasty 3000 years ago (Zhang, 1994) and spread to South Korea in the 5th century (Ma, 2010). Chinese sauerkraut, as a typical brine-salted vegetable fermented by lactic acid bacteria (LAB), is widely consumed in China (Yan, Xue, Tan, Zhang, & Chang, 2008). Unlike kimchi which uses direct salting to withdraw juice from the cabbage (dry salting), traditional Chinese sauerkraut is anaerobic, fermented in brine with a low salt concentration (2%e10%) by the indigenous microorganism on the raw cabbage (Chen, 2007). LAB plays an important role during fermentation, because they contribute to sensory characteristics and preservation (Holzapfel, 1995). Fermentation of sauerkraut can be divided into hetero-fermentation and homo-fermentation phase, and species and quantity of LAB varies with fermentation stage. The initial phase was hetero-fermentation dominated by Leuconostoc citreum, Leuconostoc mesenteroides and Weissella
* Corresponding author. State Key Laboratory of Food Science & Technology, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China. E-mail address: [email protected] (T. Xiong). http://dx.doi.org/10.1016/j.lwt.2015.12.057 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
€nen, 2008), and koreensis et al. (Park et al., 2010; Wiander & Ryha then gradually transited to homo-fermentation phase, dominated by Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus sakei et al. (Kim et al., 2002; Plengvidhya, Breidt, Lu, & Fleming, 2007). It was also reported that the fermentation process of Chinese sauerkraut with 4% salt also experienced hetero-fermentative and successively homofermentative phase (Xiong, Guan, Song, Hao, & Xie, 2012). Salt concentration of sauerkraut had an effect on saline taste and microbial structure of brine so as to directly or indirectly affect the quality and flavor of sauerkraut. High salt concentration can better inhibit the growth of spoilage bacteria in brine, meanwhile, the first hetero-fermentative phase was absent, due to the intolerance towards salt of Leuconostocs (Cagno et al., 2009; Wouters et al., 2013). Now, many researches about the effect of salinity on utilization ratio of sugar and acid production during olive and cucumber fermentation have been reported (Efstathios & Constantinos, 2006; Frederico et al., 2005; Lu, Fleming, & Mcfeeters, 2001; McFeeters & rez-Díaz, 2010). The water activity and osmotic pressure of brine Pe with different combinations of salt in Spanish olive fermentation were described in detail (Panagou, Hondrodimou, Mallouchos, & Nychas, 2011). However, there is little information regarding the influence of salt concentration on the fermentation of Chinese
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sauerkraut. The aim of this study was to determine the effect of salt concentration on sauerkraut fermentation. The changes of pH, LAB, fungi and Escherichia coli in brine as well as the contents of organic acid, sugar, and sucrose in cabbage and brine under different salt concentrations were examined.
2.4.3. Statistical analysis Data was represented as mean values (n ¼ 4) ± standard deviation of means. Analysis of variance (ANOVA) was performed on the data obtained every 12 h, followed by Student's t test using SPSS 20. Differences were considered significant at p < 0.05. Origin8.6 software was used for mapping.
2. Materials and methods
3. Results and discussion
2.1. Materials
3.1. Changes of pH value during Chinese sauerkraut fermentation
Fresh cabbage and other auxiliary materials were purchased from a local supermarket in Nanchang, Jiangxi Province, China.
T, F, E represent for 2%, 5%, 8% salt concentration sauerkraut, further referred to as T, F, E. Error bars represent the standard deviation. The pH is a critical indicator of fermentation progress, and its drop occurred mainly due to lactic acid, the metabolism by LAB (Adams, 1990; Kandler, 1983). As shown in Fig. 1, the initial pH values of sauerkraut with different salt concentrations (2%, 5%, 8%, w/v) were between 6.35 and 6.50. The pH values decreased sharply at first and followed by a slowly decrease to a stable level, reducing to 4 in the 1st, 2nd and 3.5th day, respectively. Salt had a significant (P < 0.05) influence on pH values at the first 48 h in fermentation, the lower the salt concentration was, the faster the pH decreased, which may be because the acid producing ability of LAB was restrained gradually with the increasing of salt concentration mez et al., 2012). In contrast, salt had no significant (Rodríguez-Go (P > 0.05) effect on the changes of pH at the end of fermentation, but the lower salt concentration resulted in the lower pH.
