Evaluation of Antifreeze Activity of Enzyme-Treated Extract from Mushroom Cell Wall
Article ID: 7202102
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Article ID: 7202102
Ice formation and growth during freezing in processed foods containing water can deteriorate food quality. Naturally derived antifreeze proteins and antifreeze polysaccharides are an attractive solution to this problem. Alkaline extracts from the basidiomycete Flammulina velutipes (enokitake) are known to inhibit ice crystal growth and are expected to maintain frozen food quality. In this study, polysaccharides/oligosaccharides (POS) were obtained from the readily available edible mushrooms F. velutipes, Hypsizygus marmoreus, Pleurotus eryngii, and Grifola frondosa. POS extracts were isolated by treatment of the fruiting mushroom body with the cell wall-lytic enzyme Uskizyme, then precipitated by ethanol addition. All POS showed antifreeze activity by suppressing ice crystal growth. The benefit of the POS isolated from enzyme-treated edible mushrooms towards frozen processed products quality and shelf-life for foods containing egg protein, fish protein, and rice starch was evaluated. POS derived from F. velutipes was effective in maintaining egg protein (chawanmushi) quality. For fish protein (surimi), the POS derived from F. velutipes and G. frondosa mushrooms suppressed freezing-induced increases in hardness and elasticity. However, for rice starch (shiratama), none of the POS had any effect in preventing retrogradation. This study is the first report to show that components obtained from mushroom cell walls by enzymatic treatment can be effectively used to improve the physical properties of foods. These results suggesting the possibility of new applications for mushrooms as potential cryoprotectants in the frozen food industry.
IBPs, ice-binding proteins; IRI, ice recrystallization inhibition; SFHE, soluble fraction by hot water extraction; IFHE, insoluble fraction by hot water extraction; ETF, enzyme-treated fraction; POS, poly-/oligosaccharides; PEG, polyethylene glycol
The food industry relies upon freezing as an important process to prevent food deterioration. Ensuring that foods maintain excellent condition throughout storage and transport is key to minimizing economic losses due to food spoilage. However, freezing as a method of preservation does have drawbacks, in some cases reducing food quality and appearance. This food quality reduction often arises due to the water-ice phase change during freezing and storage. To address this, cryoprotective food additives are used to minimize spoilage. For example, sugars such as trehalose, sugar alcohols such as sorbitol, phosphate, lipids, and glycerol are known to have cryoprotective effects. However, these additives may bring their own nutritional problems, such as high caloric content and nutrient absorption inhibition [1, 2].
Naturally derived antifreeze agents, including glycoproteins and polysaccharides, are an attractive option to mitigating food spoilage. These compounds are found in fish [3, 4, 5], insects [6, 7], plants [8], microorganisms [9], fungi [10], and their extracts [11, 12], and are potential means to maintain frozen food quality. Of the possible sources, microorganisms and fungi are promising due to their short growth cycles and high potential yields. Psychrophilic basidiomycetes, Typhula ishikariensis, is particularly attractive as it secretes an abundant number of ice-binding proteins (IBPs) to maintain the extracellular environment under freezing conditions. The molecular weights and amino acid sequences of these IBPs differ to fish-derived antifreeze proteins, with some inducing more potent antifreeze effects [13]. One major edible mushroom, Flammulina velutipes (enokitake), is a psychrophilic organism able to grow under snow cover. The antifreeze polysaccharides extracted from F. velutipes by alkaline solution extraction of the hot water treatment residue from mycelia and fruit body, show strong ice recrystallization inhibition (IRI) activity and are composed of mannose and xylose [14].
The primary components of mushroom cell walls are carbohydrates, such as β-1,3-/1,6- and β-1,4-glucan, heteropolysaccharides, such as xylo-mannan, and chitin, as well as proteins and glycan-protein complexes [15, 16]. Chen and Cheung analyzed components of four fractions extracted from the Pleurotus tuber-regium fruit body, isolated by boiling water and alkaline treatment, proposing a four-layer mushroom cell wall [17]. Interactions among these components are quite complex, with some requiring hot water to solubilize, while others require sufficient alkali treatment. Thus, completely breaking down the mushroom cell wall into its individual components or extracting the components is difficult.
In this study, we focus on the hot water mushroom extract residue. Hot water extracts of mushrooms are used as functional materials, with anti-cancer properties for example, but these residues are mostly disposed of. Therefore, we evaluated the action of enzymes on this residue to extract polysaccharides and/or oligosaccharides contained in the mushroom cell walls then evaluated their antifreeze activity. Also, the effect on quality retention during freezing was examined for egg protein, fish protein, and rice starch.
