Fermentation-based biotransformation of glucosinolates, phenolics and sugars in retorted broccoli puree by lactic acid bacteria

Food Chemistry 286 (2019) 616–623

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Fermentation-based biotransformation of glucosinolates, phenolics and sugars in retorted broccoli puree by lactic acid bacteria

T

Jian-Hui Yea, Long-Yue Huanga, Netsanet Shiferaw Terefeb, , Mary Ann Augustinb ⁎

a b

Zhejiang University Tea Research Institute, 388 Yuhangtang Road, Hangzhou 310058, China CSIRO Agriculture & Food, 671 Sneydes Road, Werribee, VIC 3030, Australia

ARTICLE INFO

ABSTRACT

Keywords: Broccoli Lactic acid bacteria Fermentation Glucosinolates Polyphenols Principal component analysis

This study investigated the effect of lactic acid bacteria (LAB) fermentation on the chemical profile of autoclaved broccoli puree, using 7 broccoli-derived LAB isolates (named F1-F5, BF1 and BF2). The total concentrations of glucosinolates (glucoiberin, progoitrin and glucoraphanin) and 10 major phenolics significantly increased from trace level and 289 μg total phenolics/g dry weight (DW) respectively in autoclaved broccoli to 55 to ∼359 μg/g DW and 903 to ∼3105 μg/g DW respectively in LAB fermented broccoli puree. Differential impacts of LAB isolates on the chemical composition of autoclaved broccoli were observed, with the major differences being the significant increase in phloretic acid after fermentation by F1-F5 and an elevated glucoraphanin level in ferments by F1 and BF2. LAB fermentation is a promising way to increase the content of glucosinolates and polyphenolic compounds in broccoli, making the ferments attractive for use as functional ingredients or as a whole functional food.

1. Introduction Fermentation had traditionally been used as a method of food preservation. More recently there has been increasing interest in fermentation for producing novel products or ingredients for a growing functional foods market (Kantachote, Ratanaburee, Hayisama-ae, Sukhoom, & Nunkaew, 2017; Molina, Médici, de Valdez, & Taranto, 2012). Lactic acid bacteria (LAB), are one of the most important group of microorganisms used in the food industry as they have a Generally Recognized As Safe (GRAS) status and used in the fermentation of commonly consumed products such as yoghurt, cheese and sausages (Pereira et al., 2017). LABs ubiquitously appear in food and are part of the healthy microflora of human mucosal surfaces. Lactic acid fermentation produces lactic acid and other metabolites which are responsible for the altered characteristics, and potentially the enhanced biological activities of products obtained upon fermentation (Di Cagno, Minervini, Rizzello, De Angelis, & Gobbetti, 2011; Pereira et al., 2017). Glucosinolates and phenolic compounds are important bioactives in broccoli (Pék, Daood, Nagyné, Neményi, & Helyes, 2013), which are associated with human health benefits as they have antioxidant, antitumor and cardio-protective properties (Moreno, Carvajal, LópezBerenguer, & García-Viguera, 2006; Vasanthi, Mukherjee, & Das, 2009). Glucoraphanin, glucoalyssin, 4-hydroxy glucobrassicin, glucoerucin, glucobrassicin, 4-methoxy glucobrassicin, neoglucobrassicin, ⁎

progoitrin, glucobrassicanapin and glucoiberin are glucosinolates present in broccoli florets (Bhandari & Kwak, 2015; Frandsen et al., 2014; Guo, Yuan, & Wang, 2013). Quinic acid, phloretic acid, caffeic acid and chlorogenic acid are important phenolic compounds in broccoli florets (Filannino, Bai, Di Cagno, Gobbetti, & Gänzle, 2015). Especially, glucoraphanin can be converted to the highly potent bioactive sulforaphane either by endogenous myrosinase or thioglucosidases in the microbiota of the human colon (Glade & Meguid, 2015; Guo et al., 2013). Breeding for high glucoraphanin, the precursor of sulforaphane, is an important objective for broccoli breeding (Gu, Wang, Yu, Zhao, & Sheng, 2014). Fermentation is a promising route to alter the chemical profiles of vegetables and potentially enhance their biological activities (Di Cagno, Coda, De Angelis, & Gobbetti, 2013). The influence of processing and fermentation on the bioactivities of cruciferous vegetables such as cabbage, mustard leaf and broccoli has been studied in terms of total polyphenol content, antioxidant capacity, glucosinolates and breakdown products (Ciska & Pathak, 2004; Maryati, Susilowati, Melanie, & Lotulung, 2017; Nugrahedi, Widianarko, Dekker, Verkerk, & Oliviero, 2015; Palani et al., 2016). Different LAB species have different fermentation characteristics which have been shown to produce a diverse range of different metabolites in fermented vegetable juices (Tomita, Saito, Nakamura, Sekiyama, & Kikuchi, 2017). Generally, raw cut vegetables are used for fermentation, and hence the biotransformation of

Corresponding author. E-mail address: [email protected] (N.S. Terefe).

https://doi.org/10.1016/j.foodchem.2019.02.030 Received 7 November 2018; Received in revised form 31 January 2019; Accepted 5 February 2019 Available online 14 February 2019 0308-8146/ Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved.

