Construction of a free-form amino acid database for vegetables and mushrooms

Free-form amino acids were quantitatively measured in over 30 species of vegetables and mushrooms. Significant amounts of free-form amino acids were found in each species analyzed. Additionally, there appear to be no rules or patterns of free-form amino acid distribution among species. Highly functional free-form amino acids, like γ-aminobutyric acid, are found in significant levels and suggest that amino acid analysis could play an informative role in nutrition. Our analysis should provide valuable data for the establishment of nutritional databases that include free-form amino acids and more accurately represent the entire amino acid profile of foods.


Introduction
Amino acids play significant biological roles in human. The amino acids required for human metabolism arise from the digestion and utilization of amino acids from food, and from free-form amino acids within food components (For a list of amino acid abbreviations used in this study: Table 1). Free-form amino acids may be utilized as nutrients and/or act as messengers that target specific receptors.
Free-form amino acids exist in foods derived from plants and animals. β-alanine (β-Ala), citrulline (Cit), γ-aminobutyric acid (GABA), and ornithine (Orn) usually exist as free-form amino acids and perform various biological functions including coenzyme components, control of osmoregulation, ammonia detoxification, and as neurotransmitters [1,2]. In human, tryptophan (Trp), a precursor of serotonin and melatonin, acts to regulate circadian rhythm [3]. Some free-form amino acids act as neurotransmitters: in the central nervous system GABA and glycine (Gly) function as inhibitory signals while glutamic acid (Glu) is an excitatory signal [4][5][6][7][8] and may have similar functions in non-neuronal cells [5]. The roles of free-form amino acids in dietary supplements have been extensively explored and dietary branched chain amino acids (BCAA; including valine (Val), leucine (Leu), and isoleucine (Ile)), improve exercise performance, endurance performance, and upper body power in human [9]. Moreover, GABA can reduce high blood pressure [10], and in Japan is authorized as "Tokuho", which is a "food for specified health uses", and other health foods containing GABA are widely available.
Glu receptors have been identified on taste buds and the gastrointestinal tract, including stomach cells [11], which suggests that free-form Glu may be involved in enhancing digestion, nutrient absorption, and/or utilization of nutrients [12]. The study was extended to examine the use of daily dietary Glu supplements in the meals of elderly people in hospital [12]. The results of this analysis showed that Glu supplementation improved the nutritional status of elderly and nutritionally deficient patients through improved food intake [12][13][14]. Therefore, free-form Glu is a highly effective supplement with the potential to improve quality of life (QOL) [12,15,16]. It is likely that information about the amino acid composition of both hydrolyzed samples and free-form amino acids in the original food samples are necessary for analyses of palatability and the beneficial functions of amino acids. The Japan Society of Nutrition and Food Science website lists some information about free-form amino acids in food, but the data presented was collated from literature published about 30 years ago [23][24][25][26]. Therefore, the amino acid contents listed in some of these databases are likely to be misleading or incomplete.
Amino acid analysis has advanced over the last few decades, and the time has come to renew these data using modern techniques that provide increased sensitivity and reliability. Currently, only a few databases specializing in free-form amino acids are available. To understand the amount of free-form amino acids in our daily foods, free-form amino acids must be "extracted" and then measured. Previously, we reported initial attempts to establish a free-form amino acid database for approximately 140 species of fruits and vegetables [27]. We showed that distribution of free-form amino acids is characteristic for each sample and significantly different from the amino acid distribution recorded in acid hydrolyzed databases. Here, we have extended our analysis of free-form amino acid contents to include additional vegetables and some mushroom species.

Extraction of free amino acids from foods
We examined 38 species of vegetables and mushrooms (Table  2). Some samples were purchased from local supermarkets in Shiga Prefecture and others were supplied by local farmers. The food samples came from various geographic regions. All food samples were washed in water and the edible and inedible parts, including peels, seeds, and skins, were separated. Samples were stored at -25°C until use.

Sample preparation
Samples (5-10 g) were ground to a fine powder with a mortar and pestle. The powdered samples were added to 100 mM HEPES-Na buffer, pH 7.0, and homogenized on ice with a Tissue-Tearor (Biospec). The homogenate was centrifuged at 10,000×g for 10 min and the supernatant was collected. Suitable supernatant aliquots were treated with 60% perchloric acid to remove proteins. The protein content of samples was estimated by Bradford protein assays (BioRad). Ultrafiltration was performed using CENTRICON ® 10 (Millipore) when further removal of protein contamination was necessary. After ultrafiltration, the protein content was measured by Bradford protein assay.

