Effect of Honey on the Maintenance of Taste Components in the Chub Mackerel Scomber Japonicus (Houttuyn, 1782) View PDF

Chub mackerel are small fish that migrate over a wide area and contribute to a good catch [1]. Chub mackerel are consumed throughout the world because of their good taste. Similar to that, in other fish, the major taste component of chub mackerel is inosinic acid (IMP), which is generated by the degradation of ATP-related components as follows: ATP→ADP→AMP→IMP→HxR→Hx.

ATP is degraded to IMP relatively quickly, and then, IMP accumulates in the fish muscle [2]. However, IMP is degraded to nontaste components inosine (HxR) and hypoxanthine (Hx) by IMPdegrading enzymes (IMPases) over time. Therefore, IMPase activity should be inhibited to maintain the taste of fish. IMPases consists some types of enzymes, and their composition and structure depend on fish species [3]; thus, IMPase in each fish species needs to be investigated. In addition, the conditions under which inhibit IMPase activity is inhibited in chub mackerel should also be investigated to preserve the taste.

Chub mackerel is often stored in liquid seasoning, brine, vinegar, miso, or sweet sake. Salt has been reported to inhibit IMPase activity in chub mackerel [2]; thus, processing methods using salt or liquid seasoning including salt can preserve the taste of chub mackerel. Sugar is often added to vinegar, miso, or sweet sake liquid seasoning. However, the effect of sugar on IMPase activity in chub mackerel has not been reported in detail. The main component of sugar is sucrose, which inhibits IMPase activity in the chicken grunt Parapristipoma trilineatum (Thunberg, 1793), Japanese amberjack Seriola quinqueradiata (Temminck & Schlegel, 1845), and Pacific cod Gadus macrocephalus (Tilesius, 1810) [4]. Fructose has been reported to inhibit sucrase activity in rats [5]; thus, it is expected that these saccharides will also have an inhibitory effect on IMPase activity in chub mackerel. Honey contains glucose and fructose and is one of the sugar-rich products. Honey has been reported to inhibit acetylcholinesterase, which is associated with Alzheimer’s disease [6]. It also inhibits the activity of angiotensin I-converting enzyme [7]. Therefore, honey is expected to inhibit IMPase activity in chub mackerel. Hydroxymethylfurfural (HMF), which is generated by heating honey, has been reported to exert enhanced immunostimulatory effects [8]. Melanoidin, the color component in honey, is reported to possess antioxidant activity [9]. Furthermore, HMF inhibits an increase in the growth of ethanolgenerating yeast [10]. Similarly, melanoidin inhibits carboxypeptidase [11], angiotensin I-converting enzyme, and lipase activities [12]. Therefore, it is expected that these components could also inhibit IMPase in chub mackerel. To the best of our knowledge, there have been no studies on the inhibitory effect of honey on the activity of IMPase. Therefore, in this study, we aimed to examine the effect of honey and sugar on IMPase activity in chub mackerel. Furthermore, we investigated the effects of individual components of honey, HMF, and melanoidin on IMPase activity in chub mackerel.

Materials

Chub mackerels were purchased in retail from a local market near the Tamagawa University, and Japanese Pure Hundred Flower Honey (Ou Youhouen Co., Ltd., Akita, Japan) was used for the subsequent experiments. IMPase was then extracted as an enzyme solution from the fish samples. White meat chub mackerel samples were collected and homogenized in three volume equivalents of water. The homogenate was dialyzed against ultra-pure water for 2 days at 10°C; then, the dialysate was filtered (No. 1; Advantec Co., Ltd., Tokyo, Japan) and diluted twice to adjust the concentration of the enzyme solution. This enzyme solution was then passed through a 0.20-µm filter to remove all bacteria

Methods

Effect of honey on IMPase activity in chub mackerel: IMPase activity was determined using standard reaction mixtures. A 3.5-mL reaction mixture comprising 0.5 mL of IMP (25 mM), 2 mL of buffer (50 mM maleic acid/Tris/NaOH, pH 6.0), 0.5 mL of honey prepared at 2-10-fold dilutions, and 0.5 mL of enzyme solution was incubated at 30°C for 24 h, and the reaction was stopped by adding 1.5 mL of 10% perchloric acid. The resulting precipitate was separated by centrifugation (13,040 × g, 5 min), and the level of free phosphoric acid was determined using the molybdenum blue method. The amount of phosphoric acid was considered to reflect the enzyme activity [13].

