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Food Microbiology 27 (2010) 741e748 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production Pornpan Jaichumjai a, b, Ruud Valyasevi c, Apinya Assavanig a, Peter Kurdi c, * a Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Center of Excellence for Agricultural Biotechnology: AG-BIO/PERDO-CHE, Commission on Higher Education, Ministry of Education, Thailand c Food Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand b a r t i c l e i n f o a b s t r a c t Article history: Received 10 November 2009 Received in revised form 22 March 2010 Accepted 23 March 2010 Available online 30 March 2010 The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The Hþ-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: Acid-sensitive Hþ-ATPase activity Internal pH Lactobacillus plantarum 1. Introduction Nham is a Thai-style fermented pork sausage that is made of minced pork, boiled pork rinds, cooked rice, garlic, salt, sugar, pepper, chili and sodium nitrite. It is packed in banana leaves or plastic sheets and allowed to ferment for approximately 3e4 days at ambient temperature (about 30e35 C in Thailand), after which period the pH of the product turns to 4.5e4.6 (Valyasevi and Rolle, 2002). Nham is either fermented spontaneously, or starter cultures are used to initiate fermentation for better quality control in terms of consistency and microbiological safety of the product. A hazard analysis found that the Nham fermentation’s critical limit for product safety is pH 4.6 or lower (Paukatong and Kunawasen, 2001). Lactobacillus plantarum BCC 9546 is one of the commercial Nham starter cultures available in the Thai market. Nham producers generally do not store the product at their factories, storage usually takes place at the retailer’s facilities in refrigerated conditions. Many Nham manufacturers tend to keep the product at room temperature during product handling, transportation or storage which can result in prolonged fermentation * Corresponding author. Tel.: þ66 2 564 6700x3752; fax: þ66 2 564 6590. E-mail address: [email protected] (P. Kurdi). 0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2010.03.014 (over-fermentation). This process is mainly caused by lactic acid bacteria that produce organic acids, mostly lactic acid, which contribute to a variety of undesired flavor and texture changes in Nham such as water dripping, discoloration and off-flavor development. Since Nham producers prefer reducing the product loss caused by over-fermentation there is a considerable interest in an economic solution to prevent over-fermentation. A mutated starter culture whose growth is sensitive to high acidity, i.e. pH 4.6, may offer a strategy to the manufacturer to overcome the above problem.. The potential new acid-sensitive starter strain would possibly have reduced energy metabolism and would be unable to continue the fermentation below pH 4.6, thereby over-fermentation could be prevented. There are several reports about acid-sensitive mutants of lactic acid bacteria such as an acid-sensitive Lactobacillus delbrueckii subsp. bulgaricus in yoghurt fermentation has reduced post-acidification that prolongs the viability of Bifidobacterium breve in yoghurt during refrigerated storage (Ongol et al., 2007). An Oenococcus oeni mutant that lacks malolactic activity (Galland et al., 2003), as well as an acidsensitive variant strain of Lactobacillus helveticus (Yamamoto et al., 1996) and Streptococcus bovis mutants (Miwa et al., 2000) had reduced Hþ-ATPase activity. These mutants were obtained with the use of neomycin as a selective tool for isolating Hþ-ATPase deficient 742 P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748 strains. In Escherichia coli, a correlation between resistance to neomycin (Kanner and Gutnick, 1972) and defects in ATPase activity has been reported. Some of the independently obtained aminoglycoside antibiotic-resistant ATPase mutants of Escherichia coli appeared to have membranes leaky to protons, and a decrease in proton gradient across such membranes was demonstrated (Tsuchiya and Rosen, 1975; Humbert and Altendorf, 1989). It was postulated that defective Hþ-ATPase would lead to cellular energy shortage that would result in a reduced uptake of neomycin into the cell, thereby leading to resistance to the antibiotic (Humbert and Altendorf, 1989). Hþ-ATPase plays an important role in cytoplamic pH regulation in lactic acid bacteria by expelling protons out of the cell utilizing the energy released by ATP hydrolysis. Mutants defective in Hþ-ATPase are impaired for survival at low extracellular pH (Nannen and Hutkins, 1991), likely because many cytoplasmic enzymes have their pH optima in a neutral range (Hutkins and Nannen, 1993). Besides the Hþ-ATPase there are also other enzymes that may contribute to pH homeostasis of lactic acid bacteria such as glutamate decarboxylase and arginine