Influence of garlic and pepper powder on physicochemical and sensory qualities of flavoured rice noodle

This research aimed to investigate the effect of dried garlic powder (DGP) and dried white pepper powder (DWPP) on physicochemical and sensory properties and to develop a garlic-pepper flavoured purple rice noodle (GPFRD). The garlic-pepper powder (GPP) aroma found to be comprised of pine, garlic, onion, citrus and woody characteristic. The 78 g of DGP and 20 g of DWPP provided high sensory rating score of pepper aroma (5.9 ± 0.1) and overall aroma (5.6 ± 0.2) with a high content of beta-caryophyllene (0.101 ± 0.04 mg/g powder), limonene (0.069 ± 0.02 mg/g powder), allicin (10.48 ± 0.18 mg/g powder) and piperine (0.71 ± 0.11 mg/g powder). The cooked GPFRD mixed with garlic-pepper at 2% possessed the good quality of physical and chemical properties with sensory rating score. The GPFRD using 2% of GPP provided preference rating score in the range of 6.0–6.7 with consumer acceptance at 82.0% and purchase intention at 74.0%. Consequently, the optimum ratio of DGP and DWPP provided a better spice mix for aroma, flavour, with some bioactive compound aspects. A suitable amount of GPP can provide the preferable properties of flavoured purple rice noodles.

Analysis of volatile compounds from GPP using gas chromatography flame ionization detector (GC-FID). The volatile compounds from GPP were analysed using GC-FID. The 10 g of sample was extracted in 250 ml of dichloromethane for 2 h, afterward; the solvent was taken to evaporate using a rotary evaporator (V800, Büchi, Switzerland) at 40 °C under adjusted pressure at 900 mbar. The evaporated extract has adjusted the concentration of 10 mg/ml and filtered before the analysis with GC-FID (GC-2010, Shimadzu, Kyoto, Japan). One µl of the garlic-pepper solution was analysed using the GC-FID condition as described above 15,16 . Analysis of allicin content. The allicin content was prepared from 1 g of GPP and extracted in 30 ml of distilled water at 50 °C, 2 h using the magnetic stirring rod at speed of 50 rpm. The mixed solution was filtered before analyzing the allicin content 18 . Ten ml of solution was mixed with 10 ml Trizma ® hydrochloride (1 M, pH 8) and incubated for 15 min. The 50 ml of DTNB (2 mM) which prepared 50 mM Sodium acetate trihydrate and 100 ml of L-Cysteine (20 mM) were then added into the mixture and adjusted the volume into 1000 ml with distilled water. The prepared solution was analysed under absorbance at 412 nm and calculated for allicin content in mg/g. Analysis of piperine content. The piperine content in GPP was analysed following Rameshkumar method 8 . One gram of GPP was extracted in 60 ml of 95% Ethanol at 50 °C, 2 h using the magnetic stirring rod at speed of 50 rpm. The extract solution was adjusted into 100 ml with 95% Ethanol. The prepared solution was diluted two steps by pipetted 5 ml and adjusted into 50 ml, then pipetted another 5 ml and adjusted into 25 ml using 95% Ethanol. The final prepared solution was analyzed under absorbance at 343 nm with Ethanol as blank. The piperine content was calculated and expressed in g/g dry sample.
