Nutritional composition of Cassava (Manihot esculenta) and its application to elder-friendly food based on enzyme treatment

ABSTRACT

Cassava (Manihotesculenta) is known as one of the most nutritious root vegetables, but the texture is relatively hard and tough for the elderly, making it difficult to eat. So, this study attempted to identify the nutritional properties of Cassava and the change in texture according to enzyme treatment to confirm the possibility of using Cassava as anelder-friendly food suitable for the Korean Industrial Standard (KS).To this, the nutritional composition and physiological activity wereanalyzed, and the effect of enzymes (cellulase, polygalacturonase,and α-amylase) on the texture properties of Cassavas was studied. Asa result, Cassava contained various bioactive nutrients such asminerals, essential fatty acids, antioxidants, and anti-diabetessubstances. In all experimental groups, hardness was significantly decreased as the enzyme treatment time increased. Based on KS, when Cassava was treated with cellulase + α-amylase for 3.07 h, α-amylase + polygalacturonase for 3.69 h, and polygalacturonase + cellulase for 4.06 h or more, it showed soft hardness that could be chewed with theelderly's tongue. In addition, it was observed that the microstructure changed as the shape of the tissue was deformed, arrangement became irregular, and size and number of pores increased. In conclusion, root vegetables such as Cassava can be softened tosuit KS using enzymes to promote consumption of elderly consumers andhelp balance nutrition. In addition, considering that Korea isentering a super-aged society, it is thought that this study canprovide positive help to the development of the elder-friendly food industry.

KEYWORDS:

• Cassava
• elder-friendly food
• texture

Introduction

As people get older, their physical function weakens and the need for nutrients increases.^[1] So facilitating access to nutritious food is one of the ways to positively improve the quality of life, especially for the elderly. For example, root vegetables are foods rich in minerals (Ca, K, Fe, etc.) and vitamins (vitamin A, B, C, etc.), and they are actively recommended to the elderly to prevent cardiovascular disease, osteoporosis, dementia, among others.^[2] However, it is not easy to eat root vegetables with a hard texture for the elderly who are relatively weak in chewing due to aging. Therefore, research is important to find a way to easily consume root vegetables with a hard texture to help balance the nutrition of the elderly. As part of this, Korea’s Ministry of Food and Rural Affairs quantified the hardness of food that the elderly can chew in three levels to improve their accessibility to various foods.^[3] This classification system was named the Korean Industrial Standard (KS) and was intended to be used in the development of elder-friendly foods. KS’s first level of hardness is 55,000 to 500,000 N/m², which is defined as chewable by the teeth of the elderly, and the second-level hardness is 22,000 to 50,000 N/m² that can be chewed with the gum, and the third-level hardness is less than 2,000 N/m² that can be chewed with the tongue. Therefore, research on the development of elder-friendly foods that meet KS is needed.

Accordingly, this study attempted to develop elder-friendly foods using Cassava (Manihot esculenta), which is known to have various bioactive substances.^[4] In particular, Cassava can be an excellent source of natural physiological nutrients for the elderly because it is rich in antioxidants such as phenols, flavonoids, among others. However, since Cassava, like other root vegetables, has a hard texture and a lot of tough fiber, softening is essential for the elderly to chew it easily. According to previous study, soft texture of food is an important factor in increasing balanced nutritional intake of the elderly, so it is used as an attribute of root vegetable quality evaluation.^[2] According to another previous study, research is actively being conducted to develop elder-friendly foods by applying freeze–thaw enzyme impregnation methods to produce softened root vegetable for the elderly.^[5] Therefore, this study attempted to find a way to satisfy KS, which can be easily consumed by the elderly, by comparing the potential softening effects of exogenous enzymes (cellulase, polygalacturonase, and α–amylase) on the texture change of Cassava. In addition, a microstructure approach was applied to understand the biochemical deformation characteristics of the fiber structure that may occur in Cassava’s internal tissues during the softening process by enzymes, and through this, the possibility of using Cassava as an elder-friendly food suitable for KS was sought.

