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Basic Studies on Food Science And Biochemistry
on food science and biochemistry on the functional ingredients of mushrooms
Introduction
When it comes to the functional compounds of mushrooms, mushrooms like the reishi and poria, which have been used medicinally since ancient times, might be the first to come to mind.

Meanwhile, mushrooms have long been used as food, and for centuries important forest products such as shiitake mushrooms have been grown and wild mushrooms have been collected for food. In recent years, various types of mushrooms have been cultivated for consumption and widely distributed in the market. Although it is typical for mushrooms to be displayed in the vegetable section of supermarkets in Japan, many consumers are unaware of the fact that mushrooms are microorganisms.

I, the author, became involved in research using mushrooms after transferring to his current laboratory in 2003, a time when I was exploring new research themes as a microbiologist. In this paper, I introduce some of the research findings that have unfolded based on simple questions about mushrooms, using approaches such as material production technology using cultivated microorganisms, handling techniques for microbial enzymes, and research methods on functional ingredients in food.
1. Studies on enzymes involved in the production of gamma-aminobutyric acid (GABA) in mushrooms
Gamma-aminobutyric acid (GABA) is a non-protein functional amino acid that has attracted attention in recent years and is a food ingredient that is expected to have various physiological functions such as reducing blood pressure and anti-diabetic effects.

In vivo, GABA is synthesized when the alpha-carboxyl group of glutamate is removed by glutamate decarboxylase (GAD), and many foods have been developed that enhance GABA by utilizing the activities of GAD, which is found in lactic acid bacteria, sprouted brown rice, and red mold (a fermentation starter). Edible mushrooms are also a relatively promising source of GABA, which is known to accumulate in high concentrations in fruiting bodies.

However, although enoki (Flammulina velutipes) were known to have significant amounts of GABA, there were no detailed studies or reports on the enzymes involved. Then, when the amount of GABA in the fruiting bodies of mushrooms available at supermarkets and other outlets was measured, it was discovered that the concentration was higher than that of tomatoes, which typically contain high levels of GABA (Fig. 1).
Therefore, we decided to study enzymes involved in GABA synthesis using the fruiting bodies of enoki mushrooms, maitake (Grifola frondosa), and shiitake (Lentinula edodes), but the enoki enzyme activity was localized in the sediment after cell disruption, with no activity detected in the disrupted supernatant so we stopped short of purifying the enzyme to a single entity using electrophoresis and instead focused on studying various properties using partially purified enzymes (data unpublished). On the other hand, however, enzymes from maitake and shiitake were found to be active in the supernatant after cell disruption, so we tried purification.

The enzyme derived from maitake was a dimeric enzyme with a molecular weight of about 97,000 and, together with enzymes partially purified from enoki, acts only on L-glutamic acid in a similar way to glutamic acid decarboxylase (GAD) as reported thus far.

On the other hand, the enzyme derived from shiitake mushrooms was a dimeric or trimeric enzyme with a molecular weight of approximately 131,000 and it was an enzyme that acted not only on L-glutamic acid, but also on L-aspartic acid, L-2-aminoadipic acid, and other substances. A database search based on the results of analysis of the internal amino acid sequence of the enzyme derived from shiitake mushrooms revealed high similarity to the phosphatidylserine decarboxylase (PSD) of shiitake. However, when the gene encoding this enzyme was cloned and the similarity of the primary amino acid sequence with PSD derived from other organisms was examined, the similarity to proteins with confirmed PSD activity was found to be low (Figure 2). In addition, we summarized the enzymatic properties of these enzymes that were purified or partially purified in Tables 1 and 2.
2 Research on the colour of mushrooms
2-1. Re-evaluation of pink oyster mushroom pigmentation
The pink oyster mushroom, characterized by its vivid pink fruiting body, is an edible mushroom found in various regions around the world. Takekuma et al. reported that the pink color, which resembles that of the Japanese crested ibis, is due to its protein compounds. However, we conducted a re-examination because there were doubts about the consistency between the experimental method and results described in the paper.As a result, it was confirmed that the pigment is a protein as stated in the paper; however, its form of existence was found to be not a heterodimer as described, but a monomer, and the molecular weight was also revealed to be different at 24,500.