Materials. Four edible commercially available mushrooms, Flammulina velutipes (enokitake), Hypsizygus marmoreus (buna-shimeji), Pleurotus eryngii (eringi), and Grifola frondosa (maitake), were used in this study. Uskizyme (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan), which contains β-1,3- and β-1,6-glucanase and chitinase activities [18, 19], was used to enzymatically degrade the mushroom cell wall.
Preparation of mushroom cell wall extract. The fresh mushroom fruit body (500 g) was finely cut, added to 3 L of deionized water, then boiled for 10 min. The soluble fraction by hot water extraction (SFHE) was then collected using 100-mesh nylon bag to separate the liquids from solids. This was repeated five times with the resulting liquid combined and lyophilized. The remaining solid insoluble mushroom material was homogenized using a blender, and the fraction passing through a 32-mesh sieve used as the insoluble fraction by hot water extraction (IFHE). After suspending IFHE in deionized water, 0.1 % Uskizyme was added and allowed to react for 24 h at 40 °C. The enzyme was inactivated by placing in a boiling bath for 10 min, then reaction mixture filtered through glass fiber filter paper GS-25 (Advantec Toyo Kaisha, Ltd., Tokyo, Japan). The isolated solid was washed three times with deionized water, and the filtrate used as the enzyme-treated fraction (ETF). After small molecule removal from the filtrate by dialysis (MWCO 3,500), the filtrate was then lyophilized to yield enzyme-extracted poly-/oligosaccharides (POS). The flow of POS preparation is shown in Fig. 1.
Analysis of monosaccharide composition and uronic acids in POS. POS monosaccharide composition and uronic acid levels were assessed by HPLC analysis after hydrolysis with dilute sulfuric acid [20, 21]. Hydrolysis was performed with 0.3 M sulfuric acid at 120 °C for 60 min, followed by neutralization with saturated barium hydroxide. The analytical column used was an Asahipak NH2P-40 3E (3.0 mm × 250 mm) (Shodex; Resonac Corporation, Tokyo, Japan), and ribose, fucose, xylose, mannose, glucose, galactose, glucose, and glucuronic acid were used as standards.
Analysis of molecular weight distribution of POS. POS molecular weight distributions were measured by HPLC using an OHpak SB-806M HQ (8.0 mm × 300 mm) (Showa Denko K.K., Tokyo, Japan) and an index refractometer. Pullulan (STANDARD P-82, Shodex; Resonac Corporation) was used as a standard.
IRI assay. IRI assays were performed using a partially modified sucrose sandwich method [8, 22] with polyethylene glycol (PEG) 3400 as the control. Each POS or PEG 3400 was dissolved in 23 % sucrose solution to a final concentration of 1.0 mg/mL. These solutions were sandwiched between two clean microscope cover glasses, then placed on a thermal stage 10021 (Japan High Tech Co., Ltd., Fukuoka, Japan). The sandwiched sample temperature was programmed (Linkam Scientific Instruments Ltd., Redhill, Surrey, United Kingdom) to cool from room temperature to −20 °C at a rate of −20 °C/min, hold for 30 s, and then heat to −6 °C at the same rate. Ice crystal growth at −6 °C was observed for 40 min using an optical microscope B51 (Olympus Corporation, Tokyo, Japan), and analyzed using ImageJ (Media Cybernetics, Inc., Silver Spring, MD, USA). IRI evaluation was performed by comparing average ice crystal size between test and control samples.
Physical property analysis of various foods with POS. Whole eggs were homogenized and filtered through a colander (32 mesh). To make the sample mixture, 6.7 g of the whole egg mixture and 0.05 g of salt was added to 13.25 g of deionized water and mixed. POS was added at a final concentration of 0.1 w/w%. After filling the container, the mixture was heated in a steam convection oven at 100 °C for 20 min to make savory egg custard (chawanmushi). These samples were frozen, stored at −18 °C for 4 weeks, then thawed overnight in a refrigerator at 4 °C. Sample physical properties were measured and texture analyzed using a creep meter RE2-33005C and software TAS-3305 ver.2.5 (Yamaden Co. Ltd., Tokyo, Japan) with an 8-mm diameter cylindrical plunger, a compression penetration speed of 10 mm/s, a clearance of 30 %, and evaluated by texture analysis (n = 3). The elasticity was calculated from the breaking distance (mm) when the sample was penetrated twice consecutively, as the ratio of the first and second penetration relative to the control. The breakage point at that time was also measured as the force (N).
