Dong-Hyun Kim, Department of Pharmaceutical Science, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, Korea. E-mail: email@example.com
In the preliminary study, kimchi, a traditional food fermented with Chinese cabbage, protected scopolamine-induced mouse memory deficit in passive avoidance test. Therefore, we screened protective ingredients, particularly lactic acid bacteria, from Chinese cabbage kimchi against scopolamine-induced memory deficit in mice.
Methods and Results
Lactic acid bacteria, isolated from Chinese cabbage kimchi, were identified by 16S rDNA sequence analysis, G+C content and cellular fatty acid composition and sugar fermentation test. Memory deficit was induced in mice by intraperitoneally injecting with scopolamine.
Kimchi, particularly its supernatant, protected scopolamine-induced memory deficit in mice in passive avoidance test. Of kimchi ingredients, a lactic acid bacterium, strain C29, potently protected scopolamine-induced memory deficit in mice. C29 was a gram-positive, catalase-negative, anaerobic and non-motile rod. Its pylogenetic property was near to Lactobacillus pentosus (99%) and Lact. plantarum (99%). However, C29 fermented inulin and L-rhamnose and grew in pH 3 and at 45°C in contrast with Lact. pentosus and Lact. plantarum. Therefore, it named to be Lact pentosus var. plantarum C29. The strain C29 protected scopolamine-induced memory deficit in Y-maze and Morris water maze tests. Furthermore, C29 increased hippocampal BDNF and p-CREB expressions, which were reduced by scopolamine.
Lactobacillus pentosus var. plantarum C29 may protect memory deficit by inducing BDNF and p-CREB expressions.
Significance and Impact of the Study
Lactic acid bacteria, such as Lact pentosus var. plantarum C29, may prevent memory deficit and its contained fermented foods may be beneficial for dementia.
Alzheimer's disease (AD) shows progressive loss of recent memory followed by disorders of language, praxis or visual perception (McKhann et al. 1984). A loss of cholinergic function in the central nervous system contributes significantly to the cognitive decline associated with advanced age and AD (Sarter and Bruno 2004; Terry and Buccafusco 2003). Scopolamine, an anti-cholinergic drug, causes memory impairments in healthy young humans that parallel the memory impairments seen in non-demented drug-free elderly subjects (Araujo et al. 2005; Tariot et al. 1996). Thus, scopolamine treatment represents a good model for the memory deficit, which may be better than an Aβ-treated or transgenic animal model (Araujo et al. 2005). Many attempts have been made to reverse cognitive deficit by increasing brain cholinergic activity with acetylcholinesterase (AChE) inhibitors such as donepezil and tacrine, or cholinergic agonists such as carbachol in spite of their side effects such as pain, nausea and vomiting (Doody 1999; Musial et al. 2007; Campbell et al. 1978).
Kimchi, a traditional food that is fermented with vegetables such as Chinese cabbage and radish, is usually processed with various seasonings, such as red pepper powder, garlic, ginger, green onion, salts, etc. Spontaneous fermentation without starter or sterilization leads to the growth of lactic acid bacteria, including Leuconostoc sp. (Leuc. mesenteroides, Leuc. kimchii, Leuc. citreum, etc) and Lactobacillus sp. (Lact. plantarum, Lact. sakei, Lact. brevis, Lact. pentosus, Lact. lactis, etc) (Kim and Chun 2005; Park et al. 2010).
Lactic acid bacteria (LAB) are safe micro-organisms that improve disturbances of the indigenous microflora (Campieri and Gionchetti 1999), ameliorate the development of microflora (Collins and Gibson 1999), have anti-diabetic and anti-hyperlipidemic effects (Tabuchi et al. 2003; Taranto et al. 1998), inhibit carcinogenesis (Perdigon et al. 1991), have anti-colitic effects (Daniel et al. 2006) and induce non-specific activation of the host's immune system (Perdigon et al. 1991). However, their protective effects against memory deficit have not been studied.
In the preliminary study, we found that kimchi, particularly its supernatant, protected scopolamine-induced mouse memory deficit in passive avoidance test. Therefore, we isolated LAB from the supernatant of kimchi and then measured their protective effects against scopolamine-induced memory deficit in mice.
