氨基酸与口感的文章

uc g f Te cienc hine echn Available online 11 December 2011 ac g bo in the fermentation mycelia were higher than those in the fruiting bodies. By the addition of soybean flour in the fermentation media, the flavour 50-nucleotides content in the fermentation mycelia was sig- , are a bioactive metabolite production. Furthermore, the chemical com- positions of fermentation system, i.e., volatile organic compounds (Li, Wang, & Tang, 2011), androstenol (Wang, Li, Li, & Tang, 2008), nucleosides and nucleobases (Liu, Li, Li, Wan, & Tang, 2011), and fatty acids (Tang, Li, Li, Wan, & Tang, 2011) were also investigated, and the Tuber fermentation system and fruiting 5 -inosine monophosphate (5 -IMP), 5 -guanosine monophosphate (50-GMP), and 50-xanthosine monophosphate (50-XMP), are com- monly detected in mushrooms (Mau, 2005). 50-AMP, 50-IMP, 50-GMP, and 50-XMP, which also give the umami or palatable taste, were considered to be umami 50-nucleotides (Yamaguchi, Yoshika- wa, Ikeda, & Ninomiya, 1971). Umami taste, also called the palatable taste or the perception of satisfaction, is related to anoverall flavourperception inducedor en- hanced by MSG, and considered to be the predominant flavour of mushrooms (Yamaguchi, 1979). Recently, researchers found the specific taste cell receptor on the tongue (mGluR4) for umamiwhich ⇑ Corresponding author at: Key Laboratory of Fermentation Engineering, Ministry of Education, Hubei University of Technology, Wuhan 430068, China. Tel./fax: +86 27 88015108. Food Chemistry 132 (2012) 1413–1419 Contents lists available at Food Che lse E-mail address: yajietang@hotmail.com (Y.-J. Tang). establishes an ectomycorrhizal symbiosis with trees and shrubs. Due to its characteristic aroma and delicious taste, truffles are the precious and expensive delicacies that are widely used in the famous French and Italian cuisines. Because of the decrease in the natural production of truffles combined with the increase in worldwide demand, a new way to produce truffles on a large scale is urgently needed. By taking the Chinese truffle Tuber sinense as a typical example, our group have developed a novel submerged fer- mentation process for the production of mycelia and its bioactive metabolites for the first time (Tang, Zhu, Li, Mi, & Li, 2008). This process is considered a potential alternative resource for truffles, and it may also be helpful for other mushroom fermentations for et al., 2011). The water-soluble taste components, such as free amino acids and 50-nucleotides, make important contributions to the typical mushroom flavour (Litchfield, 1967). The free amino acids impart the food taste with a smooth feeling, thereby soften a sharp taste from some substances. Therefore, the combination of free amino acids always gives rise to a unique natural flavour (Mau, 2005). In addition, monosodium glutamate (MSG)-like amino acids (i.e., aspartic acid andglutamic acid), also calledumami amino acids, give the most typical mushroom taste (Yamaguchi, 1979). The 50-nucle- otides, including 50-cytosine monophosphate (50-CMP), 50-uridine monophosphate (50-UMP), 50-adenosine monophosphate (50-AMP), 0 0 0 Keywords: Truffle Fruiting bodies Fermentation mycelia Umami Equivalent umami concentration Soybean flour 1. Introduction Truffles, belonging to Tuber genus 0308-8146/$ - see front matter � 2011 Elsevier Ltd. A doi:10.1016/j.foodchem.2011.11.130 nificantly increased, and the equivalent umami concentration of the fermentation mycelia (i.e., 608.07 g/ 100 g) was approximately 38.1–93.4 times higher than those of the fruiting bodies. From the viewpoint of umami taste, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting bodies. � 2011 Elsevier Ltd. All rights reserved. hypogeous fungus that bodies were confirmed to be partly similar in their volatile organic compounds (Li et al., 2011), androstenol (Wang et al., 2008), nucleosides and nucleobases (Liu et al., 2011), and fatty acids (Tang Received in revised