Branched-chain amino acid synthesis and glucosinolate-myrosinase system during takuan-zuke processing of radish root.

The traditional Japanese fermented pickle, takuan-zuke, is produced via dehydration and salt-aging of the Japanese winter radish root (Raphanus sativus L.). It has been reported that γ-aminobutyric acid (GABA; antihypertensive factor) accumulates during this production. Herein, the results of the comprehensive study (metabolites, enzyme activity, and gene expression levels were investigated) on the mechanism of branched-chain amino acid (BCAA) synthesis and glucosinolate (GLS)-myrosinase system of daikon dehydration have been reported. BCAAs, GLS, and their precursors were temporarily upregulated, and the genes linked to BCAA, GLS, and isothiocyanate synthesis (BCAT4, MAM3, IPMDH1, RMB1, RMB2, and GRS1) were upregulated during daikon dehydration. BCAAs and GLS accumulated in daikon during dehydration owing to the upregulation of genes, encoding these synthases as a stress response. These results suggest that the biological response of daikon adds characteristic flavor and health functionality to takuan-zuke and helps optimize the processing parameters to produce pickles with improved health benefits. PRACTICAL APPLICATIONS: Takuan-zuke is a popular pickle in Japan, which is produced from a Japanese winter radish root (Raphanus sativus L.), commonly referred to as daikon in Japan. Takuan-zuke is produced by dehydrating daikon either by the process of salt-pressing (shio-oshi) or sun-drying (hoshi). The processes influence the variations in the nutritional value of the final product. The results reveal that both the daikon dehydration processes can be followed to generate increased amounts of health-promoting components (e.g., branched-chain amino acid and glucosinolate) in takuan-zuke.

Metabolism of glutamic acid to alanine, proline, and γ-aminobutyric acid during takuan-zuke processing of radish root.

Takuan-zuke is a traditional Japanese fermented pickle, prepared by dehydration of radish root (daikon) by salt-pressing or sun-drying followed by aging with salt. We previously reported that alanine, proline, and γ-aminobutyric acid (GABA) accumulate during daikon dehydration, whereas the level of glutamic acid, their precursor, decreases. We have also reported that dehydration and salt-aging markedly influence the dynamics of free amino acids. In this study, we quantitatively analyzed free amino acid levels, enzyme activity, and gene expression to characterize takuan-zuke amino acid metabolism. Enzyme activities related to alanine, proline, GABA, and glutamic acid metabolism were sustained during dehydration. Moreover, genes encoding alanine, proline, and GABA synthases (ALT1, P5CS1, and GAD4) were significantly upregulated during dehydration. These effects may represent cellular stress responses to the dehydration process. The biological response of daikon contributes to the healthy functional aspects that characterize takuan-zuke. These findings could guide the selection of suitable vegetable varieties to produce pickled vegetables with health-promoting properties. PRACTICAL APPLICATION: The fermented pickle takuan-zuke, prepared by dehydration of radish root (daikon), accumulates amino acids, such as alanine, proline, and GABA, during preparation that provide taste and health benefits. In this study, the aforementioned amino acids were found to accumulate because of the stress response of daikon during the dehydration process and not because of the action of microorganisms during fermentation. Takuan-zuke processing is a method for improving the nutrition of daikon.

Low pH Enhances the Glucosinolate-Mediated Yellowing of Takuan-zuke under Low Salt Conditions.

This study was performed to clarify the enhancement of the 4-methylthio-3-butenyl isothiocyanate induced yellowing of salted radish root (takuan-zuke) by low pH during short-term salt-aging at low temperature and low salinity. We used two different methods to prepare the dehydrated daikon prior to salt-aging: air-drying outdoors (hoshi takuan-zuke) or salting with a stone press (shio-oshi takuan-zuke). Low salt-aging at low temperature was carried out under pH control with citrate-phosphate buffer. The yellowing of both types of takuan-zuke was accelerated below pH 5, and the color of air-dried takuan-zuke was deeper than that of salt-pressed takuan-zuke. To elucidate this phenomenon, several previously reported yellowing-related compounds were analyzed by high-performance liquid chromatography. The result showed that the production of the primary pigment, 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan, was low compared with that in previous reports. Therefore, we suggest that an unknown pigment was generated through a previously unreported pathway.

Protein components of water extracts from fruiting bodies of the reishi mushroom Ganoderma lucidum contribute to the production of functional molecules.

BACKGROUND: Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated. RESULTS: Various glycoside hydrolases (GHs), such as ß-N-acetylhexosaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-ß-1,3-glucanase (GH family 128), and ß-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated ß-oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE inhibitory activities. Finally, it was observed that extraction at 50 °C is suitable for yielding functional molecules with high ACE inhibitory activities. CONCLUSION: Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. This study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction. © 2018 Society of Chemical Industry.

Short-term mastication after weaning upregulates GABAergic signalling and reduces dendritic spine in thalamus.