Sensory evaluation of GPP. The consumer was recruited through a sensory evaluation class at Chiang Mai University, and written informed consent was obtained from all participants before the experiment was conducted. All participants signed and returned the consent form to the research team, ensured participants consent before participating in the study. The experimental ethics was exempted in this study according to Chiang Mai University's Institutional Review Board from Chiang Mai University Ethics Committee as the GPP was assessed for inclined risks toward health issues of any consumers. All methods used in this part of the experiment were carried out following the ethical of sensory evaluation guidelines and regulations 19 . The demographic details of the consumers were not collected as the main concern was the sensory acceptance toward the attribute's preference of GPP as the consumers are all 100% Thai with ranged in age from 18-25 years old. The GPP was prepared for sensory acceptance using untrained consumer (n = 60) rated for attributes preference (colour, garlic aroma, pepper aroma, and overall aroma) with 9-point hedonic scale 19 . The sample was prepared in disposable closed lid plastic cups coded with a three-digit number. The evaluation was performed in individual air-conditioned booths ( www.nature.com/scientificreports www.nature.com/scientificreports/ Preparation of GPFRD. The GPFRD was prepared using the method from Li and Vasanthan 20 with slight modification. The ingredients consisted of the white rice (75%), purple rice (25%), then mixed with GPP at the variation of 2, 4, 6% using 0% GPP as a control sample. All three ingredients were mixed and adjusted the moisture content into 48%. The mixture was left to equilibrium for 1 h before extruded in the single-screw extruder (19/20 DN, Brabender DHG, Germany) with 1.0 mm die opening and a screw speed of 200 rpm at barrel zone 1 at 60 °C, barrel zone 2 at 70 °C, and barrel zone 3 at 80 °C. The extruded noodle was cut in the length of 12 in and dried in a hot air oven at 60 °C for 1 h to dehydrate the excessed moisture inside the noodle. The product was stored in an airtight container at room temperature (28 ± 2 °C) before analysing for moisture content, water activity, and colour value (L*, a*, b*). Moreover, the GPFRD was cooked and then analysed for colour value (L*, a*, b*), main volatile compounds, allicin content, and piperine content using the method previously described. Besides, the GPFRD was also analysed for cooked characteristics (cooking loss, cooking weight, firmness, and tensile strength).
Cooking loss and cooking weight of GPFRD. Dry noodle diameter (ND) was measured with a caliper. The cooking loss and cooked weight of starch noodles were measured by a modification of AACC Method 66-50 21 . Noodles (5 g) were cut into 3-5 cm lengths and cooked in 200 ml of boiling distilled water for 1 min more than the optimum cooking time. The beaker was covered with aluminum foil to minimize evaporation losses. The optimum cooking time was determined by crushing cooked noodles between a pair of glass plates until the white hardcore in the noodle strand disappeared. This indicated that starch in the center of noodle strands was cooked/ hydrated. The cooked noodles were then filtered through a nylon screen, rinsed with distilled water, and drained for 5 min. The cooking loss (CL) was determined by evaporating the combined cooking water and rinse water to dryness at 110 °C and expressed as the percentage of solids loss during cooking. The cooked weight (CW) was calculated as the weight of cooked noodles as a percentage of dry noodle weight before cooking.
Firmness and tensile strength of GPFRD. The cooked GPFRD was measured for firmness and tensile strength using a texture analyzer (TA-XT Plus, 1125 Stable microsystem Ltd., Surrey, UK) equipped with spaghetti/noodle tensile rig (A/SPR) 20 . Several long strands of noodles were soaked in distilled water for 40 min and cooked to the optimum time. The cooked noodles were rinsed with cool water immediately over a nylon screen and placed in distilled water for testing. After removing the surface moisture from noodle strands with blotting paper, 10 noodle strands were cut to 6.5 cm in length and placed on end into the lower rig arm slot and winding the loosened arm sufficiently, to anchor the noodle end by least two revolutions of the arm. The arm is tightened, and the same procedure is performed to anchor the other noodle end of the upper arm. Tensile strength was tested with rigs arm separation at a speed of 1 mm/s. The initial distance between clamps was set at 10.0 cm. The lower and upper rigs were distanced until the strand ruptured. The maximum stress and breaking distance were recorded. The tensile strength (TS) and breaking distance (BRD) were expressed as the maximum Force before the noodle strand was ruptured (N) and the ratio of the breaking distance to the cross-section area of the noodle strand (mm/mm 2 ).
Consumer acceptance of cooked GPFRD. The participants for the consumer acceptance of the optimized formulation of GPFRD were recruited randomly at Princess Mothers Health Garden, Chiang Mai, Thailand, and written informed consent was obtained from all participants before the experiment was conducted. All participants signed and returned the consent form to the research team, ensured participants consent before participating in the study. The experiment ethics was exempted in this study according to Chiang Mai University's Institutional Review Board from Chiang Mai University Ethics Committee as all ingredient in GPFRD was assessed for inclined risks toward health issues of any consumers. All methods used in this part of the experiment were carried out following the ethical of sensory evaluation guidelines and regulations 19 . The demographic details of the consumers were not collected as the main concern was the sensory acceptance toward the attribute's preference of GPFRD as the consumers are all 100% Thai with ranged in age from 18-25 years old. The GPFRD was prepared and put in disposable closed lid plastic cups coded with a three-digit number and evaluate in an individual booth ensuring comfort and privacy. The untrained consumers (n = 100) were asked to rate the preference score for attributes preference (colour, garlic aroma, pepper aroma, and overall aroma) with a 9-point hedonic scale 19 . The product acceptance and purchase intention for the final product was added into the questionnaire on the consumer acceptance test.
