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Solid-state NMR characterization of triacylglyceroland polysaccharides in coffee beans

Noriko Kanai, Naoki Yoshihara & Izuru Kawamura

To cite this article: Noriko Kanai, Naoki Yoshihara & Izuru Kawamura (2019) Solid-state NMRcharacterization of triacylglycerol and polysaccharides in coffee beans, Bioscience, Biotechnology,and Biochemistry, 83:5, 803-809, DOI: 10.1080/09168451.2019.1571899

To link to this article: https://doi.org/10.1080/09168451.2019.1571899

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Solid-state NMR characterization of triacylglycerol and polysaccharides incoffee beansNoriko Kanai a, Naoki Yoshiharab and Izuru Kawamura a

aDepartment of Chemistry, Chemical Engineering, and Life Science, College of Engineering Science, Yokohama National University,Yokohama, Japan; bInstrumental Analysis Center, Yokohama National University, Yokohama, Japan

ABSTRACTIt is important to understand the structural characteristics of triacylglycerol (TAG), polysac-charides and trace elements in coffee beans, so that residues can be reutilized in applicationsincluding biodiesel oils. Here, we performed 1H and 13C solid-state NMR measurements onIndonesian green beans, roasted beans, and spent coffee grounds (SCGs). In the NMR spectra,there were liquid-like TAG containing linoleic acids based on observed signals of -CH=CH-CH2-CH=CH- group in an acyl chain, which play a role in decreasing TAG’s melting point. Wefound TAG was still abundant in the SCGs from NMR spectra. After lipids were removed fromSCGs, the intensity of the TAG signal decreased considerably, with approximately 64% of theTAG was successfully extracted. We described the chemical structure of TAG in coffee beansand demonstrated that it is possible quantify the amount of extracted TAG using solid-stateNMR.

ARTICLE HISTORYReceived 29 November 2018Accepted 8 January 2019

KEYWORDSCoffee; triacylglycerol;linoleic acid; polysaccharide;biodiesel

Coffee is one of the world’s most popular bev-erages, and more than 400 billion cups of coffeeare consumed each year [1]. Approximately9.5 million tons of coffee beans were consumedin 2017, and this is increasing year by year [2].Approximately 8 million tons of spent coffeegrounds (SCGs) are generated annually [3]. Theyare now classified as food industry waste, andthere is a responsibility to establish an efficientand comprehensive method for reusing or addingvalue to SCGs. Research on SCGs has sharplyincreased in the past ten years [1]. SCGs can bereused to produce fuel for industrial boilers, asa substrate for the cultivation of microorganisms,and as a raw material to produce ethanol, amongother uses [4]. Biodiesel is a source of renewableenergy, and the production of biodiesel has beendramatically increased in past ten years. Accordingto some reports, oils from SCGs have the potentialto be used in biodiesel oil [5–7]. Furthermore,coffee beans contain polysaccharides in their cellwalls such as cellulose and hemicellulose [3,6,8].The extracts from coffee oils, including SCGs, havenot been completely characterized at the molecularlevel, which is important SCGs can be used forgreen chemistry applications.

Coffee beans contain a complex mixture of hun-dreds of different compounds. Lipids are abundantin coffee beans and SCGs [3,8,9]. Lipids still repre-sent about 10–20% of the mass of SCGs [1,10,11].The major lipid in coffee beans is triacylglycerol

(TAG) [10,12], which is widely found in animalfat and vegetable oil. TAG consists of three acylchains with saturated and unsaturated fatty acids.The unsaturated fatty acids are oleic (18:1(n-9)),linoleic (18:2(n-6)) and linolenic (18:3(n-3))acids [10].

Nuclear magnetic resonance (NMR) is an effectiveand non-destructive analytical tool to identify thestructure and dynamics of molecules [13–15].Solution NMR analysis of the extracts of roastedcoffee beans (RCBs) and green coffee beans (GCBs)has been used to analyze the structure of organiccompounds and investigate their metabolomics toclassify beans from different geographic regions[16,17]. Solid-state NMR characterization is a directmethod for analyzing insoluble biomacromolecules,such as polysaccharides in plant cell walls, membraneproteins, and amyloid peptides [18–22]. Additionally,the use of solid-state NMR with magic angle spinning(MAS) does not require any complex sample treat-ment. If we could reveal detailed structural informa-tion about the lipids in coffee beans via solid-stateNMR analysis, we could estimate their amounts andthe degree of unsaturation in lipid molecules withoutneeding to extract from coffee beans. This would leadto a deeper understanding of how lipids extractedfrom SCGs can be used as raw materials to generatebiodiesel oil. Therefore, we investigated the structuresof TAG and polysaccharides from GCBs, RCBs andSCGs using 1H and 13C solid-state MAS NMRtechniques.

