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Characteristic Metabolic Changes of the Crust from Dry-Aged Beef Using 2D NMR Spectroscopy

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Characteristic Metabolic Changes of the Crust from Dry-Aged Beef Using 2D NMR Spectroscopy molecules Article Characteristic Metabolic Changes of the Crust from Dry-Aged Beef Using 2D NMR Spectroscopy Hyun Cheol Kim 1 , Ki Ho Baek 1 , Yoon-Joo Ko 2, Hyun Jung Lee 1 , Dong-Gyun Yim 1,* and Cheorun Jo 1,3,* 1 Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Korea; [email protected] (H.C.K.); [email protected] (K.H.B.); [email protected] (H.J.L.) 2 National Center for Inter-University Research Facilities, Seoul National University, Seoul 08826, Korea; [email protected] 3 Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea * Correspondence: [email protected] (D.-G.Y.); [email protected] (C.J.); Tel.: +82-2-880-4820 (D.-G.Y.); Tel.: +82-2-880-4804 (C.J.) Academic Editor: Míriam Pérez Trujillo Received: 9 June 2020; Accepted: 2 July 2020; Published: 7 July 2020 Abstract: Two-dimensional quantitative nuclear magnetic resonance (2D qNMR)-based metabolomics was performed to understand characteristic metabolic profiles in different aging regimes (crust from dry-aged beef, inner edible flesh of dry-aged beef, and wet-aged beef striploin) over 4 weeks. Samples were extracted using 0.6 M perchlorate to acquire polar metabolites. Partial least squares-discriminant analysis showed a good cumulative explained variation (R2 = 0.967) and predictive ability (Q2 = 0.935). Metabolites of crust and aged beef (dry- and wet-aged beef) were separated in the first week and showed a completely different aspect in the second week via NMR-based multivariable analyses. Moreover, NMR-based multivariable analyses could be used to distinguish the method, degree, and doneness of beef aging. Among them, the crust showed more unique metabolic changes that accelerated proteolysis (total free amino acids and biogenic amines) and inosine 50-monophosphate depletion than dry-aged beef and generated specific microbial catabolites (3-indoxyl sulfate) and γ-aminobutyric acid (GABA), while asparagine, glutamine, tryptophan, and glucose in the crust were maintained or decreased. Compared to the crust, dry-aged beef showed similar patterns of biogenic amines, as well as bioactive compounds and GABA, without a decrease in free amino acids and glucose. Based on these results, the crust allows the inner dry-aged beef to be aged similarly to wet-aged beef without microbial effects. Thus, 2D qNMR-based metabolomic techniques could provide complementary information about biochemical factors for beef aging. Keywords: 2D qNMR; wet aging; dry aging; crust; metabolomics; 1H-13C HSQC 1. Introduction Meat aging is a series of biological and physiochemical transitions from muscle to meat after slaughter that causes proteolysis and enhances meat tenderness and palatability [1,2]. These changes could be used to improve meat quality under controlled environments, such as temperature, relative humidity, aging period, and the aging method by packaging conditions [3]. The major aging methods are divided into dry and wet aging. Meat for wet aging is vacuum-packed, while meat for dry aging is exposed to air without packaging [4]. Dry-aged meat is known to generate concentrated flavors (beefy and roasted) after aging, while wet-aged meat is known to induce sour and bloody/metallic flavors [4–6]. Molecules 2020, 25, 3087; doi:10.3390/molecules25133087 www.mdpi.com/journal/molecules Molecules 2020, 25, 3087 2 of 14 Dry-aged meat is commonly produced in aerobic conditions, and its surface is dried rapidly [7]. The dried meat surface is called the crust. Significant water loss occurs in both the crust and internal meat during dry aging [3]. In general, the crust is trimmed-off and considered inedible because of not only hardness and dryness but a high concentration of microorganisms, such as mold and yeast [4,8,9]. Trimming and water losses of dry-aged meat are directly related to economic loss, even if it can create value-added meat after aging [10]. Lee et al. [3]. suggested that the microorganisms on the surface of dry-aged beef might be the primary reason for the differences between wet- and dry-aged beef, and the composition of the microorganisms may have a pivotal role in the production of characteristic flavors and textures [8]. Therefore, it could be hypothesized that these environmental differences may affect the metabolic changes of the crust and edible portion of dry-aged beef, followed by characteristic sensory properties. For a quality assessment of beef products, chemometric analyses are required to measure various metabolites, such as free amino acids, peptides, nucleotides, sugars, lipids, and aromatic compounds [11]. Among various chemometric analyses, nuclear