Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2019 Oct 12;9(11):398. doi: 10.1007/s13205-019-1926-y

Characterization and probiotic properties of Lactobacilli from human breast milk

Chanettee Jamyuang 1, Phanphen Phoonlapdacha 2, Nalinee Chongviriyaphan 2, Wasaporn Chanput 3, Sunee Nitisinprasert 1, Massalin Nakphaichit 1,✉
PMCID: PMC6790201  PMID: 31656736

Abstract

Several studies have reported a complex microbial community in human breast milk. This community impacts the shape of the infant gut microbiota and consequently impacts host health. Lactobacillus is an important probiotic and has many applications in the functional food industry. This study isolated and evaluated the potential probiotic bacteria from human milk. Two Lactobacillus species, L. plantarum and L. pentosus, were isolated from the breast milk of Thai women. L. pentosus HM04-22, L. pentosus HM04-3, L. plantarum HM04-80, L. plantarum HM04-88 and L. plantarum HM01-1 showed good adhesion activity (> 55%) and resistance in gastric (pH 2) and bile (pH 8) conditions. Characterization of the probiotic properties indicated that all selected Lactobacillus isolates had anti-adhesion properties against Escherichia coli and Salmonella Typhimurium. Lactobacillus isolates protected Caco-2 cells from pathogen adhesion at 25–40%. In addition, the five selected strains presented anti-inflammatory properties by reducing interleukin (IL)-8 expression at 0.14 ± 0.16 to 0.52 ± 0.117-fold. However, the strains had no effect on the expression of tight junction genes, including zona occludens (ZO)-1, occludin and claudin-1. In conclusion, five selected Lactobacillus isolates from human milk were candidates for use as probiotics to promote health. However, more tests in animal models and clinical trials need to be performed.

Keywords: Probiotic, Human breast milk, Adhesion, Immune

Introduction

Breastmilk contains a complex community of bacteria, which impacts the establishment of the infant gut microbiota. The development of gut microbiota in the early stage plays a role in gut barrier formation and immune system maturation. The disruption of biological processes may affect human health later in life. Firmicutes and Proteobacteria were found to be dominant phyla in human breast milk. However, milk microbiota are diverse and depend on maternal factors and behaviours, breast-feeding practice and other milk components.

Probiotics are beneficial microbes that impact host health. Most species of probiotics are in the group of lactic acid bacteria. Several studies have reported that lactic acid bacteria such as Streptococcus, Lactococcus, Leuconostoc, Weissella, Enterococcus and Lactobacillus and members of Propionibacterium and Bifidobacterium were isolated and characterized from human breast milk (Martín et al. 2003; Rajokaa et al. 2017). Lactobacillus gasseri, Bifidobacterium breve and Streptococcus salivarius isolated from human milk showed potential probiotic properties for agglutination with pathogens (Damaceno et al. 2017). The isolation of Lactobacillus crispatus and Lactobacillus gasseri from human milk indicated that the bacteria could exhibit resistance to gastrointestinal tract conditions and good adhesion on intestinal cell lines. Moreover, Lactobacillus gasseri and Enterococcus faecalis presented antimicrobial activity by bacteriocin production (Kozak et al. 2015). Compared with other species, Lactobacillus and Bifidobacterium are the most widely used probiotics in the food and beverage industry (Fenster et al. 2019; Terpou et al. 2019).

The adhesion ability of probiotics is a critical factor contributing to the persistent beneficial effects on the host. Probiotics could be localized on both intestinal mucosa and epithelium cells. Probiotics can modulate gut pathophysiology by enhancing epithelial barrier function (Anderson et al. 2010; Jose et al. 2015). Tight junctions (TJs) are formed by protein dimers that span the space between adjacent cell membranes. Major TJ-plaque proteins include zonula occludens (ZO)-1, occludin and claudins. These proteins constitute the major structure and modulate some functions of TJs (Mccall et al. 2009). Lactobacillus amylophilus D14 has been shown to improve intestinal permeability induced by Escherichia coli (ETEC) and Salmonella Typhimurium (Yu et al. 2012). Lactobacillus plantarum could protect cell junctions and mucosal barriers damaged by pathogen infection (Karczewski et al. 2010; Potoćnjak et al. 2017).

Probiotics can help maintain and restore the balance of the intestinal immune system. Intestinal epithelial cells release potent neutrophil attractant chemokines such as interleukin (IL)-8 during pathogenic infection. Although IL-8 aims to combat and eliminate pathogens, its persistent production may damage epithelial cells. Ulcerative colitis and Crohn’s disease are example diseases associated with the persistent synthesis of IL-8. Bifidobacterium infantis W52, Lactobacillus casei W56 and Lactococcus lactis W58 significantly suppressed IL-8 synthesis by Caco-2 cells when Salmonella was co-incubated (Malago et al. 2009).

Therefore, the aims of this study were to (i) isolate and screen Lactobacillus spp. from human breast milk; (ii) determine the probiotic properties of the Lactobacillus spp. including resistance to acid and bile salts, and adhesion to both mucus and Caco-2 cells as a model of epithelial cells; (iii) test the effects of the Lactobacillus isolates on the adhesion of enteric pathogens; and (iv) examine the ability of the Lactobacillus isolates to enhance epithelial barrier functions and effect immune modulation.

