*Article* **Nutrient Composition, Antioxidant Activities and Glycaemic Response of Instant Noodles with Wood Ear Mushroom (***Auricularia cornea***) Powder**

**Muhammad Kamil Zakaria 1, Patricia Matanjun 1,\*, Ramlah George 1, Wolyna Pindi 1, Hasmadi Mamat 1, Noumie Surugau <sup>2</sup> and Jaya Seelan Sathiya Seelan <sup>3</sup>**


**Abstract:** *Auricularia cornea,* or wood ear mushroom (WEM), is an edible macrofungus that is popular as a delicacy and for various biological activities. This study aims to determine the nutrient composition, in vitro antioxidant activities and the effect on postprandial blood glucose in human subjects after consuming instant noodles incorporated with 5% WEM powder. The proximate composition of WEM powder was 9.76% moisture, 2.40% ash, 7.52% protein, 0.15% fat, 37.96% crude fibre, 42.21% carbohydrate, and a total dietary fibre was 69.43%. Meanwhile, the proximate composition of 5% WEM noodles was 10.21% moisture, 2.87% ash, 11.37% protein, 0.16% fat, 5.68% crude fibre and 68.96% carbohydrates, while the total dietary fibre was 13.30%. The mineral content of WEM powder in decreasing order: potassium > calcium > magnesium > sodium > iron > zinc > manganese > copper > selenium > chromium. The incorporation of 5% WEM powder significantly (*p* < 0.05) reduced carbohydrates and increased the ash, crude fibre and total dietary fibre, antioxidant activities and total phenolic content of the instant noodles. Furthermore, the incorporation of 5% WEM significantly increased potassium, calcium, magnesium, iron, and zinc content. The addition of WEM powder reduced the postprandial glycaemic response and produced a moderate glycaemic index (GI). In conclusion, the incorporation with WEM powder could be an effective way of developing nutritious and low GI instant noodles, thus, improving nutrient intake and human health.

**Keywords:** *Auricularia cornea*; wood ear mushroom; instant noodles; glycaemic response

## **1. Introduction**

The wood ear mushroom (WEM) is a wood-decaying fungi from the genus Auricularia, commonly known as jelly or black fungus. Its distinct gelatinous ear-like shape, fruiting body and brown-to-black colouration have unique sensory features and are rich in medicinal properties [1,2]. Similar to other edible mushrooms, the WEM is low in fat and calories and a good source of protein, fibre, essential elements, and bioactive compounds [3]. The WEM has been reported to exhibit several biological activities, such as antioxidant [4], antimicrobial [5], anti-inflammatory [6], immunomodulatory [7], hypoglycaemic [8], and hypocholesterolemic effects [9], all of which contribute to good health. The WEM is a promising solution for disease prevention through incorporation as an ingredient in supplements or food products in the nutraceutical, pharmaceutical, and food industries [10]. Nonetheless, this novel ingredient remains underutilised because of consumer preferences for other edible mushrooms, such as oyster and button mushrooms. Furthermore, there is limited data on the locally grown WEM as a functional ingredient.

**Citation:** Zakaria, M.K.; Matanjun, P.; George, R.; Pindi, W.; Mamat, H.; Surugau, N.; Seelan, J.S.S. Nutrient Composition, Antioxidant Activities and Glycaemic Response of Instant Noodles with Wood Ear Mushroom (*Auricularia cornea*) Powder. *Appl. Sci.* **2022**, *12*, 12671. https://doi.org/ 10.3390/app122412671

Academic Editor: Antonio Valero

Received: 10 November 2022 Accepted: 4 December 2022 Published: 10 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The incorporation of instant noodles with various ingredients to improve minerals, fibre, and vitamin content, is a popular practice [11,12]. Conversely, the use of mushrooms as a functional element in instant noodles is relatively new. Few studies have incorporated edible mushrooms such as oyster and shitake mushrooms, but there have been no reports on WEM utilisation [13–17]. According to the literature, the incorporation of mushroom powder significantly improved the proximate composition of noodles in terms of protein and fibre content, but information regarding antioxidant activities is limited. In terms of sensory quality, several studies that used different types of mushroom species found the incorporation of more than 5% would negatively affect the sensory quality of the noodles, especially the texture and taste [14,15,18]. Furthermore, the potential of WEM polysaccharides to exhibit the hypoglycaemic effect has widely been discussed, but the scientific evidence is limited to animal studies [3,19]. Previous studies have demonstrated that the incorporation of *Auricularia* spp. into the diet could induce hypoglycaemia in genetically diabetic mice [20–22]. Similarly, in vitro studies by Vallee et al. (2017) and Wu et al. (2014) also showed the hypoglycaemic effect of *Auricularia* spp. where 5% of the *Auricularia* mushroom powder was able to attenuate in vitro starch digestion [23,24]. Therefore, this present study examined the nutrient composition, antioxidant activities, and the effect on postprandial blood glucose levels of WEM powder and instant noodles incorporated with WEM powder. The study findings could add value to the product by improving nutrient qualities, hence, providing healthier food choices for consumers.