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J.-H. Ye, et al.

components during fermentation is attributed to an array of reactions induced by endogenous enzymes, autochthonous bacteria and added microorganisms. However, the biotransformation of plant-derived components attributable to the action a single strain of microorganism is lacking. In order to isolate the effect of particular microorganisms on biosynthesis or degradation of bioactive compounds, studies need to be conducted on starting materials that have been thermally processed to inactivate endogenous enzymes and kill vegetative bacteria. This knowledge could help to guide the selection of microorganisms for producing fermented products with the desired metabolites for enhanced nutrition. In the present study, seven lactic acid bacteria strains, previously isolated from broccoli, were used for fermentation of autoclaved broccoli puree. The autoclaving step used prior to the addition of LAB isolates was intended to eliminate the contribution of endogenous enzymes and autochthonous bacteria present in fresh broccoli to the biotransformation of compounds during the fermentation process. The changes of glucosinolates, phenolic compounds and free sugars in autoclaved broccoli puree before and after fermentation were examined. Fourier Transform infrared spectroscopy (FTIR) was used for a rapid comparison of broccoli samples fermented by different LABs. Principal component analysis (PCA) was used as a multivariate statistical tool to classify and discriminate broccoli samples produced by different LAB strains based on the quantified chemical compounds or FTIR spectra.

for fermentation, viz. F1-F5 isolated from broccoli leaves and BF1 and BF2 isolated from broccoli floret. F1-F5 were identified as L. plantarum and BF1 and BF2 were identified as Leuconostoc mesenteroides by 16S rRNA sequencing. These lactic acid bacteria were deposited in the National measurement institute (NMI, Melbourne, Australia) (BF1: V17/021729, BF2: V17/021730, B1: V17/021731, B2: V17/021732, B3: V17/021733, B4: V17/021734, B5: V17/021735). Stock cultures of F1-F5, BF1 and BF2 that were kept frozen at −80 °C were activated by inoculating into MRS (De Man, Rogosa and Sharpe) medium and cultivation at 30 °C. The autoclaved broccoli puree were respectively inoculated with 10% (v/v) of different cell suspensions of F1-F5, BF1 and BF2 in sterile phosphate buffer saline, corresponding to ca. 108 cfu/g, and incubated for 15 h at 30 °C until pH below 4.4 was achieved. The pH value of autoclaved broccoli puree during fermentation was recorded using a pH data logger (MM-PIT-4U, EAI instruments, UK). The lactic acid bacteria count immediately after fermentation was determined by plating on MRS agar. The rest of the samples including 7 fermented samples, respectively termed F1-F5, BF1 and BF2, were frozen immediately after fermentation. Part of the autoclaved broccoli was immediately frozen after autoclaving and was termed control (CK). All of the samples were freeze dried for metabolic fingerprint analysis. All experiments were performed in triplicate.

2. Materials and methods

The water extracts of fermented broccoli samples were prepared for profiling glucosinolates while the methanol extracts were prepared for profiling phenolic compounds. The freeze dried broccoli sample (0.12 g) was extracted with 2 mL water, and the mixture was shaken in a thermo-shaker (Allsheng Instruments Co., Ltd., Hangzhou, Zhejiang province, China) at 80 °C and 200 rpm for 50 min. The mixture was pressed and the liquid, containing the glucosinolates, was collected. To extract phenolics, the broccoli sample (0.12 g) was extracted with 2 mL 80% (v/v) methanol solution. The mixture was shaken in a thermoshaker at 60 °C and 200 rpm for 50 min. The mixture was pressed and the liquid was collected. The collected aqueous and methanolic liquids were made up to 2 mL, and then centrifuged at 4 °C and 13,800g for 20 min. The supernatants were collected for UHPLC-MS/MS analysis.

2.4. Preparation of water and methanol extracts of broccoli samples

2.1. Standard compounds and other chemicals Glucoraphanin (≥97%) was purchased from Chengdu DeSiTe Biological Technology Co. Ltd. (Sichuan, China). Quinic acid (≥98%) and chlorogenic acid (≥98%) were purchased from Shanghai Yuanye Bio-Technology Co. Ltd. (Shanghai, China). Phloretic acid (≥98%) was bought from Dalian Meilun Biological Technology Co. Ltd. (Liaoning, China). Formic acid and methanol were of HPLC grade (Jinmei Biotech Corporation, Tianjin, China). The Milli-Q water was prepared by an EASYPure II UV Ultra Pure Water System (Barnstead International, Dubuque, IA, USA). Biochemical reagents for microbial analysis were purchased from Thermo Fisher Scientific (Australia).