Materials and methods

Sample preparation

Cassava grown in Gunma (Japan) in 2022 was purchased and selected by freshness. That is, rotten roots were sorted out, and the selected Cassavas was cut into square shapes (1 cm x 1 cm x 1 cm). Then, cellulose 0.5 g + α-amylase 0.5 g, α-amylase 0.5 g + polygalacturonase 0.5 g, and polygalacturonase 0.5 g + cellulose 0.5 g (set the total amount of enzymes to be 0.1% of the weight of the raw material) were mixed with Cassava (1 kg), respectively, and sealed in a vacuum pack. These were placed in a water bath at 55°C, which is the optimal activity temperature of the enzyme, for 0.5, 1, 2, 3, and 4 h. All of these experiments were repeated three times, and Cassava, which was not treated with enzyme, was set as a control group. The pH of cellulose used in this study is 3.5, which is used to decompose fiber components in vegetables or fruits. The pH of polygalacturonase is also 3.5, which is used to break down pectin in vegetables and fruits. The pH of α-amylase is 5.5, which is used to hydrolyze starch.

Analysis of fatty acid profiles

The fatty acid (FA) composition was analyzed using a gas chromatography (GC) method.^[6] One gram of Cassava was put into a 50 mL test tube, 2 mL of a 5% pyrogallol ethanol solution was added, and 1 mL of a standard solution (triundecanoin) was added. Ten milliliters of 8.3 M hydrochloric acid was added, decomposed for 60 min while stirring at 200 rpm at 80°C, and cooled to room temperature. After mixing 15 mL of diethyl ether, the supernatant (diethyl ether layer) was collected in another new 50 mL test tube by passing syringe filled with 5 g Na₂SO₄. Again, 15 mL of petroleum ether was put into the original test tube and the supernatant (petroleum ether layer) passed Na₂SO₄ again to combine into the previous test tube. The solvent collected in the test tube was evaporated and removed at 40°C under a nitrogen stream, and the crude fat of Cassava was extracted. 1.5 mL of a 0.5 N methanol sodium hydroxide solution was added to the extracted crude fat, and left in an 85°C water bath for 10 min. After cooling this, 2 mL of a 14% BF₃ methanol solution was added, and then left in an 85°C water bath for 10 min to make FA methyl ester (FAME). This was cooled to room temperature, and 2 mL of isooctane and 1 mL of saturated NaCl solution were added, and then cooled to room temperature. The supernatant (isooctane layer) passed through a column filled with Na₂SO₄ was used as a test solution and analyzed with GC (Agilent Technologies 7890A gas chromatograph, Palo Alto, CA, USA). The column used for the analysis was a silica capillary column (SP-2560, 100 m × 0.25 mm i.d. ×0.2 mm film thickness, Supelco), and a flame ionization detector was used as a detector. Carrier gas was used as He at a flow rate of 1.0 mL/min. The results were expressed in g/100 g.

Analysis of mineral profiles

The mineral composition of the Cassava was analyzed using an energy-dispersive X-ray fluorescence spectroscopy (EDXRF) method.^[7] 1.5 g of Cassava was put in a sample cup covered with 5-μm-thick polypropylene film (SPEX, Metuchen, NJ, USA) and the fluorescence energy of each mineral was measured using EDXRF (EDX-7000, Shimadzu, Kyoto, Japan) to measure the content thereof. The primary X-rays used in the analysis were obtained from the 50 W Rh X-ray tube, and a silicon drift detector was used. The results were expressed in mg/100 g.

Analysis of antioxidant effects

Cassava was freeze-dried and powdered, and 10 g of it and 70% ethanol of 100 mL were mixed. This mixture was incubated at room temperature for 24 h. Then it was filtered by filter (Whatman No.2) and used as an antioxidant test sample.

Total polyphenol content

Total polyphenol contents were analyzed according to the research method of Kim and Iida.^[1] Cassava extract with ethanol (40 µL) and Folin-Ciocalteu reagent (Sigma-Aldrich, Tokyo, Japan) diluted 10-fold times (800 µL) were put into a tube, and set for 5 min. Seven percent sodium carbonate aqueous solution (800 µL) was added, nano-pure water (360 µL) was added, and left at room temperature for 2 h. Its absorbance was measured using a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK) at 760 nm. Gallic acid (31.25 ~ 250 ppm) was used as the standard. The result was expressed in GAE g/100 g.