## **2. Materials and Methods**

## *2.1. Sample Preparation*

The WEM or *A. cornea* (previously known as *A. polytricha*) was cultivated and supplied by the Rural Development Corporation, a government agency under the Sabah Ministry of Agriculture and Fisheries. The fresh WEM was dried at 50 ◦C for 24 h in a dehydrator (Cabela's 80 L, Sidney, NE, USA), ground into 250 μm powder and stored in an air-tight container for further use.

The instant noodle was formulated based on Sulaiman et al. with slight modifications [25]. The ingredients were purchased from the local supermarket, and filtered tap water from the laboratory was utilised in the making of instant noodles. The formulation of the control instant noodles comprised 100% all-purpose wheat flour (Johor Flour Mill, Johor, Malaysia), 38% water, 2.5% iodide salt (Adabi, Rawang, Malaysia), and 1.2% sodium bicarbonate (Kings, Pulau Pinang, Malaysia). In the modified instant noodle, 5% of the wheat flour was replaced with the WEM powder, while the remaining ingredients were unchanged. All ingredients were weighed accordingly. Water was mixed with salt and sodium bicarbonate before the solution was added gradually into the flour until it formed a dough. The dough was rested for 30 min before the sheeting process to reduce the dough thickness to 2–3 mm and obtain a smooth surface. The dough was cut into equal lengths and air-steamed for 8 min before it was finally dried overnight (24 h) at 50 ◦C using a dehydrator (Cabela's 80 L, Sidney, NE, USA).

## *2.2. Proximate Composition Analysis*

The moisture, ash, crude fat, crude protein, and crude fibre content, were determined using the standard Association of Analytical Chemists (AOAC) methods [26]. The moisture content was determined after drying the noodles overnight at 105 ◦C in a universal oven (Binder Inc., Bohemia, NY, USA). The ash content was determined after the dried sample was incinerated in the furnace (Carbolite Gero Ltd., Derbyshire, UK) overnight at a temperature of 550 ◦C. Meanwhile, the fat content was determined via the Soxhlet method using the Soxhlet Avanti auto system and petroleum ether as the solvent (Soxtec™ 2050, FOSS, Hillerød, Denmark). The protein content was then determined after the sample was digested with concentrated sulphuric acid (H2SO4) and selenium tablet using a digester (KJELDATHERM, Gerhardt GmbH, Königswinter, Germany) before being calculated by an automated Kjeldahl machine (Kjeltec, Foss, Hillerød, Denmark). The conversion factor used

to determine the crude protein in the mushroom was 4.38, and 6.25 for general food [27]. The crude fibre content was determined based on the weight of the sample, which was digested and filtered using the machine (Fibertherm, Gerhardt, Brackley, UK), followed by overnight drying in a universal oven (Binder Inc., Bohemia, NY, USA) and 4 h of ashing using furnace (Carbolite Gero Ltd., Derbyshire, UK). Finally, the carbohydrate content was calculated from the sum of percentages of crude protein, ash, fat, and crude fibre subtracted from 100.

## *2.3. Total Dietary Fibre (TDF)*

The TDF was determined using the Total Dietary Fiber Assay Kit (Megazyme International Ireland Limited, Wicklow, Ireland) based on AOAC enzymatic-gravimetric methods 985.29. A sample was mixed with 50 mL phosphate buffer solution (pH 6.0 ± 0.1) before 50 μL a-amylase was added, and the mixture was boiled for 30 min. Then, the pH was adjusted to 7.5 ± 0.1 by adding 0.275 N of sodium hydroxide (NaOH) and 100 μL protease. The mixture was then incubated at 60 ◦C with continuous agitation for 30 min. Next, 0.325 N hydrochloric acid (HCl) was added to adjust the pH to 4.5 ± 0.2 before adding 200 μL amyloglucosidase, and the solution was incubated at 60 ◦C for 30 min. The mixture was filtered through a crucible containing fritted glass disk and Celite 545, followed by three washings of 20 mL 75% ethanol, two 10 mL 95% ethanol and two 10 mL portions of acetone before being dried overnight at 105 ◦C and weighed. Finally, the dried residue was analysed for protein content using the Kjeldahl method and a duplicate residue was analysed for ash. The weights of protein and ash were subtracted from the residue weight obtained to determine the insoluble dietary fibre.