Cabbages were cut into small pieces (2e3 cm 6e8 cm), then the pieces of cabbage were washed and drained as the raw material of sauerkraut. Fermentation were carried out in 5 L ceramic jars, each containing 1 kg sliced cabbage pieces and auxiliary materials, including crystal sugar (4%), hot red pepper (4%), garlic (3%), ginger (2%) and Chinese prickly ash (1.5%) (All percentages were calculated by the volume of sterile water). Salt (2%, 5%, and 8%, w/v) and sugar (4%) were dissolved in 2000 mL sterile water, saline solutions at the three concentrations were prepared as mentioned above. Finally cold sterile water was added into sauerkraut jar that was then water sealed. The sauerkraut jars were kept at ambient temperature (20e25 C) during experiments. 2.3. Sampling During the fermentation, brine (10 mL) and cabbage (10 g) samples were withdrawn aseptically every 12 h for 7 days, the sauerkraut jars were shaken before each sampling. A part of the brine was used for measurements of pH value and analysis of microbiological changes, the other part was stored at 20 C for HPLC analysis. The cabbage samples were pulped and diluted 10 times, 5 mL diluent was taken and then stored at 20 C for HPLC rez-Díaz, 2010). analysis (McFeeters & Pe 2.4. Analytical methods 2.4.1. Microbiological analysis 1 mL brine was aseptically added into 9 mL sterile saline (0.85% NaCl, w/v), appropriately diluted, 100 mL brine of 3 suitable gradient dilutions were respectively coated on the following flat plate, each with a parallel coating. Violet Red Bile Dextrose agar (VRBDA) for Enterobacteriaceae, the agar media were incubated at 37 C for 24 h, LAB on MRS incubated at 37 C for 48 h (Efstathios & Constantinos, 2006; Wang, Ren, Liu, Zhu, & Wang, 2013), Fungi on Yeast Extract Peptone Dextrose Medium agar (YPD) incubated at 25 C for 72 h. To avoid bacterial growth, YPD was supplemented with chloramphenicol (0.1 g L1; SigmaeeAldrich) (Wendy, Ilenys, rez, 2012). & Pe 2.4.2. Determination of organic acids, sugar and pH value HPLC (Model 1200, Agilent, USA) was used to determine the concentration of sugars and organic acids. For HPLC analysis, the brine and cabbage slurry samples were thawed and centrifuged at 10000 g for 10 min, then filtered through 0.22 mm membrane (Xiong, Li, Guan, Peng, & Xie, 2014). The pH value of the brine samples was measured using a pH meter (PHS-25, Shanghai Precision Scientific Instruments Company, China).
3.2. Microbiological changes during fermentation As shown in Fig. 2A, the original population of LAB were 3.14e3.34 log CFU/mL and then sharply increased at the first day in three fermentation, the concentration of T and F exceeded log8.0 CFU/mL at 1st and 1.5th day respectively. However, the growth of LAB in E was relatively slow, only reaching to 8.02 log CFU/mL in the 3rd day, probably due to the initiating strain of fermentation mainly was hetero-fermentative LAB with short metabolic cycle, poor salt tolerance and acid resistance (Wouters et al., 2013; Xiong et al., 2012). In the mid-late fermentation, the amount of LAB remained stable above 8.0 log CFU/ml, probably because the dominant LAB was homo-fermentative with strong salt-tolerance and acid-resistance (Chorianopoulos, Boziaris, rez-Díaz, 2010). Stamatiou, & Nychas, 2005; McFeeters & Pe Therefore, salt had a significant inhibitory effect on the growth of LAB in brine at early stage of fermentation, but the effect was not
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significant on the late fermentation of sauerkraut (Choi et al., 2003). It was reported that pectinase produced by fungi softened vegetables and led to deterioration in flavor (Etchells, Bell, Monroe, Masley, & Demain, 1958). Fungi was regarded as undesirable microorganism in sauerkraut fermentation (Wouters et al., 2013; Zhao & Ding, 2008). As shown in Fig. 2B, initial populations of fungi were about 3.75e4.00 log CFU/mL and reduced to an undetectable amount before the ripe of sauerkraut, but the specific growth rate was affected by the amount of salt. It was found that the fungi in T reached to 5.35 log CFU/mL in the 0.5th day, while that in F reached to 4.67 log CFU/mL in the 1st day, then both decreased gradually until disappeared. However, the amount of fungi in E was maintained at 3e4 log CFU/mL for 5 days, then rapidly decreased till uncountable in the 6.5th day. Heterofermentative LAB and yeast consumed sugar to produce carbon dioxide forming anaerobic environment in brine (Wendy et al., 2012; Xiong et al., 2014). It is presumed that the anaerobic environment, decreased pH, as well as elevated acidity may make fungi disappear. Salt was found to have a significant (p < 0.05) influence on the amount of fungi during fermentation. Compared with the other two salt concentration, 5% (w/v) salt not only reduced the peak of fungi, but also made fungi disappear firstly. As shown in Fig. 2C, the initial content of E. coli in three kinds of sauerkraut were 4.38e4.54 log CFU/mL, the growth curve of E. coli was similar to the fungi. The amount of E. coli in T and F rose to the
