High Pressure Induced Effects on Free Amino Acid Generation in Mung Bean Sprouts during Preservation after Trigger Pressurization

Abstract

The aim of this study was to investigate the potential for improving the quality of mung bean sprouts by using high hydrostatic pressure (HHP). Mung bean sprouts were subjected to HHP treatments of 200, 400, or 600 MPa at 25 °C for 10 min. Treated and untreated samples were preserved for 0–8 days at 4 °C, and then analyzed to determine changes in amino acid concentration. The total amino acid concentration of samples treated at 200 and 400 MPa increased linearly during the preservation period. The amino acid concentration of treated samples was 2–3 times higher than that of untreated samples after 4 and 8 days of preservation. Moreover, the trend of increases in amino acid concentrations was different; thus, HHP treatment might have improved not only proteolysis, but also amino acid metabolism in mung bean sprouts. Taken together, this study demonstrates that HHP treatment is an attractive method to develop value-added foods.

Introduction

High hydrostatic pressure (HHP) is well known as an alternative method for microbial inactivation, and does not require heating (Farr, 1990; Considine et al., 2008). Products and crops treated under HHP are preserved fresh compared to those under heat treatment, as the covalent bonds of organic compounds are maintained in this process (O'Reilly et al., 2001; Montero et al., 2002). Several studies have investigated the applicability of HHP treatment for sterilization of microorganisms in foods (Smelt, 1990; Farks and Hoover, 2011; Ishikawa et al., 2016) and processing of agricultural products and fermented foods (Sasagawa et al., 2006; Ueno et al., 2009(a); (b); (c)). HHP treatment also causes damage to the cell structure and membrane of cellular biomaterials. Mass transfer of components within the cell membrane is accelerated because of cellular membrane injury induced by HHP treatment (Eshtiaghi et al., 1994). In addition, some cellular enzymes were active during HHP treatment, despite the cell membrane being destroyed. Under these conditions, unique enzymatic reactions might continue in the treated cells. In other words, high-pressure induced transformation (Hi-pit) occurs in the cells.

In recent years, Hi-pit has been used with remarkable success to develop high value-added foods (Fujii, 2006). Genome modification technology has typically been employed to develop high value-added foods, but profound ethical issues constrain our ability to apply these versatile methods to produce food. HHP treatment gives rise to alternative biochemical reactions with new or unknown biochemical pathways, so that the metabolic products contribute to the development of value-added foods. In general, enzymes become inactivated when they encounter extreme high pressure, however, certain enzymes may continue to be active below approximately 400 MPa. Thus, it is expected that HHP treatment below approximately 400 MPa will induce new or unknown biochemical reactions in biomaterials. Ueno et al. (2009) reported that the amount of quercetin increased in onions subjected to 200 MPa of high pressure; they also suggested the potential of the high-pressure procedure to induce new unidentified metabolic pathways in turnips. Shigematsu et al. (2011) confirmed that several amino acids increased selectively in brown rice subjected to 200 MPa of pressure. Although the effects of HHP treatment on harvested crops have been demonstrated by various researchers, details of this high-pressure induced transformation are not fully understood.

Sprouted vegetables cultivated heterotrophically are expected to be effective protein factories because they can be cultivated at a high density (Tamate et al., 2015). To the best of our knowledge, the first trial investigating the effects of high pressure on a sprouted vegetable was carried out by Barba et al. (2017). Although positive results on the effects of HHP treatment were demonstrated on Brussels sprouts, high-pressure induced effects on sprouted vegetables in general remain unknown.