Materials and methods
Tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride; purity ≥99%), (−) scopolamine hydrobromide, acetylthiocholine (ATCh), 5,5′-dithiobis-[2-nitrobenzoic acid] (DTNB), AChE (electric eel type VI-S), radioimmunoprecipitation assay (RIPA) lysis buffer, and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against BDNF, p-CREB, CREB and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The protease inhibitor cocktail was purchased from Roche Applied Science (Mannheim, Germany). Immobilon-P nylon membrane was from Millipore Co. (Billerica, MA, USA). Polyvinylidene difluoride membranes (PVDF) and enhanced chemiluminescence (ECL) detection kit (Luminta™ Forte Western HRP substrate) were purchased from Millipore (Billerica). All other materials were of the highest grade available.
Isolation and identification of lactic acid bacteria
The novel bacterial strain C29 was isolated from kimchi according to the previously reported methods (Kim and Chun 2005; Park et al. 2010). The kimchi was donated from a distributor of a commercially available brand in Korea: kimchi was consisted of 1 kg of Chinese cabbage, which was immersed in 12% (W/V) NaCl (80% refined salts) solution for 10 h and then washed three times with water, 30 g green onion, 25 g garlic, 8 g ginger and 40 red pepper powder, 100 g glutinous rice paste. A 500 μl of supernatant sample, immediately obtained upon opening the kimchi container, was serially diluted, inoculated into GAM agar (Nissui Pharm. Co., Tokyo, Japan), and anaerobically cultured at 37°C for days according to the manufacturer's instructions. The isolated bacteria was suspended in 10% skim milk (BBL, Le Pont de Claix, France) containing 10% glycerol and stored at −80°C. For identification of LAB, the Gram-reaction was determined using a Gram Stain kit (bioMerieux, Grenoble, France) according to the manufacturer's instructions. Additional enzyme activities, and biochemical characteristics were determined using the API 20E and API 50CHL test strips (bioMerieux, Seoul, Korea) according to the manufacturer's instructions. The 16S rDNA was amplified by PCR using 27F, 1492R primer after purified using the plasmid kit (Bio Basic Inc., Markham, ON, Canada) and the purified 16S rDNA was sequenced using ABI 3730XL DNA analysis. The G+C content was determined by a fluorimetric method using SYBR Green and a real-time PCR thermocycler (Gonzalez and Saiz-Jimenez 2002). The identified C29 is stored in Kyung Hee University Cell Bank (KHCB-2011-25, Seoul, Korea) and Korea Culture Center of Microorganisms (KCCM10885, Seoul, Korea).
Male ICR mice weighing 28–30 g were purchased from the Orient Co., Ltd, a branch of Charles River Laboratories (Seoul, Korea). All experiments were performed in accordance with the NIH and Kyung Hee University guides for Laboratory Animals Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University. The mice were housed 5 or 6 per cage, allowed access to water and food ad libitum, and maintained at an ambient temperature of 22 ± 1°C with 50 ± 10% humidity and a 12-h diurnal light cycle (lights on 07:00–19:00 h) prior to testing. All behavioural experiments were carried out in a room adjacent to the housing room under the same ambient conditions.
Passive avoidance test
For the assay of long-term memory performance, the passive avoidance test was carried out in identical illuminated and non-illuminated boxes (Gemini Avoidance System, San Diego Instrument, CA, USA). The illuminated compartment (20 × 20 × 20 cm) contained an 100 W bulb, and the floor of non-illuminated compartment (20 × 20 × 20 cm) was composed of 2 mm stainless steel rods spaced 1 cm apart. These compartments were separated by a guillotine door (5 × 5 cm). For acquisition trial, mice were initially placed in the illuminated compartment and the door between the two compartments was opened 10 s later as has been described previously (Lee et al. 2009). When mice entered the dark compartment, the door automatically closed and an electrical foot shock (0·5 mA) of 3 s durations was delivered through the stainless steel rods. Memory impairment was induced by scopolamine treatment (0·9 mg kg−1, i.p.) 30 min after the final administration of each sample, tacrine or 10% Tween 80 solution. Control animals were administered 10% Tween 80 solution only. Twenty-four hours after acquisition trial, the mice were again placed in the illuminated compartment for the retention trials. The time taken for a mouse to enter the dark compartment after door opening was measured as latency times in both acquisition and retention trials. If a mouse did not enter the dark compartment within 180 s, it was assumed that the mouse had remembered the single training trial. Test agents, kimchi extract non-fermented kimchi ingredient mixture extract, Chinese cabbage extract (50 mg kg−1), lactic acid bacteria (1 × 109 and 1 × 1010 CFU per mice, p.o.), tacrine (10 mg kg−1, p.o.), which is a well-known memory deficit-ameliorating acetylcholinesterase inhibitor (Musial et al. 2007) or vehicle (p.o.), were orally administered to mice once a day for 3 days and their final administration was given 1 h before treatment with scopolamine. Each group consisted of six mice.