Mastication enhances brain function and mental health, but little is known about the molecular mechanisms underlying the effects of mastication on neural development in early childhood. Therefore, we analysed the gene expression in juvenile neural circuits in rats fed with a soft or chow diet immediately after weaning. We observed that the gene expression patterns in the thalamus varied depending on the diet. Furthermore, gene ontology analysis revealed that two terms were significantly enhanced: chemical synaptic transmission and positive regulation of dendritic spine morphogenesis. With respect to chemical synaptic transmission, glutamate decarboxylase and GABA receptors were upregulated in the chow diet group. The related genes, including vesicular GABA transporter, were also upregulated, suggesting that mastication activates GABAergic signalling. With respect to dendritic spine morphogenesis, Ingenuity Pathway Analysis predicted fewer extension of neurites and neurons and fewer number of branches in the chow diet group. The numbers of spines in the ventral posterolateral and posteromedial regions were significantly decreased. These results suggest that mastication in the early developing period upregulates GABAergic signalling genes, with a decrease of spines in the thalamus.

The salted radish takuan-zuke shows antihypertension effects in spontaneously hypertensive rats.

Recently, we reported that the antihypertensive compound, γ-aminobutyrate (GABA), increases over time during the dehydration of salted radish, known as takuan-zuke, a popular pickle in Japan. The objective of this study was to clarify the antihypertensive effects of takuan-zuke. We prepared two types of takuan-zuke by sun-drying (hoshi takuan-zuke) and salt-pressing (shio-oshi takuan-zuke) using dehydration processes. The oral administration of takuan-zuke lowered systolic blood pressure in spontaneously hypertensive rats (SHRs). Shio-oshi takuan-zuke (SR) demonstrated a clear antihypertension effect compared with hoshi takuan-zuke (DR), despite equal GABA concentrations in the feed. Furthermore, takuan-zuke demonstrated angiotensin converting enzyme (ACE) inhibition in vitro. These findings indicated that takuan-zuke contains unknown substances that have hypotensive actions independent of GABA. Further evaluation revealed that takuan-zuke contains polyphenols, arginine, and α-linolenic acid as possible antihypertensive factors. Collectively, our results suggest that the salty Japanese food takuan-zuke has antihypertensive effects in vivo, likely involving complex mechanisms.

Nutritional content and health benefits of sun-dried and salt-aged radish (takuan-zuke).

We investigated the nutritional characteristics of salted radish roots (takuan-zuke) prepared using different methods: takuan-zuke based on sun-drying (hoshi) or salt-pressing (shio-oshi) dehydration, different salt-aging temperatures, and salting with rice bran. We examined differences in nutritional substances in salted radish using chromatographic analysis, bioassay methods, and multivariate analysis. We previously reported that the amount of γ-aminobutyrate in takuan-zuke was increased by both dehydration treatments. In the present study, we observed that sucrose and proline were increased by sun-drying treatment, while little change occurred with salt-pressing treatment. Branched-chain amino acids were increased by both treatments. Interestingly, free fatty acids increased with salt-aging duration, irrespective of the dehydration method. Addition of rice bran to long salt-aging treatment increased the levels of niacin, glutamate, and acetate. Metabolite concentrations were higher in hoshi takuan-zuke than shio-oshi takuan-zuke. Our comprehensive analysis reveals effects of specific manufacturing conditions on beneficial components of takuan-zuke.

Transcriptomic responses of the liver and adipose tissues to altered carbohydrate-fat ratio in diet: an isoenergetic study in young rats.

BACKGROUND: To elucidate the effects of altered dietary carbohydrate and fat balance on liver and adipose tissue transcriptomes, 3-week-old rats were fed three kinds of diets: low-, moderate-, and high-fat diets (L, M, and H) containing a different ratio of carbohydrate-fat (C-F) (65:15, 60:20, and 35:45 in energy percent, respectively). METHODS: The rats consumed the diets for 9 weeks and were subjected to biochemical and DNA microarray analyses. RESULTS: The rats in the H-group exhibited lower serum triacylglycerol (TG) levels but higher liver TG and cholesterol content than rats in the L-group. The analysis of differentially expressed genes (DEGs) between each group (L vs M, M vs H, and L vs H) in the liver revealed about 35% of L vs H DEGs that were regulated in the same way as M vs H DEGs, and most of the others were L- vs H-specific. Gene ontology analysis of these L vs H DEGs indicated that those related to fatty acid synthesis and circadian rhythm were enriched. Interestingly, about 30% of L vs M DEGs were regulated in a reverse way compared with L vs H and M vs H DEGs. These reversed liver DEGs included M-up/H-down genes (Sds for gluconeogenesis from amino acids) and M-down/H-up genes (Gpd2 for gluconeogenesis from glycerol, Agpat9 for TG synthesis, and Acot1 for beta-oxidation). We also analyzed L vs H DEGs in white (WAT) and brown (BAT) adipose tissues and found that both oxidation and synthesis of fatty acids were inhibited in these tissues. CONCLUSIONS: These results indicate that the alteration of dietary C-F balance differentially affects the transcriptomes of metabolizing and energy-storing tissues.

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke).

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b(*) values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.