Effect of honey, glucose, fructose, and sugar on IMPase activity in chub mackerel:A 3.5-mL reaction mixture comprising 0.5 mL of IMP (25 mM), 2 mL of buffer (50 mM maleic acid/Tris/NaOH, pH 6.0), 0.5 mL of honey (prepared at 2-10-fold dilution) or 8%-41% of glucose, fructose, glucose + fructose (1:1), or sugar (sucrose) [14], and 0.5 mL of enzyme solution was incubated at 30°C for 24 h. The reaction was stopped by adding 1.5 mL of 10% perchloric acid. IMPase activity was determined as described above.

Effect of HMF on IMPase activity in chub mackerel: A 3.5-mL reaction mixture comprising 0.5 mL of IMP (25 mM), 2 mL of buffer (50 mM maleic acid/Tris/NaOH, pH 6.0), 0.5 mL of 1%-10 % HMF (0.5 mL), and 0.5 mL of enzyme solution was incubated at 30°C for 24 h, and the reaction was stopped by adding 1.5 mL of 10% perchloric acid. IMPase activity was determined as described above.

Effect of melanoidin on IMPase activity in chub mackerel: First, a model melanoidin solution was prepared. Briefly, 0.5 g each of the L-isotopes of cysteine, glutamine, glutamic acid, isoleucine, leucine, proline, serine, tryptophan, tyrosine, and valine, along with 50 g of glucose were dissolved in 50 mL of distilled water, and this solution was autoclaved (121°C, 15 min). A 3.5-mL reaction mixture comprising 0.5 mL of IMP (25 mM), 2 mL of buffer (50 mM maleic acid/Tris/ NaOH, pH 6.0), 0.5 mL of model melanoidin solution, and 0.5 mL of enzyme solution was incubated at 30°C for 24 h, and the reaction was stopped by adding 1.5 mL of 10% perchloric acid. IMPase activity was determined as described above.

Statistical Analysis

Data were subjected to one-way analysis of variance using the least significant difference method. T-tests were used for pairwise comparisons; statistical significance was set at p < 0.05 by Microsoft Excel.

Effect of honey on IMPase activity in chub mackerel

Figure 1 shows the effect of honey on IMPase activity in chub mackerel. The IMPase activity without the addition of honey was 28 PO4 ng?g-1min-1. However, the addition of honey inhibited the IMPase activity. Interestingly, the extent of inhibition reduced with increasing dilution of honey (2-10-fold dilution of honey [p < 0.05]), as demonstrated by the increased IMPase activity (4.7 to 18 PO4 ng?g-1min-1). The white column shows IMPase activity with no addition of honey and the grey columns show IMPase activity in the presence of honey diluted 2–10-fold (n = 3).

Effect of honey, glucose, fructose, and sugar on IMPase activity in chub mackerel

Figure 2 shows the effect of glucose, fructose, glucose + fructose (1:1), and sugar on IMPase activity in chub mackerel. The IMPase activity without any addition was 65 PO4 ng?g-1min-1. However, the addition of honey inhibited the IMPase activity. Interestingly, the extent of inhibition reduced with increasing dilution of honey (2-10- fold dilution of honey [p < 0.05]), as demonstrated by the increased IMPase activity (8.2 to 44 PO4 ng?g-1min-1). Furthermore, the IMPase activity increased from 31 to 48 ng?g-1min-1 with glucose, from 4.3 to 43 ng?g-1min-1 with fructose, from 1.1 to 42 ng?g-1min-1 with glucose + fructose (1:1), and from 34 to 69 ng?g-1min-1 with sugar, as the final concentration of these saccharides decreased from 5.9% to 1.1% (p < 0.05). There was no difference in IMPase activity between the addition of sugar at a final concentration of 1.1% and no addition of sugar (p > 0.05); however, all other test concentrations showed low enzyme activity compared with the activity without any additions (p < 0.05). Glucose and sugar addition at a final concentration of 5.9% showed higher activity than that with honey addition (p < 0.05), whereas the addition of fructose and glucose + fructose (1:1), at the same final concentration, showed lower activity than that with the addition of honey (p < 0.05). At a final concentration of 2.3%, a higher activity was observed with glucose and sugar addition than that with honey addition (p < 0.05). However, there was no significant difference in activities between the addition of fructose or glucose + fructose (1:1) and the addition of honey (p > 0.05). Similarly, at a final concentration of 1.1%, a higher activity was observed with sugar addition than with honey addition (p < 0.05). However, there was no significant difference in enzyme activity with the addition of glucose, fructose, or glucose + fructose (1:1) compared to that with the addition of honey (p > 0.05).