For the assay of short-term memory performance, Y-maze test was carried out. The Y-maze is a three-arm horizontal maze (40-cm-long and 3-cm-wide with 12-cm-high walls) in which the arms are symmetrically disposed at 120° angles from each other. The maze floor and walls were constructed from dark opaque polyvinyl plastic as has been described previously (Lee et al. 2009). Mice were initially placed within one arm, and the sequence (i.e. ABCAB, etc.) and number of arm entries were recorded manually for each mouse over an 8-mm period. An actual alternation was defined as entries into all three arms on consecutive choices (i.e. ABC, CAB or BCA but not BAB). Maze arms were thoroughly cleaned between tests to remove residual odours. Memory impairment was induced by scopolamine treatment (0·9 mg kg−1, i.p.). Mice were gently placed in the maze. Test agents, lactic acid bacteria (1 × 109 and 1 × 1010 CFU per mice, p.o.), tacrine (10 mg kg−1, p.o.) or vehicle (p.o.) were administered to mice once a day for 3 days and their final administrations were given 1 h before treatment with scopolamine. Each group consisted of 6 mice.
The percentage of alternations was defined according to the following equation:% alternation = [(number of alternations)/(total arm entries–2)] × 100. The number of arm entries served as an indicator of locomotor activity.
Morris water maze test
For the assay of spatial short-term and long-term memory performance, Morris water maze test was carried out. The Morris water maze is a circular pool (90 cm in diameter and 45 cm in height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water containing 500 ml of milk (20 ± 1°C). The tank was placed in a dimly lit, soundproof test room with various visual cues. The pool was conceptually divided into quadrants. A white platform (6 cm in diameter and 29 cm high) was then placed in one of the pool quadrants and submerged 1 cm below the water surface so that it was invisible at water level. The first experimental day was dedicated to swimming training for 60 s in the absence of the platform. During the four subsequent days, the mice were given four trials per day with the platform in place. When a mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 60 s, it was placed on the platform for 10 s as has been described previously (Lee et al. 2009). The animal was taken to its home cage and was allowed to dry up under an infrared lamp after each trial. The time interval between each trial was 30 s. During each trial, the time taken to find the hidden platform (latency) was recorded using a video camera-based Ethovision System (Nodulus, Wageningen, The Netherlands). For each training trial, mice were placed in the water facing the pool wall at one of the pool quadrants in a different order each day. One day after the last training trial sessions, mice were subjected to a probe trial session in which the platform was removed from the pool, allowing the mice to swim for 60 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed. Memory impairment was induced in mice with scopolamine (0·9 mg kg−1, i.p.) at 30 min after treatment of the test agent. Control group received 10% Tween 80 solution only. Test agents, lactic acid bacteria (1 × 109 and 1 × 1010 CFU per mice, p.o.), tacrine (10 mg kg−1, p.o.) or vehicle (p.o.) were administered to mice once a day for 3 days and their final administrations were given 1 h before treatment with scopolamine. Each group consisted of six mice.
After stimulation with scopolamine, hippocampus lysates were prepared with ice-cold lysis RIPA buffer containing 50 mM Tris–HCl (pH 8·0), 150 mmol l−1 sodium chloride, 1·0% Igepal CA-630 (NP-40), 0·5% sodium deoxycholate, 0·1% sodium dodecyl sulphate, 1% phosphatase inhibitor cocktail and a protease inhibitor cocktail. Hippocampus lysates were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immobilon-P nylon membrane. BDNF, p-CREB, CREB and β-actin were analysed using the corresponding antibodies as previously reported (Lee et al. 2009). Immunodetection was carried out using an ECL detection kit.
Values are expressed as means ± SEM For the passive avoidance test, data were analysed by a Kruskal–Wallis non-parametric anova test. If the results were significant, each treatment group was compared using the Tukey's post hoc test. Statistical significance was set at P < 0·05. For the microplate assay, data were analysed by one-way analysis of variance (anova). If the results were significant, each group was compared using the Dunnett's post hoc test. Statistical significance was set at P < 0·05.