## *2.4. Mineral Analysis*

The mineral quantification of copper, zinc, chromium, calcium, magnesium, potassium, sodium, iron, manganese, and selenium, was performed according to the AOAC method [26]. The dry samples were added with 10 mL of 65% nitric acid (HNO3) before the mixture was heated until the brown smoke was undetectable. Then, 2 mL of distilled water and 3 mL of hydrogen peroxide (H2O2) were added to the solution and heated until the total volume was reduced to 5 mL. Next, 10 mL HCl was added to the solution and reheated for 15 min. Finally, the mixture was filtered and transferred to a volumetric flask for dilution. The quantification of elements was carried out using the Inductive Coupled Plasma-Optical Emission Spectrometer (ICP-OES) (Perkin Elmer, Waltham, MA, USA).

## *2.5. Antioxidant Activities Analysis*

The samples were soaked in methanol for 24 h and agitated at 150 rpm at room temperature twice [28]. Both filtrations were combined, and the solvent was removed via evaporation with a rotary evaporator at 40 ◦C. The dried extracts were stored in a dark place at 4 ◦C until further use.

The scavenging activity of 2, 2-diphenyl-2-picryl-hydrazyl (DPPH) inhibition by the extracts was determined based on Teoh et al. with modification [29]. The extract at various concentrations was added to 2.9 mL of methanolic DPPH radical solution (6 × <sup>10</sup>−<sup>5</sup> M). The mixture was shaken vigorously and left to stand for 45 min at 25 ◦C in the dark. The mixture absorbance was then measured at 517 nm with a UV-VIS spectrophotometer (Lambda 35 UV–VIS Spectrometer, PerkinElmer, Waltham, MA, USA), and Trolox (Merck, EMD Millipore Corporation, Darmstadt, Germany) was used as the calibration standard while methanol mix with DPPH radical was the negative control. The results were reported as the IC50 Of WEM.

Next, the ferric-reducing antioxidant power (FRAP) assay of the WEM extract was determined based on the method by Yim et al. with some modifications [30]. The FRAP reagent was prepared freshly, containing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl and 20 Mm iron (III) chloride (FeCl3) at a ratio of 10:1:1 at a temperature of 37 ± 2 ◦C. The reagent (200 μL) was mixed with 20 μL methanol extract, and the absorbance at 593 nm was recorded after 10 min using a UV–VIS spectrophotometer (Lambda 35 UV–VIS Spectrometer, PerkinElmer, Waltham, MA, USA). Iron (II) sulfate heptahydrate (FeSO4.7H2O) (Systerm, Classic Chemicals Sdn. Bhd., Shah Alam, Selangor, Malaysia) was used as a standard for a calibration curve.

The total phenolic compound (TPC) of the sample extract was determined based on the Folin-Ciocalteu assay by Teoh et al., with some modifications [29]. The methanol extract (200 μL) was transferred into a test tube and mixed thoroughly with 1 mL of Folin-Ciocalteu reagent. After 3 min, 0.8 mL of 7.5% (*w*/*v*) sodium carbonate (Na2CO3) was added to the mixture and agitated for 30 min in the dark. The absorbance of the extracts and prepared blank were measured at 515 nm using a UV–VIS spectrophotometer (Lambda 35 UV–VIS Spectrometer, PerkinElmer, Waltham, MA, USA). The result was represented as mg of gallic acid equivalents (GAE) per gram extract.

## *2.6. Glycaemic Analysis (Blood Glucose Measurement and Calculation of Glycaemic Index)*

Ethical approval for this study was granted by the Medical Research Ethics Committee of Universiti Malaysia Sabah [JKEtika 2/21(16)]. The analysis was conducted at the Nutrition and Dietetic Laboratory, Faculty of Food Science and Nutrition, Universiti Malaysia Sabah. The protocol for glycaemic analysis was conducted according to Wolever et al. [31].

Ten healthy volunteers participated in this study. Participants were given a subject information sheet with details of the study protocol and had the opportunity to ask questions. They signed an informed consent before participating in the study. Inclusion criteria of study participants are as follows: age 23 to 31 years old and non-smoker, normal body mass index (BMI) (19 and 25 kg/m2), and has normal fasting blood glucose level (<5.6 mmol/L). Individuals that were pregnant, allergic to food, hypersensitive, restricted or specified diet, inflammatory or metabolic diseases, and prescribed with medications that may interfere with carbohydrate metabolism, were excluded from participating in this study.