deiminase. It was demonstrated that in Lactobacillus ssp. glutamate decarboxylase catalyze the decarboxylation of glutamate, resulting in the stoichiometric release of the end products g-aminobutyrate (GABA) and CO2 (Hanaoka, 1967). The reaction product, GABA is exported from the cell via the Glu2/GABA antiporter, leading to the decrease of cytoplasmic Hþ ion concentration and a slight increase of the internal pH. The arginine deiminase (ADI) pathway may also contribute to pH homeostasis converting arginine to ammonia, ornithine and carbon dioxide and generating 1 mol of ATP per mol of arginine. Ammonia production (contingent upon arginine availability) may contribute to survival at acidic environment by neutralizing the pH. This process has been reported in oral streptococci (Marquis et al., 1987; Casiano-Colòn and Marquis, 1988; Curran et al., 1995) as well as in Streptococcus lactis and Streptococcus cremoris (Poolman and Konings, 1988) and in a L. plantarum strain isolated from orange peel (Arena et al., 1999). All the above three mechanisms reduce acidification of the internal compartment and are important in maintaining acid resistance for the survival of lactic acid bacteria. L. plantarum is one of the most intensively studied Lactobacillus strains, however the application of its mutant as an acid-sensitive starter have not been reported to date. In this study we attempted to isolate an acid-sensitive L. plantarum strain with mutation in either Hþ-ATPase, glutamate decarboxylase or arginine deiminase and employ it in Nham production to hinder the fermentation below pH 4.6 and subsequently prolong the shelf life during storage at ambient temperature (30 C). 2. Materials and methods 2.1. Bacterial strains: acid isolation of acid-sensitive mutants L. plantarum BCC 9546 and Pediococcus acidilactici BCC 9545 were obtained from BIOTEC Culture Collection, Pathumthani, Thailand. Lactobacillus brevis LSF 8-13 was a gift from Chulalongkorn University, Thailand. Bacteria were grown in MRS medium until mid-exponential phase was reached (A660 about 0.8e0.9). Then 0.1 ml aliquots of 10-and 100-fold dilution of the culture was spread onto half strength MRS plates containing 600 or 750 mg/ml neomycin sulfate for acidsensitive mutant generation and incubated at 30 C for 2 days. After that neomycin plates were used as master plates for replica-plating on half strength MRS pH 4.5 and on half strength MRS plates. After incubation at 30 C for 2 days, the acid-sensitive colonies, identified as the ones unable to grow on half strength MRS pH 4.5 plates, were picked up from the half strength MRS replica plates. They were cultured in MRS broth for 15 h and adjusted to A660 about 0.8 then were used as inoculum (2%) for the growth experiments in MRS broth. The pH of the culture broth was monitored for 24e72 h and the isolates with the highest culture broth pH were stored in 20% glycerol at 80 C. To obtain isolates with higher acid-sensitivity, mildly acid-sensitive mutants (pH 4.2) were re-streaked onto half strength MRS containing 1500 mg/ml neomycin plates and incubated at 30 C for 24e48 h then each single colony was inoculated to MRS broth, incubated at 30 C for 15 h. The cultures were adjusted to A660 around 0.8 then inoculated (2%) to MRS broth. The pH of the culture broth was monitored for 24e72 h and the isolates with the highest culture broth pH were kept and stored in 20% glycerol at 80 C. 2.2. Measurement of growth characteristics Growth parameters such as viable count, culture pH, and titratable acidity were measured during 72 h culture in MRS broth at 30 C. Samples were taken from the culture broth at various time points and were serially diluted with sterile saline and spread onto half strength MRS plates. Viable counts were determined after 2 days of incubation at 30 C. Another portion of the sample was diluted 50-fold with CO2-free distilled water and titratable acidity was measured by the method of AOAC (2000). The pH of the culture broth was measured using a pH meter (Mettler Toledo, Switzerland). Growth was monitored by measurement of absorbance at 660 nm (Helios-a spectrophotometer, Thermo Electron Corp., UK). 2.3. Measurement of Hþ-ATPase activity Activity of Hþ-ATPase was assayed using the method of Matsumoto et al. (2004) with minor modifications. Cells cultivated in MRS broth overnight at 30 C were harvested by centrifugation (8000 g for 5 min) and washed three times in sterile saline, then harvested cells were suspended in sterile saline to an A660 around 27. Each bacterial suspension (1.0 ml) was added to 10 ml of MRS broth with different pH (4.0, 5.0, 6.0 and 7.0 adjusted with L-lactic acid) and then was incubated for 1 h at 30 C. Cells were centrifuged at 8000 g for 5 min, resuspended in 1 ml of 75 mM TriseHCl buffer (pH 7) containing 10 mM MgSO4 and permeabilized by the addition of 30 ml of toluene:acetone mixture (1:9, v:v; Lauret et al., 1996) and