Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e9, 2017 www.elsevier.com/locate/jbiosc

Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity Byung-Taek Oh,1, 2, z Seong-Yeop Jeong,3, z Palanivel Velmurugan,1 Jung-Hee Park,1 and Do-Youn Jeong3, * Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 54596, South Korea,1 Plant Medical Research Centre, College of Agricultural and Life Sciences, Chonbuk National University, Jenoju, Jeonbuk 54896, South Korea,2 and Microbial Institute for Fermentation Industry (MIFI), Sunchang, Jeonbuk 56048, South Korea3 Received 22 March 2017; accepted 20 May 2017 Available online xxx

The aim of this study was to investigate the fermentation of blueberry fruit with selected probiotic bacteria (Bacillus amyloliquefaciens and Lactobacillus brevis) and yeast (Starmerella bombicola) isolated from fermented starfish for the extraction of functionalized products for biomedical applications. All probiotic-based fermented extracts showed augmented antibacterial and antioxidant activity compared to the control. Biochemical parameters of viable cell count, titratable acidity, total phenol, total anthocyanin, total flavonoids, total sugar, and reducing sugar were analyzed during a 0e96 h fermentation period. In addition, Fourier transform infrared (FTIR) spectroscopy was performed to determine the functional groups in the control and fermented extracts and it signifies the presence of alcohol groups, phenol groups, carboxylic acids, and aliphatic amines, respectively. The well diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays determined that the S. bombicola-mediated fermented extract has excellent activity, followed by B. amyloliquefaciens and L. brevis, at a high concentration of 1.0 g/mL fermented extract. The ABTS and DPPH showed significant scavenging activity with IC50 values of (30.52 ± 0.08)/ (155.10 ± 0.06) mg/mL, (24.82 ± 0.16)/(74.21 ± 1.26) mg/mL, and (21.81 ± 0.08)/(125.11 ± 0.04) mg/mL for B. amyloliquefaciens, L. brevis, and S. bombicola, respectively. Developing a value-added fermented blueberry product will help circumvent losses because of the highly perishable nature of the fruit. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Blueberry; Probiotics; Fermentation; Functional compound; Skin bacteria; Antioxidant activity]

Blueberries are the indigo-colored fruit of perennial plants from the section Cyanococcus within the genus Vaccinium. This genus also includes cranberries, bilberries, and gooseberries (1). Species of Cyanococcus are the most common fruits sold as blueberries and are native to North America. The fruit is a berry of approximately 5e16 mm (0.20e0.63 in) in diameter with a flared crown at the end. The fruit is a pale greenish color at first, which is followed by a reddish-purple and finally dark purple colored berry when ripe. They are covered in a protective coating of powdery wax, colloquially known as the bloom (2). Blueberry (Vaccinium corymbosum L.) belongs to the Ericaceae family, whose edible fruits are rich in phenolic compounds (particularly anthocyanins, caffeic, chlorogenic, p-coumaric acid, ferulic acid, and phenolic acids) with high antimicrobial and antioxidant potential and also with the flavonoid subclass of anthocyanins, although flavonols (predominantly quercetin derivatives), phenolic acids (caffeic, chlorogenic, p-coumaric acid, and ferulic acid) and proanthocyanidins are also present (3e12). This augmented activity was due to hydrolysis of phenolic and flavonoid compounds by the probiotic microorganisms and breakdown of structural plant cell walls during fermentation (13).

* Corresponding author. Tel.: þ82 63 650 2000; fax: þ82 63 653 9590. E-mail address: [email protected] (D.-Y. Jeong). z The first two authors contributed equally to this work.

Lactic acid bacteria (LAB) used for liquid state fermentation (LSF) with plant-based material are mostly used as a traditional food fermentation method for food preservation, enhanced nutritional quality, production of bioactive compounds, and provision of health benefits beyond basic nutrition and health promotion properties (14). Liao et al. (15) and Torino et al. (16) reported that LAB fermentation of plant-based materials allows for the accumulation of the blood pressure regulator g-aminobutyric acid (GABA), and LAB-fermented soybean milk showed anti-inflammatory, anti-hypertensive, antioxidant (17,18), and anti-obesity properties (19). Sophorolipid fermentation by the yeast Starmerella bombicola utilizes glucose as a hydrophilic carbon source and a fatty acid as a hydrophobic carbon source to obtain anti-cancer, antimicrobial, dermatological, immunoregulatory, spermicidal, and antiviral compounds (20). Asian traditional fermented foods are generally fermented by LAB such as Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus kimchi, Lactobacillus fallax, Leuconostoc mesenteroides, Weissella confusa, Weissella koreenis, Weissella cibaria, and Pediococcus pentosaceus, which are considered as a probiotic source in food practice. Availability of certain specific nutrients such as vitamins and minerals and the acidic nature of fruits provide a conducible medium for LAB fermentation (21,22). The fermentation medium of Bacillus amyloliquefaciens, when present over a certain concentration, had a good antimicrobial effect on pepper pathogenic fungi. According to in vitro tests and in vivo test results, the

