Formation of Resistant Starch During Processing and Storage of Instant Noodles

Abstract

Instant noodle was prepared in a commercial noodle plant using varying steaming and frying conditions. Wheat flour had very low amount of resistant starch (RS) as 0.26 g/100 g dry solids. Dough was formed by mixing wheat flour with other adjuncts for 15–20 min, cut into about 0.5 mm strands and steamed in a tunnel for 90 to 240 s. Steaming of dough increased RS content from 0.22 to 0.49 g/100 g dry solids in 90 s and to 1.4 g/100 g dry solids in 240 s. Frying of steamed noodles at temperatures ranging from 145–170°C for 60–160 s resulted in sudden decrease in moisture content from 42.9–49.6 to 0.6–1.6 g/100 g dry solids and increase in oil content up to about 20 g/100 g dry solids. Frying of steamed noodle strands resulted in only slight increase in RS content (up to 1.2 times), depending on steaming time and temperature. Storage of instant noodles showed up to 1.4 times increase in RS content but refrigerated storage did not increase the RS content.

Keywords:

• Resistant starch
• Instant noodle
• Steaming
• Frying
• Storage effect

INTRODUCTION

Resistant starch (RS) is defined as the sum of starch and products of starch degradation that could not absorbed in the small intestine of healthy individuals.[1] Fermentation of RS in the colon by colonic bacteria producing short chain fatty acids (SCFA) has been associated with various health benefits such as preventing incidence of colo-rectal cancer.[2] The foods with significant RS level have a low glycemic index (GI) value and hence have possible protective effects against type II diabetes, obesity and heart diseases.[3] RS also appears to function as a prebiotic by supporting growth of probiotic microorganism and also has the symbiotic effect on them.[4] RS is a natural component in many native foods like cereals, legumes, fruits, vegetables and also in various processed foods up to 15% of their weight. It is classified into four types viz., RS1, RS2, RS3, and RS4.[5] RS1 physically trapped and inaccessible starch is found in whole or partly ground cereal grains, seeds, and legumes. RS2 consists of non-gelatinized condensed and crystalline resistant starch granule found in foods like green banana and native potato. RS3 is due to the recrystallization or retrogradation of amylose and amylopectin after food processing. Majority of moist heat-treated foods and processed foods contain some quantity of RS3 and is therefore of both commercial and nutritional interest. RS4 represents a group of starches that have been chemically modified in such a manner as to decrease enzyme resistance.

Instant noodles are prepared by steam cooking of dough strand under pressure and later fried in cooking oil. Steam cooking of starch-water mixture triggers a number of physico-chemical and functionality changes in starch granules, such as the loss of granular structure associated with melting of crystallites and underlying helices, and the generation of an amorphous structure. This structure may later re-acquire ordered helical or crystalline order (retrogradation) and become resistant to digestion by human α-amylase. Various investigators have reported that retrogradation following cooking results in formation of enzyme RS.[6,7] Generally, RS3 has been found to contain short and linear chains of thermally stable α-1, 4 glucans of about 10–100 chain lengths; significant double helix content and moderate crystallinity (mostly B-type).[8] Several studies have shown that high amylose starches and products made from them have high RS contents measured in vivo which correlate with enzyme-resistant values determined in vitro. [9]

Formation of RS in foods is influenced by a number of factors such as water content, pH, heating time and temperature, freezing, drying, frying, fermentation, storage, presence of constituents like protein, lipid, minerals, and inhibitors etc.[10,11] Instant noodle is a common snack food in Asian countries including Nepal. Wheat flour, the base material for noodle, is a very poor source of RS, containing less than 0.4 g RS/100 g dry solids.[12,13] Wheat flour undergoes a number of processing stages during instant noodle preparation such as dough formation, steaming, frying, and storage. The aim of the present research is to study the effect of processing during instant noodle manufacture as well as storage conditions on RS formation.

MATERIAL AND METHODS

Materials

Wheat flour and other noodle ingredients (gluten, common salt, guar gum, and alkaline salts) were purchased from the local market. Enzymes for RS analysis: pepsin, α-amylase, and amyloglucosidase were obtained from Sigma Pharmaceuticals.