Therefore, in the present study, we investigated the effects induced by HHP treatment on sprouted vegetables. Our research group previously reported the growth behavior of mung bean sprouts and changes in bioactivity occurring during cultivation (Tamate et al., 2015; Nakai et al., 2016; Tamate et al., 2016). Tamate et al. (2015) reported that the bioactivity of mung bean sprouts varies between 0 and 50 h in the early cultivation stage and between 50 and 100 h in the middle stage (Tamate et al., 2015). Additionally, they also showed that the bioactivity of middle stage mung bean sprouts is highest during the cultivation period. Therefore, in the present study, mung beans were cultivated at 40 and 70 h to investigate the dependence of bioactivity on the cultivation period. In particular, enzyme activity for the plant body might be the highest in the middle stage. Thus, in the present study, mung bean sprouts at different stages of cultivation were subjected to 200–600 MPa of HHP treatment, and the dynamics of metabolic products during the preservation period, in particular, of free amino acids, are discussed from an engineering viewpoint.

Materials and Methods

Sample preparation    Mung beans (Vigna radiata) harvested in 2015 in China were used to produce the sprouts in this study. The beans were soaked for 5 h in 38 °C Milli-Q water prior to transplantation. Sprouted beans were selected, and were planted on 0.8 % agar media including 10 mM CaCl₂·2H₂O in a cultivation bath. The volume of the bath, which was placed in a dark room at a controlled temperature of 25 °C, was 20 × 35 × 5.5 cm³.

High-pressure treatment    Several harvested sprouts were vacuum-sealed using the FCB-200 (FUJIIMPULSE Co. Ltd., Osaka Japan) in a sample bag of approximately 50 × 50 mm². Each sample bag was placed in the chamber (size: Φ3.0 mm×120 mm) filled with distilled water in the high hydrostatic pressure machine (Hikari Kohatsu Kiki Co. Ltd., Hiroshima Japan). Samples were subjected to 200, 400, or 600 MPa under 25 °C for 10 min. The speed of pressure increases was approximately 4.4 MPa/s for primary pressure, and 2.3 MPa/s for secondary pressure. Depressurization was performed within a few minutes by manual opening of a valve. Each sample was immediately placed in a 4 °C refrigerator, then stored for 0, 4, or 8 days.

Determination of amino acid concentration    The amino acid composition was analyzed using the dabsyl chloride method (Knecht, and Chang, 1986). One gram of sample was crushed using a micro spatula in a 15-mL centrifuge tube with 4 mL of Milli-Q water. Then, 1.8 mL of the sample solution was centrifuged at 5000 rpm at 4 °C for 15 min to separate the supernatant and the sediment. The supernatant of this solution was then centrifuged at 12500 rpm at 4 °C for 10 min to remove the redundant polymers in the solution. Approximately 100 µL of the centrifuged solution was derivatized using 500 µL of dabsyl chloride and 400 µL of 50 mM NaHCO³ in a 67 °C water bath for 10 min. This solution was immediately cooled in an ice bath, and then the amino acid concentration of the derivatized solutions was quantified using the HPLC system. The HPLC system in this study consisted of an auto sampler (AS-950), a degasser (DG980-50), a PDA detector (UV2075 Plus), an intelligent pump (PU-2080), and an oven (CO-2067, all from JASCO Co., Tokyo Japan). The temperature of the oven was maintained at 40 °C and a C18 column (UG120, Shiseido) was used in this study. Solvent A consisted of 25 mM sodium acetate/4 % DMF and Milli-Q water, and solvent B was 100 % acetonitrile. The injection volume was 20 µL and the flow rate was 1.0 mL/min. Amino acids were separated by 60 min-gradient elution from 85 % of solvent A to 40 % (60 % of solvent B) and the concentration was detected at the absorbance of 436 nm.

Results and Discussion

Free amino acid composition in mung bean sprouts at 40 and 70 h    Figure 1 shows the free amino acid concentrations in mung beans cultivated at 40 and 70 h. Aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala), proline (Pro), valine (Val), isoleucine (Ile), leucine (Leu), tryptophan (Trp), phenylalanine (Phe), lysine (Lys), histidine (His), and tyrosine (Tyr) were detected in both cultivation periods. Thr and Val were found at high concentrations of approximately 1.3–1.6 µmol/mg in each growth period. Total concentrations in sprouts cultivated at 40 and 70 h were 8.70 ± 1.82 and 8.20 ± 2.47 µmol/g, respectively. Amino acid concentrations, except for the concentration of Glu, and total concentration were similar between the two cultivation periods.