vigorous mixing for 5 min. Then permeabilized cells were kept at 80 C before use. Hþ-ATPase activity assay was conducted as described by Belli and Marquis (1991) with minor modifications. Permeabilized cells (100 ml) were mixed with 1 ml of 50 mM Trisemaleate buffer (pH 6.0) containing 10 mM MgSO4 and 10 ml of 0.4 M N,N0 -dicyclohexyl carbodiimide (DCCD; Wako Pure Chemicals Industries Ltd., Osaka, Japan) in ethanol or 10 ml of ethanol (as control). Mixtures were incubated at 37 C for 1 h, then a 10 ml aliquot of 0.5 M ATP (Fluka) was added to start the assay. Mixtures were incubated at 37 C for 20 min and subsequently put on ice to stop the reaction. Liberated inorganic phosphate (Pi) in the reaction mixtures was measured using the Phosphor C test kit (Wako). Results of the assay are expressed as the amount of Pi produced per minute per mg protein of permeabilized cells. Protein content of the cells was determined by the Bradford method (Bradford, 1976). 2.4. Internal pH measurement Bacterial intracellular pH was measured as outlined by Kurdi et al., (2000). Strains were cultured in MRS broth to mid-exponential phase (6e7 h) at 30 C, harvested and washed two times in ice cold 150 mM KPO4, 1 mM MgSO4, pH 7.0 buffer then resuspended in 150 mM KPO4, 1 mM MgSO4, pH 7.0 buffer to A660 w 10. The cell suspension was diluted to a final volume of 9 ml with prewarmed buffer (A660 w 0.5), P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748 and then preloaded with the membrane permeable precursor probe 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester (Molecular Probes Inc., Eugene, OR, USA) at 30 C for 1 h. After that the cell suspension was centrifuged (8,000 g for 5 min) and the cells were resuspended in 150 mM KPO4, 1 mM MgSO4 buffer, pH 7.0, then 1 M glucose was added to a final concentration of 10 mM and the mixture was incubated at 30 C for 1 h, divided into two parts and centrifuged (8000 g for 5 min). One part was resuspended in the same buffer (pH 7.0), while the other part was resuspended in the same buffer with pH 5.0. The two cell suspensions were incubated at 30 C for another 30 min. After washing once, the cells were resuspended in the same buffer followed by internal pH measurements using a spectrofluorometer (FP 6500, Jasco Co., Tokyo, Japan) with excitation and emission wavelength of 490 and 520 nm, respectively. Calibration of the fluorescent signal was carried out using de-energized cells in buffers pH 5.0 and pH 7.0. De-energization was achieved by adding valinomycin (Fluka) and nigericin (Fluka), both from a 2 mM stock to a final concentration of 2 mM, while DCCD (0.01 mM final concentration) was added to the cells in the cuvette (30 C) placed in the spectrofluorometer when its effect on the internal pH of the cells was investigated. 2.5. Measurement of arginine deiminase activity Every strain was grown to stationary phase (15e24 h) in GYPeArg broth, while L. plantarum BCC 9546 was also inoculated to GYP broth and used as Arg-free control. The GYP medium contains (g/l): 0.2, glucose; 3, yeast extract; 5, peptone; 1 ml, Tween 80; 5 ml, salt solution. The salt solution comprised of (g/l): 40, MgSO4$7H2O; 2, MnSO4$4H2O; 2, FeSO4$7H2O; 2, NaCl. Such GYP medium was supplemented with 3.484 g/l, arginine (20 mM) (GYPeArg) and both media were adjusted to pH 6.5 with 6 N HCl before sterilization at 121 C for 15 min. All L. plantarum strains and P. acidilactici BCC 9545 as positive control were harvested by centrifugation at 8000 g for 5 min at 25 C suspended in the same media to A660 around 1, then these cell suspensions were used to inoculate (1%) GYPeArg broth and GYP broth. Fifty ml samples were taken at 0, 9, 24, 48 and 72 h. The arginine deiminase activity was detected in terms of ammonia production from arginine by using NHþ 4 ion-selective electrode (Mettler Toledo, Switzerland), and was defined as the amount of ammonium ion produced per mg of protein. The protein concentrations of the cell suspensions were determined by Bradford method using bovine serum albumin as standard (Bradford, 1976). 2.6. Determination of glutamate decarboxylase activity Bacterial strains were cultured in GYPeGlu medium (per liter: glucose, 5 g; proteose peptone, 5 g; yeast extract, 5 g; Tween 80, 1 ml; salt solution (as described above), 5 ml; monosodium glutamate, 9.36 g (50 mM)) for 24 h. Cells were harvested by centrifugation (8000 g for 5 min), then incubated at A660 w 10 in 50 mM sodium acetate buffer pH 4.5 containing 50 mM monosodium glutamate for 24 h. After centrifugation (8000 g for 5 min), 10 ml supernatants were subjected to TLC analysis on Silica gel 60 F254 plates (Merck) using 1-butanol:glacial acetic acid:H2O ¼ 3:2:1 as solvent. The TLC plate was developed with 0.5% ninhydrin in acetone. A known g-aminobutyric acid (GABA) producer L. brevis LSF 8-13 was used as positive control. The amount of glutamate that Lactobacillus strains consumed was measured by a biochemical analyzer (YSI 7100MBS, YSI Inc., Yellow Springs, Ohio, USA) using glutamate specific membrane. Assuming that consumed glutamate was fully converted to GABA by the intracellular glutamate decarboxylase enzyme the GABA production of our strains was calculated. The protein concentrations of the cell suspensions were determined as mentioned above. 