Effect of HMF on IMPase activity in chub mackerel

Figure 3 shows the effect of HMF on IMPase activity in chub mackerel. The IMPase activity without any addition was 45 PO4 ng?g1 min-1. However, with the addition of HMF, the IMPase activity varied from 32 to 38 PO4 ng?g-1min-1 with final concentrations of HMF ranging from 0.14-1.4%, and no differences were observed among final HMF concentrations ranging from 0.14%-1.4% (p > 0.05). The addition of HMF resulted in lower IMPase activity that that without any addition (p < 0.05). varied between 21 and 26 PO4 ng?g-1min-1 with final concentrations of melanoidin between 0.057% and 0.11%, and there were no differences in IMPase activity at final melanoidin concentrations of 0.057% to 0.11% (p > 0.05); however, the IMPase activity decreased to 16 PO4 ng?g-1min-1 at a final melanoidin concentration of 0.14% (p > 0.05). The IMPase activity upon melanoidin addition at all tested concentrations was lower than that with no addition (p < 0.05).

Model melanoidin was prepared using 0.5 g each of L-cysteine, L-glutamine, L-glutamic acid, L-isoleucine, L-leucine, L-proline, L-serine, L-tryptophan, L-tyrosine, and L-valine and 50 g of glucose. They were dissolved in 50 mL of distilled water (n = 3).

Effect of honey on IMPase activity in chub mackerel

Honey affects the activity of several enzymes. The inhibition of angiotensin I-converting enzyme by honey [7]; inhibition of α-glucosidase by the polyphenols in honey [15]; and increased serum cardiac marker enzymes (creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and aspartate transaminase (AST)) in rats by honey [16] have been reported. Similarly, in this study, it was demonstrated that honey inhibited IMPase activity. 

Effect of honey, glucose, fructose, and sugar on IMPase activity in chub mackerel

Sucrose has been reported to inhibit IMPase activity in the chicken grunt, Japanese amberjack, and Pacific cod. The IMP decomposition rate significantly decreased from 60% to 40% when sucrose increased from 2% to 4% in the chicken grunt and Pacific cod [4]. Similarly, when sucrose was added to Japanese amberjack fish meat, the 5’-NTase activity was inhibited with increasing sucrose concentration [17]. In this study, although IMPase activity was inhibited with increasing sugar concentration, showing a similar trend to that in previous reports, sugar had the lowest inhibitory effect among all examined saccharides. Fructose showed a stronger tendency to inhibit IMPase activity than glucose. Consistent with this result, higher inhibition of enzyme activity has been reported with fructose than with glucose for peroxidase in apples and carrots [18] and for glucokinase-like enzyme activity in the liver of Atlantic salmon (Salmo salar) [19]. However, fructose did not show strong inhibition of strawberry polyphenol oxidase and peroxidase activities [20]. This suggests that the effects of glucose and fructose may differ depending on the enzymes. Fructose is a typical competitive inhibitor and glucose is a classical non-competitive inhibitor of invertase in Tropaeolum majus leaves [21]. Similarly, in Saccharomyces cerevisiae, partial non-competitive inhibition, and competitive inhibition of invertase by glucose and fructose, respectively have been reported [22]. Differences in the inhibition mechanisms of sugars in Pacific cod result in differences in the inhibitory effect of sugars on IMPase activity [23]. Similar differences in the inhibitory effect on IMPase activity was observed in this study, which could also be due to variations in the inhibition mechanisms. 