2.5. Chemical composition analyses

2.2. Preparation of autoclaved broccoli puree

2.5.1. Glucosinolate analysis The water extracts of broccoli samples were used for determination of glucosinolates, using UHPLC-MS/MS (Waters Corporation, Milford, MA, USA) according to the modified method reported by Wang, He, and Ma (2013). The UHPLC conditions were: Water ACQUITY UHPLC HSS T3 column (2.1 mm × 150 mm, 1.8 μm), column temperature 35 °C, injection volume 2 μL, mobile phase A = 0.1% formic acid + 99.9% water (v/v), mobile B = methanol. Linear gradient elution was varied linearly from 99.9% (v) A/0.1% (v) B to 98.0% (v) A/2.0% (v) B during the first 15 min, then to 45.0% (v) A/55.0% (v) B at 55 min and finally held at 99.9% (v) A/0.1% (v) B for re-equilibration until 60 min. The flow rate was 0.3 mL min−1. An electrospray ionization (ESI) technique in a negative ion mode was employed for MS scan. The ion source conditions were set as follows: capillary voltage 3000 V, cone voltage 30 V, extractor 3.0 V and RF lens 0.2 V, ion source temperature 150 °C, desolvation gas nitrogen at a flow rate of 600 L h−1 and desolvation temperature at 350 °C. Full scan ranging from 100 to 1000 atomic mass unit (amu) were recorded. Argon was used as the collision gas. The cone voltage and collision energy for glucosinolates and phenolic acids are present in Table 1. The triple quadrupole was set-up to perform daughter ion scan experiments, and the mass of parent ion was respectively set according to the detected [M−H]− (m/z) of individual compounds. The selected reaction monitoring (SRM) mode was used for quantifying individual compounds during UHPLC-MS/MS analysis, using an external standard method. The concentrations of

Broccoli puree samples were prepared as follows. To prepare the fresh broccoli puree, broccoli florets were cut into 1 cm × 1 cm cubes, and the broccoli cubes were mixed with ice-cold water at a broccoli/ water ratio of 1:1 (w/w) and homogenized for 1 min using a magic bullet kitchen blender (Nutribullet pro 900 series, LLC, USA). The broccoli puree was immediately packed in high barrier retort pouches (Caspak Australia) and autoclaved at 121 °C for 3 min to inactivate endogenous enzymes and autochthonous microorganisms. The efficacy of the autoclaving treatment for inactivating endogenous microflora was evaluated by plating the autoclaved samples on Plate Count Agar, MRS Agar, Violet Red Bile Glucose Agar, Potato Dextrose Agar for enumerating respectively total bacteria, lactic acid bacteria, enterobacteriaceae and yeast and mould counts in the samples. The microbiological results showed that the endogenous microflora in autoclaved samples were effectively inactivated, with the reduction of total plate count, LAB count, yeast and mould count and the enterobacteriaceae count to non-detectable level, because the 3 min autoclaving process exposed the broccoli puree samples to 30 min treatment at temperatures higher than 90 °C during temperature come-up and cooling in addition to the 3 min hold time at 121 °C. 2.3. Microorganisms and preparation of different broccoli samples Seven strains of lactic acid bacteria isolated from broccoli were used 617

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Table 1 The retention time, MS and MS/MS fragmentation data of identified compounds in both autoclaved and fermented broccoli samples by UHPLC-DAD-MS/MS. Cone voltage, Collision energy

[M−H]− (m/z)

MS/MS fragmentation data

Identification

References

Glucosinolates 1 2.72 2 3.35

30 V, 27 eV 30 V, 27 eV

422 388

358, 259, 195, 162 275, 259, 146

Glucoiberin Progoitrin

3

Nazif et al. (2010), Velasco et al. (2011) Kusznierewicz et al. (2013), Velasco et al. (2011) Standard; Velasco et al. (2011)

Peak

Rt (min)

30 V, 27 eV

436

194, 259, 372, 420

Glucoraphanin

Phenolic acids a 1.48 b 30.50

4.75

30 V, 25 eV 30 V, 27 eV

191 353

127 135, 161, 191

Quinic acid Chlorogenic acid

c d e f g

31.41 33.69 39.37 45.00 45.71

30 V, 30 V, 30 V, 30 V, 30 V,

27 eV 25 eV 25 eV 27 eV 27 eV

353 165 223 753 753

135, 173, 179, 191 147 164 205, 223, 529 205, 223, 529

h i j

46.58 47.18 47.88

30 V, 27 eV 30 V, 27 eV 30 V, 27 eV

723 959 929

175, 193, 205, 223, 499 205, 223, 529, 735 160, 175, 205, 223, 529, 705

Isomer of chlorogenic acid Phloretic acid Sinapic acid 1,2-Disinapoyl gentiobiose Isomer of 1,2-Disinapoyl gentiobiose 1-Sinapoyl-2-feruloyl gentiobiose 1,2,2′-Trisinapoyl gentiobiose 1,2′-Disinapyl-2-feruloyl gentiobiose

Standard; Taşkın and Taşkın (2017) Standard; Filannino et al. (2015), Qi et al. (2016) Moqbel et al. (2018), Qi et al. (2016) Standard; Filannino et al. (2015) Yasir et al. (2016) Filannino et al. (2015) Filannino et al. (2015) Filannino et al. (2015) Filannino et al. (2015) Filannino et al. (2015)

Table 2 Effect of LAB fermentation on glucosinolates in autoclaved broccoli samples (μg/g).a Glucosinolates c