Each participant completed five sessions of glycaemic index trials: three sessions for reference carbohydrates (50 g of glucose dissolved in 250 mL of water) and one session for control instant noodles and one session for instant noodles incorporated with 5% WEM as a test food. The sessions were carried out on separate days. The order of the test food, control and reference that were tested by each participant was randomised using online tool Research Randomizer (Version 4.0) [32]. The analysis was conducted in the morning after the participant fasted overnight for a minimum of 10 h. Blood glucose levels were measured in capillary blood obtained by finger prick using a lancet (ACCU-CHEK® Safe T-Pro Plus, Roche, Basel, Switzerland), blood glucose test strip (Freestyle Freedom Lite®, Abbott Laboratories, Chicago, IL, USA) and glucometer (Freestyle Freedom Lite®, Abbott Laboratories, Chicago, IL, USA). Blood sugar levels were measured at seven time points (0, 15, 30, 45, 60, 90, and 120 min). The glycaemic response or the incremental area under the curve (IAUC) for each reference and test food was calculated by the trapezoid rule [33].

The glycaemic index (GI) was calculated by using Equation (1) [31].

$$\text{GI} = (\text{IAUC of the test food} / \text{the IAUC of reference food}) \times 100\tag{1}$$

## *2.7. Data Analysis*

The experiments were carried out in triplicates, and the data were presented as average values. Statistical Package for the Social Science (SPSS) version 26 for Windows (SPSS Inc., Chicago, IL, USA) was used in the statistical analysis. First, the independent *t*-test was used to determine significant differences (*p* < 0.05) in the mean value of proximate composition and mineral content between samples. The analysis of variance (ANOVA) test was then performed to determine the significant differences (*p* < 0.05) in the mean value of antioxidant activities and total phenolic content between samples. A paired T-test was used for the mean comparison of postprandial blood glucose response within 120 min between control and incorporated instant noodles with 5% WEM. The mean of IAUC between reference and test food was compared using ANOVA and Tukey's B post-hoc for multiple comparisons.

### **3. Results and Discussion**

#### *3.1. Proximate Composition*

This study aims to utilise edible mushrooms as a functional ingredient for food products, as there are reported studies on the underutilisation of this ingredient [34,35]. Edible mushrooms are considered a superfood that is highly nutritious, low in calories and possesses other biological compounds beneficial to human health [36,37]. The proximate composition of WEM powder is shown in Figure 1, where the highest composition is a carbohydrate, followed by crude fibre, moisture, protein, ash, and fat. The fat content of WEM powder was the lowest as all edible mushrooms are well-known for the low fat content (≤5% dry weight) and were the least nutritive constituents [38].

**Figure 1.** The proximate composition of WEM (*A. cornea*) powder on a dry weight basis was expressed as means ± standard deviation (*n* = 3), WEM = wood ear mushroom.

The moisture content of dry WEM powder was 9.76 ± 0.09% which is consistent with earlier studies that reported the moisture content of dried *Auricularia* species ranged between 6 to 15% [39,40]. Most fresh edible mushrooms, including *Auricularia* species, are high in moisture (85–94%), thus providing WEM with a unique gelatinous and smooth texture, but highly perishable [41,42]. Besides mushroom variety, environmental factors such as temperature, relative humidity, and post-harvest condition, can also influence the moisture content [43]. Therefore, most commercial WEM was dried to inhibit microbial and enzymatic activities while improving shelf-life stability [41]. The dried, hard, and brittle WEM can be rehydrated for consumption without altering the flavour and texture.

The ash content recorded in the present study (2.40 ± 0.03%) was slightly lower than previous studies (3 to 9% dry weight) for cultivated *Auricularia* species in China and India [44,45]. One of the primary factors influencing ash content is the substrate where edible mushrooms grow, which may vary depending on the cultivation technique. Moreover, the substrate composition can differ in organic matter content, pH, and metal concentration [46]. Thus, the ash content of cultivated mushrooms tends to be lower than wild mushrooms, as the latter grows in a nutrient-rich environment [47,48]. Nevertheless, the ash content of cultivated mushrooms can be improved via substrate modification [39]. Mushrooms absorb minerals from the ecosystem through bioaccumulation, hence, promoting the ash content in both wild and cultivated mushrooms [27]. Additionally, climate differences and genetic

factors could alter the ash content of mushrooms [46]. Regardless of whether cultivated or wild, the ash content of edible mushrooms is higher or comparable to most vegetables [47].

The protein content of WEM in the present study was in line with *A. polytricha* (7.2%) grown in Vietnam but lower than *A. auricular* (11.38%) from China [43,45]. In addition, the protein content of *Auricularia* species was lower than other edible mushroom species, such as oyster and shitake mushrooms (wild or cultivated) as reported in a previous study [49]. The difference in protein content might be caused by the mushroom species and strain, the development of the fruiting body, substratum, and location [50]. Furthermore, recent findings reported that there is a significant difference in crude protein content between wild and cultivated mushrooms [39,50].

