Note
Homogenization in water improves the recovery rate of imidazole dipeptides in meat
2021 年 27 巻 2 号 p. 335-339
詳細
2021 年 27 巻 2 号 p. 335-339
The beneficial effects of the imidazole dipeptides (IDPs) carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) in humans are widely accepted. Meats are a rich dietary source of IDPs. Therefore, accurate and reproducible methods for IDP quantification in meat should be developed to determine the quantity of meat required to ensure IDP intake at physiologically effective levels. In the present study, we attempted to establish a novel method for estimating the IDP content in meats and quantifying IDP in various meat samples. The IDP content in the samples was estimated accurately using the novel homogenization method. We observed that the IDP content of meats differed based on the species of chicken, cooking method, muscle tissue type, and region.
Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are imidazole dipeptides (IDPs) that exert various physiological effects in humans, including pH-buffering action, metal-ion chelation, and antioxidant activity (Boldyrev et al., 2013). In recent studies, the therapeutic potential of IDP supplements has been tested in numerous diseases, such as diabetes, as well as in the associated complications, such as ocular disease, aging, and neurological disorders, and IDP supplementation has been found to alleviate fatigue and suppress cognitive decline in humans (Begum et al., 2005; Hisatsune et al., 2016).
Meats are known to have a higher IDP content than other foods. Among meats, chicken breast has the highest IDP content. Therefore, meat, especially chicken breast, when consumed in adequate quantities, can provide IDP at sufficient levels to alleviate fatigue and suppress cognitive decline. To determine the quantity of meat required for sufficient IDP intake, an accurate and reproducible method for IDP quantification in meats should be developed. Here, we established a novel method to estimate the IDP content in meat and quantified IDP in various meat samples.
Sample collection and processing Frozen breast meat samples of free-range local traditional pedigree chicken (Jidori) shipped at different ages and other meat samples purchased from various prefectures of Japan were used in this study. Among the free-range local traditional pedigree chickens in Japan, Hakata Jidori, produced in Fukuoka Prefecture, was developed approximately 30 years ago by crossing the breeds Shamo with Sazanami, and then crossing the resultant breed with white Plymouth Rock. Where indicated, these meats were cooked by baking or boiling. Two methods were used to process the meat samples: the conventional method and the homogenization method. In the conventional method, 25 mL of 10% sulfosalicylic acid (SSA) was added to the minced meat sample (6 g), and the mixture was shaken for 20 min. The volume was adjusted to 100 mL using sodium citrate (pH 2.2). The mixture was filtered using filter-paper (No. 1; Advantec, Tokyo, Japan). The filtrate was diluted five-fold using sodium citrate (pH 2.2) and filtered again using a 0.45-µm filter (Dunnett and Harris, 1992). In the homogenization method, the minced meat sample (10 g) was homogenized in 50 mL of ice-cold Milli-Q water for 1 min at 17 500 rpm using a POLYTRON homogenizer (PT1200E; Kinematica AG, Switzerland), and the volume was adjusted to 100 mL using Milli-Q water. Five milliliters of 3% SSA was added to 5 mL of the homogenate, followed by centrifugation (3 500 × g for 30 min at 4 °C), and filtration using a 0.45-µm filter.