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Please cite this article in press as: Oh, B.-T., et al., Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.011

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fresh-keeping effect of fermentation liquid was not as good as that of preservative liquid but was still stronger than that of other treatment (23). In addition, B. amyloliquefaciens-mediated fermentation showed a significant increase in total polyphenol content compared to control (24). Plant-based foods release flavonoids during the fermentation process, resulting in rich antioxidative activity; therefore, fermentation is one of the major methods to supply natural antioxidants from plant-based materials. The fermentation-induced structural breakdown of the cell walls can also liberate and/or induce the synthesis of various bioactive compounds (13,25). The total phenol content present in plant-based material is increased after fermentation, indicating potential antioxidant activity due to the increase in total phenolic compounds (26). The reducing singlet oxygen quenchers and hydrogen donors are obtained from phenolic compounds after fermentation. Overall, the high concentration of polyphenolic compounds present in plant parts, mostly in the fruits, will be a good source of antioxidants after fermentation. During fermentation of plant-based materials with suitable microbes, cell wall and/or starch are broken down via glucosidase, amylase, cellulase, chitinase, inulinase, phytase, xylanase, tannase, esterase, invertase, or lipase enzymes to facilitate the production of more useful compounds and to change the structure of phytochemicals present in the fruits or other parts of the plant. The presence of LAB in controlled fermentation contributes to the simple phenolic conversion and the depolymerization of highmolecular weight phenolic compounds (13,27). Various environmental parameters like pH, substrate, oxygen, temperature, type of microorganism, time, and extraction during fermentation play a key role in the production of enhanced antibacterial and antioxidant compounds. This work, we investigated the liquid state fermentation in blueberry fruit (V. corymbosum L.) with S. bombicola, L. brevis, and B. amyloliquefaciens to produce water-soluble extracts with potential antibacterial, phenolic composition, and antioxidant activity. Results derived from this work provide valuable information on the composition and bioactivity of fermented blueberry fruit products that can be used to develop novel functional foods.

MATERIALS AND METHODS Blueberry fruit preparation for fermentation Ten kilograms of fresh blueberry fruits were obtained directly from a farm in Suncheon, South Korea. The acquired fruits were frozen, dried using a food dehydrator, and powdered using a kitchen mixture followed by uniform sieving. Probiotic cultures, preparation of inocula, and inoculation A total of 60 isolates were picked from homemade fermented starfish, which is one of the traditional fermented foods in South Korea. One gram of fermented starfish was ground using a ceramic mortar and pestle, followed by serial dilution and plating on MRS (de Man, Rogosa, and Sharpe) agar medium and incubated. After incubation, the plates were examined for various colonies, identified based on unique colony morphology, and original colonies were streaked on fresh MRS medium to obtain pure cultures. The obtained pure cultures were subject to identification (Cosmo Genetech Laboratory, South Korea) by 16S rRNA gene sequencing according to previous methods (28,29). The strain sequences were compared with those in the National Center for Biotechnology Information (NCBI) database using the BlastN search program, and the closest matched species were identified. Pure cultures were stored at 80 C in MRS supplemented with 20% (v/v) glycerol for subsequent experimentation. Each isolate was subculture twice before each experiment to ensure purity of the isolates. The culture was inoculated into the fermentation fruit medium, and the colonies were counted at the end of fermentation. Liquid state fermentation Blueberry fruit powder (10 g) was suspended in sterile distilled water (1:5 w/v) in a 250-mL Erlenmeyer flask and autoclaved to eliminate indigenous microbiota present on the fruit. All 60 probiotic organisms (108 CFU/mL) were inoculated in each flask separately and incubated at 37 C with continuous agitation at 350 rpm for 96 h. Samples were withdrawn every 12 h to determine changes in bacterial population, and biochemical analysis. Fermented samples were centrifuged (1610 g, 15 min, 6 C), and supernatants were freeze-

dried using a lyophilizer. The lyophilized powder was subject to estimation of antibacterial and antioxidant activity. Among the 60 isolates, bacteria Bacillus amyloliquefaciens and L. brevis and one yeast, S. bombicola produced excellent antibacterial and antioxidant activity. Therefore, we selected these three probiotic microorganisms for subsequent experiments. Again, the same fermentation procedure was conducted for the three organisms with blueberry fruit, the samples were centrifuged, freeze-dried, and stored under vacuum at 20 C for further analysis. Microbial growth analysis (viable cell count) Fermentations were monitored for viable cell count by withdrawing samples at 0, 12, 24, 48 and 96 h of fermentation using plate counts. The B. amyloliquefaciens and L. brevis were counted in BHI broth supplemented with 1.5% (w/v) agar after incubation at 37 C for 96 h. The S. bombicola were counted in corncob hydrolysis agar medium after incubation at 37 C for 96 h. Cell counts are expressed as log10 CFU/g for all three organisms. Results were calculated by plotting log CFU versus time. All assays were performed in triplicate. pH measurement The pH value was recorded using a pH meter (HI 2211 pHORP Meter, HANNA Instruments, MI, USA). Periodic sampling of fermentation extract and 5 mL of control extract were transferred aseptically to a sterile glass tube, and the pH was measured by immersing the glass electrode of the pH meter in the extract. Titratable acidity Estimation of titratable acidity was determined by the titration method of 0.1 M of sodium hydroxide according to Wu et al. (30). Approximately 10 g of each sample was weighed and then mixed with 20 mL of distilled water. The samples were subjected to titration with phenolphthalein as indicator. Acidity ( T) was calculated using the following equation Acidityð TÞ ¼