form 8 November 2011 Accepted 29 November 2011 in the Tuber fermentation mycelia and natural fruiting bodies. Not only the total contents of the free amino acids and 50-nucleotides, but also the contents of umami amino acids and flavour 50-nucleotides Comparison of free amino acids and 50-n fermentation mycelia and natural fruitin Ping Liu a, Hong-Mei Li a, Ya-Jie Tang a,b,c,d,⇑ aKey Laboratory of Fermentation Engineering, Ministry of Education, Hubei University o b Lab of Synthetic Biology, Shanghai Advanced Research Institute, Chinese Academy of S cNational Key Laboratory of Biochemical Engineering, Institute of Process Engineering, C d State Key Laboratory of Bioreactor Engineering, East China University of Science and T a r t i c l e i n f o Article history: Received 9 June 2011 a b s t r a c t The profiles of free amino mycelia and natural fruitin journal homepage: www.e ll rights reserved. leotides between Tuber bodies chnology, Wuhan 430068, China es, Shanghai 201203, China se Academy of Sciences, Beijing 100080, China ology, 130 Meilong Road, Shanghai 200237, China ids and 50-nucleotides were first compared between Tuber fermentation dies. A total of 20 free amino acids and five 50-nucleotides were identified SciVerse ScienceDirect mistry vier .com/locate / foodchem (Chaudhari, Landin, & Roper, 2000). In particular, the umami taste 2.1. Tuber fruiting bodies collection and fermentation mycelia culture stry The fruiting bodies of T. sinense, Tuber aestivum, Tuber indicum, Tuber himalayense, and Tuber borchii var. sphaerospermum were purchased from the Kunming Rare Truffle Co. Ltd. (Yunan province, China), and the Tuber fruiting bodies were collected by Mr. Jian- Ming Wu, who is a very experienced wild edible truffle expert. After harvest, the truffle fruiting bodies were immediately stored in a refrigerator at �20 �C. After freeze-drying, the dried fruiting bodies were pulverised and then passed through a 250-lm stain- less sieve. The strains of Tuber melanosporum, T. sinense and T. indicum were provided by Mianyang Institute of Edible Fungi (Sichuan, Chi- na), and the strain of T. aestivum was provided by the Huazhong University of Agriculture (Hubei, China). Except otherwise men- tioned, the fermentation mycelia were cultured under the follow- ing basal media: 35 g/l sucrose, 5 g/l peptone, 5 g/l yeast extract, 0.5 g/l MgSO4�7H2O, 1 g/l KH2PO4, and 0.05 g/l Vitamin B1, and the details of the culture procedure has been previously described (Tang et al., 2008). In order to investigate the effect of media on the equivalent umami concentration (EUC) of fermentation mycelia, the fermentation mycelia of T. melanosporum was cultured in the following three proposed media, i.e., corn media: the addition of 5 g/l corn syrup in the basal media; soybean media: the addition of 5 g/l soybean flour in the basal media; the corn and soybean media: the addition of 5 g/l soybean flour and 5 g/l corn syrup in the basal media. All the freeze-dried samples were pulverised and then subjected to pass through a 250-lm stainless sieve. 2.2. Free amino acid assay of mushrooms can be synergistically increased by the combination of umami amino acids and umami 50-nucleotides (Yamaguchi et al., 1971). Through the equation derived from sensory evaluation (Yamaguchi et al., 1971), the equivalent umami concentration (EUC) has oftenbeencalculated tounderstand themushroomsumami-like taste characteristics (Cho, Choi, & Kim, 2010; Huang, Tsai, Lee, & Mau, 2006; Mau, Lin, Ma, & Song, 2001; Tsai, Weng, Huang, Chen, & Mau, 2006; Tsai, Wu, Huang, & Mau, 2007; Tseng, Lee, Li, & Mau, 2005). In addition, it was noteworthy that the EUC values and uma- mi sensory intensities exhibited the same patterns in pine-mush- rooms of different grades (Cho et al., 2007; Cho et al., 2010). However, to the best of our knowledge, the umami-like taste com- poundprofiles in the Tuber fruitingbodies and fermentationmycelia have never been investigated before. In