Fig. 1.

Free amino acid concentration and their summation in mung bean sprout cultivated at 40 and 70 h. Error bar indicates standard deviation.

Effect of HHP treatment on free amino acid concentration in mung bean sprouts    Changes in total amino acid concentration at 0–8 days of preservation of sprouts treated with 200, 400, and 600 MPa are shown in Figure 2 (Refer to Table S1 for the change in concentration of each amino acid during 0–8 days). For both cultivation period groups (40 and 70 h), the average concentration of total amino acids increased gradually from around 8 to 30 µmol/g during the preservation period for sprouts subjected to 200 and 400 MPa. Amino acid concentrations of both 200 and 400 MPa treatment groups were higher than those of the untreated sample after 4 days of preservation. In contrast, the amino acid concentration of the 600 MPa treatment group increased only slightly during the 0–8-day period, and its behavior was most similar to that of the untreated sample throughout the preservation period. Ueno et al. (2009) reported that partial destruction of cell membrane structures due to HHP treatment in the 200–400-MPa range induced certain enzymatic reactions. On the other hand, in the case of treatment above 400 MPa, it is expected that substantial enzyme inactivation would occur in the food sample. Therefore, in the present study, it is very likely that HHP treatment at 200 or 400 MPa improved enzyme reactions in mung bean sprouts during preservation, whereas this effect was not confirmed in the case of HHP at 600 MPa. Interestingly, in the case of the 70 h cultivation group, the total amino acid concentration reached a plateau after 6 days in the 400 MPa treatment group, but continued to increase in the 200 MPa treatment group. In other words, the reaction rate of mung bean sprouts with the highest bioactivity, i.e., the 70 h cultivation group in this study, was also improved by some specific HHP treatments. Peneus et al. (2006) showed that enzyme proteolysis was maximally enhanced by pressure levels of 200–300 MPa. Furthermore, Ueno et al. (2010) described the lack of homogeneity in the effects of high pressure in 200– 400 MPa treatments. In the present study, we assumed that the effects of 200 and 400 MPa treatments on enzyme reactions in mung bean sprouts would be similar. However, differences in the effects between high-pressure levels should be investigated in more detail to fully understand the enzyme reactions enabling addition useful characteristics in the future.

Fig. 2.

Course of total amino acid concentration in samples cultivated at (a)40 h and (b)70 h during preservation after 10 min pressurization.

Table S1. Free amino acid concentration of high pressure treated and untreated samples cultivated at (a) 40 h and (b) 70 h. Note that 0d, 2d. 4d and 8d in top column of Table indicate preservation period after pressurization