743 2.7. Nham fermentation One loopful of stock cultures of L. plantarum BCC 9546 and N7501 were cross-streaked on half strength MRS agar and half strength MRS agar plates containing 1500 mg/ml neomycin, respectively and were incubated at 30 C for 24e48 h. A single colony of bacteria was transferred into 5 ml of MRS broth and incubated at 30 C for 15 h. Cells were harvested by centrifugation at 25 C at 8000 g for 5 min and washed with 5 ml of 0.1% sterile peptone water. Finally, the cell concentration was adjusted to 107 cfu/ml with 0.1% sterile peptone water. Nham sausages were mainly produced at Product Development Division, Department of Livestock, Pathumthani using the standard recipe (Visessanguan et al., 2004), and transported to the laboratory for further analysis. Nham was prepared by mixing ground pork (52%), cooked pork rind (35%), sucrose (0.4%), garlic (4.3%), salt (1.9%), cooked rice (4.3%), sodium erythorbate (0.2%), sodium tripolyphosphate (0.2%), monosodium glutamate (0.2%), whole bird chili (2%), and potassium nitrite (0.01%). Half of the sausages (two batches) were prepared with a modified formula which contained the same ingredients but only 3% of garlic. The ingredients were thoroughly mixed and stuffed into plastic casings (3.0 cm diameter, approximately 200 g each). A total of four batches of Nham were prepared by combining two kinds of starter cultures (the wild-type and N750-1) with two kinds of Nham formulations (normal formulation and reduced garlic (3%) formulation). Nham sausages were incubated at 30 C for 7 days. Samples were taken at 0 h, 12 h and every 24 h up to 7 days. Nham sample (25 g) was aseptically transferred to a sterile plastic bag and pummeled at 230 rpm for 30 s in a stomacher Model 400 (Seward, England), with 225 ml of sterile peptone water. Appropriate decimal dilutions of the samples were prepared using the same diluent and 0.1 ml of each dilution was plated in triplicates on half strength MRS agar containing 0.5% CaCO3 incubated at 30 C for 1 day for total LAB count. After that, all colonies were replicaplated on half strength MRS-neomycin plates (1500 mg/ml of neomycin sulfate) containing 0.5% CaCO3 and incubated at 30 C for 2 days for neomycin-resistant mutant count. Direct pH measurements were carried out using a pH meter. 2.8. Statistical analysis Results are expressed as means standard deviations of triplicate analyses for each sample unless otherwise stated. A one-way analysis of variance and Duncan’s multiple range tests were used to establish the significance of differences among the mean values at the P < 0.05 significance level. The statistical analyses were performed using SPSS version 15.0 for Windows (2006). 3. Results and discussion 3.1. Isolation of acid-sensitive mutants of L. plantarum BCC 9546 Approximately 6,700 spontaneous neomycin-resistant mutants (600 mg/ml neomycin sulfate) were obtained with a frequency of 105e106. Only one mutant was found to have its culture broth pH near the desired pH 4.6 after 72 h of growth (strain R5 with a culture broth pH of 4.4). Whereas, among the mutants resistant to 750 mg/ml neomycin sulfate, strain N750 had a culture broth pH of 4.5 at 24 h which decreased to pH 4.19 at 72 h. To generate mutants with resistance to higher neomycin concentration, R5 and N750 were restreaked on half strength MRS plates containing 1500 mg/ml neomycin sulfate. Twenty-six colonies of R5 mutants were found not to lower the pH of the MRS broth below 4.4 during 72 h of incubation at 30 C. In addition, four of N750 mutants resistant to 1500 mg/ml neomycin sulfate had their culture pH above 4.4 after 72 h of incubation at 30 C. P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748 From the above mutants with culture broth pH not lower than 4.4 after 72 h, three representative strains were selected for further studies, namely: R5 (resistant to 600 mg/ml neomycin), R5-18 (an R5-derivative resistant to 1500 mg/ml neomycin) and N750-1 (an N750-derivative resistant to 1500 mg/ml neomycin). different pHs (pH 4e7, this pH range resembles that of in Nham between the beginning and the end of fermentation). Table 1 shows the ATPase activity of L. plantarum BCC 9546 and its acid-sensitive mutants at different acidic pHs in the presence (A) and absence (B) of DCCD, a relatively specific inhibitor of the F0 part of the F0F1ATPase. The difference in ATPase activity with or without DCCD was also calculated and since it is the activity inhibited by DCCD this value can be considered as the activity of Hþ-ATPase (Table 1C). The