The WEM powder is high in carbohydrates (42.21 ± 1.71%), which agrees with the study conducted by Shan et al. (28.38–82.8%) [45]. The carbohydrate content may vary between species, but up to 70% of total carbohydrate content were non-digestible polysaccharides in mushrooms, thus, diminishing fungi as the primary source of energy [51]. In this study, the fibre content of cultivated WEM powder (37.96 ± 1.63%) was higher than in a previous study (2.11–15.32% dry weight) [49]. Several studies have also reported no differences in fibre content between cultivated and wild WEM, and WEM also recorded the highest fibre content than other edible species [41,45,49]. In addition, the non-digestible polysaccharide of edible mushrooms is an alternative source of dietary fibre that remains underutilised compared to fruits, vegetables, and legumes [34].

Some but not all proximate compositions of WEM powder in the current study are comparable with other findings of cultivated WEM. The slight variation may be due to factors such as strain, the development stages of the fruiting body, nutrient distribution within the fruiting body, post-harvest handling and environmental conditions [47]. However, ensuring the stability of nutritional constituents is critical to incorporate WEM powder in food products [52].

A previous study by Sulaiman et al. found that the 5% of WEM incorporated in instant noodles was the best formulation among five modified formulas ranging from 5% to 25% [25]. The sensory evaluation of the 5% formulation recorded the highest score for colour, aroma, taste, texture, and overall acceptance, and there was no significant difference in any of the sensory attributes when compared with the control. This would be a good indication for the product to be accepted and its suitability for consumption. Table 1 shows the proximate composition of instant noodles incorporated with WEM and without WEM powder (control). The results showed a significant (*p* < 0.05) reduction of carbohydrates, increased ash and crude fibre, and no significant differences in moisture, protein, and fat content between the control instant noodle and noodles incorporated with 5% WEM. Specifically, the moisture content of both noodles was higher than previous findings (<5%), but the values remained within the recommended shelf-life stability (<12%) [53]. Furthermore, the current protein value of noodles did not improve with WEM inclusion, thus, contradicting past studies where the addition of mushroom powder had significantly increased the protein content of the noodles [13–15]. This finding might be attributed to the lower protein content of WEM compared to other edible mushrooms. Meanwhile, the significant increase of ash content in the noodles incorporated with 5% WEM powder indicated that this ingredient is a good source of minerals, as observed in other studies [40,46]. In addition, the current study demonstrated a significant (*p* < 0.05) increase in crude fibre content in WEM noodles, due to the presence of natural fibre in the mushroom [13–15]. Consequently, the addition of WEM powder significantly reduced the carbohydrate content. Nevertheless, according to Shams et al., long storage periods can reduce the fibre content and increase the carbohydrate content caused by the degradation of complex polysaccharides into simple sugar [53].


**Table 1.** The proximate composition of noodles incorporated with WEM and without WEM (control).

Values were expressed as means ± standard deviation (*n* = 3). Different superscripts in the same column were significantly different (*p* < 0.05).

## *3.2. Total Dietary Fibre (TDF)*

Table 2 demonstrates that the incorporation of WEM significantly increased (*p* < 0.05) the TDF of instant noodles due to the high dietary fibre in WEM (69.43 ± 1.12%). The TDF of WEM mushrooms was higher than the crude fibre content as the TDF accounted for soluble and insoluble fibres, thus reflecting the overall mushroom fibre content [34]. Moreover, the current result exhibited higher TDF content than previous studies, and the improvement was attributed to the WEM powder as a rich source of fibre, as stated in earlier findings [13,54]. Resultantly, the addition of WEM powder in noodles will lead to a sufficient intake of dietary fibre that can help improve gut health, weight management, and glycaemic response among consumers [55,56].

**Table 2.** The dietary fibre of noodles incorporated with WEM and without WEM (control).


Values were expressed as means ± standard deviation (*n* = 3). Different superscripts in the same column were significantly different (*p* < 0.05).

#### *3.3. Mineral Analysis*

Table 3 shows the mineral composition of WEM powder, consisting of 10 macro and microelements expressed as mg/kg of the dry weight. Potassium was the highest macro element found in the WEM powder, consistent with earlier studies on the *Auricularia* species [45,57,58]. Meanwhile, the calcium and magnesium in WEM were lower than previous findings from Cameroon at 886.2 mg/kg and 835.4 mg/kg, respectively [59]. The sodium content reported in this study (68.929 ± 1.72 mg/kg) was the lowest macro-element, which was lower than an earlier study (109.1 mg/kg) [59]. Furthermore, WEM contained more potassium and calcium than magnesium and sodium, which agrees with the study by Kadnikova et al. [40].