Amino acid standards
GABA, hydroxyproline (HYP), L-Gln, L-Asn, β-Ala, L-cysteine (Cys), and L-Trp were purchased from Wako Chemicals. Purchased amino acids were added to type H amino acid mixture standard solution (Wako Chemicals), to produce a working standard solution of each amino acid (100 µmol/L in 0.1 N HCl).

Amino acid analysis
Free-form amino acids extracted from food samples were analyzed on a Hitachi Ultra High Speed Liquid Chromatography system

Pleurotus eryngii
King oyster mushroom Linearity of the dose response was evaluated using an amino acid standard solution within the range of 10 to 60 pmol. Minimum and maximum correlation coefficients (r 2 ) for Asn, as an example, were 0.974 and 0.997, respectively. Ten consecutive standard solution injections were analyzed to evaluate reproducibility ( Table 3). The relative standard deviation of peak area for Tyr and His was in the range of 0.3% and 5.1%, respectively. The mean limit of detection was 0.8 pmol.

Results
Chromatograms for the amino acid mixture standard and eggplant "kamonasu" (Solanum melongena) were obtained and analyzed ( Figures  1 and 2). In our system, the typical time to baseline NBD-amino acid separation, with the exception of Pro and β-Ala, was about 8 min.
The free-form amino acid contents of the vegetables and mushrooms analyzed varied considerably (Tables 4 and 5). The total free-form amino acids found in the extracts ranged from 59.91 (Garden rhubarb) to 4,093 and 424.6 (Jew's ear) to 12,773 µmol per 100 g of vegetables and mushrooms, respectively. Of the vegetables and mushrooms analyzed, the podded pea (4,093 µmol per 100 g) and dried porcini mushrooms (12,773 µmol per 100 g) had the highest total freeform amino acids of vegetables and mushrooms, respectively. The most abundant free-form amino acids found in the vegetables examined were Asn, Gln, Glu, and GABA, all of which are important metabolites in human. In mushrooms, arginine (Arg), Glu, Gln, and Ala were the most abundant free-form amino acids. Orn was found in mushrooms, but rarely in vegetables and HYP, Cys, and methionine (Met) were found at low levels in both vegetables and mushrooms.
The free-form amino acid compositions of five different kinds of eggplant were analyzed. Slight differences were observed in the freeform amino acid contents of the five species, but Asn, Gln, and GABA were abundant in all.
BCAAs, common ingredients in drinks designed for athletes, were not detected in most foods analyzed. However, moso bamboo and some peas, including the podded pea, contained considerable amounts of Val, Leu, and Ile BCAAs.

Discussion
Here, we identified and measured significant levels of free-form amino acids in both vegetables and mushrooms. These free-form amino acids should contribute to the taste and palatability of foods. Indeed, Glu provides umami taste and has been shown to improve appetite [12,14,15], both of which contribute to QOL. Glu is particularly important in the foods of elderly and sick people, as it improves QOL by increasing nutrient intake. It is often discussed that excess intake of amino acids is toxic for human. For example, it has been warned that Glu has a potential risk of neurotoxicity like headache. However, this warning was almost eliminated after the joint report of FAO, WHO, and the Expert Committee on Food Additives (JECFA) in 2000 [28]. Thus, Glu is considered relatively safe and could be consumed without any concerns.
GABA is known for health-promoting functions, including an antihypotensive effect. Some studies suggest that supplementation with 10~20 mg (equivalent to 97~194 µmol) GABA reduces blood pressure in human [29,30]. Commercially available GABA enriched "Tokuho" foods are typically supplemented with 20 mg of GABA in Japan. Our present study suggests that two pieces of eggplant "kamonasu", equivalent to 90 g, per day provides enough GABA to maintain blood pressure at a normal level.
Free-form amino acids are significantly beneficial to human health. The presence of these amino acids in vegetables and mushrooms suggests that they also provide a benefit to those species. In plants, free-form amino acids are likely absorbed directly from soil via the roots [31]. Some of these free-form amino acids are metabolized or stored, and some are utilized as signaling molecules to regulate cellular functions including enzyme activity, gene expression, and redoxhomeostasis [32]. Pro, Arg, Met, Glu, and GABA are involved in the regulation of plant responses to various stress conditions [33][34][35]. Asn, the most abundant free-form amino acid in the foods analyzed in our study, is accumulated under stress conditions or mineral deficiencies [36]. In plants, free-form Asn also plays a central role in nitrogen storage and transport [36,37]. The roles of free-form amino acids in mushrooms are not well understood and are still being determined.
Here, we have demonstrated the presence of free-form amino acids in plants. However, there are many factors that influence     Mushroom "Honshimeji" Mushroom "Maitake" Mushroom "Shiitake" Porcini mushrooms (Dried) Winter mushroom Table 5. Contents of free-form amino acids assayed in mushrooms (µmol/100 g sample on a wet weight basis)