How Is Glutathione Used in Aquaculture?

Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine with sulfhydryl groups, and is the most important low molecular weight antioxidant synthesized in cells. It occurs in nature in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). GSH is usually referred to as GSH[1], which has a sulfhydryl group and γ-glutamine. GSH has many physiological functions, such as scavenging free radicals, enhancing immunity, and chelating and detoxification[2].

With the continuous development of high-density aquaculture, aquatic diseases are becoming more and more serious, and the state attaches more and more importance to food safety. Safe and efficient feed additives have become the key to solving the health problems of aquaculture animals and improving the yield and quality of aquaculture animals.

In recent years, the application of glutathione in aquaculture has been widely researched and become a hot spot for feed additive development. The article summarizes the latest research progress on the production and application of glutathione in aquaculture both at home and abroad and aims to provide new scientific ideas for the application of glutathione in aquaculture by elucidating the principles and effects of glutathione on different aspects of aquaculture animals.

1.Sources and Metabolism of Glutathione

1.1 Sources of Glutathione

Glutathione is mainly synthesized and reduced in the body.GSH is synthesized in liver cells by a two-step enzymatic reaction catalyzed by γ-glutamylcysteine synthetase (GSHI or γGCS) and glutathione synthetase (GSHII or GS), using glutamic acid, cysteine and glycine as substrates[3] . GSSG is synthesized in a two-step enzymatic reaction catalyzed by γ-glutamylcysteine synthetase (GSHI or γGCS) and glutathione synthetase (GSHII or GS) [3]; the GSH reduction pathway mainly involves the intracellular GSSG being reduced to GSH by glutathione reductase (GR) [4].

In addition, GSSG can generate GSH under the action of sulfotransferase, or react with ascorbic acid to generate GSH; when GSH is depleted or insufficient, mammals can utilize methionine to generate GSH [5].

1.2 Metabolism of Glutathione

Glutathione can be completely absorbed and transported by the small intestine [6], and through the bloodstream into the pancreas, liver and other parts of the metabolism, to complete the scavenging of free radicals, detoxification and nutrient transport and other functions [7], and the kidneys are able to uptake GSH from the blood, which is an important place for the removal of GSH from the blood plasma. Adverse conditions such as aging, toxicity, infection, and oxidative stress can reduce the synthesis of intracellular GSH, which can lead to a decrease in GSH levels in the body, and exogenous supplementation of GSH can alleviate or terminate these problems.

2. Production Methods of Glutathione

2.1 Direct Extraction Method

The direct extraction method mainly uses organic solvents to obtain glutathione from animal and plant tissues rich in glutathione (e.g., corn germ or wheat germ) as raw materials and then obtains glutathione through separation, concentration, and drying after treatment with amylase and protease[8]. However, the direct extraction method has the problems of low purity and low yield, which is not suitable for industrialized production and application.

2.2 Chemical Synthesis

The production of glutathione by chemical synthesis began in the 1970s. Glutathione was synthesized from glutamic acid, cysteine and glycine through the stages of group protection, condensation and deprotection[9]. This method was applied to the production of glutathione earlier, but it has many problems, such as complicated operation, high cost, long time consumption, and serious environmental problems.

2.3 Enzymatic Method

Enzymatic synthesis of glutathione refers to the synthesis process of glutathione in the organism, using L-glutamic acid, L-cysteine and glycine as the substrates, and adding two glutathione synthetases, γGCS and GS, which are obtained from natural organisms, and then synthesizing the glutathione step by step by the two synthetases, and both of these two reactions need to be added with the addition of adenosine triphosphate (ATP) to provide energy[10] . Both reactions require the addition of adenosine triphosphate (ATP) for energy[10] . Therefore, although the enzymatic synthesis of glutathione is more efficient, it is easily affected by factors such as enzyme activity and ATP price.

2.4 Microbial Fermentation

Compared with extraction, chemical synthesis and enzymatic methods, the production of glutathione by fermentation has the advantages of fast production rate, mild reaction conditions, low cost and less pollution, and has become the main method of glutathione production with great potential for development.

There are two types of strains for glutathione production, one is the high-yielding strains obtained from conventional mutation breeding, such as Saccharomyces cerevisiae, Candida utilis, and Pseudomonas fluorescens [11-13]. The second type is genetically engineered bacteria (GEBs), which use recombinant technology to introduce the genes for glutathione synthesis into another microorganism for glutathione production, and nowadays, most of them are genetically engineered bacteria using Saccharomyces cerevisiae or Escherichia coli as the carriers [14-15].

In addition to strains, the factors affecting the production of glutathione by microbial fermentation include microbial fermentation control technology and glutathione purification technology. Microbial fermentation control technology includes the optimization of culture medium, culture conditions, and the control of the fermentation process; and glutathione purification technology includes solvent extraction, hot water extraction, and high-pressure homogenization[16]. Nowadays, the technology of microbial fermentation for the production of glutathione is relatively mature, and it is capable of large-scale factory production and has great potential for development.