CONTACT Izuru Kawamura izuruk@ynu.ac.jpSupplementary data for this article can be accessed here.

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY2019, VOL. 83, NO. 5, 803–809https://doi.org/10.1080/09168451.2019.1571899

© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

Materials and methods

Sample preparation

The GCBs and RCBs were commercially availableRobusta coffee varieties in Indonesia that had beenwashed. GCBs and RCBs were ground using an elec-tric coffee grinder (Kalita, EG-45) for 70 s and for40 s respectively, to a uniform particle size. Hot waterwas then added to the ground RCBs. Wet-groundRCBs were thinly spread on a filter paper and com-pletely dried at room temperature for at least 1 day.Completely dried ground RCBs were termed SCGs.For the proton/deuterium exchange experiment,450 mg of RCBs was suspended in 5 mL of 99.5%D2O (CIL) and then the RCBs were dried at roomtemperature. TAG containing only oleic fatty acids inacyl chains (≧97%, Sigma Aldrich) was used asa reference sample.

SEM

The internal structures of the GCBs and RCBs wereobserved with field emission scanning electronmicroscope (FE-SEM) (HITACHI, SU8010). GCBswere rapidly freeze-dried and polished by embed-ding in a resin block to expose the section. RCBswere divided into a several mm-sized pieces usinga cutter and then were lyophilized by a freeze-dryer(EYELA, FDU-2100).

Extracting lipids from SCGs

The equivalent of a cup of coffee SCGs, 10 g wasgently stirred in 100 mL of n-hexane at roomtemperature for 1 day. The solvent was removedby reduced pressure evaporation at 80°C (EYELA,

SB-1200). Approximately about 70 μL of lipidswere extracted as a clear yellow brown oil in liquidform, and solid residues were filtered (Figure 1).The extraction rate of TAG was estimated by com-paring the integrated intensity ratio of the signal at2.6–2.7 ppm in the 1H-MAS NMR spectra. (Figures3(b) and 4(b))

1H and 13C solid-state NMR experiments

The GCBs, RCBs, SCGs samples as well as the solidresidue and liquid lipids extracted from the SCGswere directly packed into a 4.0 mm outer diameterzirconia NMR rotor. 1H-MAS, 13C cross polarization-magic angle spinning (CP-MAS) and 13C dipolardecoupling-magic angle spinning (DD-MAS) solid-state NMR spectra were recorded at room tempera-ture with a recycle delay time of 4 s on a 600 MHzspectrometer (Bruker Avance III) equipped witha 4.0 mm E-free MAS probe. The MAS frequencieswere at 12.0 and 10.0 kHz for the 1H and 13C NMRexperiments, respectively. The DD-MAS method pri-marily detects 13C NMR signals from the mobilecomponents based on their spin-lattice relaxationtimes compared with the recycle delay times [21,22].1H and 13C chemical shifts were referenced to tetra-methylsilane at 0.0 ppm, respectively (Figure 1).

Results and discussion

SEM observations

The cell walls in the GCBs and RCBs had stronghoneycomb structures with pore sizes of several tensof µm as shown in Figure 2. The honeycomb struc-ture in the SEM image of GCB is partly distorted

Figure 1. The experimental design of solid-state MAS NMR characterization in coffee beans [green coffee beans (GCBs); roastedcoffee beans (RCBs); spent coffee grounds (SCGs)] and solid residue and liquid lipids extracted from SCGs.

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because of the removal of moisture during lyophili-zation. However, a similar honeycomb structure ofthe cell walls is evident in the GCB when observedwith an optical microscope (Supporting InformationFigure S1). The pores of the GCB and RCB are filledwith liquid lipids, and the moisture, which was pre-sent in the GCB but not in the RCB, was believed tobe absorbed into the cell walls (see NMR sectionbelow).

Solid-state NMR characterization of TAG

Firstly, we observed 1H and 13C solid-state NMR spec-tra of GCBs, RCBs and SCGs as shown in Figure 3. Ingeneral, 1H MAS NMR signals of a solid-state sampleare quite broad due to huge 1H-1H homonuclear dipo-lar interactions [21]. However, all three 1H MAS NMRspectra of GCBs, RCBs and SCGs had similar narrow

spectral patterns, except there was a broad signal ataround 4.5 ppm in the GCBs (Figure 3(a–c)).