C  V  100 M  0:1

(1)

where C is the concentration of sodium hydroxide, V is the volume of sodium hydroxide consumed, and M is the weight of the sample. Total phenolic and total monomeric anthocyanin determination Total phenolic content (TPC) of all three extract and control extract was determined by the FolineCiocalteu method (31,32). Results are expressed as mg gallic acid equivalents (GAE) per gram of sample (mg GAE/g). Total monomeric anthocyanin was determined using a pH differential method (33e35). Total monomeric anthocyanin was expressed as mg of cyanidin-3-glucoside (cyd-glu, molar extinction coefficient of 26,900 L cm1 mol1 and molecular weight of 449.2 g mol1), and malvidin-3-glucoside (mvd-glu, molar extinction coefficient of 28,000 L cm1 mol1 and molecular weight of 463.3 g mol1). The units for total monomeric anthocyanin were mg/100 mL of sample or 100 g of standard. A POLARstar Optima microplate reader (BMG LABTECH GmbH, Ortenberg, Germany) was used to measure total monomeric anthocyanin with absorbance measured at 520 and 700 nm, and the samples were replicated three times. Total flavonoids Total flavonoid content was determined according to Samad et al. (6,36) by the following colorimetric method, where catechin was used as a standard. Catechin concentrations ranging from 0.05 to 0.5 mg/mL were used to generate the standard calibration curve. Briefly, 0.025 mL of all three extracts and control fermented blueberry (10 mg/mL) or catechin standard solution (0.05e0.50 mg/mL) were mixed with 0.125 mL of distilled water in the wells of a 96-well plate, followed by addition of 0.08 mL 5% w/v sodium nitrite solution. After 5 min, 0.015 mL of a 10% w/v of aluminum chloride solution was added and allowed to react for 6 min. Later, 0.05 mL of 1 mol/L sodium hydroxide and 0.027 mL distilled water were added to the reaction mixture. The final reaction mixture was vortexed vigorously, and the absorbance of the final solution was measured immediately at 510 nm using a UVevis spectrophotometer (Perkin Elmer Corporation, Norwalk, CT, USA). The results are expressed as the equivalent to milligrams catechin per gram (mg CE/g) of fresh weight. Total sugar and reducing sugar The total sugar and reducing sugar of the blueberry fermentation medium as well as the non-fermented control were determined according to methods published in Ranganna (37), Kulkarni and Aradhya (38), and Baskan et al. (39). Fourier transform infrared analysis Fourier transform infrared (FTIR) spectra were produced by a PerkineElmer FTIR spectrophotometer (Norwalk, CT, USA). The infrared spectra obtained from the blueberry powder (control) and blueberry fermented extract with B. amyloliquefaciens, L. brevis, and S. bombicola were analyzed. Bactericidal activity The bactericidal activity of the probiotic-mediated fermented blueberry extract and control extract was evaluated against four types of bacteria known to cause skin infections: Brevibacterium linens (KACC-14346), Propionibacterium acnes (KACC-11946), Bacillus cereus (KACC-10001), and Staphylococcus epidermidis (KACC-13234) (KACC, Korean Agricultural Culture Collection). The measured variables were zone of inhibition (ZoI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). To enrich the cultures, each was aseptically inoculated into brain heart infusion (BHI) medium and incubated. For P. acnes, the culture was incubated in an anaerobic chamber under controlled conditions (all subsequent experiments for P. acnes were performed in anaerobic conditions). Later, the antibacterial

Please cite this article in press as: Oh, B.-T., et al., Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.011

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sensitivity profile of each strain was tested by spreading a 50 mL aliquot of an overnight culture grown in BHI on Muller-Hinton agar (MHA) (MB Cell, Seoul, South Korea) plates that were seeded with a 0.5 McFarland scale bacterial suspension. Each plate was punctured with 4-mm-diameter well and filled with a different concentration (0.25 g/mL, 0.5 g/mL, 0.75 g/mL, 1.0 g/mL) of the probioticmediated fermented blueberry extract (freeze dried powder) and control extract. Plates were then incubated at 30 C for B. linens and B. cereus and 37 C for P. acnes (anaerobic) and S. epidermidis. After incubation, the plates were examined for clear zone formation. The presence of a clear zone around the samples was recorded as inhibition against the bacterial strains. The diameters of such zone of inhibition were measured, and the mean value for each organism was recorded and expressed in millimeters. Determination of minimum inhibitory concentration (MIC) was performed according to Jorgensen and Turnidge (40) and Silva et al. (8). All three probioticmediated fermented and control extracts were prepared at 160, 80, 40, 20, and 10 mg/mL and inoculated with approximately 100 mL of 108 CFU/mL mid-log culture of each separately grown test organism (skin bacteria) in 96-well microtiter plates and incubated per the specific microorganism growth conditions described above. An appropriate blank was used for the control. Following incubation, the turbidity of the growth medium was measured at 600 nm with a POLARstar Optima microplate reader (BMG LABTECH GmbH). The MIC was determined as the lowest concentration of fermented extract that entirely inhibited bacterial growth. The minimum bactericidal concentration (MBC) was determined as described by Silva et al. (8). Briefly, MIC and higher concentrations were plated in BHI agar and incubated per the specific microorganism growth conditions as described above. The MBC concentration was considered when no bacteria were detected after 24 h of incubation. Sterile BHI broth was used as the negative control and was calculated according to AATCC 100 as shown in Eq. (2).