Total flavonoid content

The total flavonoid content of Cassava was measured using the aluminum chloride colorimetric method described by Kim and Iida.^[1] Ethanol extracted Cassava (0.5 mL) was mixed with 95% ethanol (1.5 mL), 10% aluminum chloride hexahydrate (0.1 mL), 1 M potassium acetate (0.1 mL), and deionized water (DW; 2.8 mL), and incubated at room temperature for 40 min. Its flavonoid content was measured using a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK), the blank was read at 415 nm. Rutin (31.25 ~ 500 ppm) was used as the standard. The result was expressed in RE g/100 g.

Superoxide dismutase activity

Superoxide dismutase (SOD) activity was analyzed according to the method of Kim and Iida.^[1] Cassava extract extracted with ethanol (0.025 mL), 100 µM xanthine (0.025 mL), and 60 µM nitro blue tetrazolium (0.025 mL) in 0.1 M phosphate buffer (pH 7.4) were mixed. A xanthine oxidase solution (0.07 U/mL) was added to a total volume of 1 mL. After 10 min of incubation at 25°C and enzyme treatment, absorbance was read at 560 nm using a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK). The result was expressed in U/g.

DPPH radical scavenging activity

DPPH radical scavenging activity of the Cassava was measured in terms of hydrogen donating of radical scavenging ability using stable radical, 2,20-diphenyl-1-picrylhydrazyl (DPPH).^[1] After mixing an ethanolic stock solution (50 µL) and an ethanolic solution of 6 × 10⁻⁵ M DPPH (2 mL), absorbance was immediately measured at 515 nm. Then, after 1 h, the absorbance was measured again to calculate the inhibition rate with a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK). The result was expressed as percentage of inhibition.

ABTS radical scavenging activity

ABTS radical scavenging activity was analyzed according to research methods described by Kim and Iida.^[1] The scavenging effect on 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) radical cation was measured using an antioxidant assay kit (CS0790, Sigma Chemical Co., MO, USA). Trolox (5 ~ 50 ppm) was used as standard, and butylated hydroxytoluene was used as positive control. The reaction mixture (1 mL) contained Cassava ethanol diluent (10 µL), myoglobin reagent (20 µL), and ABTS reagent (150 µL; 10 mL ABTS and 25 µL 3% H₂O₂), which were left at room temperature for 10 min in a dark place, and then absorbance was measured at 405 nm using a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK). The result was expressed in TE mg/g.

Ferric ion reducing antioxidant power

Ferric ion reducing antioxidant power (FRAP) was evaluated using ferric reducing ability of plasma assay.^[1] The FRAP reagent used was prepared by mixing an acetate buffer (25 mL), 2,4,6-Tris(2-pyridyl)-s-triazine solution (2.5 mL), and ferric chloride solution (2.5 mL). This reagent (300 µL) was heated to 37°C, DW (30 µL) and Cassava ethanol extract (10 µL) were added and left for 4 min, and absorbance was measured at 593 nm using a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK). The result was expressed in μmol/g.

Analysis of antidiabetes effects

α–Glucosidase inhibitory activity

The absorbance of ethanol extracted Cassava extract (50 μL) cultured in 0.1 M phosphate buffer (100 μL; pH 7.0) was measured on a 96-well plate at 37°C for 10 min.^[1] After preculturing, 5 mM p-nitrophenyl-α-d-glucopyranoside solution (50 μL; Sigma-Aldrich, Tokyo, Japan) was added to a 0.1 M phosphate buffer (pH 7.0). The reaction mixtures were incubated at 37°C for 5 min and then absorbance was measured. Absorbance readings were recorded at 490 nm in a microplate reader (Imark, BioRad, CA, USA) before and after incubation. The result was expressed as percentage of inhibition.

α–Amylase inhibitory activity

Ethanol extracted Cassava extract (500 μL) were added to 0.02 M sodium phosphate buffer (500 μL; pH 6.9 with 0.006 M sodium chloride) containing porcine pancreatic α-amylase solution (0.5 mg/mL; Sigma-Aldrich, Tokyo, Japan) and was incubated at 25°C for 10 min.^[1] After preincubation, 1% starch solution (500 μL) in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to a tube, and the reaction mixture was cultured at 25°C for 10 min. Thereafter, 3,5-dinitrosalicylic acid color reagent (1 mL; Sigma-Aldrich, Tokyo, Japan) was added, and the reaction was incubated in a boiling water bath for 5 min and then cooled to room temperature. Afterwards, DW (10 mL) was added, absorbance was measured at 540 nm using a UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK). The result was expressed as percentage of inhibition.