(a) 40h
	Od				2d				4d				6d				8d
	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa
Asp	0.787±0.216	0.791±0.134	0.733±0.235	0.790±0.421	0.528±0.107	0.885±0.145	1.137±0.203	0.554±0.323	0.594±0.027	1.137±0.500	1.426±0.070	0.577±0.077	0.606±0.264	1.190±0.257	1.108±0.204	0.927±0.124	0.670±0.049	1.463±0.121	1.646±0.453	0.741±0.021
Glu	0.916±0.289	0.873±0.207	0.990±0.312	0.809±0.117	1.089±0.106	1.014±0.196	1.793±0.324	1.151±0.363	1.044±0.317	1.754±0.599	2.291±0.579	0.762±0.138	0.984±0.299	1.604±0.853	2.151±0.804	0.966±0.274	1.119±0.334	2.005±0.492	2.434±0.625	1.066±0.283
Thr	1.325±0.034	1.505±0.112	1.326±0.009	1.294±0.099	1.458±0.058	1.793±0.135	2.111±0.344	1.457±0.195	1.523±0.068	2.710±0.019	3.003±0.786	1.340±0.047	2.041±0.695	6.230±2.675	6.506±3.442	2.512±1.040	2.597±1.013	7.197±2.345	7.912±1.612	4.999±2.316
Ala	0.224±0.045	0.310±0.036	0.489±0.260	0.365±0.067	0.300±0.040	0.567±0.244	0.823±0.325	0.523±0.278	0.339±0.080	1.085±0.269	1.167±0.384	0.481±0.217	0.350±0.117	1.111±0.470	1.249±0.389	0.614±0.102	0.850±0.284	1.248±0.267	1.526±0.193	0.950±0.345
Pro	0.980±0.406	0.903±0.118	0.839±0.347	0.754±0.143	1.170±0.249	1.307±0.403	0.917±0.351	0.802±0.162	0.981±0.048	1.626±0.384	1.168±0.587	0.859±0.192	1.253±0.379	2.205±0.790	1.627±0.627	0.966±0.173	1.731±0.435	2.310±0.700	2.036±0.388	1.196±0.355
Val	1.560±0.553	1.863±0.178	1.683±0.734	1.461±0.238	1.940±0.425	2.622±0.772	2.580±0.443	1.563±0.538	1.794±0.282	3.669±0.009	4.101±0.836	1.764±0.412	1.927±0.619	4.162±1.452	3.928±1.271	1.914±0.233	2.730±0.551	4.546±0.829	4.813±0.759	2.385±0.852
Ile	0.535±0.085	0.655±0.232	0.667±0.137	0.582±0.049	0.780±0.117	0.981±0.169	1.134±0.164	0.641±0.099	0.730±0.122	1.406±0.178	1.501±0.276	0.617±0.072	0.858±0.273	1.830±0.571	1.984±0.801	0.763±0.162	1.262±0.338	2.054±0.422	2.677±0.605	0.878±0.437
Leu	0.668±0.066	0.753±0.203	0.705±0.267	0.698±0.074	0.708±0.154	1.139±0.171	1.488±0.157	0.901±0.200	0.819±0.112	1.898±0.651	1.993±0.368	0.891±0.062	0.855±0.163	0.390±0.254	2.309±0.623	1.027±0.172	1.369±0.436	2.287±0.380	2.772±0.439	1.373±0.286
Trp	0.100±0.056	0.203±0.137	0.211±0.102	0.078±0.042	0.156±0.066	0.200±0.10(	0.266±0.124	0.119±0.101	0.121±0.024	0.330±0.098	0.793±0.339	0.154±0.007	0.135±0.076	1.113±0.443	0.345±0.229	0.535±0.812	0.210±0.040	0.375±0.158	0.388±0.173	0.284±0.141
Phe	0.313±0.048	0.423±0.081	0.448±0.124	0.328±0.048	0.383±0.044	0.595±0.12!	0.933±0.150	0.444±0.103	0.425±0.086	1.040±0.348	1.285±0.237	0.512±0.077	0.464±0.145	2.060±0.907	1.611±0.483	0.743±0.268	0.775±0.270	1.503±0.235	1.523±0.819	0.952±0.277
Lys	0.595±10.098	0.821±0.096	0.691±0.205	0.758±0.140	0.737±0.028	1.186±0.29;	1.321±0.213	0.953±0.330	0.810±1.111	1.579±0.293	2.018±0.471	0.949±0.038	0.735±0.177	1.161±0.388	2.831±0.201	0.881±0.048	1.234±0.409	2.441±0.673	2.695±0.643	1.333±0.067
His	0.065±O.175	0.826±0.153	0.696±0.116	0.856±0.191	0.746±0.088	0.956±0.23J	1.069±0.152	0.920±0.237	0.861±0.152	1.084±0.277	1.192±0.302	0.718±0.085	0.629±0.139	2.417±0.388	1.039±0.057	0.729±0.055	0.838±0.224	1.278±0.329	1.212±0.225	0.952±0.304
Tyr	1.105±0.209	1.449±0.182	1.300±0.264	1.534±0.324	1.255±0.130	1.803±0.434	2.273±0.321	1.804±0.395	1.488±0.231	2.519±0.719	2.683±0.436	1.817±0.150	1.332±0.409	2.417±0.962	2.460±0.561	1.295±0.379	1.868±0.542	2.920±0.946	3.042±0.599	2.155±0.677