Statistical analysis. All data were analysed in triplicate and reported as a mean±standard deviation. The statistical analysis was conducted using SPSS 11.0 (SPSS Inc., IBM Corp., IL, USA) using Duncan's multiple range test (DMRT) with a significant level determined at a 95% confidence limit (p < 0.05). The regression analysis on RSM to indicate the optimized ratio of DGP and DWPP was appointed using Design Expert 6.0 (Stat Ease Inc., MN, USA).

Results and discussion
Identification of main volatile compounds of GPP using gas chromatography flame ionisation (GC-FID) coupled with gas chromatography olfactometry (GC-O). The retention of garlic-pepper aroma was detected as beta-pinene (weak to medium), allyl methyl sulfide (medium to strong), dimethyl disulfide (weak to medium), dimethyl trisulfide (weak to strong), limonene (weak), and beta-caryophyllene (weak), respectively. The results showed that the garlic aroma characteristics can be defined from allyl methyl sulfide (garlic), dimethyl disulfide (onion), and dimethyl trisulfide (fried garlic) while the white pepper aroma characteristics can be defined as beta-pinene (pine), limonene (citrus), and beta-caryophyllene (woody). Moreover, the findings of main pepper aroma compounds consisted of beta-pinene, limonene, and beta-caryophyllene 8  www.nature.com/scientificreports www.nature.com/scientificreports/ phytochemical, antioxidant and aroma compounds of Piper longum L. and Piper chaba. Those can be used as information to confirm the main aroma compounds of GPP 21,22 . Physical properties, main volatile compounds and active compounds on GPP. The moisture content, water activity, and colour value (L*, a*, and b*) were a significant difference and was in the range of 6.45% to 7.86%, 0.34 ± 0.01 to 10.36 ± 0.03, 81.56 ± 0.01 to 88.92 ± 0.03, 0.35 ± 0.10 to 0.77 ± 0.02, and 13.95 ± 0.26 to 19.53 ± 0.15, respectively. The results showed significant amount of volatile compounds from GPP as beta-pinene (0.006-0.037 mg/g), allyl methyl sulfide (0.008-0.101 mg/g), dimethyl disulfide (0.001-0.034 mg/g), dimethyl trisulfide (0.194-0.606 mg/g), limonene (0.016-0.114 mg/g), and beta-caryophyllene (0.007-0.226 mg/g). The bioactive compounds of GPP also showed a significant difference amount of allicin (garlic) as 8.45-12.65 mg/g powder, and piperine (white pepper) as 0.04-1.31 mg/g powder ( Table 2). The increasing amount of DGP and DWPP caused the volatile compounds and bioactive compounds from GPP to be increased as suggested in the findings from many pieces of research on DGP and DWPP 10,14,16 . Sensory acceptance on GPP. The GPP was evaluated for sensory acceptance (n = 60). The result showed a significant difference in colour, garlic aroma, pepper aroma, overall aroma. The sensory rating score of attributes was in the range of 3.5-6.9; colour (5.5-7.1), garlic aroma (4.7-7.2), pepper aroma (4.5-7.3), and overall aroma (3.5-7.4) as shown in Table 1. The GPP sensory scores were affected by the DGP and DWWP, resulting to affect its overall aroma. The DGP with the medium of amount DWPP showed the highest score of overall aroma because the DWPP contained that pleasant aroma toward consumers and it can enhance the aroma of DGP when it was blended together 23,24 . The optimisation and validation of GPP. There were five responses (pepper aroma, overall aroma, limonene, allicin, and piperine) that fitted to create the regression equations and can be explained altogether using Table 3 and Fig. 1.