Analysis of texture

Texture profile analysis (TPA) was performed using the Texture Analyzer (TA-XT Express 20,096, Stable microsystems Ltd., London, UK). The enzyme-treated Cassava samples (1 cm x 1 cm x 1 cm) were subjected to a two cycle compression test using a 25 kg load cell. In addition, samples were compressed using a 10 mm diameter cylindrical probe (pretest speed 3 mm/s, trigger force 5 g, test speed 3 mm/s, return speed 3 mm/s, test distance 5 mm, time 5 s). TPA recorded the hardness.

Analysis of fiber structure

Fiber structure of the enzyme-treated Cassava sample was observed using a scanning electron microscope (SEM). The SEM (JSM-IT 800, JEOL Ltd., Tokyo, Japan) used in this case is a type of electron microscope, which scans the surface with a focused electron beam to generate images of an image of a sample. Before SEM characterization, samples were thoroughly degreased and dried using a freeze-dryer. It was mounted on a stub, coated with conductive material, and photographed.

Statistical analysis

The results of the experiments were tested with a one-way analysis of variance (ANOVA). If the test results were significant, the test was performed using a Scheffe post-hoc test to analyze the significant difference between the test groups. These statistical analysis was performed using IBM SPSS statistics program (Version 23.0, GraphPad Software Inc., San Diego, CA, USA), and if the p-value was less than 0.05 (p < .05), it was judged to be statistically significant.

Results and discussion

Fatty acid composition

The total fat of Cassava was 2.79%. Cassava’s FA composition consisted of six types as shown in Table 1. Among them, α-linolenic acid (C_18:3n3; omega-3), which is an unsaturated FA, accounted for the largest proportion. Next, it contained a lot in the order of linoleic acid (C_{18:2n6c c}; omega-6), palmitic acid (C_16:0), and oleic acid (C_18:1n9c; omega-9). On the other hand, it contained relatively little saturated FAs such as undecanoic acid (C_11:0) and stearic acid (C_18:0). As such, it can be seen that Cassava is a nutritiously excellent food that can help reduce the risk of developing chronic diseases due to its high rate of unsaturated FA. Previous studies have found that people from Greece and other Mediterranean regions had a low rate of heart disease compared to other locations despite consuming a relatively high-fat diet.^[8] The reason was that it was a diet high in unsaturated FAs. According to another previous study, unsaturated FAs have been reported to help lower LDL cholesterol levels, reduce inflammation, and build stronger cell membranes in the body.^[9] They also announced that they may also help reduce the risk of rheumatoid arthritis. Compared to other root vegetables, the total unsaturated FA content was relatively higher in Cassava (55.58%) than in carrot (17.00%), one of the root vegetables considered beneficial to the health of the elderly.^[10] In particular, the proportion of unsaturated FAs called omega FAs was high, which is one of the nutrients that induce consumption of the elderly to prevent dementia caused by aging.^[11] Omega FAs are known to help maintain normal brain function by reducing the production and accumulation of amyloid plaque, a toxic waste considered to be a major cause of Alzheimer’s disease.

Table 1. Analyzed fatty acid composition of Cassava.

	Fatty acids	Content (%)^1)
Saturated fatty acids	C(11:0)	3.29 ± 0.12
	C(16:0)	21.71 ± 0.74
	C(18:0)	2.91 ± 0.19
	Total	27.91 ± 1.05
Unsaturated fatty acids	C(18:1n9c)	19.80 ± 1.15
	C(18:2n6c)	22.14 ± 0.95
	C(18:3n3)	30.15 ± 1.15
	Total	72.09 ± 3.25
Total fat		2.79 ± 0.11

1) Results are shown as mean ± standard deviation in triplicate.