Alaska pollack was prepared by filleting and skin removal, leaving only the flesh. After using a mortar and pestle to homogenize 100 g of the flesh, 3 g of salt was added and kneaded to make fish paste (surimi). POS was then added to the test sample at a final concentration of 0.1 w/w%. The surimi was packed into a 3-cm diameter cylindrical container. After heating in a steam convection oven at 40 °C for 20 min, the mixture was further steam-heated at 90 °C for 30 min to make fish cake. After cooling, the sample was cut into 3 cm lengths. Samples were frozen, stored at −18 °C for 4 weeks, then thawed overnight in a refrigerator at 4 °C. Sample strength was measured using the equipment described above. Measurements were made using a 5-mm diameter spherical plunger, a compression penetration rate of 1 mm/s, and a measured strain rate of 99 % (n = 3), and the force (N) and distance (mm) at the breaking point were calculated using the fracture strength analysis software BAS-3305 ver.2.5 (Yamaden Co. Ltd., Tokyo, Japan).
White rice flour (shiratamako) and glutinous rice flour (joshinko), 10 g each, were combined with 18 g of deionized water then kneaded. POS was added to the test sample at a final concentration of 0.1 w/w%. After adjusting to 6 g, this was heated in a steam convection oven at 100 °C for 20 min to make rice flour dumpling (shiratama). Samples were frozen, stored at −18 °C for 4 weeks, then thawed overnight in a refrigerator at 4 °C. Sample hardness was measured using the above equipmen with an 8-mm diameter cylindrical plunger, a compression penetration speed of 1 mm/s, and a measured strain rate of 99 % (n = 3), and was calculated from the penetration distance (mm) when a force of 20 N was applied from the sample contact point using the fracture strength analysis software BAS-3305 ver.2.5 (Yamaden Co. Ltd., Tokyo, Japan).
Statistical analysis. Data is presented as average values from independent experiments. Student’s t-test was used when group variance was considered within 5 %. When compared groups were clearly different, Welch’s t-test was used. Differences were evaluated as significant when p < 0.05, and trend considered significant when p < 0.1.
Extraction of POS from mushroom cell wall. For all four mushrooms, the moisture content of the undried mushroom fruit body was about 90 %, with G. frondosa having the lowest solids content at 7.3 %. IFHE, the component of solids not solubilized by hot water treatment, was 38.2 %, 57.8 %, 46.7 %, and 61.5 % of solids in F. velutipes, H. marmoreus, P. eryngii, and G. frondosa, respectively. SFHE, the hot water extracted component, was greater for F. velutipes, but lowest for G. frondosa. Next, the ETF solubilized by enzymatic degradation of IFHE was less than 50 % for all mushrooms, with P. eryngii the highest, yielding 43.7 %. Furthermore, the recovery of POS by ethanol precipitation was about 30 %. The mass of each fraction obtained from 100 g of unseasoned mushroom is shown in Table 1.
Table 1. Material balance (g) by enzyme-treated per 100 g of fruit body.
| Mushroom | Moisture | Solid contents | IFHE derivatives | POS | |||
| SFHE | IFHE | total | ETF | Residue | |||
| F. velutipes | 89.0 ± 0.61 | 6.80 ± 0.05 | 4.20 ± 0.05 | 11.0 | 1.52 ± 0.01 | 2.68 ± 0.01 | 0.44 ± 0.01 |
| H. marmoreus | 91.5 ± 0.85 | 3.59 ± 0.04 | 4.91 ± 0.04 | 8.5 | 1.39 ± 0.02 | 3.52 ± 0.02 | 0.04 ± 0.01 |
| P. eryngii | 90.0 ± 0.88 | 5.33 ± 0.03 | 4.67 ± 0.03 | 10.0 | 2.04 ± 0.17 | 2.63 ± 0.17 | 0.56 ± 0.02 |
| G. frondosa | 92.7 ± 0.96 | 2.81 ± 0.03 | 4.49 ± 0.03 | 7.3 | 1.18 ± 0.08 | 3.31 ± 0.08 | 0.23 ± 0.01 |
Monosaccharide composition and molecular weight distribution of POS. Table 2 shows relative percentages of identified monosaccharides produced by POS acid hydrolysis. For all mushrooms, the main monosaccharide component was glucose. This is ascribed to the β-1,3- and β-1,6-glucans comprising the majority of the mushroom cell wall.
