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Isolation and in-vitro probiotic characterization of fructophilic lactic acid bacteria from Chinese fruits and flowers
LWT - Food Science and Technology 104 (2019) 70–75
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Isolation and in-vitro probiotic characterization of fructophilic lactic acid bacteria from Chinese fruits and flowers
T
Hafiz Arbab Sakandara,b, Stan Kubowc, Faizan Ahmed Sadiqa,b,∗ a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China c School of Human Nutrition, Macdonald Campus of McGill University, 21111 Lakeshore, Ste. Anne de Bellevue, Quebec, H9X 3V9, Canada b
A R T I C LE I N FO
A B S T R A C T
Keywords: FLAB Probiotics Fructose Fruit Flower
This study was undertaken to focus on the isolation and probiotic characterization of fructophilic lactic acid bacteria (FLAB) isolated from various flowers and fruits belonging to China. Seventy-three FLAB isolates exhibiting fructose fermentation were grown in 30% fructose media, and only eight isolates showed the capability to grow under such high osmotic pressure. From the above isolates, 16S rRNA gene sequencing revealed two strains as Fructobacillus fructosus, three strains as Lactobacillus kunkeei, two strains as F. pseudoficulneus, and one strain as F. durionis. These strains were characterized subsequently for their carbohydrate metabolism and probiotic potential. In comparison to other isolated FLAB strains, L. kunkeei strains showed the maximum capacity to tolerate various low pH levels and bile acid concentrations together with maximal effects on cholesterol assimilation, antipathogenic activity, and hydrophobicity. The fructophilic strains preferred D-fructose over Dglucose and so these strains have potential for use in cereal fermentation to ameliorate fructose-mediated irritable bowel syndromes.
1. Introduction A large diversity of fruits and flowers provides an outstanding niche of promising health promoting compounds that can be further enhanced after fermentation with a myriad of microbes to generate various plant-based fermented foods (Leroy & De Vuyst, 2014). Lactic acid bacteria (LAB) are typically involved in most fermented foods, which involve biotransformation of natural compounds in plants through various functional and metabolic pathways (Liu et al., 2017; Väkeväinen et al., 2018). It is important to note, that different strains of LAB involved in the fermentation of foods possess varying metabolic characteristics leading to diverse portfolios of bioactive compounds (Liu et al., 2018). Moreover, fruits, vegetables, and flowers harbor a huge reservoir of LAB strains with undiscovered probiotic properties that could be exploited to generate novel health promoting fermented foods (Di Cagno, Filannino, & Gobbetti, 2016, pp. 279–291; Sakandar, Usman, & Imran, 2018). Notwithstanding such a vast repertoire of microbes, each fruit and flower harbors unique microbial communities with only a very small portion represented by LAB. Among the LAB group, fructophilic lactic acid bacteria (FLAB) are newly explored potential LAB that have been indicated to possess unique biofunctional traits (Bulgarelli, Schlaeppi, Spaepen, van Themaat, & Schulze-Lefert,
∗
2013). FLAB are unique group in LAB for their preference of D-fructose over D-glucose as a carbon source. These bacteria are primarily isolated from fructose-rich sources including flowers, fruits, flower and fruitbased fermented products, high fructose containing foods, and from the gut of honey bees (Endo, Futagawa-Endo, & Dicks, 2009). The Fructobacillus genus of FLAB is branched from the regrouping of Leuconostoc spp. based on their morphological, phylogenetic positions, and biochemical attributes (Makarova & Koonin, 2007; Van de Guchte et al., 2006). Along with poor growth on D-glucose, these species also lack ethanol production, which is an unusual attribute due to the incomplete genetic encoding of acetaldehyde dehydrogenase (Endo & Okada, 2008). To date, research on the isolation and characterization of FLAB is scarce (Antunes et al., 2002; Chambel et al., 2006; Leisner et al., 2005). Also, FLAB have not been characterized for their probiotic potential. These isolates could be potential candidates in cereal fermentation in the baking industry. Since FLAB can readily utilize fructose, their cereal fermentation can help in preventing irritable bowel syndrome (IBS) as fructose is considered as a precipitation factor in this syndrome. The present study was undertaken to investigate the isolation and probiotic characterization of FLAB from Chinese flowers and fruits.
Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. E-mail addresses: [email protected], [email protected] (F.A. Sadiq).
https://doi.org/10.1016/j.lwt.2019.01.038 Received 16 September 2018; Received in revised form 13 December 2018; Accepted 23 January 2019 Available online 23 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
concentration. A suspension of 5 mL was vortexed for 10 s and incubated at room temperature for 24 h. After 24 h interval, the autoaggregation was measured at 600 nm. The percentage of auto-aggregation was expressed as follows:
2.1. Fruits and flowers sampling Seven fruits (apple, banana, Chinese peach, plum, cantaloupe, kiwi fruit, and lychee), five flowers (narcissus, pink rose, red rose, yellow rose, and sunflower), honey, and rose petals jam were collected from gardens in Wuxi, Jiangsu Province, China in the months of May and June 2018. The samples were immediately packed to preserve their original microbiota and refrigerated at 4 °C until further analysis.
autoaggregation (%) = [(OD1 − OD2)/ OD1] × 100 Where OD1 represents the optical density at 0 h and OD2 the data after 24 h. All experiments were performed in triplicate. 2.6. Antimicrobial susceptibility of FLAB
2.2. Isolation of FLAB Selected FLAB strains and L. plantrum ATCC 14917 (control) were tested for antimicrobial susceptibility. Twenty hours old culture was used for preparing the inoculum for antimicrobial susceptibility testing. A standard concentration of 2 × 108 CFU/mL of bacterial culture was taken and inserted into an Eppendorf tube containing normal saline. The Eppendorf tubes containing inoculum were mixed thoroughly and then turbidity of each solution was compared with standard MacFarland solution. Mueller-Hinton agar plates were then inoculated with each sample with the help of sterile cotton swab, which were set aside to allow them dry. Five commercial antibiotics were taken to test the susceptibility of FLAB against them. The antibiotics were ciprofloxacin (CIP), ceftriaxone (CRO), novobiocin (NV), gentamicin (CN), and vancomycin. Sterile forceps were used to evenly dispensed and lightly pressed the antibiotic discs onto the agar surface. The assay plates were incubated at 30 °C for 24 h.
Isolation of FLAB was done according to the method of Endo et al. (2011). Briefly, fresh fruits and flowers were collected in aseptic plastic bags, squashed and 5 mL of fructose yeast peptone (FYP) broth was added in the samples. FYP broth was composed of 1% D-fructose, 0.5% polypeptone, 0.2% sodium acetate, 1% yeast extract, 0.05% Tween 80, 0.02% magnesium sulphate heptahydrate, 0.001% iron sulphate heptahydrate, 0.001% manganese sulphate tetra hydrate, 0.001% sodium chloride, 0.005% sodium azide, and 0.005% cycloheximide (pH 6.8). The broth was incubated for 24 h at 30 °C in shaking incubator. After incubation, 50 μl of each sample was inoculated into FYP broth at 30 °C for 24 h and subsequently into 30% FYP broth. The 30% FYP was used for selection of FLAB, which was supplemented with 30% D-fructose. After visible growth was seen, cultures from 30% FYP broth were streaked onto FYP agar, consisting of 0.5% calcium carbonate and 1.5% agar. These FYP agar plates were incubated at 30 °C until colonies were visible. These colonies were selected after careful morphological observation. These isolates were further grown into FYP broth and GYP (glucose yeast peptone) broth (1% D-glucose instead of D-fructose) and incubated static at 30 °C for 24 h. Isolates that showed good growth in the FYP broth but poor growth in the GYP broth were considered as FLAB.