Materials and methods

Isolation of Lactobacillus from human breast milk

Milk samples were collected from five healthy women at Ramathibodi Hospital, Thailand, who were in the stage of lactation at 2–6 months after birth. All procedures involving human subjects were approved by the Ethics Committees of Ramathibodi Hospital, Thailand (reference number: ID10-58-16). Mammary areola and nipples were cleaned with sterile water. The samples were collected in a sterile tube containing Man-Rogosa-Sharpe (MRS) (Difco™, France) broth combined with 0.05% l-cysteine (Sigma-Aldrich, USA). Milk samples were kept in an anaerobic box on ice during transportation. For isolation, the milk samples were diluted with normal saline (0.85% NaCl) and cultured on MRS agar containing 0.05% l-cysteine and CaCO3 for 24–48 h at 37 °C in an anaerobic chamber.

Bacterial isolates were selected based on morphological differences, including size, colour and acid production. The selected strains were preserved in MRS broth (Difco™, France) containing 20% (v/v) glycerol at − 20 °C. Before the activity assay, the isolates were activated twice in MRS broth at 37 °C for 24–48 h in an anaerobic chamber.

Identification of bacterial isolates

The selected isolates were observed by optical microscopy to determine their morphology and Gram-staining results. All Gram-positive isolates with rod shapes were identified to the genus level by MALDI–TOF mass spectrometry analysis. One colony of bacterial cells was spread onto a MALDI–TOF–MS steel anchor plate (BigAnchor 96-well plate; Bruker Daltonics, Germany). One microlitre of 70% formic acid (Sigma-Aldrich, USA) was added to the well plate, followed by matrix solution (4-hydroxy--cyanocinnamic acid (HCCA), Bruker Daltonics, Germany). The plate was dried at room temperature. A bacterial test standard (BTS; Bruker Daltonics) was used for MicroFlexLT mass spectrometer Bruker Daltonics calibration. The results were expressed as log score values indicating the similarity of the unknown sample, and profiles were compared using MALDI BioTyper 3.0 software (version 3.3.1.0). The cut off scores were ≥ 2.00 and 1.700–1.900 for the species and genus levels, respectively.

Mucin adhesion assay

The adhesion assay was performed according to the modified method of Sánchez et al. (2010). One hundred microlitres of a purified porcine gastric mucin type III (Sigma, USA) solution in phosphate buffered saline (PBS) (10 mg/ml) was immobilized on a 96-well sterilized polystyrene microtiter plate (F96 Maxisorp Immunoplate; Nunc, Denmark) at 4 °C for 18 h. The wells were washed twice with 200 µl of PBS and incubated with 20 g/l bovine serum albumin (BSA) (Sigma-Aldrich) at 4 °C for 2 h. To eliminate non-bound BSA, the wells were washed twice with 200 µl of PBS. Then, 100 µl of 109 CFU/ml bacterial cell suspensions was added and incubated at 37 °C for 1 h before 200 µl of PBS was used to remove the unbound bacteria from the wells after incubation by washing five times. Then, 200 µl of a 0.05% (v/v) Triton X-100 solution (Sigma-Aldrich, Singapore) was added and incubated for 2 h at room temperature to remove the attached cells. The cell suspension was thoroughly mixed with a micropipette, and 100 µl of the resulting suspensions were sampled and plated to obtain the colony forming units per millilitre or CFU/ml (NAdhere) on MRS agar. Viable cells before (NInitial) and after (NAdhere) mucin absorption were enumerated. Percent adhesion was calculated according to the following equation:

%Adhesion=logcfuNAdhere×logcfuNInitial-1×100.

Lactobacillus rhamnosus GG was used as a positive control.

Cell survival rate assay after sequential exposure to simulate the gastrointestinal tract

Tolerances of isolates to simulated gastrointestinal tract conditions were determined using the method described by Ranadheera et al. (2012) with slight modifications. Simulated gastric juice was prepared by dissolving 3 g/L pepsin (P7000) in sterile filtered NaCl 0.5% (w/v), adjusted to pH 2.0 with 4 M HCl. Simulated small intestinal juice was prepared by dissolving 1 g/L pancreatin USP (P7545) in a sterile solution of 0.5% NaCl with 0.45% bile salt, adjusted to pH 8.0 with sterile 0.1 M NaOH. Cell survival rate assays were performed by adding 1 ml of 109 CFU/ml bacterial cell suspensions to 9 ml of simulated gastric juice solution for 60 min at 37 °C. The gastric juice solution was removed by centrifugation at 8000 rpm for 5 min and subsequently resuspended in 9 ml of simulated small intestinal juice solution. Samples were further incubated for 120 min at 37 °C. The intestinal juice was then removed by centrifugation at 8000 rpm for 5 min. The cell pellet was suspended in a sterile 0.85% NaCl (w/v) solution. Survival cells were counted on MRS plates. Viable cells before (N0) and after (N1) growth in the simulated gastrointestinal tract were enumerated by the equation of Uraipan and Hongpattarakere (2015) as follows:

Survival rate%=logcfu/mlN1×logcfu/mlN0-1×100

Anti-adhesion properties of Lactobacillus isolates against pathogens on Caco-2 cells

Adhesion inhibition of Lactobacillus isolates against E. coli O157:H7 DMST 12743 and S. Typhimurium ATCC 13311 on CaCo-2 cells was investigated according to three assays: (i) protection, (ii) competitive adhesion, and (iii) displacement (Ren et al. 2012).