Table 2. Constituent monosaccharide analysis of POS (%).
| Mushroom | Glc | Gal | Man | Xyl | Fuc | GlcA |
| F. velutipes | 71.0 ± 1.3 | 10.9 ± 1.4 | 10.5 ± 0.9 | 3.49 ± 1.1 | 4.18 ± 0.4 | - |
| H. marmoreus | 59.8 ± 0.8 | 9.68 ± 0.7 | 6.03 ± 0.7 | - | 3.06 ± 0.2 | 21.4 ± 2.3 |
| P. eryngii | 87.7 ± 1.5 | 4.40 ± 1.0 | 6.26 ± 0.8 | 0.31 ± 0.9 | 1.33 ± 0.3 | - |
| G. frondosa | 84.2 ± 1.0 | 5.41 ± 0.9 | 6.67 ± 0.7 | - | 3.67 ± 0.5 | - |
Interestingly, monosaccharide relative ratios varied among mushrooms. Galactose, mannose, and fucose were present in the POS of all mushrooms. Xylose was detected only in the POS of F. velutipes and P. eryngii, being particularly abundant in F. velutipes. Glucuronic acid was only observed in the POS of H. marmoreus, at a relatively high percentage of 21.4 %. These findings indicate that mushroom cell wall components vary among mushroom species.
POS molecular weight distributions were then determined by size exclusion chromatography (SEC) (Fig. 2). Based on the pullulan standard, POS molecular weight distribution was divided into three sections: above 78.8 × 104, between 78.8 × 104 and 4.73 × 104, and below 4.73 × 104. POS isolated from G. frondosa were primarily high molecular weight (78.8 × 104 or higher), whereas POS from P. eryngii and H. marmoreus was mainly in the 78.8 × 104 to 4.73 × 104 range. For POS derived from F. velutipes, the distribution ratio of the three fractions was about equal. The POS obtained by the present method, in addition to poly/oligo-saccharides, may contain glycoproteins, since overlap of proteins and polysaccharides was confirmed by electrophoresis (data not shown). These glycoproteins may be evidenced by the high molecular weight type (78.8 × 104 or higher) observed in SEC.
Kawahara et al. extracted xylo-mannan with IRI activity from F. velutipes by alkaline treatment with an average molecular weight of 32 × 104 and a xylose to mannose ratio of 1:3 [14]. This ratio was also observed for the F. velutipes POS, suggesting xylo-mannan as the source of the high xylose content. However, the reason for the different molecular weight distribution was unclear, and may result from differences between alkali treatment and enzymatic treatment. Additionally, the outer cell wall of yeast and other organisms are composed of highly glycosylated mannoproteins [23], so peaks above 78.8 × 104 may be glycoproteins.
Ice recrystallization inhibition activity of POS. Ice crystal size changes over time in the presence of POS were observed by microscopy (Fig. 3). PEG 3400, known to have IRI activity, was used as the positive control because the ice crystals without any addition would be too large to compare on the same scale. For PEG 3400, the ice nuclei, initially small, were observed to increase over time with increasing gaps between ice nuclei. This may be due to continued growth and fusing of ice nuclei, resulting in ice recrystallization. However, in the POS solution, there was little change in the ice nuclei, suggesting ice recrystallization suppression. The results of ice crystal size measurements after 40 min are shown in Fig. 4. For the PEG 3400 control, the ice crystal coverage area per unit area (0.66 mm × 0.88 mm) was large, but the number of ice crystals was small due to fusion and growth of ice nuclei by recrystallization. However, when POS was added, the ice crystal coverage area per unit area was smaller, with an increased number of ice crystals. This supports the inhibitory activity of POS towards ice crystal growth. Polysaccharides have a high affinity for water because they have hydroxyl groups, which suggests that the movement of water molecules is inhibited, suppressing the formation of ice crystals. In addition, the sugar components of POS from F. velutipes and P. eryngii, which have a large number of small ice crystals, are xylose and mannose in common, suggesting the possibility of xylo-mannan. However, the structure of the polysaccharides needs to be examined in the future.
N is the number of ice crystals, Ave. is the average particle size, and Q1, Q2 and Q3 are expressed in ascending order as divided values (25 %, 50 % and 75 %).
Effect of POS on frozen food quality. As POS from all mushrooms induced an inhibitory effect on ice recrystallization, the influence on actual processed foods was investigated. In this study, chawanmushi, surimi, and shiratama were used as proteinaceous and starchy processed foods, with the effect of POS on their physical properties after freezing and thawing evaluated.