2.7. Antagonistic activity against pathogens by FLAB The antagonistic activity of isolates was carried out by using well diffusion assay according to the method of Sakandar et al. (2018). Briefly, FLAB were grown in MRS broth (pH 6.5). These inoculated broths were incubated at 37 °C for 18 h after that cells were harvested by centrifugation (8000 g for 20 min at 4 °C). The cell free supernatant was sterilized by filtration through a 0.22 μm filter (Millipore). The antipathogenic activity was determined against Salmonella typhimurium CMCC5011, Staphylococcus aureus NCTC8325, and Escherichia coli 25922, These pathogens were grown on Muller Hinton agar and were kept at 40 °C for 4 h. Afterwards, 100 μL of the filtered neutralized supernatants from all isolated strains were added to filter paper discs (6 mm diameter) placed on the Muller Hinton agar plate surface, previously inoculated with indicator pathogens. The plates were incubated for 24 h at 37 °C, and finally the plates were inspected for clear zones around the discs.
2.3. Identification of FLAB strains For the identification of FLAB isolates, phylogenetic analysis was done, based on 16S rDNA gene sequence. The TIANamp bacteria DNA kit (Tiangen Biotech co., LTD, Beijing, China) was used for the extraction of genomic DNA. 16 S rDNA gene sequencing was done through the commercial service of Macrogen Inc. Korea (www.dna.macrogen.com). Internet BLAST Gene database (http://www. ncbi.nlm.nih.gov) was used to analyze obtained DNA sequences. Afterwards, these sequences were submitted to the GenBank. Molecular Evolutionary Genetics Analysis (MEGA 7) software was used for the construction of phylogenetic tree by using the neighbor-joining method (Fig. 1). Accession numbers for the sequences are shown in Table 1.
2.8. In-vitro screening of the selected microbiota as candidates for probiotic properties
2.4. Biochemical analysis 2.8.1. Tolerance to low pH, cell surface hydrophobicity, bile salts tolerance, and cholesterol assimilation To determine the tolerance of FLAB strains, method of Sakandar et al. (2018) was adopted and to determine cell surface hydrophobicity and bile salt tolerance, method of Del Re et al. (2000) was used and the experiment was done in triplicate. For cholesterol assimilation analysis, method of Tomaro-Duchesneau et al. (2014) was used with minor changes as follows. Briefly, FLAB strains were cultured overnight into MRS broth. Cholesterol-PEG 600 was added into MRS broth at a final concentration of 100 μg/mL and an 1% (v/v) inoculum of each overnight grown FLAB strain was inoculated onto MRS-cholesterol-PEG 600 and incubated at 30 °C for 24 h. Afterwards, FLAB suspensions were centrifuged at 2200×g for 10 min and supernatants containing nonassimilated cholesterol were collected. For the determination of cholesterol assimilation, absorbance values were determined through UV–visible spectrophotometer at 570 nm.
Carbohydrates fermentation reactions were analyzed using API CHL 50 galleries (BioMérieux, Marcy l’Etoile, France), according to the manufacturer's instructions. Briefly, inoculum from all isolates with turbidity 0.5 OD nm at 600 nm was added into wells containing 49 sugars and incubated at 37 °C for 24 and 48 h. The obtained results were analyzed with help of manual provided with API CHL 50 galleries. 2.5. Auto-aggregation assay Auto-aggregation experiment was performed according to the method of Del Re, Sgorbati, Miglioli, and Palenzona (2000). Briefly, the FLAB isolates were cultured for 24 h in FYP broth, the cells were centrifuged for 15 min at 4 °C (5000 g). Subsequently cells were washed three times with phosphate saline buffer (PBS) of neutral pH. Further, obtained cells were suspended in sterile PBS to obtain a 108 cfu/mL 71
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Fig. 1. Phylogenetic tree of FLAB and related genera based on partial 16 S rRNA sequence. This phylogenetic tree was constructed by the neighbor-joining method.
JNGBKS4), two strains (JNGBKS1 and JNGBKS3) as F. pseudoficulneus, and one strain (JNGBKS5) as F. durionis. Three strains were isolated from fruits (peach, banana and kiwi fruit) and five from flowers (sunflower, narcissus, yellow rose and pink rose) (Table 1). FLAB strains have been previously isolated from fructose-rich sources (Verón, Di Risio, Isla, & Torres, 2017). Endo et al. (2009) have isolated FLAB from various fruits and flowers. Chambel and his coauthors (2006) isolated FLAB from ripe fig. Many FLAB have been isolated by using 30% FYP broth as strains of F. pseudoficulneus, F. fructosus, and Lactobacillus sp. showed growth in high concentrated fructose media. Growth of many LAB is inhibited in aerobic culturing conditions whereas aerobic culturing conditions has promoted the growth of FLAB, even if the strains are catalase negative (Endo et al., 2018).
Table 1 Fructophilic LAB strains. Strains
Species
Isolated from
Accession #
JNGBKS1 JNGBKS2 JNGBKS3 JNGBKS4 JNGBKS5 JNGBKS6 JNGBKS7 JNGBKS8
F. F. F. F. F. L. L. L.
Peach Narcissus Banana Sunflower Kiwi fruit Narcissus Yellow rose Pink rose
MH796215 MH796216 MH796217 MH796218 MH796219 MH796220 MH796221 MH796222
pseudoficulneus fructosus pseudoficulneus fructosus durionis kunkeei Kunkeei Kunkeei
2.9. Statistical analysis The obtained results were statistically compared and analyzed by using SPSS (Version 16.1) software and these results are expressed as mean ± SD. Tukey's test was used to determine the differences between means at significance level of p < 0.05.
3.2. Biochemical analysis All FLAB strains were fermented with only few carbohydrates (two to five, among forty-nine tested carbohydrates). Three strains of L. kunkeei (JNGBKS6, JNGBKS7, and JNGBKS8) fermented 5 out of 49 carbohydrates in the API CHL50 test gallery in 1–4 days. These three strains fermented D-fructose and D-glucose within 1 day while mannitol, sucrose, and trehalose were fermented in 3–4 days. Two strains of F. pseudoficulneus (JNGBKS1 and JNGBKS3) also fermented D-fructose and D-Glucose in one day whereas none of the other carbohydrates were fermented. Two strains of F. fructosus (JNGBKS2 and JNGBKS 4) were unable to ferment trehalose; however, D-fructose, D-glucose, mannitol, and trehalose were fermented in 1–3 days. D-fructose was fermented first, followed by D-glucose, mannitol, and sucrose. In the case of F. durionis JNGBKS, mannitol was not fermented even up to 5 days of treatment although the other carbohydrates (D-fructose, D-glucose,
3. Results and discussion 3.1. Isolation and identification of FLAB Out of the twelve analyzed flowers and fruit samples, 73 strains of FLAB with clear zones on MRS agar supplemented with CaCO3 were isolated, based on Gram staining along with oxidase and catalase tests. Among these 73 strains, only 8 FLAB strains had the capacity to grow in the 30% FYP. Sequence analyses recognized three strains (JNGBKS6, JNGBKS7 and JNGBKS8) as Lactobacillus kunkeei, two strains (JNGBKS2 and JNGBKS4) as Fructobacillus fructosus, two strains (JNGBKS2 and 72
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significant differences (p < 0.05) were observed among F. fructosus, F. pseudoficulneus, and F. durions strains. Only a minimum hydrophobicity was observed for F. pseudoficulneus JNGBKS3 and a minimum autoaggregation was observed with F. durionis JNGBKS5 (Table 2). Taken together, all the isolates tested in the present study exhibited some degree of auto-aggregation, which is a characteristic might have been adapted by these strains as protective survival mechanism in the hostile environmental conditions of the GIT. The present study findings in terms of the auto-aggregation and hydrophobicity values are in concert with previous findings of Cebrián et al. (2012), and Riaz Rajoka et al. (2017), regarding probiotic characteristics of L. rhamnosus isolated from human milk.