Caco-2 cells were cultured in Dulbecco’s Modified Eagle’s minimal essential medium (DMEM) (HyClone, USA) supplemented with 10% (v/v) foetal calf serum (HyClone, USA), 1% (v/v) nonessential amino acids (HyClone, USA) and 1% (v/v) penicillin–streptomycin (10,000 IU/ml and 10,000 μg/ml)(Invitrogen, USA), respectively. The Caco-2 cells were grown in a culture flask at 37 °C in a 5% CO2 environment until confluent. Cells were seeded at approximately 105 cells per well into 24-well tissue culture plates for 21 days post-confluence.

Lactobacillus isolates from 18 h cultures grown at 37 °C in MRS broth were harvested, washed twice with PBS and resuspended in non-supplemented DMEM to achieve a concentration of 109 CFU/ml. E. coli O157:H7 DMST 12743 and S. Typhimurium ATCC 13311 were activated in nutrient broth (NB) at 37 °C for 18 h. Bacterial cells were harvested, washed twice with PBS and resuspended in non-supplemented DMEM to achieve a concentration of 109 CFU/ml.

In the protection assay, Caco-2 cell monolayers were inoculated with 300 µl of the Lactobacillus isolates (1 × 109 CFU/ml) suspended in DMEM and incubated for 30 min at 37 °C in an atmosphere of 5% CO2 and 95% air. A total of 300 µl of the pathogens (109 CFU/ml) suspended in DMEM was added and incubated for 30 min at 37 °C in an atmosphere of 5% CO2 and 95% air.

In the competition assay, Caco-2 cell monolayers were inoculated with both 300 µl of Lactobacillus isolates (1 × 109 CFU/ml) and 300 µl of pathogen suspension in DMEM and incubated for 1 h at 37 °C in an atmosphere of 5% CO2 and 95% air.

In the displacement assay, Caco-2 cell monolayers were inoculated with 300 µl of the pathogen (1 × 109 CFU/ml) suspended in DMEM and incubated for 30 min at 37 °C in an atmosphere of 5% CO2 and 95% air. A total of 300 µl of the Lactobacillus isolates (1 × 109 CFU/ml) suspended in DMEM was added and incubated for 30 min at 37 °C in an atmosphere of 5% CO2 and 95% air.

The non-adherent cells were removed by washing 4 times with sterile PBS. The adherent cells were lysed with 0.1% Triton X-100 (Merck, Germany) for 10 min. The concentration of adhered bacterial cells was enumerated by plate counting in triplicate on xylose lysine desoxycholate (XLD) agar for S. Typhimurium and eosin–methylene blue agar (EMB) agar for E. coli and then incubated at 37 °C for 48 h. The ability of the Lactobacillus isolates to inhibit adhesion was determined by comparing the adhesion of the pathogen in the presence of the Lactobacillus isolates to that of pathogen alone, which was expressed as a percentage.

Testing the immunomodulation properties

The expression of cytokines (IL-6 and IL-8) and tight junction proteins (ZO-1, claudin-1 and occludin) of the Caco-2 cell line with and without bacterial cells was investigated. Caco-2 cell monolayers were cultured on 24-well plates as previously described. Lactic acid bacteria or pathogens were diluted in non-supplemented DMEM and incubated on Caco-2 cells for 6 h at 37 °C in an atmosphere of 5% CO2 and 95% air.

RNA was extracted from Caco-2 cells with Trizol reagent (Thermo Fisher Scientific, USA). Total RNA was purified with an RNA isolation kit, RNase (Thermo Fisher Scientific, USA). The cDNA was synthesized using the Prime Script First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Gene expression levels of the two cytokines (IL-6 and IL-8) and three tight junction proteins (ZO-1, claudin-1 and occludin) from Caco-2 cells were quantified by LightCycler® 480 real-time PCR (Roche, Germany). Primer sequences are shown in Table 1. The reaction mixture contained 0.8 µl of forward primer (10 µg/µl), 0.8 µl of reverse primer (10 µg/µl), 10 µl of LightCycler® 480 SYBR Green Master (Roche, Germany) and 2 µl of cDNA (50 ng/µl) with the volume adjusted to 20 µl by DI–water. Real-time PCR amplification was carried out under the following conditions: 45 cycles of 95 °C for 3 min, 95 °C for 10 s and 55 °C for 30 s. The β-Actin gene was used as an internal control for normalization. Each reaction was performed as five replicates. For relative quantification of the transcripts, the formula RQ = 2−ΔΔCt was used in the analysis of real-time quantitative PCR data following Livak and Schmittgen (2001).

Table 1.