Furthermore, it has been revealed through gel filtration chromatography that the yellow sugar protein forming a complex with the pigment is a low molecular weight water-soluble melanin with molecular weights of 10,800, 5,300 and 4,400.The gene encoding the pink pigment protein of the pink oyster mushroom was cloned by Fukuda et al. as a 1022 bp gene with 6 introns on the genome, and registered in the database as a novel protein PsPCP with 226 amino acid residues (Figure 3). The mRNA that encodes the PsPCP is strongly expressed during fruiting body primordium development and a correlation with fruiting body pigmentation was observed. In addition, it has been revealed that the recombinant protein obtained by heterologous gene expression of the native pigment protein has the function of stabilising the coloration of indolone (3H-indol-3-one), the main body of the pigment (Figure 4).

2-2. Enzymes involved in the identification and production of the yellow pigment of golden oyster mushroom
The yellow pigment of the golden oyster mushroom (Pleurotus citrinopileatus) is a water-soluble pigment. The color is lost when added to soups, but is known not to fade when cooked at high temperatures, such as in tempura. The molecular weight of the native pigment purified from the fruiting body is estimated to be approximately 4,200 based on size exclusion chromatography. It exhibits properties such as thermal stability, resistance to protease digestion, solubility in distilled water, slight solubility in methanol, and insolubility in most organic solvents. It has also been revealed that it is depigmented by oxidizing agents such as KMnO4 and exhibits properties closely resembling those of the yellow melanin pheomelanin, such as reducing Fe3 reduction. In many mushrooms, it has been reported that brown pigments are formed in the fruiting body by the action of phenol oxidising enzymes such as laccase and tyrosinase. Tyrosinase from the button mushroom (Agaricus bisporus), a commercially available enzyme also known as mushroom tyrosinase, is used as a standard reagent for melanin production experiments.

When the substrate specificity of enzymes purified from the golden oyster mushroom was compared to that of commercially available mushroom tyrosinase, the enzyme from the button mushroom showed reactivity not only towards L-3, 4-dihydroxyphenylalanine (L-DOPA), a precursor of melanin, but also towards various phenolic compounds. On the other hand, the enzyme from the golden oyster mushroom acted specifically on dopamine, from which the α-carboxyl group of L-DOPA and DOPA had been decarboxylated, and produced a bright yellow coloring substance (Table 3). From this, it was suggested that this enzyme may be involved in the production of the yellow pigment in the golden oyster mushroom. Furthermore, although the molecular weight of this enzyme was approximately 19,000 based on analysis by SDS-PAGE, the result of molecular weight determination by size exclusion chromatography estimated the native molecular weight to be about 350,000, with the structure of the native form still unknown at present.There are many unknown aspects of the characteristics and structure of this enzyme, and further investigation will be necessary in the future.
3. Research on the functional ingredients of mushrooms
Mushrooms have been reported to have various functional ingredients, but most of them inherently exist in mushrooms, and there are not many reports on cultivation methods to enhance functional ingredients. However, I thought that it might be possible to enhance the functional ingredient content in the fruiting bodies by controlling the cultivation conditions for functional ingredients, such as the aforementioned GABA for which the bio-synthetic pathway is clearly understood, and compounds that are absorbed into the fruiting body from the cultivation substrate. Trehalose is a disaccharide also known as mushroom sugar, and it has been reported that trehalose in the fruiting body of mushrooms is synthesized inside the mycelium during the mycelial stage and then transferred to the fruiting body.

Therefore, thinking that the amount of trehalose transferred to fruiting bodies might increase if a significant amount of trehalose is present in the mycelium at the time of fruiting body formation, I first attempted to enhance the trehalose content in the mycelium and investigated the functionality of mycelia that had absorbed it. As a result, it was found that by cultivating shiitake mushrooms on a substrate containing about 5% trehalose during mycelium cultivation a significant amount of trehalose accumulates in the mycelium, and that mycelium with high trehalose content shows improved resistance to heat treatment and freezing treatment.
Further, with the increase in trehalose content, the ability to inhibit the generation of peroxidative lipids in the mycelium of mushrooms also increased. Additionally, it was discovered that the trehalose content in the fruiting bodies can be boosted by injecting trehalose into shiitake substrate blocks just before the mushrooms start growing.

