Several species of Lactobacillus, Bifidobacterium, and other microbes have been recognized as probiotics (Salminen et al., 2005; Ljungh and Wadstrom, 2006). Probiotic bacteria are used in the development of fermented foods (Heller, 2001). They can improve the immune functions of the host and regulate systemic and intestinal immune responses. Activation of the immune response requires the close physical contact between the probiotic bacteria and immune cells on the intestinal surface (Naidu et al., 1999; Perdigón et al., 2001; Behnsen et al., 2013; Saez-Lara et al., 2015).

Probiotics have been implicated as having therapeutic potential in several immune response-related diseases, including allergies, eczema, and viral infections, and may potentiate the body’s response to vaccines (Yan and Polk, 2011). Their abilities are strain-dependent (Schrezenmeir and de Vrese, 2001), thus, the screening of appropriate strains is important to evaluate the potential value of bacterial species in the context of probiotic therapy (Ding et al., 2017).

Non-viable probiotics may elicit therapeutic immune responses. The cell wall and cytoplasmic extracts of these bacteria may improve the immune functions of the host (Tejada-Simon and Pestka, 1999; Giahi et al., 2013). However, very few studies have explored the immune response to bacterial particles. It is also necessary to clarify whether bacterial particles comprising small-sized components can elicit stronger immune responses than whole cells. Salminen et al. (1999) redefined probiotics as “microbial cell preparations or components of microbial cells that exert beneficial effects on the health and well-being of the host.” The definition does not imply that probiotics can only be used in foods, but also suggests several other applications for imparting health benefits (Salminen et al., 1999). The use of non-viable microorganisms may be economically advantageous as they increase the shelf-life and reduce the requirement for refrigeration of food products. These characteristics may expand the use of probiotics to areas where strict handling conditions cannot be met, particularly in developing countries (Ouwehand and Salminen, 1998).

Herein, we examined the immunostimulatory effects of several lactic acid bacteria (LAB) isolated from Kimchi to screen potential probiotic strains using RAW 264.7 cells. In addition, the effects of a heat-inactivated probiotic strain and derived nanosized bacterial particles on the induction of nitric oxide (NO) and cytokines were compared to confirm the use of bacterial particles as effective adjuvants for immunotherapy.

Isolation of LAB

In total, 22 LAB were isolated from several fermented Kimchi samples. To isolate LAB, 1 ml of Kimchi sample was diluted 10-fold in saline and cultured for 24 h on MRS agar (Difco) containing bromocresol purple and sodium azide. The LAB colonies were yellow due to acid production. Each colony was re-streaked on the same agar to ensure purity. Each isolate was stored in glycerol at -80°C using a model 8525 Bio Freezer Model (Thermo Fisher Scientific).

Preparation of heat-inactivated LAB isolates and nanosized bacterial particles

The LAB isolates were cultured in MRS broth at 37°C for more than 24 h. The bacteria were recovered and washed twice with phosphate-buffered saline (PBS; Welgene). The bacteria were diluted using PBS to a density of 8 × 10⁸ CFU/ml, and heat-inactivated at 80°C for 30 min for the preparation of heat-inactivated LAB isolates. Following heat treatment, the cells were immediately immersed in ice cold water, and bacterial particles were prepared by sonication for preration of nanosized bacterial patricles. To achieve this, approximately 10 ml of the heat-inactivated bacterial suspension was sonicated with or without 0.2% (w/v) dispersant (gelatin B or monoolein). Sonication was carried out at 30% amplitude with 30 sec pulses separated by 10 sec intervals using a VC 505 sonicator equipped with a tip-type probe (Sonic & Materials Inc.).

Measurement of bacterial particles

The average diameters of the whole cells and nanosized bacterial particles were measured by dynamic light scattering (DLS) using a NanoBrook 90plus Zeta apparatus (Brookhaven Instrument Corporation) equipped with a 40 mW red diode laser (640 nm) at a detection angle of 90°. Each sample was analyzed eight times and the average value was calculated.

Cell culture

RAW 264.7 cells were obtained from the Korean Cell Line Bank (KCLB). The cells were cultured in a humidified incubator with an atmosphere of 5% CO₂ (IncuSafe CO₂ incubator; PHC Holdings Corporation) in Dulbeccós modified Eagle’s medium (Welgene) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

Cell viability assay

RAW 264.7 cells were seeded in the wells of a 24-well plate (5.0 × 10⁴ cells/well). After 24 h incubation, the culture medium was removed and replaced with fresh medium containing intact bacteria (2 × 10⁹ CFU/ml) for 24 h. Following incubation, 55 μl of a 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) was added to each well. After 3 h of incubation, the precipitated formazan was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and the optical density of each well was measured at a wavelength of 540 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific). The viability of RAW 264.7 cells was calculated as follows, using the PBS-treated group as the negative control:

Cell viability (%) = (O.D. of experimental group/O.D. of the negative control group) × 100.