Instant Noodles Preparation

Instant noodle was prepared in a local continuous plant (Asian Thai Foods, Duhabi, Nepal) with an operating capacity of 1.12 ton instant noodle per h. Wheat flour (200 kg), water (41.5 L) gluten (8 kg), common salt (1.7 g), guar gum (300 g) and alkaline salts (300 g) were mixed in a horizontal mixture (locally made, double arm, 22 rpm, 0.5 ton capacity) for 20 min to form a homogenous dough. The dough was initially sheeted to 3 cm thickness and was gradually reduced to about 0.5 mm by a series of smooth stainless steel reduction rollers and further cut into noodle strands, waved and steamed. The steaming process was carried out in a steaming tunnel (15 m × 0.5 m, 2 kg pressure and maintained at 100°C) from 90 to 240 s. The weaved, steamed noodle was cut into desired shape, dipped in soup and fried in palm oil (free fatty acid value, 0.2% and peroxide value, 0.5 meq/kg) in a frying tunnel (10 m × 0.5 m) for time varying between 60 to 160 s. The frying oil temperature at the entrance of tunnel was maintained at 145°C and the exit temperature was varied form 152 to 170°C. The fried noodle was cooled and packed airtight in a low-density polyethylene bag and stored at room temperature (∼25°C) until further analysis. A subset of sample was also stored under refrigeration temperature (4°C) to study the effect of storage condition on RS content of noodle.

Resistant Starch Analysis

RS content of raw materials as well as processed noodles was determined by the method given by Goňi et al.[14] with some modifications. The samples were ground to fine particles, using a mortar and pestle and then passed through a 1 mm sieve. These sieved samples were defatted by extracting three times with petroleum ether. About 100 mg of fat free dry sample was mixed with 10 mL of KCl-HCl buffer (pH 1.5). The mixture was incubated with 0.2 mL pepsin solution (1 g pepsin per 10 mL KCl-HCl buffer) in a shaking water bath set at 40°C for 60 min. The sample was neutralized by 0.5 M NaOH, buffered (9 mL 0.1 M NaOH), and incubated with 1 mL α-amylase solution (400 mg in 10 mL Tris-Maleate buffer) in a shaking water bath set at 37°C for 16 h. The incubated tubes were centrifuged for 15 min at 3000 g. Supernatant was decanted and the residue was washed (centrifuged) again with 10 mL water. The residue was mixed with 3 mL water and 3 mL 4M KOH and shaken for 30 min. The mixture was neutralized with 5.5 mL 2M HCl and pH was adjusted to 4.75 using 3 mL 0.4 M sodium acetate buffer. The starch residue was further hydrolyzed with 80 μL of amyloglucosidase for 45 min in a shaking water bath set at 60°C. After completion of incubation, the tubes were centrifuged for 15 min at 3000 g and supernatant was transferred to 250 mL volumetric flask. The residue was further washed with 10 mL water, supernatant was transferred to the same volumetric flask, and volume was made up to 250 mL. Glucose content of the sample was determined by glucose determination kit (Span Diagnostics Ltd., India) using the method recommended by Goñi et al.[14] This value was multiplied by 0.9 to obtain the starch content in the undigested residue (or enzyme resistant starch). The RS content of the test sample was then calculated using the following equation:

Statistical Analysis

All the samples were analysed in triplicates. Data were analysed using the analysis of variance (ANOVA) by the Statgraphics package (Statistical Graphics Corporation, 1993, Manugistic Inc., USA). The multiple range test LSD (Duncan multiple rage test), with significance level at p ≥ 0.05, was applied to the results to test the significant difference.

RESULTS AND DISCUSSION

Resistant Starch Content of Flour and Mixed Dough

The flour and dough used for noodles preparation were analyzed for moisture content and RS content. The moisture contents of wheat flour and dough were 15.4 and 41.3 g/100 g dry solids, respectively. RS contents of the flour and dough was found to be 0.26 and 0.22 g/100 g dry solids, respectively. Other investigators have reported a slightly different RS values for wheat flour such as 0.38% by Kim et al.[12] and less than 0.2% by Kavita et al.[13] However, comparison of RS content from a particular starch with values obtained from other studies is difficult as enzyme digestion protocol, amount of starch present and the type/cultivar of starch (plant source) being analysed affect the RS analysis.[13] The moisture content of dough was 41% (dry basis), which was enough for dough formation, but it was unlikely to affect the RS content as there would not be any gelatinization and subsequent retrogradation due to cold mixing. Therefore, the result showed that the mixing the flour with water and other noodles ingredients (water, salts, gluten and gum) for 20 min in the process of preparing dough did not significantly (p ≤ 0.05) alter RS content of flour.