different concentrations of all three probiotic-mediated fermented and control extracts were prepared at 16, 32, and 80 mg/mL in warm DMEM from the original extract suspensions, added to the cells, and incubated for 24 and 72 h. The cells were allowed to grow for 7 days, and on the 4th day, the media was replaced with a fresh DMEM. After 7 days, the cells were rinsed with PBS on discarding DMEM followed by addition of dilution to all three probiotic-mediated fermented and control extracts and incubated for 24 h. Fresh DMEM without any extract was the control. After the incubation period, the DMEM was removed and replaced with the resazurin solution. A stock solution of 110 mg/mL resazurin was prepared in phosphate buffered saline, along with a sterilized 0.2 mm filter. This was then diluted in 1:10 ratio in fresh warm DMEM, and 600 mL of the resazurin solution was added to each, well keep it for 2 h. A 200 mL of each reduced solution was transferred into 96 well plates. The fluorescent intensity was recorded using a plate reader (lex ¼ 544 nm and lem ¼ 590 nm). Viable cells (%) were calculated according to the following equation:

Rð%Þ ¼

AB  100 A

(2)

where A is the number of bacteria recovered from the inoculated test specimen, and B is the number of bacteria according to A conditions of the antibacterial modified sample. Consequently, R (%) is the percent reduction ratio, which indicates biostatic efficiency. Each experiment was performed in triplicate, and the final values are presented as the mean  SEM. ABTS radical cation scavenging activity ABTS radical cation (ABTS þ) scavenging activity of probiotic-mediated fermented and control extracts was  measured using the ABTS þ assay described by Samad et al. (6,36). Scavenging activity was measured based on the ability of the antioxidant molecules to quench  the ABTS þ, a blue-green chromophore with characteristic absorption at 734 nm.  After the addition of 100 mL of ABTS þ solution to 100 mL of blueberry fermented extract and both standards (3.12e400 mg/mL), the decrease in absorbance at  734 nm was monitored. The ability to scavenge ABTS þ was calculated as 

Scavenge ABTS þ abilityð%Þ ¼ 

h

Ablank  Asample

.

i Ablank  100

(3)

where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. DPPH radical scavenging activity Hydrogen donating ability of the probiotic-mediated blueberry fermented extracts and control extract was determined according to Samad et al. (6) and Bhat et al. (41), based on the reduction of a methanolic solution of the colored free radical DPPH to the nonradical form. Briefly, 10 mg/mL of each probiotic-mediated fermented extract and control extract stock solution was prepared in dimethyl sulfoxide (DMSO), and 10 mg/mL of ascorbic acid was prepared in methanol as a synthetic standard antioxidant. Dilutions were performed to attain concentrations ranging from 3.12 to 1600 mg/mL. The diluted solution (100 mL) was mixed with 100 mL of freshly prepared 0.20 mol/L DPPH-methanol solution, which was then kept in the dark at room temperature for a 30-min reaction. The absorbance of the solution was determined using a UVeVis spectrophotometer at 517 nm. The percentage inhibition of free-radical DPPH was calculated as Inhibitionð%Þ ¼

h . i Ablank  Asample Ablank  100

Viabilityð%Þ ¼ 100  Absorptiontest =Absorptioncontrol

3

(5)

Statistical analysis Three treatments with three replications were used. Bacterial populations were reported as log CFU/mL. All data were expressed as mean  standard error mean (SEM). Analysis of variance (ANOVA) was performed on cell counts using the General Linear Models (GLM) procedure of SPSS software version 17.0. All experiments were performed in biological and technical triplicates on three separate days.