ATPase activity of all mutant strains were significantly lower than that of wild-type strain at all conditions and at pH 4 the ATPase activity of all mutants decreased below the values at other pHs (5e7). Since the Hþ-ATPase activity (DCCD inhibited) of the wildtype strain was significantly higher than those of all mutant strains at the above pH interval it indicates that the mutants had reduced membrane-bound Hþ-ATPase activity. It is postulated that mutants with reduced Hþ-ATPase activity do not generate sufficient energy to concentrate neomycin, therefore they are resistant to neomycin because neomycin uptake by bacterial cells is energy dependent process (Kanner and Gutnick, 1972). Such defect in the energy metabolism in our acid-sensitive mutants might explain their growth characteristics and reduced acid production in MRS broth. 3.2. Growth characteristics of the representative mutants Growth and acid production of the three acid-sensitive mutants compared to parent strain BCC 9546 are shown in Fig. 1 A and B, respectively. After 8 h of culturing, the cell number of R5-18 and N750-1 were similar to that of the wild-type strain, while the viable count of R5 was slightly lower. Afterwards, until 48 h of cultural time, the viable count of the three mutants declined by 3 to 10 fold and were lower than that of the wild-type strain. However, after 48 h the cell number of R5 and R5-18 increased again and surpassed that of the wild-type strain while the viable count of N750-1 remained stagnant (Fig. 1A). The acid production of all strains increased until 24 h culture time followed by virtually no or substantially less acid production in the next 24 h. While no more acids were produced by the parent strain and N750-1 the acidity of the culture broths of R5 and R5-18 were increased considerably between 48 and 72 h (Fig. 1B). Overall, N750-1 produced about half as much acid as the wild-type after 72 h, while the acid production of the other two mutants did not differ much from that of N750-1 until 48 h. The tendencies observed in acid production were also reflected in the pH values of the culture broths (Fig. 1B). The pH of the culture broth of all mutants differed from that of the wild-type strain by one pH unit at 8 h and the pH of their culture broths did not decrease between 24 and 48 h. Yet after 48 h the culture broth pH of R5 and R5-18 decreased nearly to the pH value of the wild-type strain’s culture. Only the culture broth pH of N750-1 (w4.5) was substantially higher than that of the wild-type at the end of the 72 h culturing period. Taken together these observations the cell number increase of R5 and R5-18 accompanied by increased acid production and culture pH reduction between 48 and 72 h suggest that the mutations that made R5 and R5-18 acid-sensitive are unstable and can be reversed after 48 h of continuous culture. This makes R5 and R5-18 not suitable for the application as Nham starter cultures. On the other hand, the acid-sensitivity of N750-1 is probably caused by a stable mutation and seem to be at the desired level, since this strain appear to reduce the pH of the culture broth no lower than about 4.5, what makes it a good candidate for a new Nham starter culture. 3.4. Measurement of internal pH In order to characterize the pH homeostasis of acid-sensitive mutants, their internal pH upon energization with glucose was investigated. Internal pH (pHi) measurements revealed that the mutants had significantly lower pHi (by about 0.2 pH unit) at external pH 7.0 than that of wild-type (Table 2). At acidic condition (pH 5.0) the internal pH of the mutants were also lower than the pHi of the wild-type however only that of N750-1 did by a significant margin. At pH 5.0 the difference between external and internal pH (DpH) of the parent strain was one unit, whereas N750-1 could maintain a DpH of only 0.43, and it is likely that acidification of the cytoplasm under acidic condition affects the physiology of the latter strain considerably. Lactic acid is a weak organic acid with increased proportion of its protonated (neutral) form at low pH that can freely pass the cell membrane, then dissociating at the more alkaline cytoplasmic pH (Kashket, 1987). This possibly results in the disturbance of the internal pH regulation in N750-1, at least, due to its reduced Hþ-ATPase activity, therefore it cannot maintain its cytoplasmic pH near neutral at external pH 5.0 as the wild-type strain. In order to reveal the overall effectiveness of other internal pH maintenance processes DCCD was applied during the internal pH measurements to inhibit Hþ-ATPase. A representative experiment shown in Fig. 2 demonstrates that after the cells were first energized by addition of glucose (10 mM), addition of DCCD resulted in 3.3. ATPase activity under acidic conditions To investigate the cause of acid-sensitivity, we measured the ATPase activity of the wild-type and the three