this work, the profiles of free amino acids and 50-nucleotides between the Tuber fermentation mycelia and natural fruiting bodies were compared. More precisely, this work includes the fol- lowing four parts: (1) the comparison of free amino acids between Tuber fermentation mycelia and fruiting bodies; (2) the compari- son of 50-nucleotides between Tuber fermentation mycelia and fruiting bodies; (3) the comparison of EUC values between Tuber fermentation mycelia and fruiting bodies; and (4) the investigation on the effect of fermentation media on the EUC value of Tuber fer- mentation mycelia. This work will serve as a useful database for the nutritional or nutraceutical evaluation of both Tuber fruiting bodies and fermentation mycelia. Furthermore, it may guide Tuber submerged fermentation process. 2. Materials and methods proves that umami is a basic taste rather than a feeling factor 1414 P. Liu et al. / Food Chemi Free amino acids were extracted and analysed as the method described by Mau, Chyau, Li, and Tseng (1997). Freeze-dried powder (500 mg) was shaken with 50 ml of 0.1 N HCl (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 45 min at ambient temperature and filtered through filter paper. The filtrate was then filtrated by using a 0.22 lm filter prior to analysis. The analysis of free amino acids was conducted with a Hitachi Model 835-50 high speed amino acid analyser (Hitachi, Tokyo, Japan). 2.3. 50-Nucleotide assay 50-Nucleotides were extracted and analysed as the method described by Taylor, Hershey, Levine, Coy, and Olivelle (1981). Freeze-dried powder (500 mg) was extracted with 25 ml of deion- ised water. This suspension was heated to boiling for 1 min, cooled, and then centrifuged at 11,000�g for 30 min. The extraction was repeated twice. The combined supernatants were then evaporated and redissolved in deionised water to a final volume of 10 ml. The supernatant was filtrated using a 0.22 lm filter prior to HPLC analysis. The analysis of 50-nucleotides were preformed on Dionex Ulti- mate 3000 system (Dionex, USA), equipped with an on-line degas- ser, two solvent delivery pumps, and a diode array detector. The column used for separation was Synergi Hydro RP-18 column (250 � 4.6 mm, 4 lm, Phenomenex) fitted with a C18 guard col- umn (Phenomenex). The optimised mobile phase was 4 mM KH2PO4 aqueous solution, whose pH was adjusted to 2.0 by H3PO4. The column oven temperature was maintained at 35 �C and the flow rate at 1.0 ml min�1. The detection wave was fixed at 254 nm. Each 50-nucleotide was identified by matching its reten- tion time with that of an authentic standard (Sigma) in the HPLC chromatogram and quantified by the calibration curve of the authentic compound. 2.4. Equivalent umami concentration (EUC) The equivalent umami concentration [EUC, g monosodium glu- tamate (MSG)/100 g] is the concentration of MSG equivalent to the umami intensity given by the mixture of umami amino acids and umami 50-nucleotides and is represented by the following addition equation (Yamaguchi et al., 1971): Y ¼ X aibi þ 1218 X aibi � � X ajbj � � where Y is the EUC of the mixture in terms of g MSG/100 g; ai is the concentration (g/100 g) of each umami amino acid [aspartic acid (Asp) or glutamic acid (Glu)]; aj is the concentration (g/100 g) of each umami50-nucleotide [50-inosinemonophosphate (50-IMP), 50-guano- sine monophosphate (50-GMP), 50-xanthosine monophosphate (50- XMP) or 50-adenosine monophosphate (50-AMP)]; bi is the relative umami concentration (RUC) for each umami amino acid to MSG (Glu, 1; and Asp, 0.077); bj is the RUC for each umami 50-nucleotide to 50-IMP (50-IMP, 1; 50-GMP, 2.3; 50-XMP, 0.61; and 50-AMP, 0.18); and 1218 is a synergistic constant based on the concentration of g/ 100 g used. 2.5. Statistical analysis The statistical data were processed and a one-way ANOVA was performed using the SPSS 16.0 software (Chicago, USA). To evalu- ate the difference of free amino acids and 50-nucleotides contents in the Tuber samples, a post hoc analysis was performed using Tukey’s test. Differences were considered significant when p < 0.05. Hierarchical Cluster Analysis (HCA) is a multivariate anal- 132 (2012) 1413–1419 ysis technique, which is used to sort samples into groups. In our study, the results were confirmed by Hierarchical Cluster Analysis (HCA), and the Between-groups linkage cluster method, the Jacquotte, Costa, & Chen, 2010); taurine has recently become a popular supplement and ingredient in functional drinks (e.g., Red stry Bull, Monster, and Rockstar). The taurine content in the fruiting bodies of T. sinense and T. indicum were at a trace levels, while Squared Euclidean distance measure and Z-scores standardisation were used to establish clusters. 3. Results and discussion 3.1. Comparison of free amino acids between Tuber fermentation mycelia and fruiting bodies Table 1 shows that a total of 20 free amino acids were identified from all the Tuber fruiting bodies and fermentation mycelia, and in which seven free amino acids (i.e., threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine) were essential ami- no acids. The total amount of free amino acids in the fruiting bodies ranged from 21.96 to 27.97 mg/g, whereas it ranged from 64.61 to 69.65 mg/g in the fermentation mycelia. The total amount of essential amino acids was 4.61–13.57 and 21.58–23.89 mg/g in the fruiting bodies and fermentation mycelia, respectively, which comprised 21–49% and 33–35% of total amino acids. Obviously, the contents of total and essential amino acids in the fermentation mycelia were approximately 2.3–3.2 and 1.6–5.2 times higher than those in the fruiting bodies, respectively. Among the five Tuber fruiting bodies, the profiles of free amino acids were different, so it was difficult to find the common major amino acids in the fruiting bodies. Typically, the amino acids with the concentration of more than 2 mg/g were alanine, valine, and c- aminobutyric acid (GABA) for the fruiting bodies of T. sinense, thre- onine, glutamic acid, and alanine for the fruiting bodies of T. indi- cum, threonine, glutamic acid, alanine, and valine for the fruiting bodies of T. aestivum, and glutamic acid, alanine, and GABA for the fruiting bodies of T. himalayense and T. borchii var. In addition, the fruiting bodies of T. sinensewere much different from the other four fruiting bodies. For example, the contents of glutamic acid and valine were 0.94 and 9.59 mg/g in the fruiting bodies of T. sinense, respectively, and occupied approximately 3% and 34% of total ami- no acids, whereas they occupied approximately 12–22% and 6–11% in the other four fruiting bodies. While, Table 1 indicates that the profiles of free amino acids in the fermentation mycelia were sim- ilar with each other. Briefly, the main free amino acids were thre- onine, glutamic acid, alanine, lysine and arginine, which occupied 55–64% of total amino acids in the fermentation mycelia. The con- tents of taurine, glycine, methionine, isoleucine, leucine, tyrosine, and ornithine were all lower than 1 mg/g, which only occupied approximately 6% of total amino acids in the fermentation mycelia. In particular, methionine is considered to be an essential pre- cursor of sulphur-containing volatile organic compounds (VOCs), which are major components for the truffle aroma (Zeppa et al., 2010). Except for T. indicum fruiting bodies, the content of methio- nine in all the Tuber fruiting bodies and fermentation mycelia was at a trace level. The content of methionine in the T. indicum fruiting bodies (i.e., 0.02 mg/g) was lower than that of the fruiting body of T. melanosporum Vitt. at the maturation stages of IV (i.e., 0.03 mg/ g), V (i.e., 0.07 mg/g), and VI (i.e., 0.08 mg/g) (Harki, Bouya, & Dargent, 2006). In addition, taurine, which is also a sulfur-contain- ing amino acid, was detected in all the Tuber samples. Evidence from the studies of mechanism and animal shows that the main biological activities of taurine include its ability to conjugate bile acids, to regulate blood pressure (BP), and to act as a potent anti- oxidant and anti-inflammatory agent (Wójcik, Koenig, Zeleniuch- P. Liu et al. / Food Chemi the taurine content in the other fruiting bodies ranged from 0.10 to 0.24 mg/g, which were similar to the chicken light meat (i.e., 0.15–0.18 mg/g), turkey light meat (i.e., 0.11–0.30 mg/g), and small shrimp (i.e., 0.11 mg/g) (Wójcik et al., 2010). On the other hand, the content of taurine ranged from 0.54 to 0.91 mg/g in the fermenta- tion mycelia, which was approximately 2.3–9.1 times higher than those in the fruiting bodies, and the values were similar to the loin of pork (i.e., 0.50–0.61 mg/g), raw chicken dark meat (i.e., 0.83 mg/ g), and beef (i.e., 0.38–0.46 mg/g) (Wójcik et al., 2010). General speaking, people often take most of taurine from foods, and the Tu- ber samples could be a taurine resource for the formulation of healthy foods. Moreover, a hypotensive agent of c-aminobutyric acid (GABA) (Kohama et al., 1987; Kushiro et al., 1996) was also found in all the Tuber fruiting bodies and fermentation mycelia. The GABA content in the fruiting bodies ranged from 0.90 to 2.82 mg/g, which was higher than the mushroom fruiting bodies of Agaricus blazei (i.e., 0.36 mg/g), Agrocybe cylindracea (i.e., 0.21 mg/g), and Boletus edulis (i.e., 0.11 mg/g) (Tsai, Tsai, & Mau, 2008), and several cultivated mushroom fruiting bodies of Clitocybe maxima (i.e., 0.42–0.45 mg/g), Pleurotus ferulae (i.e., 0.31 mg/g) and Pleurotus ostreatus (i.e., 0.25 mg/g) (Tsai et al., 2009), but lower than the mushroom fruiting bodies of Agaricus bisporus at the mat- uration stages of 1 (i.e., 4.85 mg/g), 2 (i.e., 5.79 mg/g) and 3 (i.e., 3.04 mg/g) (Tsai et al., 2007). The GABA content in the fermenta- tion mycelia ranged from 3.94 to 5.89 mg/g, which was about 1.4–6.5 times higher than those of the fruiting bodies. In addition, the GABA content in T. melanosporum fermentation mycelia (i.e., 5.89 mg/g) was similar to the aforementioned highest content of mushroom A. bisporus fruiting bodies at the maturation stages of 2 (i.e., 5.79 mg/g). Besides their palatable taste, the presence of these amino acids in Tuber samples was beneficial to people’s health because taurine and GABA are bioactive compounds. Chen (1986) conducted a series of sensory evaluations on syn- thetic mushroom extracts, prepared by the omission and addition of soluble components, found that the monosodium glutamate (MSG)-like components (i.e., aspartic and glutamic acids) were ma- jor taste-active amino acids in common mushroom. Because they give the most typical mushroom taste, MSG-like components Asp and Glu are also called umami amino acids (Mau, 2005). With regard to the contents of umami amino acids in Tuber samples, Ta- ble 1 indicates that the total contents of umami amino acids in the fruiting bodies ranged from 1.50 to 7.06 mg/g, which were similar to the fruiting bodies of T. melanosporum Vitt. at the maturation stages of IV (i.e., 2.76 mg/g), V (i.e., 6.31 mg/g), and VI (i.e., 1.35 mg/g) (Harki et al., 2006). The total contents of umami amino acids in the fermentation mycelia ranged from 8.23 to 12.58 mg/g, which was approximately 1.2–8.4 times higher than those of the fruiting bodies. Yang, Lin, and Mau (2001) divided the contents of MSG-like components into three ranges: low (<5 mg/g), middle (5–20 mg/g) and high ranges (>20 mg/g). Based on the above crite- rion, the contents of umami amino acids in both the fermentation mycelia and the fruiting bodies of T. indicum and T. aestivum were at the middle range, and the other three fruiting bodies of T. sin- ense, T. himalayense and T. borchii var. were at the low range. The Hierarchical Clustering Analysis (HCA) was used to intui- tively describe the relationship of Tuber samples on the amino acid profiles. As shown in HCA dendrogram (Fig. 1a), the fruiting bodies can be divided int