RESULTS AND DISCUSSION During the fermentation process, samples were withdrawn over 96 h to estimate the probiotic microbial load. Notably, the probiotic population remained steady (mean values of 140  1.28, 120  1.34, and 68  1.18 CFU/g) during the 72 h of fermentation for B. amyloliquefaciens, L. brevis, and S. bombicola, respectively (Fig. 1), and only a slight change in pH value was observed (data not shown). In support of our results, previous studies using probiotic bacteria in cabbage fermentation juice and guava (Psidium guajava L.) did not survive in the fermentation medium for longer periods of time and completely lost viability after longer fermentation time (41e44). A steady decrease in titratable acidity was observed in blueberry fermented samples with B. amyloliquefaciens, L. brevis, and S. bombicola from 0 to 96 h compared to the control (Fig. 2A). The percentage of acidity was gradually reduced from 0 to 96 h with L. brevis (5%), S. bombicola (4%), and B. amyloliquefaciens (1.5%) after 96 h of fermentation, respectively (Fig. 2A). Our results are consistent with the earlier report on the production of naturally green arbequina olives and Spanish-style fermentation of heat-

(4)

where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. Cell viability assay Viability of primary human dermal fibroblast (HDFs) cells after exposure to probiotic-mediated blueberry fermented extracts and control extract were determined according to Chowdhury et al. (42) based on resazurin assay. It is a fluorescent technique in which the fluorescent is based on metabolically active living cells. This assay could be performed using a microplate reader by quantifying the production of resazurin proportional to the number of viable cells. Coverslips were well cleaned and placed at the bottom of the wells of sterile 24 well tissue culture plates. The 1  104 cells per well were seeded in Dulbecco’s modified Eagle medium (DMEM) supplemented with fetal bovine serum, penicillin and streptomycin, followed by incubation in CO2 incubator supplied with 95% air 5% CO2 at 37 C for 24 h. After the incubation, the DMEM was removed, and cells (50% confluent) were briefly washed with PBS. After,

FIG. 1. Impact of blueberry fermented with probiotic and its total viable counts during the 0e96 h fermentation period.

Please cite this article in press as: Oh, B.-T., et al., Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.011

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FIG. 2. Determination of (A) titratable acidity, (B) total phenol, (C) total anthocyanin, and (D) total flavonoids during blueberry fermentation with probiotic organisms during the 0e96 h fermentation period.

shocked green olives cv. Halkidiki, as well as fermentation of raw guava fruit using yeasts and LAB (41,45,46). The total phenolic content was estimated (Fig. 2B) between 0 and 96 h for cofermentation with blueberry, B. amyloliquefaciens, L. brevis, and S. bombicola, and a gradual reduction of phenolic content was noted compared to the control. Among these, B. amyloliquefaciens showed the maximum reduction of phenolic content (Fig. 2B), while S. bombicola and L. brevis had a comparable reduction of phenolic content after 96 h of fermentation. The results are expressed as gallic acid equivalent (GAE)/g1 dry weight extract of control and fermented extract of B. amyloliquefaciens (2.52 GAE/g1 dry weight extract), L. brevis (4.11 GAE/g1 dry weight extract), or S. bombicola (4.10 GAE/g1 dry weight extract). Previously, Shen et al. (7) described four individual phenolic compounds (chlorogenic acid, ellagic acid, quercetin, and quercetin-3-galactoside) present in blueberry extracts. The reduction in phenolic content might be due to the bioconversion of phenolic compounds into bioactive

compounds by probiotic microbes due to a diverse portfolio of enzymes (41). Total anthocyanin level was found to be reduced after 96 h of fermentation with probiotics (Fig. 2C). Compared to the control, total anthocyanin was reduced during the 0e96 h fermentation period by L. brevis, S. bombicola, and B. amyloliquefaciens (Fig. 2D). The results are expressed as total monomeric anthocyanin (mg CGE/100 g dw)2 of control and fermented extract of L. brevis (6.11 mg CGE/100 g dw)2, B. amyloliquefaciens (5.12 mg CGE/100 g dw)2, or S. bombicola (4.10 mg CGE/100 g dw)2. Compared with the control, the total flavonoid content was drastically reduced by up to 5% after 96 h of fermentation. The blueberry fruit extract contains 20e30% total sugar and 0.5e10% reducing sugar (Table 1), indicating that the sugars present in the fruits were utilized by the probiotic bacteria and yeast as a carbon source. Forney et al. (47) reported that glucose and fructose were the predominant sugars in blueberry fruit and were found in equal quantities, while trace amounts of sucrose

TABLE 1. Estimation of total sugar and reducing sugar from blueberry extract during fermentation with probiotic bacteria and yeast with control. Time

Total sugar (g/100 mL1)

Control (g/100 mL)

B. amyloliquefaciens 0 24 36 48 60 72 84 96

8.2 8.1 8.1 8.0 8.0 8.0 8.0 8.0

       

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

8.2 7.6 7.2 6.8 6.5 6.0 5.6 5.0

       

0.8 0.8 0.6 0.4 0.2 0.4 0.7 0.4

L. brevis 8.2 7.2 7.0 6.2 6.0 5.8 5.6 5.5

       

0.9 0.4 0.8 0.5 0.9 0.2 0.7 0.4

Reducing sugar (g/100 mL1) S. bombicola 8.2 7.2 6.9 6.5 6.2 5.6 5.0 4.8

       

0.4 0.5 0.2 0.4 0.5 0.3 0.8 0.2

B. amyloliquefaciens 3.1 3.0 2.4 2.3 2.0 1.8 1.4 0.8

       

0.2 0.7 0.5 0.2 0.8 0.2 0.5 0.5

L. brevis 3.1 2.8 2.6 2.3 1.9 1.8 1.2 1.0

       