We confirmed that the three fatty acids in the TAGmolecule were a mixture of oleic, and linoleic acids.The 1H NMR peaks in all three spectra correspondedto A-J of TAG in Figure 3 and complete assignmentsof 1H NMR signals corresponding to A-J are summar-ized in Supporting Table S2. The F signal (2.6 ppm) inall three spectra belongs to protons directly bondedwith the carbon sandwiched between two -C=C-bonds as -CH=CH-CH2-CH=CH- in linoleic acid.Most -C=C- bonds in natural unsaturated fatty acidshave a cis-conformation, which causes a bend in thealkyl chain and molecules that are not well stacked.TAG is likely a liquid in coffee beans at room tem-perature because of the -C=C- bonds in the unsatu-rated fatty acids. All the peaks assigned to TAG in the1H MAS NMR observations were remarkably sharp

Figure 2. SEM images of cell walls in (a) GCBs and (b) RCBs.

MONITORING NMR PEAKS OF TRIACYLGLYCEROL INSIDE COFFEE BEANS 805

despite the use of natural products without any pre-treatment. It appears that the amount of unsaturatedfatty acids in coffee beans is higher than the amount ofsaturated fatty acids. The scale of the SCGs spectrumin Figure 3 (Left) was enlarged five times because theintensity was smaller than that of the GCBs and RCBs.

This indicates that some TAG was extracted duringcoffee brewing, decreasing the amount of TAG in theSCGs compared to the same volume of RCBs. Therewas a broad signal at approximately 4.5 ppm in theGCBs due to fixed moisture, which accounts forapproximately 5–10% of the dry weight [23,24]. The

Figure 3. [Left] 1H MAS NMR spectra of (a) GCBs, (b) RCBs and (c) SCGs (scale magnified five times), with letters from A toJ indicating peaks that correspond to structures in two fatty acids (oleic, and linoleic acid); and [Right] 13C DD-MAS NMR spectraof (d) GCBs, (e) RCBs, and (f) SCGs, with letters K and L indicating polysaccharides. For the 1H MAS NMR, all three spectra wereaccumulated from 200 scans and each experimental time was 7 min. For the 13C DD-MAS NMR, all three spectra wereaccumulated from 13,000 scans and each experimental time was around 11 hours.

Figure 4. [Left] Spectra from 1H MAS NMR of (a) extracted liquid TAG and (b) solid coffee residues after hexane treatment(Number of scans = 200), and [Right] spectra from 13C DD-MAS NMR of (c) extracted liquid TAG (Number of scans = 600) and (d)solid coffee residues after hexane treatment (Number of scans = 13000).

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broadness of this signal is likely due to the slow mobi-lity of water molecules absorbed into cell walls. Themoisture content of coffee beans is sharply reducedduring the roasting process; consequently, signalsderived from water in the cell walls did not appear ataround 4.5 ppm in the 1H NMR of RCBs (Figure 3(b))or SCGs (Figure 3(c)).

The 13C DD-MAS NMR spectra of the GCBs,RCBs and SCGs revealed similar patterns of chemi-cal shifts (Figure 3 right). All the sharp peaks, exceptthe broad peak of K (60–63 ppm) and L (70–77ppm), could be assigned to TAG, with the -C = C-double bond of linoleic acids at around 130 ppm(Supporting information Table S2). K and L arederived from the polysaccharides that constitutecell walls in coffee beans. Components of the cellwall have restricted their molecular mobility due tohigh crystallization, so K and L are not as sharp asthe peaks assigned to TAG.

After extracting the lipids from the SCGs, theliquid lipids and the solid residues were analyzed by1H MAS NMR and 13C DD-MAS NMR (Figure 4).The sharpness of the peaks in Figure 4(a,c) suggesta high purity and consistent quality in the extractedTAG from the SCGs. A single peak appeared at 172.3ppm in (c) indicates the absence of free fatty acidsand that is a great advantage for transesterificationprocess of biodiesel production in general [25]. Theseresults are also consistent with chemical shifts ofcommercial TAG, whose fatty acids are only oleicacids (Supporting Information Figure S2). Thebroad peak around 4 ppm in Figure 4(b) may bedue to water added to the brewing process oradsorbed water during the drying process. The peakat 110 ppm (Figure 4(c)) is from the Teflon coatingspacer used to avoid spillage of liquid lipids in thezirconia NMR rotor during the acquisition. The sig-nals marked with an asterisk (*) in Figure 4(d) arelikely the polysaccharides that constitute the cell wallsof coffee beans, which indicates that the cell wallswere not destroyed during the hexane treatment.On the other hand, the 13C NMR signals of TAG inthe dried residue (Figure 4(d)) were relatively lowcompared to those of the RCBs and SCGs (Figure3), suggesting that the TAGs were mostly extractedby the hexane treatments, but a small amount of TAGremained. The sharpness of the peaks in 1H MASNMR and 13C DD-MAS NMR spectra (Figure 4(a,c)) indicated the uniformity of extracted lipids fromSCGs, and a high level of similarity with the com-mercial triacylglycerol sample (Supporting Figure S3and Table S2) demonstrated the high purity of theTAG. Different from soybean oil, palm oil and cot-tonseed oil which are typical vegetable oils generatedbiodiesel fuel [26], SCGs does not need newly culti-vated lands nor completely compete with food. OurNMR results suggest SCGs to be promising raw

materials for generating biodiesel fuel. However,TAG seems to remain in the residues after delipidiza-tion process (Figure 4(a,c)), so an improvementextraction rate of TAG would be future issues. Inaddition, solid-state 1H MAS NMR technology islikely useful in the field of food science, as it ispossible to evaluate the quality of TAG rapidly.