Chawanmushi is a gelatinous dish characterized by its smooth texture, made by heating a combination of egg and soup stock. However, when chawanmushi is frozen, ice crystal growth occurs in the dense reticular gel structure, leaving a sparse reticular protein structure after melting. This results in a spongy texture and loss of quality.
The post-thawing elasticity and hardness of chawanmushi to which POS derived from F. velutipes was added, were significantly lower than those of the control (no POS added). On the other hand, no significant difference was observed in chawanmushi using POS derived from the other mushrooms (Fig. 5a). (control, 1.93 ± 0.11 N; F. velutipes, 1.69 ± 0.15 N; H. marmoreus, 2.00 ± 0 .08 N; P. eryngii, 1.90 ± 0.08 N; G. frondosa, 1.95 ± 0.04 N). In terms of elastic rate, the chawanmushi with F. velutipes POS was lower than the control, while other POS did not differ from the control. These results suggest that the POS from F. velutipes reduced the effects of water release or freeze denaturation and retained smoothness (control, 1.00 ± 0.34; F. velutipes, 0.31 ± 0.01; H. marmoreus, 1.05 ± 0.07; P. eryngii, 1.25 ± 0.09; G. frondosa, 0.92 ± 0.14). POS derived from F. velutipes contains more xylose and mannose than other POS (Table 2), suggesting that these polysaccharides may be effective in preventing the freeze denaturation of chawanmushi.
(a) chawanmushi, (b) surimi, (c) shiratama
(** p < 0.05; * p < 0.1)
In white fish surimi, moisture is retained in the protein muscle fiber network structure, which strongly affects the properties of the surimi, such as hardness and tissue density. However, freezing surimi causes ice crystal growth in the muscle fibers, inducing aggregation. Upon thawing, water is released, resulting in a crumbly texture. The firmness of kamaboko meat containing POS derived from F. velutipes and POS derived from G. frondosa was reduced compared to the control (Fig. 5b) (control, 39.64 ± 3.35 N × mm, F. velutipes, 32.56 ± 3.30 N × mm; H. marmoreus, 44.34 ± 11.03 N × mm; P. eryngii, 40.16 ± 12.24 N × mm; G. frondosa, 23.52 ± 3.82 N × mm). The decrease in resistance of the fish meat suggests protein denaturation suppression, resulting in moisture retention. Although the composition ratios of POS derived from F. velutipes and G. frondosa were not the same (Table 2), the presence of more than 78.8 × 104 polymers (Fig. 2) suggests that these polysaccharides or glycoproteins may have acted on the kamaboko.
Shiratama with the addition of POS from each mushroom tended to have a lower strain rate at break compared to the control, but no significant difference was observed (Fig. 5c) (control, 93.95 ± 3.57 %; F. velutipes, 91.93 ± 2.11 %; H. marmoreus, 92.28 ± 1.99 %; P. eryngii, 92.26 ± 0.37 %; G. frondosa, 93.18 ± 0.56 %). These results suggest that POS from mushrooms has no effect on freeze denaturation (starch retrogradation) of starch. Since starch retrogradation and protein denaturation have different mechanisms of action, it is suggested that mushroom-derived POS cannot inhibit starch retrogradation.
In this study, we demonstrated that the components of mushroom cell walls can be easily extracted by enzymatic treatment under mild, environmentally friendly conditions. With the aim to use the hot water extraction residue of edible mushroom fruit bodies (IFHE) as a cryoprotectant, we investigated the extraction of sugar components (POS) from mushrooms then evaluated POS antifreeze activity. IFHE was solubilized by the action of Uskizyme, and then POS recovered by ethanol precipitation. This residue was shown to have antifreeze activity. POS derived from F. velutipes showed the highest ice crystal growth inhibition activity among four kinds of mushrooms. The impact of the extracts obtained by enzyme-treated edible mushrooms on the quality of egg protein, fish protein, and rice starch over the freezing, storage and thawing process was evaluated. For chawanmushi, POS derived from F. velutipes suppressed hardness and elasticity increases. This suggests a suppression of recrystallization processes within the protein gel, preventing the formation of large voids. Additionally, in surimi, POS derived from F. velutipes and G. frondosa suppressed increases in hardness caused by muscle protein association due to freezing denaturation. However, for shiratama, no significant difference was observed due to the POS addition, indicating limited efficacy in inhibiting the retrogradation activity of starch.
Takemi Kamijo and Yuka Arai are employees of Kyowa Chemical Products Co., Ltd.