Isolation and Identification of C29 protecting memory deficit from kimchi
Kimchi, a traditional food fermented with Chinese cabbage, protected scopolamine-induced memory deficit in mice in passive avoidance test, although the extract of Chinese cabbage or non-fermented kimchi ingredient mixture did not protect the memory and learning deficits. Therefore, we separated into precipitate and supernatant fractions by centrifugation (500 g) and measured the protective effect of their extracts against scopolamine-induced memory deficit in passive avoidance test (Fig. 1a). Of them, the supernatant protected scopolamine-induced memory deficit in mice more potently than the precipitate. Therefore, we inoculated the supernatant of kimchi in MRS agar plates, isolated LAB and then measured their protective effect against scopolamine-induced memory deficit (Fig. 1b). Of them, C29 most potently protected scopolamine-induced memory deficit in mice, followed by C3. Therefore, to identify C29, we investigated its physiological and biochemical properties. The strain C29 cell was a Gram-positive rod (1·0–1·2 μm in width and 2·2–2·6 μm in length) that was non-motile (Table 1). The isolate grew under anaerobic conditions. NaCl concentration, pH and temperature ranges for growth in MRS are 0–8·0% (w/v), pH 2·0–7·0 and 10–45°C, respectively. C29 produced o-nitrophenyl-β-d-galactopyranoside hydrolysis, tryptophan deaminase, indole production, H2S production, arginine dihydroase, lysine decarboxylase, ornithine decarboxylase, and urease by API 20E and API 50CHL tests. The strain did not reduce nitrate to nitrite or nitrogen under aerobic conditions. The strain utilized l-arabinose, d-ribose, d-glucose, d-fructose, d-mannose, d-galactose, d-mannitol, sorbitol, α-methyl- d-mannoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, d-cellobiose, d-maltose, d-lactose, sucrose, melibiose, d-trehalose, inulin, melezitose, raffinose and gentiobiose, but did not utilized glycerol, d-xylose, adonitol, dulcitol, α-methyl- d-glucoside and d-arabitol. Particularly, C29 fermented inulin and l-rhamnose, which were not fermented by Lact. pentosus and Lact. plantarum (van Reenen and Dicks 1996; Zanoni et al. 1987).
Table 1. Morphological and physiological characteristics of strains C29, Lactobacillus plantarum ATCC 14917 and Lactobacillus pentosus ATCC8041 isolated from Kimchi
The strain C29 is resistant to artificial stomachic and digestive enzyme juices more potently than Lact. pentosus and Lact. plantarum. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C29 showed a high level of similarity with the type strain of Lact. plantarum ATCC14917T (99%), Lact. pentosus DSM20314T (99%), Lactobacillus sanfranciscensis ATCC 27651T (90%), Lactobacillus casei LMG 6904T (91%), Lactobacillus brevis ATCC 14869T (92%). The genomic G+C content of strain C29 was 50·7 mol%, which was different to those of Lact. plantarum and Lact. pentosus (van Reenen and Dicks 1996; Zanoni et al. 1987). The cellular fatty acid composition of the isolate C29 was consisted of 45·5% saturated fatty acids, 25·5% unsaturated fatty acids and 10·1% branched-chain fatty acid (Table 2). Its profile was significantly different to those of Lact. plantarum and Lact. pentosus. Results from the 16S rRNA gene sequence analysis, physiological and biochemical tests, and cellular fatty acid composition indicated that C29 is the type strain of a novel species of the genus Lactobacillus, for which the name Lact. pentosus var. plantarum is proposed (Fig. 2).
Table 2. Cellular fatty acid compositions of strains C29, Lactobacillus plantarum ATCC14917 and Lactobacillus pentosus DSM20314
From (van Reenen and Dicks, 1997) and (Zanoni et al. 1987).
Saturated fatty acids
Unsaturated fatty acids
Branched-chain fatty acids
C19:0 cyclo ω8c
Memory enhancing effect of Lactobacillus pentosus var. plantarum C29
Next we investigated the protective effect of C29 against scopolamine-induced memory deficit in mice in the passive avoidance test (Fig. 3a). The step-through latency of scopolamine-treated mice was significantly shorter than that of vehicle-treated normal control mice in the passive avoidance test. Orally administered strain C29 potently protected memory deficit. The strain C29 orally administered at doses of 1 × 109 and 1 × 1010 CFU per mice was shown to protect this scopolamine-induced reduction in step-through latency by 61% (P < 0·05) and 77% (P < 0·05), respectively. The protective effect of the strain C29 against memory deficit in mice was reduced to 79% of the fresh C29-treated group by heating in water bath for 30 min (Fig. 3b). During the acquisition trial, no differences in the latency, time were observed. The effect of C29 at a dose of 1 × 1010 CFU per mice was comparable to that of tacrine (10 mg kg−1), a positive control.