Table 2 Carbohydrates fermentation by fructophilic LAB isolated from Chinese fruits and flowers. Strains
D-fructose
D-glucose
Mannitol
Sucrose
Trehalose
F. pseudoficulneus JNGBKS1 F. pseudoficulneus JNGBKS3 F. fructosus JNGBKS2 F. fructosus JNGBKS4 F. durionis JNGBKS5 L. kunkeei JNGBKS6 L. kunkeei JNGBKS7 L. kunkeei JNGBKS8
1da
1d
–
–
–
1d
1d
–
–
–
1d 1d 1d 1d 1d 1d
2d 2d 2d 1d 1d 1d
2d 2d – 3d 4d 3d
3d 3d 3d 3d 3d 3d
– – 4d 4d 3d 3d
3.4. Antagonistic activity against pathogens by FLAB
a
Number of days needed for fermentation of carbohydrates and - denotes negative results.
The production of antimicrobial compounds by LAB is a pivotal attribute for probiotics to compete with, inhibit or kill pathogens (Angmo, Kumari, Savitri, & Bhalla, 2016). Anti-pathogenic activity of FLAB was evaluated against E. coli, S. typhimurium, and S. aureus (Table 3). L. plantrum ATCC 14917 exhibited significantly (p < 0.05) high anti-pathogenic activity against all tested bacterial pathogens. Among the isolated FLAB strains, maximum anti-pathogenicity was shown by L. kunkeei strains against all three pathogens whereas the lowest (4 mm) anti-pathogenic activity was exhibited by F. durionis JNHBKS5. Among all tested pathogens S. typhimurium showed the most resistance against FLAB. Overall, the present anti-pathogenic results are in concurrence with the findings of Al Kassaa, Hamze, Hober, Chihib, & Drider (2014) on the probiotic characterization of LAB isolated from vagina. The anti-microbial activity of LAB and Bacillus sp. has been attributed to a variety of inhibitory anti-pathogenic bacterial metabolites, particularly bacteriocins (Barbosa, Jurkiewicz, Landgraf, Todorov, & Franco, 2018).
sucrose, and trehalose) were fermented within 1–4 days (Table 2). All strains fermented D-fructose and D-glucose in one day except F. durionis and F. fructosus. All isolated L. kunkeei strains fermented all tested carbohydrates, which could be due to the relatively larger genome size of L. kunkeei compared to isolated Fructobacillus spp. These latter results are in concurrence with the findings of Endo and Okada (2008) who characterized FLAB from various fructose-rich sources. Similar findings have also been reported pertaining to the isolation of FLAB from ripe figs (Antunes et al., 2002). 3.3. Auto-aggregation analysis Auto-aggregation is a probiotic characteristic that pertains to entrapment of bacteria in an aggregated form, which that allows for the stability of microbial strains in the gastrointestinal tract (GIT) resulting from lesser exposure to inhospitable intestinal conditions (Sakandar et al., 2018). The adherence ability of probiotic bacteria to the intestinal epithelial cells involves various types of interactions including hydrophobicity and auto-aggregation (Botes, Loos, van Reenen, & Dicks, 2008; Duary, Rajput, Batish, & Grover, 2011). Auto-aggregation analysis revealed that significantly high auto-aggregation was observed in L. kunkeei strains while no significant difference was seen among these strains. Among the isolated FLAB, maximum auto-aggregation was exhibited by L. kunkeei JNGBKS7 (44.54 ± 0.31) following L. plantrum ATCC 14917 (64.34 ± 0.29). F. pseudoficulneus JNGBKS1, F. pseudoficulneus JNGBKS3, F. fructosus JNGBKS2, and F. fructosus JNGBKS4 did not show a significant difference (p < 0.05) in autoaggregation, which had auto-aggregation values of 31.85, 30.24, 30.89, and 29.57, respectively. Maximum hydrophobicity was observed in L. plantrum ATCC 14917 (60.86 ± 1.12) followed by L. kunkeei JNGBKS7 (39.92 ± 1.11). No significant difference in hydrophobicity was observed between L. kunkeei JNGBKS6 and L. kunkeei JNGBKS8 although
3.5. Antibiotic resistance FLAB isolates were analyzed for their ability to tolerate antibiotics due to safety considerations. None of the FLAB isolates was resistant to any of the tested antibiotics. All isolated FLAB exhibited intermediate susceptibility against novobiocin, whereas F. fructosus, F. durionis, and L. kunkeei strains showed intermediate susceptibility against gentamicin. In case of ceftrioxin, all three strains of L. kunkeei and F. pseudoficulneus JNGBKS3 showed susceptibility (Fig. 2). In this experiment the control strain (L. plantarum ATCC 14197) had significantly high (p < 0.05) susceptibility against all antibiotics tested. Minimum values of antibiotic susceptibility were observed for the L. kunkeei strains. These latter strains are phylogenetically closer to the Leuconostoc spp. As reported by Casado Muñoz, Benomar, Lerma, Gálvez, and Abriouel (2014), genetically Leuconostoc spp. have resistance against some common antibiotics and so the relatively low antibiotic susceptibility of
Table 3 Auto-aggregation, hydrophobicity, and antagonistic activity of isolates (n = 3). Strain
% Auto-aggregation
Hydrophobicity (%)
Antagonistic activity E. coli 25922
Lactobacillus plantrum ATCC 14917 F. pseudoficulneus JNGBKS1 F. pseudoficulneus JNGBKS3 F. fructosus JNGBKS2 F. fructosus JNGBKS4 F. durionis JNGBKS5 L. kunkeei JNGBKS6 L. kunkeei JNGBKS7 L. kunkeei JNGBKS8
64.34 31.85 30.24 30.89 29.57 27.97 40.43 44.54 42.37
± ± ± ± ± ± ± ± ±
a
0.29 0.14c 0.13c 0.24c 0.17cd 0.22d 0.11b 0.31b 0.31 b
60.86 29.45 28.17 30.48 29.24 30.84 37.45 39.92 37.89
± ± ± ± ± ± ± ± ±
a
a
1.12 0.54e 1.23e 1.14d 0.93e 0.87d 0.72c 1.11b 0.75c
11 ± 0.4 7.0 ± 0.4c 7.5 ± 0.6c 6.5 ± 0.5d 7.5 ± 0.2c 7.0 ± 0.1c 9.5 ± 0.3b 9.0 ± 0.3 b 9.0 ± 0.2 b
a-e
S. aureus NCTC8325 a
10 ± 0.5 7.0 ± 0.6c 6.5 ± 0.2d 7.0 ± 0.1c 7.0 ± 0.3c 6.0 ± 0.2d 8.0 ± 05b 8.0 ± 0.2b 8.5 ± 0.2b
S. typhimurium CMCC5011 10 ± 0.2 a 6.5 ± 0.3c 7.0 ± 0.1b 6.0 ± 0.4c 6.0 ± 0.4c 4.0 ± 0.2d 7.0 ± 0.3b 7.5 ± 0.2b 7.0 ± 0.3b
Different superscript letters in the same column indicate statistical differences in each strain at the level of p < 0.05 as measured by Tukey's test. All the results were obtained after 24 h and the values are represented as mean SD of three independent replicates. 73
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Fig. 2. Antibiotic susceptibility of FLAB. The antibiotic susceptibility is indicated as the diameter of the clearing zone around the isolate colonies. The values are the expressed as mean ± SD (n = 3). The data less than 15 mm indicates resistance (R); 15 mm–25 mm indicate Intermediate susceptibility (IS), 30 mm–45 mm indicate highly susceptibility (S). Significant difference (different letters) was observed between strains and antibiotics by the Tukey's test (p < 0.05).