Measurement of amino acid concentration The amino acid concentration of the processed sample was measured using a fully automated amino acid analyzer (JVC-500/V2; JEOL Ltd., Tokyo, Japan) or liquid-chromatography-mass spectrometry (LC-MS). For LC-MS analysis, solvents of LC-MS grade were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Chromatography was performed using an LCMS-8050 (Shimadzu, Kyoto, Japan) equipped with an Intrada Amino Acid column (50 × 3 mm; Imtakt, Kyoto, Japan). The gradient elution buffers were buffer A (0.1% formic acid/acetonitrile) and buffer B (100 mM ammonium formate), and the flow rate was 0.6 mL/min. The elution gradient (buffer A: buffer B, v/v) was as follows: initial conditions, buffer A: buffer B = 86:14 for 3 min, linear gradient to 100% buffer B for 7 min, and 100% buffer B for 5 min. The samples were analyzed using tandem mass spectrometry in the ESI positive-ion mode. The operating conditions were as follows: nebulizer gas flow, 3.0 L/min; drying gas flow, 10 L/min; heating gas flow, 10 L/min; interface voltage (+), 4.0 kV; DL temperature, 250 °C; block temperature, 400 °C; interface temperature, 300 °C. Carnosine was detected using mass spectrometry in the multiple reaction monitoring mode. The concentration of carnosine in meat was measured using the chromatogram of the product ion (m/z 110.1) derived from the precursor ion (m/z 227.1). Quantitative values were obtained by comparing the chromatographic peak areas to those derived for external stable isotope-labeled internal standard amino acids (Hata et al. 2019). Stable isotope-labeled carnosine [β-Ala-His (13C6, 15N3)] was purchased from Sigma-Aldrich (St. Louis, USA). Stable isotope-labeled anserine (anserine-d4) was synthesized using a previously reported method (Gopinathan et al. 2009).
Statistical analysis The experiments were performed in at least triplicate. The results are presented as the mean ± standard deviation. Statistical significance was determined using a two-sided Student's t-test, and the level of significance was fixed at p < 0.05, as shown in Fig. 2.
Spike and recovery test to validate the methods of IDP quantification in meat extracts.
Carnosine was added to the meat extracts prepared using the conventional method (A) and homogenization method (B) at the final concentration of 0.20, 0.40, and 0.80 mg/mL, and the concentration of recovered carnosine was determined.
Measurement of IDP content in chicken breast meat. The IDP concentration in chicken breast meat samples was measured using two methods: conventional method (1) and homogenization method (2).
Statistical significance was determined using a two-sided Student's t-test, the level of significance was fixed at p < 0.05 (*p < 0.05; **p < 0.01).
Improved method for IDP quantification in chicken breast samples First, we performed a spike-and-recovery test with a reference standard to assess whether the IDP concentration in the extracts prepared using the conventional method and the homogenization method could be measured precisely. Figure 1 shows that the carnosine spikes in the extracts prepared by the conventional method and homogenization method could be recovered in a reproducible and precise manner, indicating that the IDP content in these extracts could be measured accurately. The IDP (carnosine and anserine) content in the chicken breast (of Hakata Jidori) pretreated using the conventional method was approximately 1.5 g per 100 g sample when analyzed using the fully automated amino acid analyzer (Fig. 2). The conventional method of analysis is widely employed, particularly by contract research organizations or institutes. In the present study, we proposed homogenization as a novel method for processing. In this method, a Hakata Jidori breast meat sample was thoroughly homogenized in water, followed by measurement of the IDP content in the supernatant after deproteination using SSA. The IDP content was found to be approximately 2.0 g per 100 g in each sample (Fig. 2). The basic difference between the two methods is the homogenization of the breast meat samples in 10% SSA (in the conventional method) or Milli-Q water (in the homogenization method). These results suggest that homogenization in water allowed the effective extraction of IDP present in chicken breast meat; hence, this method could be considered superior to the conventional method for measuring IDP content in muscle tissue.