0.8 0.6 0.8 0.1 0.7 0.9 0.4 0.3

S. bombicola 3.1 2.6 2.4 2.0 1.9 1.6 1.2 0.9

       

0.1 0.2 0.8 0.2 0.3 0.7 0.9 0.4

Please cite this article in press as: Oh, B.-T., et al., Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.011

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FIG. 3. FTIR spectra of the non-fermented and fermented extracts of blueberry with probiotic organisms.

were present in the blueberry fruit due to invertase enzyme activity. Samples of the control blueberry extract, B. amyloliquefaciens, L. brevis, and S. bombicola-mediated fermented blueberry extracts were subjected to FTIR analysis for detection of functional groups (Fig. 3). The spectra (400e4000 cm1) were interpreted using the guidelines of Namiesnik et al. (48), Ertani et al. (49), and Panda et al. (50). The prominent absorption peaks at 3313, 2917, 1724, 1424, 1021, and 780 cm1 were found for the control blueberry extract sample. The broad peak at 3313 cm1 indicates strong stretching vibrations of eOH functional groups, and the CeOH stretching/ bending of primary or aromatic alcohols at 2917 and 1724 cm1 might be the stretching vibrations of alkene groups -C]Ce, carboxylic acid and carbonyl groups. The absorbance at 1424 cm1 is attributed to ether linkages, or eCeO-groups. The spectra of the control and L. brevis samples were similar despite slight differences in relative intensities. The spectra of S. bombicola and B. amyloliquefaciens fermented extracts showed several novel peaks, indicating that the functional biomolecules present in the fruit were altered by the microbes during the fermentation process. The main peaks were identified at 3261, 3330, 2921, 1718, 1401, 1038, and 820 cm1 for both S. bombicola and B. amyloliquefaciens. The weak shoulder at 820 and 1038 cm1 and strong shoulder at

5

1041 and 1718 cm1 could be attributed to CeOH, CeCH, and OeOH bending related to the b anomeric configuration of carbohydrates and CeO stretching, CeC stretching and CeCH bending, respectively. The peaks at 2921, 3330 and 3261 cm1 could be assigned to gallic acid, fisetin, tannic and caffeic acids, and carboxylic acids. The FT-IR peaks obtained in our study are consistent with those of Panda et al. (51), who determined that wine prepared using sapote fruit showed peaks at 3025, 2925, 2847, 1459, and 1201 cm1, assigned to ]CeH stretching, aromatic ester, secondary amides (NeH -bending and CeH) stretching, and OeH stretching compounds, respectively. The presence of aromatic primary amine groups, which is a result of fermentation by probiotic microorganisms, implies the presence of bacterial proteins in the fermented extract, which could be contributing to the anti-bacterial nature of the fermented samples (52). In previous studies, Panda et al. (51) and Ray et al. (53) reported that the peaks near 3300, 1250, and 1000 cm1 signify the presence of alcohol, phenol groups, carboxylic acids, and aliphatic amines present sapota (Achras sapota Linn.) fruits and purple sweet potato (Ipomoea batatas L.) fermented extract, respectively. The well diffusion assay showed that the antimicrobial activity of probiotic-mediated fermented blueberry extract was sensitive to the four different skin bacteria tested, with notable differences in ZoI among all four organisms (Table 1). Among the four different skin bacteria tested, P. acne and B. linens showed excellent activity with a ZoI of 6 mm at a 1.0 g/mL concentration by S. bombicola fermented blueberry extract. The B. amyloliquefaciens and L. brevis fermented blueberry extracts showed moderate activity for all tested skin bacteria (Table 1). In the control, high concentrations of blueberry extract showed a smaller ZoI for all skin bacteria tested, demonstrating that the probiotic organisms can convert phytocompounds into biologically important peptides, exopolysaccharides, secondary metabolites, and other organic compounds (54). Our results are consistent with the earlier report on V. corymbosum and Vaccinium angustifolium extracts used to control pathogenic microorganisms (5e8). The results for MIC and MBC determinations are displayed in Fig. 4A and B, and they corroborate those found in the well diffusion screening, i.e., no significant differences were found between probiotic organism-fermented blueberry extract. In addition, significantly lower MIC concentrations were noted in the fermented extracts. Fig. 4A shows that blueberry fermented with 80 mg/mL B. amyloliquefaciens and S. bombicola extract showed significant changes in the MIC (Fig. 4A) and lower MBC (Fig. 4B) percentage for P. acne and B. linens. Previously, Sousa et al. (55) reported that fruits typically rich in sugar content that is easily extractable by water

FIG. 4. (A) MIC and (B) MBC values of each probiotic-mediated fermented extract with the appropriate control.