peak in the 36th hour and then decreased gradually, but E. coli in F passed through the peak and disappeared earlier than that in T. E. coli in E were inhibited and had no significant changes in cell population till the 5th day, and then sharply decreased. Results showed that salt had an obvious inhibitory effect on E. coli in Chinese sauerkraut, probably due to the combined effect of brine pH, LAB and salt concentration. It was also reported that the growth of E. coli was inhibited by lactic acid, bacteriocins and phenyllactic acid produced by lactic acid bacteria (Kim, Song, Yook, Ryu, & Byun, 2004; Li et al., 2015). 3.3. Changes in levels of sugars and organic acids during fermentation 3.3.1. Changes in levels of sugars during fermentation Crystal sugar, mainly containing sucrose as well as a small amount of glucose and fructose (Xiong et al. 2014), was added to the fermentation as initial carbon source. As shown in Fig. 3A, the content of sucrose in cabbage was quite different from that in brine, sucrose in brine quickly decreased while the increasing concentration of sucrose in cabbage was relatively low when fermentation started. When the content of sucrose in brine decreased to the same as that in cabbage, the content of sucrose inside and outside the cabbage synchronously declined, and total sucrose was mostly consumed in the end. However, salt had a significant (p < 0.05)
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effect on sucrose content of sauerkraut in the early fermentation stage. The lower the salt concentration was, the higher the content pez, Dura nQuintana, & of LAB in brine was (Bautista, Arroyo Lo ndez, 2010). Hetero-fermentative LAB, which can Garrido Ferna produce high-activity dextransucrase, catalyzing decomposition of sucrose (Hyun, Dong, & Nam, 2007), accounted for the majority of LAB in brine (Xiong et al., 2012). Sucrose content of Tb rapidly decreased while that in Tc increased slowly, probably due to the weak inhibition on the growth of microorganisms by high water activity as well as the slow diffusion of sucrose from Tb to Tc caused by low osmotic pressure (Frederico et al. 2005; Panagou et al. 2011). In contrast, sucrose content in Fc and Ec with higher salt concentration increased rapidly at the initial 36 h. In addition, the consuming rate of sucrose slowed down in late fermentation, as a result of the metabolism of homo-fermentative LAB, which was inhibited by the accumulation of lactic acid and the decrease of nchez, Rejano, Montan ~ o, & brine pH (Chorianopoulos et al., 2005; Sa de Castro, 2001). At the end of fermentation, E had the lowest coefficient of sucrose utilization, and sucrose remained the most inside and outside the cabbage. As shown in Fig.3B, at the early stage of the fermentation, the content of glucose in cabbage was 118.1 ± 4.2 mM and quickly decreased, while glucose concentration in brine was 11 ± 0.5 mM
and increased comparatively slowly. There were two possible sources of glucose in the brine, one may be permeation from the cabbage to the brine; the other may be the transformation of sucrose in the brine (McFeeters et al., 2010; Chorianopoulos et al., 2005). In comparison, the content of glucose in cabbage decreased the most but increased the least in Tb. These results suggested that glucose was consumed by microorganism, which grew the fastest among three fermentation. In the mid-late period of fermentation, the concentration of glucose tended to be stable and the concentration of glucose in the cabbage was slightly higher than that in the brine. Although glucose is a preferred energy source over sucrose and other monosaccharide by many microorganisms (Lu et al., 2001), the residual glucose determined in the study was high, presumably because the presence of sucrose reduced glucose utilization (Fasina, Fleming, & Thompson, 2002). The initial content of fructose in cabbage was much higher than that in the brine. The content of fructose decreased slowly throughout the fermentation in cabbage while fructose concentration increased rapidly in brine at the first 60 h of fermentation and then gradually leveled off. As shown in Fig. 3C, the sum amount of fructose in cabbage and brine was 103.8e125.7 mM finally, which was higher than the starting amount 93.7 ± 2.0 mM. These results indicated that the utilization rate of fructose was relatively
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Fig. 4. Change in the levels of lactic acid and acetic acid during fermentation of cabbage and brine with three different salt concentrations.