In this study, the ratio of omega-3, −6, and −9 of Cassava was 1.52:1.12:1. Considering that the ideal ratio of omega-3, −6, and −9 is 2:1:1^[12] it is considered that the ratio of Cassava in this study showed positive result. Of course, it is important to take enough of each of them because omega-3,-6, and −9 have different unique properties and effects. Nevertheless, the ratio between them is also very important for optimal health conditions. If people consume too much omega-6 compared to omega-3, it may cause an inflammatory response. And aside from the inflammatory response, another area where the importance of this ratio shows up very clearly is in brain health. A deficit in omega-3 leads to an increase in omega-6 in brain tissue. This can result in diminished brain growth, neurotransmitter function, and altered learning. Therefore, it can be said that eating foods with an ideal ratio of omega-3, −6, and −9 such as Cassava is particularly important for the elderly whose brain cognitive function is deteriorated due to aging.

Mineral composition

The mineral composition of Cassava is shown in Table 2, and consists of eight minerals. Among them, the K content was the highest, followed by Mg, Ca, P, Zn, Fe, Cu, and Na. Compared to other root vegetables, the K content of Cassava (265.07 mg/100 g) was similar to that of the radish (233 mg/100 g) known as an excellent K source.^[10] The Mg content of Cassava (16.19 mg/100 g) was lower than that of the beet (23 mg/100 g), while that of the carrot (12 mg/100 g) was higher. The Ca content of Cassava (32.51 mg/100 g) was lower than that of lotus root (45 mg/100 g) and celeriac (43 mg/100 g), while it was about twice as high as that of the ginger root (16 mg/100 g). The P content of Cassava (27.33 mg/100 g) was similar to that of turnip (27 mg/100 g). The Zn content of Cassava (1.74 mg/100 g) was similar to that of the wasabi (1.62 mg/100 g) and was about three to four times higher than that of parsnip (0.59 mg/100 g) and shallot (0.40 mg/100 g). The Fe content of Cassava (0.20 mg/100 g) was lower than that of the kohlrabi (0.40 mg/100 g), while it was similar to that of the onion (0.21 mg/100 g). The Cu content of Cassava (0.19 mg/100 g) was slightly higher than that of sweet potato (0.15 mg/100 g) and artichoke (0.13 mg/100 g). The Na content of Cassava (10.02 mg/100 g) was lower than that of the potato (16.00 mg/100 g), while it was about twice as high as burdock root (5.00 mg/100 g). Through these research results, it was confirmed that Cassava is rich in minerals involved in various biological reactions such as enzyme reaction, energy formation, and nerve stimulation transfer. So, like carrot and radish, Cassava is thought to be a good source of mineral substances to help the elderly balance their nutrition. Previous studies have shown that people with Alzheimer’s disease have low levels of Mg in their bodies.^[13] In addition to Mg, it was suggested that there was a significant association between K and Ca intake and decreased cognitive function. As such, mineral deficiency in the elderly can cause various negative symptoms including cognitive decline. That is why a mineral-rich diet is especially important for the healthy life of the elderly.^[14]

Table 2. Analyzed mineral composition of Cassava.

Minerals	Content (mg/100 g)^1)
Fe	0.20 ± 0.01
Mg	16.19 ± 0.78
P	27.33 ± 1.12
K	265.07 ± 12.67
Na	10.02 ± 0.46
Zn	1.74 ± 0.07
Cu	0.19 ± 0.01
Ca	32.51 ± 1.63

1) Results are shown as mean ± standard deviation in triplicate.

Antioxidant effects

Total polyphenol content

Table 3 shows that the total polyphenol content of Cassava (31.95 g/100 g) is higher than that of carrot (18.41 g/100 g), known as one of the representative antioxidant foods among root vegetables.^[15] Phenols found in Cassava are functional substances commonly found in natural foods and are known to help with antibacterial, antiviral, anticancer, and anti-inflammatory functions.^[16] Furthermore, previous studies have reported that phenols have a significant effect on the brain, including the potential to protect neurons from injury caused by neurotoxins, the ability to suppress neuro-inflammation, memory, learning, and cognitive functions.^[17] So, phenols are considered one of the most promising factors in preventing cognitive decline and Alzheimer disease due to aging. Therefore, Cassava is thought to be a good source of polyphenol and can be used as an elder-friendly food.

Table 3. Analyzed antioxidant and antidiabetic effects of Cassava.