Effect of Steaming

The effect of steaming at constant pressure (2 kg) and constant temperature (100°C) on the RS content of the instant noodle is given in Table 1. The steaming process was accompanied by increase in moisture as well RS content of the dough. The increase in moisture content of the dough (41.3 g /100 g dry solids) during steaming was gradual but significant (P ≥ 0.05): 42.9 at 90 s to 49.6 g/100 g dry solids at 240 s. Considering 0.22 g RS/100 g dry solids in dough, the increase in RS content of dough was sharp. The RS value was almost doubled at 90 s (0.49 g/100 g dry solids) and increased almost six times (1.4 g/100 g dry solids) after 240 s of steaming. Trend analysis of the data indicated that more RS could be generated at higher steaming time, i.e., beyond 240 second (Fig. 1). The regression coefficient for steaming vs RS content was 0.9887% for moisture free basis 0.9867% for moisture and fat free basis. However, the longer steaming more likely to cause higher degree of gelatinization of noodle starch, causing processing difficulties in later operations, and also accompanied by lower yield of noodles. Therefore, longer steaming time was avoided in this study.

Table 1 Effect of steaming and frying on resistant starch formation in instant noodles.^{1, 2}

Steaming parameters			Frying parameters
Time (s)	Moisture (%)	RS^1	Time (s)	Temperature (^oC)	RS^2
90	42.9 ± 0.1^a	0.49 ± 0.05^a1	60	145–170	0.55 ± 0.15^a1
120	45.6 ± 0.1 ^b	0.58 ± 0.02^a1	80	145–168	0.62 ± 0.02^a2
150	47.3 ± 0.2^c	0.70 ± 0.01^b1	100	145–165	0.78 ± 0.05^ab1
180	47.9 ± 0.1 ^d	0.89 ± 0.02^c1	120	145–162	0.98 ± 0.10^b1
210	49.4 ± 0.2^e	1.21 ± 0.11^d1	140	145–158	1.36 ± 0.06^c2
240	49.6 ± 0.2^f	1.40 ± 0.06^e1	160	145–152	1.55 ± 0.28^c1
^1Resistant starch values expressed as % dry basis; ^2mean ± standard deviation (three replicates); and ^3means followed by the same letter superscripts in each row do not differ at LSD at p≥0.05; letter superscript followed by same numerical superscript in each column do not differ at p≥ 0.05.

Figure 1 Trends showing the formation of resistant during frying of steamed noodle.

The raw granular starch exists in semi-crystalline state. Heating of starch in excess water (60°C) results in irreversible swelling and solublization of starch granules, preferentially amylose component, leading to loss of double helical order and crystallinity (as shown by birefringence and X-ray diffraction), uptake of heat and increase in viscosity.[15,16] The gelatinized starch when cooled, the dispersed starch molecules undergo slow re-association forming a tightly packed structure, the process is known as retrogradation. The concentrated amylose gel during retrogradation slowly crystallizes to double helical B-form with melting temperature as high as 120–165°C.[17] The starches respond differently to heating and cooling cycle and water level of the cooked starch and amylose content. The manner in which re-association of these glucan chains during cooling/storage forming a gel largely determine the resistance of starch to enzyme digestion.[17,18]

Majority of studies have shown that heating (cooking) of starch favors the formation of resistant starch. Amylose content is found to be correlated with the increase in RS content of starchy foods during processing. Akerberg et al.[19] baked a mixture of barley (of varying amylose content) and regular wheat flour (1:2.2 ratio) by conventional method (200°C/45 min). They found that baking of dough from waxy barley (3% amylose) yields 0.6% RS, ordinary barley (23% amylose) yields 2.6% RS and high amylose barley (44% amylose) yields 3.5% RS. In another study, Berry[9] reported that autoclaving of starch with high amylose content (amylomaize, Hylon V and VII or purified potato amylose) led to significant RS formation (20–34% RS, dry basis), whereas no such effect was observed in autoclaved high amylopectin starch (waxy maize or purified potato amylopectin) (0·2–4% RS, dry basis). Sievert and Pomeranz[17] also reported the yield of RS is positively correlated with amylose content of the autoclaved and cooled test starches (wheat, maize, potatoes, waxy maize and amylomaize). For example, 21% RS was formed in amylomaize (70% amylose) compared to 7.8% in wheat starch (27% amylose). Bjorck et al.[20] also reported 6.2% RS (dry basis) in autoclaved wheat starch. The regular wheat flour used in noodle preparation contains relatively low amount of amylose (∼23%) so, there is less probability of double helices formation and crystallization of cooked starch, justifying low RS content in steamed noodle.