Primer sequences used for real-time PCR

Gene Primer References
ZO-1 (269 bp) Foreword 5′-ATCCCTCAAGGAGCCATTC-3′ Orlando et al. (2014)
Revered 5′-CACTTGTTTTGCCAGGTTTTA-3′
Claudin-1 (275 bp) Foreword 5′-AAGTGCTTGGAAGACGATGA-3′ Orlando et al. (2014)
Revered 5′- CTTGGTGTTGGGTAAGAGGTT-3′
Occludin (237 bp) Foreword 5′-CCAATGTCGAGGAGTGGG-3′ Orlando et al. (2014)
Revered 5′-CGCTGCTGTAACGAGGCT-3′
β-Actin (140 bp) Foreword 5′-CTGGAACGGTGAAGGTGACA-3′ Li et al. (2009)
Revered 5′-AAGGGACTTCCTGTAACAATGCA-3′
IL-6 (118 bp) Foreword 5′-AGCCACTCACCTCTTCAGAAC-3′ Liu et al. (2017)
Revered 5′-GCCTCTTTGCTGCTTTCACAC-3′
IL-8 (98 bp) Foreword 5′-CTGATTTCTGCAGCTCTGTG-3′ Kina et al. (2009)
Revered 5′-GGGTGGAAAGGTTTGGAGTATG-3′ Martirosyan et al. (2013)
Open in a new tab

Statistical analysis

Data were analyzed by analysis of variance (ANOVA) with significance at p ≤ 0.05. All statistical analyses were performed using SPSS version 15.0.

Results

Isolation and identification of Lactobacillus spp. from human breast milk

All Gram-positive and acid-producing isolates were selected from five human breast milk samples. Under optical microscopy, the isolates were rod and cocci shape. All rod isolates were identified as Lactobacillus by MALDI–TOF–MS. Globally, Lactobacillus were isolated from three milk samples (Fig. 1). These isolates belonged to two species, Lactobacillus plantarum and Lactobacillus pentosus. The species that were more frequently isolated from human milk were L. plantarum.

Fig. 1.

Fig. 1

Open in a new tab

Profiles of Gram-positive, acid-producing bacterial isolates from breast milk samples. Bacterial profiles defined by shape (a) and bacterial genera of rod isolates from three samples identified by MALDI–TOF–MS (b)

Screening for adhesion activity and intestinal tract tolerance

Forty-three Lactobacillus isolates were screened for adhesion properties. The data indicated different activities on mucin adhesion, as shown in Fig. 2. The percentage of adhesion activity of the Lactobacillus isolates varied from 46.12 ± 0.03 to 64.51 ± 0.36%. Lactobacillus rhamnosus GG (LGG) was used as the standard reference strain criterion with an adhesion activity of 64.64 ± 0.68%. From forty-three isolates, the adhesion activities of 24 isolates were 55%, and these 24 isolates were selected. Strains HM04-80 and HM04-88 showed the highest adhesion activity at 64.51 ± 0.36% and 62.59 ± 0.30%, respectively.

Fig. 2.

Fig. 2

Open in a new tab

Mucin adhesion ability of Lactobacillus isolates from human breast milk. Lactobacillus rhamnosus GG (LGG) was the standard control. The adhesion rate was calculated by estimating the percentage of adhesion with the number of CFUs attached to the mucin compared to the initial number of CFUs added. The adhesion rate was calculated from three replications

The survival rates of the 24 selected Lactobacillus isolates were investigated in a simulated gastrointestinal tract. The results showed that these isolates had survival rates between 40 and 63% (Fig. 3). In addition, the reference strain, L. rhamnosus GG, showed a survival rate of 54.86 ± 0.57%. The Lactobacillus isolates with the highest survival rates were selected, including L. plantarum HM04-80, L. plantarum HM04-88, L. pentosus HM04-22, L. pentosus HM04-3 and L. plantarum HM01-1 (p ≤ 0.05).

Fig. 3.

Fig. 3

Open in a new tab

Survival rate of Lactobacillus isolates from human breast milk in the simulated human gastrointestinal (GI) tract. The percent survival rate was calculated by estimating the number of living cells (CFUs) before and after incubation in GI conditions. The mean ± SD of three independent experiments are shown; *Represents a significant difference between tested isolates

Anti-adhesion against pathogens on Caco-2 cells

Selected Lactobacillus isolates were capable of inhibiting E. coli and S. Typhimurium adhesion on Caco-2 cells. The highest inhibition activity was observed in the protection assay, followed by competition and displacement (Fig. 4). The inhibition activity of the five strains showed no significant differences between strains. The inhibitory activity of the protection, competition and displacement mechanisms of the Lactobacillus isolates against S. Typhimurium were 30.56 ± 2.22–40.26 ± 1.75%, 21.77 ± 1.52–26.68 ± 4.72% and 13.72 ± 1.75–19.18 ± 1.72%, respectively. The inhibition of the Lactobacillus isolates against E. coli was lower than that of S. Typhimurium. The inhibitory activity of the protection, competition and displacement mechanisms against E. coli were 20.43 ± 1.80–25.17%, 11.49 ± 1.09–14.19 ± 1.40% and 0.20 ± 0.73–3.79 ± 4.00%, respectively.

Fig. 4.