magnetic resonance (NMR) spectroscopy and/or chromatography-based mass spectroscopy have been widely used for elucidating metabolic profiles from various biological samples together with multivariable analyses [12]. Between them, NMR can provide intact chemical information without derivatization [13]. Several NMR studies combined with multivariable analyses have been reported on the metabolic profiles of during beef aging [14,15]. However, to our knowledge, there are no studies reported about metabolic changes in the crust of beef during the aging period. Therefore, a two-dimensional quantitative NMR (2D qNMR) analysis was performed to understand characteristic metabolic changes in the crust, dry-aged, and wet-aged beef striploin after aging for 4 weeks. 2. Results and Discussion The differences among dry- and wet-aged beef and the crust over 4 weeks of aging were investigated using 2D NMR-based metabolomics. For the comparison of polar metabolites in meat samples, perchloric acid extraction was performed [13]. Based on the 2D NMR spectra, metabolites were qualified and quantified for multivariable analyses. 2.1. Multivariable Analyses Overall metabolic differences were determined using principal components analysis (PCA) based on the peak intensities from 1H-13C hetero single nuclear coherence (HSQC) NMR spectra (Figure1). PCA is usually used to discriminate differences of samples composed of multivariable datasets via variance maximization and dimension reduction [16]. As seen in Figure1, each meat sample was separated clearly by the aging period, along with PC1 (54.5%). Among all groups, the crust was more strongly correlated with PC1 than the others (wet- and dry-aged beef). However, the crust was not correlated with PC2 (12.5%) during the aging period, while dry- and wet-aged beef correlated with PC2 depending on the aging period. Dry- and wet-aged beef showed a similar appearance in the PCA. However, dry-aged beef was better-differentiated by PC1 than wet-aged beef. Wet-aged beef was hard to be distinguished between 21 and 28 days. Along with the PC2, dry- and wet-aged beef exhibited a linear correlation until 21 days, and then the correlation decreased by 28 days of the aging period. MoleculesMolecules 20202020, ,2525, ,x 3087 FOR PEER REVIEW 33 of of 14 14 Figure 1. Principal components analysis (PCA) biplot (score plots and loadings of variable) from Figurequantified 1. Principal metabolites components of beef aged analysis by diff (PCA)erent methods biplot (score and crust. plots GABA, and loadingsγ-aminobutyric of variable) acid; from TMA, quantified metabolites of beef aged by different methods and crust. GABA, γ-aminobutyric acid; trimethylamine; IMP, inosine 50-monophosphate. TMA, trimethylamine; IMP, inosine 5′-monophosphate. The loading plots of variables, which visualize variables, can be used for understanding metabolic patternsThe easilyloading [17 ].plots Most of of variables the metabolites, which were visualize associated variables with PC1, can and be were used composed for understanding of most free metabolicamino acids, patterns biogenic easily amines, [17]. Most and bioactive of the metabolites compounds, were except associated for some with free PC1 amino and acidswere (asparagine,composed of most free amino acids, biogenic amines, and bioactive compounds, except for some free amino tryptophan, and glutamine), inosine 50-monophosphate (IMP), α- and β-glucose, and glutathione acids(Figure (asparagine,1). The proportional tryptophan, increase and glutamine) in free amino, inosine acids 5′ and-monophosphate their derivatives (IMP), (biogenic α- and amines β-glucose, and andbioactive glutathione compounds), (Figure depending 1). The proportional on the aging increase period, was in free a well-known amino acids result and of thetheir aged derivatives meat [18 ]. (biogenicBiogenic amines and such bioactive as cadaverine, compounds) putrescine,, depending tyramine, on andthe trimethylamineaging period, was (TMA) a well commonly-known resultincreased of the depending aged meat on the [18] aging. Biogenic period, amines and these such compounds as cadaverine, are usually putrescine, considered tyramine, indicators and of trimethylaminefreshness in meat (TMA) samples commonly [19,20]. increased Moreover, depending not only IMPon the and agingα- and periodβ-glucose, and these are recognized compounds as arefreshness usually indicators,considered but indicator also theses of freshness metabolites in playmeat ansamples important [19,20] role. Moreover, in meat flavor not only development IMP and αduring- and β- theglucose cooking are recognized process [3, 21as]. freshness In addition indicators, to PCA, but partial also these least metabolites squares-discriminant play an important analysis role(PLS-DA) in meat was flavor performed development
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