Measurement of NO levels

The nitrite (NO₂^-) level as an indirect indicator of the immune response was measured using the Griess reaction with a commercial Griess nitrite assay kit (Promega). RAW 264.7 cells were seeded in the wells of a 24-well plate (5.0 × 10⁴ cells/well). After 24 h incubation, the culture medium was removed and the cells were incubated in fresh medium containing whole bacterial cells or derived nanosized bacterial particles for 24 h. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich) at 1.0 μg/ml was used as the positive control. A total of 50 μl of culture supernatant was mixed with 50 μl of sulfanilamide solution and N-(1-naphthyl) ethylenediamine (NED) solution for 10 min at 25°C. Absorbance was measured at 540 nm using the aforementioned Multiskan FC microplate reader. The concentration of NO in the supernatant was determined using a sodium nitrite standard curve.

Cytokine measurement

RAW 264.7 cells were seeded in the wells of a 24-well plate (1 × 10⁵ cells/well) for 24 h. The culture medium was removed and replaced with a fresh medium containing whole bacterial cells and derived bacterial particles. The cells were incubated for 24 h. A mouse enzyme-linked immunosorbent assay (ELISA) kit (Komabiotech) was used to measure tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 levels. The blocking reagent (1% w/v bovine serum albumin [BSA]) was added to each well coated with anti-mouse TNF-α or IL-6 monoclonal antibody. The wells were washed four times with 0.01 M PBS (pH 7.4) containing 0.2% (v/v) Tween-20 (PBST). Approximately 100 ml of standard cytokine and samples were added to each well and the plate was incubated at 25°C for 1 h. The plate was washed four times with PBST, followed by the addition of 100 μl of anti-mouse TNF-α or IL-6 antibody at 0.25 μg/ml and incubation at 25°C for 2 h. The plate was washed four times and treated with 50 μl of streptavidin-horseradish peroxidase conjugate (Komabiotech) at 25°C for 30 min. The plate was washed four times with PBST. Then, 100 μl of tetramethylbenzidine was added to each well for color development, followed by treatment with 100 μl of 1 M sulfuric acid to terminate the reaction. TNF-α and IL-6 concentrations were quantified based on a linear dose-response standard curve.

16S rRNA gene sequencing

The extraction of genomic DNA was performed using an AccuPrep genomic DNA extraction Kit (Bioneer). The primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (Macrogen) were used for the polymerase chain reaction (PCR)-based amplification of 16S rRNA. PCR was performed using a model PTC-100 thermal cycler (MJ Research Inc.). The PCR mixture was prepared with PCR premix (Bioneer), 4 μl of genomic DNA (Bioneer), 13 μl of distilled water, 1 μl of extracted genomic DNA, and 1 μl of 10 pmol/μl primer. PCR was performed using a pre-denaturing step at 94°C for 5 min, followed by 30 cycles (94°C for 30 sec, 52°C for 30 sec, and 72°C for 2 min) plus one additional cycle at 72°C for 5 min. PCR product was confirmed using 2% agarose gel electrophoresis. The analyzed nucleotide sequences were identified using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) on the National Center for Biotechnology Information database.

Statistical analysis

Results are expressed as mean ± standard deviation. Student’s t-test was used to evaluate significant differences (P < 0.001) between groups. The analysis was performed using SPSS software (SPSS Inc.).

Effects of different isolates of LAB on RAW 264.7 cell viability and NO production

Macrophages are the first line of defense of the immune system against pathogens. We evaluated the effects of 22 LAB isolates of Kimchi on the viability of RAW 264.7 macrophage-like cells after 24 h incubation. The LAB isolates displayed different effects on the viability of the RAW 264 (Fig. 1). The RAW 264.7 cells varied between 67% and 161% in the presence of the isolates. At relatively high concentrations (2 × 10⁹ CFU/ml), 10 samples showed no toxicity (THL2, 4, 7, 9, 11, 14, 18, 20, 21 and 22). In addition, NO production by RAW 264.7 cells in response to the various LAB isolates was evaluated. NO plays an important role in the host immune system with antibacterial and anticancer activities, and can regulate cytokine production. Therefore, the NO level serves as an indicator of immune activity. NO production significantly increased after the treatment of cells with LAB isolates THL4, THL14, and THL20 compared with the other isolates (Fig. 2). NO production was greatest with THL20 (16.25 μM), followed by THL14 and then THL4 (13.10 and 11.12 μM, respectively). Considering the cell viability experiment and the NO experiment comprehensively, THL4, THL14, and THL20 were used for primary probiotic screening to evaluate the isolates as effective adjuvants for immunotherapy.