**Table 3.** The mineral content of WEM (*A. cornea*).

Values were expressed as means ± standard deviation (*n* = 2); dw = dry weight.

Iron and zinc have been reported as the highest micro elements found in *Auricularia* species (50–100 mg/kg), while the presence of copper, manganese, chromium, and selenium, was less than 20 mg/kg of dry weight. The amount of iron and zinc in this study was the highest for microelements, but at lower levels (50 mg/kg) compared to previous

findings [41]. Likewise, the remaining trace elements have been reported in other species, such as *A. thailandica* and *A. polytricha* [58,59]. In summary, the trend of macro and microelements in the WEM powder in decreasing order is as follows: potassium > calcium > magnesium > sodium > iron > zinc > manganese > copper > selenium > chromium.

Table 4 shows the mineral content of instant noodles with WEM and without WEM (control). There was a significant (*p* < 0.05) increase in potassium, calcium, magnesium, iron, and zinc content, but no significant difference (*p* > 0.05) in the concentration of manganese, copper, and chromium, in noodles incorporated with WEM compared to the control. Moreover, the content of sodium and selenium in incorporated noodles was significantly (*p* < 0.05) lower than the control. The present result demonstrated that all analysed minerals were present in the control noodles made from wheat flour, but the addition of WEM powder increased the prevalent macro and microelements, such as potassium, calcium, magnesium, iron, and zinc. Despite that, no increment was observed for manganese, copper, chromium, and selenium, in the incorporated noodles because of the low levels recorded in the WEM powder. Sodium was the highest constituent in both instant noodles due to salt addition in the noodle-making process. According to Ibrahium et al., the increment of minerals in the incorporated food was attributed to the high contents of mineral salts in the mushroom [60]. In addition, the current potassium, calcium, and iron content were higher than the previous finding by Parvin et al. that included oyster mushrooms in instant noodles (2705.5 mg/kg potassium, 275.8 mg/kg calcium, and 52.6 mg/kg iron) [15]. Therefore, the positive outcomes of this study could be an alternative solution for nutrient deficiencies [3].

**Table 4.** The mineral element content of noodles incorporated with WEM and without WEM (control).


Values were expressed as means ± standard deviation (*n* = 2); dw = dry weight. Different superscripts in the same column were significantly different (*p* < 0.05).

Different mushroom species may accumulate minerals at different capacities from the substratum, and various analytical methods could be the factors that explained the variation of minerals [46,57]. Furthermore, high mineral values suggested that WEM is a potential source of good-quality ingredients [44]. The essential elements in WEM are comparable to other edible mushrooms and are required in varying amounts for the proper functioning of the body (biochemical reactions, metabolic growth, and enzymatic activities) [61]. Due to bioaccumulation ability of edible mushrooms, consumer should aware of the excessive mineral intake which could harm the body due to association with increasing cardiovascular risk factor and heart diseases; thus, the guideline of recommended daily intake of minerals and trace elements should be followed for safe consumption [62]. For instance, the safe limit of copper, iron, manganese, and zinc per kg of human body weight set by the World Health Organization are 40 mg/kg, 15 mg/kg, 400–1000 mg/kg, and 60 mg/kg, respectively [46]. However, the recommendation for daily intake of dietary minerals also depends on factors such as sexes, dietary practices, food preparation and processing, bioavailability, mineral interactions, and the chemical forms of the minerals [63].

## *3.4. Antioxidant Activities and Total Phenolic Contents (TPC)*

Methanol was used as the solvent for the extraction, as higher polarity solvents could increase the yield of polar phenolic compounds, leading to higher antioxidant activity [58,64]. The phenolic compounds are a large group of secondary metabolites in plants and fungi exhibiting antioxidant activities and oxidation protection [44]. Therefore, the high TPC of a mushroom suggests a higher antioxidant capacity [50,65]. Based on Table 5, the WEM powder could exhibit antioxidant activities mediated by free radical scavenging abilities and reducing power due to the presence of total phenolic content [58]. The strong DPPH radical-scavenging activity of the extracts indicated the high hydrogendonating capacity; thus, the DPPH free radicals scavenging [66]. Meanwhile, the FRAP assay demonstrates the ability of the non-enzymatic antioxidant extract associated with the presence of reductone to break the free radical chain by donating an electron to stabilise and terminate the radical chain reactions [28]. The present data demonstrated that the TPC of WEM powder is higher than the value reported in previous studies, such as ethanolic extract of *A. polytricha* (4.74 mg GAE/g), ethanolic extract of *A. auricula* (2.75 mg GAE/g) and methanolic extract of *A. thailandica* (1.99 mgGAE/g) [29,58,66]. In addition, the wild edible mushroom has been reported to contain higher phenolic compounds than cultivated mushrooms, as the stressful environment enhanced the production of secondary metabolites [44].