cardiovascular diseases (Chien, Wu, Lee, & Hwang, 2010) as it has been reported that risks of coronary disease can be reduced 2–3% by a 1% decrease in plasma cholesterol (Jeun et al., 2010). In that regard, probiotics could play a preventative role as LAB strains are indicated to be involved in cholesterol assimilation (Abushelaibi, Al-Mahadin, ElTarabily, Shah, & Ayyash, 2017; Iranmanesh, Ezzatpanah, & Mojgani, 2014). The present findings revealed that L. kunkeei strains had significantly higher (p < 0.05) cholesterol assimilation as compared to control samples. Significantly lower cholesterol assimilation was exhibited by F. durionis JNGBKS5 (Table 4). The L. kunkeei strains were previously reported to originate from honey bee hives and honey bee intestines (Endo & Salminen, 2013; Verón et al., 2017), and it can be hypothesized that honeybees are the source of these strains in flowers and fruits.
L. kunkeei could be partly attributed to their genetic makeup. Similar findings have been obtained using L. rhamnosus GG against various antibiotics (Chang, Wang, Lau, Lin, & Tang, 2009). Likewise, Cebrián et al. (2012) showed similar degrees of resistance against various antibiotics of the probiotic E. faecalis strains as well as in isolated LAB from Opuntia ficus-indica fruit (Verón et al. (2017).
3.6. Survival under GIT conditions For the probiotic characterization, a paramount factor is the survival of LAB strains in GIT. To evaluate these characteristics, isolated strains were subjected to mimicked intestinal conditions of which pH is an important probiotic characteristic. All strains showed survival at the various tested pH levels (2, 2.5, 3, and 7.4). Maximum survival was shown at pH 3 while minimum survival was revealed at pH 7.4. L. plantrum ATCC 14917 had significantly high (p < 0.05) survival rates followed by the strains of L. kunkeei and F. pseudoficuneus while minimum survival was observed for F. durionis JNGBKS5 (Table 4). Survival of LAB strains at low pH is an uncommon characteristic as previously reported and these results are in accordance to the studies of Sakandar et al. (2018). Bile salt tolerance is another important attribute of probiotics. In the present studies L. kunkeei strains had significantly high (p < 0.05) bile salt tolerance followed by L. plantrum ATCC 14917 whereas minimum tolerance was observed for the F. durionis and F. fructosus strains (Table 4). Survival rate of L. rhamnosus and L. casei under such conditions was higher than our isolates (Palaniswamy & Govindaswamy, 2016; Riaz Rajoka et al., 2017) but the present results are consistent with the findings of Verón et al. (2017). Hypercholesterolemia is the foremost cause of atherosclerosis and
4. Conclusions The present work provides evidence for opportunities to exploit fruits and flowers as relatively unexplored sources for the isolation of novel LAB strains for their potential use as probiotics. In the present study, three, out of eight strains of L. kunkeei isolated FLAB showed significant probiotic potential in terms of their anti-pathogenic activity and tendency to survive under simulated intestinal conditions. Due to preference of D-fructose utilization of these latter strains, there is the possibility that such bacteria could be used in cereal fermentation to ameliorate risk of irritable bowel syndrome. The current study provides strong support for further in-vitro and in-vivo research to identify the potentially beneficial characteristics of these FLAB isolates and their use in food for human consumption. 74
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Table 4 In-vitro screening of selected microbiota as candidate for probiotic (n = 3). Strains
pH tolerance (%) 2.0
L. F. F. F. F. F. L. L. L.
plantrum ATCC 14917 pseudoficulneus JNGBKS1 pseudoficulneus JNGBKS3 fructosus JNGBKS2 fructosus JNGBKS4 durionis JNGBKS5 kunkeei JNGBKS6 kunkeei JNGBKS7 kunkeei JNGBKS8
Bile salt tolerance (%)
2.5 a
91.20 82.00c 81.22c 78.60bc 76.40d 68.70e 84.74b 85.80b 81.35c
3.0 a
93.70 84.10b 82.00bc 79.90d 79.30d 75.40e 86.20b 87.40b 84.65b
7.4 a
94.10 85.40c 82.80bc 82.40bc 81.10c 78.40d 88.00b 87.45b 85.36c
0 a
82.20 68.35a 79.75ab 80.70a 63.25a 59.40a 60.50b 62.22b 58.35a
0.2 a
100 100a 100a 100a 100a 100a 100a 100a 100a
Cholesterol assimilation (%) 0.4
a
94.70 84.10b 83.00b 82.90c 72.40d 68.20e 83.40b 85.50b 83.40b
92.50a 72.90b 71.90c 64.20e 67.40d 52.50f 71.70c 72.00b 73.35b
41.87 32.89 29.41 27.78 30.75 25.17 34.69 31.54 32.40
± ± ± ± ± ± ± ± ±
1.47a 0.98b 1.21bc 0.78d 0.97bc 0.65e 0.53b 0.41c 15b
a-f
Different superscript letters in the same column indicate statistical differences in each strain at the level of p < 0.05 as measured by Tukey's test. All the results were obtained after 24 h and the values are represented as mean SD of three independent replicates.