Fig. 1. Cell viability analysis of macrophages RAW 264.7 cells treated with the various heat inactivated LAB isolates. The heat-inacivated LAB isolates were treated at a concentration of 2 × 10⁹ CFU/ml for 24 h. PBS, phosphate buffered saline; LPS, lipopolysaccharide. Cell viability was determined by MTT assay.Results are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005 indicate significant difference from PBS alone.

Fig. 2. Effect of various heat inactivated LAB isolates on NO production in macrophage RAW 264.7 cells. The heat-inacivated LAB isolates were treated at a concentration of 2 × 10⁹ CFU/ml for 24 h. PBS, phosphate buffered saline; LPS, lipopolysaccharide. Nitrite levels in the culture supernatants were evaluated by the Griess reaction. Results are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005 indicate significant difference from PBS alone.

Effect of LAB isolates THL4, THL14, and THL20 on cytokine release

Upon activation, RAW 264.7 macrophage-like cells secrete cell factors, including NO, TNF-α, and IL-6, which are required for host defense. TNF-α and IL-6 mediate the secretion of other cytokines and play a key role in the innate immune response. ELISA was carried out using LAB isolates THL4, THL14, and THL20, and cytokine production was compared at an appropriate concentration of 2 × 10⁹ CFU/ml to determine the immunostimulatory activities of the isolates. THL4, THL14, and THL20 elicited the production of a significant amount of TNF-α, similar to that observed with LPS (Fig. 3). No significant difference was observed in the TNF-α level. However, the IL-6 production of 3806.10 pg/ml for THL20 was higher than for the other two isolates.

Fig. 3. Effect of isolates of THL4, THL14, and THL20 on cytokine production in macrophage RAW 264.7 cells. The heat-inacivated LAB isolates were treated at a concentration of 2 × 10⁹ CFU/ml for 24 h. PBS, phosphate buffered saline; LPS, lipopolysaccharide. Results are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005 indicate significant difference from PBS alone.

Identification of the THL20 isolate

We performed 16S rRNA gene sequencing to identify the isolate THL20. Using primers 27F and 1492R, a 1,521 bp genomic DNA fragment was obtained from the isolate THL20 using PCR. We identified the isolate THL20 as Lactobacillus sakei (LS; Accession NO: MK359165) using BLAST.

Average diameter measurement

DLS was used to investigate the size of the LS (hereafter termed heat-inactivated L. sakei) and the nLS (hereafter termed nanosized L. sakei) that were sonicated without additional treatment or with the inclusion of gelatin B or monoolein as a dispersant. The nLS coated with gelatin B and monoolein were designated nLS-G and nLS-M, respectively. The mean diameter of the whole cells was approximately 1,636.0 nm. The diameter of the bacterial particles was reduced to about 500 to 600 nm due to fractionation, regardless of the addition of the dispersant. and confimed that that the intensity of light scattering increased with a decrease in the average diameter and the size is about three times smaller, indicating that the number of particles has increased by about three times (Table 1).

Table 1 Average diameter and light scattering intensity of heat-inactivated bacteria and nanosized bacterial particles

Contents	Ls	nLs	nLs-G	nLs-M
Light scattering intensity (kcps)	93.1	178.2	222.2	234.5
Size (nm)	1,636.00	592.1	613.8	525.5

LS, heat-inactivated L. sakei THL20; nLS, nanosized L. sakei THL20; nLS-G, nanosized L. sakei THL20 coated with gelatin B; nLS-M, nanosized L. sakei THL20 coated with monoolein.

Effect of nanosized bacterial particles on NO production

Changes in the immune activation ability after sonication were measured by comparing the production of NO using L. sakei THL20. LS, nLS, nLS-M, and nLS-G induced the production of NO in a dose-dependent manner from 8 × 10⁴ to 8 × 10⁸ CFU/ml (Fig. 4). However, NO production decreased (8 × 10⁶ CFU/ml) with nLS-M. At a concentration of 8 × 10⁶ CFU/ml, nLS showed stronger immunostimulatory activity than LS (7.82 mM), and resulted in the production of 18.27 mM of NO. The amount of NO produced was higher with nLS than with nLS-M and nLS-G (Fig. 4). However, in vitro experiments using RAW 264.7 cells revealed no difference in NO production between whole cells and bacterial particles derived by fractionation in the absence and presence of either dispersant at concentrations of 8 × 10⁴ and 8 × 10⁸ CFU/ml.