(b) 70h
	Od				2d				4d				6d				8d
	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa	Untreated	200MPa	400MPa	600MPa
Asp	0.505±0.064	0.521±0.091	0.776±0.205	0.494±0.151	0.601±0.030	1.262±0.340	1.032±0.344	0.761±0.305	0.410±0.105	1.068±0.044	0.871±0.077	0.484±0.031	0.632±0.265	1.242±0.309	1.123±0.258	0.588±0.189	0.889±0.338	1.239±0.372	0.884±0.113	0.635±0.288
Glu	0.307±0.039	0.352±0.112	0.537±0.124	0.245±0.028	0.370±0.048	0.975±0.213	0.972±0.252	0.580±0.150	0.566±0.199	1.068±0.065	1.584±0.641	0.499±0.168	0.539±0.179	0.932±0.223	1.907±0.649	0.509±0.116	0.841±0.258	1.512±0.550	2.015±0.546	0.801±0.151
Thr	1.416±0.264	1.371±0.069	1.404±0.122	1.300±0.079	1.129±0.187	1.838±0.517	1.802±0.277	2.337±0.708	1.402±0.032	5.193±1.786	5.647±2.772	2.695±1.862	3.996±1.393	7.145±1.070	8.203±2.398	2.908±1.501	4.474±1.709	8.325±2.100	8.333±1.767	3.562±1.798
Ala	0.290±0.006	0.255±0.052	0.299±0.047	0.246±0.063	0.400±0.248	0.502±0.099	0.524±0.108	0.390±0.094	0.512±0.356	0.909±0.105	1.069±0.533	0.533±0.303	0.768±0.203	1.038±0.086	1.566±0.417	0.513±0.163	0.789±0.234	1.200±0.315	1.166±0.393	0.635±0.182
Pro	0.811±0.062	0.616±0.112	0.526±0.141	0.456±0.144	1.024±0.248	0.736±0.350	0.661±0.184	0.648±0.071	0.999±0.422	1.406±0.552	1.272±0.565	0.733±0.287	1.331±0.259	2.222±0.282	1.814±0.286	0.700±0.291	1.385±0.335	2.007±0.832	1.213±0.763	0.882±0.302
Val	1.693±0.106	1.271±0.253	1.637±0.493	1.266±0.320	1.821±0.449	2.436±0.785	2.238±0.274	1.674±0.411	2.170±0.879	3.670±0.814	4.692±1.249	2.242±0.742	2.740±0.502	4.188±0.487	4.858±1.123	1.887±0.706	2.412±0.742	4.833±0.888	3.961±1.094	2.267±0.278
Ile	0.811±0.039	0.718±0.064	0.801±0.120	0.699±0.113	0.882±0.216	1.084±0.197	1.078±0.095	0.907±0.159	0.996±0.387	1.741±0.511	1.853±0.922	1.051±0.385	1.258±0.237	1.141±0.330	1.501±0.726	0.721±0.327	1.428±0.264	2.263±0.586	2.094±0.302	1.112±0.234
Leu	0.740±0.022	0.786±0.152	0.887±0.119	0.685±0.079	0.773±0.087	1.438±0.390	1.436±0.271	0.957±0.187	0.943±0.205	1.679±0.265	1.881±0.556	1.138±0.202	1.073±0.191	1.860±0.374	2.214±0.424	1.165±0.283	1.142±0.146	2.003±0.333	2.110±0.302	1.329±0.131
Trp	0.096±0.025	0.079±0.042	0.095±0.031	0.082±0.037	0.076±0.053	0.178±0.117	0.185±0.140	0.119±0.089	0.113±0.047	0.413±0.124	0.456±0.032	0.143±0.095	0.211±0.058	0.365±0.117	0.454±0.146	0.321±0.275	0.210±0.040	0.423±0.065	0.495±0.138	0.254±0.115
Phe	0.424±0.034	0.410±0.140	0.532±0.095	0.371±0.064	0.444±0.095	0.839±0.266	0.906±0.136	0.574±0.125	0.511±0.205	1.050±0.272	1.327±0.533	0.743±0.232	0.662±0.164	1.157±0.229	1.723±0.389	0.725±0.159	0.668±0.089	1.326±0.287	1.484±0.268	0.930±0.146
Lys	0.624±0.161	0.653±0.101	0.750±0.159	0.556±0.147	0.644±0.211	1.202±0.268	1.107±0.219	0.777±0.161	0.872±0.311	1.802±0.184	1.315±0.043	0.994±0.197	1.206±0.174	2.122±0.074	1.823±0.168	1.049±0.187	1.228±0.204	2.008±0.333	2.003±0.254	1318±0.248
His	0637±0.141	0.679±0.195	0.816±0.157	0.701±0.186	0.636±0.112	1.174±0.188	1.089±0.251	0.787±0.150	0.754±0.132	0.851±0.082	1.047±0.172	0.789±0.149	0.756±0.093	1.066±0.207	1.321±0.171	0.991±0.147	0.905±0.226	1.246±0.237	1.327±0.335	0.897±0.153
Tyr	1.304±0.5I4	1.095±0.233	1.357±0.290	1.082±0.239	1.203±0.177	2.191±0.254	2.101±0.492	1.578±0.370	1.410±0.284	2.360±0.526	2.464±0.523	1.803±0.378	1.535±0.357	2.154±0.379	2.548±0.483	1.854±0.275	1.765±0.335	2.413±0.492	2.900±0.346	2.264±0.242