The pepper aroma of the treatment 5 (DGP and DWPP at 70 g and 15 g) provided the highest sensory rating score whereas the treatment 2 (DGP and DWPP at 95 g and 5 g) provided the lowest sensory rating scores, resulting from the increasing amount of DGP and DWPP. Figure 1A showed that the increasing of DGP can decrease the sensory score on pepper aroma whereas the increasing of DWPP can increase the sensory score on pepper aroma as the regression equation of pepper aroma showed in Table 3.
The overall aroma of the treatment 5 (DGP and DWPP at 70 g and 15 g provided the highest sensory rating score where the treatment 7 (DGP and DWPP at 85 g and 0.86 g). The increase of DGP affected overall aroma to be decreased whereas the increase of DWPP affected overall aroma to be increased which can be explained in Fig. 1B. These results suggested DGP and DWPP affected the overall aroma of GPP. Besides, the interaction of  Table 2. Main volatile compounds content and bioactive compounds from GPP. *note: the different of the letter in the same column indicated the significant difference (p < 0.05). 1 Treatments are in Table 1.  www.nature.com/scientificreports www.nature.com/scientificreports/ DGP and DWPP showed an effect on overall aroma, as the increasing of GPP can enhance the overall aroma of the GPP (Table 3). The pepper aroma and overall aroma from GPP found to be affected by DWPP and DGP but in a reverse direction. This is because of the DWPP consist of a volatile compound such as beta-pinene, limonene, and beta-caryophyllene that caused citrusy, green and woody notes and woody, pungent taste in pepper over the garlic that caused sulfurous aroma. Those aromas from the pepper can be found to be a more pleasant perception toward consumers than those aromas from garlic 24 .
The limonene and beta-caryophyllene content from garlic-powder can be expressed in the relationship of DGP and DWPP using linear regression as shown in Table 3. The limonene and beta-caryophyllene content affected by DWPP because of those compounds were the main volatile compounds that can be identified from white pepper 8 . The regression equation of limonene and beta-caryophyllene content showed that DWPP demonstrated a higher effect than DGP. The increase of DWPP affected limonene and beta-caryophyllene content to be increased. That evidence can be found in the same direction from the response surface model as shown in Fig. 1C,D.
The allicin and piperine content were found to be affected by DGP and DWPP as shown in Table 3. The allicin was a bioactive compound that can be found in garlic while the piperine was a bioactive compound that can be found in white pepper 24 . The regression equation of allicin and piperine demonstrated that the increasing amount of DGP affected the allicin content to be increased while the increasing of DWPP affected the piperine content to be increased. The effect of DGP and DWPP can be explained the regression equations more clearly using response surface models as shown in Fig. 1E,F. The overlay plot of optimised GPP was generated using the high value of sensory rating score (pepper aroma and overall aroma), the volatile compounds (limonene and beta-caryophyllene), and the bioactive compounds (allicin and piperine) as the desirable properties of GPP. The optimised formula for GPP from the RSM consisted of the 78 g garlic powder and the 20 g pepper powder (Fig. 1). The finding of this optimisation was conformed to the investigation on garlic and pepper-flavoured puff snacks which concluded the garlic content and pepper content at 70 g and 18.25 g, respectively 25 . The result suggested that the suitable ratio of garlic and pepper to create acceptance in snacks and noodles was about 4:1. The observation values from the validation of GPP illustrated the highest values of responses as pepper aroma (6.9 ± 0.2), overall aroma (6.6 ± 0.1), limonene content (0.069 ± 0.02 mg/g powder), beta-caryophyllene content (0.101 ± 0.04 mg/g powder), allicin content (10.48 ± 0.18 mg/g powder), and piperine content (0.71 ± 0.11 mg/g powder).
Physical and texture properties of cooked GPFRD. All GPFRD were cooked using boiling water (98 ± 2 °C) for 1 min. The colour values (L*, a*, b*), tensile, breaking distance, cooking loss, and cooking weight of all samples were significant differences ( Table 4). The increasing of GPP affected the lightness to be increased whereas the redness and the yellowness were decreased. This result suggested that the cooked GPFRD become more loosen when engaged with the cooking process using hot water. The higher amount of GPP can be the key www.nature.com/scientificreports www.nature.com/scientificreports/ factor that rice noodle become less intact because of the fortified powder interrupted the rice starch binding during the extrusion process. The reason that this incident happened because the rice starch partially encapsulated GPP that created flavour encapsulation instead of gelatinization the whole portion of the powder mixture. This occurrence can be explained through the encapsulation in flavour retention during high-temperature short time cooking extrusion process and the extrusion can entrap aroma and flavour compound better as it is a true encapsulation 26 .