Fig. 4. Effect of nanosized bacterial particles on NO production in macrophage RAW 264.7 cells. The nanosized bacterial sample was sonicated with a concentration of 8 × 10⁴, 8 × 10⁶ and 8 × 10⁸ CFU/ml for 24 h. PBS, phosphate buffered saline; LPS, lipopolysaccharide. A, LS (heat-inactivated L. sakei THL20; B, nLS (nanosized L. sakei THL20); C, nLS-G (nanosized L. sakei THL20 coated with gelatin B); D, nLS-M (nanosized L. sakei THL20 coated with monoolein). Results are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005 indicate significant difference from PBS alone.

Effects of nanosized bacterial particles on cytokine production

NO assay results revealed an improved immune response of nLS obtained without dispersants compared to LS. Changes in the TNF-α production by nLS and LS were measured (Fig. 5); the amount of TNF-α reported in the LS-treated group was 26,040.5 pg/ml, while that observed in the nLS-treated group was 36,119.7 pg/ml. TNF-α production increased by 38% after treatment with nLS. IL-6 production in the nLS-treated group was 254 pg/ml, which was 21% higher than that observed in the LS-treated group.

Fig. 5. Effect of nanosized bacterial particles on cytokine production in macrophage RAW 264.7 cells. The nanosized bacterial sample was sonicated with a concentration of 8 × 10⁶ CFU/ml for 24 h. PBS, phosphate buffered saline; LPS, lipopolysaccharide. A, LS (heat-inactivated L. sakei THL20); B, nLS (nanosized L. sakei THL20). Results are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005 indicate significant difference from PBS alone.

As probiotics, various LABs have been used as adjuvants for immunotherapy. Some strains have immunostimulatory properties and induce cytokine expression and immune responses. Clinical and animal studies have reported that LAB induce host immune responses (Dunne et al., 2001; Fujiwara et al., 2004; Shida et al., 2006; Inoue et al., 2007; Winkler et al., 2007; Delcenserie et al., 2008; Bautista-Gallego et al., 2013). The variation in LAB properties could be reflected in differences in their effectiveness as adjuvants for immunotherapy. Therefore, we examined the immunostimulatory activities of LAB isolates from Kimchi in terms of their ability to induce cytokine secretion and NO production by macrophage-like cells and their cytotoxicity. The aim was to screen for potential probiotics with immunotherapy potential. Overall, 20 LAB isolates showed different results. Of these, isolates THL4, THL14, and THL20 displayed relatively low cytotoxicity to RAW 264.7 cells and induced the highest NO production in the RAW 264.7 cells. Many studies have reported that LAB used as adjuvants for immunotherapy induce different levels of NO production. The cytokine levels increase with increased NO production, which is proportional to the concentration of probiotics (Park et al., 1999). Therefore, the relatively high NO production is very important for screening potential probiotics (MacMicking et al., 1997).

Macrophages play a significant role in innate and acquired immune responses (Elhelu, 1983). After activation, macrophages release pro-inflammatory cytokines, such as TNF-α and IL-6, and present antigens to helper T cells (Gordon, 1998). IL-6 and TNF-α mediate the secretion of other cytokines (Klimp et al., 2002; Fujihara et al., 2003). Herein, we evaluated the production of TNF-α and IL-6 in the presence of LAB isolates THL4, THL14, and THL 20 and compared their cytotoxicity and influence on NO production. No significant difference in TNF-α production was observed. However, the production of IL-6 was the greatest after treatment of the cells with THL20. THL20 was selected as a potential probiotic candidate for immunotherapy. It was identified as L. sakei, as evident from 99% 16S rRNA sequence identity. Many studies related to L. sakei and immune response have been reported (Park et al., 2008; Reyes-Becerril et al., 2014a, 2014b; Jung et al., 2015; Quinteiro-Filho et al., 2015). Lactobacillus sakei and its constituent lipoteichoic acid induce stronger immune responses and stimulate the in vitro production of IL-12, interferon-gamma and TNF-α to a greater degree than L. plantarum (Hong et al., 2014).