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TABLE 2. Well diffusion assay (ZOI mm) of blueberry fermented extract with probiotic bacteria and yeast against skin pathogens with control. Probiotic bacteria and yeast B. amyloliquefaciens

L. brevis

S. bombicola

Control

Fermented extract and control (g/ mL) 0.25 0.5 0.75 1.0 0.25 0.5 0.75 1.0 0.25 0.5 0.75 1.0 0.25 0.5 0.75 1.0

demonstrate lower levels of activity, like promotion of microbial growth or effective interaction with bioactive compounds. Shen et al. (7) and Silva et al. (5,8) studied blueberry fruit and leaf extracts about antibacterial activity to various bacterial pathogens; however, they identified weaker activity compared to our probiotic-mediated blueberry extract activity (Fig. 4A and B and Table 2).

B. linens 1 2 2 5 1 2 4 4 2 3 5 6

           

1.26 2.14 2.28 8.56 2.35 2.24 4.86 1.85 2.64 1.22 4.62 5.27 e e 1  4.22 2  10.20

P. acne  2.34  3.82  6.23  5.11  3.46  6.52  7.21  3.24  3.21  7.21  2.50  6.83 e e 2  5.34 3  5.42

2 4 6 7 1 3 3 4 2 3 5 6

B. cereus  1.12  4.62  4.62  9.21  2.18  6.82  3.57  6.35  8.31  6.25  6.21  1.12 e e 1  6.27 1  5.87

1 2 5 5 2 3 3 4 2 2 4 5

S. epidermidis  1.18  3.38  2.56  6.32  3.31  9.35  8.24  5.48  7.26  5.20  3.38  2.34 e e 1  4.23 2  1.16

2 3 4 4 1 1 3 4 2 3 3 4

The ABTS and DPPH radical scavenging ability of the blueberry fruit fermented with B. amyloliquefaciens, L. brevis, and S. bombicola is shown in Fig. 5A and B. The ABTSþ scavenging activity of all three extracts obtained from probiotic fermented blueberry fruit occurred in a dose-dependent manner over a concentration range from 3.125 to 400 mg/mL (Fig. 5A). The B. amyloliquefaciens, L. brevis, and S. bombicola fermented extracts displayed strong scavenging activity

FIG. 5. Probiotic-mediated blueberry fermented extract, control extract, and ascorbic acid to scavenge (A) ABTS (3.125e400 mg/mL) and (B) DPPH radicals (3.125e1600 mg/mL). All data are expressed as mean  SEM, n ¼ 2. The IC50 concentrations were statistically calculated in nonlinear regression method using Graphpad prism version 5.1.0.

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at the maximum concentration (400 mg/mL), showing 98.2  0.4%, 97.2  2.1% and 95.1  1.3% scavenging activity, respectively. Nevertheless, the IC50 values for B. amyloliquefaciens, L. brevis, and S. bombicola fermented extracts were 30.52  0.08 mg/mL, 24.82  0.16 mg/mL, and 21.81  0.08 mg/mL, respectively. Ascorbic acid was used as the positive control for which scavenging activity was the highest (99.12  0.01% at 400 mg/mL with an IC50 value of 15.53  1.26 mg/mL), although the control blueberry fruit extract without probiotic bacteria or yeast (44.40  1.16 mg/mL) showed a gradual concentration-dependent decrease in activity (Fig. 5A). Chorfa et al. (56) investigated the radical-scavenging-activity and oxygen radical absorbance capacity of blueberry anthocyanins, and it resulted in excellent activity of anthocyanin. The DPPH free radical scavenging activity of blueberry fruit (control) extract and probiotic-mediated fermented extract showed higher scavenging activity with an increase in concentration (Fig. 5B). Furthermore, B. amyloliquefaciens (80.12  2.30%), L. brevis (79.11  1.86%), and S. bombicola (80.02  1.82)% fermented extracts had greater scavenging activity at 1600 mg/mL, with IC50 values of 155.10  0.06 mg/mL, 74.21  1.26 mg/mL, and 125.11  0.04 mg/mL, respectively. At a concentration range of 3.125e100 mg/mL, the control blueberry extracts and probiotic fermented blueberry

extract had lower scavenging activity than the reference ascorbic acid standard. However, all three extracts obtained from B. amyloliquefaciens, L. brevis, and S. bombicola fermented blueberry fruit extract showed a significant amount of scavenging activity at higher concentrations. Molan (57) has examined moderate antioxidant properties of aqueous extracts prepared from the berries of five rabbiteye (Vaccinium ashei) and two highbush (V. corymbosum) using ferric reducing antioxidant power and scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay. Fig. 6AeD shows the effect of different concentrations of all three-probiotic meditated fermented extract and control extract on cells cultured for 24 and 72 h, and the insert shows viable cells. The 10% lower degree of confluency of the HDFs cells has been noticed. All three extracts had significantly less cytotoxicity and 10% reduction of cell viability at a higher concentration (80 mg/mL) of each extract within 24 h (Fig. 6A) and 72 h (Fig. 6B) compared to the control. B. amyloliquefaciens had a much milder effect but still exhibited a degree of cytotoxicity when compared to L. brevis and S. bombicola fermented blueberry fruit extracts (Fig. 6A). Longer time exposure up to 72 h has significant reduction of cell viability below 10% compared to the control. Hence, we suggest there is no degree of high cytotoxicity in all the extract. When the same tests

FIG. 6. The effect of Probiotic-mediated blueberry fermented extract, control extract on human dermal fibroblasts cells. Fifty percent of confluent fibroblasts were exposed to fermented extract of B. amyloliquefaciens, L. brevis, S. bombicola and control extract for (A) 24 h and (B) 72 h, respectively. After 24 h of exposure to all the three extracts and the control, the cytotoxic effect of fermented extract of B. amyloliquefaciens, L. brevis, S. bombicola and the control extract were also evaluated against cells that were (C) >50% and (D) >80% confluent. Untreated cells served as the control. The results are represented as mean  SEM.

