2. Materials and Methods

2.1. Materials

The raw materials included mocaf (Mocafine) and pumpkin flour (Nula), which were obtained from local industries in Sleman, Yogyakarta, Indonesia, whereas high‐protein wheat flour was purchased from local markets in Yogyakarta. The mocaf flour has a moisture content of less than 12% and a particle size of approximately 80 mesh. The pumpkin flour used has a particle size that passes an 80‐mesh sieve with a moisture content of less than 10%. The food additives used include sodium chloride (NaCl) and sodium tripolyphosphate (STPP; Na5P3O10).

All chemicals of analytical grade were supplied by Merck (Darmstadt, Germany) and Sigma‐Aldrich (St. Louis, United States). The reagents comprised petroleum ether, sulfuric acid (H2SO4), sodium hydroxide (NaOH), boric acid (H3BO3), hydrochloric acid (HCl), ethanol, n‐hexane, acetone, methanol (HPLC grade), Folin–Ciocalteu reagent, sodium carbonate (Na2CO3), gallic acid standard, and DPPH. Starch fraction analysis employed α‐amylase and glucoamylase, while sodium acetate buffer and glucose standards were used for starch hydrolysis. All reagents were used as received without any further purification.

2.2. Methods

2.2.1. Making Wet Noodles

The fresh noodles were formulated using five different ratios of wheat flour: mocaf: pumpkin flour, namely 100:0:0, 80:10:10, 70:20:10, 60:20:20, and 50:25:25 (w/w). A uniform blend of wheat flour, mocaf, and pumpkin flour was obtained, followed by the addition of salt (1.5%, w/w) and STPP (0.3%, w/w), which was maintained at the same level in all formulations. Next, water was added gradually while kneading for about 10 min until a homogeneous, elastic dough formed.

The dough was then rested for 30 min at room temperature (27°C ± 2°C) to allow for the hydration of the starch and protein components. After hydration, the dough was rolled using a pasta machine to produce a dough sheet approximately 1.5 mm thick, then cut into noodle strands approximately 2 mm wide. The resulting noodles were boiled in boiling water at a water‐to‐sample ratio of 10:1 (v/w) for 3 min. Once boiled, the noodles were drained and cooled at ambient temperature for 10 min before storage at 4°C ± 1°C until analysis.

2.2.2. Proximate Analysis

The proximate composition of uncooked fresh noodles was determined according to AOAC International standards [24]. Moisture was measured by oven drying (Method 925.10), ash by combustion in a muffle furnace (Method 923.03), crude fat by Soxhlet extraction (Method 989.05), and crude protein using the Kjeldahl method (Method 991.20). Carbohydrate content was subsequently calculated by difference using the following equation:

(1) Carbohydrate%=100−moisture+ash+fat+protein.

2.2.3. Color Analysis

Color of uncooked fresh noodles was determined using a colorimeter (Konica Minolta CR‐400, Osaka, Japan) according to the CIE L_a_b* system, including lightness (L), redness–greenness (a), and yellowness–blueness (b*). Measurements were performed at three points per sample and reported as the average.

2.2.4. Texture Analysis

The hardness and deformation properties of the cooked noodles were determined using a texture analyzer (TA.XT Plus, Stable Micro Systems, United Kingdom). Samples were compressed to 50% of their initial height at a test speed of 1 mm/s, following a previously reported method Li et al. [25]. The parameters evaluated included hardness and deformation.

2.2.5. Cooking Loss

Cooking loss was determined gravimetrically using cooked noodle samples by boiling 25 g of noodle samples in 250 mL of water for 3 min, followed by filtration of the cooking water to remove insoluble residues. After filtration, the filtrate was dried at 105°C in an oven until a constant weight was obtained, and the loss was calculated as the percentage of solids released during cooking based on the initial sample weight.

2.2.6. Analysis of β‐Carotene

β‐Carotene content was determined according to the method described by Mohammed et al. [26] with slight modifications. Briefly, 5 g of dried noodle sample was extracted with 25 mL of an n‐hexane:acetone:ethanol mixture (2:1:1, v/v/v). The mixture was vortexed for 2 min and centrifuged at 4000 rpm for 10 min. The resulting supernatant was filtered through Whatman No. One filter paper prior to analysis. Absorbance was measured at 450 nm using a UV–Vis spectrophotometer (Shimadzu UV‐1800, Japan). β‐Carotene concentration was quantified using a calibration curve prepared from analytical‐grade β‐carotene standards and expressed as _μ_g/g dry matter.

2.2.7. Analysis of Total Phenolic Content (TPC)

TPC of uncooked wet noodle samples was determined using the Folin–Ciocalteu method described by Singleton et al. [27]. Briefly, 0.5 mL of the sample extract was mixed with 2.5 mL of 10% Folin–Ciocalteu reagent and 2 mL of 7.5% sodium carbonate solution. The reaction mixture was incubated at room temperature for 30 min to allow color development. Absorbance was then measured at 765 nm using a UV–Vis spectrophotometer. TPC was quantified using a gallic acid calibration curve and expressed as mg gallic acid equivalents per g sample (mg GAE/g).