mutant strains in 9.5 6.0 pH (solid lines) B 6.5 log cfu/ml A 10.0 9.0 8.5 8.0 7.5 7.0 2.5 2.0 5.5 1.5 5.0 4.5 1.0 4.0 .5 3.5 0 24 48 Time (h) 72 0.0 0 24 48 Total Acidity (%, dashed lines) 744 72 Time (h) Fig. 1. Viable cells (A), pH and acidity (B) in the MRS culture broth of L. plantarum strains. C, B: L. plantarum BCC 9546; ;, 7: R5; A, >: R5-18; -, ,: N750-1. The black and white symbols in Fig. 1 B represent pH and acidity, respectively. The symbols represent means standard deviation (error bars) obtained from three independent experiments. P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748 Table 1 ATPase activity of L. plantarum BCC 9546 and its acid-sensitive mutants at different pHs (pH 4e7). pH 4.0 pH 5.0 pH 6.0 pH 7.0 (A) ATPase activity (nmol Pi min1 mg protein1) assayed in the absence of DCCD 69.28 4.56bcC 73.99 7.83cC Wild-type 50.15 6.75aB 63.85 4.36bC R5 3.53 0.48aA 6.93 1.04bA 9.13 0.54cA 8.62 0.54cA R5-18 6.50 2.23aA 15.35 1.67bB 13.59 0.57bB 14.03 1.61bB N750-1 3.37 0.69aA 4.65 2.06abA 8.55 1.17cA 6.32 2.16bcA Strain pH 5.0 pH 6.0 (B) ATPase activity assayed in the presence of DCCD 41.61 3.56bcC Wild-type 19.70 4.21aA 37.60 3.11bC R5 0.57 0.46aB 2.41 1.42bA 3.86 0.42cA R5-18 2.37 0.60aB 9.93 2.21cB 7.15 0.72bB N750-1 1.90 1.20aB 4.21 1.86abA 6.12 1.45bAB Strain pH 4.0 pH 5.0 pH 6.0 pH 7.0 27.67 6.62aB 5.27 0.52bA 6.44 1.03aA 2.43 0.57bA 28.54 5.37aB 5.67 0.71bA 6.44 2.63aA 1.50 1.15abA a decline in internal pH. After energization the cytoplasm became more alkaline than the extracellular buffer, and assuming that DCCD inhibited proton translocating ATPase, decrease in the internal pH upon DCCD addition was likely the result of a net proton leak through the cell membrane. When the rate of internal pH decrease was measured following the DCCD addition, the results revealed that (Table 2) the wild-type had the smallest proton leak among the strains at both tested pHs, while the internal pH decrease rate was highest in N750-1 at acidic condition (pH 5.0). This also implies that the mechanisms involved in pH homeostasis excluding the Hþ-ATPase are probably the weakest in N750-1 among the strains at least at acidic conditions. Moreover, that is coupled with the lowest Hþ-ATPase activity in N750-1 which is probably the main reason behind that this strain has the smallest internal pH at external pH 5.0. Since these parameters indicate disturbed pH homeostasis and smaller cellular energy level (smallest DpH), it is reasonable to assume that all these factors combined have also a profound impact on the survival of this strain in acidic environment. Table 2 Internal pH and proton leak in L. plantarum BCC 9546 and its acid-sensitive mutants at extracellular pH (pHex) 7.0 and 5.0. Internal pHa Proton leakb pHex Wild-type R5 R5-18 N750-1 DCCD 7.20 Val 7.15 7.10 0 45.45 4.78cC 2.94 0.32bcA 7.59 1.91bcB 4.82 1.89bAB Hþ-ATPase ¼ (Total ATPase in the absence of DCCD) - (Total ATPase in the presence of DCCD). Mean values and standard deviations obtained from three independent experiments. Different capital letters (AeC) in the same column as well as different letters (aec) in the same row indicate significant differences (P < 0.05). Strain Glu 7.25 7.00 pH 7.0 DCCD inhibited (C) ATPase activity inhibited by DCCD 26.25 4.35aC Wild-type 30.45 8.09aB R5 2.96 0.45aA 4.52 1.70bAB R5-18 4.13 2.07aA 5.43 2.91aB N750-1 1.46 1.06abA 0.44 0.43aA 7.30 pH 7.0 pH 5.0 pH 7.0 pH 5.0 7.46 0.02 7.22 0.04* 7.22 0.03* 7.25 0.01* 6.02 0.16 5.83 0.38 5.58 0.23 5.43 0.03* 0.007 0.009 0.023 0.016 0.009 0.001 0.022 0.001 0.014 0.006 0.024 0.007 0.027 0.015 0.052 0.015 Results are means standard deviation obtained from three (a) or two (b) independent experiments. *Statistically different (P < 0.05) from that of the wild-type strain at the same pH. a Measured as described in Section 2. b Unit of internal pH decrease (per minute) in energized cells after a few minutes of DCCD addition, see Fig. 2. Nig 7.05 þDCCD pH 4.0 + H -leak 7.35 I nternal pH DCCD 7.40 5 10 15 Time (min) Fig. 2. Proton leakage in the mutant N750-1 at pH 7.0 upon DCCD addition. Glucose (10 mM, Glu) energized the cells preloaded with cFSE in 150 mM KPO4 buffer containing 1 mM MgSO4 followed by the addition of Hþ-ATPase inhibitor DCCD (final concentration 10 mM) that triggered an internal pH decline attributed to Hþ-leak (arrow). The DJ and DpH component of the proton motive force was dissipated by the addition of 2 mM valinomycin (Val) and 2 mM Nigericin (Nig), respectively. This figure is a representative of two independent experiments that gave similar results. 