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were conducted with cells grown for 7 days, which had confluency of about 20%, the level of cytotoxicity changed. Fig. 6C and D shows the higher number of viable cells in comparison to the cells with 50% (Fig. 6C) and 80% (Fig. 6D) confluency in all the three extracts. However, for cells with higher confluency, when the concentration of extract was increased to 80 mg/mL, significant reduction of cell viability was observed. In conclusion, B. amyloliquefaciens, L. brevis, and S. bombicola strains were successfully isolated from fermented food and identified as potential probiotic microorganisms for use as a starter culture in the fermentation of blueberry fruit. It was found that these three strains can survive in the blueberry fruit during fermentation, and the extracts exhibited excellent antibacterial and antioxidant activity. Monitoring the physicochemical properties during fermentation with probiotic organisms showed remarkable differences compared to the control during fermentation. The probiotic-mediated fermented extracts were found to have significant scavenging capacities of different free radicals including  ABTS þ and DPPH. This indicates that, during fermentation, the probiotic organisms metabolically altered the phytochemical molecules to biofunctionalized products. The FTIR data support the conversion of phytochemicals into useful products. About the antimicrobial activity, it is important to note that B. amyloliquefaciens and S. bombicola fermented extracts appeared to possess a higher antimicrobial activity against skin bacteria and lower MIC and MBC values than L. brevis fermented extracts. Further optimization of the fermentation process using other probiotic microorganisms can help in developing a ready to blend fermented blueberry product for application. ACKNOWLEDGMENTS This research was supported by 2017 Healthy Local Food Branding Project of the Rural Resources Complex Industrialization Support Program. References 1. Litz, R. E.: Litz, R. E. (Ed.), Google booksebiotechnology of fruit and nut crops, CABI Publishing, Wallingford, UK (2005). 2. Li, Y., Yin, Y., Chen, S., Bi, Y., and Ge, Y.: Chemical composition of cuticular waxes during fruit development of Pingguoli pear and their potential role on early events of Alternaria alternata infection, Funct. Plant Biol., 41, 313e320 (2014). 3. Su, M. S. and Chien, P. J.: Antioxidant activity, anthocyanins, and phenolics of rabbit eye blueberry (Vaccinium ashei) fluid products as affected by fermentation, Food Chem., 104, 182e187 (2007). 4. Castrejon, A. D. R., Eichholz, I., Rohn, S., Kroh, L. W., and Huyskens-Keil, S.: Phenolic profile and antioxidant activity of highbush blueberry (Vaccinium corymbosum L.) during fruit maturation and ripening, Food Chem., 109, 564e572 (2008). 5. Silva, S., Costa, E. M., Pereira, M. F., Costa, M. R., and Pintado, M. E.: Evaluation of the antimicrobial activity of aqueous extracts from dry Vaccinium corymbosum extracts upon food microorganism, Food Control., 34, 645e650 (2013). 6. Samad, N. B., Debnath, T., Ye, M., Hasnat, M. A., and Lim, B. O.: In vitro antioxidant and anti-inflammatory activities of Korean blueberry (Vaccinium corymbosum L.) extracts, Asian Pac. J. Trop. Biomed., 4, 807e815 (2014). 7. Shen, X., Sun, X., Xie, Q., Liu, H., Zhao, Y., Pan, Y., Hwang, C. A., and Wu, V. C.: Antimicrobial effect of blueberry (Vaccinium corymbosum L.) extracts against the growth of Listeria monocytogenes and Salmonella Enteritidis, Food Control., 35, 159e165 (2014). 8. Silva, S., Costa, E. M., Costa, M. R., Pereira, M. F., Pereira, J. O., Soares, J. C., and Pintado, M. M.: Aqueous extracts of Vaccinium corymbosum as inhibitors of Staphylococcus aureus, Food Control., 51, 314e320 (2015). 9. Garcia-Diaz, D. F., Johnson, M. H., and de Mejia, E. G.: Anthocyanins from fermented berry beverages inhibit inflammation-related adiposity response in vitro, J. Med. Food., 18, 489e496 (2015). 10. Yoon, H. H., Chae, K. S., Son, R. H., and Jung, J. H.: Antioxidant activity and fermentation characteristics of blueberry wine using traditional yeast, J. Korean Soc. Food Sci. Nutr., 44, 840e846 (2015).

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Please cite this article in press as: Oh, B.-T., et al., Probiotic-mediated blueberry (Vaccinium corymbosum L.) fruit fermentation to yield functionalized products for augmented antibacterial and antioxidant activity, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.011