Acknowledgements
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The work was supported by the grant from China Postdoctoral Organization (Grant No. 2018M642162). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.01.038. References Abushelaibi, A., Al-Mahadin, S., El-Tarabily, K., Shah, N. P., & Ayyash, M. (2017). Characterization of potential probiotic lactic acid bacteria isolated from camel milk. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 79, 316–325. Angmo, K., Kumari, A., Savitri, & Bhalla, T. C. (2016). Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. LebensmittelWissenschaft und -Technologie- Food Science and Technology, 66, 428–435. Antunes, A., Rainey, F. A., Nobre, M. F., Schumann, P., Ferreira, A. M., Ramos, A., et al. (2002). Leuconostoc ficulneum sp. nov., a novel lactic acid bacterium isolated from a ripe fig, and reclassification of Lactobacillus fructosus as Leuconostoc fructosum comb. nov. International Journal of Systematic and Evolutionary Microbiology, 52(2), 647–655. Barbosa, M. S., Jurkiewicz, C., Landgraf, M., Todorov, S. D., & Franco, B. D. G. M. (2018). Effect of proteins, glucose and NaCl on growth, biosynthesis and functionality of bacteriocins of Lactobacillus sakei subsp. sakei 2a in foods during storage at 4 °C: Tests in food models. LWT, - Food Science and Technology, 95, 167–171. Botes, M., Loos, B., van Reenen, C. A., & Dicks, L. M. (2008). Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments. Archives of Microbiology, 190(5), 573–584. Bulgarelli, D., Schlaeppi, K., Spaepen, S., van Themaat, E. V. L., & Schulze-Lefert, P. (2013). Structure and functions of the bacterial microbiota of plants. Annual Review of Plant Biology, 64, 807–838. Casado Muñoz, M. d. C., Benomar, N., Lerma, L. L., Gálvez, A., & Abriouel, H. (2014). Antibiotic resistance of Lactobacillus pentosus and Leuconostoc pseudomesenteroides isolated from naturally-fermented Aloreña table olives throughout fermentation process. International Journal of Food Microbiology, 172, 110–118. Cebrián, R., Baños, A., Valdivia, E., Pérez-Pulido, R., Martínez-Bueno, M., & Maqueda, M. (2012). Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain. Food Microbiology, 30(1), 59–67. Chambel, L., Chelo, I. M., Zé-Zé, L., Pedro, L. G., Santos, M. A., & Tenreiro, R. (2006). Leuconostoc pseudoficulneum sp. nov., isolated from a ripe fig. International Journal of Systematic and Evolutionary Microbiology, 56(6), 1375–1381. Chang, Y. T., Wang, C. Y., Lau, J. J., Lin, J. H., & Tang, S. T. (2009). A microprocessorbased biofeedback emotion system, september 7 - 12, 2009. Munich, Germany, Berlin, Heidelberg. Chien, Y. L., Wu, L. Y., Lee, T. C., & Hwang, L. S. (2010). Cholesterol-lowering effect of phytosterol-containing lactic-fermented milk powder in hamsters. Food Chemistry, 119(3), 1121–1126. Del Re, B., Sgorbati, B., Miglioli, M., & Palenzona, D. (2000). Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Letters in Applied Microbiology, 31(6), 438–442. Di Cagno, R., Filannino, P., & Gobbetti, M. (2016). Novel fermented fruit and vegetablebased products. Novel Food Fermentation Technologies. Springer. Duary, R. K., Rajput, Y. S., Batish, V. K., & Grover, S. (2011). Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. Indian Journal of Medical Research, 134(5), 664–671. Endo, A., Futagawa-Endo, Y., & Dicks, L. M. (2009). Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches. Systematic & Applied
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Isolation and in-vitro probiotic characterization of fructophilic lactic acid bacteria from Chinese fruits and flowers
T
Hafiz Arbab Sakandara,b, Stan Kubowc, Faizan Ahmed Sadiqa,b,∗ a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China c School of Human Nutrition, Macdonald Campus of McGill University, 21111 Lakeshore, Ste. Anne de Bellevue, Quebec, H9X 3V9, Canada b
A R T I C LE I N FO
A B S T R A C T
Keywords: FLAB Probiotics Fructose Fruit Flower
This study was undertaken to focus on the isolation and probiotic characterization of fructophilic lactic acid bacteria (FLAB) isolated from various flowers and fruits belonging to China. Seventy-three FLAB isolates exhibiting fructose fermentation were grown in 30% fructose media, and only eight isolates showed the capability to grow under such high osmotic pressure. From the above isolates, 16S rRNA gene sequencing revealed two strains as Fructobacillus fructosus, three strains as Lactobacillus kunkeei, two strains as F. pseudoficulneus, and one strain as F. durionis. These strains were characterized subsequently for their carbohydrate metabolism and probiotic potential. In comparison to other isolated FLAB strains, L. kunkeei strains showed the maximum capacity to tolerate various low pH levels and bile acid concentrations together with maximal effects on cholesterol assimilation, antipathogenic activity, and hydrophobicity. The fructophilic strains preferred D-fructose over Dglucose and so these strains have potential for use in cereal fermentation to ameliorate fructose-mediated irritable bowel syndromes.
1. Introduction A large diversity of fruits and flowers provides an outstanding niche of promising health promoting compounds that can be further enhanced after fermentation with a myriad of microbes to generate various plant-based fermented foods (Leroy & De Vuyst, 2014). Lactic acid bacteria (LAB) are typically involved in most fermented foods, which involve biotransformation of natural compounds in plants through various functional and metabolic pathways (Liu et al., 2017; Väkeväinen et al., 2018). It is important to note, that different strains of LAB involved in the fermentation of foods possess varying metabolic characteristics leading to diverse portfolios of bioactive compounds (Liu et al., 2018). Moreover, fruits, vegetables, and flowers harbor a huge reservoir of LAB strains with undiscovered probiotic properties that could be exploited to generate novel health promoting fermented foods (Di Cagno, Filannino, & Gobbetti, 2016, pp. 279–291; Sakandar, Usman, & Imran, 2018). Notwithstanding such a vast repertoire of microbes, each fruit and flower harbors unique microbial communities with only a very small portion represented by LAB. Among the LAB group, fructophilic lactic acid bacteria (FLAB) are newly explored potential LAB that have been indicated to possess unique biofunctional traits (Bulgarelli, Schlaeppi, Spaepen, van Themaat, & Schulze-Lefert,
∗
2013). FLAB are unique group in LAB for their preference of D-fructose over D-glucose as a carbon source. These bacteria are primarily isolated from fructose-rich sources including flowers, fruits, flower and fruitbased fermented products, high fructose containing foods, and from the gut of honey bees (Endo, Futagawa-Endo, & Dicks, 2009). The Fructobacillus genus of FLAB is branched from the regrouping of Leuconostoc spp. based on their morphological, phylogenetic positions, and biochemical attributes (Makarova & Koonin, 2007; Van de Guchte et al., 2006). Along with poor growth on D-glucose, these species also lack ethanol production, which is an unusual attribute due to the incomplete genetic encoding of acetaldehyde dehydrogenase (Endo & Okada, 2008). To date, research on the isolation and characterization of FLAB is scarce (Antunes et al., 2002; Chambel et al., 2006; Leisner et al., 2005). Also, FLAB have not been characterized for their probiotic potential. These isolates could be potential candidates in cereal fermentation in the baking industry. Since FLAB can readily utilize fructose, their cereal fermentation can help in preventing irritable bowel syndrome (IBS) as fructose is considered as a precipitation factor in this syndrome. The present study was undertaken to investigate the isolation and probiotic characterization of FLAB from Chinese flowers and fruits.
Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. E-mail addresses: [email protected], [email protected] (F.A. Sadiq).
https://doi.org/10.1016/j.lwt.2019.01.038 Received 16 September 2018; Received in revised form 13 December 2018; Accepted 23 January 2019 Available online 23 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
concentration. A suspension of 5 mL was vortexed for 10 s and incubated at room temperature for 24 h. After 24 h interval, the autoaggregation was measured at 600 nm. The percentage of auto-aggregation was expressed as follows:
2.1. Fruits and flowers sampling Seven fruits (apple, banana, Chinese peach, plum, cantaloupe, kiwi fruit, and lychee), five flowers (narcissus, pink rose, red rose, yellow rose, and sunflower), honey, and rose petals jam were collected from gardens in Wuxi, Jiangsu Province, China in the months of May and June 2018. The samples were immediately packed to preserve their original microbiota and refrigerated at 4 °C until further analysis.
autoaggregation (%) = [(OD1 − OD2)/ OD1] × 100 Where OD1 represents the optical density at 0 h and OD2 the data after 24 h. All experiments were performed in triplicate. 2.6. Antimicrobial susceptibility of FLAB
2.2. Isolation of FLAB Selected FLAB strains and L. plantrum ATCC 14917 (control) were tested for antimicrobial susceptibility. Twenty hours old culture was used for preparing the inoculum for antimicrobial susceptibility testing. A standard concentration of 2 × 108 CFU/mL of bacterial culture was taken and inserted into an Eppendorf tube containing normal saline. The Eppendorf tubes containing inoculum were mixed thoroughly and then turbidity of each solution was compared with standard MacFarland solution. Mueller-Hinton agar plates were then inoculated with each sample with the help of sterile cotton swab, which were set aside to allow them dry. Five commercial antibiotics were taken to test the susceptibility of FLAB against them. The antibiotics were ciprofloxacin (CIP), ceftriaxone (CRO), novobiocin (NV), gentamicin (CN), and vancomycin. Sterile forceps were used to evenly dispensed and lightly pressed the antibiotic discs onto the agar surface. The assay plates were incubated at 30 °C for 24 h.