The decreasing trend of tensile indicated the breaking strength and elasticity of cooked noodles were decreased, which showed that the higher amount of GPP added can cause rice noodles to break easier. In the present study, it was observed that the increase of the GPP affected tensile to be decreased whereas breaking distance, cooking loss, and cooking weight were increased. The high cooking loss indicates high solubility of starch and low cooking tolerance; results in turbid water and a sticky mouthfeel whereas low cooking weight and low tensile cause by poor water-binding capacity 27 .
Chemical properties of cooked GPFRD. The results exhibited the range of volatile compounds followed the retention time as beta-pinene (1.15-2.96 mg/g), allyl methyl sulfide (0.79-2.83 mg/g), diallyl disulfide (0.44-0.15 mg/g), diallyl trisulfide (0.44-1.15 mg/g), limonene (0.15-0.19 mg/g), and beta-caryophyllene (0.46-0.54 mg/g) ( Table 5). The cooked GPFRD also showed a significant difference of allicin and piperine content. The results of allicin content and piperine content were in the range of 35.52-57.60 mg/g and 0.014-0.211 mg/g. The increasing of GPP affected the main volatile compounds, allicin, and piperine to be increased because of the higher ratio of garlic-pepper in the GPP appeared to contain a higher amount of total volatile base value significantly 28 . Sensory and consumer acceptance of cooked GPFRD. The cooked GPFRD with 2% GPP provided the highest sensory rating score; appearance (6.8 ± 1.1), colour (6.7 ± 1.0), garlic aroma (6.3 ± 1.2), pepper aroma (6.1 ± 1.2), overall aroma (6.1 ± 1.2), texture (6.0 ± 1.5), garlic flavour (6.1 ± 1.2), pepper flavour (6.8 ± 1.2), overall flavour (6.6 ± 1.2), spiciness (6.1 ± 1.6), aftertaste (6.0 ± 1.6), and overall liking (6.3 ± 1.3) as shown in Table 4 with consumer acceptance at 82.0% and purchase intention at 74.0%. The results revealed that garlic-pepper affected on sensory rating score to be increased from non-added GPP to 2% GPP. The increase of GPP into rice noodles over 2% can cause the sensory rating score to be decreased. The amount of GPP more than 2% was exceeded the degree of acceptability of consumers, which can be exhibited the aroma and flavour too strong toward the consumer. Similarly, some investigations suggested in the sensory tasting of noodles added with moringa leaves powder that only the increased amount of moringa leaves powder to 0.3% can retain the product acceptability from the consumer. This is also suggested that only a small number of herbs and spices should be fortified into food products because only slightly quantity can provide a high volume of aroma and flavour which can be suitable for consumers 16 .

Conclusion
The findings from this experiment revealed that the GPP exhibited main aroma compounds and its characteristic from both DGP and DWPP as beta-pinene (pine), allyl methyl sulfide (garlic), dimethyl disulfide (onion), dimethyl trisulfide (fried garlic), limonene (citrus), and beta-caryophyllene (woody). The suitable ratio of DGP and DWPP for GPP was 78 g and 20 g, provided the acceptable sensory rating score of pepper  Table 4. Colour values, texture properties and sensory evaluation of cooked GPFRD. *note: the different of the letter in the same column indicated the significant difference (p < 0.05). www.nature.com/scientificreports www.nature.com/scientificreports/ aroma (6.9 ± 0.2) and overall aroma (6.6 ± 0.1). This optimum amount also provided the highest amount of limonene (0.069 ± 0.02 mg/g), beta-caryophyllene (0.101 ± 0.04 mg/g), allicin (10.48 ± 0.18 mg/g), and piperine (0.71 ± 0.11 mg/g). Moreover, the GPP at 2% was a suitable amount to produce the most preferable GPFRD using the extrusion process. A suitable amount of GPP can provide the preferable properties of flavoured rice noodles. Besides, this work can provide introductory data for further research on the correlation between volatile compounds, antioxidant compounds, bioactive compounds, and sensory perception.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.