3.Application of Glutathione in Aquatic Animals

3.1 Application of Glutathione in Growth Performance of Aquatic Animals

The application of glutathione in the growth performance of aquatic animals, such as Eriocheir sinensis [17], Mylo-pharyngodon piceus Richardson [18] and Litope- naeus vannamei [19], has shown that glutathione has a growth-promoting effect on aquatic animals. Studies in aquatic animals, such as Litope-naeus vannamei [19], have shown that glutathione has a growth-promoting effect. The growth-promoting effects of glutathione on aquatic animals may be multifaceted. Firstly, the addition of glutathione to feed has a positive effect on the endocrine secretion of aquatic animals, which can up-regulate the expression of insulin-like growth factor I and growth hormone genes, thus promoting the secretion of growth hormone and thyroid hormone, and thus further promoting the growth of aquatic animals.

Ming et al.[20] showed that the addition of 407.45 mg/kg GSH to feed could promote the expression of insulin-like growth factor I in the liver of grass carp (Ctenopharyngodon idella) and increase the weight gain rate of grass carp. Secondly, glutathione contains cysteine, which is one of the components of coenzyme a. It can break the disulfide bond of growth inhibitory hormone molecules, alleviate the inhibitory effect of growth inhibitory hormone on growth hormone, and thus promote the growth of grass carp.21 Xiao et al.[22] found that the peritoneal injection of cysteamine hydrochloride (CSH) into grass carp significantly increased the level of serum growth hormone, and then promoted the short-term growth of juvenile grass carp. In addition, GSH can be used to enhance the short-term growth of juvenile grass carp.

In addition, GSH can promote the growth of aquatic animals by enhancing the absorption of nutrients. Feng Gupan et al.[23] showed that the addition of 0.30 g/kg of GSH to the feed decreased the feed coefficient and increased the body weight of Procambarus clarkii. In rainbow trout (Oncorhynchus mykiss), the addition of 200 mg/kg of GSH to the diet was shown to increase intake, weight gain and specific growth rate.24 Wang et al.[19] showed that the addition of 75-150 mg/kg of GSH increased the feed intake and weight gain of Litope naeus vannamei. - Litope naeus vannamei (Litopenaeus vannamei) gut wall thickness and specific growth rate. The addition of 320 mg/kg GSH to the feed significantly increased the protein efficiency of GIFT Oreochromis niloticus (tilapia), thus promoting growth [25].

3.2 Application of Glutathione in Anti-Oxidative Stress in Aquatic Animals

With the continuous development of intensive aquaculture, there are more and more factors that cause stress in aquatic animals. Oxidative stress is caused by an increase in the content of oxygen free radicals in the body, which destroys the balance between oxidation and antioxidants in the body, thus forming oxidative stress[26-27]. Oxidative stress reduces the immunity of aquatic animals, causes tissue damage, slows down growth, and ultimately reduces the yield and quality of aquatic products.

As an endogenous antioxidant, glutathione plays an important role in the antioxidant system of the organism. Firstly, glutathione is the most abundant non-protein sulfhydryl compound in the organism, and the sulfhydryl group of GSH can provide a reducing electron directly to the free radicals to reduce them to non-toxic substances, and then react with another active glutathione to form GSSG, which can achieve the effect of reducing the free radicals, and maintain free radical metabolism in the organism[28]. In addition, since GSH and GSSG can be interconverted in the body, they can be used as redox buffers to maintain the balance of cellular redox state[29].

The addition of GSH to feed can increase the accumulation of GSH in aquatic animals, thereby reducing the level of oxidative stress in the body, increasing the activity of antioxidant enzymes, and enhancing the antioxidant capacity of the body. Studies in turbot (Scophthalmus maximus), Chinese crabs, mackerel, South American white shrimp, seabass (Lateolabrax japonicus) and other aquatic animals have shown that the addition of appropriate amounts of GSH to feeds can increase the antioxidant activity in the body and enhance the antioxidant capacity [5, 17-18, 30-31]. However, excessive glutathione may hurt the antioxidant capacity of aquatic animals, which may be due to the high concentration of glutathione causing DNA damage, resulting in its pro-oxidant effect [32-33].

3.3 Application of Glutathione in Aquaculture Animals' Immune Performance

In the process of aquaculture, high farming density, feed oxidation and acidification, and deterioration of the aquatic environment have led to a continuous decline in the immune performance of aquaculture animals and a significant increase in the morbidity rate [34].GSH can improve the immune performance of aquaculture animals in many ways, first of all, the decline in immunity in the animal's body is often related to oxidative stress, and GSH, as an important antioxidant in the body, can be involved in the removal of excess free radicals to inhibit oxidative stress and improve immune performance [35]. As an important antioxidant substance in the body, GSH can directly participate in the scavenging of excess free radicals, thus inhibiting oxidative stress and improving immune performance [35].