Isolation of FLAB was done according to the method of Endo et al. (2011). Briefly, fresh fruits and flowers were collected in aseptic plastic bags, squashed and 5 mL of fructose yeast peptone (FYP) broth was added in the samples. FYP broth was composed of 1% D-fructose, 0.5% polypeptone, 0.2% sodium acetate, 1% yeast extract, 0.05% Tween 80, 0.02% magnesium sulphate heptahydrate, 0.001% iron sulphate heptahydrate, 0.001% manganese sulphate tetra hydrate, 0.001% sodium chloride, 0.005% sodium azide, and 0.005% cycloheximide (pH 6.8). The broth was incubated for 24 h at 30 °C in shaking incubator. After incubation, 50 μl of each sample was inoculated into FYP broth at 30 °C for 24 h and subsequently into 30% FYP broth. The 30% FYP was used for selection of FLAB, which was supplemented with 30% D-fructose. After visible growth was seen, cultures from 30% FYP broth were streaked onto FYP agar, consisting of 0.5% calcium carbonate and 1.5% agar. These FYP agar plates were incubated at 30 °C until colonies were visible. These colonies were selected after careful morphological observation. These isolates were further grown into FYP broth and GYP (glucose yeast peptone) broth (1% D-glucose instead of D-fructose) and incubated static at 30 °C for 24 h. Isolates that showed good growth in the FYP broth but poor growth in the GYP broth were considered as FLAB.
2.7. Antagonistic activity against pathogens by FLAB The antagonistic activity of isolates was carried out by using well diffusion assay according to the method of Sakandar et al. (2018). Briefly, FLAB were grown in MRS broth (pH 6.5). These inoculated broths were incubated at 37 °C for 18 h after that cells were harvested by centrifugation (8000 g for 20 min at 4 °C). The cell free supernatant was sterilized by filtration through a 0.22 μm filter (Millipore). The antipathogenic activity was determined against Salmonella typhimurium CMCC5011, Staphylococcus aureus NCTC8325, and Escherichia coli 25922, These pathogens were grown on Muller Hinton agar and were kept at 40 °C for 4 h. Afterwards, 100 μL of the filtered neutralized supernatants from all isolated strains were added to filter paper discs (6 mm diameter) placed on the Muller Hinton agar plate surface, previously inoculated with indicator pathogens. The plates were incubated for 24 h at 37 °C, and finally the plates were inspected for clear zones around the discs.
2.3. Identification of FLAB strains For the identification of FLAB isolates, phylogenetic analysis was done, based on 16S rDNA gene sequence. The TIANamp bacteria DNA kit (Tiangen Biotech co., LTD, Beijing, China) was used for the extraction of genomic DNA. 16 S rDNA gene sequencing was done through the commercial service of Macrogen Inc. Korea (www.dna.macrogen.com). Internet BLAST Gene database (http://www. ncbi.nlm.nih.gov) was used to analyze obtained DNA sequences. Afterwards, these sequences were submitted to the GenBank. Molecular Evolutionary Genetics Analysis (MEGA 7) software was used for the construction of phylogenetic tree by using the neighbor-joining method (Fig. 1). Accession numbers for the sequences are shown in Table 1.
2.8. In-vitro screening of the selected microbiota as candidates for probiotic properties
2.4. Biochemical analysis 2.8.1. Tolerance to low pH, cell surface hydrophobicity, bile salts tolerance, and cholesterol assimilation To determine the tolerance of FLAB strains, method of Sakandar et al. (2018) was adopted and to determine cell surface hydrophobicity and bile salt tolerance, method of Del Re et al. (2000) was used and the experiment was done in triplicate. For cholesterol assimilation analysis, method of Tomaro-Duchesneau et al. (2014) was used with minor changes as follows. Briefly, FLAB strains were cultured overnight into MRS broth. Cholesterol-PEG 600 was added into MRS broth at a final concentration of 100 μg/mL and an 1% (v/v) inoculum of each overnight grown FLAB strain was inoculated onto MRS-cholesterol-PEG 600 and incubated at 30 °C for 24 h. Afterwards, FLAB suspensions were centrifuged at 2200×g for 10 min and supernatants containing nonassimilated cholesterol were collected. For the determination of cholesterol assimilation, absorbance values were determined through UV–visible spectrophotometer at 570 nm.
Carbohydrates fermentation reactions were analyzed using API CHL 50 galleries (BioMérieux, Marcy l’Etoile, France), according to the manufacturer's instructions. Briefly, inoculum from all isolates with turbidity 0.5 OD nm at 600 nm was added into wells containing 49 sugars and incubated at 37 °C for 24 and 48 h. The obtained results were analyzed with help of manual provided with API CHL 50 galleries. 2.5. Auto-aggregation assay Auto-aggregation experiment was performed according to the method of Del Re, Sgorbati, Miglioli, and Palenzona (2000). Briefly, the FLAB isolates were cultured for 24 h in FYP broth, the cells were centrifuged for 15 min at 4 °C (5000 g). Subsequently cells were washed three times with phosphate saline buffer (PBS) of neutral pH. Further, obtained cells were suspended in sterile PBS to obtain a 108 cfu/mL 71
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Fig. 1. Phylogenetic tree of FLAB and related genera based on partial 16 S rRNA sequence. This phylogenetic tree was constructed by the neighbor-joining method.
JNGBKS4), two strains (JNGBKS1 and JNGBKS3) as F. pseudoficulneus, and one strain (JNGBKS5) as F. durionis. Three strains were isolated from fruits (peach, banana and kiwi fruit) and five from flowers (sunflower, narcissus, yellow rose and pink rose) (Table 1). FLAB strains have been previously isolated from fructose-rich sources (Verón, Di Risio, Isla, & Torres, 2017). Endo et al. (2009) have isolated FLAB from various fruits and flowers. Chambel and his coauthors (2006) isolated FLAB from ripe fig. Many FLAB have been isolated by using 30% FYP broth as strains of F. pseudoficulneus, F. fructosus, and Lactobacillus sp. showed growth in high concentrated fructose media. Growth of many LAB is inhibited in aerobic culturing conditions whereas aerobic culturing conditions has promoted the growth of FLAB, even if the strains are catalase negative (Endo et al., 2018).
Table 1 Fructophilic LAB strains. Strains
Species
Isolated from
Accession #
JNGBKS1 JNGBKS2 JNGBKS3 JNGBKS4 JNGBKS5 JNGBKS6 JNGBKS7 JNGBKS8
F. F. F. F. F. L. L. L.
Peach Narcissus Banana Sunflower Kiwi fruit Narcissus Yellow rose Pink rose
MH796215 MH796216 MH796217 MH796218 MH796219 MH796220 MH796221 MH796222
pseudoficulneus fructosus pseudoficulneus fructosus durionis kunkeei Kunkeei Kunkeei
2.9. Statistical analysis The obtained results were statistically compared and analyzed by using SPSS (Version 16.1) software and these results are expressed as mean ± SD. Tukey's test was used to determine the differences between means at significance level of p < 0.05.