Physiological activities		Status^1)
Antioxidation effects	Total polyphenol content (GAE g/100 g)	31.95 ± 1.37
	Total flavonoid content (RE g/100 g)	0.85 ± 0.10
	Superoxide dismutase activity (U/g)	12.07 ± 0.53
	DPPH radical scavenging activity (%)	35.18 ± 1.12
	ABTS radical scavenging activity (TE mg/g)	36.45 ± 1.22
	Ferric ion reducing antioxidant power (μmol/g)	30.96 ± 1.18
Antidiabetes effects	α-Glucosidase inhibition (%)	69.02 ± 3.01
	α-Amylase inhibition (%)	36.30 ± 1.66

1) Results are shown as mean ± standard deviation in triplicate.

Total flavonoid content

The total flavonoid content of Cassava (0.85 g/100 g) was found to be higher than that of carrot (0.06 g/100 g), one of the representative antioxidant foods among root vegetables.^[18] A study by Zendehbad and Malla also showed that Cassava has excellent free radical scavenging ability due to its high flavonoid content.^[19] In another preceding study, it was reported that Cassava contains other beneficial bioactive substances in addition to antioxidants, which helps protect nerve cells and has a positive effect on brain function.^[20] Therefore, it is thought that Cassava is a good source of flavonoids and is worth using as an elder-friendly food.

Superoxide dismutase activity

SOD activity (12.07 U/g) of Cassava was similar to that of tuberous root (11 ~ 13 U/g), and it was found that Cassava was also one of the root vegetables as a good antioxidant source.^[21] Harmful stimulation of superoxide anions not only damages somatic cells but also causes problems such as reduced ATP production, defects in protein synthesis, cytoskeletal damage, and DNA degradation. However, it has been reported that superoxide anions may be removed by antioxidants such as vitamins, polyphenols, and flavonoids.^[4] Therefore, Cassava is not only high in polyphenol and flavonoid content but also has good SOD activity, so it is thought that it may be used as an elder-friendly food.

DPPH radical scavenging activity

Cassava’s DPPH radical scavenging activity was 35.18%, lower than that of tomato (58.16%) and pumpkin (42.96%), but similar to spinach (36.34%), which is known to be one of the good sources of antioxidants.^[22] According to previous study, it has been reported that active radicals generated by oxidation stress can damage brain cells and increase the risk of Parkinson’s disease.^[23] However, antioxidants contained in natural foods such as Cassava are known to lower the risk of developing such disease and help brain cells grow and improve cognitive function. Meanwhile, considering that the analysis conditions are slightly different for each previous study, it would be nice if further research on this could be conducted in the future.

ABTS radical scavenging activity

Cassava’s ABTS radical scavenging activity was 3645 mg/100 g, which was superior to carrot (2888 mg/100 g) known as one of the representative antioxidant foods among root vegetables.^[24] Radical scavengers contained in these natural foods inhibit the formation of reactive oxygen species, preventing somatic cell damage. According to previous study, reactive oxygen species are highly reactive and unstable compounds, which are known to cause oxidative damage to cellular components such as DNA, proteins, and lipids.^[25] Radical scavengers, however, are removed before preventing the formation of these reactive species or damaging vital components of the cell. Therefore, Cassava is expected to be valuable as an elder-friendly food.

Ferric ion reducing antioxidant power

Cassava’s FRAP was 3096 μmol/100 g, lower than carrot (5690 μmol/100 g) compared to other root vegetables, but higher than potato (2320 μmol/100 g).^[25] It is well recognized that transport to market, storage, and cooking practices affect the content of labile antioxidants in foods, and the World Health Organization considers this information into account in their recommendations for vitamin and mineral requirements in human nutrition.^[26] Therefore, the results of this study are expected to be used as basic data for the elder-friendly food industry with excellent antioxidant ability, which has recently been in the spotlight.

Antidiabetic effects

In order to confirm the anti-diabetic properties of Cassava, α-amylase and α-glucosidase enzyme inhibition rates were analyzed. As a result, the α-glucosidase inhibition rate of Cassava was 69.02%, similar to eggplant (70.78%).^[27] The α-amylase inhibition rate of Cassava was 36.30%, similar to okra (36.84%) and stem amaranth (40.82%). According to previous study, α-amylase and α-glucosidase are carbohydrate digestion-related enzymes that can rapidly increase blood glucose levels after meals by decomposing polysaccharides into glucose, and their activity has been reported to be related to phenolic content.^[28] Another preceding study also reported that antioxidant compounds such as glycosides, polysaccharides, steroids, and terpenoids present in natural foods had an effect on reducing diabetes-related enzyme activity.^[29] Therefore, it is expected that the antioxidant ability of Cassava discussed above may have a positive effect on anti-diabetic activity. And considering the anti-diabetes characteristics as well as antioxidant properties, Cassava is thought to be useful as an elder-friendly food.