low and more fructose was transformed than consumed in the brine. Fructose was not preferred carbon source of microorganisms in Chinese sauerkraut, a similar result was reported by other researchers in cucumber juice fermentation (Lu et al., 2001). 3.3.2. Changes in levels of main organic acids during fermentation The stability of the strains was directly influenced by the content of lactic acid, the main organic acid in sauerkraut (Xiong et al., 2014). As shown in Fig. 4A, the level of lactic acid increased slowly in the first 48 h and accumulated rapidly in the following 96 h, followed by a slow increase in the brine. An interesting phenomenon was observed that the content of lactic acid in cabbage was higher than in brine at the first periods, but lower at the last. While LAB reached a concentration of 108 CFU/mL in the second day and sucrose was quickly consumed at the same time, the amount of lactic acid was small. These results might be caused by two reasons, one is that fermentation was dominated by hetero-fermentative LAB at the early stage, which was poor in acid production; the other is that some microbes in brine can utilize sucrose but cannot produce lactic acid, such as yeast, mold and pathogenic bacteria (Wendy et al., 2012). In contrast, the fermentation was dominated by homo-fermentative lactic bacteria, which had better acidtolerance and acid-production ability in mid-late phase, but the accumulation of lactic acid in the last day was slow. This may be that the capacity of acid was affected by the accumulation of lactic acid and Hþ and LAB need more energy to maintain the stability of acid production (Lolkema, Poolman, & Konings, 1995; Wu et al. 2012). Salt had a significant impact on the production of lactic acid throughout fermentation in sauerkraut, the higher the salt concentration was, the lower the lactic acid production was (Zhao & Ding, 2008). Hetero-fermentative LAB produced equimolar amount of lactic acid, CO2 and acetic acid under aerobic conditions via 6P-gloconate pathway (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003). It was also found that a short acetic acid fermentation by acetic acid bacteria in early stage of fermentation in Chinese sauerkraut (Xiong, Peng, Li, Li, & Guan, 2015). Appropriate concentration of acetic acid can effectively improve the sensory properties of sauerkraut (Oliveira, Perego, Oliveira, & Converti, 2012). As shown in Fig. 4B, the level of acetic acid changed slightly faster from the 1.5th day to 4th day, then tended to be stable until the end of fermentation. The acetic acid content in cabbage was higher than that in the brine at the first day, which might be that the cabbage contains a high amount of acetic acid. Moreover, the content of acetic acid was the highest in the brine with 2% salt and varied
depending on different concentrations of salt in sauerkraut. The highest accumulation of acetic acid in T may benefit from its highest concentration of LAB from the first day to 4th day, during which fermentation was dominated by hetero-fermentative LAB (Xiong et al., 2012). In addition, the combination of acetic acid and ethanol, producing ethyl acetate, can enhance the flavor of sauerkraut. 4. Conclusion This study examined the impact of different salt concentrations (2%, 5%, 8%, w/v) on the fermentation profiles of Chinese traditional sauerkraut. Results showed that salt concentration mainly affected the early stage of sauerkraut fermentation significantly. Different salt concentrations resulted in the different osmotic pressure, water activity and strains structure as well as microbial metabolism and the rate of exchange substances (Wouters et al., 2013). Research has shown that 2% (w/v) salt accelerated the maturation of the sauerkraut, it can not effectively inhibit the growth of harmful microorganisms. In contrast, 5% salt can make fungi and E. coli in F passed through the peak and disappeared earlier. 8% salt in E not only delayed the maturation of sauerkraut but also minimized the utilization of sucrose and slowed down the metabolism of LAB. Meanwhile, although the emergence of the peak of fungi and E. coli was avoided, it failed to make them disappear rapidly. Taken together, 5% salt concentration can improve the quality of sauerkraut in traditional fermentation. To provide a reliable basis for the industrial production of Chinese sauerkraut, future researches in the field will include detailed experimental designs that will assess the effect of added salt on sensory, flavor and shelf life, in addition, accelerate the demise of harmful microorganisms in low salt sauerkraut by enhancing the hetero-fermentation. Acknowledgments The financial supports from the National Natural Science Foundation of China (NSFC, Project No. 31560449) and the National High Technology Research Development Key Program of China (863 Key Program, 2011AA100904), State Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-ZZB201309 and No. SKLF-ZZA-201303) are gratefully acknowledged. References Adams, M. R. (1990). Topical aspects of fermented foods. Trends in Food Science &
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