Fig. 4

Open in a new tab

Inhibition rate of Lactobacillus isolates from human breast milk against S. Typhimurium (a) and E. coli (b) on Caco-2 cells. Percentage inhibition was calculated from the percentage of pathogen adhesion with and without Lactobacillus isolates. The inhibition rate was calculated from three replications

Immunomodulation properties

The expression levels of intestinal tight junction proteins of Caco-2 cells with and without the Lactobacilli isolates were investigated (Fig. 5). The results indicated that the five strains had no effect on ZO-1, occludin and claudin-1 expression. On the other hand, pathogenic bacteria, including E. coli and S. Typhimurium, decreased all tight junction protein expression (p ≤ 0.05).

Fig. 5.

Fig. 5

Open in a new tab

Expression levels of ZO-1, occludin and claudin-1 protein from Caco-2 cells incubated with Lactobacillus isolates from human breast milk and pathogens. β-Actin was used as the reference gene. Data with different superscripts are significantly different at p ≤ 0.05

Stimulation of pro-inflammatory cytokines, including IL-6 and IL-8, of Caco-2 cells was monitored by real-time PCR (Fig. 6). Interestingly, five lactobacilli isolates showed significantly reduced IL-8 levels at 0.14 ± 0.16 to 0.52 ± 0.117-fold, whereas E. coli and S. Typhimurium induced the expression of IL-8 by approximately 2-2.5-fold. The reduced activity of IL-8 was not significantly different between the five strains (p ≤ 0.05). However, the expression of IL-6 in Caco-2 cells was not observed with any of the isolates tested.

Fig. 6.

Fig. 6

Open in a new tab

Expression levels of IL-8 from Caco-2 cells incubated with Lactobacillus isolates from human breast milk and pathogens. β-Actin was used as the reference gene. Data with different superscripts are significantly different at p ≤ 0.05

Discussion

Human breast milk is the best food for infants. The milk contains complex bacterial communities that impact the establishment of the infant gut microbiota. However, the milk microbiome is diverse and affected by maternal factors and milk components (Moosavi et al. 2019). Our results found that only three milk samples analyzed contained Lactobacillus. These were identified in L. plantarum and L. pentosus. Similarly, Sharma et al. (2017) identified lactobacilli bacterial strains in the breast milk of Indian women by genus-specific PCR and 16S rRNA-based sequencing. These strains belonged to five species: L. casei, L. delbrueckii, L. fermentum, L. plantarum, and L. pentosus. In addition, Taghizadeh et al. (2017) reported that 35 samples of breast milk of Iranian women contained lactobacilli (87.5%) based on phenotypic tests. All strains were confirmed using a genotypic method (PCR) as L. plantarum. Moreover, Dubos et al. (2011) reported the biodiversity of Lactobacillus spp. from 116 Chilean mothers at 55.3% with concentrations of Lactobacillus spp. at 3.33 ± 0.55 log CFU/ml. The predominant species were L. plantarum (64%), L. fermentum (16%) and L. pentosus (9%). Jara et al. (2011) found that the microbiota of 48 samples of breast milk of Mexican women consisted of L. acidophilus (52%), L. plantarum (7%), L. paracasei (30%), L. salivarius (7%) and L. curvatus (4%). Lactobacillus species in breast milk vary depending on the country and dietary habits, while the environment affects breast microbiota populations.

The species L. plantarum and L. pentosus are genotypically closely related and show highly similar phenotypes (Torriani et al. 2001). However, probiotic properties occur specifically at the strain level. Intestinal adhesion activity is one of the main selection criteria for probiotic strains. Adhesion ability is important for probiotic colonization (Blum et al. 2000). Most intestinal bacteria adhere and colonize the mucin layer (Sengupta et al. 2013). Therefore, Lactobacillus isolated from breast milk was tested for adhesion activity on mucin compared with a commercial strain, L. rhamnosus GG. Only 21% of the total Lactobacillus isolates showed adhesion activity similar to the commercial strain. The adhesion ability of bacteria depends on strain-specific mechanisms related to the bacterial cell surface components, such as adhesins, polysaccharides, and proteins (Khalili and Ahmad 2015).

The tolerance to gastrointestinal stress is key to guarantee the performance of probiotics. A large number of viable probiotics must reach the intestine in the appropriate quantity to produce a beneficial effect on consumer health (Angelis and Gobbetti 2004). Here, the survival rate in GI conditions was a secondary criterion for probiotic selection. Five strains, including L. pentosus HM04-22, L. pentosus HM04-3, L. plantarum HM04-80, L. plantarum HM04-88 and L. plantarum HM01-1, survived intestinal conditions at 54–62%, which was better than the commercial strain. Therefore, these five strains are potential candidates for application in wide use as probiotics.

The adhesion properties of probiotics on the intestinal layer help to protect against infection by pathogens. In this study, all selected Lactobacillus strains could inhibit E. coli and S. Typhimurium adhesion. The adhesion inhibition of five Lactobacillus isolates was similar, and the best effective mechanism for anti-adhesion was protection (p ≤ 0.05). In addition, Lactobacillus isolates showed better anti-adherence against S. Typhimurium than E. coli, possibly because E. coli has higher adhesion activity on intestinal cell lines than Lactobacillus strains. This result was similar to the inhibition assays of L. salivarius and L. plantarum, which could protect Staphylococcus aureus adherence to Caco-2 cells. Anti-adhesion slightly decreased the competitive and displacement mechanisms (Ren et al. 2012). These results suggest that probiotics can protect pathogen colonization on host cells by blocking pathogen adherence with intestinal cell receptors.