Texture property

Texture is a very important factor, especially for the elderly, because the decline in chewing ability due to aging in one of the causes of nutritional imbalance.^[3] As such, texture is one of the main parameters that determine the value of the sensory characteristics of root vegetables. Therefore, softening is essential for Cassava, which has a hard texture, to be ideally used as an elder-friendly food. Foods with an ideal texture for the elderly can be easily reduced to safe particle sizes when swallowed with minimal chewing effort.^[30] On the other hand, tough or hard food may be formed in a volume that is difficult to swallow because it is difficult for the elderly to chew. So, research on elder-friendly foods with a texture that can easily decomposed into the minimum strength and number of chews is needed. Therefore, this study attempted to develop Cassava as an elder-friendly food that satisfies KS by treating it with enzymes.

As a result (Table 4), Cassava treated with cellulase and α-amylase significantly softened most quickly. When treated for 1–3 h, it had the hardness of KS that could be chewed with the elderly’s gum. And in order to have a hardness to chew with the elderly’s tongue, Cassava had to be treated for at least 4 h. Second, when Cassava was treated with α-amylase and polygalacturonase, it had a hardness to chew with the elderly’s gum when treated for 2–3 h. And in order to have a hardness to chew with the elderly’s tongue, it had to be treated for at least 4 h. Third, when Cassava was treated with polygalacturonase and cellulase, it had a hardness to chew with the elderly’s gum when treated for 30 min to 1 h. And in order to have a hardness that could be chewed with the elderly’s teeth, it had to be treated for at least 2 h. So, it is expected to take at least 5 h or more to have a hardness that can be chewed with the elderly’s tongue. Considering that amylase breaks down starch, cellulase breaks down cellulose, and polygalacturonase breaks down pectin, it can be inferred that Cassava’s carbohydrate (38.1%) has the lowest pectin ratio and is composed of relatively more starch than pectin.

Table 4. Hardness of Cassava treated with enzymes by time.

Time (hour)	Hardness^1)			F-value^2) (p-value)
	Cellulase + α-amylase	α-Amylase + polygalacturonase	Polygalacturonase^2 + cellulase^1
0.0	77710.20 ± 2335.63^A	77710.20 ± 2335.63^A	77710.20 ± 2335.63^A	0.000 (1.000)
0.5	79317.75 ± 3937.54^A	75408.20 ± 1909.90^A	76435.80 ± 1462.81^A	0.757 (0.509)
1.0	48907.95 ± 2114.42^Bc	62004.95 ± 2837.78^Bb	72781.60 ± 2096.27^Aa	75.964*** (0.000)
2.0	34836.20 ± 1372.13^Cb	42921.30 ± 2098.95^Ca	46770.85 ± 1361.41^Ba	40.932*** (0.000)
3.0	22302.30 ± 902.11^Dc	28705.15 ± 1177.58^Db	32145.45 ± 898.20^Ca	75.655*** (0.000)
4.0	5730.60 ± 203.00^Ec	18367.70 ± 300.01^Eb	24067.75 ± 1141.37^Da	554.477*** (0.000)
F-value (p-value)	565.008*** (0.000)	477.247*** (0.000)	637.337*** (0.000)

1.Results are shown as mean ± standard deviation in triplicate.

2.One-way ANOVA was used, and different letters in the same row (a ~ c) and column (A ~ F) show significant differences at ***p < .001, respectively.