The mushroom fruiting body contains a variety of compounds derived from the substrate. Phenolic compounds produced from lignin in wood, particularly through the activity of mushroom decomposition enzymes, are expected to be absorbed into the fruiting body. One of these compounds syringic acid (4-hydroxy-3, 5-dimethoxybenzoic acid) has been found to exhibit anti-osteoporotic effects independent of the pathway through estrogen receptors. It has become evident that syringic acid is more abundant in spent substrate blocks than in mushroom fruiting bodies, suggesting that spent substrate blocks may have a potential use.
These findings suggest that it may be possible to control the functional ingredient levels in mushrooms by innovating cultivation methods, so moving forward we would like to actively explore ways to enhance the levels of other functional ingredients as well.
4. Amino acid fermentation liquid preparation method using protease from mushroom fruiting bodies
It is known that mushroom fruiting bodies have protease activity, and numerous studies have reported research on applying the fermentation ability of mushrooms to the production of fermented foods.

In order to obtain preliminary knowledge for establishing a brewing method for sake with a high content of umami compounds primarily based on amino acids, we examined the use of enzymes derived from mushroom mycelium. By adding a pulverised liquid from the fruiting bodies of various edible mushrooms as an enzyme source to steamed rice, preparing a moromi (fermenting mash), and then processing it at 30°C for 48 hours, the composition of free amino acids in the mash developed characteristics different from that of both the mushroom fruiting bodies and the malted rice. In particular, a pronounced increase in umami-flavor amino acids such as glutamic acid was observed in the preparation using the shiitake mushroom fruiting body as an enzyme source (Figure 5). In consideration of simplifying the preparation process and scaling up, we were able to prepare a fermentation liquid with a high free amino acid content by processing powdered maitake fruiting bodies at 50°C for 20 hours (Figure 6). The amount of serine, which indicates sweetness, and lysine, an essential amino acid, significantly increased in the fermenting mash processed from maitake fruiting bodies.

It is known that edible mushrooms have high levels of umami compounds from nucleic acids. However, by using mushroom fruiting body enzymes in the preparation process, it has been suggested that it is possible to create a fermenting mash comparable to the kombu (kelp) and katsuo (bonito) stock (typically used to give Japanese cooking its characteristic umami flavour) that is rich in umami compounds from both nucleic acids and amino acids.
5. Conclusion
Although varied, I have roughly summarized above the findings of research on mushrooms conducted thus far. These studies apply not only the applied microbiology techniques learned during my student days, but also the food science methods learned from research themes on functional ingredients of food received upon my appointment to Kindai University in 1994.
However, these studies are all still works in progress, and it is unknown how they will unfold, but I hope that as we continue to gradually clarify the value of mushrooms as food, we will encounter new facts and phenomena.
Last but not least, I am deeply honored to have received the prestigious academic society award. I would like to express my heartfelt gratitude to all the members of the Japan Mycological Society and to the many people who have helped me.
Abstract
I conducted food science and biochemical research on the functional ingredients of mushrooms and obtained the following results.
1. Enzymes related to GABA synthesis in enoki, maitake and shiitake were studied and their properties revealed.
2. The properties of the pink pigmented protein in pink oyster mushrooms were identified.
3. The yellow pigment in pink oyster mushrooms and yellow oyster mushrooms was revealed to be a water-soluble melanin substance.
4. I focused on trehalose and syringic acid as functional ingredients in edible mushrooms and demonstrated their functionality.
5. I identified the preparation method of high-amino acid fermentation liquid using protease from edible mushrooms.

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Basic Studies on Food Science And Biochemistry

on food science and biochemistry on the functional ingredients of mushrooms

Introduction

1. Studies on enzymes involved in the production of gamma-aminobutyric acid (GABA) in mushrooms

2 Research on the colour of mushrooms

2-2. Enzymes involved in the identification and production of the yellow pigment of golden oyster mushroom

3. Research on the functional ingredients of mushrooms

4. Amino acid fermentation liquid preparation method using protease from mushroom fruiting bodies