Tight junction proteins are important barriers for the spaces between intestinal cells. Our data indicated that S. Typhimurium and E. coli suppressed the expression of tight junction proteins. This might be growing evidence that an increase in the permeability of charged and uncharged molecules of intestinal cells results in activation of the immune system and secretion of inflammatory mediators (Singh et al. 2018). Other inflammatory bowel diseases might occur. There have been reports of the benefits of probiotics on gut barrier function (Blackwood et al. 2017). However, there was no increase in the expression of tight junction protein in the five selected Lactobacillus strains isolated from human milk when compared with non-probiotic treatment.

Several probiotic strains modulate innate immunity, especially by maintaining the balance between pro-inflammatory and anti-inflammatory cytokines (Perez-Cano et al. 2010; Fernandez et al. 2011; Ren et al. 2013; Plaza-Diaz et al. 2014; Tuo et al. 2018). Notably, immune modulation properties of Lactobacillus isolates from human milk were observed. The Lactobacillus isolates significantly suppressed pro-inflammatory cytokines (IL-8) when compared with the non-probiotic treatment. Levels of pro-inflammatory cytokines have been implicated in inflammatory diseases such as metabolic syndrome, obesity, diabetes and chronic inflammation in the gut (Morita et al. 2002; Alexandraki et al. 2006; Luongo et al. 2017). The application of probiotics may help to attenuate chronic inflammation and acute discomfort in the gastrointestinal tract.

In conclusion, this result indicated that breast milk is a source of probiotics for the infant gut microbiome. The isolation and selection of Lactobacillus from human breast milk obtained five potential probiotic strains, including L. pentosus HM04-22, L. pentosus HM04-3, L. plantarum HM04-80, L. plantarum HM04-88 and L. plantarum HM01-1. These strains strengthen intestinal barrier function by inhibiting the adhesion of pathogens to intestinal cells. Moreover, they showed anti-inflammatory properties and good survival in gastrointestinal tract conditions. Further work is necessary to fully characterize the performance of the Lactobacillus isolates in in vivo studies and to apply the isolates to functional food ingredients in dairy products in the future.

Acknowledgements

The authors would like to thank Assoc. Prof. Dr. Kwannan Nantavisai and Assist. Prof. Dr. Pakamon Chitprasert for the Caco-2 cell line. This study was funded by Kasetsart University Research and Development Institute fiscal year 2017.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