[unit: µmol/g]

The data was the average value from three independent measurements with the standard deviation

Free amino acid concentration in mung bean sprouts during preservation    The relationship between amino acid concentration in untreated sprouts and sprouts treated at 200 MPa is shown in Figure 3. The scatter plot was proposed by Shigematsu et al. (2010) to clarify the effect of HHP treatment for each amino acid. The slope of the regression line is between 2.28 and 2.41 at 4 days preservation, and between 3.24 and 3.12 at 8 days preservation, in the case of 40 and 70 h cultivation, respectively. The slope of the regression line was higher than 1.0, indicating that HHP treatment increased the amino acid content of mung bean sprouts during preservation. It is likely that HHP treatment of mung bean sprouts induced proteolysis, and that the changes in free amino acid concentration in mung beans cultivated at different periods occurred mainly due to the degradation of proteins.

Fig. 3.

Relationship between amino acid concentration of 200 MPa-treated and untreated samples cultivated at (a)40 h and (b)70 h during preservation after 10 min pressurization.

The slope of the regression line for 0–4 days of preservation of mung bean sprouts in the present study was compared with previous results from different crops: soy beans subjected to 200 MPa for 10 min at 25 °C (Ueno et al., 2010), brown rice subjected to 200 MPa for 10 min at 25 °C (Shigematsu et al., 2010), and brussels sprouts subjected to 200 MPa for 3 min at 5 °C (Barba et al., 2017) (shown in Figure 4). As shown in Figure 3, the slope of the regression line for amino acid concentration increased to approximately 3 at 0–4 days of preservation. This positive trend was similar for both the 40 and 70 h cultivation groups. The slope obtained from soybean and brown rice experiments also represented a positive linear increase from 1 to 2.3 at 0–4 days of preservation. The slope obtained for mung bean sprouts was higher than that of rice or soybean after 2 days preservation. In contrast, in the case of brussels sprouts, Barba et al. (2017) showed that the slope of the regression line at 0–4 days was almost constant, or even decreased during the preservation period. Therefore, it is suggested that the effect of high pressure differed between each crop. The control sample of soybean sprouts also showed an increase in amino acid concentration. This indicates that changes occurring during the preservation period were not exclusively caused by HHP treatment.