**Table 5.** The antioxidant activities and TPC of wood ear mushroom (WEM) powder and noodles incorporated with WEM and without WEM (control).


Results were expressed as means ± standard deviation. (*n* = 3). Different superscripts in the same row were significantly different (*p* < 0.05).

The present study also demonstrated that the incorporated noodles with 5% WEM powder exhibited significantly (*p* < 0.05) lower IC50 for DPPH inhibition than the control noodles. The lower IC50 value reflected the strong DPPH scavenging capacity, but a much lower value has been recorded in another study that incorporated noodles with button mushrooms (0.4 mg/mL) [53]. Meanwhile, the FRAP value for the incorporated noodles was significantly (*p* < 0.05) higher than the control noodles, indicating a strong reduction capacity. Nevertheless, the improvement of antioxidant capacity in the incorporated instant noodles was due to the increment of TPC, which agrees with past studies but with different mushroom species and functional ingredients [18,67].

The present study supports the potential of incorporating edible mushrooms in the food product due to the abundance of phytochemicals with potent antioxidant abilities and is associated with health benefits, particularly the implications of diseases caused by oxidative stress and free radicals [68]. Despite that, the impact of processing and cooking may reduce the TPC and antioxidant capacity due to the cutting, steaming, or drying process, or long-term storage, which requires further investigation [69,70]. Besides the phenolic compounds, recent studies found that mushroom polysaccharides demonstrated potent antioxidant abilities, such as free radicals scavenging, a metal chelator, and reducing power [20,71,72]. Antioxidants are essential to protect living organisms from oxidative damage by preventing or inhibiting the oxidation reaction.

## *3.5. Glycaemic Index*

The glycaemic analysis was conducted to observe the effect of incorporating instant noodles with 5% of WEM mushroom on the postprandial blood glucose level among participants in this study. Figure 2 illustrates the mean postprandial blood glucose level within two hours for 10 female subjects with normal body mass index (BMI) who consumed the test food, containing 50 g of carbohydrates. The baseline for the blood glucose level was comparable and started to rise drastically within 15 min after consuming the test meals and peaked at 30 min, whereas the control noodles recorded higher blood glucose levels than the incorporated noodles with 5% WEM powder (*p* < 0.05). Subsequently, the blood glucose level of the control noodles exhibited a drastic drop between 30 to 60 min before a steady fall at 90 min, and remained constant until 120 min. Meanwhile, the blood glucose level of the incorporated noodles saw a gradual drop from 30 min to 120 min. At 120 min, the average blood glucose level of the control noodles was higher (5.5 mmol/L) than the noodles incorporated with 5% WEM (5.1 mmol/L) (*p* < 0.05). It was confirmed that adding WEM powder reduced the peak and lowered the glucose concentration of digested WEM noodles in blood vessels, hence, exhibiting the hypoglycaemic effect. The observation was in agreement with previous findings that the incorporation of 5% of mushroom powder was able to exhibit a hypoglycaemic effect [23,65].

**Figure 2.** The comparison of postprandial blood glucose response within 120 min between control and 5% WEM instant noodles (*n* = 10), \* indicates a significant difference (*p* < 0.05).

Numerous animal studies have demonstrated the hypoglycaemic effect of WEM by observing the effect of powder supplementation in the diet of diabetic mice, which reduces the plasma glucose, insulin, urinary glucose, and food intake [8]. On a normal occasion, carbohydrates were broken down into glucose once the test food was ingested. The blood glucose level would spike drastically until reaching the highest concentration within 30 min and fall back to normal glucose level after 2 h, depending on the type of food consumed [73]. Nonetheless, the presence of fibre-rich ingredients, such as WEM powder, interferes with digestion and delays the gastrointestinal tract emptying process affecting the absorption of nutrients, particularly glucose [74]. An in vitro study mimicking the behaviour of enzymatic digestion was conducted by Wu et al. who observed that the polysaccharides of *Auricularia* mushroom could form highly viscous substances and retard glucose diffusion by forming a barrier that interferes with starch hydrolysis [24]. In addition, another study found a negative correlation between the glycaemic response and total dietary fibre of mushroom powder. High amounts of dietary fibre could lead to the alteration of physicochemical properties and the digestibility of noodles [65]. This condition could explain the graph pattern in Figure 2.

Macronutrients, such as fat and protein, could influence the postprandial blood glucose responses to food, but the effect of those nutrients is more pronounced in mixed meals in a dose-dependent manner [33]. In contrast to one other study, the incorporation of WEM powder did not increase the protein content of instant noodles, which might not contribute to the integrity of protein network in the noodles [65]. Notably, decades of extensive studies have attempted to link certain elements to the modulation of glucose homeostasis [75]. Meanwhile, other studies suggested the hypoglycaemic effect of WEM could be mediated via the polysaccharide interaction with insulin receptors on target tissues, and partially exhibited via modulation of the anti-oxidative system, but limited knowledge is available to confirm the mechanism [20,76]. Nevertheless, study findings have revealed that adding 5% of WEM powder to noodles could lower the glucose spike, demonstrating the hypoglycaemic effect among consumers.

The glycaemic response was further calculated to obtain the incremental area under the curve (IAUC) for each respondent, which reflected the changes in blood glucose levels within two hours. The mean of IAUC between reference and test foods showed a significant difference (*p* = 0.008). Meanwhile, there was no significant difference in the mean of IAUC between both test food (incorporated noodles with 5% WEM and control). The mean of IAUC of reference food was the highest at 269.83 mmol.min/L, while the mean IAUC of the test food was 196.43 mmol.min/L and 179.78 mmol.min/L for control and incorporated noodles, respectively. Moreover, for a valid GI measurement and high accuracy of GI value, the coefficient variation of reference food should be lower than 30% and for this study, the coefficient variation of reference food that was repeated three times was 15.31%, which was lower than the recommended value [31]. Therefore, the GI value of control noodles containing 50 g of available carbohydrates was high (GI = 75.84), whereas the GI for instant noodles incorporated with 5% WEM were moderate (GI = 68.91).

The GI value of food can be influenced by various factors, particularly methodological and human factors, which are crucial for glycaemic analysis [31,77]. The moderate GI value of 5% WEM noodles indicated that the blood glucose concentration increased moderately within 2 h. Most noodles have been categorized as low GI foods because the digestion process of noodles is slower and incomplete compared to other starch-based foods such as white bread [18]. One of the factors for the discrepancy is the different types of flour used in making instant noodles, where a higher amylose or fibre content can influence the digestibility rate and glycaemic response, resulting in a low GI instant noodle [78]. In addition, food processing can cause physical alterations in the starch structure and promote starch digestibility, but some studies have postulated that the step-in noodle processing, such as mixing, sheeting, and extruding, may cause the protein matrix to continuously entrap starch granules and limit the starch hydrolysis [79]. In addition, the retrogradation process during cooling may increase resistant starch, resulting in low GI food [80]. In a glycaemic study, the control of blood glucose level proves to have a huge benefit for a diabetic patient while it also could lower the risk of cardiovascular heart disease and help in weight management for a healthy individual [81,82]. Nevertheless, this study successfully utilised WEM powder as a functional ingredient in instant noodles and exhibited a hypoglycaemic effect among study participants.

## **4. Conclusions**

The highest composition in WEM powder was carbohydrate, followed by crude fibre, moisture, protein, ash, and fat. The addition of WEM powder in noodles influenced the nutrient composition, significantly reducing carbohydrates and increasing ash, crude fibre, dietary fibre content, and antioxidant activities. Furthermore, the incorporation of WEM powder significantly improved elements such as potassium, calcium, magnesium, iron, and zinc, but not manganese, copper, and chromium. Moreover, adding WEM powder to instant noodles could lower the fluctuation of postprandial blood glucose levels and reduce the GI value. In conclusion, the incorporation of WEM powder is promising in developing nutritious and low GI instant noodles that could increase nutrient intake and improve human health.

**Author Contributions:** Conceptualization, M.K.Z. and P.M.; investigation, M.K.Z.; writing—original draft preparation, M.K.Z.; writing—review and editing, P.M., R.G., W.P., H.M., N.S. and J.S.S.S.; visualization, M.K.Z.; supervision, P.M. and R.G.; project administration, P.M. and R.G.; funding acquisition, P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Universiti Malaysia Sabah with *Skim Dana Nic* (SDN), SDN0065-2019.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Medical Ethics Committee of Universiti Malaysia Sabah (JKEtika 2/21(16)).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to express their appreciation to Universiti Malaysia Sabah for the niche grant scheme, Andree Alexander Funk and Phascheyllah Erdana Au from Rural Development Corporation for the supply of mushrooms and technical support, and financial support for the publication fee funding from the Research Management Centre, Universiti Malaysia Sabah. Special appreciation is also extended to the Institute for Tropical Biology and Conservation (ITBC) especially Jaya Seelan Sathiya Seelan and Ily Azzedine Alaia Bte Mh Subari for the technical support on the mushroom species identification.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