3.2. Biochemical analysis All FLAB strains were fermented with only few carbohydrates (two to five, among forty-nine tested carbohydrates). Three strains of L. kunkeei (JNGBKS6, JNGBKS7, and JNGBKS8) fermented 5 out of 49 carbohydrates in the API CHL50 test gallery in 1–4 days. These three strains fermented D-fructose and D-glucose within 1 day while mannitol, sucrose, and trehalose were fermented in 3–4 days. Two strains of F. pseudoficulneus (JNGBKS1 and JNGBKS3) also fermented D-fructose and D-Glucose in one day whereas none of the other carbohydrates were fermented. Two strains of F. fructosus (JNGBKS2 and JNGBKS 4) were unable to ferment trehalose; however, D-fructose, D-glucose, mannitol, and trehalose were fermented in 1–3 days. D-fructose was fermented first, followed by D-glucose, mannitol, and sucrose. In the case of F. durionis JNGBKS, mannitol was not fermented even up to 5 days of treatment although the other carbohydrates (D-fructose, D-glucose,
3. Results and discussion 3.1. Isolation and identification of FLAB Out of the twelve analyzed flowers and fruit samples, 73 strains of FLAB with clear zones on MRS agar supplemented with CaCO3 were isolated, based on Gram staining along with oxidase and catalase tests. Among these 73 strains, only 8 FLAB strains had the capacity to grow in the 30% FYP. Sequence analyses recognized three strains (JNGBKS6, JNGBKS7 and JNGBKS8) as Lactobacillus kunkeei, two strains (JNGBKS2 and JNGBKS4) as Fructobacillus fructosus, two strains (JNGBKS2 and 72
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significant differences (p < 0.05) were observed among F. fructosus, F. pseudoficulneus, and F. durions strains. Only a minimum hydrophobicity was observed for F. pseudoficulneus JNGBKS3 and a minimum autoaggregation was observed with F. durionis JNGBKS5 (Table 2). Taken together, all the isolates tested in the present study exhibited some degree of auto-aggregation, which is a characteristic might have been adapted by these strains as protective survival mechanism in the hostile environmental conditions of the GIT. The present study findings in terms of the auto-aggregation and hydrophobicity values are in concert with previous findings of Cebrián et al. (2012), and Riaz Rajoka et al. (2017), regarding probiotic characteristics of L. rhamnosus isolated from human milk.
Table 2 Carbohydrates fermentation by fructophilic LAB isolated from Chinese fruits and flowers. Strains
D-fructose
D-glucose
Mannitol
Sucrose
Trehalose
F. pseudoficulneus JNGBKS1 F. pseudoficulneus JNGBKS3 F. fructosus JNGBKS2 F. fructosus JNGBKS4 F. durionis JNGBKS5 L. kunkeei JNGBKS6 L. kunkeei JNGBKS7 L. kunkeei JNGBKS8
1da
1d
–
–
–
1d
1d
–
–
–
1d 1d 1d 1d 1d 1d
2d 2d 2d 1d 1d 1d
2d 2d – 3d 4d 3d
3d 3d 3d 3d 3d 3d
– – 4d 4d 3d 3d
3.4. Antagonistic activity against pathogens by FLAB
a
Number of days needed for fermentation of carbohydrates and - denotes negative results.
The production of antimicrobial compounds by LAB is a pivotal attribute for probiotics to compete with, inhibit or kill pathogens (Angmo, Kumari, Savitri, & Bhalla, 2016). Anti-pathogenic activity of FLAB was evaluated against E. coli, S. typhimurium, and S. aureus (Table 3). L. plantrum ATCC 14917 exhibited significantly (p < 0.05) high anti-pathogenic activity against all tested bacterial pathogens. Among the isolated FLAB strains, maximum anti-pathogenicity was shown by L. kunkeei strains against all three pathogens whereas the lowest (4 mm) anti-pathogenic activity was exhibited by F. durionis JNHBKS5. Among all tested pathogens S. typhimurium showed the most resistance against FLAB. Overall, the present anti-pathogenic results are in concurrence with the findings of Al Kassaa, Hamze, Hober, Chihib, & Drider (2014) on the probiotic characterization of LAB isolated from vagina. The anti-microbial activity of LAB and Bacillus sp. has been attributed to a variety of inhibitory anti-pathogenic bacterial metabolites, particularly bacteriocins (Barbosa, Jurkiewicz, Landgraf, Todorov, & Franco, 2018).
sucrose, and trehalose) were fermented within 1–4 days (Table 2). All strains fermented D-fructose and D-glucose in one day except F. durionis and F. fructosus. All isolated L. kunkeei strains fermented all tested carbohydrates, which could be due to the relatively larger genome size of L. kunkeei compared to isolated Fructobacillus spp. These latter results are in concurrence with the findings of Endo and Okada (2008) who characterized FLAB from various fructose-rich sources. Similar findings have also been reported pertaining to the isolation of FLAB from ripe figs (Antunes et al., 2002). 3.3. Auto-aggregation analysis Auto-aggregation is a probiotic characteristic that pertains to entrapment of bacteria in an aggregated form, which that allows for the stability of microbial strains in the gastrointestinal tract (GIT) resulting from lesser exposure to inhospitable intestinal conditions (Sakandar et al., 2018). The adherence ability of probiotic bacteria to the intestinal epithelial cells involves various types of interactions including hydrophobicity and auto-aggregation (Botes, Loos, van Reenen, & Dicks, 2008; Duary, Rajput, Batish, & Grover, 2011). Auto-aggregation analysis revealed that significantly high auto-aggregation was observed in L. kunkeei strains while no significant difference was seen among these strains. Among the isolated FLAB, maximum auto-aggregation was exhibited by L. kunkeei JNGBKS7 (44.54 ± 0.31) following L. plantrum ATCC 14917 (64.34 ± 0.29). F. pseudoficulneus JNGBKS1, F. pseudoficulneus JNGBKS3, F. fructosus JNGBKS2, and F. fructosus JNGBKS4 did not show a significant difference (p < 0.05) in autoaggregation, which had auto-aggregation values of 31.85, 30.24, 30.89, and 29.57, respectively. Maximum hydrophobicity was observed in L. plantrum ATCC 14917 (60.86 ± 1.12) followed by L. kunkeei JNGBKS7 (39.92 ± 1.11). No significant difference in hydrophobicity was observed between L. kunkeei JNGBKS6 and L. kunkeei JNGBKS8 although
3.5. Antibiotic resistance FLAB isolates were analyzed for their ability to tolerate antibiotics due to safety considerations. None of the FLAB isolates was resistant to any of the tested antibiotics. All isolated FLAB exhibited intermediate susceptibility against novobiocin, whereas F. fructosus, F. durionis, and L. kunkeei strains showed intermediate susceptibility against gentamicin. In case of ceftrioxin, all three strains of L. kunkeei and F. pseudoficulneus JNGBKS3 showed susceptibility (Fig. 2). In this experiment the control strain (L. plantarum ATCC 14197) had significantly high (p < 0.05) susceptibility against all antibiotics tested. Minimum values of antibiotic susceptibility were observed for the L. kunkeei strains. These latter strains are phylogenetically closer to the Leuconostoc spp. As reported by Casado Muñoz, Benomar, Lerma, Gálvez, and Abriouel (2014), genetically Leuconostoc spp. have resistance against some common antibiotics and so the relatively low antibiotic susceptibility of