Fig. 4.

Hi-pit effect for mung beans sprout, soybean reported by Ueno et al. 2010, brown rice reported by Shigematsu et al., 2010 and Brussels sprout reported by Barba et al., 2017. Pressurization conditions; pressure level, time and temperature of Ueno et al., 2010 and Shigematsu et al., 2010 were 200 MPa for 10 min under 25 °C, and that of Barba et al., 2017 was 200 MPa for 3 min under 5 °C.

Ueno et al. (2010) showed that HHP treatment selectively enhanced specific proteolysis and metabolism functions in the case of soybeans. In the present study, although the concentrations of all amino acids increased as a result of high-pressure induced proteolysis, we confirmed that increases in the concentrations of amino acids were not similar during preservation. Changes in relative concentration during preservation of Thr, Val, and Tyr are shown in Figure 5. Notably, relative concentration was calculated using the ratio of treated to untreated concentrations. Relative concentration of Thr increased rapidly after 2 days of preservation and reached a plateau after 6 days of preservation. Overall, we confirmed a sigmoidal increase in relative Thr concentration. In contrast, we observed a linear increase in relative Val concentration, and only a slight increase in relative Tyr concentration. The concentrations of other amino acids, Glu, Trp, and Ala, also increased. The concentration of His was similar to that of Tyr, while concentrations of Asp, Pro, Ile, Leu, Phe, and Lys slightly increased during preservation. These differences in the changes in concentration showed that HHP treatment improved not only proteolysis, but also amino acid metabolism in mung bean sprouts. Namely, protein proteolysis reaction and metabolism reactions of amino acids might coexist during the preservation period. As a result, HHP treatment might alter the distribution of free amino acids in mung beans during preservation.

Fig. 5.

Example of course of relative concentration for amino acids cultivated at (a)40 h and (b)70 h. Pressurization was carried out during 10 min under 25 °C. Relative concentration was calculated using ratio of treated sample and untreated sample of 0 day.

Conclusions

This study investigated high-pressure induced changes in amino acid concentration in mung bean sprouts. Mung beans were grown on 0.8 % agar media containing 10 mM CaCl₂·2H₂O for 40 and 70 h. Mung bean sprouts were harvested, and then subjected to HHP treatment at 200, 400, or 600 MPa. Amino acid concentrations in untreated and high-pressure treated samples were determined using HPLC and compared.

Asp, Glu, Thr, Ala, Pro, Val, Ile, Leu, Trp, Phe, Lys, His, and Tyr were detected in the untreated samples; however, no significant differences were observed between the two cultivation periods. Total amino acid concentration gradually increased during the preservation period for samples treated at 200 and 400 MPa. In contrast, amino acid concentrations in the sample treated at 600 MPa were almost constant or decreased during the preservation period. To investigate the effect of HHP treatment, we calculated the ratio of amino acid concentration in untreated and treated samples for each amino acid. Total amino acid concentrations in the treated samples were 2–3-fold higher than that in the untreated samples. Thus, our results showed that HHP treatment of mung bean sprouts improved proteolysis during the preservation period. Additionally, we observed a relative increase in the concentration of specific amino acids compared to other amino acids. Taken together, the findings of this study suggest that high-pressure induced proteolysis reaction and metabolic reactions of amino acids might coexist in mung bean sprouts during the preservation period.

Acknowledgements    This study was supported by JSPS KAKENHI, Grant-in-Aid for Exploratory Research (Number: 26660101 Representative; T. Fujii).

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