In the Y-maze test, the spontaneous alteration of scopolamine-treated mice was significantly lower than that of mice treated with vehicle alone, and the strain C29 protected the lowered spontaneous alteration induced by scopolamine (Fig. 4). The strain C29 (1 × 1010 CFU per mice, p.o.) significantly protected the lowered spontaneous alteration induced by scopolamine by 65%. Its potency was comparable to that of tacrine (10 mg kg−1). We also investigated the spatial memory-protective effect of the strain C29 on memory-impaired mice induced by scopolamine in the Morris water maze test (Fig. 5). The scopolamine-treated group exhibited longer escape latencies throughout the training days than the control group. The strain C29 (1 × 1010 CFU per mice, p.o.) significantly shortened the escape latencies prolonged by scopolamine treatment. The strain C29 potently shortened the escape latencies prolonged by treatment with scopolamine.
Next we investigated the effect of the strain C29 on hippocampi cAMP response element binding (CREB) expression and brain-derived neurotrophic factor (BDNF) activation, which exhibit neuroprotective effects (Puerta et al. 2010) (Fig. 6). Treatment with scopolamine reduced BDNF expression and CREB phosphorylation (p-CREB) in the hippocampi of mice, but, when scopolamine with oral administration of the strain C29 was given, BDNF expression and CREB activation (p-CREB) were significantly higher than when treated with scopolamine alone.
Cholinergic neurons in the central nervous system (CNS) degenerate in such a manner that it correlates with functional loss in patients with AD and senile dementia. BDNF is down-regulated in the AD brain, and it may play a role in several events that constitute the pathological cascade in AD (Phillips et al. 1991).
Scopolamine has been shown to cause memory impairment (Mintzer et al. 2010). Many studies indicate that scopolamine is increasingly disruptive with increasing age and declining cognitive status (Araujo et al. 2005). Cholinergic neurons in the central nervous system (CNS) degenerate in a manner that correlates with functional loss in patients with AD and senile dementia. Many attempts have been made to reverse cognitive deficit by increasing brain cholinergic activity with AChE inhibitors such as donepezil or cholinergic agonists such as carbachol. However, number of drugs approved for the use of treatment of patients with memory impairment is limited because of their side effects. Therefore, many attempts have been made to use functional foods to reverse cognitive deficit.
Recently, numerous reports have shown that kimchi and its ingredients, particulary lactic acid bacteria, exhibit anti-allergic, anti-inflammatory, anti-cancer and immunostimulating effects. Similarly to the effects of previously reported functional foods (Lee et al. 2011), we found that kimchi may ameliorate memory deficit. Of its ingredients, lactic acid bacteria particularly Lact. pentosus var. plantarum C29 potently protected memory deficit induced by scopolamine in passive avoidance, Y-maze and Morris water maze tests. The protective effect of C29 against memory deficit remained partially (79%) in spite of heating at 100°C for 30 min. This result suggests that its protective constituent(s) may be stable for heating. Furthermore, C29 protected the expression of BDNF and p-CREB reduced by scopolamine in the hippocampus of scopolamine-treated mouse brain. Mature BDNF (12–13 kDa) facilitates long-term potentiation (Woo et al. 2005), which is believed to be responsible for synaptic plasticity and long-term memory and is coupled to the activation of CREB (Puerta et al. 2010; Alonso et al. 2005). In the present study, scopolamine reduced BDNF expression and CREB activation in the hippocampus, and their reductions were found to be proportional to memory deficit. The strain C29 (1010 CFU per mice p.o.) protected BDNF and p-CREB expression reduced by scopolamine. Thus, strain 29 protected scopolamine-induced memory deficit, and BDNF reduction, similarly to functional food ingredients, such as arctigenin and timosaponin AIII (Lee et al. 2009, 2011).
On the basis of these findings, kimchi, particularly its ingredient C29, may prevent memory deficit by inducing BDNF and p-CREB expressions.