The amount of water used during preparation was low (41 g/100 g dry solids in dough). Besides, other ingredients of noodles formulation such as gluten, guar gum, and salts also compete for water. The dough was steamed for relatively short period of time (up to 240 s) at 100°C and at 2 kg pressure. Gelatinization process warrants sufficient water to penetrate the starch granule and it is less likely to happen at this moisture level. The RS content of steamed noodle was only 1.4 g/100 g dry solids, which is much lower than 7.8% from autoclaving as reported by Sievert and Pomeranz.[17]

Eerlingen et al.[18] reported that RS content of autoclaved wheat starch-water suspension (1 g in 10 mL water) depends on the incubation temperature and time. They reported that initial incubation of autoclaved starch yield high RS at 68°C compared to 100°C but the condition is reversed at longer incubation time. At 100°C incubation, initially there was some RS formed but rose very quickly after 150 min, a maximum of 10% after 150 min. In current study, the heating/incubation period was too short to effect nucleation or propagation of starch crystals, resulting in little RS formation in steamed noodle.

Some studies have also shown that heating does not increase the RS content. Garcia-Alonso et al.[21] reported that autoclaving or boiling of starches (from corn, rice, wheat, and potato) do not increase the RS content. They also showed boiling of native potato starch reduce RS content from 69% to 1.2%, however, boiling followed by cooling increases RS up to 4.6%. Boiling or high-pressure cooking reported to decrease the RS content of rice.[22] The decrease was more noticeable in low amylose rice than high amylose rice. Lipid present in the starchy foods tends to lower the RS content due to formation of amylose lipid complex (ALC). This is because less amylose is available for the formation of double helices and crystallization.[23] Since wheat flour contains significant amount of lipid, it might also have hindered the RS formation in steamed noodle. However, earlier studies by Holm et al.[24] showed that processing of amylose with lipid or components such as lecithin, palmitic acid, oleic acid and soybean oil affect the retrogradation to a lesser extent than monoglycerides. These authors also found that pure potato amylose and oleic acid formed complexes highly resistant to amylolysis.

Effect of Frying

The frying operation is known to reduce the moisture content of potato crisps with simultaneous increase oil uptake from the frying medium.[25] The color and flavor of fried noodles also altered which is contributed by the Maillard reactions between constituents such as sugars and amino acids. In present study, to maintain the uniformity in color of fried noodles, the oil temperature was lowered with increasing frying time. The moisture content of the steamed noodle was markedly reduced during frying, e.g., from 42.9 (90 s steaming) to 1.3 g/100 g dry solids at (60 s frying) (Table 1). The loss of moisture from the noodle during frying increased with increasing frying time (Fig. 2). The loss of moisture was accompanied by the absorption of oil, 19.2 in 90 s and finally to 22.7 g/100 g dry solids in 160 s (Fig. 2). The thickness of cut-dough sheet was only ∼0.5 mm that means water in the noodle quickly dissipates thermal energy from the surrounding hot oil in the vicinity of the frying noodle. Almost a linear increase in oil uptake and moisture removal in steamed noodle is also reported in frying of potato crisp by Goñi et al.[25,26]

Figure 2 Effect of frying time on moisture and oil retention in instant noodles.

The RS content of the steamed and fried noodle at the given time and temperature (inlet and outlet) is given in Table 1. Although, the difference between the RS content of steamed and fried noodles was low, it was significantly different (p ≥ 0.05). It shows that increasing frying time (decreasing frying temperature) resulted in increase of RS content of the steamed noodles from 7 to 12%. This value is close to 12% increase in RS content in deep-fried vada as compared to non-fried control reported by Mahadevamma and Tharanathan.[27] Pinthus et al.[28] reported that frying of patties made from corn, wheat, rice or potato at 170°C increase RS content from 4.3 to 5.4% in the core region of a patty. Upon varying the amylose content in the cornstarch from 21 to 70%, the initial RS content (prior to frying) increased from 3.5 to 22.7. Goñi et al.[26] reported that frying of the potato crisps for 8 min increases the RS content of french fries, from 1% to 5% (dry basis) whereas in freeze dried crisps increase was extremely high at 32% RS. They reported that lower the moisture content of potato crisp greater the RS formation during frying and concluded that RS is produced by thermal degradation of starch leading to structural modification in the absence of water.