References

  1. Alexandraki K, Piperi C, Kalofoutis C, Singh J, Alaveras A, Kalofoutis A. Inflammatory process in type 2 diabetes: the role of cytokines. Ann N Y Acad Sci. 2006;1084:89–117. doi: 10.1196/annals.1372.039. [DOI] [PubMed] [Google Scholar]
  2. Anderson RC, Cookson AL, McNabb WC, Park Z, McCann MJ, Kelly WJ, Roy NC. Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation. BMC Microbiol. 2010;10:316–327. doi: 10.1186/1471-2180-10-316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Angelis DM, Gobbetti M. Environmental stress responses in Lactobacillus: a review. Proteomics. 2004;4:106–128. doi: 10.1002/pmic.200300497. [DOI] [PubMed] [Google Scholar]
  4. Blackwood BP, Yuan CY, Wood DR, Nicolas JD, Grothaus JS, Hunter CJ. Probiotic Lactobacillus species strengthen intestinal barrier function and tight junction integrity in experimental necrotizing Enterocolitis. Probiotics Health. 2017;5:1–20. doi: 10.4172/2329-8901.1000159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blum S, Reneiro R, Schiffrin E, et al. Adhesion studies for probiotics: need for validation and refinement. Food Sci Technol. 2000;10:405–410. [Google Scholar]
  6. Damaceno QS, Souza JP, Nicoli JR, Paula RL, Assis GB, Figueiredo HC, Azevedo V, Martins FS. Evaluation of potential probiotics isolated from human milk and colostrum. Probiotics Antimicrob Proteins. 2017;9:371–379. doi: 10.1007/s12602-017-9270-1. [DOI] [PubMed] [Google Scholar]
  7. Dubos C, Vega N, Carvallo C, Navarrete P, Cerda C, Brunser O, Gotteland M. Identification of Lactobacillus spp. in colostrum from Chilean mothers. Archivos latinoamericanos de nutrición. 2011;61:66–74. [PubMed] [Google Scholar]
  8. Fenster K, Freeburg B, Hollard C, Wong C, Laursen RR, Ouwehand AC. The production and delivery of probiotics: a review of a practical approach. Microorganisms. 2019;7:83–100. doi: 10.3390/microorganisms7030083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fernandez EM, Valenti V, Rockel C, Hermann C, Pot B, Boneca IG, Grangette C. Anti-inflammatory capacity of selected lactobacilli in experimental colitis is driven by NOD2-mediated recognition of a specific peptidoglycan-derived muropeptide. Gut. 2011;60:1050–1059. doi: 10.1136/gut.2010.232918. [DOI] [PubMed] [Google Scholar]
  10. Jara S, Sánchez M, Vera R, Cofré J, Castro E. The inhibitory activity of Lactobacillus spp. isolated from breast milk on gastrointestinal pathogenic bacteria of no socomial origin. Anaerobe. 2011;17:474–481. doi: 10.1016/j.anaerobe.2011.07.008. [DOI] [PubMed] [Google Scholar]
  11. Jose NM, Bunt CR, Hussain MA. Comparison of microbiological and probiotic characteristics of lactobacilli isolates from dairy food products and animal rumen contents. Microorganisms. 2015;3:198–212. doi: 10.3390/microorganisms3020198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Karczewski J, Troost FJ, Konings I, Dekker J, Kleerebezem M, Robert-Jan M, Brummer JM, Wells JM. Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier. Am Physiol Soc. 2010;298:851–859. doi: 10.1152/ajpgi.00327.2009. [DOI] [PubMed] [Google Scholar]
  13. Khalili AA, Ahmad MR. A Review of cell adhesion studies for biomedical and biological applications. Int J Mol Sci. 2015;16:18149–18184. doi: 10.3390/ijms160818149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kina S, Nakasone T, Takemoto H, Matayoshi A, Makishi S, Sunagawa N, Liang F, Phonaphonh T, Sunakawa H. Regulation of chemokine production via oxidative pathway in HeLa cells. Med Inflamm. 2009;1:1–5. doi: 10.1155/2009/183760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozak K, Charbonneau D, Sanozky-Dawes R, Klaenhammer T. Characterization of bacterial isolates from the microbiota of mothers’ breast milk and their infants. Gut Microbes. 2015;6:341–351. doi: 10.1080/19490976.2015.1103425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Li M, Yuqing Z, Qihui Z, Louis WF, William EF, Chen C, Yao Q. Thymosin beta 10 is aberrantly expressed in pancreatic cancer and induces JNK activation. Cancer Invest. 2009;27:251–256. doi: 10.1080/07357900802254016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu L, Chen L, Jiang C, Guo J, Xie Y, Kang L, Cheng Z. Berberine inhibits the LPS-induced proliferation and inflammatory response of stromal cells of adenomyosis tissues mediated by the LPS/TLR4 signaling pathway. Exp Ther Med. 2017;14:6125–6130. doi: 10.3892/etm.2017.5316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  19. Luongo D, Coppola A, Treppiccione L, Bergamo P, Sorrentinoa A, Ferrocinob I, Turronic S, Nevianid E, Cargo D, Cocolin L, Rossi M. Modulation of the cytokine profile in Caco-2 cells by faecal lactobacilli and bifidobacteria from individuals with distinct dietary habits. Cytokine. 2017;90:80–87. doi: 10.1016/j.cyto.2016.11.007. [DOI] [PubMed] [Google Scholar]
  20. Malago JJ, Tooten PCJ, Konink JFJG. Anti-inflammatory properties of probiotic bacteria on Salmonella-induced IL-8 synthesis in enterocyte-like Caco-2 cells. Benef Microb. 2009;1:121–130. doi: 10.3920/BM2009.0021. [DOI] [PubMed] [Google Scholar]
  21. Martín R, Langa S, Reviriego C, Jimínez E, Marín ML, Xaus J, Fernandez L, Rodríguez JM. Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr. 2003;143:754–758. doi: 10.1016/j.jpeds.2003.09.028. [DOI] [PubMed] [Google Scholar]