IDP content in breast meat samples of free-range local traditional pedigree chickens (Jidori) from various prefectures in Japan In this experiment, we used LC-MS to measure the IDP content in the samples, such that the effectiveness and reproducibility of the method could be increased and the analysis time could be reduced. LC-MS was used to measure the IDP content in the breast meat samples of free-range local traditional pedigree chickens from eight prefectures in Japan. The samples had been pretreated by homogenization (Fig. 3A). Label A indicates the IDP content in the breast meat of free-range local traditional pedigree chicken from Fukuoka Prefecture (Hakata Jidori). Labels B to H indicate the IDP content in the breast meat of free-range local traditional pedigree chickens from the seven other prefectures. Among the samples tested, the breast meat of Hakata Jidori had the highest IDP content. This result suggests that Hakata Jidori breast meat was the best source of IDP among the different samples tested, and is possibly one of the best dietary sources of IDP in Japan. Shamo, a game fowl chicken breed in Japan, is used as a mating partner in the breeding of free-range local traditional pedigree chickens in three of the four types of high-IDP producing free-range local traditional pedigree chickens (A, B, and F in Fig. 3A). This suggests that the IDP content in meat is genetically influenced and that the genetic background of Shamo plays a role in enhancing the IDP content in meat. The IDP content in meat can also be regulated by environmental factors. Sprint training was shown to increase the IDP content in human muscles (Suzuki et al., 2004). Furthermore, some foods and chemical compounds were shown to exert exercise-mimetic effects and increase the IDP content in muscles (Dib et al., 2016). These reports suggest that exercise and/or consumption of foods exerting exercise-mimetic effects can increase the IDP content in muscles. We aimed to thoroughly investigate the molecular mechanisms underlying the modification of IDP content in meat and potentially increase the IDP content in meat using genetic and environmental optimization methods.
IDP content in various meat samples. A. IDP content in breast meat of free-range local traditional pedigree chickens from various prefectures in Japan. B.
Effects of cooking on the IDP content in breast and thigh meat of Hakata Jidori. C. IDP content in chicken, pork, and beef meat samples. The black and gray bars indicate the carnosine and anserine contents, respectively.
Effects of cooking on the IDP content in breast and thigh meat of Hakata Jidori As shown in Fig. 3B, the IDP contents in baked and raw breast and thigh meat samples were almost equal. However, boiling significantly decreased the IDP content in both breast and thigh meat, suggesting that boiling led to the release of IDP from the muscle fiber into the aqueous phase. This is the first study to report the cooking-induced changes in the IDP content of breast and thigh meat samples of Hakata Jidori, one of the free-range local traditional pedigree chickens that typically has a high IDP concentration. Next, we tested the IDP content in each part of chicken, pork, and beef meats. As shown in Fig. 3C, the relative carnosine content in pork and beef meat was greater than that in chicken meat. Anserine was found to be the predominant IDP present in chicken meat, whereas carnosine was the predominant IDP present in pork and beef meats. Our results also showed that the chicken breast samples had the highest IDP content among the samples tested. Furthermore, boiling significantly decreased the IDP content in all meat samples, suggesting that this cooking method led to the release of IDP from muscle fibers. Although there are reports on individual analyses (Peiretti et al., 2011), Fig. 3C clearly shows the species- and meat-part-specific as well as the cooking-dependent changes in IDP content.
According to previous reports, 400 mg of IDP is required to suppress fatigue caused by daily human activities (Suzuki et al., 2002; Begum et al., 2005). Furthermore, we previously reported that the daily intake of 1 g of IDP preserves verbal episodic memory and brain perfusion in the elderly, as demonstrated in a double-blind randomized controlled trial (Hisatsune et al., 2015; Rokicki et al., 2015; Katakura et al., 2017). Our results show that the Hakata Jidori breast meat samples contain the highest levels of IDP among the samples tested. Therefore, the intake of IDP at physiologically effective levels could be achieved by the daily consumption of baked breast meat of Hakata Jidori.
An accurate and reproducible method for IDP quantification in meat is crucial for determining the daily meat intake that allows IDP to exert its physiological effects, such as alleviation of fatigue and suppression of cognitive decline. In the present study, we established a novel method for the measurement of IDP content in meat that was more effective than the conventional method.
Acknowledgements This study was supported by the Support for R&D to Create New Products and New Technology in Fukuoka Prefecture from Kurume Research Park Co., Ltd. (Fukuoka, Japan). Experiments to measure amino acid concentrations using LCMS 8050 were conducted at the Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University.