2.2.8. Antioxidant Activity (DPPH)

Antioxidant activity of uncooked wet noodle samples was assessed using the DPPH free radical scavenging assay following the procedure of Brand‐Williams et al. [28] For the analysis, 1 mL of the sample extract (1 mg/mL), prepared as described previously, was added to 3 mL of 0.1‐mM DPPH solution in methanol. The mixture was then kept under dark conditions at room temperature for 30 min to allow the reaction to proceed. Subsequently, absorbance was measured at 517 nm using a UV–Vis spectrophotometer. A control solution consisting of 3‐mL DPPH solution and 1‐mL methanol was used as the blank. The free radical scavenging activity was expressed as a percentage and calculated using the following equation:

(2) Radicalscavengingactivity%=1−AT/Ao×100%.

2.2.9. Characterization of Starch Fractions and Glycemic Index

The determination of starch fractions was carried out using uncooked fresh noodle samples and a validated in vitro enzymatic hydrolysis protocol adapted from Li et al. [29], which simulates physiological starch digestion conditions through sequential hydrolysis with α‐amylase and glucoamylase. Samples were dried, finely ground, and weighed (100 mg) before incubation with sodium acetate buffer (pH 5.2) containing α‐amylase and glucoamylase at 37°C in a shaking water bath. Aliquots were withdrawn at defined time intervals (20 and 120 min), and enzymatic activity was terminated using absolute ethanol. The glucose released was quantified spectrophotometrically and used to estimate starch fractions. The classification of starch fractions was based on hydrolysis duration: RDS (within 20 min), SDS (20–120 min), and RS (remaining after 120 min). The starch fractions are calculated using the following equations: where G20 = glucose released at 20 min of hydrolysis; G120 = glucose released at 120 min of hydrolysis; and 0.9 = conversion factor from glucose to starch equivalent.

(3) RDS%=G200.9×,

(4) SDS%=G12020−G×0.9,

(5) RS%=Totalstarch−RDS+SDS,

A starch hydrolysis profile was constructed by relating the amount of glucose released to incubation time. The AUC of the sample was subsequently compared with that of white bread as the reference standard to obtain the hydrolysis index (HI). The HI value was calculated using the following equation: where AUC_sample = sample hydrolysis curve area, and AUC_reference = reference hydrolysis curve area (white bread or glucose).

(6) HI%=AUCsample/AUCreference×100,

The eGI is calculated from the HI using the empirical equation developed by Goñi et al. [30] with minor modifications

(7) eGI=39.71+0.549×HI.

The eGI value is used to estimate the potential glycemic response of the produced fresh noodle products.

2.2.10. Structural Analysis (FTIR and SEM)

The molecular structure of uncooked fresh noodles was analyzed by FTIR spectroscopy (Bruker Tensor 27, Germany) in the range of 4000–400 cm−1 with a spectral resolution of 4 cm−1 to identify functional groups and potential starch–protein interactions. Microstructural characteristics were evaluated using SEM (Hitachi SU3500, Japan) at magnifications between 500× and 5000×. Samples were dried at 40°C for 24 h and gold‐coated to improve conductivity before analysis.

2.2.11. Sensory Test

A sensory preference test was conducted to assess panelists′ acceptance of the formulated cooked wet noodles. Sensory evaluation was conducted with 30 semitrained assessors recruited from undergraduate students of the Food Technology Study Program at Universitas Mercu Buana Yogyakarta, Indonesia. Participants were 18–25 years of age and reported having no known allergies to the ingredients used in the evaluated samples. The attributes of color, aroma, texture, and overall acceptance were assessed using a 5‐point hedonic rating system, where a score of 1 indicated strong dislike and a score of 5 indicated strong liking. In a sensory laboratory maintained at 22°C ± 2°C with neutral white lighting, samples were served at 45°C ± 3°C, labeled with three‐digit random codes, and presented in randomized order. Panelists rinsed their mouths with mineral water between samples to minimize carry‐over effects.

2.2.12. Research Design and Statistical Analysis

A single‐factor completely randomized design (CRD) was employed, in which the treatment variable consisted of different formulations of wheat flour, mocaf, and pumpkin flour at proportions of 100:0:0, 80:10:10, 70:20:10, 60:20:20, and 50:25:25. Each formulation was prepared in triplicate as independent production batches (biological replicates), and all analytical measurements were subsequently performed in triplicate for each batch (analytical replicates). The assumptions of normality and homogeneity of variance were verified prior to statistical analysis. Data were analyzed by one‐way ANOVA (p < 0.05) followed by Duncan′s Multiple Range Test (DMRT) using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, New York, United States).