Secondly, GSH can enhance the immune function of the body by regulating the active factors in the animal body. Zhou Yanling et al. [36] showed that the addition of 357.69 mg/kg GSH in the feed could increase the levels of lysozyme (LZM), alkaline phosphatase (AKP) and acid phosphatase (ACP) in the juvenile Pelteobagrus fulvidraco [37].  LZM, alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in larval fish, and increased immunoglobulin M and complement 4 levels. In the study of Eriocheir sinensis, it was shown that the addition of 600 mg/kg or 900 mg/kg of GSH to the feed could increase the activities of immunoenzymes such as lysozyme, reduce the activities of Toll like receptor 1 (TLR1), Toll like receptor 2 (TLR2), and TLR2 (TLR2), and increase the levels of immunoglobulin M and complement 4. Toll like receptor 1 (TLR1), Toll like receptor 2 (TLR2) and myeloid differentiation factor 88 (Myd88) to improve the immunity performance of Eriocheir sinensis[17] .

Xue et al.[21] showed that the addition of 500 mg/kg of glutathione to the feed could improve the immune performance of Chinese mitten crab (Eriocheir sinensis) by regulating the expression of Interleu - kin-1β (IL-1β), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α, TNF-α, TNF-α, TNF-α, TNF-α, and other immune factors[22] . The expression of inflammation-related genes such as Interleukin-6 (IL-6), Interleukin-6 (IL-6) and Tumor necrosis factor-α (TNF-α) reduces gill and liver inflammation caused by ammonia stress, and enhances the immunity of common carp (Cyprinus carpio).

Glutathione can enhance the vitality of immune cells and promote the proliferation of immune cells. Tests in Oreochromis ni- loticus × O. aureus showed that exogenous addition of appropriate amounts of GSH could increase the respiratory burst of macrophages in the head of juvenile Oreochromis ni- loticus, thus participating in immunomodulation[37] . Zhao Hongxia et al.[38] showed that the number of leukocytes in the blood of grass carp increased with the increase of GSH in the feed, which enhanced the non-specific immune function of grass carp and strengthened their ability to resist diseases.

3.4 Application of Glutathione in Detoxification of Aquatic Animals

The detoxification mechanism of GSH consists of two aspects: on the one hand, as an important antioxidant, GSH can scavenge excess free radicals in the organism; on the other hand, GSH can inhibit lipid peroxidation through some enzymes, to reduce the adverse effects of harmful substances caused by oxidative stress, and attenuate their toxicity. Zhou Yanling et al.[39] showed that the addition of 357.69 mg/kg GSH to the diet of juvenile Pelteobagrus fulvidraco was able to improve its antioxidant ability and increase the stress resistance to ammonia and nitrogen. In grass carp, it was shown that the addition of 407.45 mg/kg GSH could increase the activity of antioxidant enzymes, enhance the antioxidant capacity of the organism, and reduce the damage caused by microcystins, but the excessive amount of GSH also had a negative effect[20] . Studies in Oreochromis niloticus showed that GSH could alleviate lipid peroxidation, improve liver function and alleviate aflatoxin poisoning [40].

On the other hand, GSH can bind with various toxic substances and their metabolites to form non-toxic adducts, which are then excreted from the animal. Studies in Pelteobagrus fulvidraco have shown that glutathione can bind to microcystins to form more hydrophilic products, which can be excreted more easily[41] . Ren SJ et al.[42] showed that glutathione could form intermediate products with phoxim, thus reducing the residue value of phoxim in the liver of Carassius au-ratus gibelio. Experiments in Nile tilapia showed that the addition of a certain concentration of lead to the feed caused an adaptive increase in the GSH content in the hepatopancreas and a decrease in the GSH content with the increase in the lead content, which may be attributed to the ability of GSH to form complexes with metal ions, and then these complexes will be aggregated and rapidly excreted from the body, thus achieving the effect of rapid detoxification [43-44].

4.Summary and Outlook

Glutathione can be produced in various ways, among which chemical, enzymatic and biofermentation methods have been industrially applied, laying a good foundation for the application of glutathione in aquaculture.GSH has a great potential to be used in the healthy aquaculture of aquatic animals as well as the improvement of the quality of aquatic products. Researchers at home and abroad have conducted in-depth studies on the application of GSH in aquaculture, especially in aquatic animal feed. However, the development and utilization of glutathione is limited by its water-soluble and low pH properties. Therefore, in the future research, the optimization of glutathione feed processing could be strengthened and the cooperation between GSH and other nutrients could be explored to achieve the efficient utilization of glutathione.

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