3.5. Measurement of arginine deiminase activity The arginine deiminase activity may also contribute to the pH homeostasis in some lactic acid bacteria (Marquis et al., 1987) including a L. plantarum strain from orange (Arena et al., 1999) therefore the ammonium ion production from arginine by our L. plantarum strains was investigated. Fig. 3 shows that wild-type and mutant L. plantarum strains produced very small amounts of ammonium ion. Their ammonium ion production profiles were not significantly different from each other at every sampling point. The ammonium ion concentrations in the culture broths of L. plantarum strains were slightly higher after 24 h of culturing (by 30e50%) than at the beginning of the cultivation, while the culture broth pH of these strains were dropped to 4.7 at 9 h without a change onwards (data not shown). However, this small ammonium ion concentration increase might not be the result of arginine deiminase activity, since similar ammonium ion concentration pattern was observed cultivating the wild-type strain, L. plantarum BCC 9546, in arginine-free GYP medium (43% increase). The virtually no ammonia production by any of our L. plantarum strains and their 7 NH4+ (µmol/mg protein) Strain 745 6 5 4 3 2 1 0 0 24 48 72 Time (h) Fig. 3. Ammonium ion production by L. plantarum and P. acidilactici strains in GYPeArg medium at 30 C for 72 h. C: L. plantarum BCC 9546; ;: R5; A: R5-18; -: N750-1; :: P. acidilactici BCC 9545; B: L. plantarum BCC 9546 in GYP medium. The symbols represent means standard deviation (error bars) obtained from two independent experiments. 746 P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748 slightly decreasing culture broth pH is a sharp contrast to the ammonium ion production by the positive control (P. acidilactici) which also coupled with culture broth pH increase to pH 8.3 (data not shown). Therefore, considering all these findings, we conclude that arginine cannot be metabolized by our L. plantarum strains. 3.6. Measurement of glutamate decarboxylase activity The glutamate decarboxylase catalyzes the a-decarboxylation of glutamic acid to yield g-aminobutyric acid and carbon dioxide. This intracellular reaction results in the decrease of cytoplasmic Hþ-ion concentration and a slight increase of the cytoplasmic pH. Fig. 4 demonstrates that all of our L. plantarum strains were able to produce GABA from glutamate, albeit at a seemingly lower extent than the positive control L. brevis LSF 8-13. To quantify the GABA production the amount of glutamate consumed by L. plantarum strains were measured by biochemical analyzer. It was found that N750-1 showed the lowest GABA production (0.07 0.12 mmol/mg protein/24 h), while the wild-type produced nearly twice as much GABA (0.12 0.04 mmol/mg protein/24 h). The other two mutants, R5 and R5-18, produced about twofold more GABA than the wildtype strain (0.26 0.02 and 0.24 0.05 mmol/mg protein/24 h, respectively). According to the Hþ-ATPase activity measurements (Table 1C) N750-1 showed less activity than R5 and R5-18 which probably result in less available cellular energy for solute, e. g. glutamate, transport in this strain. That would provide a possible explanation for this strain’s lowest glutamate decarboxylase activity although the likelihood of down-regulation or mutation in the glutamate decarboxylase enzyme could not be ruled out. On the other hand, R5 and R5-18 had higher glutamate decarboxylase activity than that of the wild-type, which might be due to a slight up- regulation as a compensatory response to their acid-sensitivity. Nevertheless, these results indicate that the moderate/weak glutamate decarboxylase activity of these strains might play only a marginal role in their pH homeostasis and this effect might be realized over a longer period of time (24 h), therefore it might not provide effective protection against the much faster cytoplasmic acidification. This latter notion is also supported by the growth experiment data (Fig. 1), which showed almost no difference in cell number, total acidity and culture broth pH among the tree acidsensitive mutants in MRS culture up to 48 h. This suggest that in spite of glutamate most likely available in MRS broth the higher glutamate decarboxylase activity of R5 and R5-18 is not able to make these mutants more acid-resistant than N750-1 with the least glutamate decarboxylase activity (until 48 h). 3.7. Nham fermentation From among the mutants only N750-1 was selected for Nham fermentation trials because of its lowest Hþ-ATPase activity, mutation stability and because its MRS broth pH was the highest after 72 h (around pH 4.48, Fig. 1B). This mutant and the wild-type strain (WT, L. plantarum BCC 9546) were used in two formulas, (1): normal formula (using 4.3% garlic) and (2): limiting carbon source in Nham by decreasing concentration of garlic to 3%. Table 3 shows the number of total lactic acid bacteria (LAB) and of the mutant during Nham fermentation. The mutant was counted by replica-plating on half strength MRS-neomycin plates (1500 mg/ ml of neomycin sulfate) to confirm that most of the colonies that grew on these plates were N750-1. The initial inoculation level was approximately 104 cfu/g Nham in all four treatments. The viable count of total LAB and