Based on these results, a linear regression equation was derived (Figure 1), and using this, the time when Cassava should be treated with enzymes to suit KS was inferred. As a result, when treated with cellulase and α-amylase, it was inferred that the treatment for at least 1.14 h would have KS’s 1st level hardness, the treatment for 1.41 to 2.96 h would have KS’s 2nd level hardness, and the treatment for 3.07 h or more would have KS’s 3rd level hardness. In the case of treatment with α-amylase and polygalacturonase, it was inferred that the treatment for at least 1.49 h would have KS’s 1st level hardness, the treatment for 1.80 to 3.56 h would have KS’s 2nd level hardness, and the treatment for 3.69 h or more would have KS’s 3rd level hardness. In the case of treatment with polygalacturonase and cellulase, it was inferred that the treatment for at least 1.75 h would have KS’s 1st level hardness, the treatment for 2.08 to 3.92 h would have KS’s 2nd level hardness, and the treatment for 4.06 h or more would have KS’s 3rd level hardness. The results of this study are meaningful in that they confirm the excellence of nutritional characteristics and increase the accessibility of the elderly for Cassava. In addition, as the recent increase in the elderly population is drawing attention to the use of enzyme treatments that soften root vegetables for elderly consumers, such research is expected to help the growth of the elder-friendly food industry. Based on these studies, the development of elder-friendly foods with excellent physiological activity should be continuously carried out.

Figure 1. Regression equation for hardness of Cassava treated by enzyme and time. The Y-scale is hardness (N), and the X-scale is enzyme treatment time (hour). The coefficient of determination (R²) is a number between 0 and 1 that measures how well a statistical model predicts an outcome, and a higher coefficient is an indicator of a better goodness of fit for the observations. KS’s hardness of the first level is 55,000 ~ 500,000 N, the second level is 22,000 ~ 50,000 N, and the third level is less than 2,000 N.^[3] .

Electron microscopic structure

Changes in the structure and shape of the internal microstructure of Cassava before and after enzyme treatment were examined (Table 5). The control group without enzyme treatment did not have significant pores or cracks and showed a regular internal structure. On the other hand, in the enzymatically treated samples, a change to a non-uniformity and an open formation structure generally occurred, and this change is expected to alleviate the hardness of Cassava. That is, in the enzyme-treated samples, the pore size between the internal tissues increased, and the number of cracks and holes also tended to increase. In addition, it was confirmed that the microstructure was more deformed and the tissue arrangement became more irregular as the enzyme treatment time elapsed. These changes appear to be due to the fact that some cell walls are destroyed by heating and the number and size of pores and cracks in the internal tissue are increased by the chemical reaction of the enzyme during the immersion process, resulting in a large surface area for enzymes to act on and soften more effectively.^[31] Similar structural changes were observed in Tiwari et al.’s study of processing carrots using cellulose.^[32] As in this study, the SEM image of the control group showed a relatively smooth surface and some cracks. In contrast, enzyme-treated samples showed that the enzyme activation process caused the microstructure to deform, resulting in the development of a porous surface. In addition, more open pores were observed in the enzyme-treated sample, while more closed pores with more collapsed structures were observed in the control group. As such, in both this study and previous studies, the control group used as a comparison presented a morphological structure distinct from the enzyme-treated sample. It is thought that these changes in internal organization may be correlated with hardness. That is, it is expected that the enzyme treatment helped efficiently increase the softness of Cassava.

Table 5. SEM micrograph of Cassava treated with enzymes by time.

Time (hour)	Enzyme^1)
	Cellulase	Polygalacturonase	α-Amylase
0.0
0.5
1.0
2.0
3.0
4.0

1) SEM microscope filmed at 1,800 ×.

Conclusion

This study proved that Cassava contains a variety of bioactive nutrients, including minerals, essential FAs, antioxidants, and antidiabetic substances. That is, it was confirmed that Cassava could be used as an elder-friendly food, and that it could be developed as an elder-friendly food suitable for KS through enzyme treatment for softening. Compared to the control group, the hardness, which is the texture parameter of the experimental group was significantly decreased, so it seems that the enzyme treatment as a pretreatment method could act as a root vegetable softener with the ability to improve softness. And enzymes treatment can help reduce total cooking time and fuel expenditure, as well as help elderly people chew hard root vegetables like Cassava more easily and smoothly and safely swallow them. So, it seems that elderly consumers with reduced chewing and swallowing function will be able to access and consume root vegetables more easily. As such, the development of elder-friendly foods suitable for KS is very important for the healthy quality of life of the elderly. Therefore, it is expected that such research will help increase the consumption of elder-friendly foods and develop related industries.

Disclosure statement

No potential conflict of interest was reported by the authors.