Table 3 Auto-aggregation, hydrophobicity, and antagonistic activity of isolates (n = 3). Strain
% Auto-aggregation
Hydrophobicity (%)
Antagonistic activity E. coli 25922
Lactobacillus plantrum ATCC 14917 F. pseudoficulneus JNGBKS1 F. pseudoficulneus JNGBKS3 F. fructosus JNGBKS2 F. fructosus JNGBKS4 F. durionis JNGBKS5 L. kunkeei JNGBKS6 L. kunkeei JNGBKS7 L. kunkeei JNGBKS8
64.34 31.85 30.24 30.89 29.57 27.97 40.43 44.54 42.37
± ± ± ± ± ± ± ± ±
a
0.29 0.14c 0.13c 0.24c 0.17cd 0.22d 0.11b 0.31b 0.31 b
60.86 29.45 28.17 30.48 29.24 30.84 37.45 39.92 37.89
± ± ± ± ± ± ± ± ±
a
a
1.12 0.54e 1.23e 1.14d 0.93e 0.87d 0.72c 1.11b 0.75c
11 ± 0.4 7.0 ± 0.4c 7.5 ± 0.6c 6.5 ± 0.5d 7.5 ± 0.2c 7.0 ± 0.1c 9.5 ± 0.3b 9.0 ± 0.3 b 9.0 ± 0.2 b
a-e
S. aureus NCTC8325 a
10 ± 0.5 7.0 ± 0.6c 6.5 ± 0.2d 7.0 ± 0.1c 7.0 ± 0.3c 6.0 ± 0.2d 8.0 ± 05b 8.0 ± 0.2b 8.5 ± 0.2b
S. typhimurium CMCC5011 10 ± 0.2 a 6.5 ± 0.3c 7.0 ± 0.1b 6.0 ± 0.4c 6.0 ± 0.4c 4.0 ± 0.2d 7.0 ± 0.3b 7.5 ± 0.2b 7.0 ± 0.3b
Different superscript letters in the same column indicate statistical differences in each strain at the level of p < 0.05 as measured by Tukey's test. All the results were obtained after 24 h and the values are represented as mean SD of three independent replicates. 73
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Fig. 2. Antibiotic susceptibility of FLAB. The antibiotic susceptibility is indicated as the diameter of the clearing zone around the isolate colonies. The values are the expressed as mean ± SD (n = 3). The data less than 15 mm indicates resistance (R); 15 mm–25 mm indicate Intermediate susceptibility (IS), 30 mm–45 mm indicate highly susceptibility (S). Significant difference (different letters) was observed between strains and antibiotics by the Tukey's test (p < 0.05).
cardiovascular diseases (Chien, Wu, Lee, & Hwang, 2010) as it has been reported that risks of coronary disease can be reduced 2–3% by a 1% decrease in plasma cholesterol (Jeun et al., 2010). In that regard, probiotics could play a preventative role as LAB strains are indicated to be involved in cholesterol assimilation (Abushelaibi, Al-Mahadin, ElTarabily, Shah, & Ayyash, 2017; Iranmanesh, Ezzatpanah, & Mojgani, 2014). The present findings revealed that L. kunkeei strains had significantly higher (p < 0.05) cholesterol assimilation as compared to control samples. Significantly lower cholesterol assimilation was exhibited by F. durionis JNGBKS5 (Table 4). The L. kunkeei strains were previously reported to originate from honey bee hives and honey bee intestines (Endo & Salminen, 2013; Verón et al., 2017), and it can be hypothesized that honeybees are the source of these strains in flowers and fruits.
L. kunkeei could be partly attributed to their genetic makeup. Similar findings have been obtained using L. rhamnosus GG against various antibiotics (Chang, Wang, Lau, Lin, & Tang, 2009). Likewise, Cebrián et al. (2012) showed similar degrees of resistance against various antibiotics of the probiotic E. faecalis strains as well as in isolated LAB from Opuntia ficus-indica fruit (Verón et al. (2017).
3.6. Survival under GIT conditions For the probiotic characterization, a paramount factor is the survival of LAB strains in GIT. To evaluate these characteristics, isolated strains were subjected to mimicked intestinal conditions of which pH is an important probiotic characteristic. All strains showed survival at the various tested pH levels (2, 2.5, 3, and 7.4). Maximum survival was shown at pH 3 while minimum survival was revealed at pH 7.4. L. plantrum ATCC 14917 had significantly high (p < 0.05) survival rates followed by the strains of L. kunkeei and F. pseudoficuneus while minimum survival was observed for F. durionis JNGBKS5 (Table 4). Survival of LAB strains at low pH is an uncommon characteristic as previously reported and these results are in accordance to the studies of Sakandar et al. (2018). Bile salt tolerance is another important attribute of probiotics. In the present studies L. kunkeei strains had significantly high (p < 0.05) bile salt tolerance followed by L. plantrum ATCC 14917 whereas minimum tolerance was observed for the F. durionis and F. fructosus strains (Table 4). Survival rate of L. rhamnosus and L. casei under such conditions was higher than our isolates (Palaniswamy & Govindaswamy, 2016; Riaz Rajoka et al., 2017) but the present results are consistent with the findings of Verón et al. (2017). Hypercholesterolemia is the foremost cause of atherosclerosis and
4. Conclusions The present work provides evidence for opportunities to exploit fruits and flowers as relatively unexplored sources for the isolation of novel LAB strains for their potential use as probiotics. In the present study, three, out of eight strains of L. kunkeei isolated FLAB showed significant probiotic potential in terms of their anti-pathogenic activity and tendency to survive under simulated intestinal conditions. Due to preference of D-fructose utilization of these latter strains, there is the possibility that such bacteria could be used in cereal fermentation to ameliorate risk of irritable bowel syndrome. The current study provides strong support for further in-vitro and in-vivo research to identify the potentially beneficial characteristics of these FLAB isolates and their use in food for human consumption. 74
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Table 4 In-vitro screening of selected microbiota as candidate for probiotic (n = 3). Strains
pH tolerance (%) 2.0
L. F. F. F. F. F. L. L. L.
plantrum ATCC 14917 pseudoficulneus JNGBKS1 pseudoficulneus JNGBKS3 fructosus JNGBKS2 fructosus JNGBKS4 durionis JNGBKS5 kunkeei JNGBKS6 kunkeei JNGBKS7 kunkeei JNGBKS8
Bile salt tolerance (%)
2.5 a
91.20 82.00c 81.22c 78.60bc 76.40d 68.70e 84.74b 85.80b 81.35c
3.0 a
93.70 84.10b 82.00bc 79.90d 79.30d 75.40e 86.20b 87.40b 84.65b
7.4 a
94.10 85.40c 82.80bc 82.40bc 81.10c 78.40d 88.00b 87.45b 85.36c
0 a
82.20 68.35a 79.75ab 80.70a 63.25a 59.40a 60.50b 62.22b 58.35a
0.2 a
100 100a 100a 100a 100a 100a 100a 100a 100a
Cholesterol assimilation (%) 0.4
a
94.70 84.10b 83.00b 82.90c 72.40d 68.20e 83.40b 85.50b 83.40b
92.50a 72.90b 71.90c 64.20e 67.40d 52.50f 71.70c 72.00b 73.35b
41.87 32.89 29.41 27.78 30.75 25.17 34.69 31.54 32.40
± ± ± ± ± ± ± ± ±
1.47a 0.98b 1.21bc 0.78d 0.97bc 0.65e 0.53b 0.41c 15b
a-f
Different superscript letters in the same column indicate statistical differences in each strain at the level of p < 0.05 as measured by Tukey's test. All the results were obtained after 24 h and the values are represented as mean SD of three independent replicates.
Acknowledgements
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