Bacteria consist of a cell wall (or cell envelope in the case of Gram-negative bacteria) and cytoplasm (Silhavy et al., 2010). Immune responses elicited against components of the cytoplasm and, in particular, the cell wall have been previously reported (Chiang et al., 2012; Sukhithasri et al., 2013). We examined the immune stimulation after treatment of RAW 264.7 cells with bacterial particles. Macrophage activation was enhanced with smaller particles. NO production was observed after the treatment of RAW 264.7 cells with LS and nLS in a concentration-dependent manner. NO production significantly increased after treatment with nLS, compared to LS treatment at 8 × 10⁶ CFU/ml. However, it was difficult to observe any difference in NO production at very high (8 × 10⁸ CFU/ml) or low (8 × 10⁴ CFU/ml) concentrations. This reflects the detection limitations for in vitro experiments. Therefore, additional in vivo experiments are necessary. TNF-α and IL-6 levels were evaluated at an appropriate concentration (8 × 10⁶ CFU/ml). Significant differences were observed between macrophage-like cells treated with LS and nLS using ELISA. nLS was associated with 38% and 21% increased production of TNF-α and IL-6, respectively, compared with LS.

The immunostimulatory activity improved after fractionation. This may reflect the heightened tendency of nanosized bacterial particles to directly contact macrophage-like cells to better induce immune stimulation, as is evident in the experiments using dispersants. Dispersants are used to coat small-sized particles to prevent aggregation during fractionation (Pirrung et al., 2002). The reduced size of the particles obtained after fractionation was similar in the presence or absence of the dispersing agent. However, NO production was lower for nLS-G compared to nLS. Monoolein inhibits cyclooxygenase 2, an intermediate mediator of the immune response, at concentrations above 50 μM (Chang et al., 2008). We found that the use of monoolein as a dispersant to coat the bacterial particles significantly decreased NO production. As a result, it was confirmed that bacterial particles have immune functions owing to their direct contact with macrophage-like cell and there is a stronger immunostimulatory effect than the whole cells. It was reported that nanosized bacterial particles containing components of the cell wall elicit more pronounced immune responses than that by whole cells (Lee et al., 2016). In addition, nanometric L. plantarum were more easily ingested by microfold cells in Peyer's patches of the intestine than they are by whole cells, which is indicative of heightened anticancer activity (Lee et al., 2015).

An important part of this paper is to improve the ability to enhance immunity according to the application method of lactic acid bacteria used as an immunostimulant. The difference in immune activity according to components such as cell wall and cytoplasm through contact with macrophages is already known through various papers (Park et al., 1999; Tejada-Simon and Pestka, 1999; Chiang et al., 2012). Going one step further, we wanted to check the effect of immunostimulants with higher immune activity according to the manufacturing method using the selected bacteria.

In summary, L. sakei THL20 was selected as the candidate probiotic for immunotherapy among LAB isolated from Kimchi. We confirmed the increased number of particles as a result of a reduction in the average diameter and an increase in the intensity of light scattering through the sonication grinding process. and nLS (nanosized L. sakei THL 20) containing a cell wall and cytoplasm induced stronger immune responses than LS (heat-inacivated L. sakei THL 20). In addition, this increased immune activity was confirmed through dispersant application experiments that the cell wall and cytoplasmic structure should be in contact with macrophages as they are.

유산균은 효과적인 면역 치료 대체제로 사용되어지는 프로바이오틱스이다. 본 연구에서는 Raw 264.7 세포에서 Lactobacillus sakei THL20 열처리 사균체와 그 나노 분쇄물에 대한 면역 활성능을 검토하였다. 우선, 김치 유래 유산균 중 산화질소 및 사이토카인 생성 능력이 가장 우수한 L. sakei THL20를 선발하였다. 다음으로 L. sakei THL 20 열처리 사균체와 그 나노분쇄물의 사이토카인 생성능을 비교한 결과, 나노 분쇄물의 tumor necrosis factor-alpha 및 interleukin-6 생성능이 열처리 사균체 대비 각각 38%, 21% 증가함을 확인하였다. 추가적으로 분산제를 첨가하지 않고 나노 분쇄물을 제조한 경우 대식세포와의 직접적인 접촉이 증가함에 따라, 분산제를(gelatin B 또는 monoolein) 첨가한 시료와 비교하여 더 강한 면역 반응을 보였다. 본 실험 결과로 면역 치료제로서 유산균 나노 분쇄물의 잠재력을 확인할 수 있다.

None.

No potential conflict of interest was reported by the authors.