N750-1 increased until 12 h then the bacterial counts of all Nham formulations were slightly decreased until the end of incubation time (7 days). However, the number of Fig. 4. Glutamate conversion to g-aminobutyric acid by Lactobacillus strains as demonstrated with TLC. Glu: glutamate (negative control), GABA: g-amino butyric acid, L. b. 8-13: Lactobacillus brevis LSF 8-13 (positive control), WT: wild-type (L. plantarum BCC 9546). This TLC profile is a representative of three independent experiments that gave similar results. P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748 747 Table 3 Number of LAB and acid-sensitive mutant starter culture cells (log cfu/g) in Nham during 7 days at 30 C. Time (day) 0 0.5 1 2 3 4 5 6 7 N750-1-normal N750-1-3% G WT-normal WT-3% G Total LAB N750-1 Total LAB N750-1 Total LAB Total LAB 4.26 0.02a 8.41 0.34a 8.41 0.33abc 8.42 0.23abc 8.20 0.32ab 8.03 0.21b 8.08 0.12bc 8.00 0.18a 8.09 0.12a 4.00 0.06bc 8.10 0.35a 8.04 0.33c 8.08 0.05bc 7.97 0.19b 7.73 0.07c 7.85 0.11cd 7.72 0.10b 7.77 0.22b 4.25 0.03a 8.50 0.19a 8.53 0.16ab 8.42 0.29abc 8.42 0.26a 8.06 0.18ab 8.05 0.21bc 8.07 0.08a 8.06 0.11a 3.98 0.03c 8.10 0.19a 8.23 0.12bc 8.05 0.17c 8.02 0.15b 7.72 0.09c 7.74 0.10d 7.74 0.17b 7.73 0.18b 4.16 0.14ab 8.45 0.28a 8.81 0.07a 8.48 0.16a 8.43 0.04a 8.31 0.10a 8.44 0.17a 8.23 0.04a 8.06 0.11a 4.13 0.14abc 8.41 0.30a 8.55 0.28ab 8.45 0.21ab 8.32 0.17ab 8.30 0.10a 8.32 0.14ab 8.02 0.20a 7.95 0.11ab Mean values and standard deviations obtained from three independent experiments. Different letters (aed) in the same row indicate significant differences (P < 0.05). N7501-normal: N750-1 acid-sensitive starter culture was used in normal Nham formulation. N750-1-3% G: N750-1 acid-sensitive starter culture was used in 3% garlic Nham formulation. WT-normal: wild-type starter culture was used in normal Nham formulation. WT-3% G: wild-type starter culture was used in 3% garlic Nham formulation. total LAB in Nham fermentation using the wild-type or N750-1 starter were about three times higher than the viable count of the N750-1 starter culture strain in Nham. This means that the number of N750-1 starter cells represents about one third of the total LAB cell number in Nham. This affirms that in Nham inoculated by the mutant starter acid-sensitive L. plantarum cells play a major (or dominant) fermentative role. Comparing the pH of Nham (Table 4) in all trials shows that the pH values at 0 and 12 h in both wild-type and mutant fermented sausages were not significantly different but after 24 h there is a significant difference in pH of Nham fermented by different starters until 7 days. In Nham fermented by the wildtype the pH decreased lower than 4.6 and faster than Nham fermented with mutant in normal formulation. N750-1 as a new starter culture gave Nham a consistent final pH about 4.5 until 7 days (in normal formulation) while wild-type fermented Nham had a pH about 4.6 between the 1st and the 2nd days. Using 3% of garlic did not result in a product with significantly higher pH than that of produced with the traditional 4.3% garlic content neither when the acid-sensitive mutant nor when the wild-type strain was used as starter. Although, when the mutant was used as a starter at the end of 7 days of fermentation the pH of Nham formulated with 3% garlic was higher than that of Nham formulated with 4.3% garlic by about 0.2 units. This implies that garlic amount reduction in the formulation of Nham may contribute to less acid production in Nham fermented with the mutant. Concerning the safety of Nham fermented by the mutant, the sausages formulated with 3% garlic were above the critical pH limit of 4.6 (Paukatong and Kunawasen, 2001), therefore these are not considered to be safe. However the mutant fermented Nham with 4.3% garlic content had safe pH (4.6 or Table 4 Change in pH of Nham during 7 days at 30 C. Time (day) N750-1-normal N750-1-3% G WT-normal WT-3% G 0 0.5 1 2 3 4 5 6 7 6.10 0.03a 5.78 0.12a 5.21 0.09a 4.84 0.11a 4.69 0.13a 4.60 0.15a 4.55 0.18a 4.53 0.16a 4.48 0.15a 6.07 0.10a 5.81 0.17a 5.22 0.07a 4.88 0.05a 4.77 0.11a 4.68 0.15a 4.66 0.13a 4.67 0.16a 4.67 0.19a 6.07 0.06a 5.76 0.07a 4.97 0.05b 4.45 0.04b 4.32 0.05b 4.28 0.04b 4.22 0.02b 4.19 0.03b 4.19 0.02b 6.07 0.04a 5.76 0.15a 4.97 0.05b 4.47 0.08b 4.33 0.07b 4.30 0.07b 4.28 0.04b 4.25 0.09b 4.24 0.06b Mean values and standard deviations obtained from three independent experiments. Different letters (aeb) in the same row indicate significant differences (P < 0.05). N750-1-normal: N750-1 acid-sensitive starter culture was used in normal Nham formulation. N750-1-3% G: N750-1 acid-sensitive starter culture was used in 3% garlic Nham formulation. WT-normal: wild-type starter culture was used in normal Nham formulation. WT-3% G: wild-type starter culture was used in 3% garlic Nham formulation. below) from the 4th day onwards. 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