Glucoiberin Progoitrinc Glucoraphaninb Sum

Autoclaved TL TL TL TL

F1

F2 a

56.2 ± 15.5 65.9 ± 11.7a 236.5 ± 35.3a 358.7 ± 58.3a

F3 b

16.1 ± 8.5 37.3 ± 8.5bc 93.2 ± 31.5c 146.5 ± 39.8cd

F4 b

27.0 ± 9.7 39.8 ± 12.7abc 135.9 ± 16.6bc 202.7 ± 38.6bc

b

26.6 ± 1.5 36.9 ± 6.2bc 113.3 ± 8.5bc 176.8 ± 13.7bcd

F5

BF1

BF2

TL 24.5 ± 4.8c 86.7 ± 7.2cd 111.2 ± 10.9d,e

TL 25.9 ± 12.9c 29.0 ± 4.1d 54.8 ± 15.9e

35.7 ± 1.8ab 54.9 ± 10.8ab 169.1 ± 21.6b 259.7 ± 11.9b

a TL: trace level-the peak intensity was not strong enough for quantification. The data expressed as mean ± standard deviation (SD) of the measured values (n = 3). Mean values with different superscripts in the same row are significantly different (P < 0.05). b Glucoraphanin was quantified by an external standard. c Glucoiberin and progoitrin were quantified by glucoraphanin, which were expressed as μg glucoraphanin equivalent/g broccoli sample.

glucosinolates without reference standards were expressed as glucoraphanin equivalents. Whilst this does not give a quantitative total concentration of detected glucosinolates, it does demonstrate changes in glucosinolate concentration due to compositional changes between treatments.

2.6. FTIR Broccoli sample powder (10 mg) was mixed uniformly with 100 mg of spectroscopic grade potassium bromide (KBr) powder (1% w/w), and then pressed into a disc for FTIR analysis as described by Gad and Bouzabata (2017). The spectra measurement of all samples was performed on FTIR spectrometer (Nicolet AVATAR 370 from Thermo Fisher, Massachusetts, USA) equipped with a deuterated triglycine sulphate (DTGS) as a detector. The IR absorption spectra of all the samples were recorded from 4000 to 400 cm−1 with a resolution of 4 cm−1 and 32 scan per sample. Three replicates of all samples were obtained. Spectral pre-processing of spectral data was performed on TQ Analyst software 8.0 (Thermo Electron Corp., USA) as described by Sampaio and Calado (2017), including baseline correction, normalization to 0–1, multiplicative scatter correction (MSC), second-order derivative calculation and Savitzky-Golay smoothing filter, to minimize physical disturbances from spectra or enhance their information content. The preprocessed spectral data with the interval of 8 cm−1 was submitted to chemometrics analysis.

2.5.2. Phenolic acid analysis The methanol extracts of broccoli samples were used for analyzing phenolic acids, using the same UHPLC and MS conditions as Section 2.5.1. The cone voltage, collision energy and MS data for each phenolic acid are listed in Table 1. An external standard method was used for quantification of quinic acid, chlorogenic acid and phloretic acid in SRM mode, and the concentrations of other phenolic acids without standards were expressed as equivalent of quinic acid. Despite this limitation, the total value is representative of the chemical composition change. 2.5.3. Free sugar analysis To determine free sugars, one gram of freeze dried broccoli samples was extracted with 5 mL 80% methanol at 80 °C for 30 min. After centrifugation at 14,000g and 4 °C for 15 min, the supernatant was collected for HPLC analysis. A waters HPLC system with a refractive index detector was used for sugar analysis. The HPLC conditions were: Shodex Asahipak NH2P-50 4E column (4.6 × 250 mm, Showa Denko K.K., Japan), isocratic elution with 68% acetonitrile as the mobile phase, sampler temperature 5 °C, column temperature 30 °C , run time 15 min.

2.7. Statistical analyses The results of above tests were expressed as the mean value ± SD. All microbial counts were converted into the logarithmic base (log10 cfu). Statistical analysis was carried out on SAS 9.4 software (SAS Institute Inc., Cary, NC), using Tukey’s HSD test. Significant differences were accepted at P < 0.05. PCA was performed on SPSS statistics 17.0 software (SPSS Inc. Chicago, IL, USA) to explore the clustering of different fermented samples. 3-D score plots were drawn by the Origin Pro 8.5.1 software (Originlab Corporation, Northampton, MA, USA). 618

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The evolution of pH with time during fermentation and the final LAB counting were used to characterize the fermentation process. The pH value of F1-F3 and F5 samples decreased sharply during fermentation to pH 4.4 within about 340 min, whereas the fermentation by isolate F4 was slightly slower. The pH values of BF1 and BF2 samples declined slowly and took 700 min to reach pH 4.4 (Fig. S1, Supplementary material). The LAB count after fermentation was in agreement with pH reduction rate. The F1-F5 samples had higher value of log10 cfu per mL ranging from 9.29 to 9.47, compared with BF1 and BF 2 which ranged from 7.72 to 8.33 (Supplementary data 1). This could be due to the lower acid tolerance of Leuconostoc Mesenteroides (McDonald, Fleming, & Hassan, 1990). 3.1. Identification of glucosinolates and phenolic acids in fermented broccoli samples There were 3 glucosinolates and 10 phenolic acids in the autoclaved broccoli sample identified by UHPLC-MS/MS, according to authentic external standards or published MS/MS data (Filannino et al., 2015; Kusznierewicz, Iori, Piekarska, Namieśnik, & Bartoszek, 2013; Moqbel et al., 2018; Nazif, Habib, Tawfik, & Hassan, 2010; Qi et al., 2016; Taşkın & Taşkın, 2017; Velasco et al., 2011; Yasir, Sultana, & Amicucci, 2016). Table 1 lists the retention time, MS and MS/MS fragmentation data of individual compounds. The glucosinolates identified were glucoiberin, progoitrin and glucoraphanin, and the phenolic acids included quinic acid, chlorogenic acid, neochlorogenic acid, phloretic acid, sinapic acid, 1,2-disinapoyl gentiobiose, isomer of 1,2-disinapoyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2′-trisinapoyl gentiobiose and 1,2′-disinapyl-2-feruloyl gentiobiose. Other glucosinolates, such as glucoalyssin, 4-hydroxy glucobrassicin, glucoerucin, glucobrassicin, 4-methoxy glucobrassicin and neoglucobrassicin, which have all been reported to be present in broccoli (Frandsen et al., 2014; Guo et al., 2013; Redovniković et al., 2012; Rybarczyk-Plonska et al., 2016), were not detected in the autoclaved broccoli samples. This is possibly due to low thermal stabilities of glucosinolates (Oerlemans, Barrett, Suades, Verkerk, & Dekker, 2006), resulting in degradation during autoclaving. 3.2. Effect of LAB fermentation on glucosinolates Table 2 shows the concentration of selected glucosinolates in broccoli samples before and after LAB fermentation. The autoclaved broccoli sample (CK) contained trace levels of glucoiberin, progoitrin and glucoraphanin. This could be due to thermal degradation during autoclaving as well as conversion to isothiocyanates and isothiocyanate nitriles during pureeing. However, the homogenization of the broccoli florets into puree was conducted at low temperature to inhibit the myrosinase catalyzed conversions and samples were autoclaved immediately after pureeing. Thus, the reduction in glucosinolates can be attributed largely to thermal degradation during autoclaving. After LAB fermentation, the contents of glucosinolates in autoclaved broccoli puree samples significantly increased with the total concentration of glucosinolates ranging from 55 μg/g to 359 μg/g. Among these 7 fermented broccoli samples, F1 contained the highest level of total glucosinolates, subsequently followed by BF2, F3, F4, F2, F5, and BF1 contained the lowest concentration of total glucosinolates (Table 2). An increase in glucoraphanin concentration from trace levels in the autoclaved broccoli to 29 ∼ 237 μg/g after fermentation was observed with the largest increases detected in F1 and BF2 ferments. The lowest concentration of glucoraphanin was detected in the BF1 ferment. In addition, there were increases in progoitrin and glucoiberin with the increment being to 24 to ∼66 μg/g and 0 to ∼56 μg/g respectively. These observed increases were less than the increases in glucoraphanin. Glucoraphanin is the most predominant glucosinolate in broccoli,

c

b

ND: not detectable. The data expressed as mean ± standard deviation (SD) of the measured values (n = 3). Mean values with different superscripts in the same row are significantly different (P < 0.05). Quinic acid, chlorogenic acid and phloretic acid were quantified by the corresponding external standards. The phenolic acids without references were quantified by quinic acid, which were expressed as μg quinic acid equivalent/g broccoli sample.

3. Results and discussion

a

156.3 ± 40.8a 16.2 ± 2.6ab 19.5 ± 1.7ab 717.5 ± 163.3b 29.1 ± 10.9a 11.3 ± 4.2ab 4.9 ± 0.8b 9.8 ± 2.3b 4.6 ± 0.2abc 11.1 ± 2.9abc 980.2 ± 226.9b 234.2 ± 71.6a 6.5 ± 0.7b 4.6 ± 0.7e 623.9 ± 58.0b 17.9 ± 5.7ab 2.4 ± 0.5c 3.2 ± 0.6b 4.0 ± 0.8c 1.9 ± 0.1c 4.4 ± 1.2c 903.0 ± 133.6b 210.3 ± 60.1a 11.2 ± 5.4ab 8.6 ± 0.4de 2568.4 ± 288.2a ND 9.6 ± 4.2abc 4.8 ± 1.1l 7.4 ± 0.6bc 5.3 ± 1.9abc 10.1 ± 4.7bc 2835.6 ± 332.3a 209.1 ± 57.7a 9.7 ± 2.8ab 13.4 ± 2.2bcd 2143.9 ± 599a 6.3 ± 1.9b 11.4 ± 3.3ab 4.4 ± 0.7b 9.3 ± 2.0bc 7.6 ± 2.3ab 15.3 ± 3.2ab 2430.5 ± 647.5a 199.0 ± 8.4a 11.8 ± 2.1ab 13.5 ± 1.3bcd ND 31.6 ± 4a 8.8 ± 1.7bc 5.5 ± 1.5b 6.3 ± 1.9bc 3.8 ± 0.2bc 8.9 ± 0.6bc 289.2 ± 10.8b Quinic acidb Chlorogenic acidb Isomer of chlorogenic acidc Phloretic acidb Sinapic acidc 1,2-Disinapoyl gentiobiosec Isomer of 1,2-disinapoyl gentiobiosec 1-Sinapoyl-2-feruloyl gentiobiosec 1,2,2′-Trisinapoyl gentiobiosec 1,2′-Disinapyl-2-feruloyl gentiobiosec Sum

255.2 ± 31.1a 18.7 ± 6.9a 27.4 ± 3.9a 2726.7 ± 205.2a ND 18.0 ± 2.2a 10.5 ± 1.6a 19.6 ± 3.0a 9.0 ± 1.2a 20.1 ± 4.2a 3105.3 ± 223.8a

197.4 ± 69.5a 11.3 ± 0.2ab 19.0 ± 6.3abc 2288.6 ± 816.6a ND 11.2 ± 2.7ab 5.4 ± 2.1b 10.0 ± 2.0b 8.4 ± 2.6ab 17.8 ± 3.4ab 2569.3 ± 891.4a

209.2 ± 67.4a 11.0 ± 6.3ab 10.7 ± 3.0cde 2594.3 ± 163.9a ND 12.4 ± 3.6ab 6.4 ± 1.3b 9.3 ± 2.3bc 6.9 ± 2.8abc 12.9 ± 5.0abc 2873.3 ± 210.6a

BF1 F3 F2 F1 Autoclaved Phenolic acids

Table 3 Effect of LAB fermentation on the phenolic acid levels of broccoli samples (μg/g).a

F4

F5

BF2

J.-H. Ye, et al.

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Table 4 Effect of LAB fermentation on free sugars in broccoli samples (mg/g).a Sugars

Autoclaved b

Fructose Glucoseb Sucroseb Mannitolb Sum

F1 a

104.7 ± 1.2 100.9 ± 0.0a 27.2 ± 0.1a ND 232.8 ± 1.3a

ND ND 5.4 ± 3.2b ND 5.4 ± 3.2g

F2

F3 b

50.0 ± 5.8 ND 9.7 ± 0.5b ND 59.7 ± 6.3d

F4 c

32.8 ± 6.1 ND 8.0 ± 0.4b ND 40.8 ± 5.8e

F5 d

17.6 ± 9.0 ND 5.9 ± 1.3b ND 23.5 ± 7.8f

ND ND 6.7 ± 2.3b ND 6.7 ± 2.3g

BF1

BF2 d

5.8 ± 1.0 21.0 ± 0.1b ND 88.1 ± 4.9 114.9 ± 6.1b

3.7 ± 0.2d 6.6 ± 0.3c ND 82.2 ± 3.3 92.5 ± 3.1c

a ND: not detectable. The data expressed as mean ± standard deviation (SD) of the measured values (n = 3). Mean values with different superscripts in the same row are significantly different (P < 0.05). b Four free sugars were quantified by the corresponding external standards.

which is also reflected in the increased amount after fermentation. This observed increase could be attributed to increased extractability due to changed physical properties of the broccoli matrix by the action of microbial enzymes. Some lactic acid bacteria strains are known to produce cell wall degrading enzymes (Claus, 2007), which may enable the release of bound phytochemicals including glucosinolates. It is also possible that the increase of glucosinolates, especially glucoraphanin, could be attributed to the synthesis of glucosinolates by the fermenting organisms, but this hypothesis has not been verified. It is not known if the natural microbial enzymes in the lactic acid bacteria isolates that we investigated produce glucosinolates. However, it has been demonstrated that it is possible to produce glucoraphanin by reconstructing the biosynthetic pathway of glucoraphanin from methionine in Escherichia coli (Yang, Liu, Li, & Yu, 2018). Although the levels of glucosinolates in the final fermented samples were much lower than those in fresh broccoli florets (Moreira-Rodríguez, Nair, Benavides, CisnerosZevallos, & Jacobo-Velázquez, 2017), the result indicates that LABfermentation is a promising method to increase the bioaccessible glucosinolate level of broccoli. Neither the autoclaved broccoli nor any of the ferments contained sulforaphane. The absence of sulforaphane in the autoclaved broccoli could be expected due to the inactivation of broccoli myrosinase under the intense heat treatment and thermal degradation of the small amount of sulforaphane that may have formed during pureeing. Broccoli myrosinase is a thermolabile enzyme which is inactivated at temperatures higher than 70 °C (Van Eylen, Oey, Hendrickx, & Van Loey, 2007). The observation that there was no sulforaphane in the ferment from autoclaved broccoli suggests that the LAB isolates did not produce myrosinase. Some lactic acid bacteria have the ability to transform glucosinolates into sulforaphane nitrile (Mullaney, Kelly, McGhie, Ansell, & Heyes, 2013). However, no obvious peak assigned to sulforaphane nitrile was detected in the MS scan results of the autoclaved broccoli samples prior to or after fermentation, indicating that the lactic acid bacteria strains in this study don’t have the metabolic capacity for such conversion. As in the case of suforaphane, the small amount of sulforaphane nitrile that may have formed during the pureeing step may have been degraded during autoclaving.

plantarum (Barthelmebs, Divies, & Cavin, 2000). On the contrary, the concentration of sinapic acid in broccoli samples declined after LAB fermentation, with sinapic acid being found in ferments with BF1 and BF2 but none detected in ferments with F1-F5. This observation is in contrast to the study by Filannino et al. (2015) where a two fold increase in sinapic acid was observed during fermentation of fresh broccoli puree by L. plantarum strains, although the change was not statistically significant. The differences between the results in this study and that by Filannino et al. (2015) could be due in part to the differences in the strains of L. plantarum and the pretreatment process, where broccoli was not autoclaved prior to fermentation in their study (Filannino et al., 2015) whereas the broccoli puree was autoclaved in our study. Differences in the starting broccoli materials due to varietal and agronomic conditions may have also contributed to the differences in the observed trends for sinapic acid contents. The concentrations of chlorogenic acid and quinic acid in the fermented broccoli samples showed no consistent change, with increases, decreases or no change observed, depending on the LAB isolate used. This result is in line with a previous study where a decrease in both chlorogenic acid and quinic acid was observed during fermentation of fresh broccoli puree by one strain of L. plantarum and no change in chlorogenic acid and a decrease in quinic acid was observed during fermentation by another strain of L. plantarum (Filannino et al., 2015). F1-F5 fermented broccoli samples contained significantly greater contents of phenolic compounds than ferments by BF1 and BF2 (P < 0.05), due mainly to the abundant generation of phloretic acid during fermentation. 3.4. Effect of LAB fermentation on sugars As LABs use free sugars as a source of energy, LAB-fermentation is likely to impact the free sugars in broccoli samples, including glucose, fructose and sucrose (Bhandari & Kwak, 2015). Fructose, glucose and sucrose were the major free sugars in autoclaved broccoli samples, which are the main carbon source for the growth of the LAB starters. The total concentration of fructose, glucose and sucrose in autoclaved broccoli samples sharply declined from 233 mg/g to 5 to ∼ 115 mg/g after LAB fermentation (Table 4). This is because lactobacillus species feed on natural sugars found in the vegetables (Das & Goyal, 2012). However, the preference for simple sugars was different among these LAB isolates, as demonstrated by the varying levels of fructose, glucose and sucrose observed after fermentation. The composition of free sugars in BF1 and BF2 samples were clearly different from other fermented broccoli samples, which contained more glucose and less sucrose. More importantly, mannitol was only present in BF1 and BF2 samples at the level of 88 mg/g and 82 mg/g respectively. BF1 and BF2 are strains of Leuconostoc mesenteroides with the metabolic capacity for converting fructose into mannitol via the activity of mannitol dehydrogenase. The Leuconostoc mesenteroides also produce glycosyltransferases (Vasileva et al., 2012), which hydrolyze sucrose into fructose and glucose and then transform these simple sugars into oligosaccharides and polysaccharides. This explains the notable absence of sucrose in BF1 and BF2 samples. The other five isolates (F1-F5) on the other hand are members of the species Lactobacillus plantarum, which utilize glucose as

3.3. Effect of LAB fermentation on phenolics Table 3 shows the phenolic compositions of different broccoli samples. Autoclaved broccoli sample contained 289 μg/g of total phenolic acids among which quinic acid was the most abundant phenolic acid (199 μg/g, Table 3). After LAB fermentation, the concentration of total phenolic acids were greatly elevated, with the concentration ranging from ∼903 μg/g to ∼3105 μg/g. Phloretic acid was generated after fermentation at levels of 624 μg/g to ∼2727 μg/g and was mainly responsible for the increase in total phenolics. F1-F5 isolates which are strains of L. plantarum produced more phloretic acid than BF1 and BF2 isolates that are Leuconostoc mesenteroides strains. Filannino et al. (2015) also reported that the strains of L. plantarum accumulated phloretic acid in fermented cherry juice and fresh broccoli puree. pCoumaric acid can be metabolized by reductase into phloretic acid by L. 620

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3.5. Discrimination of fermented samples based on chemical compounds There were 15 compounds identified in the autoclaved broccoli sample (CK) using the described methods. The chemical composition of the samples was related to the LAB isolate used for fermentation. It is insufficient to characterize and compare these fermented broccoli samples simply based on an individual component. PCA was used as an exploratory data analysis method for classification and discrimination. Fig. 1 shows the score plots of autoclaved (CK) and fermented samples based on the concentrations of phenolic acids (A), sugars (B) and total metabolites (C). For all score plots, the first three principal components (PCs) explained above 80% in total of the data variance. No clustering was observed based on the concentrations of glucosinolates, thus the score plot and loading plot of glucosinolates was not presented. As shown in Fig. 1A, CK, BF1 and BF2 were respectively clustered and discriminated from F1-F5 based on the composition of phenolic acids, while F1-F5 were not well resolved. Based on free sugar composition, CK was clearly discriminated from fermented samples, while BF1 and BF2 were close to each other and both were well resolved from F1-F5 (Fig. 1B). The pattern of Fig. 1C was similar to Fig. 1A but a better resolution was obtained based on 17 metabolites including glucosinolates, phenolics and free sugars. Supplementary Fig. S2 (Supplementary information) shows the corresponding loading plots. Fructose, glucose and sucrose were the most important components to discriminate autoclaved broccoli from LAB-fermented samples. Mannitol was the key component for discriminating BF1 and BF2 from F1-F5 samples, while phenolic acids further enhanced the difference between BF1 and BF2 samples. The composition of glucosinolates comprising glucoiberin, progoitrin and glucoraphanin did not discriminate between broccoli samples fermented by different LAB strains. 3.6. Discrimination of fermented samples based on FTIR spectra The FTIR technique has the advantage of being rapid, accurate and non-destructive, and can be used for general classification and comparison of food materials. Fig. 2 A shows the normalized spectra of autoclaved and 7 LAB-fermented broccoli samples. All spectra showed similar profiles with different absorbance over the whole spectra. The characteristic peaks in the FTIR spectra of all samples appeared at ∼3353 cm−1 (assigned to O–H absorption) and 2800–3000 cm−1 (assigned to methyl (–CH3) and methylene (–CH2) symmetric and asymmetric stretching vibration). The peaks at ∼1550 and ∼1640 cm−1 were attributed to amide-stretching bands of protein in broccoli samples, while ∼1040 cm−1 and ∼1112 cm−1 represent –C–O alcohols and C–O–H alcohols (Arancibia-Avila et al., 2012; Bestard, Sanjuan, Rosselló, Mulet, & Femenia, 2001; Gad & Bouzabata, 2017; Survay et al., 2010). F5 sample had the strongest absorbance at 1720 cm−1, followed by F1-4, BF1 and BF2 samples, while CK showed the lowest absorbance. Great divergences in spectra were also observed at 1237 cm−1. The absorption peaks at around 1720 cm−1 and 1237 cm−1 were reported in the FTIR spectra of a phenolic compound (gallic acid) or polyphenol extract (Arancibia-Avila et al., 2012). The enhanced FTIR absorbance related with phenolic compounds was in an agreement with the increase of phenolics in chemical analysis. The normalized FTIR spectra were preprocessed using MSC and second-order derivatives (Fig. 2B). The whole spectra (4000 cm−1–400 cm−1), characteristic peak region (1800 cm−1–1200 cm−1) and the fingerprinting region (1400 cm−1–900 cm−1), were respectively submitted to PCA. The results showed that the autoclaved sample (CK) and 7 fermented samples were not resolved from each other based on the spectrum data of 4000–400 cm−1 (Fig. 2C) and 1800–1200 cm−1 range (Fig. not shown), whereas CK and the group of BF1 and BF2 were separated from F1-F5 samples based on the fingerprinting region of 1400–900 cm−1 (Fig. 2D). BF1 and BF2 were not well discriminated from each other (Fig. 2D). Thus, BF1 and BF2 samples are different from F1-F5 samples as they are different species. Gad and Bouzabata (2017) failed to

Fig. 1. The score plots of autoclaved and fermented broccoli samples based on the concentrations of phenolic acids (A), sugars (B) and total metabolites (C). CK: autoclaved broccoli; F1-F5, BF1 and BF2: the ferments of the corresponding LAB isolates.

the main carbon source. However, differences were observed even among the five L. plantarum isolates in their sugar metabolism pattern. F1 and F5 have a higher capacity for fructose utilization than F2, F3 and F4. All the L. plantarum isolates also seem to possess glycosyltransferase or invertase activity for utilization of sucrose since the level of sucrose substantially decreased after fermentation.

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Fig. 2. The FTIR absorbance spectra normalized to 0–1 (A) and second-order derivative spectra with Savitzky-Golay filter (B) of different broccoli samples and the score plots of PCA in the range of 4000–400 cm−1 (C) and 1400–900 cm−1 (D). CK: autoclaved broccoli; F1-F5, BF1 and BF2: the ferments of the corresponding LAB isolates.

Declaration of interests

discriminate the metabolic fingerprints of different turmeric species by FTIR, but the same materials were resolved by HPLC and UV spectroscopy. Candida species were generally discriminated by FTIR in combination with PCA (Wohlmeister et al., 2017). Our results suggest that the selection of FTIR spectrum region for PCA is crucial for discrimination performance. The unique FTIR fingerprint of each sample in combination with PCA is a promising method for discrimination and classification of homologous samples, with the advantages of rapid, nondestructive examination in real-time.

None. Acknowledgements This work was funded by the Fundamental Research Funds for the Central Universities (2017QNA6021) and the 111 Project (B17039) and CSIRO. Appendix A. Supplementary data

4. Conclusions

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.02.030.

This study showed that fermentation by lactic acid bacteria (LAB) beneficially impact the chemical composition of broccoli puree in species and strain dependent manner. Notably, the study for the first time showed that fermentation by some strains of LAB increases the glucosinolate content of broccoli perhaps through release of bound glucosinolates from the broccoli matrix. The LAB fermentation process also resulted in a significant increase in polyphenolic content, whereas fructose, glucose and sucrose were sharply reduced after fermentation. Different LAB isolates had different impacts on the chemical composition of autoclaved broccoli. Broccoli ferments with the strains F1-F5 of L. plantarum produced higher amounts of phloretic acid in fermented broccoli compared to L. mesenteroides strains (BF1 and BF2). Ferments with F1 and BF2 had higher glucoraphanin level than ferments produced with the other strains. PCA results showed that non fermented autoclaved sample, and BF1 and BF2 ferments were discriminated from F1-F5 ferments based on chemical composition or the fingerprinting region of FTIR. LAB fermentation is a promising route to increase the accessible (extractable) levels of glucosinolates and phenolics in broccoli. As such it enables the production of ingredients or foods with enhanced levels of bioactives for the functional food industry.

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