Comparatively low yield of RS during frying of noodles in present study could be due to higher moisture content of noodle starch and relatively short frying time. Frying of high moisture starch or potato crisps causes gelatinization of starch granules resulting leaching some amylose molecules from the starch matrix. Since, there is about ∼50% moisture present in noodles (initially), the thermal degradation of amylose molecules is prevented. As temperature is very high (∼150°C), there is less likelihood of alignment of leached amylose fractions responsible for double helices and crystalline structure resistant to enzyme digestion. Since, frying is accompanied by quick moisture removal (final moisture <1%), there is no water in fried samples to cause the retrogradation of starch. It is concluded that only slight increase in starch content of noodle in this study may be due to partial disruption of the starch granules as well as incomplete crystallization of cooked starch during short frying. Presence of salt (∼1 g/100 g dry solids) may also have lowered the formation of hydrogen bonds between the helices of amylose or amylopectin chains (of linear) as the sodium and chloride ions tend to adsorbed in these active sites.[11]

Little is known about the formation of ALC during frying of starch. It would be interesting to know the thermal properties as well as crystallinity of the ALC formed at higher temperature (∼150°C). The enzyme digestibility of this complex could be different from native granule as this involves ALC formation along with gelatinization, retrogradation, and desiccation.

Effect of Storage

Noodles and pasta products have been reported to be poor source of RS and this study conform previously reported RS values in this category of foods. Goňi et al.[14] had reported <1% RS for pasta products. Similarly, Englyst et al.[1] and Elmstâhl[29] has reported 2.7% and 1.2% RS content (based on starch content) in macaroni products, respectively. One of the reasons for low resistant content in wheat based products are they are traditionally low amylose starchy products and offer a little resistance to amylase digestion.

The change in the RS content of the instant noodles when stored at room temperature and at refrigerated condition at 0, 20, 40, and 60 days is shown in Table 2. The freshly prepared noodles contained 0.83 g RS/100 g dry solids. The proximate composition of noodles was 1.1 % moisture, 19.1% fat, 10.9% crude protein (Nitrogen x 6.25), 0.6% ash, 0.5 % crude fiber, and 67.9 % carbohydrates (all values in dry basis). There was no significant difference (p ≥ 0.05) in RS content of instant noodle stored at refrigerated or room temperature (∼25°C). The RS content of noodles increased significantly (p ≥ 0.05) with storage time, irrespective of storage conditions. The increase in RS content after 20 days was 40% and 29% for refrigerated and room temperature, respectively. There was slight but not significant increase in RS of noodles after 20 days storage. Our previous study also showed that storage of extruded regular maize starch at 4°C for 12 day results in 25% increase in RS content.[30] Kim et al.[12] found about 2.5 times increase in RS content of pastry wheat flour during for 7 days storage at 4°C. Similarly, Kumari et al.[31] prepared a range of ready to eat cereals and legumes based foods with appreciable amount of resistant starch, ranging from 2.1–3.1% (fresh weight basis). Storage of these foods resulted in an increase of RS content from 6 to 53%. Huth et al.[32] reported up to 6% increase in RS value of extruded barley flour followed by storage at 4°C or −18°C for 3–7 days with no further increase of RS when storage continued to 14 days. Kavita et al.[13] found that longer the duration of storage of gelatinized wheat flour, the greater is the formation of RS. Our study further confirmed that the gelatinized and fragmented amylose molecules undergo molecular re-association or retrogradation during storage, which results in increased RS content. The retrogradation process may occur rapidly in the beginning and slow down with time.

Table 2 Effect of storage on the resistant starch content of instant noodles.^1,2,3

Description	0 day	20 days	40 days	60 days
Room temperature	0.83 ± 0.10^a1	1.07 ± 0.09^b1	1.13 ± 0.01^b1	1.15 ± 0.04^b1
Refrigeration	0.83 ± 0.10^a1	1.16 ± 0.07^b1	1.18 ± 0.03^b1	1.20 ± 0.05^b1
^1Resistant starch values expressed as % dry basis; ^2mean ± standard deviation (three replicates); and ^3means followed by the same letter superscripts in each row do not differ at LSD at p≥0.05; letter superscript followed by same numerical superscript in each column do not differ at p≥0.05.

CONCLUSIONS

Wheat flour is a poor source of resistant starch. Processing of wheat dough during commercial noodle preparation and storage alters the enzyme-resistance of starch. Dough forming operation did not contribute significantly on the formation of RS; however, steaming of the noodle strand under pressure contributes significantly on the RS formation. Steaming followed by frying also contribute towards the increased RS content of noodle. Storage of noodles increased the RS formation significantly. The rate of increase and quantity of RS is higher at refrigerated condition compared to that of room temperature.

Acknowledgments

The authors gratefully acknowledge with gratitude to the Asian Thai Foods Pvt. Ltd.; Chowdhary Groups; Nepal Academy of Science and Technology (NAST); Central Campus of Food Technology, Tribhuvan University; CTL Pharmaceuticals Pvt. Ltd., and Deurali Janata Pharmaceuticals Pvt. Ltd; Nepal for their financial and material support during the work.