Effect of HMF on IMPase activity in chub mackerel

HMF is a heterocyclic aldehyde formed during the Maillard and caramelization reactions. It is the most consistent marker for honey freshness or quality deterioration and is used in national, regional, and international standards as a quality control parameter because its quantity increases upon heat treatment and storage. Furthermore, HMF causes cytotoxicity in humans through reduced granulocyte metabolism [24]. Therefore, the maximum limit for this parameter established by international standards is 40 mg?kg-1, and in honeys of tropical origin, the European directives, and the Codex Alimentarius allow a maximum HMF concentration of 80 mg?kg-1 [25]. In this study, although we examined the effect of HMF on IMPase activity at extremely high concentrations, we did not observe a substantial inhibitory effect. HMF promotes the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase, and inhibits the pyruvate dehydrogenase complex in the white-rot fungus Trametes versicolor [26]. HMF from instant coffee selectively inhibits the activities of mammalian DNA polymerase λ and terminal deoxynucleotidyl transferase; whereas, it does not influence the activities of replicative DNA polymerases such as α, δ, and ε, and DNA polymerase β [27]. According to these reports, HMF shows different effects on different enzymes. Here, the IMPase activity of chub mackerel was found to be slightly inhibited by HMF.

Effect of melanoidin on IMPase activity in chub mackerel

Melanoidin has been reported to inhibit carboxypeptidase A and B enzymes, with their maximum reaction rate decreasing with increasing melanoidin concentration [11], similar to that in the present study. Melanoidin has been found to exert an adverse effect on the activity of α-amylase, and its inhibitory effect increases with increasing molecular weight of melanoidin [28]. Furthermore, the enzyme inhibitory effect may depend on the constitution of melanoidin, as Maillard reaction products formed from the reaction of glucose or fructose with glutathione reportedly show greater inhibition of purified apple polyphenol oxidase activity than those formed from the reaction of hexoses with cysteine [29]. The composition of melanoidin in honey is not uniform, as it depends on the type of source flower. Melanoidin from buckwheat honey has been reported to contain multi-component polymers including the protein-polyphenol-oligosaccharide complex [9]. However, as buckwheat honey has a higher polyphenol content than honey from other nectar sources [30], polyphenols are thought to be one of the components of melanoidin. As the content of polyphenols in the Japanese Pure Hundred Flower Honey used in this study was considerably lower than that in buckwheat, melanoidin in the honey used in this study was considered general melanoidin consisting of sugar and amino acids. In this study, we investigated the effect of a model melanoidin on IMPase activity and observed that the IMPase activity decreased with increasing melanin concentration. As honey contains high-molecular weight melanoidin (65–235 kDa) [9,31], it could be one of the main components that inhibit IMPase activity. This study had some limitations. It is difficult to handle actual samples of both honey and mackerel because of their large individual differences.

In this study, we investigated the effects of honey and the major saccharides in honey, HMF, and melanoidin on IMPase activity for maintaining the taste components in chub mackerel. Fish and honey have differences in their taste. With the addition of honey, the IMPase activity decreased with increasing honey concentration, indicating that honey has an inhibitory effect on IMPase activity. Similarly, high IMPase activity inhibition was observed with fructose, a component of honey. With HMF addition, inhibition of IMPase activity was observed. However, there was no correlation between the increase in HMF concentration and inhibition of IMPase activity, and this inhibition was relatively low. The enzyme activity decreased with increasing melanoidin concentration. These results indicate that honey has an inhibitory effect on IMPase activity in chub mackerel, mainly due to fructose and melanoidin. Thus, honey is an effective ingredient for processing chub mackerels.

It is difficult to identify the specific component of honey responsible for inhibiting the activity of IMPase in chub mackerel, as it is a complex mixture of various components. However, saccharides are the main components of honey; thus, we studied the effect of major polysaccharides in honey on the activity of IMPase. The results indicated that they exerted the greatest inhibitory effect on IMPase activity. However, the components of honey depend on the type of pollen collected. Therefore, in the future, we will investigate the effect of various types of honey on IMPase activity and clarify the effect of honey on the taste components of chub mackerel in greater detail.

HMF: hydroxymethylfurfural

Hx: hypoxanthine

HxR: inosine

IMP: inosinic acid

IMPase: inosinic acid degrading enzyme

We thank Dr. Jun Nakamura. This work was supported by the Mishima Kaiun Memorial Foundation.

The authors declare that they have no conflicts of interest.

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