Solid-state NMR characterization ofpolysaccharides

The spectra from 13C CP-MAS NMR show the maincomponents of the cell walls (Figure 5). Cellulose andhemicellulose are polysaccharides that constitute theprimary cell wall. There were high intensity peaksbetween 60 and 110 ppm representing carbons, con-stitute the five- or six-membered rings of polysacchar-ides. Galactomannan, arabinogalactan and celluloseare the most common polysaccharides in the coffeebeans [8]. Using SPINASSIGN based on the RIKEN1H and 13C chemical shift database of metabolites, the13C NMR signals at 81 and 102 ppm could be assignedto the galactose group of polysaccharides [27]. Thisindicates signals at 81 and 102 ppm are from poly-saccharides included in hemicellulose. The broad sig-nals of E, F and G are likely TAG and glycoproteinsidechains [28], lignin aromatic carbons [28,29] andthe carbonyl groups of lignin, hemicelluloses and theprotein backbone [30]. The spectral similarities of theGCBs, RCBs, SCGs and solid residues indicates thatthe cell wall structure was maintained through theroasting, grinding, and delipidization processes. Thepresence of strong intramolecular and intermolecularhydrogen bonds in cellulose microfibrils and hemicel-lulose may explain the stable structure of the cellwalls [8,31]. Deuterium secondary isotope shifts on13C NMR signals can be used to identify exchangeablehydroxyl protons [32]. A comparison of the 13C CP-MAS NMR spectra of RCBs and D2O-suspended RCBsrevealed that the C6 signal of the suspended RCBsshifted partly toward a higher field shift of 1 ppm (at61.8 ppm) (Supporting Information Figure S3). Thissuggests that water molecules can be reached easily atthe cell walls and that they dynamically interact withpolysaccharides via hydrogen bonds. Cellulose canbecome an attractive source of cellulose nanofibersoxidized by water-soluble 2,2,6,6-tetramethylpiperi-dine-1-oxyl (TEMPO), which is a promising new func-tional material [33]. The production of cellulosenanofibers from coffee bean will be our next focus.

Conclusions

Thick cell walls in the GCBs and RCBs were observedto form honeycomb structures even after grinding orbrewing, and lipids were contained in pores. 1H and13C solid-state NMR measurements were performed

MONITORING NMR PEAKS OF TRIACYLGLYCEROL INSIDE COFFEE BEANS 807

to characterize the lipids and polysaccharides in cof-fee beans that had not been processed, those that hadbeen roasted, those that were left over from coffeebrewing, and those that had undergone lipid extrac-tion. The structure of lipids in coffee beans wasdetermined by NMR analysis. TAG contains oleicand linoleic fatty acids, and they were successfullyisolated in liquid form via delipidization process.The structures of major polysaccharides, such asgalactomannan, arabinogalactan and cellulose, in thecoffee beans were characterized by 13C solid-stateNMR. These characterizations will help advanceresearch on generating biodiesel fuel from the TAG.

Author Contributions

N.K. and I.K. designed the study; N.K. prepared samples;N.Y. performed SEM study. N.K. and I.K. performed solid-state NMR characterization; N.K. and I.K. wrote the paper.All authors discussed the results of the study.

Acknowledgments

This work was financially supported by ROUTE (ResearchOpportunity for UndergraduaTEs) program from YokohamaNational University to N. K. and Yokohama AcademicFoundation (674) to I. K. The authors thank Prof. KazuhikoNishitani at Tohoku University for his helpful advice aboutthe SEM observation of the cell walls. The authors also thankMr. Shinji Ishihara at Instrumental Analysis Center ofYokohama National University for his technical assistance

with the optimization of NMR spectrometer and probe per-formance. The authors wish to thank Prof. Akira Naito atYokohama National University for valuable discussions.

Disclosure statement

No potential conflict of interest was reported by theauthors.

Funding

This work was supported by the ROUTE (Research Opp-ortunity for UndergraduaTEs) program from YokohamaNational University; Yokohama Academic Foundation [674].

ORCID

Noriko Kanai http://orcid.org/0000-0002-3159-0993Izuru Kawamura http://orcid.org/0000-0002-8163-9695

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