  22. Martirosyan A, Von Bargen K, Arce Gorvel V, Zhao W, Hanniffy S, Bonnardel J, Méresse S, Gorvel J-P, Moreno E. In vivo identification and characterization of CD4+ cytotoxic T cells induced by virulent Brucella abortus infection. PLoS ONE. 2013;8(12):e82508. doi: 10.1371/journal.pone.0082508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mccall IC, Betanzos A, Dominique A, Porfirio W, Gary WN, Charles M, Parkosb A. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization. Toxicol Appl Pharmacol. 2009;241:61–70. doi: 10.1016/j.taap.2009.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moossavi S, Sepehri S, Robertson B, Bode L, Goruk S, Field CJ, Lix LM, de Souza RJ, Becker AB, Mandhane PJ, Turvey SE, Subbarao P, Moraes TJ, Lefebvre DL, Sears MR, Khafipour E, Azad MB. Composition and variation of the human milk microbiota are influenced by maternal and early-life factors. Cell Host Microb. 2019;25:324–335. doi: 10.1016/j.chom.2019.01.011. [DOI] [PubMed] [Google Scholar]
  25. Morita H, He F, Fuse T, Arthur C, Hashimoto HO, Hosoda M, Mizumachi K, Kurisaki JI. Adhesion of lactic acid bacteria to caco-2 cells and their effect on cytokine secretion. Microbiol Immunol. 2002;46:293–297. doi: 10.1111/j.1348-0421.2002.tb02698.x. [DOI] [PubMed] [Google Scholar]
  26. Orlando A, Linsalata M, Notarnicola M, Tutino V, Russo F. Lactobacillus GG restoration of the gliadin induced epithelial barrier disruption: the role of cellular polyamines. BMC Microbiol. 2014;14:19–31. doi: 10.1186/1471-2180-14-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Perez-Cano FJ, Dong H, Yaqoob P. In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk. Immunobiology. 2010;215:996–1096. doi: 10.1016/j.imbio.2010.01.004. [DOI] [PubMed] [Google Scholar]
  28. Plaza-Diaz J, Gomez-Llorente C, Fontana L, Gil A. Modulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver by probiotics. World J Gastroenterol. 2014;20:15632–15649. doi: 10.3748/wjg.v20.i42.15632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Potoćnjak M, Petra Pušić P, Frece J, Abram M, Janković T, Gobin L. Three new Probiotic Lactobacillus plantarum Strains in the probiotic toolbox against gut pathogen Salmonella enterica Serotype Typhimurium. Food Technol Biotechnol. 2017;55:48–54. doi: 10.17113/ftb.55.01.17.4693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rajokaa MS, Mehwishc HM, Siddiq M, Haobin Z, Zhua J, Yan L, Shao D, Xu X, Shi J. Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk. Lebensmittel Wissenschaft Technol. 2017;84:271–280. [Google Scholar]
  31. Ranadheera CS, Evans CA, Adams MC, Baines SK. In vitro analysis of gastrointestinal tolerance and intestinal cell adhesion of probiotics in goat’s milk ice-cream and yogurt. Food Res Int. 2012;49:619–625. [Google Scholar]
  32. Ren D, Li C, Qin Y, Yin R, Li X, Tian M, Du S, Gu HH, Liu C, Zhu N, Sun D, Li Y, Jin N. Inhibition of staphylococcus aureus adherence to Caco-2 cell by Lactibacilli and cell surface properties that influence attachment. Anaerobe. 2012;18:508–515. doi: 10.1016/j.anaerobe.2012.08.001. [DOI] [PubMed] [Google Scholar]
  33. Ren DY, Li C, Qin YQ, Yin RL, Du SW, Ye F, Liu HF, Wang MP, Sun Y, Li X, Tian MY, Jin NY. Lactobacilli Reduce Chemokine IL-8 Production in Response to TNF-α and Salmonella Challenge of Caco-2 Cells. Biomed Res Int. 2013;2013:925219–925228. doi: 10.1155/2013/925219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sánchez B, Saad N, Jean-Marie S, Bressollier P, Urdaci MC. Adhesive properties, extracellular protein production and metabolism in the Lactobacillus rhamnosus GG strain when grown in the presence of mucin. J Microbiol Biotechnol. 2010;20:978–984. doi: 10.4014/jmb.0911.11007. [DOI] [PubMed] [Google Scholar]
  35. Sengupta S, Chattopadhyay MK, Grossart HP. The multifaceted roles of antibiotics and antibiotic resistance in nature. Front Microbiol. 2013;4:1–13. doi: 10.3389/fmicb.2013.00047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sharma C, Pal B, Singh Thakur N, Gulati S, Gupta S, Mishra KS, Panwar H. Antibacterial effects of Lactobacillus isolates of curd and human milk origin against food-borne and human pathogens. 3Biotech. 2017;7:31–40. doi: 10.1007/s13205-016-0591-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Singh AP, Sharma S, Pagarware K, Siraji RA, Ansari I, Mandal A, Walling P, Aijaz S. Enteropathogenic E. coli effectors EspF and Map independently disrupt tight junctions through distinct mechanisms involving transcriptional and posttranscriptional regulation. Sci Rep. 2018;8:1–17. doi: 10.1038/s41598-018-22017-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taghizadeh M, Safaei HG, Poursina F. Identification of Lactobacillus plantarum in breast milk. Res Mol Med. 2017;5:50–60. [Google Scholar]
  39. Terpou A, Papadaki A, Lappa LK, Kachrimanidou V, Bosnea LA, Kopsahelis N. Probiotics in food systems: significance and emerging strategies towards improved viability and delivery of enhanced beneficial value. Nutrients. 2019;11:1591–1623. doi: 10.3390/nu11071591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Torriani S, Giovanna Felis E, Dellaglio F. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol. 2001;67:3450–3454. doi: 10.1128/AEM.67.8.3450-3454.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tuo Y, Song X, Song Y, Liu W, Tang Y, Gao Y, Jiang S, Fang Q, Guangqing M. Screening probiotics from Lactobacillus strains according to their abilities to inhibit pathogen adhesion and induction of pro-inflammatory cytokine IL-8. J Dairy Sci. 2018;101:4822–4829. doi: 10.3168/jds.2017-13654. [DOI] [PubMed] [Google Scholar]
  42. Uraipan S, Hongpattarakere T. Antagonistic characteristics against food-borne pathogenic bacteria of lactic acid bacteria and bifidobacteria isolated from feces of healthy Thai infants. Jundishapur J Microbiol. 2015;8:1–9. doi: 10.5812/jjm.8(5)2015.18264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yu Q, Wang Z, Yang Q. Lactobacillus amylophilus D14 protects tight junction from enteropathogenic bacteria damage in Caco-2 cells. J Dairy Sci. 2012;95:5580–5587. doi: 10.3168/jds.2012-5540. [DOI] [PubMed] [Google Scholar]

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES