Embed Size (px)
Citation preview
Journal of Functional Foods 34 (2017) 311–318
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier .com/ locate/ j f f
Construing temporal metabolomes for acetous fermentative productionof Rubus coreanus vinegar and its in vivo nutraceutical effects
http://dx.doi.org/10.1016/j.jff.2017.04.0341756-4646/� 2017 Elsevier Ltd. All rights reserved.
⇑ Corresponding author at: Department of Bioscience and Biotechnology, KonkukUniversity, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea.
E-mail addresses: [email protected] (M.Y. Lee), [email protected](H.Y. Kim), [email protected] (D.E. Lee), [email protected] (D. Singh),[email protected] (S.H. Yeo), [email protected] (S.Y. Baek), [email protected](Y.K. Park), [email protected] (C.H. Lee).
Mee Youn Lee a, Hyang Yeon Kim a, Da Eun Lee a, Digar Singh a, Soo Hwan Yeo b, Seong Yeol Baek b,Yoo Kyoung Park c, Choong Hwan Lee a,⇑aDepartment of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Koreab Fermented Food Science Division, Department of Agro-food Resource, National Institute of Agricultural Sciences, Rural Development Administration, Jeollabuk-do 565-851,Republic of KoreacDepartment of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 446-791, Republic of Korea
a r t i c l e i n f o a b s t r a c t
Article history:Received 10 January 2017Received in revised form 24 April 2017Accepted 26 April 2017
Chemical compounds studied in this article:Acetic acid (PubChem CID: 176)Citric acid (PubChem CID: 311)Phenethyl acetate (PubChem CID: 7654)Isoamyl acetate (PubChem CID: 31276)p-Cymene (PubChem CID: 7463)Verbenone (PubChem CID: 65724)Linoleic acid (PubChem CID: 5280450)Oleic acid (PubChem CID: 445639)LysoPC (22:6) (PubChem CID: 10415542)LysoPC (20:3) (PubChem CID: 53480467)
Keywords:MetabolomicsRubus coreanus vinegarDietary supplementationBone healthRat model
The temporal metabolomes associated with Rubus coreanus (RC) vinegar production were delineatedusing mass spectrometry-based metabolomic analyses with multivariate studies. We observed a clus-tered pattern of 27 discriminant metabolites: 4 organic acids, 9 sugars and sugar derivatives, 2 alcoholand carboxylic acid, 6 esters, and 6 terpenes. The levels of organic acids and alcohols were steadilydecreased throughout fermentation, whereas those of sugars and sugar derivatives, carboxylic acids,esters, and terpenes peaked at 6 days. Further, the in vivo nutraceutical potentials of RC vinegar wereexamined with its metabolomic implications for bone health in growing rat model. Intriguingly, the plas-matic metabolomes among treated rat groups were observed with lower levels of non-essential aminoacids and lysophosphatidylcholines (LysoPCs) coupled with higher levels of fatty acids and bile acids.The plasmatic metabolomes complemented with bone morphometric and clinical parameters suggestthe bone health ameliorating effects of RC vinegar.
� 2017 Elsevier Ltd. All rights reserved.
1. Introduction ponents, among the fermented beverages. Biochemically, acetous
Vinegar, a conventional seasoning consumed throughout theworld, has also been recognized for diverse health effects throughalleviating hyperglycemia and blood pressure, promoting systemiccalcium absorption as well as aiding digestion (Jo et al., 2013; Kishiet al., 1999). Hence, vinegar could equivocally be labeled asnutraceutical with its amalgam of nutritional and functional com-
fermentation involves the acetic acid bacteria (AAB)-mediatedstoichiometric transformation of ethanol to acetic acid and water(Ho, Lazim, Fazry, Zaki, & Lim, 2017). Traditionally, various rawsubstrates such as grain malts, fruits, vegetables, and wines havecommonly been employed for the acetous production of differentvinegar types. Notwithstanding the raw substrate blends,quintessential vinegar biochemistry involves two basic steps: (a)the yeast anaerobically converts sugars to ethanol, followed by(b) the AAB-mediated ethanol oxidation to acetic acid, in differentproportions (Tesfaye, Morales, Garcıa-Parrilla, & Troncoso, 2002).However, this deceptively simple biochemistry combined withthe intricate bioconversions result in auxiliary metabolites thatdetermine the nutritional and functional components as well asthe characteristic tang of commercial vinegar types. Besides acetic
312 M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318
acid proportions, the quality of commercial vinegar is determinedby the constituent peptides, volatile compounds, alcohols andpolyols, and organic acids, as well as various other compounds(Ubeda et al., 2016).
Although, the nutraceutical importance of vinegar has longbeen realized, the specific physiological effects of vinegar con-sumption on bone health are still not fully understood. A few pre-vious studies have demonstrated the positive effects of dietaryvinegar on bone health through promoting the intestinal assimila-tion of Ca+2 or its osteoblast-mediated absorption in bones usingovariectomized rat models (Kishi et al., 1999). Furthermore, Leeet al. (2016) have described the dynamic metabolic alterations inthe Rubus coreanus (RC) vinegar-fed ovariectomized rats with dis-criminant metabolites under controlled experimental conditions.The RC vinegar is made using the Korean black raspberry (bok-bunja), which has therapeutic applications in folk medicine andfunctional foods such as juices, candies, and rice cakes. These fruitsare conventionally known to have a number of health benefits suchas antibiotic, anticancer, anti-inflammatory, anti-osteoporotic, andimmunomodulatory effects, owing to the dynamic metabolitecompositions (Jung et al., 2009; Lim, Lee, & Do, 2015). In our previ-ous studies, we have probed the differential metabolite profiles forvarious commercial and traditional vinegar types made using dif-ferent raw materials including ’bokbunja’ and correlated those withcorresponding bioactivity phenotypes (Jang et al., 2015). In recentyears, food metabolomics has successfully been applied to explorethe impact of different processing methods on nutritional qualityand functionality of end products (Lee et al., 2014; Ziółkowska,Wasowicz, & Jelen, 2016). Moreover, the metabolic implicationsof dietary food supplementation can effectively be tracked in theplasma metabolomes of animal or human models using hyphen-ated mass spectrometry or spectroscopic methods (Caimari et al.,2015; Kim et al., 2013).
The temporal metabolite profiling for fermentation biopro-cesses provides insight into the complex metabolic interactionsand transformations in relation to different microbial assortmentsas well as physicochemical parameters such as pH, titratable acid-ity, and varying substrate types. In the present investigation, wehave outlined the metabolic profiles for the acetous fermentationthat occurs during RC vinegar manufacturing. The metabolomeswere further correlated with their relative levels in correspondingpathways and physicochemical parameters during the course offermentation. We further probed the potential nutraceutical prop-erties for the RC vinegar and its effects on bone health using agrowing rat model.
2. Materials and methods
2.1. Chemicals and reagents
HPLC grade methanol, acetonitrile, and water were purchasedfrom Fisher Scientific (Pittsburgh, PA, USA). Analytical grade formicacid, methoxyamine hydrochloride, pyridine, and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) were obtained fromSigma-Aldrich (St. Louis, MO, USA).
2.2. Sample information
2.2.1. Rubus coreanus vinegarThe fermentative stage specific RC vinegar samples used in the
study were procured from the rural development administration(RDA, Republic of Korea). The Korean black raspberry (R. coreanus)samples harvested from Gochang County, South Korea, were usedfor RC vinegar acetous manufacturing. The first step was carriedout by subjecting a sugar solution of 25�Bx (sugar strength) to
the yeast-mediated fermentation process at 25 �C for 10 days.The final alcohol concentration was diluted to 6%. Subsequently,the acetous fermentation was performed by adding 10% jongcho(made from RC wine), containing the AAB, Acetobacter pasteriusKACC16934. The blend with 4% titratable acidity was incubatedat 30 �C for approximately 2 weeks. The RC vinegar samples repre-senting different stages of acetous fermentation—0, 3, 6, 9, and13 days—were collected and stored at ultra-low freezing (�80 �C)conditions until further analysis. The sample pH values were mea-sured using a pH meter (Thermo Fisher Scientific, Beverly, MA,USA) calibrated at a room temperature. The titratable acidity wasestimated using the method described by Calamari, Gobbi, &Bani, (2016) with some modifications. The vinegar samples weretitrated using NaOH (0.1 N) solution with an endpoint value ofpH 8.2. All experiments were performed in triplicate. In case of ani-mal studies, RC vinegar samples collected during the 6 D acetousfermentation stage were used for dietary supplementations intreated rat groups.
2.2.2. Sample extractionsThe RC vinegar samples were mixed with methanol (1:1) using
a Twist Shaker (Biofree, Seoul, Korea) for 1 h at room temperature.The mixtures were cold centrifuged (4 �C) at 2370g for 5 min(Universal 320R, Hettich Zentrifugen, Tuttlingen, Germany) in 50-mL centrifuge tubes. The supernatant solution was filtered througha 0.2-lm polytetrafluoroethylene (PTFE) filter and concentratedusing a speed vacuum concentrator (Modulspin 31, Biotron,Incheon, Korea). The analytical replicates (n = 3) representing eachexperimental batch (n = 3) were subjected to HS-SPME-GC-TOF-MS (headspace solid phase microextraction-gas chromatography-time-of-flight-mass spectrometry) and GC-TOF-MS analyses.
2.3. Animal models
2.3.1. Experimental design and RC vinegar-based dietThe animal studies were conducted in accordance with the
approval of the Ethics Committee of the Kyung Hee University(KHUASP(SE)-14-020) and the guidelines for the care and use oflaboratory animals (National Research Council, 2010). Thirty-twoSprague-Dawley rats (3 weeks old, 45–50 g females) were pur-chased from Daehan Biolink (Eumseong, Korea) and housed incages, under standard laboratory conditions (room temperature22 ± 2 �C, humidity 55 ± 5%, 12 h light/dark cycle). The pellet chowdiet and water were provided ad libitum during a 1-week acclima-tization period. After this period, the rats were fed with an Amer-ican Institute of Nutrition (AIN)-93G diet (Research Diet Inc., NewBrunswick, NJ, USA) throughout the experimental period. Theexperimental growing rat groups were randomly divided into fourgroups as follows: control (CON, n = 8); Low-dose RC vinegar (LRV,gavage fed 2.5% vinegar at 5 mg/kg bw/day, n = 8); High-dose RCvinegar (HRV, gavage fed 5.2% vinegar at 5 mg/kg bw/day, n = 8);and casein phosphopeptide (CPP, gavage fed 1 mg/mL, n = 8) asthe positive control. The rats were gavage-fed every morning for8 weeks. During the 8-week experimental period, body weightwas measured twice a week while food intake was monitoreddaily. At the end of the experimental period, the rats were fastedovernight for 12 h and sacrificed. The rats were anesthetized andthe blood was collected by cardiac puncture into serum-separating tubes. The serum was separated by centrifugation(1006g for 15 min), followed by storage at ultra-low temperature(�80 �C) until further analyses.
2.3.2. Analysis of morphometric and clinical parametersThe femur and tibia were isolated from both right and left sides
of each sacrificed animal, and their respective lengths were mea-sured with a digimatic caliper (Mitutoyo Co., Kanagawa, Japan) to
M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318 313
hundredths of millimeters. The average lengths of right and leftside bones (femur and tibia) from each animal were used for com-parison among the groups. The alkaline phosphatase (ALP) andosteocalcin levels in serum were analyzed using an ALP analysiskit (Roche Diagnostics, Seoul, Korea) and a rat osteocalcin EIA kit(Biomedical Technology Inc., Boston, MA, USA), respectively. Thelevels of serum calcium (Ca) and phosphorus (P) were determinedusing the chemical auto-analyzer ADVIA 1650 (Bayer, Tokyo,Japan). The Ca:P ratio was calculated to estimate the adequacy ofcalcium metabolism in the animals. The experimental data wassubjected to statistical analyses using Duncan’s multiple range testat p < 0.05.
2.3.3. Rat plasma sample preparation for mass spectrometry (MS)analysis
The rat plasma metabolite profiling was performed using GC-TOF-MS and ultra-performance liquid chromatography quadrupole(UPLC-Q)-TOF-MS. The plasma samples were extracted by adding900 lL of ice-cold methanol to 300 lL of rat plasma. The mixturewas homogenized (frequency = 30 Hz) for 3 min by using a mixermill (Retsch GmbH & Co, Haan, Germany) and was subsequentlykept at -20 �C for 1 h. The resulting supernatant was filteredthrough a syringe filter (0.2 lm) and transferred to an Eppendorftube. The supernatant (1 mL) was completely dried using a speedvacuum machine (Biotron, Seoul, Korea). The dried samples werere-dissolved in methanol and filtered again using 0.2-lm PTFE fil-ters prior to hyphenated MS-analyses.
2.4. Equipment
2.4.1. HS-SPME-GC-TOF-MS analysisVolatile extraction by HS-SPME was carried out according to the
modified method described by Machiels and Istasse (2003). Thevinegar samples (2 mL) were placed in 4 mL glass headspace vialswith a small magnetic stir bar (as the extraction was carried outunder constant magnetic stirring) with a sample/headspace ratioof 1:1. The extraction of volatiles was carried out using SPME appa-ratus and carboxen-polydimethylsiloxane fibers (CAR/PDMS,75 lm). The sample volatiles were equilibrated and extracted for20 and 40 min at 70 �C, respectively. The compounds were thendesorbed from the fiber directly into the GC injector under splitless mode for 2 min at 300 �C. The HS-SPME analysis was per-formed using an Agilent 4890 GC system (Palo Alto, CA, USA) cou-pled with a Leco TOF Pegasus III mass spectrometer (Leco Corp., St.Joseph, MI, USA). The GC system was equipped with a DB-5MS cap-illary column (30 m length � 0.25 mm i.d. � 0.25 lm film thick-ness). The oven temperature was programmed as follows:initially maintained at 30 �C with 10 min hold, followed by an ele-vation to 60 �C at 3 �C/min, to 200 �C at the rate of 20 �C/min, andthen held for 2 min. The mass spectra were obtained using a mass-selective detector with an electronic impact of 70 eV and a multi-plier voltage of 1550 V, while the data was collected at rate of 1scan/s over the m/z range of 40–500. The volatile metabolites foreach stage of acetous fermentation sample extracts were puta-tively identified using the NIST digital library.
2.4.2. GC-TOF-MS analysisFor GC-TOF-MS analysis, the dried residues were methoximated
with 50 lL of methoxyamine hydrochloride in pyridine (20 mg/mL) at 30 �C for 90 min. Derivatization for silylation was performedby adding 50 lL of MSTFA at 37 �C for 30 min. The GC-TOF-MSanalysis was performed using an Agilent 4890 GC system (PaloAlto, CA, USA) coupled with a DB-5MS capillary column (30 mlength � 0.25 mm i.d. � 0.25 lm film thickness; J & W Scientific,Folsom, CA, USA) and a Leco TOF Pegasus III mass spectrometer(Leco Corp., St. Joseph, MI, USA). Helium was used as the carrier
gas at a constant flow rate of 1.5 mL/min. A total of 1 lL of thederivatized sample was injected in the split mode (10:1). The oventemperature was sustained at 75 �C for 2 min and then increased to300 �C at a rate of 15 �C/min. The temperature was then main-tained at 300 �C for 3 min. The mass data were collected in theelectron ionization (EI) mode with an ionization energy of 70 eVand mass scan (m/z) range of 45–1000 at an acquisition rate of20 scans/s. The injector and ion source temperatures were set at250 and 230 �C, respectively.
2.4.3. UPLC-Q-TOF-MS analysisThe UPLC-Q-TOF-MS analyses for the plasma samples were per-
formed on a Waters Micromass Q-TOF Premier with an UPLCACQUITYTM System (Waters, Milford, MA, USA). The chromato-graphic system was equipped with a Waters Acquity Tunable UV(TUV) detector, a sample manager (auto sampler) and a binary sol-vent manager (pump). The chromatographic operations were per-formed using the ACQUITYTM UPLC BEH C18 column (100 � 2.1 mm,1.7 lm). The mobile phase consisted of water (A) and acetonitrile(B) with 0.1% formic acid (v/v) at a flow rate of 0.3 mL/min. The sol-vent gradient condition was initially set at 5% Solvent B for 1 minfollowed by a linear gradient over 10 min ending at 100% SolventB. Five microliters of the sample volume was injected into thechromatographic system with a constant flow rate maintained at0.3 mL/min. The MS system was tuned with the following opera-tional parameters: ion source temperature, 100 �C; desolvationgas flow, 300 L/h; capillary voltage, 2.5 kV; and cone voltage,40 V. The MS analysis was carried out in both negative and positiveion modes within an m/z range of 100–1000.
2.5. Data processing and multivariate analysis
HS-SPME-GC-TOF-MS and GC-TOF-MS data files were con-verted to CDF format (⁄.cdf) using the Leco ChromaTOF softwareprogram (version 4.44, Leco Corp.). UPLC-Q-TOF-MS data files wereconverted into NetCDF format (⁄.cdf) using the MassLynx Data-Bridge (version 4.1, Waters). Following the conversion, peak detec-tion, retention time correction, and data alignments were carriedout using the MetAlign software package (http://www.metalign.nl). The aligned ⁄.cdv files were then transferred to Excel (Micro-soft, Redmond, WA, USA) data sheets for sequential multivariateand statistical analyses using the SIMCA-P + 12.0 software program(Umetrics, Umea, Sweden). Principal component analysis (PCA)was performed to obtain an overview of the MS data for metabo-lites and to identify the singularity value. Further, partial leastsquares-discriminant analysis (PLS-DA) was applied to find amodel that separated classes of observations of the Y axis (or ver-tical line) based on their X variables (or horizontal line). The PLS-DA model performance was based on the cumulative goodness offit (R2) and the cumulative goodness of prediction (Q2) for eachmodel. Metabolites with a variable importance projection (VIP)value greater than 0.7 and a p-value of less than 0.05 were selectedas significantly discriminant metabolites related to differentgroups. To visualize differences of metabolites between samples,heat map analysis was performed using MeV software (http://www.tm4.org/). The p-values for different metabolite-based clus-ter groups were determined using Statistica 7 (StatSoft, Tulsa,OK, USA). Experimental groups were compared using one-wayANOVA and Duncan’s test, with p-values < 0.05 considered signifi-cant. All histological data were analyzed using PASW Statistics 18software (SPSS, Chicago, IL, USA), at p < 0.05 (two-tailed). Followingmultivariate statistical analysis, the major metabolites were posi-tively identified by comparing the mass spectra and retentiontimes with those of standard compounds based on databases fromthe NIST mass spectral database (National Institute of Standards
314 M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318
and Technology, FairCom, Gaithersburg, MD, USA), in-houselibrary, and references.
3. Results and discussion
3.1. Temporal metabolite profiles for RC vinegar during acetousfermentation
The temporal metabolite profiling towards delineating the ace-tous fermentative stages for RC vinegar were performed using thecombined HS-SPME-GC-TOF-MS and GC-TOF-MS analyses. ThePCA and PLS-DA models obtained from GC-TOF-MS data indicatedthat the metabolites gradually changed according to the fermenta-tive stages (Fig. 1). We observed the clearly distinct and clusteredmetabolite profiles for the time points 0 days, 3–6 days, and 9–13 days following the acetous fermentation. The PLS-DA scoreplots exhibited 42.3% of the total variability (PLS1: 30.9%; PLS2:11.4%) with quality parameters R2X = 0.651, R2Y = 0.989, andQ2Y = 0.913. The PLS-DA pattern was found to be in agreementwith that of the corresponding PCA pattern. The significantly dis-criminant metabolites corresponding to varying stages of acetousfermentation were determined using the VIP value (VIP > 0.7)and p-value (p < 0.05). These discriminant metabolites were identi-fied using their retention times and mass fragmentation patternswith reference to standard compounds, National Institute of Stan-dards and Technology (NIST MS Search Program, version 2.0,Gaithersburg, MD), and in-house libraries. A total of 27 metabolitesfalling into five categories including organic acids (4), sugars andsugar derivatives (9), alcohol and carboxylic acid (2), esters (6),and terpenes (6) were identified as being significantly discriminantduring the course of fermentation (Table S1).
3.2. Correlations among metabolite levels and physiochemicalparameters during acetous fermentation of RC vinegar
The relative variations in the metabolite levels were envisagedusing a heat map (Fig. 2), where the values represent fold changesnormalized by an average of all values at each fermentative stage.Intriguingly, most of the metabolite levels spiked at 6 days exceptthose of organic acids: lactic acid, succinic acid, malic acid, andcitric acid. The organic acids are vital for the characteristic tart ofvinegar which determines vinegar quality (Hajimahmoodi et al.,2016). The levels of lactic acid, succinic acid, and malic acid wereobserved as linearly decreasing during fermentation while thosefor citric acid were reciprocally increased until day 6 and declined
Fig. 1. (A) Principal component analysis (PCA) and (B) partial least-squares discriminandata sets for acetous fermentative stages in Rubus coreanus(RC) vinegar production ( ,
steeply thereafter. Biochemically, succinic acid and malic acid arethe major intermediates for driving the energy metabolism at thebeginning of acetous fermentation while citric acid acts as anacidulant. The marginal decrease in organic acid levels coupledwith a significant rise in acetic acid content was accompanied bya mild decrease in pH and a proportional increase in titratable acid-ity (Fig. S1). The titratable acidity was substantially increased untilthe first 6 days and slightly afterwards. The Acetobacter speciesconverts ethanol to acetic acid in the presence of oxygen duringthe acetous fermentation (Ho, Lazim, Fazry, Zaki, & Lim, 2017).Simultaneously, content of other alcohols such as 2-Methyl-1-butanol decreases as the fermentation proceeds. The acetic acidis both volatile and pungent with a distinctive sour taste addingto the characteristic aroma of the vinegar end product (Akakabe,Tamura, Iwamoto, Takabayashi, & Nyuugaku, 2006). Besides aceticacid, a number of auxiliary components and metabolites affect thefunctional properties of vinegar, and contribute to its anti-dyslipidemia, anticancer, anti-obesity, and antihypertensive effects(Kondo, Kishi, Fushimi, & Kaga, 2009).
The quality of vinegar is primarily determined by its character-istic aroma, which is defined as the sum of a large number of vola-tile compounds, whose composition is specific to the variety offruits or grain blends added as substrate materials. The mostimportant among the aroma compounds, the esters, are formedduring acetous fermentation through the reaction between acidsand remaining alcohols during vinegar aging (Alcarde, Souza, &Bortoletto, 2014). The myriad of ester compounds produced duringvinegar aging includes methyl acetate, ethyl acetate, isobutyl acet-ate, isoamyl acetate, phenethyl acetate, and myrtenyl acetate. Theester compounds are mostly derived from fruits and wine added asflavor components during the artisanal steps in commercial vine-gar production (Jo et al., 2013). Most of the ester compounds wereobserved as maximally increased until 6 days followed by a sharpor gradual decrease until 13 days. Exceptionally, the phenethylacetate levels were observed as linearly increased during the entireprocess. Here, we concluded that the observed pH stability after6 days onwards resulted in the decreased rate of ester hydrolysisduring RC vinegar aging, with methyl acetate and ethyl acetateas exceptions. The reduced rates of ester hydrolysis and pH varia-tions were similarly reported earlier in cases of yeast fermentation(Malowicki, Martin, & Qian, 2008).
The terpenes are mostly produced by a variety of plants as theprimary components of essential oils, contributing to their flavor,scent, and color. Hence, terpenes are often employed in food man-ufacturing, stress aromatherapy, perfumery, and seldom as antimi-crobial agents (Lokeshkumar et al., 2015). In this study, we
t analysis (PLS-DA) score plots based on the GC-TOF-MS and HS-SPME-GC-TOF-MS0 days; , 3 days; , 6 days; , 9 days; , 13 days).
Fig. 2. Heat map representation of the relative levels of significantly discriminant metabolites detected using the GC-TOF-MS and HS-SPME-GC-TOF-MS data sets of the Rubuscoreanus (RC) vinegar acetous fermentative stages. Each data point was normalized by an average of all values.
M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318 315
observed that levels of the terpene compounds cymene, camphe-none, terpineol, myrtenal, myrtenol, and vebenone were variouslyincreased until 6–9 days of acetous fermentation, followed by agradual decrease until 13 days. Hence, the observed variation inthe levels of terpenes can be correlated with distinctive berry fla-vors as well as the varietal aroma derived from bokbunja. Further-more, terpenes are reportedly known for a wide range ofpharmacological and biological activities including anti-oxidant,anticancer, and antinociceptive effects (Lokeshkumar et al., 2015;Sliva et al., 2014). In general, these compounds are rapidlyincreased until 6 days followed by a decrease during the laterstages of acetous fermentation. Hence, we hypothesized a meta-bolic propensity between the decreasing levels of alcohols (after6 days) and the enhanced biochemical activities of Acetobacter spe-cies under alcohol-limiting conditions. We observed two-phasevinegar production where the levels of primary substrate, i.e., 2-methyl-1-butanol, rapidly fall during the initial 6 days, followedby the reduction of volatile compounds during the subsequentstages. Hence, the commercial quality of RC vinegar generallydepends upon its differential aging as well as the resulting metabo-lite compositions.
3.3. In vivo nutraceutical effects of dietary RC vinegar
We examined the in vivo nutraceutical effects of RC vinegar on agrowing rat model through evaluating clinical parameters: body
weight, bone morphometrics, serum biochemistry, and associatedmetabolomes. The body weights of LRV- and CPP-treated ratgroups were relatively increased while those for HRV-treatedgroups marginally decreased in comparison to the CON group(Fig. S2). Acetic acid, a primary component of vinegar, is reportedto inhibit body fat accumulation and thus helps to reduce bodyweight and BMI (Kondo, Kishi, Fushimi, Ugajin, & Kaga, 2009).Additionally, exogenously supplemented acetic acid in rat modelshas been described to alter lipid metabolism in both skeletalmuscles and liver through inhibiting the expression of lipogenicgenes, and thus may suppress obesity and obesity-linked type 2diabetes (Yamashita, 2016). Moreover, the RC contains antho-cyanins such as cyanidin-3-glucoside, cyanidin-3-rutinoside, andpelargonindin-3-glucoside, with associated antioxidant propertieshelpful in preventing obesity (Choi, Shim, & Kim, 2015; Lee,Dossett, & Finn, 2013). Recently, Jeong et al. (2015) demonstratedthe in vivomodulation of thermogenic genes affecting the differen-tiation of brown adipocytes using high-fat diet (HFD)-fed micesubjected to dietary supplements of RC extracts. In this study, weobserved that the body weights of rats fed on different doses ofdietary RC vinegar were not significantly affected. Nevertheless,the data suggest that the dietary consumption of RC vinegar hasmarginal anti-obesity effects making it a non-obesogenicnutraceutical beverage.
We further examined the effects of RC vinegar ingestion in ratmodels through analyzing the serum biochemical markers of bone
316 M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318
formation and resorption (Table 1). The ALP levels, which deter-mine the bone formation, were observed comparatively higheramong the treated rat groups. However, serum osteocalcin (OC)was marginally increased in HRV as compared to LRV and CPPgroups. Intriguingly, the serum OC levels in HRV group werealmost the same as those detected in CON. Functionally, OC repre-sents the major non-collagenous proteins produced by bone osteo-blasts, which precipitate calcium and hydroxyapatites (Barrère,van Blitterswijk, & de Groot, 2006). On the other hand, the compa-rable levels of serum Ca and P were observed among the differentrat groups including the positive control (CPP).
Among the experimental rat groups, the bone mineral mass wascorrelated with an increase in bone length. We evaluated the bonelength of the femur and tibia in growing rats subjected to varyingdoses of RC vinegar. There was no significant difference in femurlengths; however, the tibia lengths were considerably increasedamong the treated rat groups (HRV > LRV > CPP) as compared toCON. Arguably, we correlate the effects of RC vinegar on tibialength with respect to its relative acidity and metabolite levelsfor dietary supplements. The acidity of HRV was similar to thatat 6 days of acetous fermentation with a concomitant compositionof metabolites related to nutritional, taste, aroma, and quality com-ponent of the vinegar. Our results are in agreement with therecently published studies describing the positive effects of dietaryRC products on bone health (Lee et al., 2016; Ma et al., 2013).
3.4. Plasma metabolomic profiles for rat models subjected to dietaryRC vinegar
The analyses of rat plasma metabolite profiles from GC-TOF-MSand UPLC-Q-TOF-MS datasets indicated four distinct metaboliteclusters along PLS1 and PLS2 components in PLS-DA score plots(Fig. 3). The data of PCA model presented in SupplementaryFig. S3. A total of 13 and 10 significantly discriminant plasmaticmetabolites (VIP > 0.7) from experimental rat groups were selectedbased on the GC-TOF-MS and UPLC-Q-TOF-MS datasets, respec-tively (Tables S2 and S3). The relative plasmatic levels of putativelyidentifiedmetabolites—amino acids (3), organic acids (2), sugar (1),fatty acids (4), bile acids (2), lysophosphatidylcholines (6)—andthose unidentified (5) are expressed using a heap map (Fig. 4).Among the putatively identified metabolites, the levels ofornithine, glucose, tyrosine, oleic acid, monopalmitin, glycocholicacid, LysoPC 22:6, LysoPC 20:3, and LysoPC 22:4 were observedas significantly discriminant (p < 0.05) among each rat groups.
The concentrations of different amino acids are affected bymetabolism, lifestyle, genetic factors, and diet (Scalbert et al.,2009). In general, the higher plasmatic levels of non-essentialamino acids including serine, ornithine, and tyrosine are oftenassociated with idiopathic osteoporotic conditions (Pernow et al.,2010). Intriguingly, we observed that the plasma levels of amino
Table 1Clinical and bone morphometric parameters studied for the different growing rat groups
Outcome variablesa CON L
SerumAlkaline phosphatase (U/ L) 68.6 ± 11.26 7Osteocalcin (ng/ mL) 28.4 ± 3.94 2Calcium (mg/dL) 11.0 ± 0.35a 1Phosphorus (mg/dL) 7.0 ± 0.73 7Serum Ca/P (mg/dL) 1.6 ± 0.14 1
Bone lengthFemur (mm) 32.4 ± 2.00 3Tibia (mm) 35.9 ± 2.17b 3
CON, Control; LRV, treated with low-dose RC vinegar (2.5% acidity, 5 mg/kg bw/day); Hwith casein phosphopeptide (1 mg/mL). Data are expressed as mean ± SD (n = 8). The starat groups were evaluated using (a) one-way ANOVA and (b) Duncan’s multiple rangestatistical tests.
acids were marginally decreased among the dietary RC vinegar-treated groups as compared to CPP and CON, which could be corre-lated with their increased bone mineral mass, i.e., tibia length aswell as higher mineral density. Similarly, the reduced glucoselevels detected in treated groups may also be linked with bonehealth, since higher levels of blood glucose are often associatedwith age or disease-related bone loss (Krakauer et al., 1995). Inaddition, the high levels of glucose in the obesity model are oftencorrelated with dysregulated carbohydrate metabolism, diabetes,as well as high BMI and atherosclerosis (Park, Sadanala, & Kim,2015). Isidro and Ruano (2010) have proposed a relationshipbetween diabetes and bone diseases manifested through the mul-tiple pathway alterations regulating insulin levels and calciumuptake leading to increased risk of bone fractures. The lower levelsof plasma fatty acids are often correlated with bone loss (Baggio,2002), with simultaneous effects on other vital physiological func-tions such as energy storage, cell membrane function, oxygentransport, and regulation of inflammation and cell proliferation(Coletta, Bell, & Roman, 2010). In our study, we observed that thelevels of palmitoleic acid, linoleic acid, and oleic acid wereincreased in both the RC vinegar-treated rat groups (LRV, HRV)as well as the positive control group (CPP). In particular, the higherlinoleic acid levels are reportedly known to enhance the trabecularsurface, tissue level bone formation rates, and serum ALP(Schlemmer, Coetzer, Claassen, & Kruger, 1999).
In our study, we observed that the levels of two conjugated bileacids, taurocholic acid and glycocholic acid, were increased follow-ing the dietary vinegar treatment among the LRV and HRV ratgroups as in the CPP group (positive control). These elevations inplasma levels of conjugated bile acids, which represent the majorfraction of bile acids in the blood, are primarily associated withnormal hepatic metabolism (Vessey, 1978). These bile acids nor-mally regulate cholesterol homeostasis, glucose metabolism, lipidsolubilization, and metabolic signal induction of genes involvedin energy metabolism and uncoupling (Chiang, 2013). Further, bileacids enhance calcium absorption in mammals, which in turnincreases fat (fatty acid) absorption.
The lysophosphatidylcholines (LysoPCs), as potential plasmamarkers, are reportedly decreased in various obesity models fol-lowing weight loss (Heimerl et al., 2014). We observed the reduc-tion in plasma levels of LysoPC (20:4), LysoPC (22:6), LysoPC (16:0)LysoPC (20:3), and LysoPC (22:4) among the treated rat groups(LRV, HRV, and CPP) as compared to the untreated group (CON).In earlier work, Drobnik et al. (2013) reported a reduction inLysoPC levels following anti-inflammatory treatments throughrapid and transient mobilization of intracellular calcium. Addition-ally, Kwak et al. (2004) and Mebarek et al. (2013) describedLysoPCs as significant biomarkers of bone health and bone forma-tion because of their active role in osteoclast inhibition in mam-mals. Quintessentially, various metabolites and their relative
subjected to dietary Rubus coreanus (RC) vinegar treatment.
RV HRV CPP
4.5 ± 14.57 77.1 ± 19.97 71.3 ± 12.406.5 ± 3.35 28.8 ± 5.40 24.7 ± 3.650.9 ± 0.27ab 10.8 ± 0.26b 10.9 ± 0.34ab
.0 ± 0.15 7.1 ± 0.79 6.8 ± 0.61
.6 ± 0.06 1.5 ± 0.15 1.6 ± 0.13
2.6 ± 1.91 32.4 ± 1.47 32.6 ± 1.377.4 ± 1.27ab 38.2 ± 2.04a 37.5 ± 1.35ab
RV, treated with high-dose RC vinegar (5.2% acidity, 5 mg/kg bw/day); CPP, treatedtistical significance for the various test parameters studied among the experimentaltests, at p < 0.05, with value superscripts (a, b, and ab) indicating their respective
Fig. 3. Partial least-squares discriminant analysis (PLS-DA) score plots derived from the (A) GC-TOF-MS and (B) UPLC-Q-TOF-MS datasets for plasmatic metabolomic profilesfrom the dietary Rubus coreanus (RC) vinegar-administered growing rat groups. ▲ CON, Control, LRV, treated with low-dose RC vinegar (2.5% acidity, 5 mg/kg bw/day),HRV, treated with high-dose RC vinegar (5.2% acidity, 5 mg/kg bw/day), CPP, treated with casein phosphopeptide.
Fig. 4. Heat map representation for plasmatic levels of significantly discriminant metabolites based on the GC-TOF-MS and UPLC-Q-TOF-MS datasets from the growing ratgroups subjected to dietary Rubus coreanus (RC) vinegar treatment. Each data point shown on the heat map was normalized to the control group (CON). Red representsupregulation, and blue indicates downregulation compared with the CON. *p < 0.05 compared to CON. The numbers in parentheses are the metabolite numbers as shown inTables S2 and S3.
M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318 317
contents in plasma are involved in calcium homeostasis and boneformation in body.
4. Conclusions
The study presented herein outlines the temporal metabolicprofiles during the acetous fermentation of RC vinegar. Variousnon-volatile and volatile metabolites were found to be significantly
discriminant for specific stages of fermentation. In particular, somevolatile compounds such as esters and terpenes were increase untilthe 6 day of fermentation. Furthermore, we investigated thechanges in morphometric parameters, clinical factors, and plasmametabolome profiles for the RC vinegar-fed rat models in orderto determine the nutraceutical applications of RC vinegar. Thisin vivo study demonstrated that dietary supplementation of RCvinegar positively affects bone health and body weight amongthe growing rats. We also suggested potential biomarker metabo-
318 M.Y. Lee et al. / Journal of Functional Foods 34 (2017) 311–318
lites such as non-essential amino acids and LysoPCs in rat plasma,which can be putatively correlated with vinegar intake and itspotential health effects.
Acknowledgements
This work was supported by the Bio-Synergy Research Project(NRF-2015M3A9C4075815) of the Ministry of Science, ICT andFuture Planning through the National Research Foundation of Korea(NRF), and by the Bio & Medical Technology Development Programof the National Research Foundation (NRF) funded by the Koreangovernment (MSIP&MOHW) (No. 2016M3A9A5923160). This workwas also carried out with the support of ‘‘Cooperative ResearchProgram for Agriculture Science & Technology Development(Project No. PJ00982603)” Rural Development Administration,Republic of Korea.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jff.2017.04.034.
References
Akakabe, Y., Tamura, Y., Iwamoto, S., Takabayashi, M., & Nyuugaku, T. (2006).Volatile organic compounds with characteristic odor in bamboo vinegar.Bioscience Biotechnology and Biochemistry, 70, 2797–2799.
Alcarde, A. R., Souza, L. M., & Bortoletto, A. M. (2014). Formation of volatile andmaturation-related congeners during the aging of sugarcane spirit in oakbarrels. Journal of the Institute of Brewing, 120, 529–536.
Baggio, B. (2002). Fatty acids, calcium and bone metabolism. Journal of Nephrology,15, 601–604.
Barrère, F., van Blitterswijk, C. A., & de Groot, K. (2006). Bone regeneration:Molecular and cellular interactions with calcium phosphate ceramics.International Journal of Nanomedicine, 1, 317–332.
Caimari, A., Puiggròs, F., Suárez, M., Crescenti, A., Laos, S., Ruiz, J. A., ... Arola, L.(2015). The intake of a hazelnut skin extract improves the plasma lipid profileand reduces the lithocholic/deoxycholic bile acid faecal ratio, a risk factor forcolon cancer, in hamsters fed a high-fat diet. Food Chemistry, 167, 138–144.
Calamari, L., Gobbi, L., & Bani, P. (2016). Improving the prediction ability of FT-MIRspectroscopy to assess titratable acidity in cow’s milk. Food Chemistry, 192,477–484.
Chiang, J. Y. L. (2013). Bile Acid Metabolism and Signaling. ComprehensivePhysiology, 3, 1191–1212.
Choi, M. H., Shim, S. M., & Kim, G. H. (2015). Phenolic acids and quercetin fromKorean black raspberry seed protected against acetaminophen-inducedoxidative stress in mice. Journal of Functional Foods, 19, 404–416.
Coletta, J. M., Bell, S. J., & Roman, A. S. (2010). Omega-3 fatty acids and pregnancy.Reviews in Obstetrics & Gynecology, 3, 163–171.
Drobnik, W., Liebisch, G., Audebert, F. X., Frohlich, D., Gluck, T., Vogel, P., ... Schmitz,G. (2013). Plasma ceramide and lysophosphatidylcholine inversely correlatewith mortality in sepsis patients. Journal of Lipid Research, 44, 754–761.
Hajimahmoodi, M., Khanavi, M., Sadeghpour, O., Ardekani, M. R. S., Afzalifard, S., &Ranjbar, A. M. (2016). Application of organic acid based artificial neural networkmodeling for assessment of commercial vinegar authenticity. Food AnalyticalMethods, 9, 3451–3459.
Heimerl, S., Fischer, M., Baessler, A., Liebisch, G., Sigruener, A., Wallner, S., &Schmitz, G. (2014). Alterations of plasma lysophosphatidylcholine species inobesity and weight loss. PLoS ONE, 9, 1–7.
Ho, C. W., Lazim, A. M., Fazry, S., Zaki, U. K., & Lim, S. J. (2017). Varieties, production,composition and health benefits of vinegars: A review. Food Chemistry, 221,1621–1630.
Isidro, M. L., & Ruano, B. (2010). Bone disease in diabetes. Current Diabetes Reviews,6, 144–155.
Jang, Y. K., Lee, M. Y., Kim, H. Y., Lee, S., Yeo, S. H., Baek, S. Y., & Lee, C. H. (2015).Comparison of traditional and commercial vinegars based on metaboliteprofiling and antioxidant activity. Journal of Microbiology and Biotechnology,25, 217–226.
Jeong, M. Y., Kim, H. L., Park, J., Jung, Y., Youn, D. H., Lee, J. H., ... Um, J. Y. (2015). RubiFructus (Rubus coreanus) activates the expression of thermogenic genes in vivoand in vitro. International Journal of Obesity, 39, 456–464.
Jo, D., Kim, G. R., Yeo, S. H., Jeong, Y. J., Noh, B. S., & Kwon, J. H. (2013). Analysis ofaroma compounds of commercial cider vinegars with different acidities usingSPME/GC-MS, electronic nose, and sensory evaluation. Food Science andBiotechnology, 22, 1559–1565.
Jung, J., Son, M. Y., Jung, S., Nam, P., Sung, J. S., Lee, S. J., & Lee, K. G. (2009).Antioxidant properties of Korean black raspberry wines and their apoptoticeffects on cancer cells. Journal of the Science of Food and Agriculture, 89, 970–977.
Kim, H. W., Lee, A. Y., Yeo, S. K., Chung, H., Lee, J. H., Hoang, M. H., ... Lee, S. J. (2013).Metabolic profiling and biological mechanisms of body fat reduction in mice fedthe ethanolic extract of black-colored rice. Food Research International, 53,373–390.
Kishi, M., Fukaya, M., Tsukamoto, Y., Nagasawa, T., Takehana, K., & Nishizawa, N.(1999). Enhancing effect of dietary vinegar on the intestinal absorption ofcalcium in ovariectomized rats. Bioscience Biotechnology and Biochemistry, 63,905–910.
Kondo, T., Kishi, M., Fushimi, T., & Kaga, T. (2009). Acetic acid upregulates theexpression of genes for fatty acid oxidation enzymes in liver to suppress bodyfat accumulation. Journal of Agricultural and Food Chemistry, 57, 5982–5986.
Kondo, T., Kishi, M., Fushimi, T., Ugajin, S., & Kaga, T. (2009). Vinegar intake reducesbody weight, body fat mass, and serum triglyceride levels in obese Japanesesubjects. Bioscience Biotechnology and Biochemistry, 73, 1837–1843.
Krakauer, J. C., McKenna, M. J., Buderer, N. F., Rao, D. S., Whitehouse, F. W., & Parfitt,A. M. (1995). Bone loss and bone turnover in diabetes. Diabetes, 44, 775–782.
Kwak, H. B., Lee, S. W., Li, Y. J., Kim, Y. A., Han, S. Y., Jhon, G. J., ... Lee, Z. H. (2004).Inhibition of osteoclast differentiation and bone resorption by a novellysophosphatidylcholine derivative, SCOH. Biochemical Pharmacology, 67,1239–1248.
Lee, J., Dossett, M., & Finn, C. E. (2013). Anthocyanin fingerprinting of true bokbunja(Rubus coreanus Miq.) fruit. Journal of Functional Foods, 5, 1985–1990.
Lee, M. Y., Kim, H. Y., Singh, D., Yeo, S. H., Baek, S. Y., Park, Y. K., & Lee, C. H. (2016).Metabolite profiling reveals the effect of dietary Rubus coreanus vinegar onovariectomy-induced osteoporosis in a rat model. Molecule, 21, 1–19.
Lee, S. Y., Lee, S., Lee, S., Oh, J. Y., Jeon, E. J., Ryu, H. S., & Lee, C. H. (2014). Primary andsecondary metabolite profiling of doenjang, a fermented soybean paste duringindustrial processing. Food Chemistry, 165, 157–166.
Lim, H. K., Lee, H. R., & Do, S. H. (2015). Stimulation of cannabinoid receptors byusing Rubus coreanus extracts to control osteoporosis in aged male rats. TheAging Male, 18, 124–132.
Lokeshkumar, B., Sathishkumar, V., Nandakumar, N., Rengarajan, T., Madankumar,A., & Balasubramanian, M. P. (2015). Anti-oxidative effect of myrtenal inprevention and treatment of colon cancer induced by 1, 2-dimethyl hydrazine(DMH) in experimental animals. Biomolecules & Therapeutics, 23, 471–478.
Ma, B., Liu, J., Zhang, Q., Ying, H., A, J., Sun, J., ... Liu, Y. (2013). Metabolomic profilesdelineate signature metabolic shifts during estrogen deficiency-induced boneloss in rat by GC-TOF/MS. PLoS One, 8, 1–10.
Machiels, D., & Istasse, L. (2003). Evaluation of two commercial solid-phasemicroextraction fibres for the analysis of target aroma compounds in cookedbeef meat. Talanta, 61, 529–537.
Malowicki, S. M., Martin, R., & Qian, M. C. (2008). Volatile composition in raspberrycultivars grown in the Pacific Northwest determined by stir bar sorptiveextraction-gas chromatography-mass spectrometry. Journal of Agricultural andFood Chemistry, 56, 4128–4133.
Mebarek, S., Abousalham, A., Magne, D., Do, L. D., Bandorowicz-Pikula, J., Pikula, S., &Buchet, R. (2013). Phospholipases of mineralization competent cells and matrixvesicles: Roles in physiological and pathological mineralizations. InternationalJournal of Molecular Sciences, 14, 5036–5129.
National Research Council (2010). Guide for the care and use of laboratory animals(8th ed.). Washington, D.C.: The National Academy Press.
Park, S., Sadanala, K. C., & Kim, E. K. (2015). A metabolomic approach tounderstanding the metabolic link between obesity and diabetes. Moleculesand Cells, 38, 587–596.
Pernow, Y., Thorén, M., Sääf, M., Fernholm, R., Anderstam, B., Hauge, E. M., & Hall, K.(2010). Associations between amino acids and bone mineral density in menwith idiopathic osteoporosis. Bone, 47, 959–965.
Scalbert, A., Brennan, L., Fiehn, O., Hankemeier, T., Kristal, B. S., van Ommen, B., ...Wopereis, S. (2009). Mass-spectrometry-based metabolomics: Limitations andrecommendations for future progress with particular focus on nutritionresearch. Metabolomics, 5, 435–458.
Schlemmer, C. K., Coetzer, H., Claassen, N., & Kruger, M. C. (1999). Oestrogen andessential fatty acid supplementation corrects bone loss due to ovariectomy inthe female Sprague Dawley rat. Prostaglandins. Leukotrienes and Essential FattyAcids (PLEFA), 61, 381–390.
Sliva, R. O., Salvadori, M. S., Sousa, F. B. M., Santos, M. S., Carvalho, N. S., Sousa, D. P.,... Medeiros, J. R. (2014). Evaluation of the anti-inflammatory andantinociceptive effects of myrtenol, a plant-derived monoterpene alcohol, inmice. Flavour and Fragrance Journal, 29, 184–192.
Tesfaye, W., Morales, M. L., Garcıa-Parrilla, M. C., & Troncoso, A. M. (2002). Winevinegar: Technology, authenticity and quality evaluation. Trends in Food Science& Technology, 13, 12–21.
Ubeda, C., Callejón, R. M., Troncoso, A. M., Moreno-Rojas, J. M., Peña, F., & Morales,M. L. (2016). A comparative study on aromatic profiles of strawberry vinegarsobtained using different conditions in the production process. Food Chemistry,192, 1051–1059.
Vessey, D. A. (1978). The biochemical basis for the conjugation of bile acids witheither glycine or taurine. Biochemical Journal, 174, 621–626.
Yamashita, H. (2016). Biological function of acetic acid-improvement in obesity andglucose tolerance by acetic acid in type 2 diabetic rats. Critical Reviews in FoodScience and Nutrition, 56, S171–175.
Ziółkowska, A., Wasowicz, E., & Jelen, H. H. (2016). Differentiation of winesaccording to grape variety and geographical origin based on volatiles profilingusing SPME-MS and SPME-GC/MS methods. Food Chemistry, 213, 714–720.
